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Proton NMR studies of human lumbar intervertebral discs Rind, Teresa 1990

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P R O T O N N M R STUDIES OF H U M A N L U M B A R I N T E R V E R T E B R A L DISCS Teresa Rind B. Sc. (Physics) University of Victoria  A THESIS S U B M I T T E D THE  IN P A R T I A L  FULFILLMENT  REQUIREMENTS FOR T H E D E G R E E OF M A S T E R OF  SCIENCE  in THE  FACULTY OF GRADUATE  STUDIES  DEPARTMENT OF PHYSICS  We accept this thesis as conforming to the required standard  THE  UNIVERSITY  O F BRITISH  October 1990  © Teresa Rind  COLUMBIA  OF  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 forfinancialgain shall not be allowed without my written permission.  Department of Physics The University of British Columbia 1956 Main Mall Vancouver, Canada  Date:  Abstract  Problems with the low back resulting from diseases of the lumbar disc account for much human suffering and medical expense. Through early identification of changes in the disc, Magnetic Resonance Imaging offers a potential method for recognition of those at risk of low back trouble. An understanding of the chemical and structural basis of the MR image will therefore be of great help in understanding the degenerative mechanism itself. With this in mind, NMR studies of human intervertebral discs were performed to extract data that could be correlated with different stages of degeneration. In vitro samples of anulusfibrosusand nucleus pulposus of grades II and IV discs were examined. The Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence was used to obtain T  2  relaxation data, which were analysed with a non-negative least squares algorithm and presented as a continuous spectrum of exponentials. An inversion-recovery sequence was used to obtain T i data which were analysed with the same algorithm, then presented as a discrete exponential spectrum. The same procedures were used to produce relaxation times from samples of isolated disc collagen and proteoglycan. Also, T re2  laxation data were obtained with a twelve-echo CPMG imaging sequence from healthy volunteers, and these data analysed with the same procedure used for in vitro data. In vivo and in vitro results  were compared and found to agree in terms of the T  2  relaxation values. Most change between grades occurred in the nucleus, where both Ti and T values decreased from grade II to grade IV. The results of the proteoglycan 2  and collagen studies support the theory that structure, not chemical composition, is responsible for the observed changes in relaxation times. Based on estimates of the percentages of protons in the nucleus due to water, proteoglycan and collagen, tentative ii  assignments were made to the components of the T spectra for grades II and IV 2  nucleus, and these were supported by the results of all parts of the study.  iii  Acknowledgements  I wouldfirstlike to thank my supervisor Alex MacKay, who made this project possible. He provided guidance and encouragement, and shared his expert knowledge of the workings of the Room 100 magnet and spectrometer. He also set up and developed the partnership with Richard Pearce's group in the UBC Department of Pathology, found many valuable reference papers, offered advice on this thesis, and obtained all of the MRI CPMG data. I enjoyed working with Dick Pearce, Joyce Mathieson, and Gwen Bebault from the Department of Pathology at the UBC Hospital, and greatly appreciate all the time and thought they contributed. Specifically, I would like to thank Dick for sharing his extensive knowledge of the intervertebral disc and offering suggestions on the direction of the project, as well as providing all of the samples and many valuable reference papers. I would also like to thank Joyce who prepared in vitro disc samples and taught me how to prepare the collagen and proteoglycan samples; and Gwen who shared thoughtful insights on the study of degenerative discs. The crew in Room 100 have been very supportive and provided help in solving day-to-day problems with the equipment and computing system. Thanks to Clare Morrison, our system manager, the computing system in the lab has been running well and has enabled me to do experimental work and writing without interruption. Also, the relaxation results would not have been possible without Ken Whittall's NNLS program. Finally, I would like to thank Julia Wallace for showing me the ropes in Room 100.  iv  Table of Contents  Abstract  ii  Acknowledgements  iv  List of Tables  vii  List of Figures  viii  1  Introduction  1  2  The Intervertebral Disc  3  2.1  Composition of the Disc  5  2.2  Disc Degeneration  7  2.3  Disc Grading Scheme  9  3  4  5  Proton N M R Theory  12  3.1  Basic NMR and Relaxation Theory  12  3.2  NMR of Water in Biological Samples  17  Application of N M R to Intervertebral Discs  23  4.1  Advantages of Magnetic Resonance  23  4.2  Purpose of This Work  24  Experimental Methods  25  5.1  25  Experiments Performed  v  5.2  Sample Preparation  27  5.3  Data Collection  27  5.3.1  T and the CPMG Sequence  28  5.3.2  Ti and the Inversion Recovery Sequence  29  5.3.3  In Vivo CPMG  30  5.4  6  7  2  Data Analysis  30  5.4.1  Analysis of CPMG Data  31  5.4.2  Analysis of Inversion-Recovery Data  32  Results  37  6.1  T and Tx of Disc Samples  37  6.2  In Vivo MRI T Values  46  6.3  Solid Signal in Disc Samples  51  6.4  Ti and T of Collagen Samples  51  6.5  T and T of Proteoglycan Samples  53  2  2  2  x  2  Discussion  55  Bibliography  61  vi  List of Tables  5.1  Disc Samples  26  5.2  Collagen Samples  5.3  Proteoglycan Samples  26  6.1  T Values for Nucleus and Anulus of Disc Samples  38  6.2  Ti Values for Nucleus and Anulus of Disc Samples  38  6.3  Average T Values for Grades II and IV Anulus and Nucleus  40  6.4  Average Tj Values for Grades II and IV Anulus and Nucleus  41  6.5  T Values of In Vivo MRI Samples  46  6.6  Average In Vivo T Values for Each Lumbar Disc 2  47  6.7  Ti and T Values for Collagen Samples  53  6.8  T i and T Values for Proteoglycan Samples  53  7.1  Tentative Assignments to the T Spectrum  59  • • • 26  2  2  2  2  2  2  vn  List of Figures  2.1  The Lumbar Spine and The Intervertebral Discs  4  2.2  Components of the Disc  4  2.3  Disc Grading System by Morphology and MRI  10  5.1  Examples of Discrete and Continuous NNLS Spectra  34  5.2  NNLS Spectra With Different Values of Facmu  35  5.3  Example of NNLS Fit to CPMG Data  36  6.1  Typical T2 Spectra for In Vitro Disc Samples  39  6.2  T2 Values for In Vitro Disc Samples — Nucleus  42  6.3  T Values for In Vitro Disc Samples — Anulus  43  6.4  Ti Values for In Vitro Disc Samples — Nucleus  44  6.5  Ti Values for In Vitro Disc Samples — Anulus  45  6.6  T Values for In Vivo MRI Samples  48  6.7  Graph of Mean MRI T Values for Each Disc  49  6.8  Histogram of T Values for MRI Samples  50  6.9  Solid Signal in Disc Samples  52  2  2  2  2  6.10 Proteoglycan Ti and T Relaxation Rates Versus Concentration . . . . 2  viii  54  Chapter 1 Introduction  Problems with the spinal column in humans are widespread, costly, and serious. Sixty to eighty percent of all adults have experienced back pain at some time in their lives, with most of these symptoms occurring in the lower portion of the spine, the lumbar region. The most common disorders giving rise to the pain are instability and misalignment, disc degeneration, spinal stenosis, and facet disease. Accompanying the pain are sensory changes and weakness in the body. The social impact of these phenomena is striking when one considers that back trouble leads to many worker's compensation claims, time lost from work, and great hospital costs. In fact, degenerative disorders of the spine are the leading cause of disability in the working years; over any twelve month period approximately 25% of working men will have back trouble. In addition, these disorders are among the most common causes of impairment in general, and account for a major portion of medical expenses. [1,2] Most scientists believe that lower back pain is often due to disc degeneration, as alterations in disc structure may cause pain and impairment by affecting nerves and the spinal cord. Extensive research has been conducted in the epidemiology, anatomy, biomechanics, biochemistry and neuromechanics of the disc and spine, but the pathophysiology of disc degeneration and the role of biochemical changes are poorly understood, and complex. Autopsy results show that by the fifth decade of life, 85-95% of the population have degenerate discs. Possible contributing factors to disc degeneration are thought to be genetic makeup and biomechanics. Indeed, disc failure occurs in the 1  Chapter 1.  Introduction  2  areas of the spine that undergo the most mechanical stress; the role of genetics is not so easy to observe or define. [1,2,3] What is known is that degeneration begins early in life and occurs in everyone, to different extents among individuals [2,4]. A method of early detection of problem discs would help in preventing unnecessary injury and pain; a better understanding of the causes and mechanisms of disc degeneration would be the first step in finding an effective prevention and/or cure. Magnetic resonance may be the solution to both of these needs. MRI could be used to non-invasively detect early degenerate discs, and knowledge of the theory behind the images could lead to an understanding of the degenerative process.  Chapter 2 The Intervertebral Disc  Intervertebral discs play a remarkable role in stabilizing the spine by anchoring the vertebrae to one another, allowing a fair amount of movement of the spine, absorbing loads, and always regaining their original size and shape. Discs vary in size, from the smallest cervical discs in the neck region, to the largest lumbar discs in the lower back (Figure 2.1). They contain special types of cartilage which are noted for their strength and resilience. The tensile strength comes from collagen fibrils, and the resilience is due to large proteins called proteoglycans. In this type of tissue, it is the extracellular material that is important, rather than the cells themselves. In fact, the disc is mostly water and'extracellular matrix, including some of the largest proteins made by any cell in the body. [3,5] The body of a healthy disc is composed of two distinct regions: the inner gelatinous nucleus pulposu3 surrounded and contained by thefibrousrings of the anulus fibrosus.  