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Proton spin relaxation as a means of characterizing the pathology of multiple sclerosis Stewart, Wendy Anne 1989

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PROTON SPIN RELAXATION AS A MEANS OF CHARACTERIZING T H E PATHOLOGY OF MULTIPLE SCLEROSIS By Wendy Anne Stewart B. Sc. (Chemistry) University of Dundee M . Sc. (Chemistry) University of British Columbia A T H E S I S S U B M I T T E D I N P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F D O C T O R O F P H I L O S O P H Y in T H E F A C U L T Y O F G R A D U A T E S T U D I E S C H E M I S T R Y -We accept this thesis as conforming to the required standard T H E U N I V E R S I T Y O F B R I T I S H C O L U M B I A March 1989 © Wendy Anne Stewart, 1989 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia Vancouver, Canada DE-6 (2/88) A b s t r a c t Multiple sclerosis (MS) is a demyelinating disease of the central nervous system; the cause(s) of the disease and its pathological progression are at present unknown. Magnetic resonance imaging (MRI), in particular the spin-echo radiofrequency pulse sequence, proved a powerful tool in the study of MS. The question addressed in this thesis, is whether quantitative measurement of the spin-lattice (Xi) and spin-spin (T2) relaxation times can provide a means of characterizing the pathology of MS in vivo; the final goal being to use these parameters to differentiate acute inflammatory lesions from more chronic demyelinating and gliotic ones. Quantitative MR imaging methods, using two-point determinations of Ti and T2, were first used to study post mortem and fixed brain tissue from MS patients. These data demonstrated differences in Ti and T 2 with varying degrees of demyelination and gliosis; however, chronic MS pathology is similar in all cases, and provides little information on the earlier stages of the disease process which are believed to be immune-mediated. The early immune-mediated lesion was then examined by studying experimental allergic encephalomyelitis (EAE), an animal model for MS, in primates. The data obtained from these studies confirmed that the values of Ti and T 2 obtained from these two-point determinations are sensitive to variable pathology, but cannot be used to distinguish acute from chronic lesions. An in-depth in vitro T2 study was then carried out on guinea pigs inoculated to produce EAE; this was followed by repeat measurements on the formalin fixed tissue. T2 measurements were also carried out on samples from the fixed MS tissue already studied using quantitative imaging methods. The relaxation data were analyzed using two different methods; the first assuming a fixed number of exponentials (minuit), and the second a continuous distribution of relaxation ii times (NNLS). It is clear from these studies that the models currently used to describe the relaxation behaviour of water protons in tissue are inadequate to explain the results observed. Both analysis methods were sensitive to the different types of pathology present; however, assuming a continuous distribution of relaxation times not only provides a better fit to the data, but also represents a more realistic picture of the complex physical system being studied. These results show that T2 data obtained from tissue are reproducible, and can distinguish differing degrees of inflammation. The same trends observed in vitro were also observed in the fixed tissue; comparison of the fixed tissue data from inflammatory lesions with that from MS tissue showed that T2 can be used to differentiate acute and chronic lesions. This implies that quantitative M R measurements, together with the sophisticated methods of analysis employed in this thesis, can be used to follow the pathological progression of MS; this would provide insight into the disease process and lead eventually to an understanding of the cause(s) of the disease. i i i Table of Contents Abstract ii List of Tables vii List of Figures xiv List of Abbreviations xxv Acknowledgements xxx 1 General Introduction and Thesis Goal 1 1.1 General Background 1 1.2 Basic Principles of Magnetic Resonance 9 1.3 M R Imaging and Image Enhancement 13 1.3.1 Slice Selection 15 1.3.2 Image Enhancement 17 1.4 Relaxation Measurements 19 1.4.1 Measurement of T i 19 1.4.2 Measurement of T2 20 1.4.3 Imaging Methods 21 2 M R I of Multiple Sclerosis 24 2.1 Introduction 24 2.2 Methods 30 iv 2.3 Results 3 5 2.3.1 Case 1 35 2.3.2 Case 2 42 2.3.3 Case 3 46 2.3.4 Case 4 50 2.4 Summary of Results 54 2.5 Discussion 59 3 MRI of Experimental Allergic Encephalomyelitis in Primates 64 3.1 Introduction 64 3.2 Methods 66 3.2.1 Induction of EAE 66 3.2.2 MRI Protocol . 69 3.2.3 Pathology 73 3.3 Results 7 4 3.3.1 Case 1 . . . 74 3.3.2 Case 2 80 3.3.3 Case 3 8 3 3.3.4 Case 4 91 3.3.5 Cases 6 and 7: Gd-DTPA Study 98 3.3.6 Case 8 98 3.3.7 Case 9 1 0 6 3.3.8 Case 10 1 1 3 3.4 Summary of Results 121 3.5 Discussion • • • 124 v 4 In Vitro Quantitative Relaxation Measurements 130 4.1 Practical Aspects 130 4.2 Tissue Relaxation Models 131 4.3 Methods 138 4.3.1 T-i Measurements 138 4.3.2 Ti Measurements 140 4.3.3 Analysis of Relaxation Data 141 4.3.4 Spectroscopy 143 4.3.5 Protocol for Studying Tissue 143 4.4 Results 148 4.4.1 Guinea Pig Tissue 148 4.4.2 Primate Tissue 164 4.4.3 Fixed MS Tissue 169 4.5 Discussion 176 5 Conclusions and Suggestions for Further Work 191 5.1 Conclusions . . . 191 5.2 Suggestions for Further Work 194 A Extraction of Myelin Basic Protein from CNS Tissue 196 B Results of N N L S Analysis of Guinea Pig CNS Tissue 197 References 200 vi List of Tables 2.1 Table showing details of scoring for pathology of sections taken from the inferior surface of each 10 mm thick brain slice. M N C - mononuclear cells, M O - macrophages 38 2.2 Pathological score for the three lesions seen in the right cerebral hemi-sphere of slice 3, case 1 40 2.3 Quantitative M R I data from fixed brain in case 1. T\N and T2N correspond to the average values of T\ and T2 from normal appearing white matter in the same slice 41 2.4 Correlation between lesion area as seen on post mortem and fixed brain M R I and the actual lesion area seen on the inferior surface of each 10 mm thick brain slice in case 2 42 2.5 Pathological score for section taken from the inferior surface of slice 4, case 2 44 2.6 Ti and T2 values from M R I number 5, which is equivalent to the inferior surface of fixed brain slice 4, case 2. The area from which these values were obtained corresponds to that from where the section was taken. Cadaver T1N = 342± 30 ms(18), cadaver T2N = 84± 7 ms(17), fixed brain TlN = 217± 10 ms(6) and fixed brain T2N = 66± 7 ms(22). The numbers in brackets give the number of areas from which the mean and standard deviation were determined 45 vii 2.7 Correlation between lesion area as seen on post mortem and fixed brain M R I and the actual lesion area seen on the inferior surface of each 10 mm thick brain slice in case 3 46 2.8 Pathological score for sections taken from fixed brain slices 5 and 6, case 3. L - left cerebral hemisphere and R - right cerebral hemisphere 49 2.9 T\ and T2 values from areas examined microscopically. Post mortem TIN = 368± 21(24), T2N = 91± 9(7), and fixed brain T1N = 218± 10(18), T2N = 71± 6(24). Bracketed numbers denote the number of areas from which the mean and standard deviation were determined. P M - post mortem, F B - fixed brain 49 2.10 Comparison of lesion area seen on the post mortem and fixed brain M R I in case 4. Slice 1 is the sagittal pilot scan and is not included 50 2.11 Pathological score for lesions taken from slices 5 and 6, case 4. L - left cerebral hemisphere, and R - right cerebral hemisphere 53 2.12 Ti and T2 values from area examined microscopically in case 4. Post mortem TIN = 381± 12(14), T2N = 77± 9(20) and fixed brain T1N = 207± 14(20), T2N = 65± 5(24). The numbers in brackets give the number of areas from which the mean and standard deviation were determined. P M - post mortem, F B - fixed brain 53 2.13 Quantitative area measurements from individual lesions. P M - post mortem, F B - fixed brain, B R A I N - data from digitized brain slice 54 2.14 Quantitative M R I data from normal appearing white matter as seen on the post mortem and fixed brain SE M R I 55 viii 2.15 Ti and T 2 values from computed images, correlated with severity of de-myelination in cases 1-4. Lesions are denoted by the same numbers as those used in the text. P M - post mortem, F B - fixed brain, A R E A - size and number of lesions 56 2.16 Ti and T 2 values from computed images, correlated with severity of gliosis in cases 1-4. Lesions are denoted by the same numbers as those used in the text. P M - post mortem and F B - fixed brain 57 2.17 Ti and T 2 values from computed images, correlated with severity of in-flammation in cases 1-4. Lesions are denoted by the same numbers as those used in the text. P M - post mortem, F B - fixed brain, A R E A -denotes size and number of lesions 58 2.18 Mean intensity and standard deviation from regions consisting of 18 pixels on a computed T 2 image, from regions of normal appearing white matter. P M - post mortem and F B - fixed brain 60 2.19 Post mortem Ti and T 2 values from normal appearing white matter in case 4, showing the effects of multi-slice interference. The numbers in brackets give the number of ROI used to determine the mean and error 63 3.20 Clinical scoring for monkeys inoculated to produce E A E 67 3.21 T i and T 2 values from lesions in cases 1, 2, 3, 4 and 9 obtained at the time of death 123 4.22 Pathological scoring for guinea pig CNS tissue 145 ix 4.23 Results from minuit analysis of CPMG data obtained from the cervical spine of normal control guinea pigs. T2 (ms) values are given together with the percentage contribution of each component to the signal observed. Pathological examination of this tissue confirmed that each section was normal 150 4.24 Results from minuit analysis of CPMG data obtained from the cervical spine of guinea pigs inoculated to produce EAE. T2 (ms) values are given together with the percentage contribution of each component to the signal observed 151 4.25 Pathology from the cervical spine of guinea pigs inoculated to produce EAE. PVI-SS - perivascular inflammation in the subarachnoid space, PVI-VR - perivascular inflammation in the Virchow-Robin spaces, PCI - parenchymal cellular infiltration, D - demyelination and E - edema. . . 152 4.26 Results from minuit analysis of CPMG data from the upper thoracic spine of normal control guinea pigs. T2 (ms) values are given together with the percentage contribution of each component to the total signal observed. . 152 4.27 Results from minuit analysis of CPMG data from the upper thoracic spine of guinea pigs inoculated to produce EAE. T2 (ms) values are given to-gether with the percentage contribution of each component to the total signal observed 153 4.28 Pathology from the upper thoracic spine of guinea pigs inoculated to pro-duce EAE. PVI-SS - perivascular inflammation in the subarachnoid space, PVI-VR - perivascular inflammation in the Virchow-Robin spaces and PCI - parenchymal cellular infiltration 153 x 4.29 Results from minuit analysis of CPMG data from the lower thoracic spine of normal control guinea pigs. T 2 (ms) values are given together with the percentage contribution of each component to the total signal observed. . 154 4.30 Results from minuit analysis of CPMG data from the lower thoracic spine of guinea pigs inoculated to produce EAE. r 2 (ms) values are given to-gether with the percentage contribution of each component to the total signal observed 154 4.31 Pathology from the lower thoracic spine of guinea pigs inoculated to pro-duce EAE. PVI-SS - perivascular inflammation in the subarachnoid space, PVI-VR - perivascular inflammation in the Virchow-Robin spaces and PCI - parenchymal cellular infiltration 155 4.32 Results from minuit analysis of CPMG data from the lumbar spine of normal control guinea pigs. T2 (ms) values are given together with the percentage contribution of each component to the total signal observed. . 155 4.33 Results from minuit analysis of CPMG data from the lumbar spine of guinea pigs inoculated to produce EAE. T2 (ms) values are given together with the percentage contribution of each component to the total signal observed 156 4.34 Pathology from the lumbar spine of guinea pigs inoculated to produce EAE. PVI-SS - perivascular inflammation in the subarachnoid space, PVI-V R - perivascular inflammation in the Virchow-Robin spaces and PCI -parenchymal cellular infiltration 156 4.35 Results from minuit analysis of CPMG data from the sacral spine of normal control guinea pigs. T 2 (ms) values are given together with the percentage contribution of each component to the total signal observed 157 xi 4.36 Results from minuit analysis of CPMG data from the sacral spine of guinea pigs inoculated to produce EAE. The T2 (ms) values are given together with the percentage contribution of each component to the total signal observed 157 4.37 Pathology from the sacral spine of guinea pigs inoculated to produce EAE. PVI-SS - perivascular inflammation in the subarachnoid space, PVI-V R - perivascular inflammation in the Virchow-Robin spaces and PCI -parenchymal cellular infiltration 158 4.38 Results from minuit analysis of CPMG data from the left cerebral hemi-sphere of guinea pigs inoculated to produce EAE. T2 (ms) values are given together with the percentage contribution of each component to the total signal observed 158 4.39 Pathology from the left hemisphere of guinea pigs inoculated to produce EAE. PVI-SS - perivascular inflammation in the subarachnoid space, PVI-V R - perivascular inflammation in the Virchow-Robin spaces and PCI -parenchymal cellular infiltration 159 4.40 Results obtained from minuit analysis of CPMG data from fixed guinea pig lumbar spine. T2 values are given together with the percentage con-tribution of each component to the signal observed 165 4.41 Results obtained from minuit analysis of CPMG data from fixed guinea pig lower thoracic spine. T2 values are given together with the percentage contribution of each component to the signal observed 166 4.42 Results from minuit analysis of CPMG data from primate case 8. T2 (ms) values are given together with the percentage contribution of each component to the total signal observed. W M - white matter, G M - grey matter, L - lesion, LMB - left midbrain 166 xii 4.43 T 2 values obtained using minuit to analyze CPMG data from fixed MS tissue. Lesions are numbered as they were in chapter 2, and W M - nor-mal appearing white matter, G M - normal appearing grey matter and G M / W M - mixture of grey and white matter. Two values are given for case 4 W M , the data were obtained six months apart 169 4.44 Results from minuit analysis of CPMG data from the homogenized brain and spine of a normal guinea pig. T 2 values (ms) are given together with the percentage contribution of each component to the total signal observed. B - brain tissue and C/T - cervical/thoracic spine. Two sets of data are given for C/T, determined 4 hours apart 180 xiii List of Figures 1.1 Diagram of a nerve cell or neuron 1 1.2 The behaviour of spins in the presence of two orthogonal magnetic fields. A. Precession about BQ. B. Behaviour of spins after the application of a second magnetic field, B\, perpendicular to Ba 10 1.3 Diagram showing the variation of the static magnetic field, Ba in the pres-ence of a linear field gradient, Gx 13 1.4 Two-dimensional Fourier transform spin-echo pulse sequence for slice se-lective imaging 14 1.5 Diagram showing selective excitation of a slice. A. Selective rf pulse and slice select gradient. B. Magnetization in the z-direction after application of selective pulse. C. Selected slice 16 2.6 Diagram showing the simultaneous excitation of normal and abnormal tissue in a 10 mm thick slice due to the irregular shape of the lesion. . . 29 2.7 A. Multi-slice spin-echo pulse sequence showing variable time parameters. B. Multi-slice inversion-recovery pulse sequence, n = the number of slices in both sequences 31 xiv 2.8 A . Sagittal pilot scan obtained post mortem. Grid shows the center of each of the 12 transverse images to be obtained. B . Sagittal pilot scan from fixed brain. Grid shows the center of each of the 12 coronal images to be obtained. The coronal images of the fixed brain are equivalent to the transverse images obtained in situ. T E = 26 ms and T R = 400 ms in both images 33 2.9 Comparison of the inferior surface of a 10 mm thick brain slice in case 1 with the corresponding MRI . A . Spin echo M R I level 3, T E = 40 ms and the T R = 2 sec. B . Inferior surface of fixed brain slice at the same level. 36 2.10 Display of SE M R I level 3 together with the equivalent digitized negative of fixed brain slice on the Vax 11/750 computer. The lesions are outlined on both the image and the digitized brain slice. Lesion area on M R I = 489 mm 2 and actual lesion area on brain slice = 562 mm 2 37 2.11 Comparison of gross pathology with the spin echo M R I . A . SE M R I level 4, T E = 40 ms and T R = 2 sec. B . Inferior surface of 10 mm thick fixed brain slice at the equivalent level 39 2.12 Section taken from slice 3, case 1 stained with P T A H . The section shows lesion 8, with cystic spaces 40 2.13 Comparison of the SE M R I with the gross pathology. A . Post mortem SE M R I at level 4. T E = 40 ms and T R = 2 sec. B . Fixed brain SE M R I at equivalent level. Parameters as in A . C. Inferior surface of fixed brain slice at the equivalent level 43 2.14 Inferior surface of fixed brain slice 4, case 2. The box shows the position of the section taken for microscopic examination 44 2.15 Comparison of SE image with the gross pathology. A. Post mortem SE MRI at level 5. TE = 40 ms and TR = 2 sec. B. Fixed brain SE MRI at the equivalent level. Parameters as in A. C. Inferior surface of fixed brain slice at the equivalent level 47 2.16 Comparison of SE image with the gross pathology. A. Fixed brain SE MRI at level 6. Tau = 40 ms and TR = 2 sec. B. Fixed brain SE MRI at the equivalent level. Parameters as in A. C. Inferior surface of fixed brain slice at the same level 48 2.17 SE MRI from case 4. A. Post mortem MRI level 4. B. Fixed brain MRI at the same level. In both images tau = 40 ms and TR = 2 sec 51 2.18 A. Inferior surface of fixed brain slice 5, case 4. Box shows the position of the section that was removed for microscopic examination. B. Fixed brain slice 6, case 4, showing the two sections removed for microscopic examination 52 2.19 Computed Ti and T 2 images from the fixed brain in case 3. A. T\ image. B. T 2 image 61 3.20 A. Sagittal pilot scan of monkeys brain. The grid shows the position of the 8 transverse slices to be obtained. B. Sagittal pilot scan of fixed monkey brain. The grid shows the position of the 8 coronal slices to be obtained. The coronal slices of the fixed brain are equivalent to transverse images in vivo. In both images TE = 26 ms and TR = 800 ms 70 3.21 Image of monkey brain showing lesions outlined using a trackball 71 3.22 Serial SE20oo/4o MRI obtained from case 1. A. Day 15 after inoculation. B. Day 18 after inoculation 75 xvi 3.23 Comparison of the gross distribution of lesions with the equivalent MRI in case 1. A. SE2000/40 image on day 23 showing lesion in the brain stem. B. The inferior surface of the 5 mm thick brain slice at the same level. . 77 3.24 Histopathology of lesion in case 1 A. H & E showing diffusion of polymor-phonuclear cells, lymphocytes and macrophages into the tissue around the vessel. B. LFB- PAS-H) showing LFB-positive myelin debris at the centre of the lesion. C. Holmes' stain showing swelling, beading and destruction of axons at the centre of the lesion 78 3.25 ORO-H of frozen section showing macrophages filled with granular su-danophilic lipids in case 1 79 3.26 Plot of T\ and T2 versus time after inoculation in case 1. Ti and T2 values are given from normal appearing white matter plus or minus two standard deviations 79 3.27 Comparison of the SE MRI with the gross pathology in case 2. A. SE2000/40 MRI on day 22 after inoculation. B. The inferior surface of 5 mm thick brain slice at the same level 81 3.28 Histopathology of lesion in case 2. Section stained with H & E showing the collar of hemorrhage around the vessel displacing the exudate peripherally. 82 3.29 Serial SE2000/40 MRI from case 3. A. Day 11 after inoculation. B. Day 12 after inoculation 84 3.30 SE MRI from fixed brain. The lesions in both hemispheres are pale com-pared to the bright areas seen on the in vivo images 85 3.31 Comparison of the SE MRI with the gross pathology in case 3. A. SE2ooo/6o MRI slice 4. B. The inferior surface of 5 mm thick brain slice at the same level 86 xvii 3.32 Comparison of SE M R I with the gross pathology in case 3. A . SE2ooo/40 M R I at level 3. B . The inferior surface of 5 mm thick brain slice at the same level 87 3.33 Comparison of the SE M R I with the gross pathology in case 3. A . SE2ooo/4o M R I slice 5. B . The inferior surface of 5 mm thick brain slice at the same level 88 3.34 Histopathology of lesion observed in the left cerebral hemisphere at M R I level 3. Section stained with H & E showing compact exudate similar to that seen in cases 1 and 2 89 3.35 Plot of Ti and T 2 versus time after inoculation for case 3. The dashed line denotes data from normal tissue plus or minus two standard deviations. 90 3.36 SE20oo/4o obtained from case 4 on day 16 after inoculation 91 3.37 Serial SE20oo/40 M R I from case 4. A . Day 16 after inoculation. B . Day 18 after inoculation. C. Day 19 after inoculation 92 3.38 Fixed brain SE20oo/40 M R I from case 4 showing abnormal area in the left cerebral hemisphere. This gliotic lesion appears as a bright area of in-creased T 2 , just as was seen in the fixed brains of chronic MS patients. . 93 3.39 Comparison of the SE M R I with the gross pathology in case 4. A . Post mortem SE20oo/6o M R I at level 5. B . The inferior surface of 5 mm thick brain slice at the same level 94 3.40 Histopathology of posterior and medial part of lesion in the left visual radiation of case 4. Section stained with gallo-DR 96 3.41 Histopathology of lesion in the corpus callosum of case 4. Section stained with gallo-DR 97 xviii 3.42 Plot of Ti and T 2 versus time after inoculation for case 4. Ti and T 2 values from normal appearing white matter are given plus or minus two standard deviations 97 3.43 SE MRI from case 7 on day 22 after inoculation. A. Slice 5. B. Slice 6. . 99 3.44 Gd-DTPA scan from case 7 showing breakdown in the blood brain barrier where lesion is enhanced in slice 5. A. Slice 5. B. Slice 6. In both images TE = 26 ms, TR = 500 ms and number of PE increments = 256 100 3.45 SE MRI showing preclinical lesions on day 26 after inoculation in case 8. A. MRI level 4. B. MRI level 5. C. MRI level 6 101 3.46 SE MRI at level 5 from case 8, showing spread of lesions into left cerebral hemisphere 102 3.47 Plot of total lesion area versus time after inoculation in case 8 103 3.48 Plot of total slice lesion area versus time after inoculation for two SE MRI slices in case 8 104 3.49 Plot of Ti versus time after inoculation for for two areas of the lesion in slice 5, case 8 104 3.50 SE MRI from case 9. A. Slice 3 on day 26 after inoculation. B. Stice 3 on day 29 after inoculation. C. Slice 5 on day 29 after inoculation 107 3.51 Plot of individual lesion area versus time after inoculation in case 9. Le-sion in left cerebral hemisphere slice 3. Lesion in left cerebral hemisphere slice 4 108 3.52 Plot of total lesion area versus time after inoculation in case 9 108 3.53 Comparison of the MRI with the gross pathology. A. SE MRI at level 4. B. The inferior surface of 5 mm thick brain slice at the equivalent level. . 109 3.54 A. Plot of Ti versus time after inoculation for lesion in slice 4. B. Plot of T 2 versus time after inoculation for the same lesion I l l xix 3.55 A . Plot of Ti versus time after inoculation for cerebellar lesion in slice 7. B . Plot of T2 versus time after inoculation for the same lesion 112 3.56 SE M R I on day 52 after inoculation. A . Slice 4. B . Slice 5 114 3.57 A . SE M R I level 4 on day 86 after inoculation, showing new lesion in the right cerebral hemisphere. B . SE M R I level 4 on day 103 after inoculation. In both images T E = 120 ms and T R = 2 sec 115 3.58 Plot of individual lesion area versus time after inoculation in slice 4, case 10. This plot shows the appearance of the new lesion in the left cerebral hemisphere on day 86 116 3.59 Plot of total lesion area versus time after inoculation for case 10. Data is shown to day 135; little change was observed in the brain after this point. 117 3.60 A . Inferior surface of fixed brain slice showing area of relapse. B . The inferior surface of fixed brain slice equivalent to M R I level 5 118 3.61 A.Plot of Ti versus time after inoculation for the new lesion in the right cerebral hemisphere in M R I slice 4. B . Plot of T2 versus time after inoc-ulation for the same lesion 120 4.62 Vector diagram showing the interaction of two spins 132 4.63 Diagram showing the possible transitions for two coupled spins. 0 - zero quantum transition, 1 - single quantum transition and 2 - double quantum transition 134 4.64 C P M G decay curve for CNS tissue 139 4.65 FID from lesion 1, case 4, using the modified IR pulse sequence. The pretrigger and data regions are labelled, together with the region of interest chosen. The tau value was 1 ms 140 4.66 Plot of signal amplitude versus tau for lesion 1 from case 4. . . 141 xx 4.67 A. Diagram showing division of spinal cord into five sections denoted by their approximate level in the spinal column. B. The tissue was gently rolled onto the small diameter glass rod and placed in the bottom of the 10 mm NMR tube. C - cervical, UT - upper thoracic, LT - lower thoracic, L - lumbar, S - sacral and LH - left hemisphere 144 4.68 Diagram showing the right cerebral hemisphere of the superior surface of brain slice 4 in case 1. This is the opposite face to that shown in figure 2.29, page 38. Sections were taken from areas corresponding to lesions 8 and 10. Samples of normal appearing grey and white matter were also removed 146 4.69 A. Diagram showing the superior surface of slice 7 in case 4, the opposite face to that shown in figure 2.18B, page 52. Sections were taken from the areas corresponding to lesions 1 and 3 as indicated. Samples of normal appearing grey and white matter, as indicated on the MRI, were also removed. B. Diagram showing the superior surface of slice 6 in case 4, the opposite face to that shown in figure 2.18A, page 52. Sections were taken from the areas corresponding to lesion 2 as indicated 147 4.70 Histologic section stained with H & E, from the sacral spine of guinea pig 14, showing a large cuff of perivascular inflammation in the subarachnoid space 149 4.71 Histologic section stained with H & E, from the upper thoracic spine of guinea pig 13, showing a perivascular cuff in a Virchow-Robin space. . . 149 4.72 Histologic section stained with H & E, from the lower thoracic spine of guinea pig 16, showing extensive parenchymal cellular infiltration. Com-parison of this section with the one shown in figure 4.72 shows the hyper-cellularity in the tissue around the vessel 150 xxi 4.73 Summary of minuit data for spinal cord. Plot of percentage contribution to signal versus T 2 value for guinea pig spinal cord. A . Cervical. B . Upper thoracic. C. Lower thoracic. D. Lumbar. E . Sacral. tissue from normal controls, abnormal tissue 160 4.74 Plot of amplitude versus T 2 value for the lower thoracic spine of guinea pig 16 162 4.75 Plot of amplitude versus T 2 value for the lumbar spines of guinea pigs 2, 7 and 9 (normal) and 13, 16 and 11 (abnormal) 163 4.76 Plot of amplitude versus T 2 value (sec) for fixed guinea pig lumbar spine. Guinea pig number is given in each spectrum; numbers 2, 7 and 9 are nor-mal, and 13, 16 and 11 are abnormal. The peak around 1 sec corresponds to the T 2 of free water in the formalin solution around the tissue. The magnitude and T 2 of this peak varied, depending on the amount of water present 167 4.77 Plot of amplitude versus T 2 value for normal appearing white matter in primate case 8 168 4.78 Plot of amplitude versus T 2 value for lesion 3 in primate case 8 168 4.79 Plot of amplitude versus T 2 value for the left midbrain in primate case 8. 168 4.80 Plot of amplitude versus T 2 value for normal appearing white matter in case 1 170 4.81 Plot of amplitude versus T 2 value for normal appearing grey matter in case 1 170 4.82 Plot of amplitude versus T 2 value for normal appearing grey/white matter mixture in case 1 171 4.83 Plot of amplitude versus T 2 value for lesion 8 in case 1 171 4.84 Plot of amplitude versus T 2 value for lesion 10 in case 1 172 xxii 4.85 Plot of amplitude versus T2 value for normal appearing white matter in case 4 172 4.86 Plot of amplitude versus T2 value for normal appearing grey matter in case 4 173 4.87 Plot of amplitude versus T2 value for lesion 1 in case 4 173 4.88 Plot of amplitude versus T2 value for lesion 2 in case 4 174 4.89 Plot of amplitude versus T2 value for lesion 3 in case 4 174 4.90 Plot of amplitude versus T\ value for normal appearing white matter in case 1 175 4.91 Plot of amplitude versus T\ value for lesion 1 in case 1 175 4.92 A . Plot of amplitude versus Ti value for normal appearing white matter in case 4. B . Plot of amplitude versus T\ value for lesion 1 in case 4. . . 175 4.93 Plot of log of signal amplitude versus time after the 90° pulse in a C P M G sequence, demonstrating non- exponential behaviour of brain water proton relaxation. A . Manganese chloride solution. B . Brain tissue data fitted to one exponential. C. Brain tissue data fitted to two exponentials. D. Brain tissue data fitted to three exponentials. 177 4.94 Plot of percentage contribution to signal versus T2 for normal and ab-normal guinea pigs. predominantly perivascular inflammation in the subarachnoid and Virchow-Robin spaces. extensive parenchymal cel-lular infiltration. normal tissue 180 4.95 Plots of amplitude versus T2 value for a smooth fit to guinea pig lumbar spinal cord C P M G data. The figure on each spectrum denotes the guinea pig number; numbers 2,7 and 9 are normal and 13,11 and 16 are abnormal. 182 4.96 Plot of maximum and minimum amplitude versus guinea pig number show-ing differences between normal and abnormal tissue 183 xxni 4.97 A . Proton spectrum from the lumbar spine of guinea pig number 13. B . Proton spectrum from the fixed lumbar spine of guinea pig 13. In both spectra tau = 400 ps 185 4.98 Plot of amplitude versus T 2 value for fresh and fixed left hemisphere. The numbers denote the guinea pig and F implies fixed tissue. The peak around 1 sec in the fixed tissue data corresponds to the T 2 value for free water in the formalin solution around the tissue. The magnitude and T 2 of this peak varied, depending on the amount of water present 186 4.99 Plot of amplitude versus T 2 value for a smooth fit to normal appearing white matter in primate case 8, showing two distributions of T 2 187 4.100Plot of amplitude versus T 2 value for normal and abnormal fixed tissue. A . Normal spinal cord. B . Normal white matter from MS patient. C. Inflammatory lesion from guinea pig spine. D. Partially demyelinated and gliotic lesion from MS patient 190 XXLV List of Abbreviations A angstroms, unit of measurement. B0 static magnetic field strength. Bx rotating magnetic field perpendicular to Ba. B B B blood brain barrier. Bz magnetic field in the z-direction. C cervical spine. CNS central nervous system. CP Carr-Purcell modified spin-echo pulse sequence. C P M G Meiboom-Gill modification of C P pulse sequence CSF cerebrospinal fluid. C T X-ray computed tomography. 