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Cortical activity following restoration of elevated intraocular pressures to normal pre-laser pressures… Jim, Janey 2003

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CORTICAL ACTIVITY FOLLOWING RESTORATION OF E L E V A T E D INTRAOCULAR PRESSURES TO N O R M A L PRE-LASER PRESSURES IN A PRIMATE M O D E L OF G L A U C O M A by J A N E Y JIM fi.Sc, The University of British Columbia, 2001 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE F A C U L T Y OF G R A D U A T E STUDIES (Department of Ophthalmology and Visual Sciences, Neuroscience Program) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH C O L U M B I A January 2004 © Janey Jim, 2003 Library Authorization In presenting this thesis in partial fulfillment of the requirements for an advanced degree at the University of British Co lumb ia , I agree that the Library shal l make it freely avai lable for reference and study. I further agree that permiss ion for extensive copying of this thesis for scholar ly purposes may be granted by the head of my department or by his or her representat ives. It is understood that copying or publication of this thesis for f inancial gain shal l not be al lowed without my written permiss ion. J a n e y J i m 10/01/2004 N a m e of Author (please print) Date (dd/mm/yyyy) Title of Thes is : Cort ical Activity Fol lowing Restorat ion of E levated Intraocular P ressu res to Normal P re -Lase r P ressures in a Pr imate Mode l of G l a u c o m a Degree: Master of Sc ience Yea r : 2004 Department of Ophthalmology and V isua l S c i e n c e s , Neurosc ience Program The University of British Co lumb ia Vancouver , B C C a n a d a Abstract Glaucoma is a group of diseases that affects the optic nerve, causing debilitating visual deficits with no known cures. The primate model of glaucoma was developed to enable investigators to study this disease. Research in glaucoma has examined the disease at the level of the eye and optic nerve head. Recent evidence has also emerged extending glaucomatous injury to the central nervous system. While changes in metabolic activity in response to elevated intraocular pressure (IOP) have been reliably demonstrated, few studies exist that examine metabolic activity following the lowering of elevated levels of IOP to normal values. This study looks at the effects of lowering IOP, following elevated IOP levels of 2 and 4 weeks, by comparing the metabolic activity levels between ocular dominance bands in the primary visual cortex. Elevated IOP (> 30 mmHg) was induced by unilateral lasering of the trabecular " meshwork of primates. The animals' IOP was periodically monitored over a period of 2, 4, 8, and 16 weeks. The first set of animals was sacrificed following the designated period of elevated IOP. A second set of animals had their IOP returned to normal pre-laser values (< 20 mmHg) by trabeculectomy following deprivation. They were allowed to survive for several months with normal pre-laser IOP values before being sacrificed. A l l animals' cortices were histochemically processed for cytochrome oxidase (CO). OD ratios comparing the CO density values of ocular dominance bands were obtained. CO density was found to be lower in deprived eye columns than in non-deprived eye columns as early as 2 weeks post elevated IOP, suggesting that a decrease in metabolic activity in the eye subjected to elevated IOP occurs. Further, OD ratios comparing the metabolic activity between deprived and non-deprived eye bands are affected by the duration of deprivation. Animals subject to longer periods of elevated IOP had ratios further away from values of normal animals. Moreover, animals without pressure lowering therapy had lower OD ratios compared to animals with trabeculectomy performed. Our results suggest that lowering elevated IOP to normal levels has an effect on metabolic activity within the primary visual cortex. Table of Contents Abstract i i Table of Contents iv List of Tables vii List of Figures . . . v i i i List of Abbreviations x Acknowledgments xii 1. General Introduction 1 A. Central visual pathway 2 B. Definition of glaucoma 6 i . Definition of IOP 6 i i . Effects of elevated IOP on the visual pathway 9 a. Optic nerve head 9 b. Retinal ganglion cells 16 c. Lateral geniculate nucleus 20 d. Visual cortex 22 i i i . Types of glaucoma 24 iv. Method of diagnosis 24 v. Current therapies 24 vi. Efficacy of IOP lowering interventions 25 C. Primate model of glaucoma . . .28 D. Dynamic metabolic organization revealed by CO 30 i . Cytochrome oxidase histochemistry 30 i i . Layers 2/3 of the visual cortex 32 i i i . Layers 4 of the visual cortex 34 iv. Consequences of monocular deprivation 35 v. Recovery of CO activity 37 E. Effect of trabeculectomy on the adult primary visual cortex following elevated IOP - A Hypothesis 39 2. Materials and Methods 44 A . Animal preparation 44 i . Surgery 44 B. Tissue preparation 48 i . Blocking, flattening, sectioning 48 C. Histochemistry 54 i . Cytochrome oxidase 54 D. Data collection and analysis 55 i . Predictors for glaucoma- measurements 55 i i . Image capturing 56 i i i . Intra-animal analysis 56 iv. Statistical analysis 59 3. Results 63 A . Controls 63 i . Over and under reaction control 63 i i . Sham operated animal 66 i i i . Normal control animals 66 B. Metabolic activity experimental animals - Intra-animal comparisons 71 i . Optic nerve transection 71 i i . Short-term chronic glaucoma vs. short-term transient glaucoma 72 a. Two weeks post elevated IOP 72 • Qualitative analysis 72 • Plot Profile analysis 75 " Quantitative analysis 78 • Statistical analysis 85 b. Four weeks post elevated IOP 85 • Qualitative analysis 85 • Plot Profile analysis 90 • Quantitative analysis 90 • Statistical analysis 92 c. Combined 2 and 4 weeks post elevated IOP..92 i i i . Long-term chronic glaucoma vs. short-term transient glaucoma 95 a. Eight and 16 weeks post elevated IOP 95 • Qualitative analysis 95 • Plot Profile analysis 98 • Quantitative analysis 98 b. Chronic vs. transient glaucoma analysis .... 103 iv. Analysis of predictors of OD ratios 106 a. Two and 4 weeks NO T R A B I l l b. Two and 4 weeks TRAB I l l 4. Discussion 117 A. CO histochemistry detects early glaucomatous change 117 B. Optical density ratios '. 119 i . Metabolic activity differences 119 i i . Dependence on duration of elevated IOP 120 v C. Comparison of chronic (NO TRAB) and transient (TRAB) Glaucoma Animals 122 i . Trabeculectomy arrests the progression of glaucomatous effects in the visual cortex 122 i i . The visual cortex recovers from metabolic activity depression following trabeculectomy 124 D. Limitations to the study 127 E. Future directions 129 i . Alternative criteria for evaluating glaucomatous damage in the primate model 129 i i . Future applications for the evaluation of metabolic activity by OD ratios 132. F. Conclusions from this study 134 5. References 135 6. Appendix 150 A. Central visual pathway 150 i . Visual neuroanatomy 150 a. The Retina 150 b. The L G N 153 c. The visual cortex 154 i i . The Retinotopic map ' 154 i i i . Organization of the primary visual cortex 157 B. Types of glaucoma '. 163 i . Open-angle glaucoma 163 i i . Normal-tension glaucoma 166 i i i . Chronic narrow-angle glaucoma 166 iv. Closed-angle glaucoma 167 v. Congenital glaucoma 167 vi. Secondary glaucomas 168 C. Method of diagnosis for glaucoma 168 D. Current therapies for glaucoma 169 i . Medications . . , 169 i i . Lasering 169 i i i . Filtration surgery 170 iv. Drainage implants 171 v. Cyclodestructive procedures 171 vi List o f Tables Table 1: Animal intraocular pressure history. Table 2: Experiment OD ratios. Table 3: Experiment mean OD ratios. Table 4: Predictors of OD ratios. vii L is t of Figures Figure 1: The central visual pathway Figure 2: Circulation of aqueous humour in the anterior chamber of the eye. Figure 3: Structural changes in the trabecular laminae with increasing age and in cases of primary open-angle glaucoma. Figure 4: Optic nerve heads in normal and glaucomatous eyes. Figure 5: Depiction of a classic glaucomatous nerve fiber bundle defect in the nasal visual field. Figure 6: Diagrammatic example of a trabeculectomy. Figure 7: Schematic representation of the experimental protocol Figure 8: Flattened primary visual cortex. Figure 9: Measurement of optical density ratio. Figure 10: CO staining of V I . Figure 11: Over and under reaction control. Figure 12: ONT and experimental control animals. Figure 13: ONT and experimental control animals plot profiles Figure 14: Short-term (2 weeks) chronic and transient glaucoma. Figure 15: Short-term (2 weeks) chronic and transient glaucoma plot profiles. Figure 16: Comparison of OD ratios in relation to the experimental group type after 2 and 4 weeks of elevated IOP. Figure 17: Short-term (4 weeks) chronic and transient glaucoma. Figure 18: Short-term (4 weeks) chronic and transient glaucoma plot profiles. Figure 19: Comparison of OD ratios in relation to the experimental group type in short-term glaucoma. Figure 20: Long-term chronic glaucoma. Figure 21: Long-term chronic glaucoma plot profiles. Figure 22: OD Ratios in relation to duration of deprivation. Figure 23: Comparison of OD ratios of long-term chronic glaucoma (NO TRAB) animals to transient glaucoma (TRAB) animals. Figure 24: Comparison of mean OD Ratios in relation to duration of deprivation. Figure 25: Correlation of OD ratio predictors with OD ratios in NO T R A B animals. Figure 26: Correlation of OD ratio predictors with OD ratios in T R A B animals. Figure 27: Pathway of light through the retina. Figure 28: Termination of L G N afferents in the visual cortex. Figure 29: Visual field representation on the visual cortex. Figure 30: Modular organization of the primary visual cortex. Figure 31: Blockage of aqueous outflow in primary open-angle glaucoma. Figure 32: Example of procedure for calculating IOP Ratio. List of Abbreviations ALT -argon laser trabeculoplasty ATP -adenosine triphosphate CO -cytochrome oxidase COR -cytochrome oxidase reactivity cup:disc -ratio describing the appearance of the optic nerve. Ratios exceeding 0.5 or asymmetric ratios are considered suspicious for glaucoma. cupping ratio -(CCUp:disc/GCUp;disc), comparison of the optic disc cupping ratio of the control eye (C) to the treated eye (G) DG -2-deoxyglucose fMRI -functional magnetic resonance imaging IOP -intraocular pressure IOP ratio - (QOP/GIOP) , comparison of the "IOP area under the curve" measurements of the control eye (C) to the treated eye (G) K -koniocellular pathway LGN -lateral geniculate nucleus M -magnocellular pathway Na -sodium NO TRAB -experimental animals subject to a period of elevated IOP (>30-35 mm Hg) and sacrificed OD -optical density ODC -ocular dominance columns ONT . -optic nerve transection P -parvocellular pathway PB -phosphate buffer -phosphate buffered saline -primary open-angle glaucoma -positron emission tomography -experimental animals subject to a period of elevated IOP (>30-35 mm Hg), given a trabeculectomy to restore IOP to normal pre-laser values (<20 mm Hg) and allowed to survived at normal IOP levels for at least months before sacrifice. -tetrodotoxin Acknowledgments I am grateful for the continuous support, guidance, and encouragement that I received from my research supervisor Dr. Joanne Matsubara during my studies and the preparation of my thesis. I would also like to thank Eleanor To and Al l i son Ning for their assistance in the lab. Finally, acknowledgements are due to the members of our collaborating lab, Dr. Paul Kaufman and his staff at the University of Wisconsin, Department of Ophthalmology. x i i 1. General Introduction Glaucoma is considered to be the third largest cause of blindness worldwide after cataract and trachoma. Estimates prepared by the World Health Organization in 1998 put the total number of suspect cases of glaucoma worldwide at around 105 million, of which more than 80% of the blind and suspect cases live in the developing world. Glaucoma is often referred to as the "sneak thief of sight" because no symptoms are evident until vision is already lost. This group of diseases has certain common features including, in particular, cupping and atrophy of the optic nerve head, characteristic visual field loss, and, often increased intraocular pressure (IOP). Earlier studies have focused on understanding this disease at the level of the retina and optic nerve head (Fechtner and Weinreb, 1994). More recently, evidence has emerged extending glaucomatous injury to the central nervous system, potentially offering additional insight into the nature of the disease and vision loss (Gupta and Yucel, 2001). Studies demonstrating transneuronal degeneration at the level of the L G N and visual cortex in response to elevated IOP have introduced an additional level of the visual system that glaucoma therapies must consider. Current pharmaceutical and surgical therapies for glaucoma are largely focused on lowering elevated IOP. Efficacies of these treatments however are only evaluated at the level of the eye through pressure measurements, cup to disc ratios, visual field testing, or anterior angle measurements. In consequence, functional alterations in the central nervous system are largely ignored. Furthermore, the impact of lowering pressures in glaucoma oh visual activity has not yet been demonstrated at the level of the L G N or visual cortex. This information is pertinent 1 for justifying current and upcoming therapies for glaucoma. Treatments focused at the level of the eye may not be sufficient for addressing the changes occurring in the L G N or visual cortex as a consequence of elevated IOP. Unilateral elevated IOP was induced in primates by laser scarification of the trabecular meshwork. Elevated IOP was maintained for 2, 4, 8, or 16 weeks. One group of animals was sacrificed, while a second group of animals was given trabeculectomies to lower the IOPs to normal pre-laser values. After a minimum of six months at normal IOPS, the animals were sacrificed. A l l animals' cortices were histochemically processed for cytochrome oxidase (CO). Metabolic activity differences between deprived and non-deprived eye bands were evaluated by optical density ratios. Results from this study, demonstrate changes in metabolic activity within the primary visual cortex in response to visual deprivation by elevated IOPs. Optical density ratios also reveal quantitatively the potential for visual activity recovery following intervention by lowering pressures. Information obtained in this study contributes to the current understanding about the impacts of elevated IOP on the activity of the visual cortex. A . Central visual pathway Light enters through the pupil of the eye and is focused through the lens onto the retina at the back of the eye (Figure 1). A higher number of retinal ganglion cells are devoted to the central region of the visual field compared to the periphery. The absorption of light is transduced into chemical signals that leave the eye via the optic nerve. The axons of the retinal ganglion cell travel through the nerve fiber layer to the optic disc head where it becomes bundles of axons that exit the eye (Kuffler, 1953; Wald, 2 1968). Some axons within the optic nerve cross at the optic chiasm and project to layers 1, 4, and 6 of the contralateral lateral geniculate nucleus (LGN) within the thalamus. Remaining axons remain uncrossed and project to layers 2, 3 and 5 of the L G N on the ipsilateral side. The L G N is therefore a bilateral thalamic structure, which receives input from the ipsilateral eye's temporal retina and the contralateral eye's nasal retina. The topographical arrangement of ganglion receptive field is conserved in each of the six layers of the L G N . The retinotopic map is conserved at the level of the L G N where more cells within the nucleus are devoted to the central area of the retina compared to the periphery. Projection cells from the L G N send their axons to layer IV of the visual cortex. Area VI (primary visual cortex) receives the strongest input from the L G N . In addition, each hemisphere receives information from the contralateral visual field. The retinotopic map is conserved again within the visual cortex. Regions of the cortex devoted to the central region of the visual field are magnified (Berne and Levy, 1983). The primary visual cortex is organized by layers and by columns. The layers are segregated according to its neuronal input and output activity. The columns are modules of organization responsible for colour, orientation selectivity and ocular dominance properties. Segregation of input of one eye (ie. L G N layers 1, 4, 6) from the other (ie. L G N layers 2, 3, 5) is the basis for ocular dominance columns observed under models for monocular deprivation (Hubel and Wiesel, 1965; Wiesel et al., 1974; Hubel and Wiesel, 1977; Livingstone and Hubel, 1984). Please refer to the appendix for a more thorough description of visual neuroanatomy. 3 Figure 1: T h e c e n t r a l v i s u a l pathway. V i s u a l i n f o r m a t i o n p r o c e s s e d i n the t e m p o r a l r e t i n a travels d o w n the o p t i c tract a n d c o n t i n u e s to the i p s i l a t e r a l lateral g e n i c u l a t e n u c l e u s ( L G N ) . R e t i n o g e n i c u l a t e afferents synapse i n layers 2 , 3 , a n d 5 o f the L G N . V i s u a l i n f o r m a t i o n p r o c e s s e d i n the n a s a l r e t i n a travels d o w n the o p t i c tract, crosses at the o p t i c c h i a s m a n d c o n t i n u e s to the contralateral L G N w h e r e it synapses w i t h layers 1,4, a n d 6 o f the L G N . G e n i c u l o c o r t i c a l afferents f r o m layers 1 a n d 2 o f the L G N b e l o n g to the m a g n o c e l l u l a r ( M ) p a t h w a y , whereas afferents f r o m layers 3 , 4 , 5 , a n d 6 b e l o n g to the p a r v o c e l l u l a r p a t h w a y (P). A f f e r e n t input f r o m e a c h eye r e m a i n s segregated as it travels to the v i s u a l c o r t e x f o r m i n g o c u l a r d o m i n a n c e c o l u m n s . 4 Left Eye Right Eye Left Eye Right Eye Column Column Column Column Left Visual Cortex Right Visual Cortex (Layer 4C) (Layer 4C) Figure 1 B. Definition of glaucoma Glaucoma is a group of diseases defined by damage to the optic disc. It is often characterized by elevated IOP, which leads to retinal ganglion cell degeneration and eventual vision loss. Patients afflicted with this disease, both in the presence or absence of elevated IOP will demonstrate optic disc damage. Therefore, both the damage to the retinal ganglion cell layer and the damage to the optic disc are hallmarks of this disease. i . Definition of IOP To understand the different types of glaucoma, it is pertinent to understand IOP. IOP is "the rate at which aqueous humor enters the eye (inflow) and the rate at which it leaves the eye (outflow)" (Shields, 1992). Inflow is dependent upon the rate of aqueous humor production and outflow depends on the flow of aqueous humor from the eye via the trabecular meshwork. Under normal conditions, the ciliary processes of the ciliary body form aqueous humor. The fluid enters the posterior chamber and passes to the anterior chamber via the pupil. Following exchange with surrounding tissues within the chamber angle, the aqueous humor leaves the anterior chamber by passing through the trabecular meshwork. The trabecular meshwork is a structure consisting of the Schlemm's canal, intrascleral channels and episcleral and conjunctival veins (Figure 2). It is important to understand this structure because many ocular problems encountered in glaucoma involve the trabecular meshwork. Most of the aqueous humor leaves the eye through the trabecular meshwork and Schlemm's canal. This outflow is pressure dependent and its resistance can be modulated by ciliary muscle contraction (Drecoll and Rohen, 1994). In old eyes, the number of trabecular cells decreases with age, and the 6 Figure 2: Circulation of aqueous humour in the anterior chamber of the eye. Aqueous humour formed by the ciliary processes enters the posterior chamber and passes to the anterior chamber via the pupil. Following exchange with surrounding tissue within the anterior chamber angle, the aqueous humour leaves the anterior chamber via the' trabecular meshwork. (Figure adapted from Lutjen-Drecoll and Rohen, 1994) underlying basement membrane thickens (Rohen and Lutjen-Drecoll, 1982). In addition, the sheaths surrounding the elastic-like fibers of the cribiform network thicken causing the spaces within the network to be smaller (Figure 3). Since the aqueous path area is reduced, outflow resistance increases (Lutjen-Drecoll and Rohen, 1994). Normal IOP in a general population with no known eye disease is recorded at 15.5 +/- 2.57 mm Hg (Armaly, 1968; Johnson, 1966; Perkins, 1965; Segal and Skwiercynska, 1967). In the aging population, IOP increases with age, but not significantly because the increase in outflow resistance is partly compensated for by a decrease in aqueous humor production. However, i f additional materials lead to further obstruction of the aqueous pathway through the trabecular meshwork, the equilibrium is disturbed and IOP may rise substantially, resulting in glaucoma (Drecoll and Rohen, 1994). Increases in IOP can be influenced by factors including genetics, age, sex, refractive error, large optic disc cups and race (Armaly, 1967; Armaly, 1968; Klein and Klein, 1981; Tomlinson and Philips 1970).1 i i . Effects of elevated IOP on the visual pathway a. Optic nerve head The primary consequence of elevated IOP within the eye is progressive atrophy of the optic nerve head. This pathologic alteration leads to irreversible loss of vision. Changes in optic disc patterns and the retinal nerve fiber layer characterize glaucomatous optic disc atrophy. In glaucoma, the optic nerve head is defined as the distal portion-of the optic nerve that is directly susceptible to elevated IOP. It extends from the retinal surface to the myelinated portion of the optic nerve that begins just behind the sclera. 