Outside the disc body are the cartilage endplates and vertebral bodies, as illustrated in Figure 2.2. The nucleus pulposus (NP) is a proteoglycan-rich matrix interspersed with a relatively small number of randomly oriented collagenfibres.Cell density in this region is only 4000 cells/mm , being mostly chondrocytic andfibroblasticin type, with a smaller 3  number of notochordal cells. [1,3] The anulusfibrosus(AF) is made up of 15-20 concentric layers of collagen fibres, organized so that the fibres of adjacent laminae are at 40-90° to one another. The cells 3  Chapter 2. The Intervertebral Disc  4  Intervertebral discs  Intervertebral foramina Lumbar curvature  Sacrum  Figure 2.1: The lumbar spine and the intervertebral discs.  This drawing of the lumbar spine shows the positioning of the intervertebral discs between the vertebrae. (From Human Anatomy by K. M. Van De Graaff, Wm. C. Brown Publishers, Iowa, 1988. pl71.)  anulus  End-plate  Figure 2.2: The components of the healthy intervertbral disc.  The drawing illustrates the anulusfibrosus,nucleus pulposus, endplates and surrounding vertebral bodies.  5  Chapter 2. The Intervertebral Disc  of the outer anulus are fibrocytic while the inner cells are chondrocytic. [1,3] Nutrient transport in the disc is unique, due to the fact that only the outermost layers contain blood vessels, lymphatics and nerves.  In fact, discs are the largest  volumes of tissue in the body without nerves and vessels. Nutrients reach the cells in the disc by moving through the matrix from the blood vessels at the edges. Some transport is achieved by entrainment in moving fluid under changing loads, and another means is active transport by chondrocytes, but the main mode of nutrient travel through the matrix is diffusion. Thus, the maximum concentration of any nutrient or solute in the matrix may be controlled by the local proteoglycan content. [3,5,6]  2.1  Composition of the Disc  Collagen  Collagen is found throughout the disc, in various fibril concentration, di-  ameter, and organization. Ten types of collagen have been found in the disc, with types I and II being the most abundant, followed by type VI. Type I, found mostly in the anulus, increases from 0-80% abundance towards the outer layers, while type II collagen increases 0-80% in the opposite direction, being found mostly in the nucleus. Type VI is found in both regions, 10% in the A F and 15-20% in the NP. In adult lumbar discs, the dry mass in the outer anulus fibrosus is 60-70% collagen, and 10-20% collagen in the nucleus pulposus. As far as structure goes, thefibrilsin the outer anulus are arranged in tight concentric layers at angles to one another; the inner anulus fibrils are less dense and less organized; and in the nucleus they form a loose network with no organization. [2,3] The general structure of a collagen fibril starts with procollagen, the precursor. This is a protein with about 1100 amino acids, every third one being glycine and one third of the others being proline or hydroxyproline. The procollagen molecule twists into  C h a p t e r 2.  T h e Intervertebral  Disc  6  a compact left-handed helix; three of these form a right-handed superhelix, a rod-like macromolecule approximately 1.4 nm in diameter and about 300 nm long. Groups of these superhelixes form thefibrils,which are from 10 to 200 nm in diameter, and of varyied length. They possess great tensile strength as a result of the multiple twisting and grouping. [5] Proteoglycan  Like collagen, proteoglycans are present throughout the disc. They  are similar to the proteoglycans of articular cartilage in many ways, but differ in several respects that may be important in the degenerative process. [1] Different regions of the disc contain one or more of five distinct species of proteoglycans, though the relationships amongst these species are not yet defined. One population of disc proteoglycans form large macromolecular aggregates while the rest do not, and the proportions of these two groups differ between anulus and nucleus. Their relative amounts also change with aging: some elaborate PG aggregates are found in newborns' discs but not in mature spines. Another grouping of the proteoglycans is in terms of their components. One family is rich in keratan sulfate while another contains large amounts of chondroitin-6 sulfate. These repeating disaccharides are two of the main constituents of proteoglycans in general, as described below. [3,7,8,9] The  central molecule of a proteoglycan is the repeating disaccharide hyaluronic  acid, to which are attached amino acid chains called core proteins. The attachment is achieved without chemical bonding, by use of a link protein and a good fit with a globular shape formed from the end of the core protein. Each core protein then has many polysaccharide chains attached to it. Three regions are defined on the core protein by virtue of what is attached: (1) the linkage region containing the globular end and a few small sugar chains; (2) a region having keratan sulfate chains and small o-linked oligosaccharides; and (3) a region with chondroitin sulfate chains. There are  7  Chapter 2. The Intervertebral Disc  approximately 50 keratan sulfate chains and 100 chondroitin sulfate chains on a core protein, and there may be up to 100 core proteins on a central strand of hyaluronic acid when large aggregates form. The overall weight of such an aggregate may be many tens of millions of atomic units. [5,6] Water  Acting with collagen and proteoglycans to give discs their unique proper-  ties is water, which makes up about 70% of the healthy disc. The repeating disaccharides chondroitin sulfate and keratan sulfate contain sugars which carry negative charges, forming a large ordered array of electronegativity. Since water is a dipole, H 2 O molecules become organised in layers around these proteoglycan charges. When the disc is under pressure from loading, the water is forced away from the negative charges and they become closer together. Electrostatic repulsion prevents further compression; as soon as the load is reduced the H 0 returns to its former arrangement 2  and the structure is restored. Thus water plays a major role alongside proteoglycans in providing the disc with resilience. The movement offluidsalso helps to nourish the tissue as explained in Section 2. [3,5] While a proteoglycan molecule can structure an amount of water many times its own weight, this is not the only way H 0 is stored in the disc: some is trapped in the 2  interstices of the proteoglycan and collagen matrix. The structured water, however, is reponsible for the important disc properties. [5] 2.2  Disc Degeneration  As the disc ages or degenerates, structural and chemical changes occur affecting all components of the tissue. As the disc becomes more fibrous and disorganised, the distinction between anulus and nucleus becomes less clear. All collagen fibrils increase in diameter and more chondrocytic cells appear, while proteoglycan and water content  Chapter 2. The Intervertebral Disc  8  decrease. Water loss runs to 30%, with the majority lost from the nucleus. Disc volume can shrink dramatically, in some cases from 15 cc to 1 cc, causing secondary effects from increased forces at the facet joint, such as misalignment, arthritic changes and potential fractures. [1,2,3,4] Some of the water loss is related to changes in-the proteoglycan molecules; a decrease in the molecular "weight of proteoglycan aggregates in the nucleus leads to a decrease in water-structuring capacity. Not only do the aggregates lose size, but there are fewer of them, and the remaining proteoglycans become smaller. The small scale chemical structures of the proteoglycans do not change, but the relative amounts of the components do. For example, the ratio of chondroitin sulfate to keratan sulfate increases in some parts of the disc and decreases in others. Also, a diminished association with collagen results in some loss of tensile strength. [2,3] Data seem to indicate that proteoglycan is lost from all discs of a spine before any morphologically recognizable degeneration takes place. This loss of proteoglycan is thought by some to stem from a long term decrease in nutrient supply. This may be partly due to the fact that less vessels penetrate the anulus as the disc ages. It may also be related to an accumulation of large macromolecular fragments which the disc cannot remove, resulting in abnormal biomechanical and biophysical tissue properties. The cellular feedback mechanism would become confused and the matrix would not function properly or maintain itself. This is one possible explanation of degeneration. [1,3,4,8] The extracellular matrix does seem to have a limited ability for resynthesis and repair, though it is not known if the repair is good enough to restore long term function. Most restructuring occurs in the anulus with the collagenous material being resynthesized most easily; repair in the nucleus is minimal. [3]  Chapter 2. The Intervertebral Disc  9  One of the main problems in finding an explanation for disc degeneration is that the relationships have not been well defined between age-related changes in the disc and the development of clinical syndromes or change in function. Normal aging and abnormal degeneration are difficult to separate. [3] 2.3  Disc Grading Scheme  A disc grading scheme has been devised by J. P. Thompson and associates [3, page 193] [10,11] for assessment by gross morphology and Magnetic Resonance Imaging. The scheme delineatesfivestages of degeneration based upon the appearance of midsagittal sections of the lumbar disc. Features affecting the classification are the nucleus pulposus, anulusfibrosus,cartilaginous and bony end-plates, and the periphery of the vertebral body. Figure 2.3 illustrates the appearances of thefivegrades by morphology and MRI. Grade I discs (found in the young) are identified by a bulging gel nucleus, a distinct fibrous anulus with discrete layers of collagenousfibres,and a uniformly thick hyaline endplate. In the MR T2-weighted spin echo image with T E = 90 ms, the nucleus appears uniformly bright and the anulus uniformly dark grey, with the endplate appearing as a single dark line. Grade II discs (the healther discs examined in this project) still have a bulging nucleus with gel at its centre, but whitefibroustissue appears around the edges, and the anulus contains mucinous material between thefibrouslaminae. The endplate is no longer uniform in thickness. MRI shows some loss of brightness in the central horizontal plane of the nucleus in the form of dark bands, and light areas in the anulus. The infiltration offibroustissue into the nucleus and the invasion of mucinous material into the anulus increase through grades III and IV, with the clear demarcation  Chapter 2.  