2D two-dimensional. 2DFT two-dimensional Fourier transformation. D demyelination. D self diffusion coefficient. d dose of myelin basic protein or steroid. DW dwell time. E edema. A E energy difference between spin states. E A E experimental allergic encephalomyelitis. XXV f,fb fraction of bound water. fst fraction of structured water. /„, fraction of free water. Af bandwidth of selective radiofrequency pulse. F B fixed brain. F C A Freunds' complete adjuvant. F E frequency encoding. FID free induction decay. G gliosis. Gx,y,z magnitude of field gradient in x-, y- or z- direction. Gr magnitude of read gradient. AGP magnitude of phase encoding increment. gallo-DR gallocyanin combined with darrow red. Gd-DTPA gadolinium diethylenetriamine penta-acetic acid. GP guinea pig. H & E hematoxylin and eosin. h Plancks constant. I spin quantum number. I2r signal intensity at time 2r after the 90° pulse in a spin-echo sequence. Ir signal intensity an inversion-recovery sequence with an inter-pulse delay r. Ia equilibrium value of the magnetization. IR inversion-recovery. xx vi k Boltzmann constant; delay between data acquisition and reapplication of inversion-recovery pulse sequence. L lumbar spine. L F B - C V luxol fast blue combined with creosil violet. L H left cerebral hemisphere. LT lower thoracic spine. MO macrophages. m weight of monkey. M B P myelin basic protein. MHz megahertz. M N C mononuclear cells. M R magnetic resonance. M R I magnetic resonance imaging. A n equilibrium population difference between spin states. N M R nuclear magnetic resonance. Na population of spin state alpha. population of spin state beta. v0 precession frequency in hertz. ORO-H oil red 0 combined with hematoxylin. P perivascular inflammation. PCI parenchymal cellular infiltration. P E phase encoding, p.m. post meridien. fi magnetic moment. xx vii P M post mortem. P T A H phosphotungstic acid hematoxylin. P V I perivascular inflammation. PVI-SS perivascular inflammation in the subarachnoid space. P V I - V R perivascular inflammation in the Virchow-Robin spaces. r distance separating two interacting nuclei, rf radiofrequency. p proton density. ROI region of interest. S sacral spine. cr screening constant. SE spin echo. S(LD) signal from 2DFT pulse sequence in the frequency domain. S(t) signal from 2DFT pulse sequence in the time domain. S0 ratio obtained from two images. SW sweep width. Ti spin-lattice relaxation time. Tip spin-lattice relaxation time in the rotating frame. T 2 spin-spin relaxation time. Tib spin-lattice relaxation time of bound water. Tist spin-lattice relaxation time of structured water. Tiw spin-lattice relaxation time of free water. TIN spin-lattice relaxation time of normal appearing white matter. T2N spin-spin relaxation time of normal appearing white matter. xxviii v Tiobs observed spin-lattice relaxation time. T temperature in degrees kelvin. Ta length of the primary lobe of a sine function. t time after the 90° pulse in a C P M G pulse sequence. At dwell time. tp pulse duration; also time of phase encoding increment in 2DFT imaging sequence. tr delay after data acquistion and reapplication of C P M G pulse sequence, r inter-pulse delay. r c correlation time. T E time of the echo in a spin-echo pulse sequence. T R total time elapsed before reapplication of a pulse sequence; referred to as the repeat time. 8 angle between vector joining two nuclei and the static magnetic field; also flip angle of rf pulse. U T upper thoracic spine. v volume of micro-osmotic pump. w weight of MBP/steroid; also correction factor for 180° pulse in an IR sequence. Ax slice thickness. xxix Acknowledgements I would like to thank my supervisor, Professor L . D . Hall , for his support throughout this thesis, and for introducing me to magnetic resonance imaging. I would also like to thank my collaborator and friend Dr. D.W. Paty, whose consistence support and encouragement throughout this project have been invaluable and much appreciated. I would like to thank the members of my lab for their support and helpful discussions. Thanks also go to the members of the MS-MRI research group for their friendship and interest in my weekly updates. I especially thank Keith Cover for his help in battling with lATgX, and Ed Grochowski for his assistance regarding the use of the programs on the Vax 11/750. The use of the whole body imaging system in the extended care unit required interac-tion with the M R I technicians responsible for clinical operation of the scanner. I would like to Cathy Jardine, Sharon Hall , Lesley Costley and Karen Smith for their help and cooperation throughout this project; and for giving me time on the scanner at a decent hour whenever possible! In a multi-disciplinary project such as that described in this thesis, the work could not have been completed without the assistance of the pathologists I have collaborated with. I would like to thank Dr. Ken Berry, Dr. Wayne Moore (Department of Neuropathology, V G H ) , Dr. Buster Alvord (Department of Neuropathology, University of Washington) and Dr. Kaz Watabe (UBC), for their assistance and support for the project as a whole; I especially thank Wayne for overloading the histology lab with my tissue so that I had the data for my thesis. Thanks also go to the technicians who carried out the embedding and staining of my never ending number of samples. XXX I must also extend thanks to Professors Myer Bloom and Alex Mackay and their graduate students for their helpful discussions and for the use of their spectrometer. I especially thank Julia Wallace for her friendship and patience with my questions; Ed Sternin and Clare Morrison for allowing me 10,000 blocks on the microvax, (which I constantly filled!) and for being there when the computer baffled me; Cindy Aroujo, Teresa Rind, Frank Nezil and everyone else in Hennings room 100 for adopting me as part of the lab; it made my last year a very enjoyable one. Ken Whittall wrote the NNLS programs that I used to analyze my relaxation data; I would like to thank him, not only for allowing me to use them, but also for the time he spent with me trying to optimize the analysis and for his interest in the project. Working with animals is both a difficult and time consuming operation; I extend my heartfelt thanks to Sharon Barwick, without whose technical assistance, friendship, patience and support, this thesis could not have been completed. Animals also require a lot of care, and the technicians in the animal facilities at U . B . C . have been terrific; I thank them all for being so readily available and helpful, and for sharing with me the laughter and the hard times during the project. Thanks also go to Sarka Hruby (Neuropathology, University of Washington) for teaching me how to handle primates and how to produce the animal model for MS in this species. I would also like to thank Bev Gray, Nicholas Mair and Anneke Ress for their help in the preparation of this thesis and for their encouragement throughout the project. Last but not least, I would also like to thank all my friends at Granville Chapel for their support, encouragement and prayer throughout my Ph.D.; and Anneke Rees for feeding me during the hard times and the fun times, it kept me going. xxxi Chapter 1 General Introduction and Thesis Goal 1.1 General Background Multiple sclerosis (MS) is a demyelinating disease of the central nervous system (CNS) affecting 1 in 1000 people in British Columbia, most of whom are females between the ages of 20 and 45 years. Exactly as the name suggests, demyelination results in the destruction of the myelin sheath surrounding the nerve axons (figure 1.1). The myelin sheath is composed of alternate lipid and protein layers and acts as an "insulator", allowing the fast conduction of an electric potential along the axon's length. The results of myelin destruction are many and varied, and depend on the location and extent of the damaged area: any part of the CNS can be affected. MS is a multi-phasic disorder, being most commonly characterized by relapses and remissions. The manifestation of many different clinical symptoms may mimic a large number of monophasic disorders, dendrites axon Figure 1.1: Diagram of a nerve cell or neuron. 1 Chapter 1. General Introduction and Thesis Goal 2 making MS a difficult disease to diagnose [1]. Other diseases also show demyelination in the CNS, such as acute disseminated encephalomyelitis [2]. The causes of, and adequate treatments for MS have eluded researchers for decades: even with the sophisticated immunological and pathological techniques available today, the puzzle remains unsolved. One of the main obstacles preventing any progress in this area is the lack of knowledge about the pathological progression of the disease; this problem exists because the only pathological data available, except for a few biopsies, are post mortem, by which time the disease is usually in the chronic stage. Pathological and clinical studies are numerous [3,4,5,6], yet little is known about the intermediate stages in the disease process. In addition to clinical examination, a number of paraclinical techniques are used to support the clinical diagnosis of MS. These techniques have been in use for many years, and include: evoked potentials, which examine the ability of neurons to generate and conduct electric potentials; oligoclonal banding in the cerebrospinal fluid (CSF)(a characteristic protein banding pattern seen when the fluid is subjected to electrophoresis); and X-ray computed tomography (CT), an imaging technique which provides images dependent on the tissue density. These techniques are useful in diagnosis, but provide very little additional information about the nature of the disease, apart from the detection of blood brain barrier (BBB) breakdown using contrast-enhanced CT scanning [7]. In addition, these techniques examine only one variable, and serial scanning is not possible using CT, because of the problem of repeated exposure to X-rays. The introduction of magnetic resonance imaging (MRI) to clinical use in 1981 rev-olutionized the study of MS in vivo. Young et al were first to publish images from MS patients, using the inversion-recovery (IR) pulse sequence [8]. These images clearly demonstrated the anatomical detail that was available using MRI. The multi-variable nature of MRI also allowed the manipulation of the radio-frequency pulse sequences Chapter 1. General Introduction and Thesis Goal 3 to provide different contrasting in the final image [9,10]. Subsequent studies of MS patients showed that the spin-echo (SE) pulse sequence was much more sensitive to the number and extent of MS lesions in the brain [11,12] than the IR pulse sequence. Comparisons between MRI and other proven paraclinical techniques used to aid diag-nosis, have repeatedly shown the important role this technique has in studies on MS [13,14,15,16,17,18,19,20,21,22]. MRI is already being used as a quantitative index of disease activity in serial studies of MS patients [23,24,25,26]. Such studies provide a comparison of disease activity with clinical activity, and have shown that there is no correlation between lesions seen on the MRI and the frequency of clinical relapse; thus, many of the new MRI lesions seen in these studies are clinically asymptomatic. MRI is also being used to monitor the effects of drug therapy on disease activity [27]. This type of information on MS was not accessible before the advent of MRI, and it has had an enormous impact on the MS research community [28,29]. The multi-variable nature of MRI means that it can provide not only structural infor-mation, but also dynamical information. The proton signal observed in a MR experiment is dependent on the spin-lattice relaxation time (Ti), the spin-spin relaxation time (T2), the spin diffusion coefficient (D), the proton density (p), and various correlation times (tc) describing the interactions of the active proton spins with other spins, macromolecules and other surfaces with which they come in contact. The proton relaxation times are dependent on the molecular environment of the spins and are also sensitive to their re-stricted motion. Thus, any changes in the molecular environment of the spins should in turn affect the relaxation parameters. Relaxation time measurements are frequently used in chemistry to provide structural, conformational and functional information on complex molecules in solution [30,31,32] and the applications of such measurements to biological samples are already underway. In 1973, Damadian demonstrated differences in the relaxation times in normal versus Chapter 1. General Introduction and Thesis Goal 4 tumour tissue [33]. Those early data demonstrated the future use of M R in the dif-ferentiation of normal from abnormal tissue and subsequently many relaxation studies have been carried out on various tissues [34,35,36]. The results to date show it unlikely that simple Ti and T 2 measurements can be used to specifically characterize tissue and, perhaps more importantly, pathology. Increases in the values of these relaxation times are seen in many different diseases [37,38,39,40,41,42] some of which MS can mimic, such as multiple infarcts and primary brain tumors [43,44,45]. However, this is not perhaps as disappointing as it first seems since it is unlikely that M R I alone would be used to diagnose disease regardless of its success. So, in the event of clinical diagnosis, one poses the question; can the measurement of M R parameters provide any useful information on the disease process? Thus, the specific question being addressed in this thesis is whether M R can provide information on the pathological progression of MS in vivol This requires that M R I can provide information, not only on the location and extent of MS lesions in the CNS, but also on the type of lesion visible on the M R I . The following approach has been taken to answer this question: quantitative M R I at 0.15 Tesla on a whole body imaging system has been used to study post mortem and fixed CNS tissue from MS patients. Those data are presented in chapter 2, and demonstrate that there are different intensities on the M R I , corresponding to different values for the relaxation times; microscopic examination of these areas showed them to be due to different pathology. Variations in lesion M R I characteristics have also been seen in a study on chronic, but stable, MS patients [46]. The application of this type of measurement to study early immune-mediated lesions in vivo was then tested by following the evolution of experimental allergic encephalomyelitis (EAE) , an animal model for MS, in primates and then comparing the quantitative M R I data with the pathology at the time of death. Those data corroborated the evidence provided from the studies on MS tissue. However, as will be described in detail later in Chapter 1. General Introduction and Thesis Goal 5 this chapter, quantitative measurements on most commercial MRI instruments, including the one used in these studies, involve the calculation of Ti and T2 values from only two points on the signal decay curve; hence, the accuracy of those T\ and T2 values and how real the differences were for different pathology, were in question. In addition, the areas of abnormality studied in the human and primate tissues consisted of mixtures of pathology, thus preventing the isolation of the effects of each type of pathology on the quantitative MR measurements. The pathology observed in the CNS of guinea pigs after induction of EAE is less complex than that observed in primates, with some lesions being primarily inflammatory. In vitro relaxation measurements, using the conventional spectroscopic methods described in the next section, were therefore carried out on normal and abnormal CNS tissue from guinea pigs inoculated to produce EAE. Studies were repeated after the tissues were fixed in 10% neutral buffered formalin. Those studies showed that small differences in T2 are reproducible from animal to animal and that differences are observed when pathology is present. Even though the samples studied were mixtures of normal and abnormal tissue, differences were seen for different degrees of inflammation involving different parts of the CNS. T2 values were obtained for primate CNS tissue from an animal that had also been followed in vivo. As a last comparison, T\ and T2 values were obtained from samples of the fixed MS tissue already studied using quantitative MRI. The results from these studies are presented in chapter 4. Two different methods were used to analyze the data, both of which demonstrated clearly that CNS water proton spin relaxation does not follow simple mono-exponential behaviour. The data also show that relaxation measurements on tissue can be reproduced, with errors around 5%. Spectroscopic studies carried out simultaneously, showed no contribution to the MR signal from lipid protons. The fixed tissue showed the same trends observed in vitro: this is important since most of the MS tissue available is already fixed, and these results show that useful information can Chapter 1. General Introduction and Thesis Goal 6 be obtained from the study of fixed tissue. The studies on fixed MS tissue showed differences between the areas that were completely demyelinated and gliotic, and those only partially demyelinated and gliotic. These data also differed from the primarily inflammatory lesions studied in the guinea pigs. The multi-disciplinary nature of this thesis implies a large plurality of readers; there-fore, although this is an integrated thesis, the author has chosen to subdivide the data into separate chapters, thereby allowing the specialist reader to access easily the information of interest to him. The multi-disciplinary nature of the thesis also means it includes many medical terms that may be unfamiliar to some readers; the large number of terms precludes the inclusion of a glossary, but they are listed below and the reader is referred to Dorlands Medical Dictionary, which is available in the University of British Columbia bookstore and in the Woodward Biomedical library. In addition, where possible, the terms are defined briefly as they appear in the text of this thesis. Chapter 1. General Introduction and Thesis Goal 7 List acute akinesia allergic anaesthesia anorexia anterior fissure arachnoid membrane astrocytes(fibrous, protoplasmic) ataxia atropine axon blood brain barrier brainstem central nervous system cerebellum cerebral cortex cerebral hemispheres cerebrospinal fluid chronic chronic-progressive corpus callosum corticomedullary corticosteroids decerebration decortication of Medical Terms demyelination dendrites dexamethasone phosphate disseminated dorsum septum dura-mater edema encephalomyelitis euthanyl exudate fibrin formalin Freunds' complete adjuvant gitter cells glia/glial gliosis gross pathology hemiplegia hemorrhage histiocytes histology hyperacute hypercellularity hyperplasia immunization immunology incontinence infarct inflammation inoculation in situ internal capsule intracardially in vitro in vivo ketamine lamellae lesion leucocytes leucoencephalopathy lymphocytes lysosome' macrophage microglia microscopic pathology midbrain mononuclear cells multiple sclerosis myelin myelin basic protein Chapter 1. General Introduction and Thesis Goal 8 necrotic periventricular r ompun neuroglia perivascular cuffing strabismus neurological phagocytes subarachnoid space nystagmus pia-mater subc l i n i c a l oligoclonal plaque subcortical oligodendrocyte p lasma cells subpial optic chiasm polymorphonuclear cells sudanophilic lipids p a r a c l i n i c a l post mortem synapse paraplegia presynaptic thalamus parenchyma ptosis tremor paresis quadriplegia Virchow-Robin spaces pathogenesis remitting-relapsing visual r a d i a t i o n perivenous remyelination W a l l e r i a n degeneration Chapter 1. General Introduction and Thesis Goal 9 Before presenting the results of the studies briefly described above, the reader is now introduced to the basic concepts of the pulse Fourier transform M R experiment [47]. Detailed M R theory is not presented, since many excellent references already exist on the subject [48,49]; however, the information given should enable the reader to understand the description of M R I which follows in the next section. 1.2 Basic Principles of Magnetic Resonance When a spin system is placed in a static magnetic field, the system becomes ordered and the spins may take up one of 21 + 1 orientations in the magnetic field (where J is the spin quantum number). For protons, J = 1/2; thus there are two energy levels, and the energy difference between them is given by equation 1.1; where 7 is the gyromagnetic ratio and h is Planck's constant. By convention, the direction of the static magnetic field strength, BQ, is assigned to be along the z-axis. The spins precess about the direction of B0 (figure 1.2), and since AE = hv, the frequency of the precession is given by equation 1.2 [47]; lB0 v = (1.2) 2TT At thermal equilibrium, the populations of these two energy levels are given by the Boltzman distribution (equation 1.3). Radiation corresponding to the energy difference between these two states will induce transitions in both directions. wa = "d-tr* ( L 3 ) where k = Boltzman constant, Np = population of upper state and Na = population of lower state. However, since there is initially a greater population in the lower energy Chapter 1. General Introduction and Thesis Goal 10 A B Figure 1.2: The behaviour of spins in the presence of two orthogonal magnetic fields. A . Precession about BQ. B . Behaviour of spins after the application of a second magnetic field, Bi, perpendicular to BQ. state, there is a net absorption of energy. If there were no relaxation processes, the populations of the two states would soon become equal, a process known as saturation. Although this can occur (and is often used in experiments [47,50]), it is usually possible to observe an absorption continuously when the appropriate resonance frequency is ap-plied. This indicates that non-radiative relaxation processes are occurring that prevent saturation. These processes are characterized by the spin-lattice relaxation time, T\, and the spin-spin relaxation time, T2, the two time constants associated with the spin system's return to its equilibrium state. Relaxation is considered further in chapter 4. Transitions are induced by an oscillating radiofrequency (rf) pulse, by applying a current in a coil wound perpendicular to the direction of B„. This transmitter generates a linear field oscillation along the x-axis which is equivalent to two counter-rotating field vectors in the xy plane, but interaction only occurs between the precessing nuclear magnetization and the vector rotating in the same direction [50]. When the frequency of Chapter 1. General Introduction and Thesis Goal 11 B\ is equal to the precession frequency of the nuclear spins, this is known as the resonance condition and maximum signal is observed. Application of B\ produces a torque which moves the magnetization towards the xy plane. The final position of the magnetization will depend upon the length of time for which the rf is applied. The B\ field is applied as a pulse of duration tp, which usually lasts a few microseconds. The angle, 0, (usually referred to as the flip angle or tip angle) through which the magnetization is tipped from the z-axis is easily calculated from equation 1.4, 9 = iBxtp (1.4) The magnitude of B\ is chosen to be large enough to tip the magnetization from the z-axis into the xy plane in a few microseconds (figure 1.2). It usual to refer to the pulse magnitude in terms of the flip angle rather than the pulse duration, since the former is a reproducible parameter. After the application of a 90° pulse, the precessing magnetization induces in the receiver coil an oscillating current which can be detected. This signal is called the free induction decay (FID): free of the influence of the rf field, induced in the coil, and decaying back to equilibrium. Since the signal decays away after excitation, it is referred to as a transient The N M R signal observed must be digitized in order to be processed by the computer: this is referred to as analogue- to-digital conversion. This process involves the conversion of a voltage at a particular moment, to a binary number; this number is then stored as a 'data point' and represents the signal amplitude at that point in time. The signal is sampled at regular intervals, defined by the dwell time, DW (equation 1.5); D W = - k ( L 5 » where SW is the spectral width. The data points must be collected at a sampling rate greater than or equal to twice the signal frequency, if the frequency of a signal is to be Chapter 1. General Introduction and Thesis Goal 12 represented correctly. The data collected in this manner, represent the variation of the signal over time; for useful information to be obtained this signal must be converted to the frequency domain. This is achieved using the mathematical process of Fourier trans-formation [51], which converts a function of time f(t) into the corresponding function of frequency, F(u>), as shown in equation 1.6. F(u) = [+°° f(t)e~iutdt (1.6) J —oo The inverse relationship also holds; F(u)e'utdu (1.7) oo Due to the effects of a phenomenon referred to as chemical shift, the field experienced by a nucleus, i, is not exactly B0 but; Bi = B0{l-<r) (1.8) where o is the screening constant, and characterizes the effects of the spatial electronic distribution around nucleus i. The resonance frequency of the nucleus is then given by; Ut = i { 1 ~ a ) B ° (1-9) The range of frequencies of interest is usually known for the nucleus being studied; thus, the spectral width can be defined by setting the dwell time to an appropriate value (equa-tion 1.5). For protons, the majority of signals occur over a narrow range of frequencies; these signals are excited simultaneously using a broadband rf pulse. Subsequent Fourier transformation of multiple resonances then gives rise to the familiar N M R spectra; the frequency differences between the resonances being determined by cr for each nucleus. At low magnetic field strength, the effects of chemical shift are negligible; however, as the field increases, so too the effects of chemical shift. For the CNS studies carried out Chapter 1. General Introduction and Thesis Goal 13 Figure 1.3: Diagram showing the variation of the static magnetic field, BQ in the presence of a linear field gradient, Gx. in this thesis, chemical shift was not a problem; this is due to the fact that the images obtained were of tissue water. There are protons in other electronic environments (such as lipid proton); however, these nuclei are bound up in membranes and on the time scale of the imaging experiment, are not observed due to their short T2-values. 1.3 MR Imaging and Image Enhancement The basic principles of M R I are the same as those for pulse F T spectroscopy described above. The difference is the addition of linear field gradients to spatially define the object under investigation. In the presence of such a magnetic gradient, Gx, the magnetization along the z-axis is given by equation 1.10: Bz(x) = BQ + Gx.x (1.10) This is demonstrated in figure 1.3. The resonance frequency of the system also becomes spatially dependent, as shown in equation 1.11; nfB0 7<7x.x + (1.11) 2ir 2IC where vx is the resonance frequency of nuclei at position x during the application of gradient Gx. Conventional N M R spectroscopy examines the whole sample, while imaging Chapter 1. General Introduction and Thesis Goal 14 RF G: selective 90° A 180° echo G> G x-Figure 1.4: Two-dimensional Fourier transform spin-echo pulse sequence for slice selective imaging. methods examine a section, or sections, of a sample [52,53,54]. Selection of a slice is achieved by using three orthogonal linear field gradients and a selective rf pulse. The combination of one gradient with the selective pulse provides a slice of known thickness with an in-plane resolution that is defined by second and third gradients. The use of the F T method in imaging was first demonstrated by Kumar, Welti and Ernst [55], and is an extension of the two-dimensional F T (2DFT) spectroscopic methods [56] used in the elucidation of complex molecular structures [57]. A typical 2DFT pulse sequence for imaging is shown in figure 1.4. The x- and y-gradients in figure 1.4 spatially define the plane of the slice. One is referred to as the read gradient, which is a constant value, and the other as the phase-encoding gradient, which is incremented in amplitude for each successive excitation. Together they produce a 2D matrix whose size and resolution are Chapter 1. General Introduction and Thesis Goal 15 determined by the number of phase encoding increments chosen. The signal observed, S(t) can be written as S(t) = J J p(r)s(r,t)dxdy (1.12) where s(r,t) is the contribution from the area dxdy, volume averaged over the slice thickness at position r, and p{r) is the spin density. 2DFT of S(i) gives 5(u) = 5 K , w „ ) (1.13) such that S(u) = J J S{t)exp{iut)dtxdty (1.14) The field of view (FOV) from which the image is obtained, is defined by equations 1.15 and 1.16, which describe the dependency of the F O V on the read or frequency encoding (FE) and phase encoding (PE) gradients [58]. where Gr is the magnitude of the F E gradient and At is the duration of the dwell time. PE= ,A2?V (1.16) where AGP is the magnitude of the P E increment and tp is the duration of the increment. 1.3.1 Slice Selection In all the experiments carried out in this thesis, slice selection was achieved by application of a sinc-shaped frequency-selective (narrow bandwidth) rf pulse in the presence of a linear field gradient (figure 1.5) [53]. As the diagram shows, only those spins that are precessing with a frequency contained in the narrow band of rf used to excite the slice will be detected. The other spins remain unperturbed and do not contribute to the Chapter 1. General Introduction and Thesis Goal 16 Figure 1.5: Diagram showing selective excitation of a slice. A . Selective rf pulse and slice select gradient. B . Magnetization in the z-direction after application of selective pulse. C. Selected slice. Chapter 1. General Introduction and Thesis Goal 17 signal observed. The position of the selected slice is dependent on the direction of the applied gradient and the frequency band excited by the selective rf pulse. Multiple slices can be obtained simply by off-setting the frequency of the rf pulse to excite a different frequency range. The slice thickness, A x , is defined by equation 1.17. A x = ^  (1.17) lGz where A / is the bandwidth of the selective pulse which is given by A / = 2 x i (1.18) where T0 is the length of the primary lobe in the sine function. The F O V and slice thickness are chosen to optimize the signal-to-noise ratio and resolution in the final image of the sample under study. 