9 Figure 3: Structural changes in the trabecular lamellae with increasing age and in cases of primary open-angle glaucoma. Trabecular lamella is displayed in A) young, normal eyes, B) older eyes, and C) glaucomatous eyes. In older eyes (B), long-spacing fibrils increase. A thickening of the basement membranes is also observed. In glaucomatous eyes (C), thickening of basement membranes is observed with clusters of long-spacing collagen. An increase in elastic-like fiber sheaths is also present. Together, these changes increase aqueous outflow resistance. (Figure adapted from Lutjen-Drecoll and Rohen, 1994). 10 trabecular cell central core basement of lamella membrane elastic- l ike fibre with sheath central core elastic- l ike fibre with sheath clusters of long-spacing col lagen trabecular cell thickened basement membrane elastic-l ike fibre with sheath clusters of long-spacing col lagen Figure 3 The terms "optic nerve head", "optic disc" or "optic papilla" are used interchangeably, when referring to this portion of the optic nerve head that is visible by ophthalmoscopy. The optic nerve head is composed of the nerve fibers that originate in the ganglion cell layer of the retina. The fibers converge upon the nerve head from all areas of the retina and the axons leave the eye globe through a fenestrated scleral canal known as the lamina cribrosa. The lamina cribrosa consists of sheets of connective tissues and elastic fibers that provide collagen support for the disc. Within the nerve head, the axons are grouped into fascicles. Axons from the macula and from the nasal retina terminate in the central portion of the optic nerve. Fibers from the temporal periphery of the retina terminate in the superotemporal and inferotemporal aspects of the optic nerve. It is these fibers that are the most susceptible to glaucomatous damage (Minckler, 1980; Radius and Anderson, 1979). The central portion of the optic disc contains a depression called the cup and an area of pallor, which is an area completely devoid of axons by the border of the lamina cribrosa. Tissue between the cup and the disc margin is referred to as the neural rim. The bulk of the axons are located in this area, which is orange-red in color due to the associated capillaries. The optic nerve head is the site of entry and exit of the retinal vessels. These ride up the nasal wall of the cup, kink at the cup margin and cross the neural rim to the retina (Shields, 1998). Under normal conditions, the optic nerve head is generally a vertical oval in appearance (Figure 4). However, there is considerable variation in size and shape in the normal population without glaucoma (Jonas et al., 1988a; Jonas et al., 1988b). In the early stages of glaucoma, deepening of the optic cup is sometimes observed. This occurs 12 Figure 4: Optic nerve heads in normal and glaucomatous eyes. Normal optic nerve heads (A) are symmetrical between the two eyes. Neural rims are also even for 360°. In contrast, glaucomatous optic nerve heads (B) first experience inferior enlargement of the cup away from the original cup margin. A polar notch is created due to glaucomatous optic atrophy. (Figure adapted from Shields, 1998). 13 v e s s e l s k i n k Figure 4 when the lamina is not yet exposed to glaucomatous damage (Portney, 1976). Mechanical pressure on the lamina cribrosa and possible optic nerve head ischemia cause the neural rim of the optic disc to thin as bundles of axons atrophy in the inferotemporal and superotemporal areas of the optic disc (Jonas, 1993; Jonas et al., 1988; Morgan, 2000; Radius et al., 1978; Shin et al., 1992). This leads to enlargement of the cup in a vertical or oblique direction. As glaucomatous damage continues, atrophy in the temporal neural rim usually follows with the nasal quadrant being the last to go (Jonas et al., 1993). Eventually, all neural rim tissue is lost and the ultimate result is total cupping (Spaeth et al., 1976; Hitchings and Spaeth, 1976). In addition to the optic nerve head, atrophy of ganglion cell axons can be observed in the retinal nerve fiber layer (RNFL). Defects observed in this layer may be the earliest signs of glaucoma, preceding changes to the optic nerve head and the visual field (Airaksinen and Alanko, 1983; Sommer et al., 1991c). The first detectable RNFL abnormality is a diffuse loss of axons (Tuulonen and Airaksinen, 1991). The retina appears mottled, but it is usually hard to detect. As the disease progresses, localized defects are well outlined by surrounding healthy nerve fiber bundles. The loss of axons appears wedge shaped with a defect area in the retinal nerve fiber layer and a notch at the optic disc margin (Figure 5). Ganglion cell atrophy cannot be clinically visualized, but an isolated scotoma (visual field deficit) corresponding to localized ganglion cell dropout can be detected. As mentioned before, the first glaucomatous damage affects the upper and lower temporal areas of the optic disc. The affected fibers originate from ganglion cells close to the temporal raphe and correspond to the nasal visual field. 15 As the disease progresses, the retinal nerve fiber layer defect becomes wider and extends towards the optic disc as the axons become damaged. The visual field defect extends towards the blind spot, when proximally originating axons have degenerated and the RNFL defect connects with the optic disc margin (Tuulonen and Airaksinen, 1994). b. Retinal ganglion cells There are three classes of retinal ganglion cells: M (parasol)-cells, P (midget-cells, and K (koniocellular)-cells. These cells function in parallel, and simultaneously convey different aspects of visual sensation. The M-cells comprise about 10% of the ganglion cell population and are the largest ganglion cells. They have large receptive fields, large-diameter axons with higher conduction velocity and are not sensitive to color. M-cells respond best to high temporal and low spatial frequency. They project to the ventral, magnocellular layers (layer 1 and 2) of the lateral geniculate body (LGN). . The P-cells, which comprise about 80% of the population, are smaller cells that project to the dorsal parvocellular layers (layer 3, 4, 5, 6) of the L G N . They have greater sensitivity to color, have smaller receptive fields, smaller-diameter axons, and slow conduction velocity. They respond best to high spatial and low temporal frequency. The remaining 10% of ganglion cells (koniocellular) terminate in the interlaminar layers of the L G N . This pathway's characteristics are the least well known, but is hypothesized to be associated with colour vision (Leventhal et al., 1981; Rodieck et al., 1985; Dacey and Petersen, 1992; Hendry and Yoshioka, 1994). A major mechanism contributing to the death of retinal ganglion cells is the strangulation of their axons at the optic disc. Shear forces in the tissues of the lamina 16 Figure 5: Depiction of a classic glaucomatous nerve fiber bundle defect in the nasal visual field. (A) The loss of axons appears wedge shaped with a defect area in the retinal nerve fiber layer (RNFL) and a notch at the optic disc margin. Corresponding defect in the visual field is displayed below. G-ganglion cell drop-out, D-RNFL defect, N-optic disc notch, VFD-visual field defect. In (B), the development of an optic disc notch and a wedge-shaped retinal nerve fiber layer defect is depicted three dimensionally. (Figure adapted from Higginbotham, 1994). 17 RNFL defect cribrosa obstruct the to-and-fro transport between axon terminal and ganglion cell soma. This results in a reduction or cessation in the normal flow of intracellular materials between the retinal ganglion cells and their target neurons. Damaged axons undergo retrograde degeneneration, leading to eventual cell death under elevated IOP conditions (Anderson and Hendrickson, 1974; Gaasterland et al., 1978; Quigley and Addicks, 1980; Mickler and Spaeth, 1981). The ganglion cell axons that are first affected under these circumstances are those that enter the dorsal and ventral aspect of the optic disc. These axons relate to cells from the temporal retina that receives input from the peripheral nasal visual hemifield. The loss of ganglion cells progresses towards central vision as the disease advances (Kitazawa et al., 1977; Harwerth et al., 1992). Retinal ganglion cell degeneration is first associated with structural abnormalities in the dendritic arbor. These changes include a thinning of the proximal and distal dendrites, abrupt reductions in dendritic process at branch points, and a general decrease in complexity of the dendritic tree. Anatomic changes at the level of the cell soma occur at relatively the same time or slightly after. Axon diameter changes follow those at the level of the dendritic tree and cell soma. A possible explanation for these observed changes is that in response to injury, retinal ganglion cells pare their distal dendrites in an effort to conserve energy and homeostasis at the level of the cell soma. As the disease progresses, damaged axons undergo retrograde degeneration, further depriving the neuron from trophic materials residing within the axons. As the trophic stores deplete, the dendritic field degenerates further until finally the cell itself begins to shrink. At some yet undefined point in the process of degeneration, the injured neuron will undergo apoptosis when an intracellular signal is activated (Quigley et al., 1995; Nickells, 1996). 19 Studies on changes in axon diameter (Quigley et al., 1987; 1988), soma size (Glovinsky et al., 1991; 1993), and neurofilament content (Vickers et al., 1995) have reported that cells of all sizes are lost, but large ganglion cells appear to be affected most severely under advanced stages of glaucomatous damage. This is speculated to be due to the fact that the axons in the inferior and superior poles have a greater portion of larger . than normal nerve fibers (Quigley et al., 1987, 1988). In addition, the flow of fast axonal transport ganglion cells appears to be preferentially affected over the slow transport ganglion cells (Dandona et al., 1991). During mild and moderate stages of the disease, both classes of cells have similar reductions in dendritic field size, but a reduction in soma size was not prevalent (Glovinsky et al, 1991, Weber et al. 1998). In contrast,. chronic elevation of IOP results in a significant reduction in dendritic field size and axon diameter in parasol (M) cells. Similarly, parasol cells have smaller dendritic field and axon diameters compared to midget cells when cup:disc ratios equals or exceeds 0.6 (Quigley, 1993; Weber et al. 1998). c. Lateral geniculate nucleus Following the loss of afferent fibers in the central nervous system, target neurons are known to become atrophic and then die by a process of transneuronal degeneration (Kupfer, 1965; Matthews et al., 1960; Cowan, 1970). Transynaptic changes due to the degeneration of retinal ganglion cells can be seen at the level of the thalamus. Studies have found that both the M-pathway (movement and form) and the P-pathway (detail and color vision) are impacted by glaucoma and that elevated IOP has a profound effect on the neuronal size, density and its number of neurons, as well as the laminar volume 20 within the L G N . These cellular changes are present in all L G N layers, but the deprived eye layers are more severely affected. Weber et al. (2000) found that long durations of elevated IOP at low to moderate levels result in larger reductions in mean cell size of Inl-and P- cells within the L G N . Further, high mean levels of IOP also shorten the time required for the cell soma to reduce in size. The mechanism for a reduction in mean soma size may be due to shrinkage or cell loss. It was found that both cell loss and cell shrinkage contributes to the decrease in mean soma size within both M - and P-layers in glaucoma. Therefore, cell size rather than cell class may be the main factor for glaucomatous change in the L G N . This finding is consistent with the results reported in primate retina (Glovinsky et al. 1993; Glovinsky et al. 1991). However, it was also found that cell shrinkage plays a greater role in the P-layers whereas cell loss is the primary mechanism in the M-layers that determine the final mean soma size after glaucomatous damage. This was confirmed through estimates on the number of neurons. M-cell loss was four times greater than P-cell in glaucomatous animals (Weber et al. 2000). Conflicting evidence exists regarding changes in cell density due to glaucomatous damage. In an earlier study, Chaturvedi et al. (1993) found a significant decrease in cell density in the M - but not P-cells of human eyes affected by glaucoma. Weber et al. (2000) in contrast found that there was an overall increase in cell density in all layers, and the increase was 2 times greater in the P-layer compared to the M-layer in primate eyes. This difference is attributed to animal-human variation. Further, it is proposed that laminar shrinkage causes the appearance of increased cellular density in the P-layers, where cell loss is not occurring. The M-layers appear to have less of an increase in cellular density because the volumetric shrinkage is balanced by cell loss. As suspected, 21 the decrease in laminar volume was threefold greater in the P-layers compared to the M -layers. This observation is in concordance with the observations of Ahmad and Spear (1993). This initial decrease in P volume most likely reflects an early loss of axonal material (Glees et al., 1941). Finally, the degree of cell loss is similar between M and P layers. This was demonstrated histologically through labeling of L G N relay neurons (Yucel et al., 2000) and through decreased staining density of CO histochemistry and synaptophysin (SYN) inmmunohistochemistry (Crawford et al., 2000; Vickers et al., 1997) in primates. However, the onset of cell loss in P-layers is delayed approximately 1.5 weeks relative to the M-layers. This differential pattern in cell loss is suspected to be due to a difference in sensitivities of subpopulations of the neurons to optic nerve injury (Goldby, 1957; Saini and Garey, 1981; Weber et al., 2000). d. Visual cortex The effects of elevated IOP on the visual cortex have been demonstrated through cytochrome oxidase reactivity (COR). Experimental glaucoma reduces retinal ganglion cell activity. The reduction in the metabolic drive to downstream targets can be revealed through COR. It was found that afferent input to the primary visual cortex is dramatically altered due to metabolic activity changes in the L G N . Experimental glaucoma for approximately a one-year period reduces COR activity in the deprived eye bands in layer 4 of the visual cortex. Further, elevated IOP appears to affect the P-cell input to V I layer 4Cp more than in the M-cell input layer 4Ca (Crawford et al., 2000). These findings are in conflict with those of Vickers et al. (1997). In their study, they 22 found that experimental glaucoma in monkey affects the metabolism in both divisions of the geniculocortical afferent pathway, but detrimental early effects were stronger in the M-cell pathway. In layers 2 and 3, the target of differential input from the two parallel pathways into layer 4C, COR was uniformly reduced in blobs (areas that normally have intense CO staining) with input from the glaucomatous eye. The glaucomatous blobs were on average reduced by half. The degree of loss was also consistent to the degree of loss of afferent inputs (Crawford et al., 2001). Preliminary research into the potential for functional recovery of the visual cortex has been reported recently. Lam et al. (2003) investigated the expression patterns of neurochemicals associated with cortical synaptic plasticity in primates following elevated IOP. Activity-dependent changes, or plasticity, in the strength of neuronal signaling play a critical role in modifying information transmitted and encoded in existing neuronal connections. Expression levels of G A B A a receptor protein, GAP43, and S Y N immunoreactivity were lower in glaucomatous eye bands compared to normal eye bands. In contrast, CAMKIIot immunoreactivity was higher in glaucomatous eye bands compared to the normal eye bands. Lam et al. also found that GAP-43 and S Y N had an overall upregulation and G A B A a receptor protein had an overall downregulation in the visual cortex of glaucomatous animals compared to normal animals. These findings suggest that the redistribution of synaptic plasticity neurochemicals may support functional recovery of cortical neurons after damage to retinal ganglion cells induced by elevated IOP. 23 i i i . Types of glaucoma Glaucoma can be classified into primary and secondary forms. In primary glaucoma, cause of outflow obstruction and subsequent IOP elevation is not related to other ocular or systemic disorders. There are two major types of primary glaucoma: chronic, Or open-angle glaucoma, and acute closed angle glaucoma. Other variations that are less common include normal-tension and congenital glaucoma. These maladies are all confined to the anterior chamber. Secondary glaucoma is usually acquired due to ocular or systemic disorders that are unrelated to glaucoma. These also include congenital or open-angle or closed angle glaucoma as well. Detailed descriptions on the four types of glaucoma are available in the appendix. iv. Method of diagnosis The best way to prevent vision loss from glaucoma is early diagnosis and treatment. High risk factors for glaucoma include: high intraocular pressures, family history, ethnic background, age, and optic nerve appearance. There are four main tests for glaucoma: tonometry, ophthalmoscopy, perimetry and gonioscopy. Descriptions of each test can be found in the appendix. v. Current therapies The general therapeutic approach to glaucoma is to significantly reduce intraocular pressure. This is done either by increasing drainage from the eye, by decreasing the amount of fluid produced in the eye, or a combination of the two. An initial reduction of 30% is a common target. The target is then adjusted depending on the 24 response to therapy and the anticipated iatrogenic risk of the next intervention (Anderson, 1989). Medications are the first line of therapy, followed by laser trabeculoplasty, then surgery (trabeculectomy presently being the procedure of choice), then implantation of a drainage device, and finally ciliodestructive procedures (Garratt, 1989, Glaucoma Laser Trial Research Group, 1990; Migdal and Hitchings, 1986; Molteno, 1986; Perkins, 1994). Detailed descriptions of these therapies are available in the appendix. vi . Efficacy of I O P lowering interventions Elevated IOP is present in the majority of glaucomatous cases (Sommer, 1989; Sommer et al., 1991b). It has been demonstrated in animal models that elevated IOP causes a glaucomatous pattern of damage (Quigley and Addicks, 1980). Based on these observations and historical clinical evidence, lowering IOP by medical means is assumed to be helpful in the treatment of glaucoma. Many studies contribute indirect evidence to lend support for the beneficial effects of lowering IOP. However, this assumption has been challenged due to a lack of controlled clinical trials demonstrating that treating glaucoma alters the course of the disease. Nevertheless, evidence from a collation of clinical observations, studies of conditions related to glaucoma and small retrospective studies of glaucoma therapy do support a relationship between the lowering of IOP and the disease progression. It has been shown that decreasing IOP may lead to reversal of optic nerve cupping (Spaeth, 1983; Spaeth, 1985). Evidence also suggests that lowering of IOP in ocular hypertensives is associated with less progression of optic nerve and visual field changes (Shin et al, 1976; Epstein et al, 1989; Kass et al, 1989). 