The Intervertebral Disc  Figure 2.3: The morphologic and MRI grading system. Photographs of actual disc slices are shown at left, and M R images of corresponding grade are shown at right. (Courtesy of R. H. Pearce)  10  Chapter 2. The Intervertebral Disc  11  between nucleus and anulus dissolving at the grade III level. In a grade IV nucleus there are also horizontal clefts parallel to the endplate, and the anulus contains focal disruptions. In the MR image the nucleus and anulus are indistinguishable, with increasing regions of bright and dark signal and decreasing grey areas. Focal defects appear in the line of the endplate in the grade IV disc. Finally, the grade V disc is severely degenerate, with clefts extending through the remaining tissue (nucleus and anulus are indistinguishable), and diffuse sclerosis in the endplate. The MR image reveals defects and thick areas in the endplate, extreme loss of overall disc height, and dominant bright and dark signals in the disc body.  Chapter 3  Proton N M R Theory  A brief overview of basic NMR theory leading to the Ti and T relaxation processes 2  is contained in the following section, which has been summarized from C. P. Slichter's Principles of Magnetic Resonance [12]. For more details please refer to this source. The second part of this section describes briefly some relevant aspects of the NMR of water in biological tissues, including water structuring, proton transfer, cross-relaxation, and compartmentation. 3.1  Basic N M R and Relaxation Theory  A nucleus has total magnetic moment fl and total angular momentum J related by (3.i)  p= j 7  Define a dimensionless operator / such that J = %I and it follows that (Im\fj, i\Im') =  yh(Irn\I i\Im')  x  x  where m and m' are spin quantum numbers. If one applies afieldH, the interaction energy of the nucleus will be —jl-H. The Hamiltonian is thus H =  -fi-H  Taking thefieldto be H along the z-direction gives a Hamiltonian of 0  Ti = —yhH I 0  12  z  Chapter 3. Proton NMR Theory  13  The eigenvalues are thus multiples of the eigenvalues of I : for / = 1/2, we have z  E = -j%H m,  m = -1/2,  0  +1/2  To detect these energy levels, one needs an interaction that causes transitions between these levels, i.e. one needs AE = hu  where io is an angular frequency. In NMR one uses an alternating magnetic field applied perpendicular to a static field Ho. The perturbing term in the Hamiltonian is H rt = — vt  jhH®I cosujt x  where H® is the amplitude of the alternatingfieldand u is the frequency of that field. I has matrix elements between states m and m' which disappear unless m' — m i l . x  Allowed transitions are between adjacent levels: huj = AE = ~/h,H  0  so that CJ =  -yH  Q  Thus we can find u needed to observe a resonance, if we know 7. To estimate 7, a classical picture is used, by computing the magnetic moment and angular momentum of a charged particle moving in a circular path. Then by comparing the expressions for fj. and J and considering equation 3.1, we get 7 = e/2mc, where e is the charge of the particle, m is the mass, and c is the speed of light. A general result of this is that large masses have low 7's. For magnetic fields around 2 Tesla, hydrogen nuclei (protons) resonate at about 90 MHz.  Chapter 3. Proton NMR Theory  Spin-Lattice Relaxation: m = +1/2,  14  Consider nuclei of spin 1/2, each with two states,  —1/2, called |+) and |—) . Some nuclei will be in the |+) state, and  some in the |—) state at a given time. Call the number in the |+) state N+, and the number in the |—) state JV_. To see that there must usually be a coupling of the spins to another system (the lattice), consider the magnetization of an unmagnetized piece of material: a net number of transitions from upper to lower state is required, so the spins give up energy, which is accepted by another system, a reservoir (the "lattice"). Let W | be the probability per second that the coupling will induce a spin transition from |+) to |—) (higher energy) and let W[ be the probability for the reverse process. W\ and W[ are unequal, seen by considering what is happening in the coupled system (Slichter, page 8). Then if n is defined as N+ — 7V_, the population difference, and N is the total population, using dN+ , = NJWldt  N+W1  we get dn no — n It ~~ Tj  (3.2)  where W[ -  n =N 0  W[  W]\  + WT  and  The solution is n = n + 0  Ae~  t,Tl  where A is a constant of integration, no is the population difference at thermal equilibrium, and T i is a characteristic time related to the approach to equilibrium. T is a  called the "spin-lattice relaxation time".  Chapter 3. Proton NMR Theory  15  Effect of Alternating Field H i ( t ) :  An alternatingfieldcan be considered as two  rotating components of equal amplitude, rotating in opposite directions. One rotates in the same sense as the precession of the spins, but the other, going the opposite way, will not be "seen" by the spins. A spin sees only Hi(t) — Hi(icosto t z  + jsinu t). z  The  equation of motion for a spin infieldsHo and H\ (t) is  *  = / ? x p o + ii(0] 7  One can eliminate the time dependence by working in a rotating coordinate system (rotating about the z-axis with frequency u ).  Then both H and Hi will be time  z  0  independent. If we set up the x-axis along Hi, we have |  Near resonance, u> + jH z  0  + iHo) + HHI  = /iX  m 0 (set LO = —u = —jH ). z  0  The above equation can also  be written as  where H  eff  =k (H -u/y)  + iHi  0  So in the rotating system, the moment will precess in a cone about H g at angular t  frequency ^H g. At exact resonance (u> = 7^0) we have H g = Hit, and fl will precess e  t  in the y-z plane. Application of Hi then for a time t (applying a "pulse") will cause p  the moment to precess through an angle  e = 7H1 t  p  One can choose Hi and t to produce any angle 6; for example, a "180° pulse" is when p  Chapter 3. Proton NMR Theory  16  Thus to put spins into the x-y laboratory plane, one must apply a ninety degree pulse (6 = 7r/2); in the lab frame, spins will then precess about the z-direction. This is detected through an induced emf in the same coil used to apply the pulse. Interactions of spins with each other and with the lattice cause decay of the signal (emf) detectable in the x-y plane. This decay is called the "Free Induction Decay" (free of H\) and may be exponential in nature, though this is not generally the case. Bloch Equations for Free Induction Decay: Knowing that M = 7?in/2 and z  using equation 3.2, one expects that dM  z  _  M -M  dt  ~  T  Q  Z  x  where MQ is the magnetization at thermal equilibrium. Also, the x and y components of the magnetization are reduced over time due to spin-spin interactions, and this decay is represented by the following equations: dM ~dT  M  x  x  =  dMy _  My  ~dT ~ ~ ~T^  where T is introduced as a different relaxation time than T i . This is because while 2  the longitudinal (Ti) decay involves a change of energy (transferred to the lattice), the transverse decay conserves energy. The exponential form of the transverse decay is arbitrary but is useful to describe some important phenomena. It is important to note that the processes causing T relaxation are irreversible processes. 2  Spin Echoes: For liquids, whose T values are on the order of tenths of a second or 2  seconds, inhomogeneities in the Hofieldcan dephase spins at a rate higher than the transverse relaxation rate, thus "covering up" the real T value which one wants to 2  observe. To eliminate the effect of the magnet inhomogeneity, spin echoes are used.  17  Chapter 3. Proton NMR Theory  A spin echo is created by applying a 90° pulse to put the magnetization into the x-y plane, followed by a 180° pulse a time r afterwards. Before the 180° pulse, the spins will have dephased due to the inhomogeneities; some will be "ahead" and some will be "behind", by the angle 6 = fSHr  where 8H = H — H , the difference in localfieldfrom H . After the 180° pulse, the 0  0  spins that were ahead will now be behind, and vice versa, so that after another time r they will line up again. At this time, 2r from the initial 90° pulse, a spin echo will be observed as an isolated peak in the detected transverse magnetization amplitude. Note that during the period 2r the effects of T relaxation will continue unaffected by 2  the 180° pulse, so that the amplitude of the echo signal will be M(2r) =  M e~ ^ 2T  2  0  for single exponential relaxation behaviour. 3.2  N M R of Water in Biological Samples  Most or all of an NMR or MRI signal from a biological sample is due to protons of water, so understanding the behaviour of this water is vital to any analysis of relaxation results. Some signal may come from the proton-rich solutes which are a source of relaxation events, but this is fairly insignificant compared to the contribution of water protons. [15] States of Water in Tissues: Water in biological tissues behaves differently from bulk water, in that it is structured around macromolecules as described in Section 2.1. A small fraction of the H 0 molecules interact with slow-moving constituents such 2  Chapter 3. Proton NMR Theory  18  as proteins and cell walls via electrostatic interactions and hydrogen bonding, forming a well-defined hydration layer in solutions, hydratedfibressuch as collagen, and all biological samples such as intracellular H2O and tissues. The hydration layer is characterized by water molecules that are oriented and restricted in motion, thus with different properties from bulk water. [13,14] Most fat-free tissues can be considered to contain three water fractions, based on their location and behaviour: bound water consists of H 0 molecules hydrogen-bonded 2  to fixed sites on macromolecules; structured water is motionally perturbed by the macromolecules but not bonded to them; and bulk water's motion is solely determined by the interactions between H 0 molecules. Each fraction has its own relaxation rates, 2  governed by the motion of the molecules, proton transfer processes, and spin exchange. [15] Interactions between H 0 and macromolecules: 2  The most important source of  relaxation time shortening in tissues is due to changes in the motion of water molecules near the macromolecular surface. This restricted motion in the hydration layer is not uniform, leading to anisotropic diffusion of H 0 molecules. Macromolecules with dif2  ferent protein content also have different water-perturbing capacities, so the behaviour of hydration water depends strongly on the nature of the hydrated species. [13,15] Magnetic dipole-dipole coupling is the important interaction in proton water studies, and this is affected by molecular