1.3.2 Image Enhancement The contrast observed in the final image is dependent on the variables in the rf pulse sequence used, and on the relaxation parameters in the different areas of the sample. If we consider a SE pulse sequence (1.19) [59], for example, then the signal intensity observed is dependent on 2r and T2, according to equation 1.20. (90° — r — 180° — r — Acquisition of echo) (1.19) I2r = I0exp(-2T/T2) (1.20) where 7 2 t is the signal intensity at time 2r and IQ is the equilibrium value of the mag-netization. Similarly, for an IR pulse sequence (1.21), the signal intensity observed is given by equation 1.22. (180° — T — 90° — Data Acquisition - Delay) (1.21) Chapter 1. General Introduction and Thesis Goal 18 IT = I0[l - 2exp(-r/T1)} (1.22) where IT is the intensity of the signal observed after the 90° pulse when the inter-pulse delay is equal to r . Variation of the parameters in these pulse sequences will provide different contrast in the final image. The delay time before reapplication of the pulse sequence should be at least bT\ if the system is to return to its equilibrium state. If the delay time is shorter than this, then the signal magnitude is also dependent on Ti according to equation 1.23; / = J 0 [ l - 2exp(-T/Tl) + expiTR/Tr)} (1.23) where TR is the total time elapsed before reapplication of the pulse sequence. For many years, scientists utilized the ability of paramagnetic metal ions to alter the relaxation times of various nuclei [60] and in the past few years attention has focused on the possibility of using such ions to enhance M R I contrast in vivo. The ion most com-monly used is gadolinium ( G d 3 + ) ; it is administered in the form of a complex which the body can excrete, and which is not harmful to the vital organs. The preferred complex is gadolinium diethylenetriaminepenta-acetic acid (Gd-DTPA) [61,62,63,64]. Enhance-ment occurs due to a reduction in the Ti and T2 values of the water protons in the presence of the dominating dipolar interaction with the unpaired electrons on the para-magnetic ion. This is due to the large local fields produced by the unpaired electrons, which have magnetic moments that are much greater than those of the water protons. The rf pulse sequence is chosen to utilize the decrease in Ti, and is usually a SE sequence with a small r-value and a short T R [65]. The signal intensity dependence on the T R is given in equation 1.23 above. Areas penetrated by the paramagnetic complex will then be brighter in the image than normal tissue, which, in most tissues, is impermeable to the complex. In normal human brain tissue, there is no enhancement after intravenous Chapter 1. General Introduction and Thesis Goal 19 administration of Gd-DTPA because the blood brain barrier (BBB) prevents the con-trast agent reaching the cerebral extravascular space [66]. The pituitary gland and stalk and intracavernous portions of cranial nerves III-VI, which have an incomplete B B B , do enhance with Gd-DTPA. In many diseases of the CNS, the B B B is damaged in local areas; these areas are then permeable to the Gd-DTPA complex and will be enhanced on the M R I [67]. Image enhancement studies on MS patients [68,69,70] have shown that lesions do have different characteristics; some enhance very quickly while others form an outer ring of enhancement which eventually fills the lesion. It is reasonable to assume that the pathology of these two sets of lesions is likely to be different; areas which en-hance rapidly are probably inflammatory lesions, in which it is believed the B B B is most permeable. The other areas are probably demyelinating and gliotic, where it is common to see inflammation on the periphery; this would explain the ring of enhancement rapidly observed around the lesion before all of it is enhanced. Contrast enhancement studies JK; were carried out on three of the primate cases presented in chapter 3. 1.4 Relaxation Measurements 1.4.1 Measurement of Ti There are a number of methods available for measuring the T i of magnetic nuclei [71, 72,73,74]. The one most commonly used is the IR method; this method employs the IR pulse sequence 1.21; as described above the magnitude of the signal obtained after a 90° pulse is given by equation 1.22 (page 16). As previously mentioned, a delay time after data acquisition should be at least 5Ti to allow the spin system to return to equilibrium. The r-value is varied, and T i can then be determined from a three-parameter exponential fit to the data (equation 1.24) [74]. Ir = / „ ( ! - [1 + w(l - expi-k/TiWexpi-T/Ti)) (1-24) Chapter 1. General Introduction and Thesis Goal 20 where I0 is the signal intensity at T >^ T i , k is the waiting period between the data acquisition and reapplication of the 180° pulse and w is the correction factor for the 180° pulse. Detailed error analysis in the use of this method has been carried out [75], and shows it to be a reliable method when conditions are optimized. 1.4.2 Measurement of T2 The first measurements of T2 were carried out by Hahn [59] in 1950; he demonstrated that the intensity of the signal obtained from a sample after a spin echo pulse sequence (1.19) had been applied is dependent on T2, as given by equation 1.20 (page 16). Carr and Purcell then showed that in the presence of a field gradient, the signal intensity is also dependent on the diffusion coefficient, as shown in equation 1.25 [76]. I2r = I0exp(-2r/T2)exp(-l<y2G2DT3) (1.25) where G is the magnitude of the field gradient and D is the diffusion coefficient. For larger values of T 2 the effects of diffusion are much more pronounced, due to the r 3 dependence. The effects of diffusion can be reduced by using the Carr-Purcell (CP) pulse sequence (1.26), which is a modification of the Hahn spin-echo method. (90° - r - 180° - r - echo - r - 180° - r - echo - ...) (1.26) Further modifications to this sequence have been made by Meiboom and Gi l l (CPMG) [77], as shown in 1.27; that method overcomes the accumulation of errors due to pulse imperfections, which occur when the C P method is used. (90° - r - 180°y - r - echo - T - 180°y - r - echo - ...) (1.27) If a short value of r is used in this sequence, problems arise due to effective spin-locking of the magnetization about the y-axis. If spin locking occurs, the measurement of T2 is Chapter 1. General Introduction and Thesis Goal 21 then equivalent to T i p , the Ti in the rotating frame [47]. This led Freeman and Hi l l to a further modification of the method, given in 1.28 [78]. (90° — T — 18(£ — T — echo - r - 1801,-r-e c h o - r - 18(£ - ...) (1.28) In the limit of short interpulse spacing, the Freeman-Hill modification does not lead to spin locking. In all of the above methods, T 2 can be determined from an exponential fit to equation 1.20 (page 16), assuming monoexponential relaxation behaviour and neglecting the effects of diffusion. 1.4.3 Imaging Methods As described briefly in the general background, commercial imaging methods for deter-mining Ti and T 2 generally involve obtaining at least two images of the same slice using two different pulse sequences. Each pulse sequence has two variables, thereby allowing Ti and T 2 values to be determined by taking the ratio of the signal intensity in each pixel from both images [79,80]. Others methods employ a single sequence that provides data to determine Ti and T 2 simultaneously [81,82,83]. The relaxation times are usually obtained for the whole slice, pixel by pixel, or from a chosen region of interest (ROI). The whole body M R I system used for many of the experiments described in this thesis, is a Picker International N M R Cryogenic '2000' system operating at 0.15 Tesla. Compu-tations to produce Ti and T 2 images were performed as follows, using the manufacturers software: Ti computation: one IR and one SE sequence are required to compute T i ; for optimal results, T R and T E should be equal in both images. The intensities observed in the IR and SE images are given in equations (1.29) and (1.30). The ratio of these images is Chapter 1. General Introduction and Thesis Goal 22 then taken (equation 1.31), which eliminates the dependence on the equilibrium magne-tization, I0, and T?,. Ti is then obtained from a look-up table, which is a plot of S0 versus T i . Studies of water doped with either copper (II) sulphate, or manganese (II) chloride, demonstrated that the computed Ti values are reproducible and comparable to the value obtained using conventional spectroscopic methods [84]. As previously mentioned, the disadvantage of this two-point computational method is that it is not possible to make error estimates on the values obtained; only the reproducibility can be determined. How-ever, the values obtained on the same instrument using the inversion-recovery method [74] show the errors to be no greater than 10-12%. IIR = I0exp{-2t/T2)[l - (2 - 2exp(-k/T1) + exp({-k - r ) /T i ) ]exp( - i /T 1 ) (1.29) where I 0 is the equilibrium magnetization, 2t is the echo delay (TE) for the spin-echo readout after the 90° pulse in the IR pulse sequence, k is the time between data acquisition and reapplication of the pulse sequence and r is the time between the 180° pulse and the 90° pulse in the IR pulse sequence. ISE = I„exp(-2r/T2)[l - 2exp(-k/T1) + exp{-TR/T{)] (1.30) s = I m = 1 - (2 - 2exp{-k/Ti) + exp(-r - k/T^expj-tm ° ISE (l-2exp(-k/T1) + exp(-TR/T1) l ' ' T2 computation: two SE images with equal T R and different T E are required to com-pute T2. Once again, the ratio of these two images is taken (equation 2.26) eliminating the dependence on I0 and T\. Just as for T i , T2 is obtained from a look-up table which Chapter 1. General Introduction and Thesis Goal 23 is a plot of S0 versus T2. Phantom studies were also carried out to check the T2 values obtained using this two-point computational method [84]. The computed T2 values have been shown to be reproducible and comparable to values obtained using the spin-echo method [59]. Just as for T\, it is not possible to make error estimates on these computed T2 values; however, values obtained using the SE method show the error to be less than 12- 15% for the range typical of CNS tissue T2 values. S =I_SEL= [1 - 2exp(-k1/Tl) + exp(-TR/T1)]exp(2r1/T2) ° ISE2 [l-2exp(-k2/T1) + exp(-TR/T1)]exp(2T2/T2) K ' ) These two-point computational methods for determining T\ and T2 do not provide any information about the relaxation behaviour of the water protons; that is, whether it is mono-exponential or multi-exponential, and provide no indication as to the environment of the protons giving rise to the signal seen in the MRI . Proton spin relaxation behaviour in tissue is the subject of chapter 4, where the results of multi-exponential relaxation studies on CNS tissue are presented. Chapter 2 M R I of Multiple Sclerosis 2.1 Introduction The data presented in this chapter were obtained from immediate post mortem and fixed brain tissue from MS patients, and address two questions: 1. How accurately does M R I reflect the extent of disease in the CNS? and 2. What do variations in signal intensity on the M R I correspond to pathologically? There are essentially two classes of cells in the CNS: the nerve cells (or neurons) and the neuroglial cells (or glia). As shown in the general introduction, the typical neuron has four morphological regions; the cell body, dendrites, axon and presynaptic terminals of the axon. Neurons interconnect with one another via chemical or electrical means, and the point of contact is known as the synapse. The neurons are responsible for sending motor commands to the muscles and for the brain receiving sensory information from the different parts of the body. Glial cells, however, have a supportive role and also segregate groups of neurons from one another. They are also believed to have nutritive functions. In addition, there are certain glial cells that make myelin, the fatty insulating sheath around large axons. The features of the demyelinating disease known as MS can be related to the destruction and loss of a single type of glial cell, the myelin-producing oligodendrocyte [85]. The areas depleted of these cells are usually large enough to be seen with the naked eye as discrete plaques; they tend to be multiple, and to occur, often symmetrically, in particular locations which include the optic nerves 24 Chapter 2. MRI of Multiple Sclerosis 25 and chiasm, periventricular white matter, the corticomedullary junction, subpial regions of the cerebral cortex and brain stem, and certain parts of the spinal cord, such as the dorsum septum, subpial regions and on either side of the anterior fissure. For anatomical information, the reader is referred to references [86,87]. In all regions, lesions are almost invariably perivenous in location. Microscopically, the lesions show a loss of oligodendrocytes and myelin, other structures including axons, nerve cells and blood vessels tending to be preserved. Astrocytes, another type of glial cell, can be divided into two subclasses; protoplasmic and fibrous, each being associated with different parts of the neuron. Fibrous astrocytes contain many filaments and are found extensively in areas of the CNS containing myelinated axons (these areas are known as white matter because of the white appearance of the myelinated axons in unstained, freshly cut brain sections). The protoplasmic astrocytes contain few filaments and are associated with the cell bodies, dendrites and synapses of neurons, which are located in the grey matter (so called because of the grey appearance in brain sections). Both types of astrocytes contact blood capillaries and are therefore thought to have a nutritive function; in MS, these cells exhibit non-specific reactive changes. Perivascular cuffs of lymphocytes and plasma cells are common in subacute lesions. A third class of glial cell, the microglia, are small, ovoid cells that are capable of acting as phagocytes; that is they remove the myelin and axonal debris that arises from demyelination and other destructive processes in the CNS. There now follows an in-depth description of the complex pathology of MS which will provide the reader with some insight into the complexity of solving the MS puzzle and to make them aware of the conflicting theories that exist about the pathogenesis of the disease. The effects of different pathology on the M R signal observed are then discussed. The pathological features of MS resemble other disorders such as post-infectious and sporadic perivenous encephalomyelitis and the human [88] and experimental forms of Chapter 2. MRI of Multiple Sclerosis 26 allergic encephalomyelitis [89]. Theories about the pathogenesis of MS are usually con-cerned with the selective destruction of myelin and oligodendrocytes in affected white matter in this disease. There is no general agreement about the nature of the myelin destruction or how it occurs. Many authors have examined MS tissue at various stages of the disease process; some describe myelin breakdown occurring in the absence of an increase in the number of cells, including microglia [90,91,92,93]. Others have described hypercellularity of the tissue as an accompanying feature of commencing myelin break-down [94,95]. A third pattern was recognized on examination of plaques with bordering hypercellular white matter [96,97]; these cells were identified as oligodendrocytes, based on their nuclear morphology and oxidative enzyme activity. These authors concluded that oligodendrocytes may be the source of increased protease activity present at plaque margins and that microglial cell infiltration is a secondary response to myelin breakdown. Electron microscope studies have failed to confirm the existence of a type of myelin breakdown that occurs in the absence of infiltrating macrophages. It is now known for certain that oligodendrocyte hyperplasia does occur in areas of active myelin break-down but this is believed to be due to co-existing remyelination. The first electron microscope studies of periplaque white matter revealed changes in myelin and oligoden-drocytes in the absence of infiltrating cells. The changes were interpreted as evidence of a toxic or degenerative process primarily affecting oligodendrocytes, with myelin break-down occurring as a secondary event [98,99,100,101,102,103]. Two different types of myelin breakdown were observed frequently in the above studies: one involving granular vacuolar degeneration, consisting of focal splits containing granular osmiophilic debris, myelin fragments and 600 A diameter particles in sheathes of normal thickness; this change is now known to occur in normal white matter. The second involved compact myelin internodes and paranodes that were abnormally thin and/or exhibited premature termination of superficial or deep lamellae. Granular vacuolar degeneration as well as Chapter 2. MRI of Multiple Sclerosis 27 Wallerian degeneration was also observed in white matter remote from plaques, together with conspicuous secondary lysosome formation in oligodendrocytes, astrocytes and mi-croglia, which was also interpreted as possibly indicating a primary glial cell alteration causing secondary myelin breakdown [104]. The lesions examined in the above studies were mostly quiescent plaques with little evidence of recent myelin breakdown, suggesting that the changes observed may have been reactive or regenerative in nature. In 1975 Prineas [105] reported an electron microscope study of 16 'active' plaques from cases where fixation was achieved by in situ perfusion within a few minutes of death. It was found that hypercellularity was mainly due to the presence of infiltrating microglia. Most myelin sheaths in the infiltrated zone, including many directly contacting microglial cells, appeared normal. Some however, were abnormally thin, with superficial or deep lamellae terminating prematurely along the internode. Lymphocytes were present in the Virchow-Robin spaces but none were observed in the parenchyma. It was not uncommon however, to find plasma cells in the parenchyma. It was concluded that myelin thinning in the presence of infiltrating microglia of uncertain origin was a major pattern of myelin destruction in MS. More recent studies support this picture of myelin breakdown in the presence of microglia, and have also provided further evidence for localized areas of remyelination [106,107,108]. The water in tissues may be in many different environments. For the most part, how-ever, the exchange between these environments will be rapid compared to the timescale of the M R experiment. The result of this is the observation of a single relaxation time which is a weighted average of the values from all the different environments. Changes in the number of cells present in an area of the CNS may therefore lead to changes in the ratio of water with restricted motion (that is, associated with macromolecules, organelles or other surfaces) and water in the cytoplasm, which has relaxation characteristics more Chapter 2. MRI of Multiple Sclerosis 28 like that of bulk water. The relaxation rate observed is given in equation 2.33 [109]; — = J- + Izl (2.33) Tx T» Tlf K ' where / is the fraction of "bound" or restricted water, Tu, is the T\ of this water and Tij is the Ti of the free or unrestricted water. If the tissue exhibits multiexponential relaxation behaviour, hypercellularity could affect both the relaxation rates of the dif-ferent components and the relative contribution to the M R signal from each component. This is demonstrated clearly in the data presented in chapter 4. These changes will be affected by the location and numbers of cells involved. Myelin breakdown products will be present in the extracellular space for a short time before they are consumed by macrophages. These breakdown products comprise the proteins and lipids that combine to form the myelin sheath. Their presence in the extracellular space could affect free and bound water ratios, and could also give rise to a M R signal from the lipid protons, which would have different relaxations characteristics from those of water. In normal CNS tissue, lipids are bound up in membranes; as a result, the lipid protons behave like a solid, having a very short T2. On the timescale of the M R I experiment, the signal from these lipids has already decayed to zero and is not observed. The destruction of the myelin sheath gives rise to free lipid that has a longer T2 and can be observed. Spectroscopic studies on primates with E A E have shown a large lipid signal in the lesions resulting from the disease process [110], and this could also be the case with MS. It is clear that a technique such as M R I is not sensitive enough to detect the different mechanisms that may be causing myelin breakdown. However, these plaques can be seen grossly, and the changes are occurring at a molecular level. Thus, it is not inconceivable that M R I could detect and differentiate between an inflammatory response, involving perivascular cuffing and possible infiltration of inflammatory cells into the surrounding Chapter 2. MRI of Multiple Sclerosis 29 {10mm Figure 2.6: Diagram showing the simultaneous excitation of normal and abnormal tissue in a 10 mm thick slice due to the irregular shape of the lesion. parenchyma, and more chronic demyelinating and gliotic plaques. In addition, it may be possible to differentiate degrees of demyelination and gliosis. If this were the case, the pathogenesis of the disease could be followed over time, providing insight into the different mechanisms that are involved in each stage of the disease process. This chapter describes the initial quantitative M R I studies on MS tissue that prompted further study in this area, and provided the first evidence that M R I may be sensitive to different types of MS pathology. The approach taken was as follows: abnormal areas on the M R I , as indicated by increased signal intensity on the SE image [11], were compared with the gross pathology seen in the fixed brain. Qualitative visual comparisons of all the data were made; in addition, where appropriate, the areas of abnormality on the M R I and the actual pathology seen in the fixed brain were quantified and compared. This is not a trivial comparison, since the M R I are volume-aver aged over a 10 mm slice thickness. This means that the resultant image is an average over this thickness, and the appearance of a particular area may be dependent on more than one tissue type. The lesion borders are therefore sometimes difficult to define because the lesion may be irregularly shaped through the slice (figure 2.6). The average Ti and T2 values for regions of interest (ROI) were obtained using quantitative imaging methods. Sections Chapter 2. MRI of Multiple Sclerosis 30 were then taken for microscopic examination, and the data compared with the Ti and T 2 data obtained from the equivalent area on the M R I . Volume-averaging also affects these comparisons, since the area seen on the M R I may consist of normal and abnormal tissue, especially if the lesion is seen in only one 10 mm thick slice. The data shows M R I to be an accurate indicator of diseases extent in the brain. The quantitative M R I did show a correlation between the degree of gliosis and the Ti-values. The problems associated with such studies and their implications are identified and discussed. 2.2 Methods M R I data were collected on a Picker International N M R Cryogenic '2000' system, op-erating at a field strength of 0.15 Tesla, corresponding to a proton resonance frequency of 6.4 MHz. A receiver coil with an aperture of 30 cm was used to acquire the images. Multi-slice SE pulse sequences were employed, together with single and multi-slice IR pulse sequences (figure 2.7). The multi-slice sequences provided 12 contiguous 10 mm thick slices. The single slices were also 10 mm thick. Echo-delay times (TE) of either 40 and 60 ms or 40 and 80 ms and an inter-pulse delay (TI) of 400 ms were used in the SE and IR sequences respectively. In all sequences, the repeat time (TR) was 2 sec. These sequences were chosen to allow comparison with in vivo MS M R I data and to allow the computation of spin-lattice (Ti) and spin-spin (T 2) relaxation time images. The M R I data were transferred from magnetic tape to a Vax-11/750 computer. A n interactive computer program is available for outlining ROI using a trackball and cursor, and calcu-lating their area. The areas of increased signal intensity on the SE images were outlined using this program and their areas determined. The ROI were then transferred to the computed Ti and T 2 images; the intensity obtained from these regions corresponds to the average Ti and T 2 value from that area. In addition to this, ROI were chosen from the Chapter 2. MRI of Multiple Sclerosis 31 A. selective 180° selective 90° A e c h o selective 90° — J \ / — B. selective 180° selective 180° selective 90° A echo • A — T E T R tau selective 180° J n Figure 2.7: A . Multi-slice spin-echo pulse sequence showing variable time parameters. B . Multi-slice inversion-recovery pulse sequence, n = the number of slices in both sequences. Chapter 2. MRI of Multiple Sclerosis 32 centre of the lesions corresponding to those examined microscopically, and transferred to the computed images. In an attempt to minimize the effects of volume averaging, the Ti and T2 data given in the results section are from these central regions. The centre of the lesions is also more likely to be pathologically homogeneous. Direct comparison of the Ti and T2 values between patients was questionable since the elapsed time after death before scanning varied [111,112,113]. Although literature studies show that the Ti and T2 values remain stable for up to 24 hours, changes in temperature could lead to differences in relaxation time values, particularly Ti [114]. The length of time for fixation also varied [112,115]. The effects of fixation have only been studied over a few days, and the data on brain tissue show a tendency for Tj and T2 to decrease over time. In the studies described here, tissue had been fixed in 10% neutral buffered formalin for up to 3 years. It seems likely that Ti and T2 would eventually stabilize. However, to overcome this potential problem, values of Ti and T2 for normal appearing white matter were obtained from each slice. At least 10 ROI, each with an area of 14 pixels, were chosen on the SE images and transferred to the computed images. The ratios of lesion Ti and T2 to normal white matter Ti and T2 were then calculated. This method, of course, makes the assumption that the effects of elapsed time and temperature changes after death and time of fixation affect normal and abnormal tissue to the same extent; this is not necessarily the case. Three chronic cases of MS were scanned as soon after death as possible. A sagittal pilot scan was obtained before using the sequences described above, to allow the posi-tioning of the first transverse slice on the superior surface of the brain (figure 2.8A). This is important for later comparison with pathology. The brains were then removed and placed in 10% neutral buffered formalin. These fixed brains together with one other that was already fixed, were then scanned. The fixed brain was placed on its superior surface and a sagittal pilot scan obtained. This allowed comparison of the positioning with that Chapter 2. MRI of Multiple Sclerosis 33 Figure 2.8: A. Sagittal pilot scan obtained post mortem. Grid shows the center of each of the 12 transverse images to be obtained. B. Sagittal pilot scan from fixed brain. Grid shows the center of each of the 12 coronal images to be obtained. The coronal images of the fixed brain are equivalent to the transverse images obtained in situ. TE = 26 ms and TR = 400 ms in both images. Chapter 2. MRI of Multiple Sclerosis 34 in situ. The first coronal slice was then positioned on the superior surface of the brain and the data acquired(figure 2.8B). The coronal scans of the fixed brain corresponded to the transverse slices in the post mortem scans. Using the sagittal pilot scans to visually orient the brain, horizontal, 10 mm thick brain slices were cut using a standard brain knife guided by 10 mm outside diameter rods; the inferior surface of each brain slice then corresponded, as closely as possible, to the centre of the equivalent M R I . Black and white photographs of the inferior surface of each 10 mm thick brain slice were obtained and digitized so that they could be displayed on the Vax. Focussing was carried out on the largest brain slice, and thereafter kept constant. The brain size was scaled on the computer using a grid that was photographed at the same time with the same focus setting as that used for the brain. Lesions were identified on the actual brain slices, and if they were visible on the digitized image of the slice, they were outlined using the program described above, and the lesion areas determined. After comparison of the gross lesions with the M R I , appropriate blocks were taken for microscopic examination. They were embedded in paraffin and stained with Haema-toxylin and Eosin (H k E) , Phosphotungstic Acid Haematoxylin (PTAH) and Luxol Fast Blue- Cresyl Violet ( L F B - C V ) . Occasional sections were also stained with Bodian's stain for axons and Marchi's stain for neutral fat [116]. The following criteria were used to differentiate the pathology present in each lesion: 1. Inflammation: sections stained with H & E were examined for the presence of perivascular inflammation. Lesions were then graded according to the number of perivascular cuffs present. 2. Demyelination: L F B was absent in areas of total demyelination and pale in areas of partial demyelination. Chapter 2. MRI of Multiple Sclerosis 35 3. Gliosis: using sections stained with P T A H , lesions were examined for fibrillary gliosis. The more fibrocytes present, the denser the gliosis was considered to be. The detailed method of evaluation is given in table 2.1. The pathology of each section was then compared with the quantitative M R I data obtained from the same area. 2.3 Results 2.3.1 Case 1 A post mortem scan was not obtained from this patient. Comparison of the inferior surface of the 10 mm thick brain slice with the equivalent M R I (figure 2.9) showed a remarkable similarity between the two. Quantification of these areas showed the accuracy with which M R I could detect the extent of disease in this case (figure 2.10). Comparison of a slice at a lower level with the equivalent M R I again corresponded well with one another (figure 2.11). A section was removed from the three lesions seen in the right cerebral hemisphere, just above the level of the lateral ventricles. Microscopic examination of these lesions showed them to be completely demyelinated and gliotic. The degree of gliosis varied in each lesion, and in lesion 8 cystic spaces were also present (figure 2.12). The detailed histopathology of each lesion is given in table 2.2. On the M R I , the lesions did not all have the same intensity. The Ti and T 2 values reflected this observation (table 2.3). Chapter 2. MRI of Multiple Sclerosis 36 B. Figure 2.9: Comparison of the inferior surface of a 10 mm thick brain slice in case 1 with the corresponding MRI. A. Spin echo MRI level 3, TE = 40 ms and the TR = 2 sec. B. Inferior surface of fixed brain slice at the same level. Chapter 2. MRI of Multiple Sclerosis 37 Figure 2.10: Display of SE M R I level 3 together with the equivalent digitized negative of fixed brain slice on the Vax 11/750 computer. The lesions are outlined on both the image and the digitized brain slice. Lesion area on M R I = 489 mm 2 and actual lesion area on brain slice = 562 mm 2 . Chapter 2. MRI of Multiple Sclerosis 38 I. Demyelination (D) Number and Size (0-4) Severity 0. none 1. single small foci 2. several small foci 3. one large confluent area 4. several large confluent areas. a. none b. identified only microscopically c. macroscopically identified but mild. d. marked but partially preserved myelin. e. totally demyelinated (no stained myelin). II. Perivascular Inflammation (P) Number (0-4) Severity 0. none 1. 1-3 vessels 2. 4-6 vessels 3. 