25 The best evidence for the efficacy of lowering IOP to arrest the progression of optic nerve and visual field damage come from reviewing studies that have examined the effectiveness of the three modes of glaucoma therapy: medical, combined medical and surgical, and surgical. The results for long-term medical treatment have been poor. A number of studies have shown that over half of medically treated patients continue to suffer progressive visual field loss when followed long term. However, the loss of visual field progression in treated patients was much less than un-treated patients (Garratt, 1989). In addition, other studies demonstrated a strong correlation between low IOP and retention of fixation in visual fields of advanced glaucoma patients (Leydhecker, 1983). Stronger evidence exists for reducing IOP to provide significant protection against disease progression in studies combining medical and surgical management of glaucoma. Odberg (1987) demonstrated a dose-response relationship for IOP in causing visual field deterioration in a group of advanced glaucoma patients. Only patients with IOP consistently <15 mm Hg had a better than 50% chance of remaining stable. Together with other studies, these findings noted that pressure in advanced stages of glaucoma dictate the outcome. Pressures in the high teens often led to blindness whereas vision was usually retained when pressures remained in the lower teens (Kolker, 1977; Quigley and Maumenee, 1979; Grant and Burke, 1982). Even more convincing evidence for the effectiveness of lowering IOP comes from studies on the efficacy of surgery, mainly trabeculectomies in stopping visual field damage (Rollins and Drance, 1981). When combined with steroid use following trabeculectomy, progression of visual field loss is dramatically reduced in patients with 26 IOPs <15 mm Hg and a 6% incidence of visual field progression over a 5-year period. In contrast, patients without steroids had an average pressure of 19.3 mm Hg and a 58% disease progression (Roth et al., 1991). Other studies on trabeculectomy suggest the same dose-response effect of IOP (Kidd and O'Connor, 1985). Further, several studies recognize trabeculectomy as superior to medical treatment due to its consistency in lowering IOP with less variation. Werner et al. (1977) noted that 70% of patient with peak IOP of >21mm Hg progressed, while only 21% of patients with peak IOP of <22 mm Hg showed further visual field loss. Similarly, Greve and Dake (1979) found that 56% of patients progressed with mean IOPs > 21mm Hg, but only 29% progressed with mean IOPs of <21mm Hg. Together, these studies demonstrate some evidence that lowering IOP is beneficial and i f brought low enough, most patients may be protected from deterioration. Since pressure is considered as a risk factor for damage in glaucoma, the aim of therapy is to decrease this risk (Anderson, 1989). Some clinicians have concluded that primary surgery offers better protection to the optic nerve than medical therapy (Rollins and Drance, 1981). In addition to its ability to lower IOP quickly and effectively, surgery compared to medical therapy, have fewer disadvantages that may adversely affect success. Medications have the disadvantage of requiring patient compliance. Further, diurnal variations in IOP are not dampened as greatly with medications as with surgery (Migdal and Hitchings, 1986; deJong et al., 1989). The importance of timely intervention is also emphasized by several studies that observed a greater rate of visual field deterioration in early glaucoma (Holmin and Storr-Paulsen, 1984; Mao et al., 1991; O'Brien et al., 1991). This is contrary to earlier reports 27 noting that early glaucoma tended to progress more slowly (Grant and Burke, 1982). In addition, it is well accepted that little benefit of pressure control is seen in advanced stages of glaucoma unless pressure is reduced to very low levels (Leydhecker, 1983). Together, these findings hold important therapeutic ramifications and must be considered in future therapeutic models for glaucoma. Trabeculectomy is the most popular form of intervention for lowering elevated IOPs (Smythe and Herschler. 1994). Since it was found that earlier stages of glaucoma may experience greater deterioration, and that advanced stages of glaucoma can only benefit with therapies where the IOP is significantly lowered, the importance of a timely and reliable intervention is stressed. Trabeculectomy seems to be a suitable intervention for both cases. C . Primate model of glaucoma The monkey eye and visual processing system is very similar to humans and therefore is probably the best animal model for glaucoma. In comparison to other animal models such as in rabbits, dogs and chickens, the primate model offers the advantage of having similar iridocorneal angle anatomy to humans (Lee et al., 1985). The primate model of glaucoma, first proposed by Gaasterland and Kupfer (1974), demonstrates a method to provide sustained elevated IOP in the non-human primate. To mimic conditions of glaucoma, an argon laser is applied to the trabecular meshwork. This structure is blocked after lasering and causes a reduction in aqueous outflow. Subsequently, IOP elevates, leading to an experimental approximation of primary open-angle glaucoma. 28 This technique allows the investigator to produce sustained levels of elevated IOP with minimal ocular inflammation. The elevation in IOP is sufficient to cause selective damage to retinal ganglion cells. In addition, it mimics the damage found in open-angle glaucoma. This procedure causes cupping of the optic nerve head, changes in the nerve fiber layer (Quigley and Pease, 1996; Yucel et al., 1998), blockage of fast axonal transport (Dandona et al., 1991), and characteristic optic nerve changes (Varma et al., 1992; Yucel et al., 1999) in the primate. The primate model of glaucoma allows investigators to examine the damage inflicted by elevated IOP at the level of the retina, the L G N , and the visual cortex. Anatomical damage observed in glaucoma patients and glaucomatous (with elevated IOP) monkeys was quite similar (Schumer and Podos, 1994; Quigley et al., 1995; Dreyer et al., 1996; Nickells, 1996). Under chronic experimental glaucomatous conditions, physical optic disc changes and pattern of ganglion cell loss in a monkey model mimicked those found in humans (Quigley and Addicks, 1980; Quigley et al., 1984, 1987). With these similarities, the model has facilitated further understanding of the pathophysiology of glaucoma (Varma et al. 1992; Quigley et al 1995; Dreyer et al 1996). Finally, this model offers the advantage of examining the effects of elevated IOP over a complete time line, from early to end stage glaucoma. This aspect is very important since human glaucomatous tissue is only available post-mortem; therefore research on the impact of glaucoma on the geniculocortical pathway within the central nervous system is often inhibited otherwise. 29 D. Dynamic metabolic organization revealed by C O As mentioned before, alternating cortical columns receiving afferent input from the left or the right eye exist in the primary visual cortex. These columns code ocular dominance properties important for binocular interaction and depth perception. An ocular dominance column is an organized unit composed of similar intracortical connections (Hubel and Wiesel, 1972). The separate columns run parallel to each other and they meet perpendicular to the extrastriate area (area V2) (Hubel and Wiesel, 1972). The clear segregation in structure allows comparison of activity differences between the two eyes. i. Cytochrome oxidase histochemistry Cytochrome oxidase is a marker of neural functional activity (Wong-Riley, 1989). It is an ideal candidate marker for functional activity in the cortex because the brain depends on aerobic metabolism for its energy supply in which CO plays a critical role (Wong-Riley et al., 1978; Wong-Riley 1979). It can withstand a wide range of pH, temperatures and limited aldehyde fixation (Seligman et al., 1968; Wong-Riley, 1979b, Wikstrom et al., 1981). It is also more stable than other soluble proteins in the cytoplasm since it is an integral membrane protein of the inner mitochondrial membrane.- The continual reoxidation of cytochrome-c by CO creates an accumulation of a visible reaction product (Wong-Riley, 1979a). Therefore, the intensity of the products demonstrates the cytochrome oxidase activity in the tissue (Hevner and Wong-Riley, 1990) and can be quantified by optical densitometric measurements (Wong-Riley and Kageyama, 1986). 30 This mitochondrial membrane protein is essential for brain oxidative metabolism. It catalyzes the last step in the formation of adenosine triphosphate (ATP), the energy source for neuronal function (Wikstrom et al., 1981). CO is particularly important in the brain, where neuronal energy is almost exclusively derived from the oxidative pathway (Erecinska and Silver, 1989). CO activity is dependent on the metabolic demand of activated neurons (Wong-Riley and Carrol, 1984; Wong-Riley et a l , 1989a, 1989b; Di Rocco et al., 1989; DeYoe et al., 1995; Hendrickson and Tigges, 1985). This is displayed by the inhomogeneous distribution of CO content in brain tissue and neuronal cellular compartments (Carrol and Wong-Riley, 1984; Wong-Riley et al., 1988; 1989a; 1993). ATP is used for a variety of neuronal functions, but the majority of energy consumed is for membrane repolarization. The pumping of Na+ and other cations out of the cell through the energy-dependent ATPase system occurs largely along dendritic membranes, the major receptive sites for depolarizing input (Lowry et al., 1954; Bachelard, 1975; Lowry, 1975). The level of CO is consistently high in dendrites (Wong-Riley, 1989a, Wong Riley et al., 1989b), low in cell bodies of neurons receiving mainly inhibitory (hyperpolarizing) synapses (Mjaatvedt and Wong-Riley, 1988), low in axon trunks and bundles in the white matter that propagate action potentials, and variable in axon terminals with varying energy usage (Wong-Riley, 1989; Wong Riley et a l , 1989b). CO is useful for demonstrating change in metabolic activity following functional alterations. Histochemical changes of CO in the L G N and the cortex have been detected within 8 to 14 hours following functional alterations (Mawe and Gershon, 1986; Liu et al., 1990; Trusk et al., 1992). There is also evidence that CO activity and functional activity are correlated under normal conditions and adaptive adjustments of cellular 31 levels of CO are made after altered neuronal activity (Wong-Riley, 1989). Energy metabolism and neuronal activity are coupled tightly (Lowry, 1975), but neuronal activity controls energy metabolism as well as energy-generating enzymes such as CO (Wong-Riley, 1989). CO can therefore, not only reveal a cell's capacity for aerobic metabolism, but also the relative levels of energy-dependent activity averaged over a period of time. Since CO is regulated at local levels, sites of high CO activity are perceived as sites of intense energy demand, and most likely constant membrane depolarization. Active depolarization of certain classes of neuronal cell bodies and presynaptic terminals also display elevated levels of CO (DiRocco et al., 1989). With these benefits, CO has the capacity to label metabolically active dendrites and axonal terminals that elude routine electrophysiological recordings (Carroll and Wong-Riley, 1984; Wong Riley and Carroll, 1984a; Wong-Riley et al., 1989b). CO has also provided investigators with a technique to examine the functional architecture of the visual cortex. Previously, investigators utilized autoradiography, electrophysiology, and/or Nissl staining to demonstrate the cytoarchitecture of the visual cortex. The development of the cytochrome oxidase staining method revealed major organizational features of primate striate cortex, most significantly regions of intense metabolic activity known as the CO blobs (Horton and Hubel, 1981; Humphrey and Hendrickson, 1983; Wong-Riley, 1979a). i i . Layers 2/3 of the visual cortex Distinct patterns of high CO activity have been found in laminae 2, 3, 4 A , 4C, and 6 of primates (Horton and Hubel, 1981; Humphrey and Hendrickson, 1983; Carroll and 32 Wong-Riley, 1985). In layers 2 and 3, CO histochemistry has led to the discovery of "puffs" and "interpuffs", which were previously undetected by electrophysiology, autoradiography, and Nissl staining. CO rich zones (puffs) and non-CO rich zones (interpuffs) are separated with regular spacing (Carroll and Wong-Riley, 1984). The puffs are globular in structure and have a nonhomogeneous reticulated interior. Neurons within puffs do not share uniform staining and therefore are not likely to have identical functional attributes. Two averages have been reported for diameter of puffs in the macaque monkey. Wong-Riley and Carroll (1984) measured approximately 262 by 377 pm, whereas Horton (1984) reported lower values of 150 by 250 pm. The puffs for this species forms rows that are in register with the ocular dominance columns found in Layer 4C. The centers of these puffs are spaced approximately 500pm apart within these rows (Carroll and Wong Riley, 1984). The puffs are present throughout the entire primary visual cortex and extend through the supragranular layers 1-3 (Horton 1984; Hendrickson et al., 1981; Horton and Hubel, 1981; Horton, 1984; Trusk et al., 1990; Wong Riley and Carroll, 1984). Puffs and interpuffs are similar in neuronal and neuropil composition, but CO has revealed quantitative differences between the two. First, interpuffs have a smaller mean size of neurons compared to puffs (Carroll and Wong-Riley, 1984; Trusk et al., 1990). Second, the levels of CO activity in both neurons and neuropil are much lower in interpuffs than puffs. Finally, puffs are likely to be dominated by excitatory inputs, and interpuffs by inhibitory interactions. This would be consistent with the higher CO activity observed in puffs (Sillito, 1975; Sillito et al., 1980). 33 iii. Layer 4 of the visual cortex Like layers 2/3, layer 4's heterogeneity is revealed through CO histochemistry. Layer 4 of primates is subdivided into several sublaminae, each with distinct connections and functional properties. Layers 4A and 4C have significantly darker staining than 4B (Caroll and Wong-Riley, 1984). Tangential sections of layer 4A reveal a 'honeycomb-like' pattern of CO staining (Caroll and Wong-Riley 1984; Hevner and Wong-Riley, 1990; Horton, 1984). In layer 4B, staining is much lighter due to a lack of geniculocortical input. However, periodic blobs exist that are in register with the puffs in layer 2 and 3 (Livingstone and Hubel, 1982). Layer 4C is very rich in CO activity and enzyme amount (Hevner and Wong-Riley, 199.0). This layer is further divided into a 4Ca and 4Cp where CO activity is higher in layer 4Cct than in 4CP (Liu and Wong-Riley, 1990; Wong-Riley and Carroll, 1984). Layer 4Cp is also homogeneous in CO staining. The difference in staining between upper and lower layers of 4Cp may be due to the difference in afferents and efferent interactions with different layers of the cortex (Fitzpatrick et al., 1985; Liu and Wong-Riley, 1990). In addition to laminar segregation, layer 4C is partitioned into vertical ocular dominance columns with connections exclusively to the left eye or the right (Hubel and Wiesel, 1977). There is a wide range in periodicities of ocular dominance columns between adult monkeys; however, periodicities of contralateral and ipsilateral cortices within each individual animal have been found to be virtually identical (Horton and Hocking, 1996). Experimental visual deprivation by monocular enucleation, lid suture, and retinal impulse blockade has been shown to lead to a downregulation of CO in the affected ocular dominance column (ODC) of adult monkeys (Hendrickson and Tigges, 34 1985; Hendry and Jones, 1986; Horton and Hubel, 1981; Horton, 1984; Trusk et al., 1990; Wong-Riley and Carroll, 1984; Wong-Riley et al., 1989a). The clear activity discrepancy between left and right eyes allows direct comparisons between deprived and non-deprived eyes in a monocular deprivation paradigm. This has proven to be useful in the primate model of glaucoma in which unilateral elevated IOP (a form of monocular deprivation) is performed. iv. Consequences of monocular deprivation The pattern of CO histochemistry has been examined under several adult models of monocular deprivation. Differences in CO staining are observed depending on the severity of the deprivation. Whether under a mild (lid suturing) or severe (enucleation) form of visual deprivation, CO bands can be detected in Layer 4 (Horton, 1984; Trusk et al., 1990). Until the introduction of 2-DG autoradiography and CO histochemistry, it was commonly accepted that mature cortical neurons were refractory to change providing that their geniculate afferents were not physically damaged. Functional deprivation presumably leads to decreased neuronal activity. Loss of activity in turn reduces energy and eventually the level of metabolism within the neuron is reduced as a consequence of decreased neuronal activity. The functionally induced metabolic plasticity has been reliably demonstrated by CO histochemistry and immunohistochemistry (Wong-Riley, 1994). Results from studies examining monocular deprivation with intact retinal ganglion cell afferents is of interest in particular because the impact of glaucoma is usually gradual, where retinal activity is lost over time. From these studies, one can 35 interpret that the process of loss of metabolic activity in the visual cortex is gradual when retinal ganglion cell activity is deprived. Further, the restoration of this activity has the potential of reversing the loss of activity observed in the deprived eye bands of the visual cortex. In a monocular lid suture paradigm, retinal ganglion cells retain their spontaneous activities. Horton (1984) found that the L G N still responded to diffuse light through the lid for at least 6 months. CO staining in the L G N confirmed this retention of activity. No changes were observed at the L G N . However, faint CO bands were seen in layer 4 eleven weeks after monocular lid suturing (Horton, 1984; Trusk et al., 1990). The effects also become more prominent with longer deprivation periods of 1-3 years (Trusk et al., 1990). Transneuronal labeling studies demonstrated that there is no sprouting or contraction of geniculocortical afferents in the adult animal (Horton 1984; LeVay et al., 1980). This model of deprivation reveals that mild forms of monocular deprivation have gradual effects on the loss of CO activity in the deprived eye bands of layer 4. Monocular retinal impulse blockage by tetrodotoxin induces a more drastic reduction in cortical activity compared to monocular lid suturing. Tetrodotoxin (TTX) is an advantageous method for monocular deprivation. It is a specific blocker for voltage-dependent sodium channels that will not cause cell death or interfere with axoplasmic transport (Wong-Riley and Riley, 1983). Under this model, light can still enter both the injected and non-injected eyes; therefore normal visual activity is maintained. This is important since there is no permanent damage to the system. TTX injection is reversible and enzymatic activity can return to normal subsequent to removal (Wong-Riley and Riley, 1983; Wong-Riley et a l , 1989). 36 The effects of intravitreal TTX injections can be observed in layer 4C within fourteen hours. Monocular retinal impulse blockade induces a drastic reduction in cortical activity in the affected eye's ocular dominance columns. A dramatic decrease in CO levels is observed through the pale ocular dominance columns of the affected eye (Wong-Riley, 1994). Changes in CO protein content is observed first in layer 4C, followed by layers 2/3 puffs, 4A, 5, and 6. The alternating rows of pale shrunken puffs and bands in 4C become increasingly prominent as deprivation periods increase (Trusk et al., 1992). The reduction in CO activity is most severe in layer 4Cp and 4A, followed by 4Cct, 5 and upper 6. Layer 4B and lower 6 are the least affected. The light and dark ocular dominance columns in layer 4C are equal in width (Trusk et al., 1990), and cell counting reveals that there is no significant cell death in layer 4C after T T X injection (Hendry and Jones, 1988). Unlike enucleation, there is also no evidence of cortical tissue shrinkage (Trusk et a l , 1990). However, evidence of mean cell size shrinkage exists for nonpyramidal, type C cells found in cortical puffs. Mature neurons with the highest baseline level of CO activity were the most severely affected by chronic intravitreal TTX treatment. The numerical and areal densities of mitochondria were dramatically decreased at 2 weeks following TTX injection (Wong-Riley et al., 1989a). v. Recovery of CO activity The detrimental effects of retinal impulse blockage are reversible according to the TTX paradigm (Wong-Riley and Riley, 1983; Carroll and Wong-Riley, 1987b; Wong-Riley et al., 1989a). The pupillary light reflex returns within 1 week, but the CO activity within the cortex requires a longer period of time for full recovery. Normal patterns of 37 CO labeling will return in layer 4C and puffs given an adequate recovery period of at least 4 to 11 weeks (Wong-Riley, 1994). The length of recovery required is related to the period of inactivation. The longer the interval of inactivation, the longer it takes for full metabolic recovery (Wong-Riley, 1989; Wong Riley et a l , 1989a). It is also interesting to note that packing density of mitochondria is reduced within 2 weeks of T T X and returns to control levels following 4 weeks of TTX. This implies that chronically quiescent neurons have the capacity to regain normal levels of mitochondrial volume density even though mitochondria are temporarily converted to a smaller, less-reactive variety (Wong-Riley et al., 1989a). The adverse effects of T T X blockade also reveal the capacity for metabolic plasticity within neurons. For example, certain neurons or parts of neurons maintain a high level of oxidative ability to meet active functional demands. However, when energy demand is drastically reduced, these neurons can rapidly adjust their local enzyme levels (Wong-Riley et al., 1989b; 1992). The process of recovery in CO activity following retinal impulse blockage may be due to the synthesis of new enzyme molecules and their subsequent assembly into functional holoenzymes in the mitochondria. The establishment or strengthening of synapses may also play a role (Wong-Riley et al., 1989b; 1992). Another explanation for recovery may be due to an expansion of influence from the non-deprived eye. Evidence for this was found in humans in which supernormal vernier acuity was observed in non-deprived eyes following long-term monocular deprivation (Freeman and Bradley, 1980; Moran and Gordon, 1982). Finally, recovery may simply reflect a reconsolidation of input from the previously inactivated eye (Wong-Riley, 1994). 