motions such as rotation, which varies the orientation of local fields, and translation. Magnetic interactions are partially averaged by the rotations, and when water molecules diffuse through regions of different orientations. Averaging also occurs when proton transfer takes place, and this process is enhanced along a continuous hydrogen-bonded path in the hydration layer of a macromolecule. [13,15]  Chapter 3. Proton NMR Theory  19  Relaxation rates in tissues: True T decay is caused by random dephasing of the 2  x-y magnetization due to rapid molecular tumbling motion and the resulting changing magnetic field. There is also a contribution to T caused by dephasing from a "static" 2  component of the magnetic coupling between proton pairs: large macromolecules rotate slowly on the NMR timescale, appearing to be motionless from the NMR point of view. The relatively short T in tissues is partly a result of this static dephasing. [15] 2  In general, tissue relaxation rates are affected by: (1) the strength of local magnetic interactions between nuclei, (2) molecular motion (anisotropic rotation and translation), (3) water perturbing capabilities of the macromolecules, (4) proton transfer rates, (5) diffusion rates, (6) water content, and (7) spin exchange or cross-relaxation rates [13,15]. The first three items affect intrinsic relaxation rates, while the remaining four characteristics affect the observed rates, which may differ from the intrinsic rates. Compartmentation is based on the assumption that sites of different relaxation characteristics exist and the protons at these sites interact with each other in various ways [14,15]. For example, the protons of different sites may be exchanged (proton transfer) or water molecules from one compartment may diffuse to other regions with different local relaxation rates. Water content affects the observed relaxation rates by determining the ratio of bulk water to structured water, both of which have different intrinsic rates. Restricted water motion near the macromolecules gives the structured water a smaller T value [13]. When water content decreases and a larger proportion 2  of water is structured, the observed T value is shortened. 2  Cross-relaxation (spin exchange):  Cross-relaxation between protein and solvent  protons also makes an important contribution to the relaxation rates of hydration water. Spin exchange occurs when the motion of protons from the hydration layer and protons from macromolecules is correlated. Under this condition, there will be a  Chapter 3. Proton NMR Theory  20  mutual exchange of spin magnetization between these protons. When T <C Tj and 2  slow molecular diffusion is present, energy exchanges much faster within the system of spins than between the spin system and the lattice. Note that since the motion of macromolecules depends upon the molecular weight and shape, and cross-relaxation in turn depends on the motion of the macromolecules, then relaxation times of protein solutions are related to the size and shape of the proteins. There may also be cross-relaxation arising from rotations of components of the macromolecule. [13,15] An example of cross-relaxation is seen in studies of collagen, where a short T  x  component is seen due to spin exchange between water and proteins. To determine this, inversion pulses of various lengths were used in a T experiment. The short T of a  2  collagen causes dephasing of the spins during the pulse, so the longer the pulse, the less protons in the protein are inverted. This means there are also more "solid" protons with which water protons must share their reservoir of excited spins. After equilibrium is reached between the water and protein protons, the slope of the decay is the true average T of the solution; the slope of the initial steep decay curve gives information a  about the spin exchange rate. This is known as intermolecular cross-relaxation. [15] Fast and slow exchange:  If the spin exchange rates of water protons between dif-  ferent areas in a sample are fast compared with the instrinsic relaxation rates ("fast exchange"), then an average water resonance signal is seen. If, on the other hand, the exchange rate is relatively slow ("slow exchange"), then a resonance signal characteristic of water protons in each site may be seen. [13] Under conditions of fast exchange between two regions, the observed T i value will be:  where Xj is the fraction of free or bulk water, Xh is the fraction of hydration water, and  21  Chapter 3. Proton NMR Theory  T\f, T\h are the spin-lattice relaxation rates of free and hydration water, respectively [13]. In soft tissue, the observed relaxation rate is thus the weighted average of all components within a diffusion radius of a water molecule [15]. Slow exchange occurs often in fatty tissues, where the lack of hydrogen-bonding sites limits cross-relaxation between water and fat. In this case the two compartments relax separately. [15] Compartmentation: When a tissue is compartmented, T is shorter than Ti and 2  shorter than T of bulk water, and is often multiexponential in nature [14]. There 2  is, however, disagreement over the number of H 0 compartments and the relaxation 2  characteristics of each compartment. A continuous model has been proposed, but often three compartments are sufficient to describe the relaxation behaviour of biological systems. [14,15] The fact that in compartmented systems Ti is usually single exponential and T  2  is multi-exponential is explained via the rates of exchange processes between compartments: the rates are fast on a Tj timescale but slow on a T timescale. This is not a 2  universally accepted theory, however, and other explanations have been proposed, such as a diffusion dominated process where the distance to an interface (between compartments) cannot be traversed within the intrinsic relaxation time of the diffusing species, so one observes a variety of relaxation times. [14] This view is presented by Brownstein and Tarr [16], who assume that all relaxation is due to magnetization sinks either contained within an active volume or on an active surface to which the molecule must diffuse. The general solution is: CO  M(t) =  J2l exp(-t/T , ) n  j n  n=0  where M(t) is the appropriate magnetization at time t, I is the intensity of the n n  th  22  Chapter 3. Proton NMR Theory  component, and j = 1,2. Only the first two or three terms are important for systems with only active surfaces (cell walls, for example). The Ti's, T 's and l's depend on 2  the local geometry, diffusion coefficient, and the strength of the relaxation sink. This model is best applied to systems with compartments on the order of micrometres across containing localized sinks such as walls, though there may be many other systems yet to be studied where this model fits. [14,16]  Chapter 4  Application of N M R to Intervertebral Discs  4.1  Advantages of Magnetic Resonance  The main problem of studying intervertebral discs is inaccessibility. Many techniques for gleaning information about the disc do not provide enough data or are insensitive to changes within the disc body, while others are invasive and risky to the patient. Magnetic Resonance Imaging possesses none of these problems and is now the "state of the art" technique for examining discs. The reasons are obvious when the abilities of this method are weighed against those of plainfilmradiography, myelography, contrast discography and computed tomography. [1,3] Plain film radiography shows advanced changes only, such as disc space narrowing, endplate sclerosis, and osteophyte formation, but does not reveal changes in the disc itself. Myelography only shows bulging of the disc, herniation, and spinal stenosis. Discography is invasive, dangerous, and controversial, and computed tomography is insensitive to changes that occur before the onset of structural changes. [1] On the other hand, MRI gives soft tissue contrast, excellent spatial resolution, and provides multiplanar imaging. The anulus and nucleus can be distinguished, unlike with any of the other techniques. The latest surface coil technology, cardiac gating, gradient refocussing and contrast agents have allowed for much enhancement of these features. [1,2] Most importantly, MRI is very sensitive to early changes in the disc, reflecting the  23  Chapter 4. Application of NMR to Intervertebral Discs  24  structural and chemical changes of degeneration. The signal appears to be correlated with proteoglycan content and distribution, and seems to reflect these attributes even better than gross morphology; this will help enormously with the investigation of the exact relationship between proteoglycan content and degeneration. [1,3] The role of magnetic resonance could go to actual tissue characterization of biochemical change and pathology. An early example of this sort of application is a study of the so-called "hamburger effect", where a low-intensity horizontal strip is seen in the middle of the nucleus in T2-weighted mid-sagittal images. Chemical breakdowns of the regions of different signal intensities showed no disparity in chemical concentrations, suggesting that tissue distribution and structure are responsible for the effect, not chemical composition. Further studies will investigate the relationship between tissue structure and T values tofindout what is different about the "hamburger" region. 2  Through this type of work a full understanding of the degenerative mechanism and its causes may be possible. [1,2] 4.2  Purpose of This Work  The objective of this work is to take the first step in attempting to relate the degenerative changes described in Section 2.2 to the magnetic resonance image of the disc. This would lead to a possible understanding of the degenerative mechanism and also make MRI a reliable diagnostic tool for many types of back trouble. The first step is to obtain a base of NMR data from discs in different stages of degeneration, from which initial inferences can be made and general trends can be observed. This project focuses on the discs of the lumbar spine, where most trouble occurs.  