6+ vessels 4. almost all vessels III. Gliosis Astrocytosis (G) a. none b. only a few MNC c. 1-2 layers of MNC d. 3-5 layers of MNC e. 5+ layers of MNC 0. none 1. slight, marginal 2. slight, diffuse 3. moderate, diffuse 4. marked (densely gliosed) IV. Others f. fibrillary dominant p. protoplasmic dominant 1. MO : presence of MO 2. Ax : axonal degeneration Table 2.1: Table showing details of scoring for pathology of sections taken from the infe-rior surface of each 10 mm thick brain slice. MNC - mononuclear cells, MO - macrophages. Figure 2.11: Comparison of gross pathology with the spin echo MRI. A. SE MRI level 4, TE = 40 ms and TR = 2 sec. B. Inferior surface of 10 mm thick fixed brain slice at the equivalent level. LESION D E M Y E L I N A T I O N I N F L A M M A T I O N GLIOSIS 8 4e l b 2f 9 4e l b 3p 10 4e 4d,M0 3-4p Table 2.2: Pathological score for the three lesions seen in the right cerebral hemisphere of slice 3, case 1. Chapter 2. MRI of Multiple Sclerosis 41 Lesion Zi(ms) XiTv(ms) T2(ms) T2 Ar(ms) T2/T2N 8 460 186± 17 2.25 110 54± 6 2.06 9 690 186± 17 3.63 120 54± 6 2.26 10 430 186± 17 2.25 100 54± 6 1.81 Table 2.3: Quantitative M R I data from fixed brain in case 1. TIN and T2AT correspond to the average values of T\ and T 2 from normal appearing white matter in the same slice. Chapter 2. MRI of Multiple Sclerosis 42 2.3.2 Case 2 The gross pathology seen on the inferior surface of the 10 mm thick brain slices again corresponded well with the areas of increased signal intensity on the SE M R I (figure 2.13). The total lesion area seen on the fixed brain was quantified and compared with that seen on the SE M R I (table 2.4). A section taken from brain slice 4 in this case (figure 2.14) showed lesions similar to that seen in case 1. The detailed histopathology POST M O R T E M Sli ce Area(mm2) F I X E D B R A I N Slice Area(mm 2) DIGITIZED B R A I N Slice Area(mm 2) 2 3 4 5 6 7 8 9 10 11 12 0 11 90 560 415 575 587 371 209 9 119 2 3 4 5 6 7 8 9 10 11 12 0 19 141 632 799 465 501 671 93 35 37 1 2 3 4 5 6 7 8 9 10 11 39 115 342 748 709 607 481 889 296 108 24 Table 2.4: Correlation between lesion area as seen on post mortem and fixed brain M R I and the actual lesion area seen on the inferior surface of each 10 mm thick brain slice in case 2 is given in table 2.5, and the quantitative data for this lesion are given in table 2.6. Chapter 2. MRI of Multiple Sclerosis 43 Figure 2.13: Comparison of the SE M R I with the gross pathology. A . Post mortem SE M R I at level 4. T E = 40 ms and T R = 2 sec. B . Fixed brain SE M R I at equivalent level. Parameters as in A . C. Inferior surface of fixed brain slice at the equivalent level. Chapter 2. MRI of Multiple Sclerosis Chapter 2. MRI of Multiple Sclerosis 44 Figure 2.14: Inferior surface of fixed brain slice 4, case 2. The box shows the position of the section taken for microscopic examination. LESION D E M Y E L I N A T I O N I N F L A M M A T I O N GLIOSIS 7 4e l b 2f Table 2.5: Pathological score for section taken from the inferior surface of slice 4, case 2. Chapter 2. MRI of Multiple Sclerosis 45 SLICE LESION 2i(ms) r 2(ms) Tx/TlN T2/T2N 5,PM 7 520 100 1.41 1.22 F B 7 460 140 2.14 2.13 Table 2.6: Ti and T2 values from M R I number 5, which is equivalent to the inferior surface of fixed brain slice 4, case 2. The area from which these values were obtained corresponds to that from where the section was taken. Cadaver TIN = 342± 30 ms(18), cadaver T2N = 84± 7 ms(17), fixed brain TJAT = 217± 10 ms(6) and fixed brain T2N = 66± 7 ms(22). The numbers in brackets give the number of areas from which the mean and standard deviation were determined. Chapter 2. MRI of Multiple Sclerosis 46 2.3.3 Case 3 The areas of increased signal intensity on the SE images corresponded well with the gross pathology see on the inferior surface of each brain slice (figure 2.15 and 2.16). The areas of abnormality on the SE images were quantified and compared with the actual pathology seen on the fixed brain slices, and the data are given in table 2.7. Sections POST M O R T E M F I X E D B R A I N DIGITIZED B R A I N Slice Area(mm 2) Slice Area(mm 2) Slice Area(mm 2) 4 41 3 0 1 9 5 79 4 47 2 73 6 515 5 242 3 213 7 877 6 943 4 1003 8 575 7 555 5 494 9 448 8 254 6 430 10 245 9 148 7 130 11 138 10 201 8 69 12 0 12 0 9 32 Table 2.7: Correlation between lesion area as seen on post mortem and fixed brain M R I and the actual lesion area seen on the inferior surface of each 10 mm thick brain slice in case 3. were taken from brain slices 5 and 6 in this case; sections were removed from the white matter around the posterior horns of the both lateral ventricles in slice five, and from the right posterior horn in slice 6. Microscopic examination of these sections showed demyelinated and gliotic lesions similar to those in cases 1 and 2. The histopathology of each lesion is given in table 2.8. The lesions were clearly seen on the post mortem and fixed images from this patient. The quantitative M R I data from the three areas studied microscopically are given in table 2.9. Chapter 2. MRI of Multiple Sclerosis 47 Figure 2.15: Comparison of SE image with the gross pathology. A . Post mortem SE M R I at level 5. T E = 40 ms and T R = 2 sec. B . Fixed brain SE M R I at the equivalent level. Parameters as in A . C. Inferior surface of fixed brain slice at the equivalent level. Chapter 2. MRI of Multiple Sclerosis 48 Figure 2.16: Comparison of SE image with the gross pathology. A . Fixed brain SE M R I at level 6. Tau = 40 ms and T R = 2 sec. B . Fixed brain SE M R I at the equivalent level. Parameters as in A . C. Inferior surface of fixed brain slice at the same level. Chapter 2. MRI of Multiple Sclerosis Chapter 2. MRI of Multiple Sclerosis 49 SLICE LESION D E M Y E L I N A T I O N I N F L A M M A T I O N GLIOSIS 5,L 4 3e 3c 2f 5,R 5 3e 4a,M0 3p 6,R 6 3e 3b 2f Table 2.8: Pathological score for sections taken from fixed brain slices 5 and 6, case 3. L - left cerebral hemisphere and R - right cerebral hemisphere. SLICE LESION r,(ms) T2(ms) Tr/TlN T2/T2N 7,PM 4 666 300 1.83 3.18 6,FB 4 460 180 2.07 2.47 7,PM 5 600 320 1.66 3.39 6,FB 5 450 180 2.01 2.46 8,PM 6 540 120 1.45 1.41 7,FB 6 300 100 1.43 1.39 Table 2.9: T\ and T 2 values from areas examined microscopically. Post mortem TIN = 368± 21(24), T2N = 91± 9(7), and fixed brain T1N = 218± 10(18), T2N = 71± 6(24). Bracketed numbers denote the number of areas from which the mean and standard deviation were determined. P M - post mortem, F B - fixed brain. Chapter 2. MRI of Multiple Sclerosis 50 2.3.4 Case 4 The MR images in this case showed massive periventricular lesions throughout the cere-brum (figure 2.17). When the brain was cut, these lesions could not be seen grossly (figure 2.18). The areas of increased signal intensity on the post mortem and fixed brain spin echo images were quantified and compared (table 2.10). Sections were taken POST MORTEM FIXED BRAIN Slice Area(mm2) Slice Area(mm2) 2 0 2 0 3 0 3 0 4 264 4 157 5 827 5 646 6 1,218 6 1,743 7 1,064 7 629 8 924 8 721 9 916 9 881 10 363 10 228 11 159 11 0 12 0 12 0 13 0 13 0 Table 2.10: Comparison of lesion area seen on the post mortem and fixed brain MRI in case 4. Slice 1 is the sagittal pilot scan and is not included. from slices 5 and 6 (figure 2.18). Histologic examination showed the areas of increased signal on the SE images to be large areas of partial demyelination with gliosis present. The detailed histopathology is given in table 2.11. The quantitative MRI data showed variations where the histopathology varied (table 2.12) and were different from the val-ues obtained from the totally demyelinated and gliotic lesions seen in the previous three cases. Chapter 2. MRI of Multiple Sclerosis 51 Figure 2.17: SE MRI from case 4. A. Post mortem MRI level 4. at the same level. In both images tau = 40 ms and TR = 2 sec. B. Fixed brain MRI Figure 2.18: A . Inferior surface of fixed brain slice 5, case 4. Box shows the position of the section that was removed for microscopic examination. B . Fixed brain slice 6, case 4, showing the two sections removed for microscopic examination. Chapter 2. MRI of Multiple Sclerosis 53 SLICE LESION D E M Y E L I N A T I O N I N F L A M M A T I O N GLIOSIS 6,R 1 3d Oa 2 P 5,R 2 3d 2b 3p 6,L 3 3e l b 3p Table 2.11: Pathological score for lesions taken from slices 5 and 6, case 4. L - left cerebral hemisphere, and R - right cerebral hemisphere. SLICE LESION Ti(ms) T2(ms) Tr/T1N T2/T2N 7,PM 1 510 90 1.36 1.31 F B 1 340 150 1.63 2.42 6,PM 2 830 180 2.10 2.14 F B 2 540 200 2.66 3.03 7,PM 3 640 130 1.71 1.87 F B 3 340 190 1.60 2.94 Table 2.12: T\ and T 2 values from area examined microscopically in case 4. Post mortem TlN = 381± 12(14), T2N = 77± 9(20) and fixed brain T1N = 207± 14(20), T2N = 65± 5(24). The numbers in brackets give the number of areas from which the mean and standard deviation were determined. P M - post mortem, F B - fixed brain. Chapter 2. MRI of Multiple Sclerosis 54 2.4 Summary of Resul ts A summary of the quantitative results from all 4 cases is given in the following tables. Examples of individual lesion areas quantified from the SE M R I and the actual fixed brain slices are given in table 2.13. Quantitative M R I data from normal appearing white matter are given in table 2.14; the data from pathological areas are then presented in order of increasing degrees of demyelination, gliosis and inflammation (tables 2.15, 2.16 and 2.17). P M M R I F B M R I B R A I N 361 209 396 206 266 397 184 139 15 56 - 11 63 149 203 132 149 203 132 149 156 184 105 254 Table 2.13: Quantitative area measurements from individual lesions. P M - post mortem, F B - fixed brain, B R A I N - data from digitized brain slice. Chapter 2. MRI of Multiple Sclerosis 55 C A S E . I \ ( ms) T2( ms) P M F B P M F B 1 186±17 54±6 2 342±30 217±10 84±7 66±7 3 368±21 218±10 91±9 7 1 ± 6 ( 4 381±12 207±14 77±9 65±5 Table 2.14: Quantitative M R I data from normal appearing white matter as seen on the post mortem and fixed brain SE MRI . Chapter 2. MRI of Multiple Sclerosis 56 LESION A R E A S E V E R I T Y T^ms) T2(ms) 1,PM 3 d 510 90 F B 3 d 340 160 2 ,PM 3 d 830 180 F B 3 d 540 220 3 ,PM 3 e 640 130 F B 3 e 340 190 4 ,PM 3 e 670 300 F B 3 e 460 180 5,PM 3 e 600 320 F B 3 e 450 180 6,PM 3 e 540 120 F B 3 e 300 100 7,PM 4 e 520 100 F B 4 e 460 140 8,FB 4 e 460 130 9,FB 4 e 690 130 10,FB 4 e 430 100 Table 2.15: T\ and T 2 values from computed images, correlated with severity of demyeli-nation in cases 1-4. Lesions are denoted by the same numbers as those used in the text. P M - post mortem, F B - fixed brain, A R E A - size and number of lesions. Chapter 2. MRI ol Multiple Sclerosis 57 LESION S E V E R I T Y T Y P E TaCms) T2(ms) 4 ,PM 2 f 670 300 F B 2 f 460 180 6 ,PM 2 f 540 120 F B 2 f 300 100 7,PM 2 f 520 100 F B 2 f 460 140 8,FB 2 f 460 130 1,PM 2 P 510 90 F B 2 P 340 160 3 ,PM 3 P 640 130 F B 3 P 340 190 2 ,PM 3 P 830 180 F B 3 P 540 220 5,PM 3 P 600 320 F B 3 P 450 180 9,FB 3 P 690 130 10,FB 3-4 P 430 100 Table 2.16: Ti and T 2 values from computed images, correlated with severity of gliosis in cases 1-4. Lesions are denoted by the same numbers as those used in the text. P M -post mortem and F B - fixed brain. Chapter 2. MRI of Multiple Sclerosis 58 LESION A R E A S E V E R I T Y r,(ms) T2(ms) 1,PM 0 a 510 90 F B 0 a 340 160 3 ,PM 1 b 640 130 F B 1 b 340 190 7 ,PM 1 b 520 100 F B 1 b 460 140 8,FB 1 b 460 130 9,FB 1 b 690 130 2 ,PM 2 b 830 180 F B 2 b 540 220 6 ,PM 3 b 540 120 F B 3 b 300 100 4 ,PM 3 c 670 300 F B 3 c 460 180 5 ,PM 4 a 600 320 F B 4 a 450 180 10,FB 4 d 430 100 Table 2.17: T\ and T 2 values from computed images, correlated with severity of inflam-mation in cases 1-4. Lesions are denoted by the same numbers as those used in the text. P M - post mortem, F B - fixed brain, A R E A - denotes size and number of lesions. Chapter 2. MRI of Multiple Sclerosis 59 2.5 Discussion Two questions were asked at the beginning of this chapter: (1) How accurately does M R I reflect the extent of disease in the CNS? and (2) What do variations in signal intensity on the M R I correspond to pathologically? The problem of volume averaging and the shape of the lesion within the slice suggest that comparisons between the M R I and the actual pathology would not be very impressive. The data showed clearly that this is not the case. The qualitative visual comparisons showed an excellent correlation between the gross pathology and the areas of increased signal intensity seen on the SE MRI . When these areas were quantified, the difference observed between the abnormal areas on the SE image and the actual pathology seen in the brain was 5-60% when individual lesions were considered. When total brain lesion areas were compared, the range difference was considerably lower, 20-30%. There are a number of reasons for these discrepancies. It is extremely difficult to define the margins of lesions in the subcortical white matter of both fresh and fixed tissue, since the grey matter has a T2 value of the same order as many of the abnormal areas. In the fixed tissue, the lateral ventricles often appear white, making periventricular lesions margins difficult to define. It is also difficult to define small lesions in these areas, giving rise to large errors, and the presence of many small plaques could lead to large cumulative errors. If these small plaques do not fill the slice, the signal intensity will be much lower than it should be, due to the excitation of both normal and abnormal tissue within the 10 mm slice thickness. These studies show that M R I does reflect the extent of MS in the brain, with problems arising when there are small lesions and/or subcortical lesions whose margins cannot be defined. Some small lesions may be missed completely using the 10 mm slice thickness. However, unless an MS case consisted only of such lesions, the resulting errors would be small compared to the errors involved in quantifying areas over time in a drug efficacy trial, for example. Chapter 2. MRI of Multiple Sclerosis 60 Question two is more difficult to answer. The first point to note is that the effects of fixation do seem to stabilize over time, as shown in table 2.14. The values for normal appearing white matter overlap for the cases studied; this suggests that direct comparisons can be made between the quantitative data from the lesions in each case, although this assumes that the effects of fixation on different types of pathology are the same; for the present time this is assumed to be the case. Literature data have shown that fixation decreases the T\ and T2 values obtained from tissue; this was the true for all cases studied here except case 4; the T2 values from the fixed brain M R I data were higher than those obtained post mortem. No explanation has yet been found for this observation. When the summarized data in tables 2.15, 2.16 and 2.17 are examined, the T2 values do not seem to follow any trend. This is thought to be due in part to the computation used to determine T2. The computed images produced are often extremely noisy (figure 2.19), and when average values are obtained over a ROI, the standard deviation within these regions can vary substantially (22-33%), as shown in table 2.18. The effects of demyelination and inflammation on the values of Ti do not TISSUE T2 (ms) Std. Dev. P M 83 17.9 P M 81 26.9 F B 84 18.7 F B 91 23.4 Table 2.18: Mean intensity and standard deviation from regions consisting of 18 pixels on a computed T2 image, from regions of normal appearing white matter. P M - post mortem and F B - fixed brain. seem to follow any particular trend. In these chronic inactive lesions, however, the Chapter 2. MRI of Multiple Sclerosis 61 Figure 2.19: Computed Ti and T 2 images from the fixed brain in case 3. B. T 2 image. A. Ti image. Chapter 2. MRI of Multiple Sclerosis 62 extent of inflammation is minimal and would not be expected to have major effects on the relaxation times. It is not certain what the effects of demyelination are, since for the cases studied here, it cannot be separated from the effects of gliosis. How real the differences are between the partially demyelinated and completely demyelinated lesions is questionable, since most of the completely demyelinated lesions were only seen in one slice. The values for the relaxation times from these areas are likely to be a mixture of normal and abnormal tissue, giving rise to lower values for the relaxation times than expected. The T\ and T 2 values for case 4 are probably the most accurate, since these lesions extended throughout both hemispheres and the data were obtained from slices where the lesions could be seen in the two adjacent slices. The data on gliosis indicate that the more gliotic a lesion is the higher its T\ value. Although the T\ values from lesions 9 and 10 are not as high as that from lesion 2, lesions 9 and 10 are small and could have lower values than expected due to volume averaging as described above. A n increase in T\ with increasingly severe gliosis makes sense, since it is known that the water content of tissue increases as the tissue becomes more gliotic [118]. It was also noted in these studies that the effects of multi-slice interference contribute to the observed values of the relaxation times. In the multi-slice pulse sequences used, excitation of the different slices is interleaved to minimize the effects of slice interference; that is, the order of excitation is slice 1,3,5,7,9,11,2,4,6,8,10,12. However, in case 4, for example, data from normal appearing white matter showed differences as high as 15% (table 2.19). Fixed tissue was less affected due to the shorter values for the relaxation times, which means a more rapid return to equilibrium after excitation and data acquisition. It is clear that multi-slice interference could have a significant effect on the lesion T\ and T 2 since they have even longer T\ values than normal white matter. The lesions in odd numbered slices would tend to show lower values than expected. The values for lesions 5, 9 and 10, which are all present in odd numbered slices, would then have longer T\ and T 2 values Chapter 2. MRI of Multiple Sclerosis 63 Slices Average TIN Average T2N 4,6 5,7 389±12(17) 367±12(18) 84±5(20) 71±8(21) Table 2.19: Post mortem Tj and T2 values from normal appearing white matter in case 4, showing the effects of multi-slice interference. The numbers in brackets give the number of ROI used to determine the mean and error. than those determined. In conclusion, there is no doubt that M R I is an accurate indicator of the extent of MS in the CNS. However, there are limits to the useful quantitative information that can be obtained using quantitative M R I methods to determine T\ and T2. The reasons for this are threefold: firstly, the effects of different pathology cannot be separated from one another. In addition, the pathology in chronic MS is similar in each case and provides no information on the early lesions, which are believed to be immune-mediated; secondly, volume averaging and multi-slice interference give rise to lower relaxation time values than expected and lastly, the observed T\ and T2 values obtained using the two-point computational method provide no indication as to the source of the signal in the tissue. This means that it is not possible to determine whether the signal follows a mono- or multi-exponential decay. In the next chapter, the first of these limits is addressed by studying an animal model for MS in primates. The disease was followed in vivo using M R I with a slice thickness of 5 mm, and the histology compared with the quantitative M R I data at the time of death. Chapter 3 M R I of Experimental Allergic Encephalomyelitis in Primates 3.1 Introduction Experimental allergic encephalomyelitis (EAE) is an animal model for MS in which at-tempts have been made to understand the mechanisms of development of early, as well as late, immunologically-rnediated, inflammatory and demyelinating lesions in the CNS. E A E can be produced in susceptible species and strains by sensitization to white matter or to myelin basic protein (MBP) in Freund's complete adjuvant (FCA) [117]. M B P makes up 30-35% of the proteins in normal CNS myelin. Manipulation of the exper-imental conditions for induction of E A E allows the production of not only hyperacute and acute forms but also chronic and remitting-relapsing courses of the disease [119]. As previously described in chapters 1 and 2, MS can run several clinical courses: acute, chronic progressive, chronic relapsing-remitting, or various combinations thereof. Histologically, the disease is characterized by relatively sharply defined demyelinative le-sions with preservation of axons. E A E can also present with a variety of clinical courses. The classical form is usually an acute disorder with inflammatory and/or demyelinating-necrotic processes depending on the species studied: in guinea pigs and rats it is almost purely inflammatory, whereas in rabbits and monkeys it is both inflammatory and de-myelinating, and in monkeys even necrotic. The early lesions are relatively small and perivascular but they may coalesce into large, irregularly shaped ones. Monkeys sensitized with M B P in F C A may show all three forms: hyperacute, acute 64 Chapter 3. MRI of Experimental Allergic EncephalomyeUtis in Primates 65 or chronic, the last being the least common unless treatment is given to prolong the course. Thus, most monkeys sensitized to MBP in FCA die with either hyperacute or acute EAE. Clinically, the acute form is characterized by rapid onset of abnormalities in vision and paralysis, leading to death within a few days. Pathologically, hyperacute EAE in monkeys is marked by severe inflammation (with neutrophils, hemorrhage, edema and fibrin), demyelination and varying degrees of necrosis. Although many of these changes resemble acute necrotizing hemorrhagic leucoencephalopathy in humans, some lesions resemble acute MS. Acute primate EAE was studied in order to observe the evolution of acute, immunologicalry-mediated, MRI-visible, CNS lesions. Alvord et al manipulated the acute model to produce remissions by treatment with myelin basic protein (MBP) and corticosteroids, followed by relapse on withdrawal of therapy [120]. At post mortem, these relapsing primates frequently show demyelinating lesions with preservation of axons, similar to demyelinating plaques seen in MS. Four animals were treated in this manner, and although changes were observed both clinically and on the MRI, the monkeys did not go on to fully recover and relapse. Prior to this time, it has not been possible in primate EAE, to mimic the spontaneous relapsing-remitting course seen in MS (both clinically and on the MRI). However, the results from case 10 describe the development of subclinical E A E with MRI lesions showing both resolution and a relapse, demonstrating that spontaneous dissemination of lesions in time and space in EAE is possible following a single immunization and without subsequent treatment. This chapter describes the use of both qualitative and quantitative MRI to study acute and chronic primate EAE. Chapter 3. MRI of Experimental Allergic Encephalomyelitis in Primates 66 3.2 Methods 3.2.1 Induction of E A E Acute E A E Permission for carrying out these studies was granted by the Animal Care Committee at the University of British Columbia. Four male Macaco, fascicularis monkeys, weighing 3-5 kg, were fed a regular diet of monkey chow and fresh fruit before and after induction of E A E . E A E was induced by intradermal injection of 0.15 ml of a water-in-oil emulsion containing 0.5 mg of Mycobacterium tuberculosis and 15 mg of M B P , divided among three sites in the hind foot pads [120]. The M B P was extracted using the batch method of Diebler et al. [121](see appendix A for details of extraction process). Clinical observations of the monkeys were made 3 times daily beginning 7-9 days after inoculation (Table 3.20) [122]. Production of Chronic E A E As mentioned in the introduction, acute E A E can be treated to produce remission fol-lowed by a relapse on withdrawal of therapy [120]. Four animals were treated at the appearance of definite neurological signs with M B P (2 mg/kg/day) and dexamethasone phosphate (0.5 mg/kg/day). The M B P and steroid were dissolved in 10% DMSO in saline to facilitate absorption, and placed in Alzet mini-osmotic pumps (model 2001, Alza, Palo Alto, Ca. 94304) with known volumes and flow rates. The calculation of amounts of M B P and steroid required is shown in equation 3.34. dmv w (3.34) / where w = weight of M B P or steroid required, d = dose of steroid or M B P (mg/kg/day), m = weight of monkey (kg), v = volume of micro-osmotic pump ((A) and / = flow rate Chapter 3. MRI of Experimental Allergic EncephalomyeHtis in Primates 67 1. ?: Prodromal Signs 2. ± : Mild Neurological signs Anorexia (food pellets scattered on the floor of the cage) Weight loss Inactivity Slow response to startle Irritability Yawning 3 . +: Moderate Neurological Signs Pupillary abnormalities (unequal, dilated, poorly reactive to light) Ptosis Strabismus Tremor Seizure Paresis Incontinence Body twisting Blindness Ataxia Akinesia Drooling Nystagmus Transient tremor Clumsiness Head tilt Apathy (indifference) "Headache" (acute distress) 4. -f+: Severe Neurological Signs Paraplegia Hemiplegia Quadriplegia Lethargy or somnolence 5. ++: Moribund 6. ++++: Death Semicoma Coma Decerebration Decortication Table 3.20: Clinical scoring for monkeys inoculated to produce E A E . Chapter 3. MRI of Experimental Allergic EncephalomyeHtis in Primates 68 of pump (/zl/day). The pumps were then implanted subcutaneously in the backs of the animals between the shoulder blades. Placement of the pumps in this area prevented the animals tampering with the stitches, which would inhibit healing and could cause infection. After the first four cases were completed, Alvord and co-workers altered the experi-mental protocol to use intradermal injections in the hind legs rather than the footpads. This modification was shown to produce a milder form of E A E with later onset. This protocol was adopted for subsequent studies on the primates. Case 5 was inoculated using the new protocol with the same dose of M B P and F C A as previous cases. However, the disease still progressed quite rapidly and the pathology observed had a hemorrhagic component, which is not analogous to MS. Treatment to produce remission was not successful. The data from case 5 were similar to that obtained from cases 1-4, therefore the data are not included in the results section. In an attempt to produce both a milder form of the disease, which would increase the chances of successful treatment to produce remission, and to alter the pathology observed, cases 6 and 7 were inoculated with two thirds of the M B P dose given to previous animals (total M B P dose of 10 mg). These two cases again progressed quite rapidly, and although they did respond to treatment, they did not fully recover. The M B P dose was reduced still further to a total dose of 5 mg; cases 8, 9 and 10 were inoculated using this dose. On the appearance of clear neurological signs, case 8 was nursed to see if she would recover, but survived only 10 days from the onset of neurological signs. Case 9 was treated with steroid and M B P as in the previous cases, but again failed to recover completely. Case 10 developed subclinical E A E without treatment, and went on to develop neurological signs at a later date. Cases 6-10 were monitored daily from day 8 after inoculation for the onset of clinical signs. Chapter 3. MRI of Experimental Allergic EncephalomyeHtis in Primates 69 3.2.2 M R I Protocol M R I data were collected on the Picker International imaging system described previously; a receiver coil, (saddle design, built by Picker International) with an aperture of 15 cm was used to obtain the images. Multi-slice SE and IR pulse sequences were employed (figure 2.7, page 31), both of which provided 8 contiguous, 5 mm thick slices. Echo-delay times (TE) of either 40 and 60 ms or 40 and 80 ms and an inter-pulse delay (TI) of 400 ms were used; in all sequences the repeat time (TR) was 2 sec. Cases 6-10 were scanned using multi-slice multi-echo pulse sequences; T E values of 40/80 ms or 40/120 ms were employed. Spin-echo sequences are referred to as SETR/TE m a U figures. The monkeys were positioned in the scanner using the laser available for patient alignment, such that the inner canthus of both eyes were aligned perpendicular to the direction of the static magnetic field; the appropriate flexion of the neck was determined visually. For comparisons to be made, repositioning was critical in the serial scans: a sagittal pilot scan was obtained in 0.8 min, allowing the position of the monkey to be checked and positioning altered if necessary. The pilot scan also allowed the centre of the first transverse slice to be positioned on the superior surface of the brain (figure 3.20A); this was important for subsequent comparison with the gross and microscopic lesions seen pathologically. The pulse sequences were chosen to allow direct comparison with human MS data, and also to allow the computation of the spin-lattice relaxation time (T\) and the spin-spin relaxation time (T2); the latter computations were performed using the manufacturers software as described in chapter 1, page 21. The quantitative M R I data were transferred to a Vax 11/750 and using the program described in chapter 2, T i and T 2 values were obtained from the computed images by outlining an area of 14 pixels at the centre of the lesions or from an area of normal appearing white matter. The same lesions were followed serially and, where possible, Chapter 3. MRI of Experimental Allergic Encephalomyehtis in Primates 70 Figure 3.20: A . Sagittal pilot scan of monkeys brain. The grid shows the position of the 8 transverse slices to be obtained. B . Sagittal pilot scan of fixed monkey brain. The grid shows the position of the 8 coronal slices to be obtained. The coronal slices of the fixed brain are equivalent to transverse images in vivo. In both images T E = 26 ms and T R = 800 ms. Chapter 3. MRI of Experimental Allergic EncephalomyeHtis in Primates 71 Figure 3.21: Image of monkey brain showing lesions outlined using a trackball. lesions that extended through three slices were chosen to minimize the effects of volume-averaging. In addition to obtaining quantitative M R I data, for cases 8-10 total lesion area, slice lesion area and individual lesion area were followed over time on the SE images (figure 3.21). The monkeys were anaesthetized for scanning using a 12:1 combination of ketamine hydrochloride (Roger/STB Inc. Chemical name ±-2-o-chlorophenyl-2-methylaminocyclo-hexanone hydrochloride, 100 mg/ml) and rompun (Haver-Lockhart. Chemical name 2-(2,6-di-methylphenylamino)-4H-5,6-dihydro-l,3-thiazine, 20 mg/ml) (0.3 mg/kg). Since the monkeys were supine while being scanned, atropine (Clark Rogers Ltd . 