38 E. Effect of trabeculectomy on the adult primary visual cortex following elevated IOP - A Hypothesis The importance of early diagnosis and intervention for preventing vision loss in glaucoma is well accepted (Higginbotham, 1994b). Surgical intervention to ensure restoration of normal IOP is shown to be effective through studies examining its effects on optic disc and visual field changes. Studies on the long-term efficacy of trabeculectomy indicated that patients with the procedure conducted had lower levels of IOP on average and a slower progression of visual field loss compared to patients without surgery (Werner et al., 1977; Greve and Dake, 1979; Rollins and Drance, 1981; Kidd and O'Connor, 1985; Roth et a l , 1991). While it is convincing that surgical intervention in glaucoma is effective in delaying the progress of the disease, current glaucoma therapies are limited to this effect. Therapies for vision recovery after glaucomatous damage remain to be discovered. However, since there are no experimental therapies for transplantation, or replacement, of retinal ganglion cells currently available, the next focus for glaucoma therapy is on neuroprotection. Glaucoma therapeutic interventions have concentrated its efforts at the level of the retina and optic nerve head (Fechtner and Weinreb, 1994). However, it is important to recognize that glaucomatous damage extends beyond this level. The potential for transneuronal degeneration in the L G N and subsequently the visual cortex, has been demonstrated (Weber et al., 2000; Yucel et al., 2000; 2001). Moreover, neuronal atrophy at these levels is apparent at early, moderate and advanced stages of glaucoma that correlate with the extent of optic nerve fiber loss (Yucel et al., 2001). Therefore, loss of vision due to glaucoma may be due to pathobiological consequences at any of these 39 levels. Despite recent trends in recognizing the impact of glaucoma at the level of the L G N and visual cortex, knowledge about the nature of glaucomatous damage and vision loss is still incomplete (Gupta and Yucel, 2001). Secondary injury to neurons in the L G N and visual cortex after optic nerve fiber loss may contribute directly to glaucomatous progression (Weber et al., 2000; Yucel et al., 2000; 2001). However, lesions of the L G N or primary visual cortex have also been shown to induce retinal ganglion cell degeneration; possibly by reduced trophic support (Pearson et al., 1992; Johnson and Cowey; 2000). The bi-directional secondary damage by loss of afferents or by trophic support may accelerate the ongoing damage at each site and may play an important role in the progression of glaucoma. Therefore, it is pertinent for glaucoma therapies to interrupt this cascade. Current glaucoma therapies address pathobiological consequences at the level of the eye. However, knowing that glaucomatous damage may be bi-directional, true efficacy of intervention at the level of the eye only exists i f the glaucomatous impact on the central nervous system is attenuated or reversed as well. The effects of glaucomatous intervention have yet to be demonstrated at the level of the visual cortex. Showing evidence that loss of visual cortical activity is arrested, or has the potential to recover after elevated IOP, will be significant for justifying research into neuroprotective strategies in glaucoma. Evidence for metabolic recovery in the visual cortex following retinal impulse interruption is seen in the TTX model of monocular deprivation. Recovery in CO activity was observed following TTX removal. Parallels can be drawn between the primate model of glaucoma and the TTX model because in both models, retinal activity 40 can be disrupted temporarily. Evidence for restoration of normal axonal transport subsequent to the return to normal IOP following chronically elevated IOP exists (Minckler et a l , 1977; Quigley and Addicks, 1980). Further, since glaucomatous damage is a gradual process, like the T T X model, retinal ganglion cell afferents are likely maintained during the earlier stages of glaucoma. Moreover, transneuronal shrinkage is only observed under severe forms of monocular deprivation such as enucleation; therefore, cortical neurons may be preserved during early stages of glaucoma (Wong-Riley, 1994). In contrast, in the monocular lid suture model, consisting of a very mild form of deprivation, no indications of sprouting or contraction of geniculate afferents were found in adult animals (LeVay et a l , 1980; Horton, 1984). Providing that all of the geniculate afferents in the optic nerve are not lost, I predict that, restoration of IOP to normal levels will affect the metabolic activity changes observed within the visual cortex in response to ocular hypertension. Further, like the TTX model, the CO activity within the visual cortex will recover to some degree in the primate model of glaucoma following trabeculectomy. The process of recovery of CO activity following lowered IOPs may be due to a number of reasons including the synthesis of new enzyme molecules and their subsequent assembly into functional holoenzymes in the mitochondria, the establishment or strengthening of synapses, or an expansion of influence from the non-deprived eye. Finally, recovery may simply reflect a reconsolidation of input from the previously inactivated eye. 41 Table 1: Animal intraocular pressure history. Intraocular pressure (IOP) was measured in all animals and cytochrome oxidase histochemistry was performed on primary visual cortex block 1, the most superficial region of the operculum. G= glaucomatous eye. C= control eye. 42 ID Length of unilateral elevated IOP (> 30 mm Hg) Pre-TRAB IOP "area under curve" Length of time post-TRAB (<20 mm Hg) Post-TRAB IOP "area under curve" Cumulative IOP "area under curve" A1 2 weeks none Not applicable G:|1017 C:|342 A2 2 weeks ,.; * y none Not applicable G:|851 C:|334 A3 2 weeks ' '•: .. ; none Not applicable G:|891 C:|693 A4 4 weeks * - ; , none Not applicable G:|2136 C:|680 A5 4 weeks none Not applicable G: 12422 C:|927 A6 4 weeks none Not applicable G: 13222 C: 12289 B1 2 weeks G: |4941 9 months G:|3241 G:|8182 C:|2581 C:|4401 C: 16982 B2 2 weeks G: 13939 9 months G:|5028 G:|8967 C: 11607 C:|4070 C: 15677 B3 2 weeks G:|4705 9 months G:|5522 G:|10227 C: 13306 C:|4928 C: 18234 B4 4 weeks G:|4342 9 months G:|4661 G: 19003 C: 11754 C:|4455 C:|6209 B5 4 weeks G:|4824 8 months G:|4523 G:|9347 C: 11639 C: 13758 C:|5397 B6 4 weeks G:|5615 8 months G:|4096 G:|9711 C: 12442 C:|4287 C: 16729 C1 4 week Optic Nerve Transection .• none Not applicable G:|280 C:|336 C2 4 week Optic Nerve Transection none Not applicable G:|378 C:|434 D1 8 weeks G: 3301 none Not applicable G:|3301 C: 2624 C: 12624 D2 8 weeks G: 3383 none Not applicable G: 13383 C: 1425 C:|1425 D3 16 weeks G: 5390 none Not applicable G: 15390 C: 2832 C:|2832 D4 16 weeks G: 5122 none Not applicable G:|5122 C: 2453 C: 12453 D5 16 weeks G: 5188 none Not applicable G:|5188 C: 1892 C:|1892 N1 Normal Animal none Not applicable C:|5211 C: 14994 N2 Normal Animal none Not applicable C:|2787 C:|2728 S1 4 mths none Not applicable G: 12666 C: 12582 Table 1 4 3 2. Materials and Methods A total of 22 monkeys were used for this study (Table 1). In overview, animals were treated under three different experimental conditions. Two groups of animals were subject to elevated levels of IOP to mimic glaucomatous conditions. Trabeculectomies were then performed on one group of animals and was given a recovery period at normal IOPs (Group B). The second group of animals was sacrificed immediately following the period of elevated IOP (Group A and D). Finally the third group was subject to optic nerve transections to simulate conditions in which all visual activity is lost (Group C). Control animals were also used for comparison (Group N and S). CO histochemistry revealing metabolic activity differences within the visual cortex was observed and compared between these groups. A. Animal preparation i. Surgery A l l monkeys (Macaca mulatto: or Macaca fascicularis) were treated by qualified staff at our collaborating lab, at the University of Wisconsin, Department of Ophthalmology. Eighteen animals were treated to unilateral elevated intraocular pressures (IOP) for periods of 2, 4, 8 or 16 weeks by laser ablation to their trabecular meshwork. A standard clinical argon laser and slit lamp delivery system was used to produce a series of focal lesions to the trabecular meshwork in one eye (75 to 250 spots, 50 pm spot diameter, 1-1.5 W, 0.5 seconds duration). IOP was monitored every 3-7 days with a tonometer after treatment, and if not consistently above 30-35 mm Hg, additional 44 laser treatments were performed until a stable ocular hypertension was achieved. IOP was checked every few days thereafter to assure stability. Additional laser treatment therapy to assure stability or IOP lowering therapy was applied as needed to control the IOP levels. The opposite eye served as a normal control eye. In addition, one monkey had its trabecular meshwork ablated, but its IOP levels were normal. This animal served as a sham-operated control (SI). Animals that were not selected for trabeculectomy (Group A and D- NO TRAB, chronic glaucoma) were sacrificed immediately after their designated periods of elevated IOP. Following 2 or 4 weeks of elevated IOP, some animals (Group B- T R A B , transient glaucoma) were selected to have trabeculectomies performed to lower IOP to normal values of <20 mm Hg. In trabeculectomy, an incision is made in the conjunctiva and Tenon's layers, and a subconjunctival space is created between Tenon's layer and sclera. A half-thickness scleral flap is created, with the hinge of the flap based in the corneoscleral junction. An ostomy is created at the base of the flap, allowing aqueous to flow from the anterior chamber to the subconjunctival space. The flap is resutured loosely to the scleral bed, so that enough resistance to aqueous flow is created to avoid hypotony. The conjunctival/Tenon's incision is closed, trapping the fluid into a bleb (Figure 6). The aqueous is reabsorbed by the normal conjunctival and scleral veins, allowing a lower intraocular pressure postoperatively than before the surgery. These animals were maintained at normal IOP values for a minimum of six months following trabeculectomy before they were sacrificed. Two monkeys (Group C- ONT) underwent a complete transection of the optic nerve in one eye, while preserving the central vessels as verified by an absence of a 45 F i g u r e 6: Diagrammatic example of a trabeculectomy. (A) An example of blockage of aqueous outflow (arrows) from the anterior chamber is depicted, caused by contact between the lens and iris. (B) Trabeculectomy with a partial thickness flap in place allows for aqueous to drain through the edges of the flap into the subconjunctival space. (Figure adapted from Skuta, 1994; Smythe and Herschler, 1994). 46 4-7 hemorrhage by indirect ophthalmoscopy. Under pentobarbital anesthesia, the intracorneal space was entered by gentle dissection and the optic nerve was exposed. A sickle knife was used to make a 3 mm linear incision in the dura parallel to the nerve. Neurosurgical angled fine scissors were used to extend the incision posteriorly several millimeters, inserted into the dural sheath, and completely transected the nerve (2 cuts each, 2/3 through the nerve). The retina was observed by ophthalmoscopy to ensure that central retinal artery occlusion did not occur. The wounds were closed and the animals were treated with systemic benzathine and procaine penicillin for 5 days, and were sacrificed at 4 weeks following the surgery. As a normal control (Group N), two monkeys did not have any surgical procedures performed. After the designated experimental periods, animals were euthanized and perfused intracardially with 750 ml phosphate buffer saline (PBS), followed by 1 litre of 4% paraformaldehyde and again with 200-300 ml PBS. The brains were excised, and shipped in 10% sucrose phosphate buffer at 4°C to UBC (Figure 7). B. Tissue preparation ii. Blocking, flattening and sectioning The pia was first removed from the surface of each brain in preparation for flattening the striate cortex. The method of flattening as described by Horton and Hocking (1996) demonstrates major geographical features in the visual cortex. The cortical area at the operculum can be flattened so that ocular dominance columns in these 48 Figure 7: Schematic representation of the experimental protocol. Normal Animals Experimental Animals No lasering IOP measured by a tonometer every 3-7 days Unilateral IOP induced by laser scarification of the trabecular meshwork target > 30 mm Hg IOP measured by a tonometer every 3-7 days X Elevated IOP monitored and maintained for 2 or 4 weeks Animals without Animals with Trabeculectomy Trabeculectomy Trabeculectomy at 2 or 4 weeks post elevated IOP to lower IOP to normal values < 20 mm Hg I IOP measured by a tonometer every 3-7 days I IOP monitored and maintained for at least 6 months at < 20 mm Hg Animals euthanized and perfused intracardiac with 4% paraformaldehyde and cryoprotected n i Visual cortex blocked and stored at -80° C III 50u.m tangential sections cut through all cortical layers III Sections stained for CO and Nissl Figure 7 5 0 areas can be analyzed histochemically. The operculum, located at the most posterior aspect of the occipital lobe, is a fairly flat and smooth piece of cortex that is separated from the calcarine sulcus and calcarine fissure into a single cortical region which we have labeled block 1 and block 2. Block 1 represents the most superficial region of the operculum and block 2 represents the internal folds of the calcarine sulcus. The remaining area of the visual cortex located within the calcarine sulcus and along the calcarine fissure (LeVay et al., 1985; Kennedy et al., 1975; LeVay et al., 1980; Van Essen et al., 1984) can be divided further into blocks 3 and 4. A 'hinge' area separates the operculum surface from the calcarine block which consists of the 'roof (block 2) of the calcarine sulcus, the ventral bank (block 3) and the dorsal bank (block 4) of the calcarine fissure. A #10 scalpel blade was used to make an incision to extend the calcarine sulcus, parallel to the opercular surface. With a metal spatula, the lunate sulcus and the inferior occipital sulcus (two of the sulci that border the posterior occipital lobe) were probed gently. At the most lateral tip of block 1, an incision was made to extend the lunate sulcus to the inferior occipital sulcus so that the opercular part of the visual cortex could be removed. The operculum (block 1 and 2) was separated from the rest of the cortex by cutting down the calcarine sulcus along the white matter. The resulting block was a relatively flat triangular piece of cortex. Further, the rest of VI was dissected out by spreading apart the calcarine fissure. Blocks 3 and 4 that were parallel to the pia surface of the fissure were dissected out (Figure 8). 51 Figure 8: Flattened primary visual cortex. A flattened representation of the primary visual cortex is shown. Borders of blocks 1, 2, 3, and 4 are defined by dotted lines. Block 1 (operculum) represents the central visual field and is separated from blocks 2, 3, and 4 by the calcarine sulcus. When flattened, blocks 2, 3, and 4 represent the roof of the calcarine sulcus, the ventral bank of the sulcus and the dorsal bank of the sulcus. These blocks represent the peripheral visual field. Cortex representing the central 8-10° of vision is lost during the flattening procedure. 52 roof of calcarine sulcus C . Histochemistry Histology on all monkey brains was not performed at the same time; therefore upon arrival at U B C , the tissue was blocked and frozen until histology was performed. A l l blocks of tissue including cortex were cryoprotected in 20% sucrose in phosphate buffer (PB) overnight. The visual cortical blocks were gently flattened tangentially between 2 glass slides before being frozen on dry ice and stored at -80°C. A l l sections were cut at 50um on the freezing microtome. Sections were reacted for cytochrome oxidase (CO). i . Cytochrome oxidase The methodology for the cytochrome oxidase reaction was adapted from Wong-Riley (1989) and Boyd and Matsubara (1996). First, 20 mg of diaminobenzidine (DAB) (Sigma-Aldrich Co) was dissolved in 50 mL of distilled water. Once dissolved, 50 mL (0.1M pH 7.2) PB, 2g sucrose, 30 mg cytochrome C (Sigma-Aldrich Co) and 20 mg of catalase (Sigma-Aldrich Co) derived from bovine heart were added to the D A B solution. Then, 5ml of 1 % nickel ammonium sulfate was added drop wise followed by approximately 1 ml of 1% cobalt chloride until the solution appeared slightly opaque. Each section was placed into 1.5 ml of the filtered cytochrome oxidase solution and incubated at 40°C until the reaction product was dark enough and alternating dark and light bands were optimally contrasting to be clearly visualized in layer 4C. Sections were then placed in PB to stop the reaction and washed three times for 5 min each in PB, mounted, dehydrated in a series of graded alcohols and xylene, and coverslipped with permount. 54 An experiment to determine the effect of the length of time on the CO staining was also conducted. Since termination of the reaction was qualitatively determined, incubating for under, or over, the ideal reaction time may affect the results. Therefore, adjacent sections from the same brain were selected for a shorter (30 minutes less) than normal incubation period, and longer (60 minutes more) than normal incubation period in CO. OD ratios from these sections were compared with data obtained from sections incubated for a normal period of time to ensure that differences in incubations times do not affect the optical density ratios used in these studies. D. Data collection and analysis i. Predictors for glaucoma measurements In order for us to examine the severity of elevated IOP in the glaucomatous eye compared to the control eye, the area under the curve of a graph plotting IOP levels vs. the number of days post-lasering was determined. The "area under the curve" was determined because it is a more accurate measure of fluctuating IOP over a period of time. The cumulative area during the deprivation period for the control eye was compared to the area of the glaucomatous eye. The difference is expressed as the IOP ratio in which the IOP area of the control eye is compared to the glaucomatous or treated eye. For an example, please see the appendix, pg 137. Cupping ratios compare the cup to disc ratio of the control eye to the glaucomatous or treated eye. Cup to disc ratio and optic nerve damage was evaluated at the University of Wisconsin. 