Chapter 5 Experimental Methods  5.1  Experiments Performed  The main portion of this study involved the examination of anulus and nucleus in vitro samples from two MRI grades of disc: II and IV. From six discs, both anulus and nucleus were studied, while from another five discs only the nuclei were used. This was decided after little difference was found between the results from the two grades of anulus. For thefirstsix disc samples, Tjand T data were collected, as well as the 2  free induction decays; for the five nucleus samples only T values were found, as this 2  became the focus of the study. The ratio of solid to liquid signal was also obtained for nucleus and anulus. Table 5.1 lists all the disc samples and their statistics. In a parallel experiment, in vivo T data were collected by MRI from the discs of 2  volunteers aged 20-70 years with no history of back problems, in conjunction with a study by Pearce and Flak [1]. All lumbar disc nuclei (L1-L2 to L5-S1) were examined for 19 volunteers. Some analysis of disc tissue components was also carried out in the hopes of obtaining more insight into the origins of the T data. Two partly denatured collagen 2  samples from grades II and IV discs were analysed for Ti and T values, compared and 2  contrasted (Table 5.2). Proteoglycan also was examined from anulus and nucleus in three different concentrations in saline solution (Table 5.3).  25  Chapter 5. Experimental Methods  26  Table 5.1: Disc Samples Sample Spine # HD1 C16 HD2 U36 HD3 C13 HD4 C14 HD5 C23 HD6 C25 HD7 C28 HD8 C18 HD9 C27 C30 HD10 HD12 C30  Disc L5-S1 L5-S1 L3-L4 L5-S1 L5-S1 L5-S1 L2-L3 L5-S1 L1-L2 L1-L2 L2-L3  Grade II IV II IV II IV II IV II IV IV  AF  NP x/  V V V V  V V V s/ V V V V V  Table 5.2: Collagen Samples Sample Spine # Disc Grade AF NP C16 L3-L4 II V ^ CI C2 U36 L2-L3 IV 1/ 1/  Table 5.3: Proteoglycan Samples Amount of Concentration Proteoglycan (mg/ml) Sample AF/NP Dry Wt. (mg) Solution (ml) PGN1 NP 20.4 0.1314 155.3 NP 20.4 0.2756 74.0 PGN2 NP 20.4 0.4093 49.8 PGN3 14.1 0.0982 143.6 PGA1 AF AF 14.1 0.1970 71.6 PGA2 AF 14.1 0.2815 50.1 PGA3  Chapter 5. Experimental Methods  5.2  27  Sample Preparation  For the grades II and IV disc study, lumbar discs from a number of spines were dissected by Pearce et al.  1  to obtain samples of anulus and nucleus about 0.2 cc, small enough  to fit into an 8 mm diameter N M R tube. The spines had been obtained within 24 hours of death and kept frozen at —80°C. The N M R samples were kept frozen, and thawed to room temperature only for the data collection period. Consistent results were found for those samples refrozen, then rethawed for repeat data collection, so this method of storage was assumed to be appropriate. For the collagen samples, intervertebral disc collagen (remaining after extraction of proteoglycan with 4 M guanidinium chloride) was washed four times with a solution of phosphate buffered saline (pH 7.4) and soaked overnight. The residue left after the final centrifuge was placed in an N M R tube and kept frozen, until data collection at room temperature. The proteoglycan was dissolved in the saline solution at initial concentrations of 155 mg/ml and 143 mg/ml for the nucleus and anulus proteoglycan, respectively. Subsequent lower concentrations were achieved by adding further amounts of saline solution to the same samples after data collection. Samples were kept refrigerated while in storage.  5.3  Data Collection  Data were collected on a custom-built data acquisition system comprised of a Nicolet Explorer Digital Oscilloscope, DB32016 slave computer (National Semiconductor Corporation), modified Bruker SXP-100 Spectrometer, custom pulse programmer, custom interface board, and the main computer, a D E C MicroVAX. The configuration was 1  UBC Department of Pathology  Chapter 5. Experimental  Methods  28  designed and put together by E. Sternin. [17] Samples in 8mm NMR tubes were placed inside the probe of a 2.0 Tesla electromagnet, tuned at 90 MHz for water. Magnet inhomogeneity was reduced as much as possible by shimming. The images for the MRI (in vivo) data were collected on the 0.15 Tesla whole body imager at the UBC Hospital.  5.3.1  T and the C P M G Sequence 2  T data were obtained with the Carr-Purcell-Meiboom-Gill pulse sequence which uses 2  the concept of spin echoes described in section 3.1. The basic sequence is the following: 90° - r/2 - 180° - r - 180° - r Each scan consisted of two CPMG pulse trains, first the above sequence, then the following: 90°_ - r/2 - 180° - r - 180° - r x  which produces a negative signal, after which the two data sets are subtracted to cancel out coherent noise and baseline offset. One hundred scans were performed and averaged to give good signal-to-noise. Tau values used in this case were 200, 400 and 800/is, to check for any tau dependence. Early results showed none, so only 200 and 400/is were used in subsequent data collection. At each spin echo between 180° pulses, four data points were collected in the vicinity of the echo peak, to be averaged later. To get an optimal representation of both fast and slow decay rates, every echo was recorded for the first 224 echoes, then every eighth echo out of the next 4096, after which the remaining FID was collected on a free-running time base. Baseline was also collected before the sequence, for 1400/is. The details of the data point collection are as follows:  Chapter 5. Experimental Methods  29  90° - T/2 - 180° - 37r/80 - collect lus T x 3 - 35r/80 - 180° (— — l^s) — collect lfis Ninety degree pulse lengths were set so that two in succession were equal to one 180° pulse, rather than just halving the determined 180° length. This avoided errors due to the rounding of the pulse edges, which would cause a pulse whose length was half of a 180° to be less than 90°. (The longer the 180° pulse, the closer the 90° will be to half the duration.) Actual pulse lengths used ranged from 5.5^s to 7.8/xs for a 180° pulse, varying from week to week as the available rf power varied.  5.3.2  T i a n d the Inversion R e c o v e r y Sequence  The spin-lattice (Ti) relaxation data were collected using the following inversion-recovery sequence: —(trigger scope) — 50/xs baseline — 90° — collect — 10s recovery 180° — r — (trigger scope) — 50/xs baseline — 90° — collect — 10s recovery The amplitude of the signal right after the ninety degree pulse in the second portion of the sequence should be proportional to (1 — 2e~^  ^ ),  T+50fls  accounting for T effects  Tl  a  only. To eliminate effects of variations with time, a single 90° pulse was first applied to record the original longitudinal magnetization amplitude, then the inversion-recovery sequence was performed and that data subtracted from the single 90° data. One can see that complete inversion recovery would yield a zero signal after subtraction. In other words, this sequence found the difference in longitudinal magnetization from before the 180° pulse to a time T' afterwards, which is 2M (r=0)e '/ _T  z  Tl  where r' = r + 50/^s.  One hundred scans were taken for each r value, and eleven different r values were  Chapter 5. Experimental Methods  30  used, logarithmically covering an extensive time period: lms, 5,10,20,100,500,1000,2000,3000,4000,5000ms 180° pulse lengths were the same as for the CPMG sequence. Signal from both the x and y channels was collected so that any phase offset could be set to zero later by data manipulation. 5.3.3  In Vivo C P M G  A twelve-echo imaging sequence was written for in vivo MRI by A. MacKay, with 20 ms echo spacing (r), four averages per echo, and TR = 2000 ms. The sequence was applied to a single 10 mm sagittal midline slice, with the 30 cmfieldof view broken down into a 128 x 256 matrix of signal intensities for each echo image. Using a DEC PDP 11/34 graphics package with a RAMTEK image analysis system, a trackball was used to highlight the nucleus of each disc in thefirstecho image; then the average signal intensity, standard deviation, and total area were recorded for each of the twelve images, calculated from the same region of interest each time. Signal intensities were then written to VAX/VMS files, listed opposite their corresponding echo times, ready for T analysis. 2  5.4  Data Analysis  Several pre-written and some new Fortran 77 programs were utilized on the Micro VAX system for analysis of the data in various states. CPMG analysis programs were written by A. MacKay , K. Whittall , and H. Le, and Ti data handling routines were written by 1  2  A. MacKay. New programs written for this project were designed to facilitate handling of results output by the other programs. Physics, Geophysics and Astronomy Depts, UBC. 2  Chapter 5. Experimental Methods  5.4.1  31  Analysis of C P M G Data  The raw in vitro CPMG data were converted to a more useful form by the program CPMGANL2, written by A. MacKay and H. Le. This program averaged the four points for each echo, calculated the corresponding echo times, subtracted baseline, and took only the top 99% of the signal. From the baseline the noise was estimated with a standard deviation calculation. The output consisted of a list of positive echo amplitudes and their corresponding times, as well as the number of echoes and the standard deviation of the baseline. To assign T values using this form of the data, a non-negative least squares (NNLS) 2  program written by K. Whittall [18] was applied to produce a spectrum of T values 2  using linear inverse theory. The echo amplitude wasfittedwith a series of exponentials in the form S(t) = £ > , e x p ( - i / T ) 2 t  t=i  where S(t) is thefittedsignal amplitude at time t, N is the number of exponentials used in the fit (120 in this project), A{ is the weighting factor for the i  ih  is the i  component, and T 2l  T value in the spectrum. A discrete or continuous fit could be chosen. A set  th  2  of N T times was created prior to running the program, logarithmically covering a 2  range wide enough to include all possible transverse relaxation times for the materials being studied. For the disc samples, a scale from 10~ s to 10°s was adequate, while a 3  wider range of times (to 10s) was needed for the proteoglycan and collagen samples. Examples of both discrete and continuousfitsare shown in Figure 5.1. The continuous fit was chosen for all T analysis as this gave unambiguous results. For this type of 2  fit, an adjustable parameter called "facmu" controlled the amount of curvature allowed in the spectrum by weighting the first derivative. The smaller the value of facmu, the less defined were the spectrum peaks, and vice versa. Figure 5.2 illustrates the effect  32  Chapter 5. Experimental Methods  of changing facmu from 100 to 500. In the study, facmu was chosen so that each peak in the spectrum was distinct, if not separated, from its neighbours. An indication of this was the symmetry of a peak; i.e., how close the weighted average T value was to 2  the value with the largest amplitude (at the top of the peak). T results quoted in this 2  report are the weighted average values, though as stated above these were very close to the maximum amplitude values. The goodness of fit was indicated by the x value calculated by the NNLS program. 