0.5 mg/ml) was also administered at the time of anaesthesia (0.2-0.4 ml) to prevent oral fluid accu-mulation and aspiration. Chapter 3. MRI of Experimental Allergic EncephalomyeHtis in Primates 72 Serial M R I of Acute E A E The monkeys were scanned before inoculation and then daily from day 9 after inocula-tion. Before each set of scans was obtained, the monkeys were checked for evidence of neurological impairment. Following the earliest detection of changes on the M R I (as in-dicated by increased signal intensity on the SE images) [13], images were obtained 3 times daily until death. T\ and T2 computations were carried out for each set of M R images and the values recorded for the abnormal areas. The ranges of T\ and T2 corresponding to normal-appearing white matter or brainstem (case 1) were obtained from statistical analyzes on 8-24 areas from the equivalent slice in 3 serial scans; both are comparable to those seen in humans [123]. When each monkey became incapacitated, being unable to feed or drink by itself, it was sacrificed using 3 ml of euthanyl ( M T C Pharmaceuticals) intracardially while under anaesthetic. When possible, immediate pre-mortem and post-mortem scans were obtained. The brain was then removed and fixed in 10% neutral buffered formalin. After a period of at least 1 week a set of M R images was obtained from the fixed brain. The brain was positioned on its superior surface and a sagittal pilot used to check its positioning. The centre of the first horizontal slice was positioned on the superior surface of the brain (figure 3.20B), thereby allowing direct comparison between the images of the fixed brain with both the in vivo images and the gross and microscopic pathology. Serial M R I of Chronic E A E A baseline scan was obtained at the time of inoculation, and serial M R I data were ob-tained every few days beginning any time between day 9-15 after inoculation. Following the earliest detection of changes on the M R I , the monkeys were scanned daily. Contrast enhanced scans were obtained from cases 6, 7 and 10 after intra-venous injection of Gd-Chapter 3. MRI of Experimental Allergic Encephalomyehtis in Primates 73 D T P A [61,62,63,64]. Multi-slice SE pulse sequences were used to obtain these contrast enhanced scans, with T E = 26 ms and T R = 500 ms. This sequence provided 4-6 slices, each 5 mm thick. When clear neurological signs were observed, mini-osmotic pumps were implanted as described above, and daily scanning continued until death. Pre-mortem or immediate post-mortem scans were obtained from each animal. As for the acute cases, the brains were removed and fixed in 10% neutral buffered formalin, and a set of M R I obtained from the fixed brain. 3.2.3 Pathology Using the sagittal pilot scan to visually orient the brain, horizontal brain slices, each 5 mm thick, were cut using a standard brain knife guided by 5 mm outside diameter glass rods; the inferior surface of each slice then corresponded as closely as possible to the centre of the equivalent MRI . After comparison of the gross lesions with the M R I data, appropriate blocks were taken for microscopic evaluation, embedded in paraffin and stained with Hematoxylin and Eosin (H & E), Luxol fast blue combined with periodic acid-Schiff and hematoxylin (LFB-PAS-H), with Holmes' axon stain (LFB-Holmes) or with Nissl's stain (LFB-Nissl) and Gallocyanin for myelin combined with Darrow red (gallo-DR). In addition, frozen sections were stained with oil red 0 and hematoxylin (ORO-H) [116]. Chapter 3. MRI of Experimental Allergic Encephalomyelitis in Primates 74 3.3 Results 3.3.1 Case 1 M R I and Clinical Observations A n area of increased signal intensity on the SE image was observed in the brainstem (midbrain) 15 days after inoculation. This change was associated with the onset of lethargy and bilateral ptosis. Over the course of the next few days there was little change in the M R I (figure 3.22), and the monkey remained clinically stable; hence the monkey was only scanned once a day on days 17 through 21. On day 21 after inoculation, the MRI-visible lesion spread rostrally and caudally. This change was associated with worsening of the ptosis in the left eye. On day 22, the M R I remained positive in the same three slices and the ptosis in the left eye was worse. By late evening of the same day, the monkey was off balance and the right arm appeared spastic. On day 23 the monkey lay prostrate in the cage. He was nursed all day to see if he would recover but had to be sacrificed at 7.35 p.m. A n immediate post mortem set of scans was obtained. The brain was removed and immediately placed in 10% neutral buffered formalin. The lesion, previously easily seen on the M R I (both SE and IR), could not be seen in the images of the fixed brain. This is in contrast to the studies on brains from humans with chronic MS as described in chapter 2, in which the demyelinated and gliotic lesions were easily and reproducibly seen on both immediate post mortem and fixed brain scans. Figure 3.22: Serial SE2000/40 M R I obtained from case 1. A . Day 15 after inoculation. B . Day 18 after inoculation. Chapter 3. MRI of Experimental Allergic Encephalomyelitis in Primates 76 Comparison of the M R I with the Gross Distribution of Lesions Photographs of the 5 mm thick brain sections and the histologic preparations were com-pared with the equivalent M R images, as shown in figure 3.23. A 10 x 10 mm lesion, mainly in the midbrain and extending into the rostral pons, could be seen on the gross sections. As was observed on the M R I , the lesion was present in three 5 mm thick brain sections. Another small lesion consisting of inflammation surrounding about 1 dozen blood vessels over an area of 5 x 2 mm could be seen in the left occipital subcortical white matter; this small lesion was not detected on the M R I . Histology The initial area of increased signal observed on the SE image was found to correspond to a confluent, perivascular inflammatory lesion, in which the vessel walls were infiltrated by white blood cells diffusing out into the tissue (figure 3.24A). Most of the cells were polymorphonuclear leucocytes with a small number of lymphocytes and a large number of macrophages; most of the macrophages were large round cells (gitter cells) filled with granular sudanophilic lipids (figure 3.25) and LFB-positive myelin debris (figure 3.24B). There was no hemorrhage in this lesion but there was evidence of subtotal necrosis with swelling, beading and destruction of axons. At the edges of the lesion, the myelin sheaths were remarkably swollen (figure 3.24C). Quantitative M R I The Ti and T2 values from the lesion were measured as a function of time after inoculation (figure 3.26) and were shown initially to increase, then stabilize while the monkey was clinically stable. Figure 3.23: Comparison of the gross distribution of lesions with the equivalent M R I in case 1. A . SE2ooo/4o image on day 23 showing lesion in the brain stem. B . The inferior surface of the 5 mm thick brain slice at the same level. Chapter 3. MRI of Experimental Allergic Encephalomyelitis in Primates 78 A. B. Figure 3.24: Histopathology of lesion in case 1 A . H & E showing diffusion of polymor-phonuclear cells, lymphocytes and macrophages into the tissue around the vessel. B. L F B - PAS-H) showing LFB-positive myelin debris at the centre of the lesion. C. Holmes' stain showing swelling, beading and destruction of axons at the centre of the lesion. Chapter 3. MRI of Experimental Allergic EncephalomyeHtis in Primates 79 "IH ' ' ' ' • ' ' r-15 16 17 18 19 20 21 22 23 Time after inoculation (days) Figure 3.26: Plot of Ti and T 2 versus time after inoculation in case 1. Ti and T 2 values are given from normal appearing white matter plus or minus two standard deviations. Chapter 3. MRI of Experimental Allergic Encephalomyelitis in Primates 80 3.3.2 Case 2 MRI and Clinical Observations A n area of increased signal on the SE image was observed in the left cerebral hemisphere on day 22 after inoculation (figure 3.27A). A non-contrast C T scan obtained the same day did not show any evidence of hemorrhage or low-density. The monkey was sacrificed on that day and an immediate post mortem scan obtained. The appearance of this abnormal area was not associated with any neurological signs apart from a small loss in weight. The lesion was not visible on the images of the fixed brain. Comparison of the MRI with the Gross Distribution of Lesions Photographs of the 5 mm thick brain section and the histologic preparation were com-pared with the equivalent MRI . The left cerebral hemispheric lesion present on the M R I was seen clearly on the gross section (figure 3.27B), as mildly hemorrhagic. On the histologic preparation the lesion was 1 0 x 8 mm, centred around the genu of the internal capsule, extending slightly into the striatum (caudate and putamen) and into the nucleus ventralis anterior thalami. This lesion consisted of small areas of inflammation which did not coalesce. Histology The lesion had many of the same characteristics as the lesion in case 1, but it was less confluent and was hemorrhagic. The medial half of the lesion consisted of smaller perivascular lesions, while the lateral half consisted of larger haemorragic lesions. Within this lesion there were collars of hemorrhage around the blood vessels, displacing the exudate peripherally (figure 3.28). The exudate consisted mostly of polymorphonuclear cells. A small amount of hemorrhage was seen in the anterior portion of the lesion, but Figure 3.27: Comparison of the SE M R I with the gross pathology in case 2. A . SE 2 0oo/40 M R I on day 22 after inoculation. B . The inferior surface of 5 mm thick brain slice at the same level. Chapter 3. MRI of Experimental Allergic Encephalomyelitis in Primates 82 Figure 3.28: Histopathology of lesion in case 2. Section stained with H & E showing the collar of hemorrhage around the vessel displacing the exudate peripherally. the major component was posterior and lateral, and these two areas coalesced. At the site of thrombosed venules there were many polymorphonuclear cells. Some of the smaller lesions that comprised the large lesion seen grossly, had as many polymorphonuclear leucocytes but no hemorrhage; others were composed of predominantly mononuclear cells (lymphocytes and a few plasma cells) distending the Virchow-Robin spaces and infiltrating diffusely into the parenchyma. Chapter 3. MRI of Experimental Allergic Encephalomyehtis in Primates 83 3.3.3 Case 3 M R I and Clinical Observations Areas of increased signal intensity on the SE images were seen in both cerebral hemi-spheres on day 11 after inoculation. There were no neurological signs observed at that time. Twelve hours later, the lesions in both hemispheres had enlarged (figure 3.29A, B) . The monkey's movements were slow and he was urinating frequently. A further twelve hours later, the lesions had spread dramatically in both hemispheres. The monkey was clumsy and his peripheral vision was diminished. Seven hours later, he was found dead in his cage. A set of scans was obtained post mortem, and the brain removed and placed in 10% neutral buffered formalin. After 10 days of fixation, a set of scans was obtained from the fixed brain; the lesions were barely visible on the images of the fixed brain (figure 3.30). Comparison of the M R I with the Gross Distribution of Lesions Comparison of the photographs of the 5 mm thick brain sections and the histologic preparations with the equivalent M R I showed that the lesions in both hemispheres corre-sponded well with the M R I appearance (figure 3.31A, B) . At M R I level 3 (figure 3.32A, B) , two lesions could be seen. The left cerebral hemispheric lesion was present in the white and grey matter of the occipital lobe, and measured 12 x 15 mm. The right-sided lesion was more anterior, and present predominantly in the white matter; it measured 12 x 11 mm. At a lower level (MRI number 5) the lesions were larger (figure 3.33A, B) . The left cerebral lesion involved the temporal cortex and white matter, and measured 17 x 10 mm. The lesion included the choroid plexus of the trigone and extended to the posterior edge of the putamen. Isolated perivascular lesions could also be seen at the level of the Figure 3.29: Serial SE20oo/4o M R I from case 3. A . Day 11 after inoculation. B . Day 12 after inoculation. Chapter 3. MRI of Experimental Allergic Encephalomyehtis in Primates 85 Figure 3.30: SE M R I from fixed brain. The lesions in both hemispheres are pale compared to the bright areas seen on the in vivo images. anterior commissure and beneath the ependyma overlying the head of the caudate. The right cerebral lesion measured 18 x 12 mm and involved the lateral edge of the thalamus (lateral geniculate and pulvinar), the posterior edge of the globus pallidus and the posterior half to two thirds of the putamen; it also included the external and extreme capsules and the anterior portion of the visual radiation. Histology In M R I number 3, corresponding to brain slice 2 (figure 3.32A, B) there were two lesions, one in each hemisphere. The left lesion consisted of two areas: posteriorly the lesion was more hemorrhagic, being diffusely so in the white matter; the hemorrhage occurred around small vessels that were only slightly inflamed. The lesions in the white matter were large compared to those in the cortex, which were not hemorrhagic. The exudate was predominantly polymorphonuclear cells. The anterior portion of the lesion was mostly present in the white matter and it was similar to the posterior portion with a Chapter 3. MRI of Experimental Allergic Encephalomyehtis in Primates Figure 3.31: Comparison of the SE M R I with the gross pathology in case 3. A . SE M R I slice 4. B . The inferior surface of 5 mm thick brain slice at the same level. Chapter 3. MRI of Experimental Allergic EncephalomyeUtis in Primates 87 Figure 3.32: Comparison of SE MRI with the gross pathology in case 3. A. SE20oo/4o MRI at level 3. B. The inferior surface of 5 mm thick brain slice at the same level. Figure 3.33: Comparison of the SE M R I with the gross pathology in case 3. A . SE M R I slice 5. B . The inferior surface of 5 mm thick brain slice at the same level. Chapter 3. MRI of Experimental Allergic Encephalomyehtis in Primates 89 Figure 3.34: Histopathology of lesion observed in the left cerebral hemisphere at M R I level 3. Section stained with H & E showing compact exudate similar to that seen in cases 1 and 2. large exudate mainly containing polymorphonuclear cells. The lesion in the left cerebral hemisphere was a mixture of hemorrhagic and nonhemorrhagic areas. The hemorrhagic areas were found on the periphery of the lesion with very little inflammation; in the centre of the lesion there were areas of perivascular inflammation with exudate similar to that in cases 1 and 2 (figure 3.34). The right cerebral lesion in M R I number 3, corresponding to brain slice 2 contained many axons which showed swelling and fragmentation. A large number of axons re-mained intact in spite of the large size and compactness of the exudate. In the larger lesions within this area the axons were almost completely destroyed. There was no su-danophilic debris observed in this lesion. The hemorrhagic areas were only present in the white matter. In M R I number 5, corresponding to brain slice 4 (figure 3.33A, B) , lesions were again seen in both cerebral hemispheres. The left-sided lesion was quite hemorrhagic, being Chapter 3. MRI of Experimental Allergic EncephalomyeUtis in Primates 90 Time after inoculation (days) Figure 3.35: Plot of T\ and T 2 versus time after inoculation for case 3. The dashed line denotes data from normal tissue plus or minus two standard deviations. more so laterally, and the most anterior portion of the lesion was not hemorrhagic. The exudate was, again, predominantly polymorphonuclear cells. The right-sided lesion was hemorrhagic laterally, with no hemorrhage medially. The exudate was similar to that in the left hemisphere, being predominantly polymorphonu-clear cells. The lesions in case 3 were more hemorrhagic and necrotic than the lesions in cases 1 and 2. In addition, the areas of inflammation and hemorrhage were more confluent than in cases 1 and 2. Quantitative M R I The T. and T 2 values of the left hemisphere lesion in M R I slice 4 were measured as a function of time after inoculation and were shown to increase from the time of detection until death. The increase was not dramatic (figure 3.35). Chapter 3. MRI of Experimental Allergic EncephalomyeHtis in Primates 91 Figure 3.36: S E 2 O o o / 4 0 obtained from case 4 on day 16 after inoculation. 3.3.4 Case 4 M R I and C l i n i c a l Observations A n area of increased signal intensity on the SE image was observed in the left cerebral hemisphere on day 16 [124] after inoculation (figure 3.36). This lesion was not associated with any neurological signs, apart from a small loss in weight. The lesion spread laterally, medially, superiorly and inferiorly over the course of the next 3 days (figure 3.37 A , B , C). On day 17 after inoculation, the monkey appeared spastic in his right arm which became progressively more severe. Later the same day he seemed to neglect his right visual field, suggesting a right hemianopia; on day 18 his left leg was affected. Twentyfour hours later he was prostrate in his cage and his whole right side appeared spastic. He was sacrificed at 2 a.m. on day 20 after inoculation. A n immediate post mortem scan was obtained, and the brain was removed and placed in 10% neutral buffered formalin. Only parts of the lesion could be seen on the M R I of the fixed brain (figure 3.38). Figure 3.37: Serial SE20oo/4o M R I from case 4. A . Day 16 after inoculation. B . Day 18 after inoculation. Chapter 3. MRI of Experimental Allergic EncephalomyeHtis in Primates 93 Figure 3.38: Fixed brain SE20oo/40 MRI from case 4 showing abnormal area in the left cerebral hemisphere. This gliotic lesion appears as a bright area of increased T 2, just as was seen in the fixed brains of chronic MS patients. Comparison of the MRI with the Gross Distribution of Lesions Comparison of photographs of the 5 mm thick brain sections and the histologic prepa-rations with the equivalent MRI, showed a large hemorrhagic lesion in the left cerebral hemisphere (figure 3.39 A, B). The lesion measured 21 x 18 mm and involved the genu and posterior limb of the internal capsule, the posterior two-thirds of the putamen, the external and extreme capsules and the insular cortex. It also involved the temporal cortex and white matter anteriorly and extended back to the anterior portion of the visual radiation, the tail of the caudate and the lateral thalamus (lamina medullaris or internus thalami). Smaller inflammatory lesions were also seen in the splenium of the corpus callosum, the columns of the fornix and in the right anterior thalamic nucleus. Smaller sub-ependymal lesions were also present at the head of the caudate on the left side. At lower levels the lesion also involved the lateral geniculate nucleus, the optic tract and the lateral half of the amygdaloid nucleus. A smaller lesion, measuring 6x3 Figure 3.39: Comparison of the SE M R I with the gross pathology in case 4. A . Post mortem SE2ooo/60 M R I at level 5. B. The inferior surface of 5 mm thick brain slice at the same level. Chapter 3. MRI of Experimental Allergic EncephalomyeHtis in Primates 95 mm, was present in the right cerebral hemisphere; it involved the retrolenticular part of the internal capsule and was visible on the M R I . Histology The larger lesion in the left cerebral hemisphere was quite edematous, necrotic and hem-orrhagic laterally and anteriorly with extensive destruction of axons. More posteriorly and medially, involving the thalamus, it was only slightly hemorrhagic. The most poste-rior part of the lesion involving the visual radiation (figure 3.40) was more demyelinated than necrotic, with abundant myelin debris, many polymorphonuclear cells and lympho-cytes. There was almost complete preservation of axons with only a few axon swellings. A n old infarct could also be seen in the anterior temporal lobe and cortex; it showed gliosis with Wallerian degeneration of the underlying white matter, and only slight in-flammation. This old infarct was the region clearly seen on the images of the fixed brain (figure 3.38). In the corpus callosum (figure 3.41), the exudate displaced most of the axons to the periphery, but a few myelinated axons coursed through the exudate. The smaller lesion in the right hemisphere showed moderate destruction and displacement of axons, but many myelinated and demyelinated axons coursed through the exudate. The exudate was made up of predominantly polymorphonuclear cells with a large number of lymphocytes and histiocytes. Quantitative M R I The Ti and T2 values of the lesion first observed on day 16 were measured as a function of time after inoculation (figure 3.42); both sets of values increased from the day of detection until death. Chapter 3. MRI of Experimental Allergic EncephalomyeHtis in Primates 96 Figure 3.40: Histopathology of posterior and medial part of lesion in the left visual radiation of case 4. Section stained with gallo-DR. Chapter 3. MRI of Experimental Allergic Encephalomyehtis in Primates 97 Figure 3.41: Histopathology of lesion in the corpus callosum of case 4. Section stained with gallo-DR. 700 600. £ «00 Ij LESION Tj mmi 4VA 16 17 11 W 20 Time after inoculation (days) Figure 3.42: Plot of Ti and T2 versus time after inoculation for case 4. Ti and T 2 values from normal appearing white matter are given plus or minus two standard deviations. Chapter 3. MRI ol Experimental Allergic EncephalomyeUtis in Primates 98 3.3.5 Cases 6 and 7: G d - D T P A Study M R I Data Cases 6 and 7 were inoculated together and developed identical areas of increased signal intensity on day 22 after inoculation, in slices 4, 5 and 6 (figure 3.43). Later the same day, the lesions had spread rostrally and caudally into slices 3 and 7. Without removing the animals from the coil during their respective scans, Gd-DTPA was administered I.V. (0.16 mmole/kg to case 6 and 0.3 mmole/kg to case 7) and serial images obtained over the next 45 minutes. In both cases, this data showed breakdown in the blood brain barrier (figure 3.44). Two mini osmotic pumps, containing 50 mg M B P (54 mg) and 15 mg (14 mg) steroid respectively for case 6 (case 7), were implanted in each animal after this scan, together with an intra-peritoneal (LP.) injection of 1 mg of steroid dissolved in saline. Eight hours later, the monkeys were rescanned; case 6 died during the procedure; case 7 developed pulmonary edema during the procedure. She seemed to recover, but then developed heart failure and could not be revived; a post mortem scan was obtained from each animal. It was not possible to obtain pathology from these two brains, due to an error in the fixation process. 3.3.6 Case 8 M R I and Clinical Observations Bilateral areas of increased signal intensity were first observed on day 23 after inoculation, in slices 4,5 and 6 (figure 3.45). These lesions were not associated with any neurological signs. On day 24, the lesions had spread and were now seen in slices 3,4,5 and 6 in the right cerebral hemisphere, and slices 4 and 5 in the left cerebral hemisphere (figure 3.46). No clinical signs were apparent at this time. On day 26 after inoculation, the lesions had enlarged further (figure 3.47), but only prodromal signs were present clinically. She Figure 3.43: SE M R I from case 7 on day 22 after inoculation. A . Slice 5. B. Slice 6. Chapter 3. MRI of Experimental Allergic Encephalomyehtis in Primates 100 Figure 3.44: Gd-DTPA scan from case 7 showing breakdown in the blood brain barrier where lesion is enhanced in slice 5. A . Slice 5. B . Slice 6. In both images T E = 26 ms, T R = 500 ms and number of P E increments = 256. Chapter 3. MRI of Experimental Allergic Encephalomyelitis in Primates A . B. Figure 3.45: SE M R I showing preclinical lesions on day 26 after inoculation A . M R I level 4. B . M R I level 5. C. M R I level 6. Chapter 3. MRI of Experimental Allergic Encephalomyehtis in Primates 102 Figure 3.46: SE M R I at level 5 from case 8, showing spread of lesions into left cerebral hemisphere. Chapter 3. MRI of Experimental Allergic EncephalomyeHtis in Primates 103 Figure 3.47: Plot of total lesion area versus time after inoculation in case 8. was watched for the next few days, and when scanned on day 29, some of the lesions had receded while others had increased in size (figure 3.48). On day 32 after inoculation, an occipital lesion was no longer visible; others had increased in size in parts and receded in others. There were also changes in signal inten-sity. Clinically, her pupils were dilated and poorly reactive to light; she was apathetic and her movements were slow. Later the same day, her left leg was affected and she would not eat by herself. She was nursed over the next few days to see if she would recover, but she continued to deteriorate chnically. On day 34, some areas were brighter on the SE images, but had decreased in size. Dramatic increases were seen in T i on slice 5 (figure 3.49), and the lateral ventricles appeared deformed on slice 4. The lesion size and intensity continued to change over the next 9 days (figure 3.47). Although nursing was continued throughout this time, she continued to deteriorate chnically and died on day 43 after inoculation. A post mortem scan was obtained. The brain was removed, and sections taken for in vitro study based on the M R I as follows: Chapter 3. MRI of Experimental Allergic Encephalomyehtis in Primates 104 Area (sq.mm) 24 26 29 32 34 35 Time (days) 37 39 43 Figure 3.48: Plot of total slice lesion area versus time after inoculation for two SE M R I slices in case 8. 25 30 35 Time (days) 40 45 Figure 3.49: Plot of Ti versus time after inoculation for for two areas of the lesion in slice 5, case 8. Chapter 3. MRI of Experimental Allergic Encephalomyelitis in Primates 105 1. Two areas of normal appearing white matter. 2. Two areas of normal appearing grey matter. 3. Section taken from the brain at approximately M R I level 5 , close to the corpus callosum, and labelled lesion 1. 4 . Section taken from the white matter at approximately M R I level 3 / 4 , adjacent to the right lateral ventricle, and labelled lesion 2. 5. Section taken from the right corpus callosum area at approximately M R I level 6, and labelled lesion 3 . 6. Section taken from the left midbrain. 7. Section taken from the corpus callosum area containing both grey and white matter. The results of this in vitro study are presented in chapter 4 . Chapter 3. MRI of Experimental Allergic Encephalomyehtis in Primates 106 3.3.7 Case 9 M R I and Clinical Observations A n area of increased signal was seen on the SE M R I on day 26 after inoculation (figure 3.50A); no clinical signs were apparent at this time. Two days later, the disease had progressed dramatically, and areas of increased signal intensity were now seen in slices 2,3,4,5,6,7 and 8 (figure 3.50B). Clinically, her pupils were dilated and she had developed nystagmus, tremors and appeared to be completely disoriented, having no concept of her position in relationship to her surroundings; her hearing may also have been affected. Later the same day, two mini-osmotic pumps, containing 43 mg of M B P and 11 mg steroid respectively, were implanted subcutaneously and 1 mg steroid dissolved in saline was administered L P . On day 30 after inoculation, she was completely disoriented and had developed ataxia; her pupils were still dilated. Later the same day, she improved clinically, being able to sit up and reach to grasp food in her hand. By day 31 after inoculation, she had again deteriorated, being completely disoriented and ataxic; she could also be lethargic. Six hours later, she was found dead in her cage; a post mortem scan showed large lesions in both hemispheres and in the cerebellum and brainstem. Individual lesions showed changes in size as shown in figure 3.51, and the total lesion area varied as shown in figure 3.52. Comparison of the M R I with the Gross Distribution of Lesions The areas of increased signal intensity in the SE images corresponded well with the lesions seen grossly on the fixed brain slices (figure 3.53). A large lesion in the left hemisphere extended from slice 2 to slice 6; lesions were also seen in the cerebellum and brainstem, just as was present in the M R I . Figure 3.50: SE MRI from case 9. A. Slice 3 on day 26 after inoculation, day 29 after inoculation. C. Slice 5 on day 29 after inoculation. B. Slice 3 on Chapter 3. MRI of Experimental Allergic Encephalomyehtis in Primates 108 400 Area ( sg . mm) 26 29 30 31 Time (days) Figure 3.51: Plot of individual lesion area versus time after inoculation in case 9. Lesion in left cerebral hemisphere slice 3. Lesion in left cerebral hemisphere slice 4. 2000 1800 1600 1400 1200 Area (sq.mm) 1000 o 14 26 29 30 31 Time (days) Figure 3.52: Plot of total lesion area versus time after inoculation in case 9. Figure 3 . 5 3 : Comparison of the M R I with the gross pathology. A . SE M R I at level 4 . B. The inferior surface of 5 mm thick brain slice at the equivalent level. Chapter 3. MRI of Experimental Allergic Encephalomyelitis in Primates 110 Histology Sections were taken from the centre of the large lesion in the left cerebral hemisphere and from the lesion in the cerebellum. Histologic examination of these areas showed them to be mainly inflammatory, with many perivascular cuffs containing predominantly polymorphonuclear cells; lymphocytes and mononuclear cells were seen in smaller num-bers. Some areas contained dense collections of polymorphonuclear cells wiping out the parenchyma, with only fragments of axons present. The primary process in both lesions was necrosis; this resembles the pathological features of acute hemorrhagic leu-koencephalitis. A few perivascular cuffs were predominantly mononuclear cells, which is consistent with the early autoimmune disease process. Quantitative M R I Changes in T\ and T 2 over time for the lesions studied histologically are shown in figures 3.54 and 3.55. Chapter 3. MRI of Experimental Allergic Encephalomyelitis in Primates 111 Figure 3.54: A . Plot of Tx versus time after inoculation for lesion in slice 4. B . Plot of T2 versus time after inoculation for the same lesion. Chapter 3. MRI of Experimental Allergic Encephalomyehtis in Primates 112 A . 380 -I 1 1 1 1 1 1 1 1 . 26 2 6 . 5 27 27 .5 28 28 .5 29 2 9 . 5 30 30 .5 31 T ine (days) B. T2 (ms) 26 2 6 . 5 27 27 .5 28 28 .5 29 2 9 . 