55 i i . Image capturing Images were captured on a light box with a Nikon Coolpix 990 digital camera. The settings on the camera remained constant throughout image capturing. NIH Image 1.62 was used to obtain density profiles and to measure the periodicity of ocular dominance columns. Raw OD values were converted to a linear index standardized by using neutral density filters. i i i . Intra-animal analysis Our analysis consisted of densiometric measures of CO staining between ocular dominance columns found in layer 4C within one animal. First, profile plots of animals were obtained. Within NIH Image 1.62, plot profile scales and density range were standardized. Images were also adjusted with filters to minimize irregular noise from blood vessels. Banding patterns were assessed using contrast-enhancement if necessary. It is important to note that contrast enhancement within this program did not change the optical density values of the image. A 2.5 mm transect was drawn through approximately three to four sets of bands with the measurement tool. A l l plots were charted with a common range of 0.34 in optical density values so that differences in values between the deprived and non-deprived eye bands could be compared. In addition, all transects were standardized to 2.5 mm, however differences in banding periodicities between animals, is to be expected (Horton and Hocking, 1996). Next, transects centred over dark immunostained bands and in adjacent light immunostained bands were obtained, taking care to avoid border regions (Figure 9). Since we were comparing the density between adjacent deprived and non-deprived eye 56 Figure 9: Measurement of optical density ratio. Diagram of protocol for determining optical density ratio measurement within a tangential section through block 1 of the primary visual cortex. A section from a monocularly deprived animal is displayed. This figure shows the ocular dominance bands in the L tangential plane after unilateral n elevated IOP. Transects were taken through the light and dark bands as shown. A ratio was then calculated. Ratio = 0 D L O D D D = Dark band L = Light band Figure 9 bands, a mean optical density ratio for an animal was calculated by dividing the mean value of 5 transects from a light or deprived eye band by the mean value of 10 transects (5 transects each) from the dark or non-deprived eye bands adjacent to it. For each animal, ten different sets of bands were measured with a total of 5 pairs of transects collected from each band, therefore amounting to at least 50 pairs of transects. The average optical density ratio was calculated for each experimental animal. In animals in which the ocular dominance bands were not evident, such as the sham operated and normal controls, the puffs and interpuffs in layers 2/3 were used to estimate potential band locations in layer 4C (Figure 10). The puffs form rows that are in register with the ocular dominance columns found in Layer 4C (Carroll and Wong Riley, 1984) and therefore can be used to approximate the direction of elongation of ocular dominance columns found in layer 4C. In addition, images were contrast enhanced to ensure that a weakly labeled or less contrasted banding pattern did not exist. This comparison is important because some animals subject to elevated IOP appear to have uniform staining. Subtle banding patterns become apparent after their images are contrast enhanced. These banding patterns can not be observed within normal control animals. It is important to note that optical density values were still obtained from raw non-contrast enhanced images for these animals. iv. Statistical analysis A l l data are presented as means ± SE. The Kruskal-Wallis test followed by the Tukey test were used to assess differences in optical density ratios between groups. The Kolmogorov-Smirnov test was used to assess differences between animals without 59 Figure 10: CO staining of V I . Comparison of cytochrome oxidase staining pattern within coronal sections of primary visual cortex from a normal animal and a deprived animal. The six layers of the cortex have different staining intensities depending on the metabolic reactivity of the layer. Layer 4 is the most severely affected when the animal undergoes monocular deprivation. Ocular dominance bands are established in layer 4 and are visualized by alternating areas of light and dark staining (red arrows). (Adapted from Wong-Riley et al., 1998). 60 Figure 10 6\ trabeculectomy and with trabeculectomy after short-term glaucoma. The one-tailed Mann-Whitney test was used to detect differences between animals without long-term chronic and short-term transient glaucoma. Non-parametric tests were chosen due to our small sample size and because the assumption of normality required by parametric tests could not be met. In all cases, P=0.05 was used as the level of significance. 62 3. Results A . Controls i. Over and under reaction control Since CO histochemistry involves the continual reoxidation of cytochrome-c by cytochrome oxidase to create an accumulation of a visible reaction product, it can be argued that decreasing or extending optimal incubation times can change the appearance of ocular dominance bands in this study. To ensure that optical density ratios do not change as a consequence of varying incubation times, control sections were incubated. For this control, a 4 week elevated IOP "NO T R A B " animal was used. Figure 11 shows adjacent sections from the same animal that were over-reacted (C) or under-reacted (A). Normal incubation times were determined as the shortest length of reaction in which defined bands can be seen clearly using a low power dissecting microscope. Under-reacted sections were determined to have the indication of bands, but were not yet clearly defined. Over-reacted sections remained in the incubating solution for an extra hour, in which a darker reaction product was observed as a result. Mean OD Ratios obtained from these sections were compared to those of sections incubated for the normal, optimum period of time. The OD ratio for the tissue undergoing under reactions was 0.76 ± 0.07. Tissue reacted for the 'optimal' amount of time resulted in an OD ratio of 0.74 ± 0.02. Tissue reacted longer than the optimal time had an OD ratio of 0.78 ± 0.04. Statistical significance does not exist among the three groups. 63 Figure 11 : Over and under reaction control. Adjacent.sections from the same animal were reacted for over (C) and under (A) the normal (B) incubation time for cytochrome oxidase histochemistry. Optical density ratios remained consistent even when the incubation time varied. Therefore, under or over-reacting should not affect the OD ratio measurements. 64 A) Under B) Normal C) Over t = 3 hrs 30 min t = 4 hrs t = 5 hrs OD Ratio: 0.76 ±0.07 OD Ratio: 0.74 ± 0.02 OD Ratio: 0.78 ±0.04 Figure 11 65 ii. Sham operated animal (SI) The sham-operated animal underwent laser scarification, but maintained normal IOP levels in each eye following treatment. The CO staining in layer 4C appears uniform and no dark and light CO bands in layer 4C were observed (Figure 12 and 13). The IOP ratio comparing the IOP of the control eye to the glaucomatous eye is 0.97 and is similar to the ratio of the normal (no treatment) animal's of 0.98. The plot profile lacks periodic fluctuations in optical density, therefore bands were not observed in a 2.5 mm transect across a tangential section through layer 4C of the visual cortex (Figure 13). The transect is relatively flat with some random fluctuations that are attributed to regular noise caused by blood vessels and cellular profiles. Together, these results indicate that in this primate model, a sufficiently elevated IOP is required to cause the characteristic changes in metabolic activity levels of the visual cortex evaluated in this study. Finally, the OD ratio obtained for this animal is 0.95 ± 0.05 (Figure 12). iii. Normal control animals (NI and N2) The normal control animals did not undergo any surgical procedures, and therefore are expected to have normal IOP levels in each eye. The IOP ratios comparing the IOP of the control eye to the glaucomatous eye are 0.96 and 0.98. Animals with normal IOP values in both eyes are expected to have a value of 1, however, differences between eyes in an individual occur naturally (Nilsson and Bi l l , 1994), therefore a 0.98 value is not surprising. The cupping ratio of 1.00 was available for one of the two normal animals. This indicates that no cupping differences exist between the two eyes. The CO staining in layer 4C appears uniform (Figure 12) and no evidence of dark and light CO 66 Figure 12: ONT and experimental control animals. CO staining of animals subjected to optic nerve transection (A,B), no treatment (C,D), or sham operation (E) is displayed. 50 pm tangential sections through layer 4C of the visual cortex were stained for CO activity. Enhanced images to optimize viewing of ocular dominance bands are displayed. Raw non-enhanced images were used to obtain OD ratios that compare metabolic activity of adjacent dark and light bands. The IOP ratio comparing the area under the curve, of a graph charting IOP over the number of days post lasering, between the control eye and the glaucomatous eye is listed. Available data on cupping (cup:disc) ratios is also listed comparing the control eye to the glaucomatous eye. IOP= IOP ratio, cup- cupping ratio. 67 ONT A ID : C1 ID : C2 OD : 0.68 +/- 0.07 OD : 0.67 +/- 0.03 IOP: 1.20 IOP: 1.15 cup : 1.00 cup : 1.00 C D ^ ~ - I ID : N1 ID :N2 ID :S1 OD : 1.00 +/-0.01 OD : 1.00 +/-0.01 OD : 0.95 +/- 0.05 IOP: 0.96 IOP: 0.98 IOP: 0.97 cup : 1.00 cup : not available cup : not available Figure 12 Figure 13: ONT and experimental control animals plot profiles. Representation of CO staining of animals subjected to optic nerve transection (A,B)> no treatment (C,D), or sham operated (E). Images contrast-enhanced by "NIH Image 1.62" are displayed to dramatically highlight ocular dominance bands observed in layer 4C of the visual cortex. Transects were taken across ocular dominance bands to show the relative contrast between light and dark bands. Plot profiles for the ONT animals (A,B) show optical density fluctuations along the 2.5 mm transect (white line). Control animals (C,D,E) do not display ocular dominance bands or periodic fluctuations in the plot profiles. 69 ONT Figure 13 10 bands in layer 4C exists (Figure 12 and 13). The plot profile analysis also indicates a lack of ocular dominance columns in layer 4C (Figure 13). The plots of a 2.5 mm transect taken across this layer is relatively flat, with no indication of periodic fluctuations in optical density. Small dips in the profile can be attributed to blood vessels within the tissue. The mean OD ratio in the normal animals was 1.00 ±0 .01 . B. Metabolic activity of experimental animals - Intra-animal comparisons i. Optic nerve transection Group C animals (n=2) were subject to complete optic nerve transections to demonstrate the metabolic activity changes under an extreme condition of deprivation. These animals were sacrificed 4 weeks following surgery and they did not experience any elevated levels of IOP. The IOP ratio, comparing the IOP of the control eye to the treated eye, was 1.20 and 1.15 (Figure 12). An untreated animal is expected to have an IOP ratio of 1. Unfortunately, data for these animals was limited. The mean IOP value over four weeks was the only information available, therefore fluctuations in IOP during this period could not be accounted for accurately. As a result, the IOP ratio in this case is greater than 1, meaning that the control eye IOP was greater than the IOP of the treated eye. This discrepancy may be due to inherent animal differences, or that the treated eye may have a slightly decreased level of IOP as a consequence of the transection. The CO staining in layer 4C revealed alternating dark and light CO bands. The OD ratios for the ONT animals were 0.68 ± 0.07 and 0.67 ± 0.03, with a mean ratio at 0.67 ± 0.01. The plot profiles for these animals also indicate evidence of differential activity between the control and treated eyes (Figure 13). 71 ii. Short-term chronic glaucoma vs. short-term transient glaucoma Please note that "chronic glaucoma" animals are subject to "NO T R A B " treatments and that "transient glaucoma" animals are subject to " T R A B " treatments. These defining terms will be used interchangeably throughout the results and discussion for emphasis. a. Two weeks post elevated IOP 1. Qualitative Analysis Results of animals sacrificed immediately (NO TRAB) following 2 weeks of elevated IOP were variable. Alternating light and dark bands were evident in layer 4C in all three animals (Figure 14 and 15), however contrast was apparent between the dark and light bands for only two of the three animals ( A l and A2). The IOP ratio comparing the IOP between the control and treated eyes yielded the values of 0.34 and 0.39 respectively. The third animal (A3) had some evidence of banding, but the effects were not as dramatic. Evidence of these bands was visualized better when the contrast was manipulated (Figure 14-C). Alternating dark and light areas were indicative of bands. The IOP ratio for this animal was 0.78. As expected, since banding was not as apparent for this animal, discrepancy between the control and glaucomatous eye was closer to normal animals' value of approximately 1. The cupping ratio for this animal was also determined to be 1.00, thereby indicating that no significant disc cupping existed in the glaucomatous eye compared to the control eye. In the TRAB group following two weeks of deprivation, alternating light and dark bands were observed in all three animals. The contrast between the bands was low in two 72 Figure 14: Short-term (2 weeks) chronic and transient glaucoma. CO staining of animals at 2 weeks following elevated IOP. 50u.m tangential sections through layer 4C were stained for CO activity after 2 weeks of elevated IOP. The "NO T R A B " (A,B,C) figures show visual cortex from animals sacrificed immediately after 2 weeks. The " T R A B " (D,E,F) figures are from animals that underwent trabeculectomy after 2 weeks of elevated IOP to reduce IOP, and allowed to survive with IOP <20 mm Hg for a minimum of 6 months. Enhanced images to optimize viewing of ocular dominance bands are displayed. Raw non-enhanced images were used to obtain optical density (OD) ratios comparing the adjacent dark and light bands. The IOP ratio comparing the area under the curve, of a graph charting IOP over the number of days post lasering, between the control eye and the glaucomatous eye is listed. Available data on cupping (cup:disc) ratios is also listed comparing the control eye to the glaucomatous eye. IOP= IOP ratio, cup= cupping ratio. 73 2 WEEK NO TRAB ID :A1 ID :A2 ID A3 OD : 0.74 +/- 0.02 OD : 0.87 +/- 0.06 OD : 0.96 +/- 0.02 IOP : 0.34 IOP : 0.39 IOP : 0.78 cup : not available cup : not available cup : 1.00 2 WEEK TRAB D • j, F • *V • ID : B1 ID : B2 ID : B3 OD : 0.92 +/- 0.03 OD : 0.92 +/- 0.03 OD : 0.67 +/- 0.04 IOP: 0.85 IOP: 0.63 IOP: 0.81 cup : 0.80 cup : 0.29 cup : 0.50 Figure 14 7 4 (Bl and B2) of the three animals, but one animal (B3) exhibited a strong contrast in ocular dominance columns (Figure 14 and 15). The IOP ratio for the two animals with low contrast is 0.85 and 0.63. The third animal had a ratio of 0.81. These ratios are closer to the normal value of 1 compared to the NO TRAB group. The IOP ratio does not correlate with the qualitative results observed in this set. Further, the cupping ratio comparing the cup to disc ratio of the control eye to the glaucomatous eye is 0.80, 0.29 and 0.50. Again, these ratios do not represent a clear correlative trend with the banding images observed. 2. Plot Profile Analysis In both the NO T R A B and TRAB group, the plot profiles all showed evidence of banding (Figure 15). Periodic fluctuations of optical density were observed on a 2.5 mm transect across the alternating deprived and non-deprived eye bands. Deprived eye bands had a lower optical density measurement compared to the non-deprived eye bands. Optical density differences between pairs of dark and light bands were fairly consistent for each animal. The amplitude difference between deprived and non-deprived eye bands was different between the NO TRAB and the TRAB group. A l l plot profiles are displayed with the same range so that the optical density differences between dark (peak) and light bands (trough) can be compared between animals. In the NO T R A B group two of the three animals had plot profiles with big fluctuations (Figure 15-A and 15-B). The third animal had a flatter plot profile, with greater resemblance to the plot profile from control animals', however periodic fluctuations can still be observed that correlate with the 75 Figure 15: Short-term (2 weeks) chronic and transient glaucoma plot profiles. Representation of CO staining of animals following 2 weeks of elevated IOP. The "NO T R A B " (A,B,C) figures show visual cortex from animals sacrificed immediately after 2 weeks. The " T R A B " (D,E,F) figures are from animals that underwent trabeculectomy after 2 weeks of elevated IOP to reduce IOP, and allowed to survive with IOP <20 mm Hg for a minimum of 6 months. Images contrast-enhanced by "NIH Image 1.62" are displayed to dramatically highlight ocular dominance bands observed in layer 4C of the visual cortex. Transects were taken across ocular dominance bands to show the relative contrast between light and dark bands. Plot profiles show the OD fluctuations along the 2.5 mm transect (white line). 76 Figure 15 banding pattern through the transect (Figure 15-C). In the T R A B group two of the three animals had a flatter plot profile with periodic fluctuations (Figure 15-D and 15-E). The third animal from this group had steeper fluctuations (Figure 15-F). 3. Quantitative analysis In the NO TRAB group, the OD ratio obtained for each animal were: 0.74 ± 0.02, 0.87 ± 0.06, and 0.96 ± 0.02 (Table 2 and Figure 14). The lower OD ratios were correlated with the presence of banding. As expected, the third animal had bands with very low contrast and therefore had a higher, and closer to normal, OD ratio. The average OD ratio for the three animals of this group was 0.86 ± 0.06 (Table 2). In the TRAB group, the OD ratios obtained for each animal were: 0.92 ± 0.03, 0.92 ± 0.03, and 0.67 ± 0.04 (Table 2 and Figure 14). Again, the presence of bands with low contrast was confirmed by the higher, closer to normal OD ratios measured in those two animals (Bl and B2). The average OD ratio for the three animals was 0.84 ±0.08 (Table 3). The OD ratios for the three animals in the NO T R A B group were distributed within the range of 0.72 to 0.98 (Figure 16-A). The values did not overlap with each, other. For the T R A B group, two of the three animals had overlapping values covering the range of 0.89-0.95. The third animal had a significantly lower OD ratio compared to the other two. The range of values presented in the TRAB group was 0.63-0.94. Both experimental groups had OD ratios that overlapped with the OD ratio range of the control animals' of 0.90-1.01. The actual mean OD ratio of the NO T R A B group was larger (0.86 ± 0.06) than the T R A B group (0.82 ± 0.15), however taking into account the error bars, the two groups overlap in values. At 2 weeks elevated IOP, the mean OD ratio for 78 Table 2: Experimental OD ratios. Optical density (OD) ratios compared the dark and light bands of ocular dominance columns. NO TRAB animals (red) were sacrificed immediately following the designated deprivation period. T R A B animals (blue) were given a trabeculectomy following the designated deprivation period and allowed to recover for at least 6 months at normal IOP values of <20 mm Hg. ONT (brown) animals were subject to complete optic nerve transections. CONTROL animals (green) were not subject to treatment or were sham operated. Note that OD ratios of 1 represent those of normal animals. 79 ID Length of unilateral elevated IOP (> 30 mm Hg) Group OD Ratio OD Ratio Range 2 weeks NO TRAB 0.74 +/- 0.02 0.72 - 0.76 0.87 +/- 0.06 0.81 - 0.93 A4 A5 G L B1 2 weeks TRAB 0.92 +/- 0.03 0.89 - 0.95 B2 2 weeks TRAB 0.92 +/- 0.03 0.89 - 0.95 B3 2 weeks TRAB 0.67 +/- 0.04 0.63 - 0.71 B4 4 weeks TRAB 0.85 +/- 0.03 0.82 - 0.88 B5 4 weeks TRAB 0.93 +/- 0.01 0.92 - 0.94 B6 4 weeks TRAB 0.85 +/- 0.02 0.83 - 0.87 C I 4 weeks ONT 0.68 +/- 0.07 0.61 - 0.75 C2 4 weeks ONT 0.67 +/- 0.03 0.64 - 0.70 p_ D2 D3 I 1.00 +/- 0.01 0.99 - 1.01 none none 1.00 +/- 0.01 0.99 - 1.01 tH S H A M 0.95 +/- 0.05 Table 2 8o Table 3: Experimental mean OD ratios. Mean optical density (OD) ratios compare the dark and light bands of ocular dominance columns. NO T R A B animals (red) were sacrificed immediately following the designated deprivation period. T R A B animals (blue) were given a trabeculectomy following the designated deprivation period and allowed to recover for at least 6 months at normal IOP values of <20 mm Hg. ONT (brown) animals were subject to complete optic nerve transections. CONTROL animals (green) were not subject to treatment or were sham operated. Note that OD ratios of 1 represent those of normal animals. 81 Length of unilateral elevated IOP (> 30 mm Hq) Group Mean OD Ratio OD Ratio Range 0.80 - 0.92 0.59 - 0.83 2 weeks TRAB 0.84 +/- 0.08 0.76 - 0.92 4 weeks TRAB 0.88 +/- 0.02 0.86 - 0.9 4 weeks ONT 0.67 +/- 0.07 0.60 - 0.74 8 weeks NO TRAB 0.68 +/- 0.07 0.61 - 0.75 16 weeks NO TRAB 0.66 +/- 0.03 0.63 - 0.69 none CONTROL 0.98 +/- 0.01 0.97 - 0.99 0.71 - 0.95 2 and 4 weeks TRAB 0.86 +/- 0.04 0.82 - 0.90 8 and 16 weeks NO TRAB 0.64 - 0.70 Table 3 82 Figure 16: Comparison of OD ratios in relation to the experimental group type after 2 and 4 weeks of elevated IOP. Optical density ratios of animals in the "NO T R A B " group (red) were compared to the " T R A B " group (blue) after (A) 2 weeks of elevated IOP and (B) 4 weeks of elevated IOP. Note that the OD ratio mean value between the two groups were roughly overlapping after 2 weeks. After 4 weeks, the OD ratio mean of the " T R A B " group was larger and closer to 1 than the "NO T R A B " group. Note that OD ratios of 1 represent those of normal animals. 83 the NO TRAB group covered the range of 0.80-0.92. The mean OD ratio for the TRAB group covered the range of 0.76-0.92. In comparison, the mean range of the control group's OD ratio was 0.97-0.99. Neither groups' mean overlapped with the control group's actual mean of 0.98 ± 0.01. At the 2 week deprivation time point, the mean OD ratios of the chronic (NO TRAB) and transient (TRAB) groups overlapped with each other, but neither group overlapped with the mean range of the control group's. 4. Statistical analysis The NO T R A B , TRAB and control groups were compared with each other with the Kruskal Wallis test. The groups were not statistically different from each other. b. Four weeks post elevated I O P 1. Qualitative analysis Alternating light and dark bands were evident in layer 4C of all three animals. However, the contrast between ocular dominance bands was weaker in one of them (Figure 17 and 18). The animals with an apparent banding pattern had an IOP ratio of 0.32 and 0.71 (A4 and A6). The animal with less severe banding had an IOP ratio of 0.38 (A5). Cupping ratio for the animals with significant banding is 0.30 and 0.22. The cupping ratio was not available for the animal with less banding. In the TRAB group following 4 weeks of deprivation, alternating light and dark bands were observed in all three animals. The contrast between the bands was evident but subtle in all 3 animals (Figure 17). When contrast enhanced, the banding patterns 85 Figure 17: Short-term (4 weeks) chronic and transient glaucoma. 