2  A good fit should have x close to the number of degrees of freedom (n/); i.e. in this 2  case the number of points being fit. This number depended on the number of echoes in the top 99% of the signal, and this number fell within the range 90-190. The value of x depended greatly on the estimated noise, or baseline standard deviation. In most 2  cases, x / f 2 n  ^  e n  between 0.9 and 1.2, while sometimes just one or two errant points  in the early data could be enough to blow up x • Fit to the data was checked by 2  plotting the fit along with the data points, and this always showed an excellent match (Figure 5.3). Changing facmu did not alter x dramatically. 2  5.4.2  Analysis of Inversion-Recovery Data  The raw T i data was transformed into an array of values (M(0) — M(r)) versus r, by a program written by A. MacKay. For each of the eleven different r value data sets, the program subtracted the baseline and output the average intensity for a data window following the 90° pulse. A noise estimator was also calculated from the baselines of all eleven data sets, by finding their standard deviation. With the inversion-recovery data represented as a decreasing function of time (in the form 2M (r=0)e" / ) they could be analysed with the NNLS program in a similar T Tl  2  procedure to the T analysis. For the T i data, however, the relaxation was nearly 2  single exponential, and the discrete fit was chosen with no ambiguity of results. This  Chapter 5. Experimental Methods  33  allowed for easy extraction of the Ti value by looking at the spectrum, and without the bother of adjusting facmu. The Ti time scale for the 120 exponential fit covered the range 10 s to 10 s. The \ goodness of fit was expected to be near 11 (the number of -3  1  2  degrees of freedom) but was often much lower due to overestimation of noise. Plotting the calculated data with the actual data showed an excellent fit as in the case of T analysis.  2  Chapter 5. Experimental Methods  Figure 5.1: Examples of N N L S spectra, fitted to C P M G data. (a) A discrete spectrum, (b) A continuous spectrum. The continuous spectrum has less ambiguity for multiexponential relaxation, and does not differ between data sets from the same sample.  34  Chapter 5. Experimental Methods  Figure 5.2: The effect of changing facmu on an N N L S continuous spectrum. (a) Facmu=100. (b) Facmu=500. Facmu is a variable parameter that controls the amount of curvature allowed in the spectrum, by weighting the first derivative. More peaks are visible and each one is narrower when facmu is larger.  35  Chapter 5. Experimental  Figure  5.3:  Methods  A plot of an NNLS fit to C P M G data. The actual data are shown as "o" symbols and the calculated signal is a solid line; the two are a close match.  36  Chapter 6 Results  6.1  T and T of Disc Samples 2  a  Table 6.1 lists all the T values and their percent contributions to the continous spectra 2  obtained for the nucleus pulposus and anulusfibrosussamples of grades II and IV discs. With the exception of HD8N, results for r = 200/JS and r = AOOus were very close, so both tau value results are shown for HD8N. Some typical T spectra are also shown 2  in Figure 6.1 to illustrate the origin of the results in Table 6.1. All T-i results are summarized in Table 6.2, which lists the components of the discrete spectra and their percent contributions. Figures 6.2-6.5 are plots of the results in Tables 6.1 and 6.2, with the dominant contributions (above 20%) shown as solid symbols and the lesser components as empty symbols. For the case of HD8N, the tau value whosefittedx / f 2 n  w  a  s  closest to one  was chosen for plotting in Figure 6.2. T — Nucleus Pulposus: Figure 6.2 clearly shows a difference in T values between 2  2  grade II and grade IV nucleus samples. The dominant contributions (solid symbols) for grade II mostly lie above 100 ms while the T values for grade IV fall in the range 2  20-70 ms. The only overlap is from sample 5 where there are two components over 20%, the lesser one (31%) at 55 ms and the larger one (49%) at 156 ms; and sample 9, where there are two components over 20% also, the smaller at 220 ms and the larger at 69 ms. Sample 9 was also the one for which the r = 200/15 and r = AOOus results 37  Chapter 6. Results  Table 6.1: T Values for Nucleus and Anulus of Disc Samples 2  T Values (ms) and % Contributions Sample AF/NP Grade 1. HD1N NP II 5.5 (2%) 36 (8%) 105 (23%) 255 (67%) 2. HD3N NP II 6.4 (4%) 38 (12%) 101 (84%) 3. HD5N NP II 4.6 (2%) 20 (5%) 101 (93%) 4. HD7N NP II 2.5 (1%) 15 (4%) 156 (95%) 5. HD9N NP II 4.5 (5%) 15 (15%) 55 (31%) 156 (49%) 6. HD2N NP IV 3.6 (8%) 23 (92%) 7. HD4N NP 2.4 (2%) 12 (7%) 47 (90%) 498 (1%) IV 8. HD6N NP 9 (2%) 42 (96%) 247 (1%) IV 9. HD8N NP IV 1.7 (2%) 18 (9%) 69 (52%) 220 (37%) 9. HD8N NP IV 9 (5%) 104 (95%) 10. HD10N NP IV 5.4 (2%) 49 (98%) 49 (98%) 11. HD12N NP IV 4.8 (2%) 1. HD1A AF II 2.9 (14%) 13 (51%) 43 (33%) 392 (2%) 2. HD3A AF II 4.0 (12%) 15 (41%) 38 (47%) 3. HD5A AF II 2.9 (22%) 10 (52%) 29 (26%) 4. HD2A AF IV 1.7 (1%) 11 (6%) 64 (91%) 562 (2%) 5. HD4A AF IV 3.5 (22%) 12 (57%) 39 (21%) 6. HD6A AF IV 4.8 (10%) 22 (90%) 2  a a  "HD8N is the only sample for which CPMG results from the two different tau values did not agree, so both are shown here, in order of increasing r.  Table 6.2: T i Values for Nucleus and Anulus of Disc Samples T Values (s) #. Sample AF/NP Grade and % Contributions NP II 0.62 (2%) 1.4 (98%) 1. HD1N 2. HD3N NP II 0.69 (34%) 1.5 (66%) HD5N NP II 0.25 (5%) 1.6 (95%) 3. rv NP 0.64 (100%) 4. HD2N NP TV 0.83 (89%) 1.2(11%) 5. HD4N 6. HD6N NP IV 0.75 (98%) 3.5 (2%) 1. HD1A AF II 0.69 (83%) 1.3 (17%) 0.47 (51%) 1.2(49%) 2. HD3A AF II AF II 0.70 (93%) 1.4 (7%) 3. HD5A HD2A 1.10 (92%) 1.9 (8%) 4. AF IV AF 0.78 (95%) 1.5 (5%) IV 5. HD4A AF 0.69 (100%) IV 6. HD6A x  Chapter 6. Results  39  Figure 6.1: Typical T spectra for in vitro disc samples. (a) Grade II nucleus, (b) Grade IV nucleus, (c) Grade II anulus. (d) Grade IV anulus. Facmu=500 in all cases. 2  40  Chapter 6. Results  did not agree. Thus this sample may be suspect (due to mis-grading or an undetected problem with the data collection that day) and the overlap between grades II and IV only occurs for sample 5. Sample 5 had the largest contributing component in the range above 100 ms, so the problem may originate with the arbitrary cut-off point of 20% for a "dominant" T component. 2  Every sample had a short T component with less than 20% contribution to the 2  signal, regardless of grade, though the grade II samples have a consistent second small component between 15 and 40 ms that is only present in two of the grade IV samples. Table 6.3 lists the average T values for grades II and IV, for the dominant components 2  and the lesser components. Table 6.3: Average T Values for Grades II and IV Anulus and Nucleus 2  AF/NP NP NP NP NP AF AF  Grade Contribution 4f of Samples Mean T (ms) Std. Dev. (ms) II 138 49 > 20% 5 11 42 IV 5 > 20% II shortest 5 4.7 1.5 shortest 4.5 2.6 IV 5 6 II 3 23 > 20% 35 25 IV > 20% " 3 2  T — Anulus Fibrosus: Unlike the nucleus samples, there was no significant dif2  ference between the dominant contributions to T relaxation of grades II and IV, as 2  seen in Figure 6.3 and Table 6.3. Due to this observation, further studies of the anulus fibrosus were discontinued in order to devote more time to the nucleus pulposus, where most change occurs with degeneration. The nucleus is also the region examined most closely in MRI grading procedures as it usually dominated the image. As with the nucleus pulposus, all anulus samples had a short T component of 2  small amplitude, with the relaxation time falling between 1 and 5 ms. Generally, the  Chapter 6. Results  41  variation between samples is greater for grade IV than for grade II, though one must take into account the small number of samples when evaluating such trends. T i — Nucleus Pulposus:  The T] values follow a similar pattern to the T results, 2  with a large decrease from grade II to grade IV. Table 6.4 lists the average T times for a  the dominant contributions to the spectrum; note the factor of two difference between grades. The division is clearer between grades than it is for T , and no overlap occurs 2  between the two groups. The Tj relaxation is simpler than the T , having only one or two components in 2  the spectra. This is expected for biological systems, as discussed in section 3.2. Table 6.4: Average Tj Values for Grades II and I V Anulus and Nucleus  AF/NP Grade Contribution # of Samples Mean T (s) Std. Dev. (s) 0.20 NP II 3 1.41 > 20% NP 0.74 0.10 IV >20% 3 NP II 2 0.44 0.26 <20% 2 1.60 NP IV <20% 2.40 AF II >20% 3 0.74 0.08 AF IV >20% 3 0.86 0.21 AF II < 20% 2 1.35 0.07 AF IV 2 1.70 0.30 <20% x  T i — Anulus Fibrosus:  The Ti values for the anulus are the same as those of  the nucleus, except that the relative proportions of contributions to the spectrum are reversed: the dominant components have shorter Tj values, and the small components have longer relaxation times. Table 6.4 shows the average Ti values for large and small components of the spectra for grades II and IV disc anuli. Not all of the samples were found to have two components, or a large and small component — some had two contributions above 20%, for example.  Chapter 6. Results  42  300  LEGEND Gr. II Gr. IV O <20% A <20% • ^20% A ^20%  250 200 H  T ,2  (ms)  150H 100 n  50  0  o  o ^ ?  2  3  O ?  4  O ?  5  •  6  Sample -  A  7  8  9  A  10 11  Nucleus  Figure 6.2: Plots of T values for in vitro disc nucleus samples. Grade II and grade IV nucleus are displayed together (see legend). All components greater than or equal to 20% are shown as solid symbols, and the rest as empty symbols. 2  Chapter 6. Results  43  i  70  LEGEND Gr. II Gr. IV  60 50 T  H  • •  0 <20% • ^20%  <20% ^20%  40  2  (ms)  30 20•  10-  0  •  •  1  2  •  3  Sample  4  5  6  Anulus  Figure 6.3: Plots of T values for in vitro disc anulus samples. Grade II and grade IV anulus are displayed together (see legend). A l l components greater than or equal to 20% are shown as solid symbols, and the rest as empty symbols. Note that the vertical scale is much smaller than for the nucleus samples. 2  Chapter 6.  Results  44  i  4.0  LEGEND  3.5 3.0  Gr.  II  Gr. IV  O <20%  •  <20% A ^20%  A  ^20%  2.5 2.0  (s)  1.5 A  1.0  •  0.5 0.0  1  2  Sample  3  4  5  6  Nucleus  Figure 6.4: Plots of T i values for in vitro disc nucleus samples.  Grades II and IV nucleus are displayed together (see legend). Symbols have the same meaning as in the corresponding T plot. 2  Chapter 6.  