5 30 30.5 31 Time (days) Figure 3.55: A . Plot of Ti versus time after inoculation for cerebellar lesion in slice 7. B . Plot of T 2 versus time after inoculation for the same lesion. Chapter 3. MRI of Experimental Allergic Encephalomyelitis in Primates 113 3.3.8 Case 10 MRI and Clinical Observations Serial M R I data were obtained every few days from day 11 to day 26 after inoculation. No lesions were visible on the M R I until this point, and the monkey was chnically asymp-tomatic. Daily clinical monitoring was continued until day 52 after inoculation, at which time she was scanned at 0.15 T using the multi-slice sequences. Bilateral hemispheric lesions were identified on these scans. The SE images showed the right hemisphere lesion to extend from slice 3 to slice 7, with the largest area occurring in slice 5 (155 mm 2). The lesion was irregularly shaped and had variable intensity throughout the hemisphere. The extent of the lesion could also be seen clearly on the IR images, on which, lesions appear as dark areas of increased T i . Areas of increased signal intensity could also be seen in the left cerebral hemisphere in slices 4 and 5 (figure 3.56). These areas were seen to increase and decrease in size over time. On day 86 after inoculation, a new lesion appeared in the left cerebral hemisphere in an area previously unaffected (figure 3.57A). This lesion could be identified on both the SE and the IR images. Over a period of 8 days the lesion changed in size and intensity and after 23 days was barely discernable on either SE or IR images (figure 3.57B, 3.58). By day 109 post inoculation, all areas of increased signal intensity had almost completely disappeared, apart from pale areas still discernible on slices 4 and 5 in the right cerebral hemisphere. Until this point, no clinical signs were apparent in this animal; periodically however, she demonstrated possible left arm weakness; this was not marked and did not affect her agility. Subtle signs of disease were food scattering, irritability, weight loss and a period of anorexia from day 54 to day 58; these signs are considered prodromal ones, and are not specific for E A E [123]. This animal maintained the use of all four limbs, and vision did not deteriorate. Daily clinical monitoring and serial scanning were continued. Figure 3.56: SE M R I on day 52 after inoculation. A . Slice 4. B . Slice 5. Figure 3.57: A . SE M R I level 4 on day 86 after inoculation, showing new lesion in the right cerebral hemisphere. B. SE M R I level 4 on day 103 after inoculation. In both images T E = 120 ms and T R = 2 sec. Chapter 3. MRI of Experimental Allergic Encephalomyehtis in Primates 116 160 Time (days) Figure 3.58: Plot of individual lesion area versus time after inoculation in slice 4, case 10. This plot shows the appearance of the new lesion in the left cerebral hemisphere on day 86. On day 167 after inoculation, she demonstrated obvious clinical signs, holding her left arm to her chest; she did not use her left hand to grasp or eat. This clinical development was not associated with any obvious change in the M R I . She subsequently recovered, then on day 197 after inoculation, a new lesion or an exacerbation of an existing lesion was seen in the right temporal lobe in slice 7; this was associated with the onset of / apathy and disuse of her left arm. She once again recovered, and was clinically stable until sacrificed on day 352 after inoculation. Intra-venous injection of Gd- D T P A during the pre-mortem scan caused no enhancement in the lesion areas. The variation in total lesion area versus time after inoculation is shown in figure 3.59. Comparison of the M R I with the Gross Distribution of Lesions The brain was cut into 5 mm thick slices as described in the methods section. Grossly, there was little pathology to be seen. Figure 3.60A shows the slice in which the new lesion Chapter 3. MRI of Experimental Allergic EncephalomyeHtis in Primates 117 Figure 3.59: Plot of total lesion area versus time after inoculation for case 10. Data is shown to day 135; little change was observed in the brain after this point. was observed on day 86 after inoculation; a small abnormal area can be seen grossly. The lesion in the left cerebral hemisphere was not clearly discernible (figure 3.60B), however other areas, which had previously been abnormal on the SE M R I , appeared to be gliotic, and sections were taken for histologic examination from these areas, together with the other areas of interest already mentioned. Histology The left hemisphere lesion corresponding to the new lesion seen on day 86 after inocu-lation, consisted of minimal perivascular inflammation; the area was mildly gliotic and hypercellular. In one area, the number of oligodendrocytes was decreased and reactive astrocytic cytoplasm could be seen; the lesion was obviously old and inactive. The lesion still visible in the left hemisphere in M R I slice 5, showed one perivascular Figure 3.60: A . Inferior surface of fixed brain slice showing area of relapse. B . The inferior surface of fixed brain slice equivalent to M R I level 5. Chapter 3. MRI of Experimental Allergic Encephalomyelitis in Primates 119 inflammatory cuff; reactive astrocytes were seen and one area was intensely gliotic. The lesion was located close to the lateral geniculate body, as was seen in the M R I , and extended into the cortex. The other areas, which had previously been abnormal on the SE M R I , showed areas of hypercellularity and mild gliosis. These areas were located close to the posterior horn of the left lateral ventricle and in the right occipital lobe. Just as for the lesions described above, these areas were clearly old and inactive, with no evidence of recent inflammation. Quantitative M R I The changes in T\ and T 2 for the new lesion seen in the right cerebral hemisphere on day 86 were plotted, as shown in figure 3.61. Chapter 3. MRI of Experimental Allergic EncephalomyeHtis in Primates 120 A. 400 T 395 390 385 T l (ms) 380 375 370 365 360 -70 B. 70 80 90 100 110 120 130 140 Time (days) Figure 3.61: A.Plot of Ti versus time after inoculation for the new lesion in the right cerebral hemisphere in M R I slice 4. B . Plot of T2 versus time after inoculation for the same lesion. T2 (ms) A. wv 95 90 85 80 •7 S Chapter 3. MRI of Experimental Allergic EncephalomyeHtis in Primates 121 3.4 Summary of Results 1. M R I detected acute E A E lesions in primates when only prodromal signs were present chnically. This M R I change was due to an increase in the Ti and T 2 values in the lesion area. Preclinical lesions have also been seen in the CNS of guinea pigs with E A E [125]. 2. M R I was an accurate indicator of the gross extent of the lesions of acute E A E [126]. 3. The oldest lesions were the most hemorrhagic, but the hemorrhage occurred in two patterns, one near vessels displacing the exudate peripherally and one at a slight distance from a much less inflamed vessel. Only the former pattern is consistent with the classical belief that hemorrhage is a secondary event in E A E . 4. In lesions with varied histologic components the M R characteristics of the lesions also varied (table 3.21). Conversely, microscopically similar lesions had the same M R characteristics. The order of T\ and T 2 values given in table 3.21, corresponds to increasing degrees of inflammation, demyelination and hemorrhagic necrosis. 5. As the disease progressed, the M R I characteristics also changed. Since the T\ and T 2 values are sensitive to the molecular environment around the protons, these M R I changes are indicative of progressive lesion evolution at a molecular level. 6. Acute inflammatory lesions were not clearly defined on the M R I of the fixed brain. Formalin fixation of these lesions must give rise to a molecular environment that is indistinguishable from normal tissue using M R I . However, just as seen in MS tissue, the small gliotic lesion in case 4 could be clearly seen in the images of the fixed brain. Chapter 3. MRI of Experimental Allergic Encephalomyehtis in Primates 122 7. The results show, for the first time, that it is possible to produce spontaneous relapsing-remitting E A E in a primate following a single immunization and without treatment. This mimics the M R I and clinical course observed in the serial studies of relapsing MS patients. 8. Just as for acute E A E , lesions were detected before the onset of clinical signs. The lesions in case 10 were notably less intense that those seen in the acute cases; this corresponds to a lower T2 value. At the time of death, these areas corresponded to mildly gliotic lesions with little evidence of recent inflammatory activity. 9. Treatment with dexamethasone phosphate causes measurable changes in the size and intensity of lesions, but for the cases presented here, was unsuccessful in pro-ducing complete clinical remission followed by relapse upon withdrawal of therapy. 10. Gd-DTPA studies showed breakdown in the B B B within a few hours of disease onset, and before clinical signs were apparent. No enhancement was seen in the lesions in case 10, which were mildly gliotic. 11. The location of lesions in case 9 could be correlated with the clinical symptoms observed; this is not always possible. Chapter 3. MRI of Experimental Allergic Encephalomyelitis in Primates 123 C A S E Ti(ms) T2(ms) 1 500 120 9 500 120 9 520 160 2 460 110 3 540 260 4 600 270 Table 3.21: T i and T 2 values from lesions in cases 1, 2, 3, 4 and 9 obtained at the time of death. Chapter 3. MRI of Experimental Allergic EncephalomyeHtis in Primates 124 3.5 Discussion The studies show that MRI can detect EAE in primates before the onset of neurological signs; similarly, in MS, MRI-visible lesions may appear in the CNS unaccompanied by any new clinical symptoms. It remains to be proved whether the first lesion in EAE is pathologically similar to the chnically asymptomatic lesions in MS. It is recognized that hemorrhage is not frequently seen in the CNS of MS patients; however, it may be that the perivascular inflammatory lesions, which may or may not be confluent, could be similar in both acute EAE and MS. As mentioned previously, serial MRI studies carried out on MS patients at U.B.C. have shown asymptomatic MRI detected lesions to come and go with a frequency that is much higher than the development of clinical signs; most of these lesions become smaller and some disappear over time. The time relations make it unlikely that these new and disappearing lesions are demyelinating; it is likely that they are primarily inflammatory. One must be wary of assuming a complete recovery in these areas of the brain, since the disappearance of a lesion on the MRI does not necessarily indicate that the tissue has returned to normal, as was clearly demonstrated in case 10. In cases 1-4 (acute EAE), the monkeys developed clinical signs within 12-24 hours after the areas of increased signal intensity were seen on the SE images. These earlier cases were also characterized by very bright areas of increased signal intensity on SE images and correspondingly dark areas on the IR images due to the significant increases in T 2 and T i . The lesions in case 10 were notably less intense. This is an important point, since this animal did not go on to develop clinical signs until much later in the course of the disease, even though the lesion size was significant, and comparable to the lesion size in all other animals. The pathology of the lesions in this case was different from previous cases, being mildly gliotic; evidence of the earlier inflammation was observed in the lesion still visible on the MRI at the time of death. Chapter 3. MRI of Experimental Allergic EncephalomyeUtis in Primates 125 It has been interesting to note the disappearance of the inflammatory lesions on the MRI of fixed tissue. It appears that as the tissue becomes more severely affected by the disease process, then the lesions become more visible on the fixed brain SE MRI. The reaction believed to be of major importance in the formaldehyde fixation of tissue, is the formation of methylene bridges between the e-amino group of lysine and available amide groups in peptide linkages of other proteins [116]. The proteins present in the exudate of the inflammatory lesions will be cross- linked on fixation, which must give rise to a relationship between bound and free water similar to that seen in normal fixed tissue. This is in contrast to fixed demyelinated and gliotic lesions, which are both MRI visible. In vitro quantitative MR studies on fresh and fixed tissue are described in chapter 4 [127]. These studies provide more detailed information regarding the pathological changes which give rise to the changes in Ti and T 2. The role that demyelination plays in the relaxation mechanisms of the tissue water protons is still not clear. The studies on human MS tissue presented in chapter 2, show that demyelinating lesions cause an increase in Ti and T 2, but how this change compares with the effects of inflammation is not yet established. The areas of abnormality seen grossly on the fixed brain slices in EAE correspond 0 = C H C — ( C H 2 ) 4 N H 2 + HOCHgOH + H ~ N NH I (3.35) Chapter 3. MRI of Experimental Allergic EncephalomyeHtis in Primates 126 well with the areas of increased signal intensity seen on the SE images. This has im-portant implications when studying the evolution of the pathological process over time in both E A E and MS. The technique used to measure the extent of disease must be both accurate and reproducible and if disease is being followed over time using M R I , repositioning of the subject in follow-up studies is critical. Studies in MS patients at U . B . C . have shown that a 5 mm deliberate offset in the slice position causes an over-all lesion area change of 11%; changes in individual slice lesion area can be as high as 50%, particularly when small lesions are present [23]. Repositioning is also critical for the quantitative measurement of M R I parameters. The images of the monkeys were obtained by selectively exciting a slice 5 mm thick. Within this thickness there may be water protons in different compartments or in different types of tissue which are not rapidly exchanging; as previously mentioned, this may give rise to multi-exponential re-laxation behaviour [128,129]. The signal contribution from each proton source depends on its volume and on the pulse sequence used to excite the slice, and the resulting image intensity in each pixel is the sum of the signal contributions through the slice. For a SE image the observed intensity would be given by equation 3.36; hr = J2l0exp(-2T/T2i)[l - exp(TR/Ti)] (3.36) i=i where n is the number of exponentials required for an adequate description of the data. This means that the computed images can only provide observed Ti and T2 values, giving no indication as to their source. Accurate repositioning is essential, since variations in Ti and T2 may be due to variations in the contributions to the signal from different compartments within the slice. In addition, the lesion may increase in size, filling the slice: this gives rise to an "observed" increase in the relaxation times because of the increased contribution to the signal from the abnormal area. The results of the quantitative M R I studies show that the Ti and T2 values increase Chapter 3. MRI of Experimental Allergic Encephalomyelitis in Primates 127 over time; when clinical signs were stable, as in case 1, the T\ and T2 values also stabilized. This finding seems to indicate that the pathological progression of the disease also halted, since T\ and T2 are sensitive to changes in the molecular environment. It must also be noted, however, that small changes in Tx and T2 (< 10%) will not be observed, and some molecular changes may cancel each other out in terms of these observed two-point determinations of T\ and T2. When studying the development of E A E lesions in case 4, it was noted that T\ began to increase before the lesion became visible on the SE image. This was also seen in the other cases of E A E in primates; in particular, the new lesion seen on day 86 in case 10, and indicates that the initial pathological changes affect T\ rather than T2. At different points in the study of case 10, the changes in T i were more dramatic than T2, showing small dark areas of increased T i on the IR images. As a results of this, parts of the lesions were more easily seen on IR than SE images. The initial increase in T\ is small, and falls within the error boundaries; therefore, although it is reproducible, it is questionable whether it could be used to predict the appearance of a lesion using these simple quantitative M R I methods. In order to use T\ and T2 to distinguish between different types of lesions, the changes in the measurements must be large compared to the errors involved. Of the nine E A E cases presented here, the most dramatic changes in T\ and T2 were observed in case 4, corresponding with lesions that were more demyelinated and hemorrhagic than those in the other cases. The difference in T\ between a lesion with no hemorrhage and little demyelination (case 1) and the lesions of case 4 is 100 ms. This difference is large enough to be considered significant. If we look at table 3.21, we see that the relaxation times for case 2 are lower than might be expected, based on the type of pathology that was present; these low values can be explained in terms of volume averaging. The lesion in this case, consisted of small inflammatory areas which did not coalesce; the area of increased signal intensity on the SE image is therefore not homogeneous, and any area chosen to obtain Chapter 3. MRI of Experimental Allergic Encephalomyehtis in Primates 128 values for the relaxation times will consist of normal and abnormal tissue, giving rise to the lower T\ and T2 values. A t the present time, the pathological process primarily responsible for the difference in M R I parameters in the lesions seen in these four cases cannot be identified. Further studies on demyelinating lesions will be necessary before we can state which pathological process gives rise to the different changes in Ti and T2. From the post mortem MS studies, the data indicates that the most gliotic lesions have the highest Ti values. Where inflammation fits into this picture is not yet understood. Both inflammatory and gliotic lesions are associated with an increase in water content [118], which in turn affects the relaxation parameters. It may be that inflammation and gliosis will have similar M R I characteristics; they could, however, be distinguished using the length of time they have been present in the brain. In addition, case 10 shows that old inactive areas do not show enhancement with Gd-DTPA; this could be due to the lack of severity of these areas but does suggest that gliotic lesions will behave differently from inflammatory ones. Treatment of E A E with steroid and M B P shows significant changes in the size and intensity of the lesions, as seen in case 9. Case 10 has shown, for the first time, that spontaneous relapsing and clinically asymptomatic M R I lesions are possible following a single immunization to produce E A E in a primate. The observations made in this case were comparable to the M R I changes seen in serial studies of MS patients. In conclusion, it is clear that there are measurable differences in the relaxation times when different pathology is present. However, just as for the studies on MS tissue, prob-lems arise due to limitations in the slice thickness possible (causing volume-averaging), and in the inability to isolate the effects of inflammation, demyelination and hemorragic necrosis on the relaxation times. The limits of the quantitative M R I methods used on this system have already been alluded too. The next chapter describes quantitative in vitro T\ and T2 measurements on CNS tissue; these studies demonstrate the lack of Chapter 3. MRI of Experimental Allergic EncephalomyeUtis in Primates 129 information available using these two-point computations to determine the relaxation times. Chapter 4 In Vitro Quantitative Relaxation Measurements 4.1 Practical Aspects Measurement of relaxation times in tissue presents many problems; not least of these is the complexity of the tissues themselves. In addition, measurements on tissue in vitro may be affected by sample preparation; which includes the effects of blotting, squashing, freezing and thawing, elapsed time after death and excision, and fixation; ideally, the tissue should also be studied at physiological temperature (37°C), but this is not always practical. Many of these factors have been studied, and the data can be summarized as follows: Beall et al carried out extensive studies on many different tissues, and have made numerous suggestions for successful relaxation measurements to be obtained from tissue samples [131]. Any action which causes a change in the water content of the sample should be avoided since this could potentially alter the values of T i and T 2 . Samples should therefore be kept in tightly sealed containers and should not be rinsed with saline or blotted with any absorbant substance, since this could also affect water content. When removing tissue, it should be cut firmly and quickly with a sharp scalpel; cutting with scissors is not recommended as it forces fluid out of the tissues. Likewise, soft tissue should not be forced into the N M R tube, since this will also result in fluid loss. It is important that the same piece of tissue studied using N M R should be sent for pathological examination, since tissue heterogeneity may lead to erroneous results when compared with the pathology. A group of samples will display a certain variation, 130 Chapter 4. In Vitro Quantitative Relaxation Measurements 131 therefore as much data as possible should be collected; Beall also recommends that extensive reproducibility studies be carried out before acquisition of final data. This is not always practical, since samples may be obtained at short notice and although other authors have shown that the effects of elapsed time on T\ and T2 after excision are minimal up to 24 hours [111,112,113], tissue deterioration begins immediately upon excision and it is important that measurements are carried out as soon after death as possible if comparisons are to be made with data obtained in vivo. Studies on the effects of temperature show a linear dependence of T\ from 20 — 50°C; T 2 showed very little dependence on temperature in this range [114]. The many different methods available for determining Tj and T 2 in vivo and the many different magnetic field strengths being employed, result in large variations in the values obtained from the same tissue type. The lack of any standard measurement and the field dependence of T i thus make it difficult to evaluate the true potential and usefulness of making such measurements; the large variation in values of the relaxation times also complicates the interpretation of the data. 4.2 Tissue Relaxation Models Spin relaxation occurs due to fluctuating magnetic fields at the site of the nucleus. For relaxation in tissue, where for the most part we are looking at tissue water, the most important mechanisms for this relaxation are dipole-dipole interactions and rotational and translational motions. Correlation times for these interactions must therefore be considered in mechanistic models for relaxation in tissue. This includes exchange rates for water molecules which are bound (hydrogen bonded to hydrophilic regions) to macro-molecules with those that are in the bulk water. When placed in a static magnetic field, B0, all particles with spin (that is, species Chapter 4. In Vitro Quantitative Relaxation Measurements 132 Figure 4.62: Vector diagram showing the interaction of two spins. containing nuclei having spin or unpaired electrons) have magnetic moments, and will therefore produce local magnetic fields. In the M R experiment, the spins are perturbed from their equilibrium state using a rotating field, B\, perpendicular to B0. After perturbation, due to the motion of these particles, a particular nucleus will experience a fluctuating field from neighbouring spins, due to changes in r and 8 (figure 4.62). When these fluctuations occur at the resonance frequency corresponding to a spin transition, energy transfer will occur: the spin relaxing to the lower state and the energy quantum being dissipated as thermal energy in the bulk of the sample. If A n is the disturbance from the equilibrium population difference at a time t, and An0 is the value of A n at time r 0 , then: A n = An 0e^T (4.37) where Ti is the spin-lattice relaxation time, and the magnetization decay is mono-exponential. The rate, or efficiency of dipole-dipole relaxation depends on both the strength and frequency of the fluctuating fields. These in turn depend on three factors, Chapter 4. In Vitro Quantitative Relaxation Measurements 133 namely: a) the distance between the nuclei involved and the angle between the vector that joins the nuclei and B0, b) the effective correlation time, T c , of the vector that joins the nuclei and c) the nature of the nuclei themselves. For solutions, we make the assumption that molecules tumble isotropically, that is, tumbling occurs equally easily in all directions. For large molecules and macromolecules, this may not be possible, in which case the tumbling is said to be anisotropic [132]. This is important in tissue, where the water molecules are, at least part of the time in association with membranes and macromolecules in the cytoplasm, and this in turn affects the relaxation rates. For two interacting spins undergoing isotropic reorientation with a single correlation time and exponential correlation function, the dipole-dipole relaxation rates are given by equations 4.38 and 4.39 [133,134]. 1 -K[—^—^r-^—) (4.38) T1DD ll + w 2 r c 2 l + 4u; 2r 2 1 K p T c + r P ^ + T r ^ } (4-39) T2DD 1 l + ^ T , ? where K = 3/x 2/i 27 4/1607r 2r 6, and is a constant for a particular nucleus; u0 = 2irv0 is the resonance frequency and T c is the correlation time, both these parameters are variables. At 100 MHz (which is close to the resonance frequency at which the measurements described in this chapter were carried out), u> = 6.4 x 1085ec -1; since r c for free water is approximately 1 0 - 1 2 sec, the product UTC = 6.4 x 1 0 - 4 , which is much less than 1 and (T2U>2) is even smaller. Thus the above equations reduce to; ± = K[Tc-r4Tc] = 5KTc (4.40) ±? = K[3TC + 5r c + 2rc] = = 5Krc (4.41) ±2 * giving 1/Ti = I/T2 = 5KTC for noviscous liquids. Relaxation requires magnetic field fluctuations at the Larmor frequency, u0. Since these fluctuations are produced by Chapter 4. In Vitro Quantitative Relaxation Measurements 134 2 aa Figure 4.63: Diagram showing the possible transitions for two coupled spins. 0 - zero quantum transition, 1 - single quantum transition and 2 - double quantum transition. proton tumbling at a rate (T c ) - 1 , relaxation involving single quantum transitions (figure 4.63) is most efficient when U>0TC ~ 1. Not all molecules tumble at the same rate, and they are continually colliding thereby changing their rate and direction of tumbling. At temperatures below room temperature, molecules will undergo slow reorientation, giving rise to a long r c; conversely, higher temperatures give rise to short r c. The relaxation time increases with increasing temperature, and decreases with increasing correlation time; this increase with temperature is due to the decrease in r c at higher temperatures. The number of protons rotating at each frequency across a range of frequencies is defined as the spectral distribution, J w (equation 4.42); where G(T) is the correlation function. For large molecules or viscous solvents, where U0TC is greater than 1, slow tumbling cannot give rise to fields at the Larmor frequency. As a result, single quantum relaxation processes cease to be efficient. When UOT0 is close to 1, the spectral density, or the intensity of appropriate field fluctuations, is at a maximum, and so relaxation is also most efficient. Since u>0 depends on the field strength, the relaxation rate of a particular /•+00 Ju = G(T)exp(iuT)dr J—oo (4.42) Chapter 4. In Vitro Quantitative Relaxation Measurements 135 spin is field dependent in molecules, or parts of molecules, where U>QTC ~ 1. Zero quantum transitions (figure 4.63) involve a mutual spin-flip between two nuclei that have similar energy levels. Such transitions do not give rise to spin-lattice relaxation, but involve the excess energy being transferred from one spin to another. When u>0tc 1, zero quantum transitions are efficient. Thus, spin-lattice relaxation is slow, but the life-time of an individual spin state is short because the excitation passes via mutual spin-flips to other spins. As a result, Ti is long but T 2 is short, giving rise to the broad N M R signals seen from solids; the T 2 for solids is therefore field independent for r c X 1 0 - 5 . As the internuclear distance between two particles increases, the intensity of the field produced by their interaction decreases, due to the r 6 dependence, and so the efficiency of relaxation also decreases. As was described in section 1.3.2, the presence of paramagnetic species also affects relaxation, since ^electron is about 1000 times bigger than 71//. The most common paramagnetic species is dissolved oxygen and in its presence the nuclear relaxation rate will increase; in tissue, changes in amounts of dissolved oxygen may affect relaxation rates in tumors for example [135]. The application of the relaxation theory described above to tissue, is complicated by the heterogeneity in the sample; protons may be in different compartments or in different chemical environments within a compartment, with exchange processes between these different types. The observed Ti and T 2 values for tissue could not be explained in terms of a simple solution to the Bloembergen, Purcell and Pound theory; however, the data could be explained if a small fraction of cell water was assumed to be immobilized on the surface of macromolecules, thus giving it a correlation time of the order of ice. Fast exchange between the free and bound water then gives rise to an observed relaxation time dependent on the fraction of bound water; Chapter 4. In Vitro Quantitative Relaxation Measurements 136 This is referred to as the fast exchange two-state (FETS) model of Zimmerman and Brittin [109]. Other models invoke three or more separate water states with variable rates of exchange between pairs of states [147,148]. A n example of such a model will now be considered; from relaxation measurements on protein solutions at different frequencies, Grosch and Noack [137] believe a three compartment model is sufficient to fit their data; this is supported by other literature data [138,139]. This three-compartment model consists of the following; 1. Bound water - water directly hydrogen bonded to a fixed polar or ion site on the macromolecule. 2. Structured water - water motionally perturbed by being in the vicinity of a macro-molecule, but not bonded to it. 3. Bulk water - water in the bulk phase where molecular motion is determined solely by the interaction characteristics of the water molecule. The water spin-lattice relaxation rate is then given by; m = ^ - r ^ - + ^- (4.44) J-lw -list *lb where /„,, fst and fb are the fractions of bulk, structured and bound water respectively, and there is fast exchange between the three fractions. Studies on lysozyme have shown that the bound water fraction, /„,, determined by an N M R titration method[140], con-sists of water molecules directly hydrogen bonded to the macromolecule with typically two or more ligands. These bound water molecules are in intimate contact with the macromolecule, being spatially close to it. The mean separation of bound water from polar portions of a polypeptide is only 2.9 A, while water molecules next to nonpolar sur-faces are at a distance of 3.7 A [1.41]. As described above, the dipole-dipole interactions Chapter 4. In Vitro Quantitative Relaxation Measurements 137 vary as 1/r6; therefore the influence of macromolecular motion by cross-relaxation will be visible in 1/Tib but not 1/Ti, t [142]. The correlation times determined using the N M R titration method are comparable to those obtained by dielectric study [136]. Just as for the F E T S model, the observed relaxation time in this three-compartmentment model is dominated by the fraction of bound water; it is also dependent on the molecular weight of the protein, which determines the rate at which the molecule tumbles [143]. Other investigators propose models assuming a continuous distribution of water pro-ton correlation times [144,145] to simultaneously explain Ti and T 2 observations. More recently, relaxation data has been analyzed in terms of a continuous distribution of relax-ation times [146]; this approach is the most valid when the complex nature of the tissues is considered. The water molecules will sample many different environments during the time course of the M R experiment; depending on the correlation times for the interac-tions involved, a single or multi-exponential decay will be observed. The data presented here support the recent literature data; simple models involving compartmentalization of water in tissue are inadequate to explain the observed results. Many literature stud-ies have shown that a single exponential fit to relaxation data is insufficient to describe the decay observed; a number of authors report three-exponential fits for some tissue data, ascribing the values to bound (~ 10 ms), intracellular (~ 70 ms) and extracellular (~ 200 ms) water [147,148]. Such compartmentalization is unlikely, due to the effects of diffusion, since water can diffuse readily through the cell membrane. Recent observa-tions on model systems for edema have shown that cell membranes alone are insufficient to produce multi-exponential relaxation behaviour [149]. These studies showed that for suspensions of red blood cell ghosts, Ti and T 2 had mono-exponential decays; multi-exponential decay was only observed in samples with the cells packed in layers, and was only observed for T 2 . For normal CNS tissue, there is no extracellular space, suggesting Chapter 4. In Vitro Quantitative Relaxation Measurements 138 that a maximum of two exponentials will be observed; the data presented here demon-strate that such a simple interpretation does not explain the observations. The data presented here are analyzed in terms of a fixed number of exponentials and in terms of a continuous distribution of relaxation times; the advantages and disadvantages of both models and possible explanations for the observed data are addressed in the discussion. 