50LUTI tangential sections through layer 4C were stained for CO activity after 4 weeks of elevated IOP-The "NO T R A B " (A,B,C) figures show visual cortex from animals sacrificed immediately after 4 weeks. The " T R A B " (D,E,F) figures are from animals that underwent trabeculectomy after 4 weeks of elevated IOP to reduce IOP, and allowed to survive with IOP <20 mm Hg for a minimum of 6 months. Enhanced images to optimize viewing of ocular dominance bands are displayed. Raw non-enhanced images were used to obtain optical density (OD) ratios comparing the adjacent dark and light bands. The IOP ratio comparing the area under the curve, of a graph charting IOP over the number of days post lasering, between the control eye and the glaucomatous eye is listed. Available data on cupping (cup:disc) ratios is also listed comparing the control eye to the glaucomatous eye. IOP- IOP ratio, cup= cupping ratio. 86 OD : 0.52 +/- 0.08 B ID : OD : 0.93 +/- 0.04 C ID_ OD A 6 _ _ ^ ^ 0.67 +/-0.01 IOP: 0.32 IOP 0.38 IOP 0.71 cup 0.30 cup : not available cup 0.22 4 W E E K T R A B D ID : B4 ID : B5 ID : B6 OD : 0.85 +/- 0.03 OD : 0.93 +/- 0.01 OD : 0.85 +/- 0.02 IOP: 0.69 IOP: 0.58 IOP: 0.69 cup : 0.60 cup : 1.00 cup : 0.30 Figure 17 Figure 18: Short-term (4 weeks) chronic and transient glaucoma plot profiles. Representation of CO staining of animals following 4 weeks of elevated IOP. The "NO T R A B " (A,B,C) figures show visual cortex from animals sacrificed immediately after 4 weeks. The " T R A B " (D,E,F) figures are from animals that underwent trabeculectomy after 4 weeks of elevated IOP to reduce IOP, and allowed to survive with IOP <20 mm Hg for a minimum of 6 months. Images contrast-enhanced by "NIH Image 1.62" are displayed to dramatically highlight ocular dominance bands observed in layer 4C of the visual cortex. Transects were taken across ocular dominance bands to show the relative contrast between light and dark bands. Plot profiles show the OD fluctuations along the 2.5 mm transect (white line). 88 4 WEEK TRAB Figure 18 become clear (Figure 18). The IOP ratios for these animals are 0.69, 0.58, and 0.69. These values on average are closer to the control values of 1 compared to the NO T R A B group. The cupping ratios for this set of animals were 0.60, 1.00 and 0.30. 2. Plot profile analysis In the NO TRAB and TRAB group, the representative plot profile (Figure 18) again showed evidence in fluctuations of optical density across the alternating deprived and non-deprived eye bands. A l l of the profiles display periodic fluctuations that correlate with the banding patterns. Deprived eye bands had a lower optical density measurement compared to the non-deprived eye bands. The amplitude difference between deprived and non-deprived eye bands was variable for the NO T R A B group. Fluctuation amplitude for this group ranged from flatter profiles, to profiles with extreme optical density differences (around 0.30) between the dark and light bands. The TRAB group had plot profiles with amplitude differences similar to the two of the three profiles from the NO TRAB group. The amplitude difference from the TRAB group was significantly smaller than the third animal in the NO TRAB group. 3. Quantitative analysis In the NO T R A B group, the OD ratios obtained were: 0.52 ± 0.08, 0.93 ± 0.04, and 0.67 ± 0.01. The higher OD ratios are correlated with the more subtle ocular dominance bands (Figure 18). In these animals, the staining pattern is closer to the uniform staining observed in the control animals. The lower OD ratio values have a 90 staining pattern closer to the ONT animals, which also have low OD ratio values. The mean OD ratio for the three animals is 0.71 ±0.12 (Table 3). In the T R A B group, the OD ratio obtained for each animal were: 0.85 ± 0.03, 0.93 ± 0.01, and 0.85 ± 0.02 (Table 2). Again, the severity of banding observed in layer 4C correlates with the OD ratio value. The mean OD ratio for the three animals was 0.88 ± 0.02 (Table 3). The OD ratios for the three animals in the NO T R A B group were distributed within the range of 0.52-0.93 (Figure 16-B). Two of the three animals had overlapping range (A4 and A6) covering optical density values of 0.44-0.68. The third animal was distinct from the other two animals covering the range of 0.89-0.97 (A5). For the T R A B group, two of the three animals (B5, B6) had overlapping values covering the range of 0.82-0.88. The third animal had a higher OD ratio compared to the other two covering the range 0.92-0.94 (B5). The range of OD ratios for this group were from 0.82-0.94. Both experimental groups had OD ratios that overlapped with the OD ratio range of the normal animal. The actual mean OD ratio of the NO TRAB group was smaller (0.71 ± 0.12) than the T R A B group (0.88 ± 0.02). After 4 weeks of elevated IOP, the mean OD ratio for the NO T R A B group was within the range of 0.59-0.83 and the mean OD ratio range for the TRAB group was 0.86-0.90. At this time point, the NO T R A B group had lower mean OD ratios compared to the TRAB group. Again, neither group overlapped with the range of mean values of the control group (0.97-0.99). The means of the NO T R A B and TRAB group do not overlap with each other, or the control group's at this time point. 91 4. Statistical analysis The NO T R A B , TRAB and control groups were compared with each other with the Kruskal Wallis test. Statistical difference was detected between the groups (H>5.6, PO.05). The Tukey test revealed statistical differences between the control group and the NO T R A B group, the control group and the TRAB group (Q<3.314, PO.05). No statistical difference was found between the NO TRAB and T R A B group. c. Combined 2 and 4 weeks post elevated I O P Figure 19 displays the combined data of 2 and 4 weeks post elevated IOP animals to compare the difference in NO TRAB animals (red) compared to T R A B animals (blue). The T R A B animals have an OD ratio range of 0.52-0.97. The NO T R A B animals have an OD ratio range of 0.44-0.98. Sham control animals (green) have an OD ratio range of 0.90-1.00. Normal, no treatment control animals (green) have a range of 0.99-1.01. The grouped OD ratio range of the control animals was therefore 0.90-1.01. The mean OD ratio for the NO T R A B group was 0.78 ± 0.07 and the mean OD ratio for the T R A B group was 0.86 ± 0.04. The mean OD ratio for the control groups was 0.98 ±0 .01 . Collectively, the NO TRAB group had a smaller mean OD ratio compared to the T R A B group. The range of the optical density ratios in the NO T R A B group was 0.71-0.85, whereas the range of the TRAB group was 0.82-0.90. When the means of all three groups were compared, the NO TRAB mean OD ratio did not overlap with the control group's range of 0.90-1.01. In contrast, the mean OD ratio of the T R A B group overlaps slightly with the control group. Even though the mean OD ratios of the T R A B group is larger, and closer to normal values, compared to the NO T R A B group, mean values from 92 Figure 19: Comparison of OD ratios in relation to the experimental group type in short-term glaucoma. After 2 and 4 weeks of deprivation, the combined mean OD ratio of the " T R A B " group (blue) was larger and closer to 1 compared to the "NO T R A B " group. Note that OD ratios of 1 represent those of normal animals. 93 CONTROL o o NO TRAB I M • 0.00 0.20 0.40 0.60 Optical Density Ratio 0.80 1.00 • 2 WK NO TRAB A 4 W K NO TRAB • 2 WK TRAB A 4 W K TRAB X NO TRAB MEAN X T R A B MEAN • SHAM CONTROL • NORMAL CONTROL * CONTROL MEAN Figure 19 these groups still overlap. No statistically significant difference was found between these three groups after short-term glaucoma (2 or 4 weeks of elevated IOP). Statistically significant difference was also not found in the direct comparison between NO T R A B and TRAB animals after short-term glaucoma. iii. Long-term chronic glaucoma vs. short-term transient glaucoma Animals with longer terms of elevated IOP (8 and 16 weeks elevated IOP and sacrificed) were also compared to animals with trabeculectomies after 2 and 4 weeks of elevated IOP and a survival period at normal IOPs of <20 mm Hg for at least 6 months. This comparison is made to demonstrate any potential differences between animals with surgical intervention for glaucoma to animals without any intervention. For this section "NO T R A B " animals will be referred to as "chronic" glaucoma animals and " T R A B " animals will be referred to as "transient" glaucoma animals. a. Eight and 16 weeks post elevated IOP 1. Qualitative analysis Dark and light bands were apparent after 8 and 16 weeks of elevated IOP (Figure 20). After 8 weeks of elevated IOP, the IOP ratios were at 0.79 and 0.42. After 16 weeks of elevated IOP, the IOP ratios were at 0.53, 0.48, and 0.36. From this, we can see that longer deprivation periods yielded a greater difference of IOP between the control and glaucomatous eye, and thus lower IOP ratios. The cupping ratio was available for only one 8 week animal (DI). The value 0.38 indicates that severe cupping existed in the glaucomatous eye since a normal cupping ratio is usually 1. 95 Figure 20: Long-term chronic glaucoma. CO staining of animals after 8 (A,B,C) and 16 (D,E,F) weeks of elevated IOP. 50pm tangential sections through layer 4C were stained for CO activity. These "NO T R A B " figures show visual cortex from animals sacrificed immediately after 8 or 16 weeks of elevated IOP. Enhanced images to optimize viewing of ocular dominance bands are displayed. Raw non-enhanced images were used to obtain optical density (OD) ratios comparing the adjacent dark and light bands. The IOP ratio comparing the area under the curve, of a graph charting IOP over the number of days post lasering, between the control eye and the glaucomatous eye is listed. Available data on cupping (cup:disc) ratios is also listed comparing the control eye to the glaucomatous eye. IOP= IOP ratio, cup= cupping ratio. 96 IOP cup 0.75 +/- 0.06 0.79 0.38 IOP cup : 0.61 +/-0.04 0.42 not available C I D ID D3 ID : D4 ID : D5 OD 0.65 +/- 0.03 OD 0.73 +/- 0.08 OD 0.61 +/- 0.04 IOP 0.53 IOP 0.48 IOP 0.36 cup not available cup not available cup not available Figure 20 <?7 2. Plot profile analysis Periodic fluctuations that correlated with the bands through 2.5 mm transects were observed for all five animals at 8 or 16 weeks post elevated IOP. Amplitude difference between the dark and light bands varied between animals, but on average remained constant within each animal (Figure 21). The amplitude difference between dark and light bands ranged from 0.24 to 0.34, which indicated a larger difference in CO staining intensity of non-deprived bands compared to the deprived bands. When compared to earlier time points of 2 and 4 weeks of elevated IOP, the difference between dark and light eye bands is worth noting. 3. Quantitative analysis The OD ratios for animals following 8 weeks of elevated IOP were 0.75 ± 0.06 and 0.61 ± 0.04 (Table 2 and Figure 20). After 16 weeks of elevated IOP, the OD ratio values were 0.65 ± 0.03, 0.73 ± 0.08, and 0.61 ± 0.04 (Table 2). The mean OD ratio after 8 weeks of elevated IOP was 0.68 ± 0.07, and the mean OD ratio after 16 weeks of elevated IOP was 0.66 ± 0.03 (Table 3). Figure 22 displays the results of the comparison between all of the chronic and transient glaucoma animals. The OD ratios of chronic glaucoma animals after 8 weeks of elevated IOP were within the range of 0.57-0.81. The OD ratios for the chronic animals after 16 weeks of elevated IOP were within the range of 0.57-0.81. It is important to note that the OD ratio ranges for the animals subject to 8 and 16 weeks of elevated IOP are well below that of the control group's range of 0.90-1.01. 98 Figure 21: Long-term chronic glaucoma plot profiles. Representation of CO staining of animals following 8 (A,B,C) and 16 (D,E,F) weeks of elevated IOP. These "NO T R A B " figures show visual cortex from animals sacrificed immediately after 8 or 16 weeks. Images contrast-enhanced by "NIH Image 1.62" are displayed to dramatically highlight ocular dominance bands observed in layer 4C of the visual cortex. Transects were taken across ocular dominance bands to show the relative contrast between light and dark bands. Plot profiles show the OD fluctuations along the 2.5 mm transect (white line). 99 Figure 21 Figure 22: OD ratios in relation to duration of deprivation. Durations of 2, 4, 8 and 16 weeks are compared. OD ratios of chronic (NO TRAB) animals are represented in red, transient (TRAB) animals are represented in blue, and optic nerve transection (ONT) animals in maroon. Distribution of OD ratios of animals at 4, 8, and 16 weeks of deprivation as well as ONT animals are roughly within the same range (0.52-0.75). Transient glaucoma animals appear to have OD ratio closer to normal (green). 101 18 16 c/j . -sc 14 3 9 4 12 5 10 CL <u a o e o a 8 6 4 2 I H» • 1 ( - • -I • 1 I • 1 -A—I 1 1 mki 1 1 I I A I I i • 1 1.00 0.00 0.20 0.40 0.60 0.80 Optical Density Ratio • 2 WK NO TRAB • 2 WK TRAB A 4 WK NO TRAB A 4 WK TRAB A 4 WK ONT • 8 WK NO TRAB • 16WK NO TRAB • SHAM CONTROL • NORMAL CONTROL Figure 22 I 0 2 . Statistical analysis comparing differences in OD ratios between control groups and groups subject to 2, 4, 8, and 16 weeks of elevated of IOP was significant (H>8.2, PO.05). Further significant difference was found between: the control group and the groups subject to 2 weeks of elevated IOP, between the control group and the group subject to 2 weeks of elevated IOP, between the control group and the group subject to 4 weeks of elevated IOP, between the control group and the group subject to 8 weeks of elevated IOP, and between the control group and the group subject to 16 weeks of elevated IOP (Q>2.807, PO.05). In the comparison between the 2 weeks of elevated IOP group with the 4, 8, and 16 weeks groups, statistically significant differences were found (Q>2.807, P<0.05). Comparisons were made between the 2 and 4 weeks groups, the 2 and 8 weeks groups, and the 2 and 16 weeks groups. Comparisons made between the 4 and 8 weeks groups, the 4 and 16 weeks groups, and the 8 and 16 weeks groups were not statistically different. b. Chronic vs. transient glaucoma analysis Figure 23 displays a direct comparison of chronic long-term glaucoma animals with transient short-term glaucoma animals. The chronic glaucoma group consists of the NO TRAB animals subject to 8 and 16 weeks of elevated IOP. In the transient glaucoma group, the T R A B animals were subject to 2 and 4 weeks of elevated, subject to trabeculectomies and allowed a recovery period at normal IOP levels for at least 6 months. This comparison is used to demonstrate any potential differences in optical densities observed between animals with and without surgical intervention. The mean of the chronic glaucoma group was 0.67 ± 0.03, while the mean of the transient group was 103 Figure 23: Comparison of OD ratios of long-term chronic glaucoma (NO TRAB) animals to transient glaucoma (TRAB) animals. Chronic animals were subject to 8 or 16 weeks of elevated IOP (red). Transient animals were subject to 2 or 4 weeks of elevated IOP, given a trabeculectomy, and allowed a survival period at normal IOP levels for a minimum of 6 months (blue). Note that the mean OD ratio of the "TRANSIENT" group was larger and closer to the control group (green), compared to the "CHRONIC" group. Further, mean difference between the chronic and transient groups was statistically significant (U>25, P<0.05). 104 CONTROL TRANSIENT| Q-=1 8 O CHRONIC X 1 M ! I I I I I ' ' 1 1 1 I ! 1 ! ! 1 1 ! 1 | ! 1 1 1 f • " 0.00 0.20 0.40 0.60 0.80 1.00 Optical Density Ratio • 8 WK NO TRAB • 16WK NO TRAB • 2 WK TRAB A 4 WK TRAB X NO TRAB MEAN X T R A B MEAN • SHAM CONTROL • NORMAL CONTROL X CONTROL MEAN Figure 23 0.86 ± 0.04. For comparison, the control group's mean was 0.98 ± 0.01. Compared to the chronic groups, the transient group's mean was closer to the control group's mean. The OD mean range of the chronic (0.64-0.70), transient (0.71-0.85), and control groups (0.97-9.99) did not overlap. Further, statistically significant mean difference was found between the chronic and transient group (U>25, PO.05). Figure 24 displays the mean OD ratios of all of the animal groups observed in this study. The 4 week ONT animals, 8 week and 16 week chronic glaucoma animals all had similar mean OD ratios at 0.67 ±0 .01 , 0.68 ± 0.07 and 0.66 ± 0.03 respectively. These mean values did not overlap with those of the normal animal's at 0.98 ± 0.01. In comparison, the mean OD ratios of the chronic glaucoma (0.86 ± 0.06) and transient glaucoma animals at 2 weeks overlapped with each other and both means covered a relatively similar range. These means at 2 weeks had OD ratios closer to the control group's mean than the ONT, 8 weeks and, 16 weeks elevated IOP groups. After 4 weeks of elevated IOP, the mean OD ratio range of the chronic glaucoma (0.71 ±0.12) and transient glaucoma (0.88 ± 0.02) groups were distinct from each other. Moreover, the transient glaucoma group had closer to control mean OD ratios compared to the chronic group. iv. Analysis of predictors of OD ratios Three predictors of glaucomatous damage were evaluated for its strength in determining the severity of deprivation as reported by OD ratios. These were IOP ratios, cupping ratios and optic nerve damage. The IOP ratio represents the comparison of IOP experienced by the control (or un-treated) eye compared to the glaucomatous (or treated) 106 Figure 24: Comparison of mean OD ratios in relation to duration of deprivation. Durations of 2, 4, 8 and 16 weeks of elevated IOP are compared. Mean OD ratios of "NO T R A B " animals are represented in red, " T R A B " animals are represented in blue, and optic nerve transection (ONT) animals are represented in brown. Distribution of the mean OD ratios of animals after 4 weeks of ONT, 8, and 16 weeks of elevated IOP were roughly within the same range (0.66-0.68). The mean OD ratios of " T R A B " and "NO T R A B " animals after 2 weeks of elevated IOP were within the same range and were not significantly different. After 4 weeks of elevated IOP the "NO T R A B " animals have a mean closer to those of animals with severe deprivation (ONT, 8 and 16 weeks of elevated IOP). In comparison, " T R A B " animals after 4 weeks of elevated IOP appear to have a mean OD ratio closer to control animals (green) compared to the "NO T R A B " group. The distribution of the means for the "NO T R A B " and " T R A B " groups after 4 weeks of elevated IOP did not overlap. 107 0.00 0.20 0.40 0.60 0.80 1.00 Optical Density Ratio • 2 WK NO TRAB MEAN • 2 WK TRAB MEAN X 4 WK NO TRAB MEAN X 4 W K TRAB MEAN A 4 W K 0 N T MEAN • 8 WK NO TRAB MEAN • 16 WK NO TRAB MEAN xCONTROL MEAN Table 4: Predictors of OD ratios. IOP ratios compare the IOP data between control (un-treated) and glaucomatous (treated) eye. IOP data is evaluated as the area under the curve of a graph recording IOP over the number of days post lasering. IOP ratios are compared with the pre-TRAB (trabeculectomy) history, post-TRAB history, and the cumulative experimental IOP history. Values closest to 1 indicate the least discrepancy in IOP between the two eyes. Cupping ratios comparing the cup:disc ratio between the control eye and the treated eye was also evaluated. A value of 1 indicates no optic disc cupping difference between the two eyes. Optic nerve damage data is also presented. "O" represents no damage, "+" mild damage, and "++" moderate to severe damage. Measured OD ratios are also presented for reference. Blocked out cells represent non-applicable or unattainable data. Both the IOP ratio and cupping ratio compares the values for the control eye to the treated eye by: C/G, C= control eye G= glaucomatous eye. 109 eye. IOP was determined by calculating the area under the curve of a graph that recorded IOP over the number of days following lasering of the trabecular meshwork. Values closest to 1 represent normal animal values, which have the least difference in IOP between the two eyes. The cupping ratio represents the comparison of the cup:disc ratio of the control eye compared to the glaucomatous eye. A value of 1 represents no optic cupping difference between the two eyes. Finally, optic nerve damage was evaluated as either having none (0), mild loss (+) and moderate to severe loss (++) (Table 4). a. Two and 4 weeks N O T R A B IOP ratios and cupping ratios were evaluated as predictors for OD ratios for the animals without trabeculectomy following 2 and 4 weeks of elevated IOP. In Figure 25-A, the IOP ratio is compared to the OD ratio measured within our study. Variability exists within this comparison, however it is noted that as IOP ratios increase to 1, so do OD ratios. Figure 25-B compares the cupping ratios with OD ratios. Again, while this relationship is not directly linear, cupping ratios closer to 1 are correlated to the more "normal" OD ratio usually at 1. b. Two and 4 weeks T R A B For the 2 and 4 weeks of elevated IOP with trabeculectomy animals, IOP ratios were evaluated before the trabeculectomy (pre-TRAB), after the trabeculectomy (post-TRAB) and for the whole experimental period (cumulative) (Figure 26-A). For the pre-TRAB analysis, IOP ratios were smaller and further away from 1 as OD ratios 111 Figure 25: Correlation of OD ratio predictors with OD ratios in NO T R A B animals. Comparison of predictors for severity of damage with OD ratios of NO T R A B animals following 2 and 4 weeks of elevated IOP is made. OD ratio values of 1 represent no deprivation and uniform staining in layer 4C. In A) the IOP ratio was compared with actual OD ratios obtained. Values closest to 1 indicate the least discrepancy in IOP between the two eyes. In B) the cupping (cup:disc) ratios of the animals was compared with actual OD ratios obtained. A value of 1 indicates no optic disc cupping differences between the two eyes. 11 Figure 25 Figure 26: Correlation of OD ratio predictors with OD ratios in T R A B animals. Comparison of predictors for severity of damage with OD ratios of T R A B animals following 2 and 4 weeks of elevated IOP. OD ratios of 1 represent no deprivation and uniform staining in layer 4C. In A) the IOP ratio is compared with actual OD ratios obtained. IOP ratios are compared with the pre-TRAB (trabeculectomy) history, post-TRAB history, and the cumulative experimental IOP history. Values closest to 1 indicate the least discrepancy in IOP between the two eyes. In B) the cupping (cup:disc) ratios of the animals are compared with actual OD ratios obtained. A value of 1 indicates no optic disc cupping difference between the two eyes. In C) optic nerve damage is presented. "O" represents no damage, "+" mild damage, and "++" moderate to severe damage. 