Results  45  2.5 2.0  i  LEGEND G r . II • <20% • ^20%  G r . IV 0 <20% • £20%  1.5 H T.  (s)  •  •  1.00.50.0  1  2  3  4  5  6  Sample — Anulus Figure 6.5: Plots of T i values for in vitro disc anulus samples. Grades II and IV anulus are displayed together (see legend). Symbols have the same meaning as i n the corresponding T plot. Note the scale difference from the nucleus T i plots. 2  Chapter 6.  Results  46  Table 6.5: T Values (in ms) of In Vivo M R I Samples 2  Volunteer # 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19  6.2  L1-L2  Lumbar Disc Names: L3-L4 L4-L5 L2-L3  134 131 126 43 50 57 81 90 90 118 120 56 128 108 129 25, 97 122 85 76 58 46 44 44 49 94 51, 159 151 136 135 178 152 129 137 93 40, 136 48 124 124 131 62, 234 87 65 62 69, 197 55, 139 104 93 36,117 52 87 116 104 62 69 104, 395 117 175  L5-S1  143 110 44 27 83 40 47 35 57 148 157 119 86 58, 181 57 52 144 60 111 129 41 33, 168 68 83, 255 104 35, 175 49 78 55, 313 55, 147 46 98 41 52 104 73 69 35, 156  In Vivo MRI T Values 2  A l l of the i n vivo M R I T values for the lumbar disc nuclei of the 19 volunteers stud2  ied are displayed in Table 6.5. In most cases where two components existed, they contributed fairly equally to the spectrum, so the actual percentages are not shown. Spectra were less complicated than those for in vitro samples because only twelve data points were available for fitting with the N N L S program. Several of the volunteers were found to have self-consistent T values among discs, 2  such as volunteer #1 (average T was 129 ms with a standard deviation of 12 ms), 2  #2 (average 44 ms, standard deviation 11 ms), #8 (49 ms; 6 ms) and #13 (118 ms;  Chapter 6. Results  47  Table 6.6: Average In Vivo T Values for Each Lumbar Disc 2  Disc: Mean T & Std. Error (ms) 2  L1-L2 L2-L3 L3-L4 L4-L5 L5-S1 104 ± 8 101 ± 8 91 ± 9 95 ± 1 0 81 ± 9  12 ms). This phenomenon can be seen in thefirstplot of Figure 6.6, which is a series of plots displaying all T components found for each type of lumbar disc. 2  The mean T values for each disc type are shown in Table 6.6. The weighted average 2  T values for two-component spectra were used in the calculations. Figure 6.7 provides 2  a graphic illustration of those results, showing a trend towards shorter T values from 2  L1-L2 to L5-S1. The error bars represent the standard error of the mean and are slightly larger for the lower lumbar discs. Figure 6.8 is a histogram plot of all the T components found among the M R I 2  volunteers, and shows clearly that there is a division of the values into two groups. The first group is centered around 50 ms, while the second is approximately centered on 120 ms. These numbers are not in disagreement with the average dominant T values 2  of in vitro nucleus samples found in Table 6.3.  Chapter 6.  400H  Results  J  I  I  L  J  L 1 - L 2 (•) L 2 - L 3 (*)  300-  (ms)  48  l  I  I  l  1_  L4-L5  200-  •  *  1000400-  1  1  * * a  •  D  *1  •  T  _i  o *  a  n i  i_  i i  • * *  A *  -i  i r i i  i i  L3-L4  1  L 5 - S 1i i  * ^  A  i  1  1  i  i  ^  A  1  i  1  i  1  i  1  i  300-  (ms)  200100-  o  o ° °  o o o  —i 2  0  o  °  °  o  0  0  r  -  1  1  1  1  1  1  1  4 6 8 10 12 14 16 18  Sample  0 « *  1  2  1  I  1  o  o  0  I  I  o  0  I  I  I  4 6 8 10 12 14 16 18  Sample  (disc)  (disc)  Figure 6.6: T values for MRI samples. Results for each disc are shown, with L 1 - L 2 and L 2 - L 3 overlaid to show similarity between discs of the same spine. The same vertical scale is used for all plots. 2  0  r  Chapter 6. Results  i  1  1  r  L1-L2 L2-L3 L3-L4 L4-L5 L5-S1  Disc Figure 6.7: Graph of mean MRI T values for each disc. This plot demonstrates the shortening of T values from L1-L2 to L5-S1. Error bars represent the standard errors of the means. 2  2  Chapter 6. Results  0  50  50  100 150 2 0 0 2 5 0 3 0 0 3 5 0 4 0 0 4 5 0  T  2  (ms)  Figure 6.8: Histogram of MRI T values. A l l discs are included in this histogram, which demonstrates the grouping of T values into two ranges. 2  2  Chapter 6. Results  6.3  51  Solid Signal in Disc Samples  The Ti inversion-recovery data show the signal decay at times immediately after the 90° pulse. For very short tau values one may sometimes observe a rapidly relaxing component which can be attributed to protons of solids in the sample. Figure 6.9 shows inversion-recovery-data from samples of grade II and IV nucleus, with r = 1 ms. The grade II sample showed very little solid component (3.8%), while 8.5% of the signal from the grade IV sample was due to solid protons. This was calculated by separating the two decay slopes and interpolating back to t = 0. Although not shown, inversionrecovery data from anulus samples had a solid component making up 12% of the signal at t — 0, for both grades of disc.  6.4  T i and T of Collagen Samples 2  Only one collagen sample from each of grades II and IV discs was examined, and these results are listed in Table 6.7. There did not appear to be any definite pattern to the relaxation times in this small sample group, but a few general observations were made. The Ti values for the collagen were very similar for all samples (grades II and IV anulus and nucleus), but the grade IV samples had a longer T component that was x  not present with the grade II samples. The Ti values for grade II anulus and nucleus were identical. As far as T went, the grade IV relaxation times were longer than those 2  for grade II samples. A longer T timescale was used for NNLS fits to accomodate the 2  slow rate of relaxation.  Chapter 6. Results  52  40 30 H co  • i-l  (a)  20 10 0  cd  0 cd  £  B <  ~50  100  150  200  250  300  35 ^  302520 1  (b)  5  10 0  f  0  50  100 150 200 T i m e (/xs)  250  300  Figure 6.9: Inversion-recovery data showing solid signal for in vitro disc samples. (a) Grade II nucleus pulposus. (b) Grade I V nucleus pulposus. The first 50/is is baseline. The solid portion of the signal shows up as the steeper slope immediately after the pulse.  53  Chapter 6. Results  Table 6.7: T and T Values for Collagen Samples x  2  T Values (ms) T Values (s) Sample AF/NP Grade and % Contributions and % Contributions NP C1N II 1.5(100%) 76 (37%) 183 (63%) C2N NP IV 1.3 (81%) 3.1(19%) 327 (100%) CIA AF II 1.5(100%) 9 (1%) 99 (99%) C2A AF IV 1.0 (64%) 2.1(36%) 80 (72%) 1180(28%) 2  a  6.5  T i and T of Proteoglycan Samples 2  Both T i and T relaxation for proteoglycan samples in saline were single or almost 2  single exponential. Table 6.8 lists the relaxation times. As proteoglycan concentration decreased, relaxation times increased for both anulus and nucleus proteoglycan, with the Ti and T values shorter for anulus than nucleus at similar concentrations. Fig2  ure 6.10 illustrates the changes of relaxation rates with proteoglycan concentration; the relationship is roughly linear for the three concentrations studied. In all cases, the T values are much longer than those of the disc samples. Table 6.8: T i and T Values for Proteoglycan Samples 2  Concentration Sample AF/NP (mg/ml) PGN1 NP 155.3 PGN2 NP 74.0 PGN3 NP 49.8 PGA1 AF 143.6 PGA2 AF 71.6 AF PGA3 50.1  T (ms) 666 1100 1340 440 666 750 2  00  2.00 2.42 3.20 1.42 1.74 —  2  54  Chapter 6. Results  0.9  • Nucleus PG A n u l u s PG A  (a)  • 0.2  30  50  .  •  • 70  •  90  110 130 150 170  PG Concentration (mg/ml) 3.0  1  • Nucleus PG A A n u l u s PG  2.6 2.2 1.8  (b)  1.4 1.0 0.6 H 0.2  30  50  70  90  110 130 150 170  PG Concentration (mg/ml)  Figure 6.10: Graphs of proteoglycan relaxation rates versus concentration in saline solution. (a) 1 / T i versus concentration for anulus and nucleus P G . (b) I/T2 versus concentration for anulus and nucleus P G .  Chapter 7 Discussion  Correlation of relaxation times with grade: The results of the in vitro disc studies show that relaxation times correlate with disc grade. T and T i values of the 2  nucleus change significantly with grade, decreasing by approximately a factor of two between grades II and IV. Furthermore, the MRI T values decrease with distance down 2  the lumbar spine, which would be expected if T were inversely proportional to disc 2  grade: the lower discs of the lumbar region are generally the most degenerate. Another fact supporting the notion that T is a sensitive indicator of degeneration is that the 2  results for the grade IV nucleus look the same as those of both grades of anulus. This ties in with the fact that as discs degenerate, the nucleus becomes more and more like the anulus until they are indistinguishable, as noted in Section 2.2. Comparison of in vitro and in vivo relaxation times:  The in vivo T values 2  agree with those found in vitro, being grouped into two ranges of relaxation times that match the grades II and IV in vitro T values. These results agree despite the differ2  ences in sample temperature and NMR frequency during data collection. The in vitro sample data were collected at approximately 23°C while the in vivo samples remained at 37°C. An increased temperature would be associated with faster thermally activated processes and increased diffusion rates. Correlation times decrease with increased thermal activity, but this should not affect T unless slow motions are present. The lack of 2  dependence of T on CPMG tau value suggests that slow motions do not play a large 2  55  Chapter 7. Discussion  56  role in relaxation. The change in diffusion rate would shorten T if the relaxation were 2  diffusion-limited, but here that does not seem to be the case, as the T times agree for 2  room temperature and body temperature. As for frequency differences, the in vitro experiments were carried out at 90 MHz, while the MRI data were collected at 6.4 MHz. This would not be expected to affect T values since the fundamental mechanism for 2  transverse relaxation is dipole-dipole, which is independent of field strength. Ti on the other hand should be much shorter at the MRI frequencies compared to 90 MHz, and this is the case: the shortest in vitro T value is 740 ms, while in vivo values are x  typically 370 ms [19]. Distribution of in vivo T values: 2  The two peaks in the distribution of in vivo  relaxation times are relatively wide (Figure 6.8, indicating the disparity among individuals' T values. Separate consideration of the data for each subject suggests that 2  relative, not absolute, evaluations of T data would be most useful from a clinical point 2  of view — often one disc in a spine will have a much lower T value than any of the 2  others, while another spine will have these "low" T values for every disc, even though 2  no other evidence of degeneration appears. For an example of this, compare samples 5 and 8 in Table 6.5. The L5-S1 disc of sample 5 has a