4.3 Me thods 4.3.1 T 2 Measurements A l l measurements were carried out on a modified Bruker SXP 4-100 N M R spectrometer operating at 2.1 Tesla (90 MHz for protons). The signal was acquired using a Nicolet 2090AR dual channel digital oscilloscope and successive scans accumulated by a National 32016 microcomputer serving a microVax II computer. The 90° pulse varied from 3—4 pis, and the 180° pulse was equal to twice the 90° pulse length. Before measurements were made on tissue, the spectrometer was set up using water doped with copper II sulphate. The phase, amplitude and pulse lengths were optimized using the rf pulse sequences shown in 4.45, 4.46 and 4.47. (90° — data acquisition — tT—)„ (4.45) (90° — data acquisition — tr—)„ (4.46) (90°_x — data acquisition — tr—)„ (4-47) This was then repeated for the tissue samples before the experimental run. T 2 mea-surements were made using the C P M G pulse sequence 4.48; the phase of the 90° pulse was alternated 180° for each excitation. Four points were collected for each of the first 224 echoes, followed by every eighth echo up to 512, giving a total of 736 echoes. The Chapter 4. In Vitro Quantitative Relaxation Measurements 139 Figure 4.64: C P M G decay curve for CNS tissue interpulse delay, r = 400 /xs, acquisition time was 1.7 sec and a delay, tr, of (90:(90°_x) - T- - 180°. - (r - 180°.-)„ - r - 180p (4.48) 10 sec was left to allow the system to return to equilibrium before reapplication of the pulse sequence. A number of samples were also studied using a r-value of 800 [is to de-termine whether the measurements were tau- dependent. Fifty transients were collected for each sample, each odd numbered transient being added and each even numbered transient substracted to give the final data. The baseline was determined from the raw C P M G data and the data outputted as follows; 35 points at 1 point intervals, 50 points at 2 point intervals and every 5th point to the end of the data. Each data set then consisted of 110-130 points to be analyzed using the programs described in section 4.3.3. A typical decay curve is shown in figure 4.64. Measurements were carried out at room temperature (24°C). Chapter 4. In Vitro Quantitative Relaxation Measurements 140 3000 2500 -c 2000 -• ? 1500 -(0 1000 -w c 500 -- 5 0 0 —I I I i I I ' ,' pretrigger d a t a ROI - i — i — | — i — i — i — i — j — i — i — r — i — | — i — i — i — i — ] — i — i — i — • — i — i i-50 100 150 200 250 Time (ps) 300 Figure 4.65: FID from lesion 1, case 4, using the modified IR pulse sequence. The pretrigger and data regions are labelled, together with the region of interest chosen. The tau value was 1 ms. 4.3.2 Ti Measurements For these measurements, a modified IR sequence was used (4.49). The signal obtained after the first (second) 90° pulse was added (subtracted) to (from) the accumulated signal in memory. (~tr - 90° -tr - 18C£ - r - 90°- )„ (4.49) The delay tr was 10 sec, the 90° and 180° pulse lengths were 3.1 ps and 6.2 ps respectively and 30 values of tau were used, varying from 1 ms to 3 sec. Data for each tau were collected in a random order to average out any systematic errors in the data acquistion. The number of transients, n = 16, the block size was 512 and the dwell time 0.5ps. The FID from one sample is shown in figure 4.65. The pretrigger region was used to determine the baseline noise, and a 10 point region of interest chosen in that part of the decay curve corresponding to mobile protons, from which the average signal intensity was determined. A plot of signal intensity versus tau then gives the result shown in figure Chapter 4. In Vitro Quantitative Relaxation Measurements 141 1. D 1 .5 T a u v a l u e ( s e c ) Figure 4.66: Plot of signal amplitude versus tau for lesion 1 from case 4. 4.66. This modified IR sequence was used since it allowed the Tj data to be analyzed using the same programs as those used for analysing T 2 data; it is also less susceptible to systematic error than the conventional IR method. 4.3.3 Analysis of Relaxation Data Minuit The C P M G data were analyzed using minuit, a non-linear functional minimization program [150]. The program allows the number of exponentials to be specified and the optimum number for each sample was chosen to be the minimum number that ad-equately described the data. The values of T 2 obtained must also be separated by at least a factor of three. For three exponentials, the data are fitted to equation 4.50; It = Aexp{—^-) + Bexp(-^r) + Cexp(-^r-) ±2a J-2b J-2c (4.50) where It is the signal intensity for a given time t after the 90° pulse, and A , B and C are the respective contributions to the signal for each T 2 value denoted by a, b and c. Chapter 4. In Vitro Quantitative Relaxation Measurements 142 Non-Negative Least Squares (NNLS) In contrast to minuit, this method analyzes the data in terms of a continuous distribution of relaxation times. This has already been done as a linear problem by Provencher [151] and first applied to M R by Kroeker and Henkelman [146]. For the data presented here, least squares and linear programming algorithms were used to interpret relaxation in terms of a spectrum of relaxation times [152]. These stable methods are non-iterative and avoid all of the difficulties associated with nonlinear optimization schemes. In addition, they are not restricted by any assumptions which limit the number of possible relaxation times. The general integral equation describing multi-exponential relaxation is; y(U)= fas(T)e^TdT (4.51) Jb where i = 1,2, . . . iV, the N data yi are measured at times and s(T) is the unknown amplitude of the spectral component at relaxation time T. The limits on the integral are restricted to the finite values a and 6, which are chosen to contain the values of T\ and T2 expected for the physical system being analyzed. The approach taken is to assume a large number of known times Tj, and then solve for the corresponding amplitudes, s,, some of which may be zero. For computer imple-mentation, equation 4.51 must be discretized. The general form of the discrete version of equation 4.51 is given in equation 4.52; Af y. = E ^ i 5 i (4-52) where i = 1,2, ...N. The matrix system defined by equation 4.52 can then be solved using the NNLS algorithm of Lawson and Hanson [153]. Chapter 4. In Vitro Quantitative Relaxation Measurements 143 4.3.4 Spectroscopy As mentioned previously, breakdown products of myelin and other CNS components may give rise to a signal from mobile lipid components. In order to determine whether the signal observed was only from water, or from lipid and water, spectra were collected at points along the CPMG pulse train; this was achieved using the sequence shown in 4.53. (90° - I _ 180°. - (r - 180°,-)n - T- - data acquisition) (4.53) In the sequence, n was varied to allow echoes to be collected 0.4-1000 ms after the 90° pulse. The data were then Fourier transformed and examined. 4 .3.5 Protocol for Studying Tissue EAE was produced in 14 Hartley guinea pigs by intradermal injection of a water-in-oil emulsion containing homogenized whole CNS tissue and FCA [117]; five controls were inoculated with FCA only. The animals were monitored daily for the onset of clinical signs; typical signs are weight loss, a slow righting reflex, and hind leg weakness which can progress to complete paralysis of the hind legs. Clinical signs may appear at any time after day 7 post inoculation. Once signs were detected, the animals were sacrificed at different stages of the disease; that is, some with hind leg weakness, some with a slow righting reflex and others that were completely paralyzed. The CNS was then removed intact. The spinal cord was divided into 5 sections, using a sharp scalpel, denoted by their approximate level in the spinal column: cervical, upper thoracic, lower thoracic, lumbar and sacral (figure 4.67). Care was taken to ensure the tissue was not damaged. The outer-most membrane (the dura) around the CNS tissue was removed, and the inner two membranes (the arachnoid and the pia-mater) were left intact. A section was also removed from the centre of the left cerebral hemisphere and similarly placed in an NMR tube. Once the T2 measurements were completed, the tissue samples were placed in 10% Chapter 4. In Vitro Quantitative Relaxation Measurements 144 C UT LT L S 1 LH B ( T f glass tissue nmr tube rod Figure 4.67: A . Diagram showing division of spinal cord into five sections denoted by their approximate level in the spinal column. B . The tissue was gently rolled onto the small diameter glass rod and placed in the bottom of the 10 mm N M R tube. C - cervical, U T - upper thoracic, LT - lower thoracic, L - lumbar, S - sacral and L H - left hemisphere. i neutral buffered formalin; after at least one week of fixation, the T2 measurements were repeated on the fixed tissue. The fixed samples were then embedded in paraffin and stained with hematoxylin and eosin (H & E) [116]. Samples were then graded according to table 4.22. E A E in Primates T2 data were obtained from the tissue samples taken from case 8 in chapter 3. Once the studies were completed, the tissue was fixed in 10% neutral buffered formalin in preparation for any histology required. Fixed MS Tissue In order to determine accurate values for the tissue aleady studied using quantitative M R I methods, samples were taken from two of the cases described in chapter 3; case 1 and case Chapter 4. In Vitro Quantitative Relaxation Measurements 145 Perivascular Inflammation in the Subarachnoid Space (PVI-SS) + 1 or 2 perivascular cuffs ++ 3-6 vessels involved with widening of subarachnoid space +++ Many vessels involved and subarachnoid space widened and filled with inflammatory cells Perivascular Inflammation in the Virchow-Robin Spaces (PVI-VR) + 1 or 2 perivascular cuffs ++ 3-6 vessels involved ++-f Many vessels showing perivascular cuffing Parenchymal Cellular Infiltration (PCI)  + A few cells invading parenchyma from 1 or 2 cuffs -f + Invasion of parenchyma by cells from several cuffs +++ Many cells infiltrating parenchymal, but more normal than abnormal tissue ++-f-+ Extensive cellular infiltration into parenchyma, more abnormal than normal tissue Demyelination (D) + Possible ++ Probable +++ Definite Edema (E)  + Possible ++ Probable +++ Definite Table 4.22: Pathological scoring for guinea pig CNS tissue. Chapter 4. In Vitro Quantitative Relaxation Measurements 146 grey Figure 4.68: Diagram showing the right cerebral hemisphere of the superior surface of brain slice 4 in case 1. This is the opposite face to that shown in figure 2.29, page 38. Sections were taken from areas corresponding to lesions 8 and 10. Samples of normal appearing grey and white matter were also removed. 4. These two cases contained lesions which varied in the degree of demyelination and of gliosis. Samples were taken from the opposite face to that from which the block was taken for histologic examination. For case 1, samples were removed from the superior surface of slice 4 as shown in figure 4.68. Tissue samples were also removed from 2 slices in case 4. The sections taken are shown in figure 4.69. Chapter 4. In Vitro Quantitative Relaxation Measurements Figure 4.69: A . Diagram showing the superior surface of slice 7 in case 4, the opposite face to that shown in figure 2.18B, page 52. Sections were taken from the areas corresponding to lesions 1 and 3 as indicated. Samples of normal appearing grey and white matter, as indicated on the M R I , were also removed. B . Diagram showing the superior surface of slice 6 in case 4, the opposite face to that shown in figure 2.18A, page 52. Sections were taken from the areas corresponding to lesion 2 as indicated. Chapter 4. In Vitro Quantitative Relaxation Measurements 148 4.4 Results 4.4.1 Guinea Pig Tissue Fresh Tissue The data from normal and abnormal tissue using minuit to analyse the data, are given in the following tables. The data were divided into the part of the spinal cord and the cerebral hemisphere, and the tissue was examined and given a pathological score according to table 4.22. The histologic appearance of each section was graded according to three degrees of inflammation (figures 4.70, 4.71 and 4.72) and for the presence of demyelination and edema. H k, E is not the ideal stain for identifying demyelination, but where possible it was noted. The pathology for each section is given for each sample after the C P M G data for the same tissue section. Chapter 4. In Vitro Quantitative Relaxation Measurements 149 Figure 4.70: Histologic section stained with H & E , from the sacral spine of guinea pig 14, showing a large cuff of perivascular inflammation in the subarachnoid space. Figure 4.71: Histologic section stained with H & E , from the upper thoracic spine of guinea pig 13, showing a perivascular cuff in a Virchow-Robin space. Chapter 4. In Vitro Quantitative Relaxation Measurements 150 Figure 4.72: Histologic section stained with H & E, from the lower thoracic spine of guinea pig 16, showing extensive parenchymal cellular infiltration. Comparison of this section with the one shown in figure 4.72 shows the hypercellularity in the tissue around the vessel. GP T2a % T2b % T2c % 1 8.68 11.99 71.51 56.02 201.27 31.98 2 11.25 13.31 82.22 61.70 230.02 24.99 3 10.61 11.77 82.23 61.91 222.02 26.32 7 10.04 12.85 79.43 61.39 233.93 25.76 7 9.98 12.18 76.40 59.65 211.37 28.17 9 10.08 13.04 74.93 59.56 224.73 27.39 18 8.41 9.67 70.99 58.75 190.49 31.58 19 10.27 11.48 75.91 59.11 192.66 29.41 Table 4.23: Results from minuit analysis of CPMG data obtained from the cervical spine of normal control guinea pigs. T2 (ms) values are given together with the percentage contribution of each component to the signal observed. Pathological examination of this tissue confirmed that each section was normal. Chapter 4. In Vitro Quantitative Relaxation Measurements 151 GP T2a % T2b % T2c % 4 9.04 11.21 73.34 60.56 209.13 28.23 8 8.69 12.01 74.87 63.60 213.97 24.39 8 8.69 11.74 72.24 60.33 189.87 27.93 11 8.51 8.21 67.38 49.44 162.64 42.35 12 10.79 10.44 78.73 66.79 209.79 22.77 12 10.86 11.01 81.10 66.91 212.56 22.08 13 8.84 8.64 72.57 56.39 169.49 34.97 14 8.96 9.37 81.85 65.34 219.27 25.28 15 8.89 9.69 75.09 59.51 204.08 30.79 16 9.69 9.60 76.59 59.74 186.26 30.65 17 9.75 9.21 77.59 64.26 199.38 26.52 Table 4.24: Results from minuit analysis of C P M G data obtained from the cervical spine of guinea pigs inoculated to produce E A E . T2 (ms) values are given together with the percentage contribution of each component to the signal observed. Chapter 4. In Vitro Quantitative Relaxation Measurements 152 GP PVI-SS PVI-VR PCI D E 4 + 11 +++ ++ + - -12 +++ ++ + ++ ++ 13 +++ 4-+ ++ + -14 +++ +++ + - -16 ++ ++ + + -Table 4.25: Pathology from the cervical spine of guinea pigs inoculated to produce E A E . PVI-SS - perivascular inflammation in the subarachnoid space, PVI-VR - perivascular inflammation in the Virchow-Robin spaces, PCI - parenchymal cellular infiltration, D -demyelination and E - edema. GP T2a % T2b % T 2 c % 1 8.15 12.69 70.03 52.15 204.23 35.15 2 10.40 12.71 75.90 57.01 214.26 30.28 3 10.37 12.02 79.69 59.93 219.05 28.04 9 7.72 10.68 67.81 53.47 198.19 35.85 18 9.32 10.71 75.95 60.89 199.96 28.39 19 9.82 11.28 72.02 56.72 189.54 31.99 Table 4.26: Results from minuit analysis of CPMG data from the upper thoracic spine of normal control guinea pigs. T2 (ms) values are given together with the percentage contribution of each component to the total signal observed. Chapter 4. In Vitro Quantitative Relaxation Measurements 153 GP T2a % T2b % T2c % 4 9.01 12.27 72.35 58.35 198.71 29.38 8 7.78 10.48 68.29 57.27 179.35 32.25 11 9.81 9.82 78.22 60.52 192.88 29.66 12 9.90 11.10 73.30 55.70 178.99 33.2 13 7.58 7.58 62.72 47.26 154.39 45.16 14 10.48 9.92 82.60 63.08 212.01 26.99 15 11.26 11.46 76.89 60.31 206.07 28.23 16 9.74 10.31 74.68 52.56 177.31 37.13 17 10.79 10.22 79.09 62.42 194.78 27.36 Table 4.27: Results from minuit analysis of C P M G data from the upper thoracic spine of guinea pigs inoculated to produce E A E . T2 (ms) values are given together with the percentage contribution of each component to the total signal observed. GP PVI-SS P V I - V R PCI D E 4 + 11 ++ +^  + - -12 + + 4 - + - -13 +++ ++ + + -14 +++ + + - -16 ++ ++ + 4- -Table 4.28: Pathology from the upper thoracic spine of guinea pigs inoculated to pro-duce E A E . PVI-SS - perivascular inflammation in the subarachnoid space, P V I - V R -perivascular inflammation in the Virchow-Robin spaces and PCI - parenchymal cellular infiltration. Chapter 4. In Vitro Quantitative Relaxation Measurements 154 G P T2a % T2b % T2c % 1 10.38 14.59 70.40 48.39 206.19 37.01 2 11.17 15.66 74.23 52.32 223.15 32.02 3 11.61 14.36 79.69 56.14 228.66 29.49 7 11.44 15.19 75.06 54.61 234.90 30.20 9 11.04 15.11 75.39 55.76 227.55 29.13 18 11.87 14.23 81.66 61.37 235.33 24.39 19 9.07 12.12 67.42 53.83 196.44 34.05 Table 4.29: Results from minuit analysis of C P M G data from the lower thoracic spine of normal control guinea pigs. T2 (ms) values are given together with the percentage contribution of each component to the total signal observed. GP T2a % T2h % T 2 c % 4 9.95 13.13 73.71 53.36 210.76 33.52 11 9.67 10.73 73.44 53.89 182.34 35.38 12 10.20 11.60 74.10 57.10 182.7 31.2 13 11.03 8.98 80.99 51.27 178.25 39.74 14 11.64 12.62 87.77 64.75 232.94 22.63 15 11.27 12.54 74.87 55.67 202.36 31.79 16 10.44 6.99 102.04 69.52 256.75 23.49 17 10.44 11.27 77.63 58.58 203.85 30.15. Table 4.30: Results from minuit analysis of C P M G data from the lower thoracic spine of guinea pigs inoculated to produce E A E . T2 (ms) values are given together with the percentage contribution of each component to the total signal observed. Chapter 4. In Vitro Quantitative Relaxation Measurements 155 GP PVI-SS P V I - V R P C I D E 4 ++ 11 + + - - -12 +++ +++ ++ ++ ++ 13 +++ +++ +++ +++ -14 +++ + + - -16 +++ ++.+ ++++ +++ +++ Table 4.31: Pathology from the lower thoracic spine of guinea pigs inoculated to pro-duce E A E . PVI-SS - perivascular inflammation in the subarachnoid space, P V I - V R -perivascular inflammation in the Virchow-Robin spaces and PCI - parenchymal cellular infiltration. GP T2a % T2b % T2c % 2 11.17 14.79 74.92 54.16 223.87 31.04 3 10.36 12.42 76.83 54.86 220.38 32.72 7 10.96 15.43 74.89 53.46 226.13 31.10 9 10.76 15.18 77.21 55.93 235.22 28.88 18 11.19 13.42 84.23 63.07 232.79 23.51 19 10.99 13.19 71.71 53.26 196.61 33.55 Table 4.32: Results from minuit analysis of C P M G data from the lumbar spine of normal control guinea pigs. T2 (ms) values are given together with the percentage contribution of each component to the total signal observed. Chapter 4. In Vitro Quantitative Relaxation Measurements 156 G P T2a % T2b % T2c % 4 10.48 12.87 80.33 55.86 223.76 31.27 8 10.43 10.93 80.93 59.89 185.69 29.19 11 10.16 11.51 81.19 60.67 201.41 27.82 12 9.20 10.05 66.50 48.98 160.98 40.95 13 10.22 7.12 96.22 68.80 220.50 24.07 14 10.07 10.40 78.74 58.26 203.48 31.34 15 10.94 10.85 78.55 60.72 210.55 28.43 16 9.05 6.60 95.02 66.95 210.41 26.45 17 11.91 10.80 84.87 62.05 205.72 27.15 Table 4.33: Results from minuit analysis of C P M G data from the lumbar spine of guinea pigs inoculated to produce E A E . T2 (ms) values are given together with the percentage contribution of each component to the total signal observed. GP PVI-SS P V I - V R PCI D E 4 ++ + + 11 ++ + + - -12 +++ +++ ++ - -13 +++ +++ ++ ++ -14 +++ ++ + ++ + 16 +++ +++ +++ +++ Table 4.34: Pathology from the lumbar spine of guinea pigs inoculated to produce E A E . PVI-SS - perivascular inflammation in the subarachnoid space, P V I - V R - perivascular inflammation in the Virchow-Robin spaces and PCI - parenchymal cellular infiltration. Chapter 4. In Vitro Quantitative Relaxation Measurements 157 GP T2a % T2i % T2c % 1 10.08 10.48 75.19 54.48 198.19 35.04 2 10.38 11.35 78.78 61.06 211.19 27.59 3 11.41 10.82 85.03 61.22 215.81 27.96 7 10.35 12.91 81.19 61.71 232.92 25.38 9 8.85 10.79 82.63 65.34 246.76 23.86 18 10.15 9.34 80.95 60.33 201.99 30.33 19 9.11 9.77 73.69 57.17 190.68 33.06 Table 4.35: Results from minuit analysis of C P M G data from the sacral spine of normal control guinea pigs. T2 (ms) values are given together with the percentage contribution of each component to the total signal observed. GP T2a % T2b % T2c % 4 9.30 10.71 83.70 60.75 224.66 28.54 8 8.73 10.15 73.19 61.96 183.71 27.89 11 9.36 8.28 72.91 52.78 168.15 38.94 12 9.09 8.73 76.60 61.65 181.29 29.60 13 8.04 6.76 74.53 55.28 168.97 37.96 14 9.96 8.53 81.66 60.37 191.51 31.10 15 9.72 7.96 74.49 52.54 176.23 39.49 16 8.76 6.95 77.68 50.99 158.48 42.05 17 9.55 8.12 82.50 61.01 190.00 30.86 Table 4.36: Results from minuit analysis of C P M G data from the sacral spine of guinea pigs inoculated to produce E A E . The T2 (ms) values are given together with the per-centage contribution of each component to the total signal observed. Chapter 4. In Vitro Quantitative Relaxation Measurements 158 GP PVI-SS P V I - V R PCI D E 4 + + 11 ++ + + - -12 +++ ++ ++ - -13 +++ ++ + - -14 ++ - - - -16 +++ +++ ++ ++ -Table 4.37: Pathology from the sacral spine of guinea pigs inoculated to produce E A E . PVI-SS - perivascular inflammation in the subarachnoid space, P V I - V R - perivascular inflammation in the Virchow-Robin spaces and PCI - parenchymal cellular infiltration. GP T 2 a % T 2 6 % 4 9.42 4.23 95.65 95.78 7 7.00 4.15 88.50 95.85 9 8.17 5.03 92.28 94.97 11 15.64 4.03 96.73 95.96 13 13.11 5.01 91.55 94.99 14 7.31 3.01 96.31 96.99 16 14.93 5.15 96.47 94.84 17 16.33 5.67 94.03 94.33 18 14.80 4.03 94.39 95.97 Table 4.38: Results from minuit analysis of C P M G data from the left cerebral hemisphere of guinea pigs inoculated to produce E A E . T 2 (ms) values are given together with the percentage contribution of each component to the total signal observed. Chapter 4. In Vitro Quantitative Relaxation Measurements 159 GP PVI-SS P V I - V R PCI D E 4 ++ 11 - + + - -13 + + + + -14 - + + -16 - ++ - -Table 4.39: Pathology from the left hemisphere of guinea pigs inoculated to produce E A E . PVI-SS - perivascular inflammation in the subarachnoid space, P V I - V R - perivascular inflammation in the Virchow-Robin spaces, and PCI - parenchymal cellular infiltration. Chapter 4. In Vitro Quantitative Relaxation Measurements 160 A 7 0 • 0 80 40 SO SO Faroant u n R B 7 0 • 0 Feroant 50 40 30 20 10 0 c : Farcant Farcant Faroant 70 •0 50 40 30 20 10 0 E 7 0 60 50 40 30 20 10 0 SO 100 ISO 200 250 - r f ^ — r-j — ^ t * -© SO 100 • 150 200 250 3£ SO 100 150 200 250 300 _• ^ -6— 1 — 1 • so 100 150 200 250 i t 0 SO 100 150 tOO 250 *2 <•»> Figure 4.73: Summary of minuit data for spinal cord. Plot of percentage contribution to signal versus T2 value for guinea pig spinal cord. A . Cervical. B . Upper thoracic. C. Lower thoracic. D. Lumbar. E . Sacral. • tissue from normal controls, • abnormal tissue. Chapter 4. In Vitro Quantitative Relaxation Measurements o 161 NNLS Examples of the results of NNLS analysis of C P M G data from fresh guinea pig tissue are given in figures 4.74 and 4.75. The rest of the data can be found in appendix B . Chapter 4. In Vitro Quantitative Relaxation Measurements 162 -I I I I I X J U o a-, =3 E 1 H _A_ 10 - T — i — i — i — i i 11 10" T 2 ( s ) 10 to Figure 4.74: Plot of amplitude versus T2 value for the lower thoracic spine of guinea pig 16. Chapter 4. In Vitro Quantitative Relaxation Measurements 163 1.0 -€> 0.8 -0.6 -Q. E 0.4 -< 0.2 -0.0 -10"3 1.0 « 0.8 J 0.6 O. E 0.4 < 0.2 -0.0 -11 • I l I 10-3 IO"2 10"1 10° 1.0 -• 0.8 | 0.6 £ 0.4 < 0.2 0.0 -16 | 10"3 IO"2 10"1 10° Figure 4.75: Plot of amplitude versus T2 value for the lumbar spines of guinea pigs 2, 7 and 9 (normal) and 13, 16 and 11 (abnormal). Chapter 4. In Vitro Quantitative Relaxation Measurements 164 F i x e d G u i n e a P i g Tissue Minuit Examples of the data from fixed normal and abnormal lumbar and lower thoracic spine are shown in tables 4.40 and 4.41. NNLS Figure 4.76 shows the results of NNLS analysis of fixed lumbar spine for normal and abnormal tissue. 4.4.2 P r ima te Tissue Minuit The results obtained after analysis using minuit are shown in table 4.42. NNLS Typical examples of the results obtained after NNLS of the primate tissue C P M G data are shown in the figures 4.77-4.79. Chapter 4. In Vitro Quantitative Relaxation Measurements 165 GP T2a % T2h % T2c % 2 24.82 26.33 98.74 45.63 774.77 28.04 4 25.89 26.46 92.75 45.89 723.33 27.65 7 26.73 26.81 95.33 30.78 839.70 42.42 9 12.00 12.49 84.09 63.23 492.00 24.28 11 22.19 24.15 76.68 59.92 561.89 15.92 12 20.25 22.12 76.80 66.52 551.99 15.92 13 27.91 15.93 111.96 61.08 868.28 22.99 14 19.98 22.09 71.65 66.93 402.43 10.98 16 22.12 10.34 94.51 77.38 405.39 12.27 18 16.86 22.97 57.99, 66.58 268.22 10.45 19 15.20 18.02 53.16 68.54 213.50 13.44 Table 4.40: Results obtained from minuit analysis of C P M G data from fixed guinea pig lumbar spine. T2 values are given together with the percentage contribution of each component to the signal observed. Chapter 4. In Vitro Quantitative Relaxation Measurements 166 GP T2a % T2b % T2c % 2 25.21 27.88 98.95 43.34 767.69 28.78 4 27.09 28.24 94.48 39.29 754.20 32.46 7 24.14 32.12 85.76 38.29 852.08 29.59 9 11.65 13.35 79.37 67.70 488.42 17.95 11 23.09 26.50 78.16 59.58 558.28 13.92 12 20.58 23.28 76.56 65.50 560.32 11.22 13 26.47 16.67 105.85 50.22 869.56 33.11 14 17.90 20.34 65.74 68.07 326.11 11.59 16 22.82 12.22 101.65 75.78 511.59 11.99 19 16.95 23.08 55.39 65.36 253.67 11.55 Table 4.41: Results obtained from minuit analysis of C P M G data from fixed guinea pig lower thoracic spine. T2 values are given together with the percentage contribution of each component to the signal observed. Tissue T2a % T2b % X 2 W M 1 13.34 18.30 102.60 81.70 3848 W M 2 13.11 17.08 88.42 82.90 25011 G M 6.31 5.04 93.80 93.90 1157 G M / W M 17.04 7.97 90.90 92.00 7871 L I 8.50 10.40 93.30 89.6 2796 L2 13.10 18.40 95.50 81.60 9864 L3 14.20 13.68 110.40 86.30 17116 L M B 11.80 15.97 92.84 84.00 6258 Table 4.42: Results from minuit analysis of C P M G data from primate case 8. T2 (ms) values are given together with the percentage contribution of each component to the total signal observed. W M - white matter, G M - grey matter, L - lesion, L M B - left midbrain. Chapter 4. In Vitro Quantitative Relaxation Measurements 167 8000 -•§ 6000 i . 4000 E < 2000 „ 3000 J 2000 CL | 1000 _ -Fl 1 L i 1 10" 3 10" 2 10" 1  2 (s) 10° 101 2000 T3 | 1500 §" 1 0 0 0 < 500 6000 F16L F9L - x> D 4000 -"Q. E < 2000 1 \ I 0 10~3 IO" 2 10" 1 i (s) 10° 101 10" 10-2 10" 1 T, (s) 10° 101 Figure 4.76: Plot of amplitude versus T 2 value (sec) for fixed guinea pig lumbar spine. Guinea pig number is given in each spectrum; numbers 2, 7 and 9 are normal, and 13, 16 and 11 are abnormal. The peak around 1 sec corresponds to the T 2 of free water in the formalin solution around the tissue. The magnitude and T 2 of this peak varied, depending on the amount of water present. Chapter 4. In Vitro Quantitative Relaxation Measurements 168 Figure 4.77: Plot of amplitude versus T2 value for normal appearing white matter in primate case 8. Figure 4.78: Plot of amplitude versus T 2 value for lesion 3 in primate case 8. Figure 4.79: Plot of amplitude versus T 2 value for the left midbrain in primate case 8. Chapter 4. In Vitro Quantitative Relaxation Measurements 169 C A S E TISSUE T2a % T2b % T2c % j ! W M 17.66 32.65 53.66 67.35 1 G M 56.06 81.08 177.52 18.92 - -1 W M / G M 48.43 59.64 108.72 40.35 1 8 15.75 4.08 96.44 79.71 1280 16.22 1 10 9.03 1.48 112.03 73.92 256.80 25.91 4 W M 4.22 6.28 25.29 67.96 72.06 25.75 4 W M 4.75 6.40 26.41 70.90 86.83 22.69 4 G M 57.54 69.87 133.50 30.13 4 1 10.69 6.64 70.40 69.86 179.94 23.50 4 2 16.35 4.64 100.96 78.57 251.51 16.79 4 3 40.21 7.67 150.36 71.76 392.33 20.57 Table 4.43: T2 values obtained using minuit to analyze C P M G data from fixed MS tissue. Lesions are numbered as they were in chapter 2, and W M - normal appearing white matter, G M - normal appearing grey matter and G M / W M - mixture of grey and white matter. Two values are given for case 4 W M , the data were obtained six months apart. 4.4.3 F i x e d M S Tissue Minuit The T 2 data obtained after analysis of C P M G data from the fixed MS fixed are presented in table 4.43. NNLS The T2 and Xi values for the fixed MS tissue were analysed using the NNLS program. The results are given in figures 4.80-4.92. Chapter 4. In Vitro Quantitative Relaxation Measurements 170 T , ( s ) Figure 4.80: Plot of amplitude versus T 2 value for normal appearing white matter in case 1. T , ( s ) Figure 4.81: Plot of amplitude versus T2 value for normal appearing grey matter in case 1. Chapter 4. In Vitro Quantitative Relaxation Measurements 171 X -8 H -J • • ' ' • 1 * - I 1 1—I—I I I I I 1F I l r l—T 1 t 10 T, (s) Figure 4.82: Plot of amplitude versus T 2 value for normal appearing grey/white matter mixture in case 1. 50 40 H • • • I 1 I—I I 1—1—' I I I 111 1—1—I I I I 11 o X 30 T3 E 10 -1 1 1 ! I I f l' | 1 1 1 I I | K>" 10 ' T, (s) 10 Figure 4.83: Plot of amplitude versus T2 value for lesion 8 in case 1. Chapter 4. In Vitro Quantitative Relaxation Measurements 172 12 -x a> -a 4 _i i i i ' < < • _j i i i • ' • '' 10 J I I I ' ' ' ' ' -T 1 1 1—I—I—I 10 ' T, (s) 10" 10 Figure 4.84: Plot of amplitude versus T 2 value for lesion 10 in case 1. 5 --S1 E 2 _J 1 1—I—I—1 I —I 1 J I I ' ' ' A •A- -i—i—i—i—i r T 2 (s) Figure 4.85: Plot of amplitude versus T2 value for normal appearing white matter in case 4. Chapter 4. In Vitro Quantitative Relaxation Measurements 173 12 O X T3 =3 CL E 4 _] I I L—I ' ' ' 10 _l I I 1 I l_ 10 T, (s) 10 • • 1 L__I—L-10 Figure 4.86: Plot of amplitude versus T2 value for normal appearing grey matter in case 4. 10 _i i i i i • * •' —i 1—i i i i i 11 _i 1 i i i > • • o X 6 CD CX. E 2 H I' ' 1 1 III!*] 10" T 2 (s) 10 Figure 4.87: Plot of amplitude versus T 2 value for lesion 1 in case 4. Chapter 4. In Vitro Quantitative Relaxation Measurements 174 12 O X 8 H -I I—1—1—1—1-ZD 6 C L E 2 H i i I _i i 1—J-• 1 It 1 I I | —1 1 1 1 I 1 T, (s) Figure 4.88: Plot of amplitude versus T 2 value for lesion 2 in case 4. 