114 0.00 0.00 0.20 0.40 0.60 0.80 1.00 OD Ratio • cumulative • pre-TRAB • post-TRAB Linear (post-TRAB) Linear (cumulative) Linear (pre-TRAB) 0.00 0.20 0.40 0.60 0.80 1.00 OD Ratio 0.00 0.20 0.40 0.60 0.80 1 00 OD Ratio Figure 26 approached 1. In contrast, the post-TRAB analysis yielded results where the IOP increased as the OD ratio approached 1. It is important to note that the IOP still departs from 1 since the ratios exceeds 1 as OD ratios approaches 1. IOP ratios greater than 1 represent histories where the control eye had greater IOP areas compared to the glaucomatous eye. Finally, in the cumulative IOP ratio analysis animals with OD ratios of 1 had lower IOP ratios compared to animals with OD ratios around 0.65. In the comparison between cupping ratio with OD ratio, cupping ratios closer to 1 yielded OD ratios closer to 1 (Figure 26-B). These results suggest that smaller differences in cup:disc ratio between eyes have smaller differences in CO staining between eye bands. Finally, animals subject to 2 and 4 weeks of elevated IOP followed by a trabeculectomy and a minimum of 6 months recovery at normal IOP levels had little or moderate loss of optic nerve axons. However, our results do not suggest a clear relationship in the comparison of optic nerve axonal loss to OD ratio (Figure 26-C). 116 4. Discussion The objective of this study was to examine anatomical correlates of metabolic activity within the primary visual cortex following the lowering of elevated IOP to normal values of <20 mm Hg in the primate model of glaucoma. Metabolic activity was measured as optical density ratios that compared the CO staining intensity of the ocular dominance bands in Layer 4C associated with the non-deprived and deprived eye. CO histochemistry in the primate primary visual cortex was compared between animals that had chronic glaucoma (NO TRAB) or transient glaucoma (TRAB). From the results, evidence for an attenuation of the effects of elevated IOP on metabolic activity within the visual cortex following trabeculectomy was found. In addition, the potential for recovery of metabolic activity in the visual cortex following a survival period of at least 6 months at normal (<20 mm Hg) IOP levels is suggested by the results. Interpretations of the results that would lead to these conclusions will be discussed. A . C O histochemistry detects early glaucomatous change The appearance of light and dark staining in layer 4C of the visual cortex confirmed changes in metabolic activity following elevated IOP. Layer 4C stained uniformly dark in normal animals without deprivation. The alternating dark and light bands observed in the visual cortex in animals after unilateral elevated IOP was due to differential metabolic activity of the two eyes. Deprived eye bands appeared light in CO staining, while non-deprived eye bands appeared dark. These results agree with previous data observing differential CO staining in the brain following chronic elevated IOP (Weber et al., 2000; Yucel et al., 2000; 2001). 117 However, the effects of glaucomatous deprivation were not uniformly observed amongst all of the animals. The effects of monocular deprivation were variable in the visual cortex in the chronic glaucoma group (NO TRAB), after 2 and 4 weeks of IOP consistently above 30-35 mm Hg. Banding was observed in all of the animals, but some animals revealed distinct bands while others revealed very low contrast bands. The absence of obvious bands can suggest that CO activity does not have the capacity to change following such a short period of deprivation. However, this possibility is invalid since distinct bands were observed in animals subject to the same treatment periods. Moreover, previous studies have demonstrated that changes in CO activity can be detected as early as fourteen hours after TTX injection (Trusk et al., 1992). The variability in severity can also be attributed to the pathophysiology of glaucoma due to ocular hypertension. Unlike severe forms of monocular deprivation, loss of retinogeniculocortical afferents due to elevated IOP is a partial and gradual process. Axoplasmic transport is partially interrupted in response to elevated IOP (Anderson and Hendrickson, 1974). Also, there is a selective effect to certain areas of the optic nerve during the early stages of glaucoma (Kitazawa et al., 1977; Harwerth et al., 1992). The variability observed in CO staining in Layer 4C may reflect the partial damage that is characteristic of glaucoma. If animals have varying degrees of glaucomatous damage at the level of the retina, at the termination of the deprivation period, CO staining may also be varied as a result. Other investigators have also observed substantial interanimal differences in response to mild long-term monocular deprivation (Trusk et al., 1990; Wong-Riley, 1994). 118 B. Optical density ratios i . Metabolic activity differences Optical density ratios were useful in evaluating the difference in metabolic activity between deprived and non-deprived eyes. Because optical density relies on CO histochemistry, the length of the reaction time affects it. In this study, a ratio on the optical density was calculated. The ratio has the benefit of effectively normalizing the data and making it resistant to differences that are consistent within an animal caused by varying incubation times. Furthermore, in a control experiment, no significant differences were found in the optical density ratios obtained from sections that were under or over-reacted compared to the normal incubation times. However, this comparison is only applicable within reasonable incubation durations. Extending reaction times may potentially lead to the over reaction of tissue in which cytochrome-c continually reacts with cytochrome oxidase until the reaction product for the deprived eye bands begin to have the same intensity as the non-deprived eye bands. This scenario however is unlikely since the CO reaction solution is known to be light sensitive and expires in reactivity with time. Furthermore, the continual reoxidation of cytochrome-c by cytochrome oxidase is also limited by the concentration of cytochrome-c within the solution. Since our over-reacted control was visibly darker than the normal section without any compromise to the banding pattern, it is assumed that our control is still within the reasonable incubation duration. Therefore, from our analysis, the optical density ratios appear correlated to the degree of difference in CO staining observed in the visual cortex, between ocular dominance bands, rather than to reaction incubation times. 119 i i . Dependence on duration of elevated IOP Optical density ratios vary in accordance to the period of monocular deprivation. OD ratios of 1 represent a uniformly dark stained layer 4C. Animals with shorter periods of deprivation had larger OD ratios, which were closer to the OD ratio of 1 in normal animals. Animals with longer periods of monocular deprivation had smaller OD ratios. This is due to the stronger, and higher contrast, banding pattern reflecting deprived and non-deprived eye activities in layer 4C. It is important to note however that the OD ratio did not fall below 0.52. Therefore, the results suggest that there is a maximum degree of deprivation as evaluated by OD ratios. This is confirmed by the fact that the mean OD ratios of animals within 4, 8 and 16 weeks of elevated IOP have a similar mean OD ratio to animals observed 4 weeks after an optic nerve transection (ONT). An ONT procedure is a very severe form of deprivation, and should produce maximal effects in deprivation because all retinogeniculate afferents is terminated. The amount of deprivation observed in layer 4C after 4 weeks of elevated IOP can be as severe as those observed after ONT. In addition, an OD ratio value of 0.52 suggests that non-deprived eye bands retain some optical density, and that optical densities of deprived eye bands are never less than half of the optical densities measure in the adjacent non-deprived eye bands. Since images are captured by a digital camera and then converted to calibrated grayscale values, it can be argued that inherent colour from the tissue converted to grayscale may be the factor responsible. However, since the OD ratios are measured by comparing bands within the same section of tissue, we assume that any underlying colour artifacts of the tissue will be found in both dark and light bands. This additional grayscale value will be negated when calculating the OD ratio because the mean optical density value of the 120 light band is divided by the mean optical density value of the dark bands. Rather, it is more likely that some mitochondrial background activity remain in the deprived eye bands and may be the factor responsible for the minimum OD ratio. Wong-Riley et al. (1989a) found evidence that mitochondria are temporarily converted to a smaller, less-reactive variety when animals are subject to TTX injection. Since neuronal activity controls energy use and energy generating enzymes like CO (Wong-Riley and Carrol, 1984, Wong-Riley et al., 1989a, 1989b; Di Rocco et al.,' 1989; DeYoe et al., 1995; Hendrickson and Tigges, 1985), it is likely that the optical density values of the deprived eye bands represent neuronal activity in the absence of input from the L G N . It is important to note that the mean NO TRAB ratios after 2 weeks of elevated IOP was closer to normal than those observed at longer periods of deprivation. This observation may be interpreted as the importance of early intervention in glaucoma. Higher optical density ratios represent less difference in metabolic activity between eye bands. Therefore, the results suggest that early intervention may prevent further impact on the visual pathways. Even though interanimal variability in the severity of deprivation, as measured by OD ratios, was observed at all time points, the degree of deprivation on average is less in animals with shorter periods of deprivation. It can be argued however, that the mean OD ratios are an artifact of averaging animals with severe deprivation with animals with no deprivation at all. This situation is not likely because ocular dominance bands were observed in all NO TRAB animals. Even though the OD ratio reported values that were sometimes close to the control animal values of 1, it was known that deprivation was evident since layer 4C was not uniformly stained. 121 In addition to the OD ratio being directly affected by the duration of deprivation, TRAB animals also have an average mean OD ratio higher than those of chronic, NO TRAB animals. This trend is stronger after 4 weeks of elevated IOP. This is very interesting and suggests two possibilities regarding the significance of lowering pressures in glaucoma. The first possibility is that intervention by trabeculectomy limits the extent of damage inflicted by elevated IOP to the degree experienced immediately before surgery. The second possibility is that intervention during the early stages of glaucoma not only stops the disease from progressing, but it reverses the damage inflicted on the neurons of the visual pathway. These two possibilities will be discussed subsequently. C. Comparison of chronic (NO TRAB) and transient (TRAB) glaucoma animals i. Trabeculectomy arrests the progression of glaucomatous effects in the visual cortex The possibility that trabeculectomy limits the extent of deprivation observed in the visual cortex, as measured by OD ratios, to the degree observed prior to surgery is plausible. It is well documented that trabeculectomy is a useful and safe procedure for lowering IOP. Moreover, studies have found that: it is a very effective method for lowering severely elevated IOP, pressures remain low following surgery, that there is only a progressive field loss in 18% of study subjects, and that continuous damage occurs in only one third of eyes with moderate to severe primary open-angle glaucoma (Kidd and O'Connor, 1985; Nouri-Mahdavi et al., 1995). In our study, this dramatic effect was observed by the comparison between our chronic glaucoma (NO TRAB) animals with our transient glaucoma (TRAB) animals. The T R A B animals were subject to 2 or 4 122 weeks of elevated IOP and given a trabeculectomy followed by a survival period of at least 6 months. Cumulatively, each animal in the TRAB group was examined for the duration of at least six and a half months. If the effects of elevated IOP continued following the trabeculectomy, one would expect that differences between the deprived and non-deprived eye bands would be comparable to those of the NO T R A B animals subject to a deprivation period longer than 4 weeks. This was not observed. Even though some animals exhibited banding in layer 4C, the OD ratio values of these animals were much higher than those of NO TRAB animals subject to 8 and 16 weeks of elevated IOP. Testing for significant difference between the NO T R A B and T R A B group following 2 and 4 weeks of elevated IOP indicated that there was no difference between the two populations. However, the NO TRAB group with elevated IOP for 8 and 16 weeks were statistically different from the TRAB group following 2 and 4 weeks of elevated IOP. Together, these results strongly suggest that trabeculectomy does have an impact in arresting or delaying progressive glaucomatous damage in the visual cortex, inflicted during the 2 or 4 weeks of deprivation. These findings contribute to the emphasis on early intervention in glaucoma (Higginbotham, 1994b). To date, evaluation of the efficacy of surgical treatment of glaucoma in slowing or preventing further deterioration of visual function cannot be addressed quantitatively. Moreover, study results cannot be compared directly to individuals without intervention due to ethical restrictions (Nouri-Mahdavi et al., 1995). Since the visual cortex is responsible for central vision processing, intact visual function is reflected in the activity at this level. Ocular dominance columns have been demonstrated by optical imaging, a non-invasive, high spatial resolution approach for studying functional brain activity. However, this 123 study is unique from previous studies in that the results from this study are the first known empirical quantitative demonstration on the benefits of lowering elevated IOP by . trabeculectomy for visual function. In the clinical setting, variable and unknown rates of disease progression are frequently encountered (Nouri-Mahdavi et al., 1995). In our results, it can be interpreted that the variation in OD ratios observed in the TRAB group is due to the deprivation experienced prior to trabeculectomy. This suggests that the sooner IOPs can be lowered in glaucoma, the less the impact will be on the central visual pathways. The severity in deprivation will be arrested at the degree in which intervention occurs. Consistent with these observations, trabeculectomy has the potential to inhibit visual field and optic nerve loss progression in glaucoma (Kidd and O'Connor, 1985; Nouri-Mahdavi et al., 1995). Further, the results from this study may also indicate that any potential secondary damage due to interruptions in cortical activity is not severe enough to motivate a cycle of progressive secondary damage. ii. The visual cortex recovers from metabolic activity depression following trabeculectomy The potential for recovery is suggested by the results from our study. Unfortunately, mean differences comparing our NO T R A B (no recovery) and T R A B (recovery) groups, with the same durations of elevated IOP, did not prove to be statistically significant, therefore this portion of our study can only be discussed in terms of general trends of mean OD ratios. These results can only be interpreted as preliminary 124 results. Limitations of this study and future suggestions for improving our study will be discussed later. The lack of contrast observed in layer 4C of the T R A B animals can be explained by three possibilities. The first explanation proposes that the animal had a deprivation effect, but metabolic activity was re-established subsequent to lowered IOPs. This recovery phenomenon is only valid under the assumption that retinal ganglion cell death has not occurred. The second possibility proposes that the initial deprivation effect was not strong enough to induce the ocular dominance bands detected by CO staining. Finally, the third, and most unlikely possibility proposes that the non-deprived eye lost activity and thus its optical density value was reduced after trabeculectomy, causing an increase in OD ratio. In the TRAB animals that did not have dramatic bands in layer 4C, the second possibility may seem likely, however evidence for the first explanation exists from the results of animals with well defined ocular dominance bands in layer 4C. Obvious bands were observed in all of the animals within the group subject to 4 weeks of elevated IOP group (TRAB). However, the contrast between the bands in these animals were not as dramatic as those of animals without trabeculectomies performed. This observation is reinforced by the OD ratio measurements. Animals with obvious banding had higher OD ratios compared to the OD ratios of NO T R A B animals with comparable banding patterns. This indicates that the CO reactivity difference between the deprived and non-deprived eye bands in transient glaucoma animals is closer to that of a normal animal's, than of an animal with chronic glaucoma. After 4 weeks of elevated IOP, the mean OD ratio was higher for the animals with trabeculectomy compared to animals without. Further, the error ranges for these means 125 were distinct from each other. A higher OD ratio can be interpreted as recovery of metabolic activity to near normal values. After 2 weeks of elevated IOP, the relationship is not as clear. The T R A B mean was found to be a lower OD ratio compared to the NO TRAB group. This observation may be attributed to the pathophysiology of glaucoma. It is a gradual, progressive disease and the extent of the impact of elevated IOP varies between individuals. Animals that are inherently more resistant to deprivation caused by elevated IOP may affect the results of this comparison. In addition, the exceptionally low OD ratio of one animal subject to trabeculectomy (see Fig 23) may contribute to the variable results observed after 2 weeks of elevated IOP. Assuming that recovery is dependent on the presence of intact retinal ganglion cells or retinogeniculocortical afferents, the extremely low OD ratio observed may be due to the fact that the extent of deprivation exceeded this point. Consistent with this hypothesis, Shirakashi et al. (1998) found that the degree of deterioration in cupping from baseline before the induction of glaucoma may be an important determinant of the degree of cupping reversal during subsequent reductions in IOP. As a result, this animal may have experienced too much irreversible damage prior to trabeculectomy. In consequence, results from this animals caused a lower final mean OD ratio for the experimental group. Further, results from this study suggest that the potential recovery effect may be sensitive to early stages of deprivation. In the comparison of the plot profiles of 2 and 4 weeks T R A B animals (Fig 15 and 18), those that were subject to trabeculectomy after 2 weeks of elevated IOP have a greater capacity to recover the metabolic activity in deprived eyes to control levels compared to animals after 4 weeks of elevated IOP. The range between maximum and minimum optical density values, representing the 126 alternating deprived