T value of 57 ms which is half 2  as long as the values for the other discs, yet the discs of sample 8 all have T values 2  equal to or lower than 57 ms. Proteoglycan and collagen studies:  The changes in T with grade are probably 2  related more to structure than to chemical composition, as noted in section 4.1. The results of the proteoglycan studies support this theory: though proteoglycan appears to be lost from the disc during degeneration, simply decreasing the concentration of isolated proteoglycan does not decrease the T value; rather, it increases, contrary to 2  57  Chapter 7. Discussion  the behaviour in discs when pg content is lowered. This is not unexpected, however, because when less proteoglycan is in solution, the water behaves more like bulk water, and the T times should increase. These results are in agreement with a recent study 2  by Weidenbaum et. al [20]. Collagen studies also demonstrate this contradictory behaviour, with T values being longer for grade IV collagen extract than for grade II. 2  As suspected, studies of these macromolecules in isolation cannot be directly applied to disc studies. Instead, one must consider the structure of the cartilage as a whole. If proteoglycan aggregates and their subspecies are fragmenting with degeneration, this may shorten T by reducing the volume of bulk water between molecules, i.e. decreasing 2  the mean diffusion path for a water molecule. The decreasing water content would add to this effect. In addition, collagen increases in the nucleus with degeneration, reducing bulk water and increasing the amount of structured water. Details of T spectra: The components of the T spectra are at different T values 2  2  2  for grades II and IV, rather than having peaks at the same T values with different 2  relative contributions to the total area. This rules out a simple model of compartments relaxing separately, with relative volumes changing during degeneration. The only common component of the spectrum is the very short (less than 10 ms) T peak, which 2  contributes about 5%. The following calculation roughly estimates the percentages of protons in the water, proteoglycan and collagen of the grade II nucleus, for comparison with the components of the T spectra. First of all, the healthy disc is about 70% water. Collagen makes 2  up 10-20% of the dry weight in the nucleus (Section 2.2), so the remaining 80-90% would be proteoglycan. This means that of the total wet weight, there would be about 70% water, 4.5% collagen, and 25.5% proteoglycan. Water has 2 protons per 18 atomic units, or 0.11 protons per unit weight. Rough estimates of the number of protons  58  Chapter 7. Discussion  per unit weight for the proteoglycan and collagen are 0.12 and 0.067, respectively. So, multiplying these fractions by the weight percentages of water, pg and collagen in the nucleus gives the following percentages of protons: 78% from water, 17% from proteoglycan, and 5% from collagen. For a grade IV nucleus, the percentage from water would decrease, as water content drops by 30%; the percentage from proteoglycan would be increased by water loss and decreased due to proteoglycan loss; and the percentage from collagen would increase both from decreased water content and increased collagen content. The solid component of the spectrum as shown in Figure 6.9 accounts for 3-4% of the signal in the grade II nucleus, so it seems likely to originate from the protons of the collagen. This theory is supported by two results: the solid component increases to 8-10% in grade IV nucleus, and collagen is known to increase with degeneration; also, the solid signal in the anulus was the largest at 12%, which also agrees with the known chemistry of the disc. Further evidence comes from the slope of the solid signal, which increases from grade II to grade IV, suggesting a more rigid structure is responsible for the signal; collagen fibrils are known to increase in diameter with degeneration. The shortest T component in the grade II and grade IV nucleus spectra con2  tributes 1-7% and thus could be attributed to bound water or proteoglycan, with cross-relaxation possibly playing a role. The second short component in the grade II nucleus spectra ranges from 4-15% contribution, with T values from 15 to 38 ms, 2  and this might be due to the protons of the proteoglycan, which are estimated at 17% abundance. The large components of the grade II spectra most likely originate from the bulk and hydration water. For the grade IV nucleus, the largest component of the spectra has T values in the range 23-50 ms and no third T component, so it appears 2  2  that for grade IV, the proteoglycan and bulk water protons are indistinguishable in terms of T values. Table 7.1 lists tentative assignments of all components of the T 2  2  Chapter 7. Discussion  59  Table 7.1: Tentative Assignments to the T Spectrum for Disc Nucleus 2  Constituent Collagen  Assignment TJ, 20-40/zs (solid signal) T , 1-6 ms  Grade II Proportion Grade IV Proportion of Protons of Protons 8-10% 3-4%  Bound water or proteoglycan Proteoglycan T , 15-50 ms or water Water T , >100 ms 2  2  1-5%  2-7%  4-14%  82-89%  48-92%  2  Total=100%, including solid spectra for the disc nucleus, grades II and IV. Cross-relaxation between some or all of these constituents is probable and leaves several models open for investigation. Details of Tj spectra:  The T spectra differ from the T spectra in that they do have x  2  common values that occur in different relative contributions. Different processes seem to be at work for Ti relaxation, probably because cross-relaxation processes would have more time to act on a T timescale. The large difference between Tj and T indicates x  2  that fast motions with correlation times less than 1/90 MHz are of importance to the relaxation processes. The complex T spectra are probably more useful for studying 2  details of the degenerative mechanism, though the Ti results are necessary to complete the picture. Future work: More accurate assignment of the components of the T spectrum to 2  specific molecules and processes in the disc will require further experiments with in vitro and in vivo disc samples. First of all, more disc samples should be measured, both in vitro and in vivo, to gain a larger sample of the population from which robust statistics can be extracted. As well, the same samples should undergo chemical analysis  Chapter 7. Discussion  60  so that the exact amounts of water, proteoglycan and collagen are known for each sample. This would allow precise comparisons between chemistry and NMR data, and contrast between samples of different grades. Histological analysis would also be valuable in correlating disc structure with the Ti and T results. Meanwhile, in vitro 2  data collection should be attempted at 37°C on samples that have not been previously frozen, to eliminate the possibility of structural differences between in vivo and in vitro tissues. Conclusions: This study has formed the framework for continuing research regarding the application of magnetic resonance to the intervertebral disc. The results of this investigation indicate that relaxation measurements, in particular T , are sensitive to 2  disc degeneration and could be used as the main tool for studying the details of the degenerative mechanism. The T i and T spectra, the in vivo T values, and the solid 2  2  signal measurements strongly support the suggested roles of collagen, proteoglycan and water in NMR measurements of the intervertebral disc. Future work will be based on verifying and expanding the model proposed here.  Bibliography  [1] R. H. Pearce, J. P. Thompson, B. Flak, A. MacKay. "Biochemical and Radiologic Studies of Degeneration in the Human Intervertebral Disc". A research proposal to the Arthritis Society, December 1989. [2] M. T. Modic in Magnetic Resonance Imaging of the Spine. Chapter 3: "Degenerative Disorders of the Spine", pp75-119. Yearbook Medical Publishers, Chicago, London, Boca Raton, 1989. [3] D. Eyre, P. Benya, J. Buckwalter, B. Caterson, D. Heinegard, T. Oegema, R. Pearce, M. Pope, J. Urban. New Perspectives on Low Back Pain. Part B: Basic Science Perspectives. Eds. J. W. Frymoyer, S. L. Gordon. Park Ridge, IL. American Academy of Orthopaedic Surgeons, ppl47-207, 1989. [4] R. H. Pearce, B. J. Grimmer, M. E. Adams. "Degeneration and the Chemical Composition of the Human Lumbar Intervertebral Disc". J. Orth. Research, 5:198205, 1987. [5] A. I. Caplan. "Cartilage". Sci. Am., 251:84-94, 1984. [6] E. D. Harris, Jr. Scientific Bases of Rheumatology Chapter 16: "Biology of the Joint." (Futher information unavailable) [7] J. J. DiFabio, R. H. Pearce, B. Caterson, H. Hughes. Biochem. J., 224:27-33, 1987.  61  62  Bibliography  [8] R. H. Pearce, B. J . Grimmer. "The Concentration of Proteoglycan Fractions in Healthy Young Human Lumbar Intervertebral Discs". Preprint, 1989. [9] G. M. Bebault, R. H. Pearce. "The Assay of Keratan Sulfate as Anion-ExchangerBound Hexose". Preprint, October 1989. [10] J. P. Thompson. "Classifications of Gross Morphologic and Magnetic Resonance Images of Human Intervertebral Discs". M.Sc. Thesis, UBC,  1987.  [11] J. P. Thompson, R. H. Pearce, M. T. Schechter, M. E. Adams, I. K. Y. Tsang, P. B. Bishop. "Preliminary Evaluation of a Scheme for Grading the Gross Morphology of the Human Intervertebral Disc". Submitted to Spine, October 1989. [12] C. P. Slichter. Principles of Magnetic Resonance ppl-42. Springer-Verlag, Berlin, Heidelberg, New York, 1980. [13] R. Mathur-De Vre. "The NMR Studies of Water in Biological Systems" Proc. Biophys. Mol. Biol, 35:103-134, 1979.  [14] P. S. Belton, R. G. Ratcliffe. "NMR  and compartmentation in Biological Tissues"  Progress in NMR Spectroscopy, 17:241-279, 1985. [15] G. D. Fullerton in Magnetic Resonance Imaging. Chapter 3: "Physiologic Basis of Magnetic Relaxation", pp36-55. C. V. Mosby Company, St. Louis, Washington, D. C. Toronto, 1988. [16] K. R. Brownstein, C. E . Tarr. "Importance of classical diffusion in NMR of water in biological cells" Phys. Rev. A., 19:2446-2453, 1979. [17] E . Sternin. Ph.D Thesis, UBC,  1988. pp34^5.  [18] K. P. Whittall, A. L. MacKay. J. Magn. Resonance, 84:64, 1989.  studies  Bibliography  63  [19] A . MacKay, private communication. [20] M . Weidenbaum, R. J . Foster, B. A . Best, E . L . Nickoloff, A . Ratcliffe, R. Jelsma, J . H . Newhouse, V . C . Mow. "Magnetic resonance imaging studies of water a n d proteoglycan content of the intervertebral disc". p338, 36th Annual Meeting, Orthopaedic Research Society, New Orleans, Louisiana, February 5-8, 1990.  

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