15 -m CD X Figure 4.89: Plot of amplitude versus T2 value for lesion 3 in case 4. Chapter 4. In Vitro Quantitative Relaxation Measurements 175 R e l a x a t i o n T i m e T1 ( s e c ) Figure 4.90: Plot of amplitude versus Ti value for normal appearing white matter in case 1. to 10 RELAXATION TIME T1 ( S E C ) Figure 4.91: Plot of amplitude versus Ti value for lesion 1 in case 1. A. B. otloo Mmm T l <B*C) Roboxotion Tim* T l (aec) Figure 4.92: A . Plot of amplitude versus Ti value for normal appearing white matter in case 4. B . Plot of amplitude versus Ti value for lesion 1 in case 4. Chapter 4. In Vitro Quantitative Relaxation Measurements 176 4.5 Discussion The results show that T 2 values from tissue are reproducible, with ~ 5% error. Using minuit, three exponentials provided the best fit to spinal cord data, and two exponentials to brain data. A two exponential fit to brain tissue was not an adequate fit to the data (figure 4.93), but three exponentials provided numbers which could not be resolved, being separated by less than a factor of three. For the best fit, \ 2 should be equal, on average, to the number of data points; the number of data points for fresh tissue was usually around 120, and \ 2 varied from 188 to 1738. The high values of x2 were only obtained for two animals; 65% of the samples studied were in the range 200-400. Much better fits were obtained using the NNLS analysis, where the x2 w a s approximately equal to the number of points. It is not possible to determine the reproducibility of the abnormal T2 values, since no two samples are absolutely identical. The data will now be considered in turn, followed by a general summary at the end of the discussion. The results obtained from the fresh guinea pig tissue, showed that the lesions were primarily inflammatory; perivascular inflammation could be seen in the subarachnoid space and in the Virchow-Robin spaces. In a few animals, extensive parenchymal cellu-lar infiltration was also seen (figure 4.72). The type of models which have been invoked to explain the relaxation data obtained from tissue were described in the introduction; using a similar approach, possible explanations for the data presented above will now be considered. The minuit data demonstrated that three exponentials provided an ade-quate fit to the spinal cord T 2 data; the question then is what do these three exponentials represent? As already indicated, literature relaxation models refer to bound water, intra-cellular and extracellular water as different compartments; since there is no extracellular space in normal CNS tissue, the source of the third exponent is not clear. From a purely anatomical standpoint, there are only two possible signal sources, the central canal of Chapter 4. In Vitro Quantitative Relaxation Measurements 177 Figure 4.93: Plot of log of signal amplitude versus time after the 90° pulse in a CPMG sequence, demonstrating non- exponential behaviour of brain water proton relaxation. A. Manganese chloride solution. B. Brain tissue data fitted to one exponential. C. Brain tissue data fitted to two exponentials. D. Brain tissue data fitted to three exponentials. Chapter 4. In Vitro Quantitative Relaxation Measurements 178 the spinal cord or the subarachnoid space; both these areas contain CSF. The T 2 value observed for the third component was in the range 189 — 246 ms for normal guinea pig spine; in abnormal tissue, the range was 154 — 257 ms. The variations seen in the long T 2 component did not coincide with the samples studied late in the experimental procedure; had this been the case, then the differences could be attributed, at least in part, to sample dehydration. If the source of the third exponent is taken to be the spinal canal, then this value would remain constant, since inflammatory cells do not infiltrate this area. However, if the source of this exponent is taken to be the subarachnoid space, the larger range observed in abnormal tissue could be explained as follows; perivascular inflammation in the subarachnoid space releases exudate from the blood vessels into the surrounding CSF; the exudate contains inflammatory cells and proteins resulting in a de-crease in the observed T 2 value due to the increase in the protein concentration [154,155]. This interpretation then implies the middle component is intracellular water; this model works for normal tissue, but in the abnormal tissue, the middle component changes with parenchymal cellular infiltration. The value of T 2 and the percentage contribution to the signal both increase; hypercellularity in the tissue would lead to an increase in the signal contribution from intracellular water, but does not readily explain the increase observed in T 2 . Such a change in T 2 would require changes in cell diameter, thereby increasing the proportion of free water to bound water, or a decrease in the bound water fraction. CNS cells vary in size from 6 — 8 fim to 60 — 80 pm, the largest cells being the Purkinje cells in the cerebellum and the anterior horn cells of the spinal cord [156]. The cells that infiltrate the area are actually smaller than neurons, therefore this simple explanation is insufficient to explain the observations. The self diffusion coefficient of free water at 37°C is 3.05 x 1 0 _ 9 m 2 s _ 1 [157]; the mean squared average distance travelled during time Chapter 4. In Vitro Quantitative Relaxation Measurements 179 r in three dimensions is given by equation 4.54 [158]; r 2 = 6DT (4.54) where D is the self diffusion coefficient. For r = T2, a water molecule would travel 1.83 x 1 0 - 9 m 2 ; this corresponds to at least 40 pm in one dimension. The CNS cell diameters are of the order of tens of microns; thus compartmentalization cannot be explained in terms of intracellular structures. Relaxation studies on protein and other macromolecular solutions show a single exponential decay; likewise the studies on red blood cell ghosts show that the cell membrane is permeable to water on the time scale of the M R experiment; thus, rather than intra- and extracellular water, a range of relaxation times will be observed due to the water sampling the different cells and extracellular space created in abnormal tissue. The rate of diffusion will be affected when the water molecules cross membranes and when they come into contact with macromolecules, both resulting in slower diffusion; this could lead to multi-exponential relaxation behaviour. [159]. Differences were observed in the minuit data between animals having primarily perivas-cular inflammation in the subarachnoid and Virchow-Robin spaces, and those also having extensive parenchymal infiltration. If the two extremes are considered (figure 4.94), the normal tissue T2 data he between them. Thus, the T2 values must pass through normal as the tissue becomes more inflamed, and the abnormal area becomes infitrated with inflammatory cells. This supports observations made by Karlick et al., that normal values of T2 were seen with parenchymal cellular infiltration [160]; in that study, data were fitted to a single exponential. When tissue was homogenized, a three-exponential fit the data was no longer acceptable, since the T2 values of the two slower relaxing com-ponents were separated by less than a factor of three (table 4.44). Even though the fit is questionable, it is interesting that the bound fraction does not change even when Chapter 4. In Vitro Quantitative Relaxation Measurements 180 Plot of Percentage Contribution to Signal versus T2 for Normal and Abnormal Guinea Pig CNS Percent 70 60 50 40 30 20 1 • Y • • • -ft 50 100 150 T2 (ms) 200 250 300 Figure 4.94: Plot of percentage contribution to signal versus T2 for normal and abnormal guinea pigs. Ik predominantly perivascular inflammation in the subarachnoid and Virchow-Robin spaces. D extensive parenchymal cellular infiltration. • normal tissue. Tissue T2a % T2b % T2c % B 4.0 2.8 83.4 80.7 133.9 16.5 C / T 9.8 10.9 70.6 60.1 176.3 28.8 C / T 9.5 11.3 70.6 61.4 169.7 27.3 Table 4.44: Results from minuit analysis of C P M G data from the homogenized brain and spine of a normal guinea pig. T2 values (ms) are given together with the percentage contribution of each component to the total signal observed. B - brain tissue and C / T -cervical/thoracic spine. Two sets of data are given for C / T , determined 4 hours apart. Chapter 4. In Vitro Quantitative Relaxation Measurements 181 the tissue is homogenized. It provides further evidence that this component is real, and future studies must include experiments to identify the source of this proton signal; literature data do not explain what the bound water represents. The homogenized tissue data also shows that multi- exponential relaxation behaviour does not arise from cellular organization. The minuit data are sensitive to the pathological changes in the tissue, but in terms of understanding the relaxation of water protons in tissue, is an inadequate model, particularly in this instance where the samples consisted of normal and abnormal tissue. The NNLS analysis provided better fits to the data, but the small changes in the Ti and in the amplitude of each component made it difficult to interpret the discrete fits. Two peaks were often seen on either side of the Ti value obtained using minuit (figure 4.74 and 4.75, page 160); this suggests a distribution of T2 values rather than a discrete value. It was also noted, that abnormal tissue consisted of less peaks in the discrete fit; this is seen clearly if the data from the lumbar spine data from three normal and three abnormal guinea pigs are compared (figure 4.75). A smooth fit was applied to the lumbar spine of three abnormal guinea pigs, and three normal pigs. The data are shown in figure 4.95. The data shows an apparent decrease in the bound water in the abnormal samples; this is more likely to be an increase in the longer components. In addition, the ratio of the two distributions is 6 in normal tissue (GP7L) and increases to 15 in abnormal tissue (GP13L); this is a significant change that could be used to differentiate the normal from the abnormal tissue. The distribution of the longer components is also narrower in abnormal tissue, just as was suggested by the discrete fit to the data. This can be explained in terms of probability distributions; in abnormal tissue, at any given time, the probability of finding a water molecule in a long Ti compartment is much higher than in normal tissue. This is due to the destruction of tissue, thereby forming extracellular space; the water molecules in abnormal tissue will also cross less boundaries Figure 4.95: Plots of amplitude versus T2 value for a smooth fit to guinea pig lumbar spinal cord C P M G data. The figure on each spectrum denotes the guinea pig number; numbers 2,7 and 9 are normal and 13, 11 and 16 are abnormal. Chapter 4. In Vitro Quantitative Relaxation Measurements 1.01 183 0.9 0.8 c c 0.7 _o o o k_ u. _ 0.6 D "o V CL 00 0.5 0.4 6 6 16 2 7 9 13 11 Pig N u m b e r Figure 4.96: Plot of maximum and minimum amplitude versus guinea pig lumber show-ing differences between normal and abnormal tissue. in a given time than in normal tissue, for the same reason. In addition, the cell type in an abnormal area will tend to be more homogeneous in size than in normal tissue; all of these changes will lead to an observed increase in T2 and to a narrower distribution of T2 values. The smooth fits shown here are similar to those obtained by Kroeker and Henkelmen [146]. The ability to use the appearance of the smooth fit to differentiate normal from abnormal tissue was further tested; a program which maximises or minimises the observed amplitude in specified regions was applied to the data. The lumbar data from guinea pigs 2, 7 and 9 (normal) and guinea pigs 13, 11 and 16 (abnormal) were maximized in the region 65 — 150 ms and minimized in the region 30 — 300 ms; the results are shown in figure 4.96. The data shown are the maximum and minimum values of signal amplitude in these two regions, determined with 95% confidence limits. It was Chapter 4. In Vitro Quantitative Relaxation Measurements 184 not possible to convert the normal into the abnormal, or vice versa; this shows that the differences between the normal and abnormal tissue are real. The fact that the data from guinea pig 11 overlaps the normal data is not unexpected since this tissue was only 10% abnormal, compared to 50 — 60% for 13 and 16; this demonstrates very clearly the problem of volume averaging, and provides a method for determining at what ratio of normal to abnormal tissue it is can no longer possible to differentiate normal from pathology. The NNLS analysis provides a model which can explain the results observed, and just as for minuit, this method is sensitive to T2 differences between normal and abnormal tissue. The spectra obtained from the guinea pig tissue showed no evidence of a signal contribution from mobile lipid protons (figure 4.97). The appearance of the spectra was determined by the position of the tissue in the N M R tube. The shoulder observed in the spectra shown appears to be due to magnetic susceptibility; positioning the tissue parallel to the direction of the magnetic field gave rise to a water peak with no shoulder. The fixed guinea pig tissue relaxation data are more complicated; this is due to the presence of free formalin in many of the samples, resulting in a long T2 component not observed in normal tissue. A three parameter fit to minuit is therefore insufficient to differentiate normal from abnormal tissue. The bound water fraction appears to be different from that seen in fresh tissue, but this may simply be due to the different fit obtained because of the free formalin. NNLS analysis of a few normal and abnormal samples showed that the information obtained from the fresh tissue is still observed in the fixed tissue. The discrete fit to fixed tissue data also showed a tendency to be less complex in the abnormal tissue (figure 4.76). This is an important observation, since literature data have shown that fixed tissue relaxation measurements cannot be used as an indicator of relaxation behaviour in vivo or in vitro. These data show that this is not the case; even in the presence of the contaminating formalin solution, the NNLS program Chapter 4. In Vitro Quantitative Relaxation Measurements 185 A . B. Pr«qua&07 (kHz) Frequency (kHz) Figure 4.97: A . Proton spectrum from the lumbar spine of guinea pig number 13. B . Proton spectrum from the fixed lumbar spine of guinea pig 13. In both spectra tau = 400 its. could still access the information on the tissue water. The bound water fraction is still present in the fixed tissue, and varies from 8 — 18 ms. Comparison of the NNLS analysis for data from fresh and fixed left hemisphere shows a remarkable similarity between the two (figure 4.98). The data obtained from the primate tissue in case 8 in chapter 2, showed little dif-ference between the normal and abnormal samples; this was true for both minuit and NNLS analyses. The most likely explanation for this is that the tissue is in fact mostly normal; cutting fresh tissue is difficult, and although attempts were made to correlate the samples taken with abnormal areas on the M R I , it is possible that the lesions were missed. This data does however provide supporting evidence for using a distribution of relaxation times rather than a discrete exponential fit; the x2 obtained using minuit was high compared to the number of data points, but increasing the number of expo-nentials to three was not possible, because the separation between components was too Chapter 4. In Vitro Quantitative Relaxation Measurements 186 10" 3 10"2 1CT1 10° T 2 (s) Q) 12000 -.•3 8000 4000 0 Q. E < P 9 L H 10~3 10" 2 10"1 10° 101 T 2 (s) 4000 •S 3000 ZD ! . 2000 < '1000 -F7LH V 6000 F9LH X> ZD +; 4000 - "Q. -- E < 2000 I I I I . 0 A . A, 10~3 IO"2 10"1 10° 101 T 2 (s) 10" 3 IO"2 10"1 10° 101 T 2 (s) Figure 4.98: Plot of amplitude versus T2 value for fresh and fixed left hemisphere. The numbers denote the guinea pig and F implies fixed tissue. The peak around 1 sec in the fixed tissue data corresponds to the T 2 value for free water in the formalin solution around the tissue. The magnitude and T2 of this peak varied, depending on the amount of water present. Chapter 4. In Vitro Quantitative Relaxation Measurements 187 E .6 -.O .1 -R o l a x c r t i o n T i m e T2 Figure 4.99: Plot of amplitude versus T2 value for a smooth fit to normal appearing white matter in primate case 8, showing two distributions of T 2 small. The x2 obtained using NNLS varied from 99 to 438, and the number of points was approximately 112 for each sample. A smooth fit to the data using boundaries as described above for the guinea pig data, gives a ratio of two distributions like that seen for guinea pig tissue, as shown in figure 4.99. The fixed MS tissue relaxation data showed marked differences between normal and abnormal tissue, using both minuit and NNLS analyses. The minuit data shows fixation to give rise to a short T 2 component that is much longer than that observed in fresh tissue. However, the NNLS indicates that a component around 12 ms is still observed in both normal and abnormal fixed tissue; just as for fresh tissue, two exponentials are insufficient to describe the data, but a three exponent fit gives T 2 values that are only separated by a factor of 2. The T\ data from the same tissue also shows a distribution of relaxation times. In lesion 8, case 1, a long T 2 of 1.2 sec was observed; this lesion had cystic spaces, which are holes in the tissue filled with fluid, and would be expected to give rise to a longer T\ and T 2 . The fully demyelinated and gliotic lesions had longer T\ and T 2 components than the partially demyelinated and gliotic lesions from case 4; this supports the trend seen in chapter 2, where it appeared that the most gliotic lesions had the highest T\ values and from the signal intensity on the images seemed to also have higher T 2 values. The results from Chapter 4. In Vitro Quantitative Relaxation Measurements 188 normal appearing white matter in case 4 suggest that the tissue was not in fact normal; increased water content and other changes have been observed in normal appearing white matter from MS patients [161]; in addition, some authors have reported a small increase in the observed T\ relaxation time of white matter in MS patients compared to normal controls [162]. The results observed suggest that M R is sensitive to these changes, which may be the first stages in the disease process, or a secondary process; further study is necessary in this area to interpret the data. A recent publication by Kroeker and Henkelman [163] describes the problem of spin-locking using the C P M G pulse sequence; this results in the measurement of T\p (the rotating frame spin-lattice relaxation time) rather than T2. The fixed tissue data pre-sented here showed no tau dependence, and a tau value of 400 ps is longer than the correlation times of both solid and mobile protons; this indicates that T2 is the param-eter being measured. It should also be noted however, that even if T\p were being measured, if the data are reproducible and differentiates between the pathology observed in MS, then this would still be an acceptable measurement to use in the study of MS patients. In summary, it is clear from the above results, that relaxation measurements can be used to differentiate not only normal from abnormal tissue, but the different types of pathology present in the tissue. Although the differences observed between the guinea pigs with varying degrees of inflammation were not large, it must be remembered that for the majority of samples studied the tissue was mainly normal. Larger differences would be expected if pure pathological tissue were studied; this comes back to the same problem of volume averaging described in chapters 2 and 3. It must be stressed again that quantitative studies should be carried out on as small a volume of tissue as possible, and preferrably from the centres of lesions. The measurements on fixed tissue appear to show the same trends as those seen in vitro; this means that the differences observed in the T2 values obtained from the fixed MS tissue should be indicative of the changes that \ Chapter 4. In Vitro Quantitative Relaxation Measurements 189 would be seen in fresh MS tissue; both minuit and NNLS analysis of relaxation data can then be used to differentiate differing degrees of demyelination and gliosis. In addition, the inflammatory lesions in the guinea pig tissue showed different relaxation behaviour from demyelinated and gliotic lesions (figure 4.100). It is clear from these in depth T 2 studies that it is possible to use quantitative M R measurements to characterize MS pathology; further studies must be completed before this goal can be realized. Recent technical improvements in commercial M R I systems allow C P M G data to be collected from a localized volume; it is also possible to obtain multi-echo images of the same slice, from which the relaxation decays of chosen ROI can be determined; these data can then be analyzed using the NNLS programs employed in the present studies. Relaxation studies on animal models for MS in vivo, with follow-up in vitro studies, would confirm the validity of the in vitro results described in this thesis. Other investigators have con-cluded that in vitro studies are indicative of the changes observed in vivo [164], although small differences are seen between the in vivo and in vitro relaxation times. Chapter 4. In Vitro Quantitative Relaxation Measurements 190 E i c • i I n f t o M w m i t w y L * a l o n : ... K 1 K " T , ( s ) Figure 4.100: Plot of amplitude versus T2 value for normal and abnormal fixed tissue. A . Normal spinal cord. B . Normal white matter from MS patient. C. Inflammatory lesion from guinea pig spine. D. Partially demyelinated and gliotic lesion from MS patient. Chapter 5 Conclusions and Suggestions for Further Work 5.1 Conclusions 1. MRI at 0.15 Tesla is an accurate indicator of disease extent in both MS and EAE in primates. Comparison of the abnormal areas on the SE MRI with the actual pathology seen on the fixed brain slices themselves showed differences in individual lesion areas of 5 — 60%, and for total brain lesion area, 20 — 30%. These differences are attributed to the effects of volume averaging over the 10 mm slice thickness used to obtain the images; in addition, the margins of periventricular and subcortical lesions are difficult to define, contributing a further source of error. MRI can detect lesions in EAE before the onset of clinical signs and allows the disease progression to be followed in vivo. For the first time, it was demonstrated that spontaneous relapsing-remitting EAE can be induced in a primate following a single inoculation and without treatment; the course of the disease in this animal followed that seen in serial MRI studies in MS patients. 2. The results show that quantitative MRI using the two- point computational method available on the Picker International system provides reproducible values of the spin-lattice and spin-spin relaxation times (T\ and T2 respectively) for normal ap-pearing white matter in both MS and EAE. For MS, typical values obtained ' post mortem are Ti = 381 ± 12 ms and T2 = 77 ± 9 ms; for fixed MS tissue Ti = 207 ± 14 ms and T2 = 65 ± 5 ms. For EAE, typical in vivo values of normal 191 Chapter 5. Conclusions and Suggestions for Further Work 192 appearing white matter relaxation times are T\ = 3 9 5 ± 2 5 ms and T2 = 7 7 ± 5 ms. The values obtained from primate tissue are essentially equal to those obtained from human tissue, as expected. It is clear from the in vitro studies that the imaging pulse sequences used to determine T2 are only detecting the most mobile protons; that is, the protons with a T2 value around 100ms. The fast relaxing component around 10ms is not observed. These quantitative M R I methods are sensitive to differences in pathology in both MS and E A E ; the changes observed were dependent on the degree of gliosis present in MS tissue and on the severity of inflammation, demyelination and hemorrhagic necrosis in E A E . The most gliotic MS lesion present in three slices caused a 120% increase in Ti and a 130% increase in T2 compared to normal post mortem white matter; the same lesion fixed gave rise to a 160% in Ti and a 230% change in T2. In primate E A E the maximum changes observed were 41% in Ti and 133% in T2. Volume-averaging and the effects of multi-slice interference resulted in lower than expected Ti and T 2 values from small lesions observed in only one slice image. The relaxation data obtained using this two-point method are useful for comparative purposes, but are not sensitive enough to provide the discrimination necessary to differentiate between acute MS lesions and more chronic demyelinating and gliotic lesions. 3. In vitro quantitative M R measurements on CNS tissue show that multi-exponential data analyses of C P M G data can discriminate between differing degrees of inflam-mation in guinea pigs inoculated to produce E A E , even when a maximum of only 50—60% of the sample is affected. Three exponentials provided the best fit to spinal cord C P M G data, and two exponentials the best fit to brain tissue C P M G data; analysis of fresh brain tissue data from a primate showed similar findings. Analysis of fixed guinea pig tissue using a fixed number of exponentials was complicated by Chapter 5. Conclusions and Suggestions for Further Work 193 the presence of free formalin solution; it was not possible to distinguish normal from abnormal tissue. Analysis of fixed tissue data using a continuous distribution of relaxation times provided better fits than the fixed number of exponentials; this analysis method could also discriminate between normal and abnormal fixed tissue. Relaxation studies on fixed MS tissue showed that both minuit and NNLS methods of analyses could distinguish between normal and abnormal tissue, and between differing degrees of gliosis and demyelination. Once again, analysis assuming a distribution of relaxation times provided the best fit to the data. In all the tissue studied, it was clear that two distributions were present; one centred around 10 ms and the other around 100 ms. The source of the short component is unknown, but the latter distribution probably arises from exchange between different cell en-vironments and, in abnormal tissue, also between intra- and extra-cellular water. The ratio of these distributions changes from 6 to 15 in the presence of extensive parenchymal cellular infiltration; the T 2 distribution at longer times also narrows. These changes could occur due to an increase in the probability of finding water molecules in long T 2 environments, and the decrease in the boundaries that the water molecules have to cross in abnormal tissue. In addition, the cellular environ-ment is more homogeneous in abnormal tissue. The technological advances being made in the field of M R I will enable the in vitro studies described in this thesis to be reproduced in vivo. Although these experiments must first be carried out on animal models, there is little doubt that the evolution of the disease processes, that are known as MS, can be followed using M R I . This information could lead to an understanding of the mechanisms involved in the disease progression, which would lead to better treatments and ultimately to understanding the cause(s) of the disease. Chapter 5. Conclusions and Suggestions for Further Work 194 5.2 Suggestions for Further Work One of the main questions arising from the results reported here is the source of the two distributions observed for T2 in CNS tissue. While it seems likely that the distribution at longer times arises from intra- and extra- cellular water exchange, the source of the bound water is not so obvious. One way to determine how real this fast relaxing component is, would be to study its behaviour with the amount of solid material present in the sample. Quantitative M R studies on wood [165] show that the moisture content can be determined from the FID by extrapolation to zero, and subsequent determination of the solid components proton contribution to the signal. Similar analysis could be carried out on tissue; this would show whether the solid component changes, and/or whether the fast relaxing water component is constant or variable. It would also be possible to determine whether there is exchange between the solid protons and the bound water molecules using T\ measurements. Studies on rocks using a pulse gradient M R method, show that the range of pore sizes available to the water protons can be determined [166]. Such a study on tissue would provide supportive evidence for the increase in cellular homogeneity in the abnormal tissue, which may be one of the causes for the changes observed in the guinea pig data. This is complicated by the rate of diffusion of water molecules in tissue; however, such pulsed gradient methods also allow the determination of the self diffusion coefficient, which would be expected to vary between normal and abnormal tissue due to changes in the number of boundaries encountered by the water molecules. A n obvious extension to this work is to increase the T\ data base for both fresh and fixed tissue. Just as for T2 this would be carried out initially in vitro, with eventual application in vivo. The guinea pig data presented here were primarily from inflamma-tory tissue; it is possible to produce more chronic forms of E A E in the strain 13 guinea Chapter 5. Conclusions and Suggestions for Further Work 195 pig. The pathology observed in these animals more closely resembles the demyelinating lesion seen in MS patients; a colony of these animals has been set up at U . B . C . to carry out such a study. This would provide information on the effects of demyelination on relaxation measurements separate from the effects of gliosis. This information could not be obtained from the studies presented here. As indicated in the discussion in chapter 4, the work presented here must be validated by an equivalent study in vivo, on E A E in guinea pigs for example. Spectroscopic studies in vitro and in vivo may also provide further information to aid in the interpretation of the relaxation data from CNS tissue. This again would involve the use of animal models for MS, but could also include a parallel study on MS patients. Such a study would show whether mobile lipid protons contribute to the M R signal observed, particularly during periods of relapse, which may be unaccompanied by clinical signs. The data presented in this thesis required the interaction of people in a number of different fields. Medically oriented projects, such as this, require a merging of science and medicine for them to succeed. The author has found this an exciting project, particularly when the long term implications for MS are considered; it is not the usual project carried out by chemistry graduate students, but the most interesting observation the author has made is that each area of medicine encountered (neurology, immunology, pathology, his-tology, anatomy, pharmacology and physiology) all have their basis in molecular science. This has meant that a chemistry background was probably the most useful for such a venture, and made the understanding of these different areas that much simpler. The near future promises to be an exciting time for M R scientists in medicine, and the author looks forward to seeing a solution to the MS puzzle in the not too distant future. Appendix A Extraction of Myelin Basic Protein from CNS Tissue Myelin basic protein was prepared from primate brain tissue as follows; one whole primate brain was homogenized in 4 litres of a 2:1 mixture of chloroform and methanol. This was left to stir overnight at 4°C. The homogenate was filtered and washed twice with the 2:1 chloroform/methanol mixture and suspended in 2 litres of cold acetone. The residue was once again filtered and suspended in 4 litres of cold water, and left to stir for just under 1 hour. The suspension was filtered and the residue suspended in 0.03M HC1 (2 vol per gram of tissue). 1.0M HC1 was added as required to reduce the pH to 3. The suspension was stirred for 1 hour, then filtered. To the filtrate was added enough 8M urea solution to dilute it to 2M urea. The resulting solution was then titrated to pH 9 by adding cellulose DE-52. Having completed this stage, the mixture was allowed to stir for 30 min. The suspension was filtered under vacuum through Whatman 41 filter paper, and the clear foamy filtrate placed in dialysis tubing (Spectrapor tubing, 32 mm diameter. Mol . wt. cutoff 6,000-8,000), and dialyzed overnight in 4 litres of water at 4°C with two water changes. 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