and non-deprived eye bands, is smaller after 2 weeks of elevated IOP compared to after 4 weeks of elevated IOP. The plot profiles of animals after 2 weeks of elevated IOP were flatter looking than those of animals after 4 weeks of elevated IOP. Again, this effect is variable between animals and therefore should be investigated further. The inconsistent results in this model of glaucoma are not unique. Interanimal variation was also found among animals with long-term monocular lid suture. Trusk et al. (1990) suggested that this variation may be due to differences in eyelid thickness or to some form of functional recovery. D. Limitations to the study Several assumptions were made in this study that are critical to the validity of our conclusions from this study. First, the measurements of CO reactivity in layer 4C operate under the assumption that differences between layer 4Cct and layer 4Cp are not significant enough to impact the OD ratios. Since tangential sections are not always cut directly parallel to the layers of the cortex, it is very likely that tissue sections contain both layers of 4C. Layer 4Ca receives mainly magnocellular input whereas layer 4Cp receives mainly parvocellular input. Crawford et al. (2000) observed a pattern of uniformly weaker staining in layer 4CP that was consistent with varying degrees of visual field loss after one year of elevated IOP. This differential staining was observed after a few months of elevated IOP. Publication on shorter periods of elevated IOP do not exist, therefore it is uncertain whether, this preferential effect would be observed in our experiment. However, differences between layer 4Cct and layer 4Cp were not found in studies from our lab with animals after 1 and 2 months of elevated IOP (Lam et al., 127 unpublished data). It would be ideal to be able to obtain OD ratios from only layer 4Cct or layer 4Cp, however it is difficult since obvious differences between these two layers could not be seen. Further, layer 4C is approximately 36 pm thick (Peters, 1994) and 50 pm sections were used in this study, therefore data was already limited without the segregation of the two layers. As a preventative measure, OD ratios were obtained from bands directly adjacent to each other with uniform staining. Patchy areas were avoided so that measurements would not be taken from potentially different layers. Since OD ratios compared adjacent bands directly, variables such as patchy staining or incomplete layers should be avoided so that the assumption can be made that the OD ratio is measured from one layer. It can be argued that OD ratio measurements were obtained from both layers of 4C because bands were picked randomly over several sections of tissue. Since the final OD ratio is the average of ten separate OD ratios, we assume that i f differences exist between layer 4Ca and layer 4C(3, measurements were obtained from both layers. The random selection of bands from different sections allows us to assume that the two layers are equally represented in our final average OD ratio for the animal. In our analysis, we must also exclude the possibility that the effects that we observed were due to cell shrinkage. Past studies have led us to exclude this potential. In the TTX induced model of monocular deprivation, only type C neurons in puffs (of layer 2/3) were found to have a reduced mean cell size (Luo et al., 1989). In addition, evidence for cell shrinkage in layer 4C only exists for animals subject to monocular enucleation (Matthews et al., 1960). The animals in our study did not experience deprivation as . 128 severe as an enucleation, therefore we can assume that cell shrinkage will not be a factor that will affect our results within a deprivation period of 2, 4, 8 and 16 weeks. Shrinkage of cortical tissue during processing is also a possibility in this experiment. However, shrinkage effects should be negligible. The sections of tissue from one animal were processed simultaneously, OD measurements were obtained from several sections, and OD ratios were measured from bands directly adjacent to each other one section. Any potential shrinkage should occur by the same factor throughout the section, therefore the optical density measurements of the bands should vary by the same degree if any variation did exist. E . Future directions i. Alternative criteria for evaluating glaucomatous damage in the primate model While this study has provided evidence supporting the efficacy of trabeculectomy in lowering elevated IOP, many questions remain due to the limitations of the experimental design. First, the variability in OD ratio observed in response to elevated IOP needs to be addressed. By increasing the sample size, a better approximation into the general trend of each group can be evaluated. However, this may not be completely feasible as the primate model is expensive and laborious. An alternative would be to redefine the criteria for determining the severity of deprivation so that direct comparisons between TRAB and NO T R A B groups can be made more accurately. In the primate model of glaucoma, the severity of glaucoma experience byan animal is usually determined by the length of time elevated IOP was measured. However, it is evident from this study that this is not a strong enough predictor during the 129 very early stages of glaucoma; since NO TRAB animals did not all have high contrast bands following 2 and 4 weeks of elevated IOP. In addition, animals subject to trabeculectomy experience a period at normal levels of IOP before they are sacrificed. The period at normal levels of IOP may potentially have an effect on the metabolic activity observed in the visual cortex. Moreover, these animals do not fit into the traditional model for glaucoma where the animals experience elevated IOPs until they are sacrificed. Traditional methods in grouping animals by duration of elevated IOP may therefore be inappropriate for the comparisons made in this study. Additional information such as data on the cup:disc ratios, optic nerve atrophy, and visual field function may serve as better predictors for the severity of deprivation observed in glaucoma. A rough preliminary analysis for some of these predictors was conducted on available data for animals subject to 2 or 4 weeks of elevated IOP. While there was a large spread of data points in the graphs comparing IOP ratios with OD ratios, and cupping ratios with OD ratios, glaucomatous eyes with comparable IOP and cup:disc ratios to those of the control eyes', tend to have a closer to normal OD ratio (Figure O and P). The trend was stronger for cupping ratios compared to IOP ratios. These findings were not surprising since Quigley and Pease (1996) observed that the optic disc changed in response to elevated IOP. It is also interesting to note that these trends were better correlated in NO T R A B animals compared to T R A B animals. For example, in the comparison of cupping ratios to OD ratios for T R A B animals, data points were clearly distributed in which higher cupping ratios were correlated with higher OD ratios. However, for NO T R A B animals, 130 there was a larger spread of cupping ratio data points at OD ratios closest to 1. Again, this observation could support our hypothesis that intervention by lowering IOPs may potentially allow metabolic activity to recover within the visual cortex, even under conditions where significant glaucomatous cupping damage has already occurred at the level of the eye. Consistent to our observations, Quigley and Addicks (1980) also reported observations that optic disc cup size increase precedes visual field loss in many eyes experiencing early glaucoma. A trend is harder to determine in the comparison of rough data determining the extent of optic nerve damage to OD ratios. However, studies in the past have reported significant linear correlation between changes in cup:disc ratios with change in the number of optic nerve fibers. An increase in cup:disc ratio of 0.1 was associated with a 10% loss of optic nerve fibers (Varma et al., 1992). Histomorphometric analysis data including cross-sectional areas of optic nerve tissue components, nerve fiber density and nerve fiber numbers may be more suitable for our study (Yucel et al., 1999). In addition, evaluating the effects of elevated IOP for TRAB animals was also complicated since the glaucomatous eye changed from increased pressures to extremely lowered pressures in which conditions sometimes existed where the glaucomatous eye had lower eye pressures compared to the control eye. This may have had an impact on recovery of metabolic activity. By redefining the criteria for grouping animals, experimental groups can be divided more appropriately so that clearer comparisons between T R A B and NO T R A B animals can be made. 131 ii. Future applications for the evaluation of metabolic activity by OD ratios From this study, we determined that the comparison of metabolic activity between eye bands, with OD ratios, was an effective method for evaluating the severity of deprivation in the visual cortex. These findings could be applied as an alternative method for evaluating visual field function in clinical glaucoma. Presently, the effectiveness of pressure lowering procedures is evaluated by comparing factors such as tonometry, angles of the eye, cup:disc ratios, and perimetry testing. Perimetry testing, and other specialized tests of visual function, are largely complicated. Unfortunately, these methods involve long periods of patient monitoring with a specified protocol, in which only a small number of individuals undergo confirmed glaucomatous progression over time. Furthermore, a high degree of variability is present (Johnson, 1997). Metabolic activity differences between the deprived and non-deprived eye bands in the visual cortex can be demonstrated according to the results from our study. Strategies into observing this in vivo may become a useful tool. Currently two noninvasive methods are available for measuring metabolic activity in the brain. Functional imaging of brain activity by PET and fMRI however are limited in spatial 3 3 resolution at 5-10 mm and 3 mm respectively. Viewing of ocular dominance columns requires a higher resolution. Blood-oxygenation level-dependent (BOLD) fMRI appears more promising as a tool for localizing brain function in vivo. However, the ability to map cortical columnar structure is highly controversial. BOLD fMRI has the ability to indicate a pattern of overall activation in the visual cortex, but is less suited to discriminate active from inactive columns (Kim et al., 2000). Until these issues are resolved, a non-invasive method for evaluating differential metabolic activity between 132 eye bands is not yet possible. However, optical imaging is still available as an alternative method for measuring OD ratios in an in vivo model. Grinvald et al. (1991) found that this technique allowed the mapping of ocular dominance columns, which are 400-500 um in width, in the V I of awake monkeys. In addition, Vnek and colleagues (1999) further refined this technique to yield imaging of 100-200 urn functional domains in the visual cortex. While this imaging technique is not feasible in humans, it can be applied in the primate model of glaucoma. The ability to observe changes in metabolic activity in vivo may allow a more comprehensive understanding about the effects of IOP on visual activity within the primary visual cortex. 133 F. Conclusions from this study: 1. Unilateral elevated IOP for 1-4 weeks resulted in light CO stained ocular dominance bands associated with the glaucomatous eye. 2. Optical density ratios were lower for animals subject to longer periods of elevated IOP compared to animals with shorter periods of 2 and 4 weeks of elevated IOP. 3. Short-term chronic glaucoma (less than 2 weeks) and transient glaucoma animals had OD ratios closer to the normal OD ratio of 1. 4. Optical density ratios of animals subject to chronic glaucoma had significantly lower OD ratios compared to those with transient glaucoma. -Pressure-lowering therapies such as a trabeculectomy are an effective intervention for preventing the progressive loss of visual cortical activity.. -Potential bi-directional secondary damage does not significantly impact the visual cortex following trabeculectomy. 5. Optical density ratios improved (approached 1) in animals with transient glaucoma compared to animals with chronic glaucoma after 4 weeks of elevated IOP. -This result suggests that lowering pressures during the early stages of glaucoma may reverse the loss of metabolic activity of the neurons in layer 4C belonging to the deprived eye. 134 5. References Ahmad A , Spear PD. Effects of aging on the size, density and number of rhesus monkey lateral geniculate neurons. J Comp Neurol. 1993;334:631-643. Airaksinen PJ, Alanko HI. Effect of retinal nerve fiber loss on the optic nerve head configuration in early glaucoma. Graefes Arch Clin Exp Ophthalmol 1983;220:190-196. Araie M , Sekine M , Suzuki Y , Koseki N . Factors contributing to the progression of visual field damage in eyes with normal-tension glaucoma. Ophthalmology. 1994:101 (8): 1440-1444. Armaly MF. The genetic determination of ocular pressure in the normal eye. Arch Ophthalmol.'1967;78:187-192. Armaly MF. Ocular pressure and aqueous outflow facility in siblings. Arch Ophthalmol. 1968;80:354-360. Anderson DR. Glaucoma: The damage caused by pressure. X L V I Edward Jackson Memorial Lecture. A m J Ophthalmol 1989;108:485-495. Anderson DR, Hendrickson A. Effect of intraocular pressure on rapid axonal transport in monkey optic nerve. Invest Ophthalmol. 1974;13:771-783. Bachelard HS. Energy utilized by neurotransmitter, in Inguan DH, Lassen N A (eds): Brain Work, Alfred Symposium VIII. New York: Academic Press, 1975.79-81. Bailey C H , Gouras P. The Retina and Phototransduction. In. Kandel ER, Schwartz J. Principles of Neural Science. 2nd ed. New York: Elsevier Science, 1985. Berne R M , Levy M B (eds.). Physiology. St. Louis: Mosby, 1983. Boyd J, Matsubara J. Laminar and columnar patterns of geniculocortical projections in the cat: relationship to cytochrome oxidase. J Comp Neurol. 1996;19;365(4):659-682. Carroll EW, Wong-Riley MTT. Quantitative light and electron microscopic analysis of cytochrome oxidase-rich zones in the striate cortex of the squirrel monkey. J Comp Neurol. 1984;222:1-17. Carroll EW, Wong-Riley M . Recovery of cytochrome oxidase activity in the adult macaque visual system after termination of impulse blockage due to tetrodotoxin. Soc Neurosci Abst. 1987a;13:1046. Carroll EW, Wong-Riley MTT. Neuronal uptake and laminar distribution of tritiated aspartate, glutamate, gamma-aminobutyrate and glycine in the prestriate cortex of 135 squirrel monkeys: correlation with levels of cytochrome oxidase activity and their uptake in area 17. Neuroscience. 1987b;22:395-412. Cartwright MJ , Anderson DR. Correlation of asymmetric damage with asymmetric intraocular pressure in normal-tension glaucoma (low-tension glaucoma). Arch Ophthalmol. 1988;106(7):898-900. Chaturvedi N , Hedley-Whyte E, Dreyer EB. Lateral geniculate nucleus in glaucoma. Am J Ophthalmol. 1993;116:182-188. Chauhan BC, Drance SM. The relationship between intraocular pressure and visual field progression in glaucoma. Graefes Arch Clin Exp Ophthalmol. 1992;230(6):521-526. Congdon N , Wang F, Tielsch JM. Issues in the epidemiology and population-based screening of primary angle-closure glaucoma. Surv Ophthalmol. 1992;36(6):411-423. Cowan W M . Anterograde and retrograde transneuronal degeneration in the central and peripheral nervous system. In Nauta WJH, Ebbesson SOE (eds.): Contemporary Research Methods in Neuroanatomy. Berlin: Springer 1970. p217-249. Cowey P, Stoerig A. Blindsight: neurons and behaviour. Prog Brain Res. 1993;95:445-459. Crawford M L , Harwerth RS, Smith EL, Shen F, Carter-Dawson L. Glaucoma in primates: cytochrome oxidase reactivity in parvo- and magnocellular pathways. Invest Ophthalmol Vis Sci. 2000;41(7):1791-802. Crawford M L J , Harwerth RS, Smith EL, Mills S, Ewing B. Experimental glaucoma in primates: Changes in cytochrome oxidase blobs in V I cortex. Invest Ophthalmol Vis Sci. 2001;42(2)358-364. Crichton A , Drance SM, Douglas GR, Schulzer M . Unequal intraocular pressure and its relation to asymmetric visual field defects in low-tension glaucoma. Ophthalmology. 1989;96(9): 1312-1314. Dacey D, Peterson MR. Dendritic field size and morphology of midget and parasol ganglion cells in the human retina. Proc Natl Acad Sci. 1992;89:9666-9670. Dandona L, Hendrickson A, Quigley HA. Selective effects of experimental glaucoma on axonal transport by retinal ganglion cells to the dorsal lateral geniculate nucleus. Invest Ophthalmol Vis Sci. 1991 ;32(5): 1593-1599. deJong N , Greve EL, Hoyng PFI, Geilssen HC: Results of a filtering procedure in low tension glaucoma. Int Ophthalmol 1989:13:131-138. 136 DeYoe EA. Trusk TC. Wong-Riley MT. Activity correlates of cytochrome oxidase-defined compartments in granular and supragranular layers of primary visual cortex of the macaque monkey. Visual Neuroscience. 1995;12(4):629-39. Di Rocco RJ, Kageyama GH, Wong-Riley MTT. The relationship between CNS metabolism and cytoarchitecture: A review of 14C-deoxyglucose studies with correlation to cytochrome oxidase histochemistry. 1989;13:81-92. Dreyer EB, Zurakowski D, Schumer RA, Podos SM, Lipton SA. Elevated glutamate levels in the vitreous body of humans and monkeys with glaucoma. Arch Ophthalmol. 1996;114(3)299-305. Epstein DL, Krug JH, Hertzmark E, et al. A long-term clinical trial of timolol therapy versus no treatment in the management of glaucoma suspects. Ophthalmology. 1989;96:1460-1467. Erenciska M , Silver IA. ATP and brain function. J Cereb Blood Flow Metab. 1989;9:2-19. Fechtner RD, Weinreb RN. Mechanisms of optic nerve damage in primary open angle glaucoma. Surv Ophthalmol 1994;39:23-42. Finley SK, Kritzer MF. Immunoreactivity for Intracellular Androgen Receptors in Identified Subpopulations of Neurons, Astrocytes and Oligodendrocytes in Primate Prefrontal Cortex. J Neurobiol. 1999;40:446-457. Fitzpatrick D, Lund JS, Blasel GG. Intrinsic connections of macaque striate cortex. Afferent and efferent connections of lamina 4C. J Neurosci. 1985;5:3329-3349. Freeman RD, Bradley A. Monocularly deprived humans: Nondeprived eye has supernormal vernier acuity. J Neurophysiol. 1980;43:1645-1653. Gaasterland D, Kupfer C. Experimental glaucoma in the rhesus monkey. Investigative Ophthalmology. 1974;13(6):455-457. Gaasterland D, Tanishima T, Kuwabara T. Axoplasmic flow during chronic experimental glaucoma, I, light and electron microscopic studies of the monkey optic nerve head during development of glaucomatous cupping. Invest Opthalmol Vis Sci. 1978; 17:838-851. Garatt S (ed): Primary Open-Angle Glaucoma, Preferred Practice Plan. San Francisco: American Academy of Ophthalmology, 1989. Gilbert CD, Wiesel TN. Morphology and intracortical projections of functionally characterized neurones in the cat visual cortex. Nature 1979;280:120-125. 137 Glaucoma Laser Trial Research Group. The glaucoma laser trial (GLT) 2. Results of argon laser trabeculoplasty versus topical medicines. Ophthalmology 1990; 97:1403-1413. Glees P, Le Gros Clark WE. The termination of optic fibers in the lateral geniculate body of the monkey. J Anat 1941;75:295-308. Glovinsky Y , Quigley HA, Dunkelberger GR. Retinal ganglion cell loss is size dependent in experimental glaucoma. Invest Ophthalmol Vis Sci. 1991;32:484-490. Glovinsky Y , Quigley HA, Pease M E . Foveal ganglion cell loss is size dependent in experimental glaucoma. Invest Ophthalmol Vis Sci. 1993;34:395-400. Goldby, F. A note on transneuronal atrophy in the human lateral geniculate body. J Neurol Neurosurg Psychiatry 1957;20:202-207. Graham PA. Epidemiology of simple glaucoma and ocular hypertension. Br J Ophthalmol. 1972;56(3):223-229. Grant M . Further studies on facility of flow through the trabecular meshwork. Arch Ophthalmol. 1958;60:523-533. Grant W M , Burke JF. Why do some people go blind in glaucoma? Ophthalmology. 1982;89:991-998. Grinvald A , Frostig RD, Siegel R M , Bartfeld E. High-resolution optical imaging of functional brain architecture in the awake monkey. Proc Natl Acad Sci USA. 1991;88:11559-11563. Greve EL, Dake CL. Four-year follow-up of a glaucoma operation. Prospective study of the double flap Scheie. Int Ophthalmol.l979;l:139-145. Gupta N , Yucel Y H . Glaucoma and the Brain. J Glauc. 2001;10(Suppll)S28-29. Harwerth RS, Smith EL III, DeSantis L. Visual fields of monkeys with experimental glaucoma. Invest Ophthalmol Vis Sci. 1992;33:1162. Heijl A . Perimetry, in Kaufman PL, Mittag TW (eds.): Textbook of Ophthalmology: Glaucoma, Vol . 7. London: Mosby, 1994. 5.1-5.2. Hendrickson A E , Tigges M . Enucleation demonstrates ocular dominance columns in Old World macaque but not New World squirrel monkey visual cortex. Brain Res. 1985;333:304-344. Hendrickson A E , Hunt SP, Wu JL. Immunocytochemical localization of glutamic acid decarboxylase in monkey striate cortex. Nature. 1981;292:605-607. 138 Hendry SH, Jones EG. Reduction in number of immunostained GABAergic neurons in deprived-eye dominance columns of monkey area 17. Nature. 1986;320(6064):750-753. Hendry SH, Jones EG. Activity-dependent regulation of G A B A expression in the visual cortex of adult monkeys. Neuron. 1988;1:701-712. Hendry SH, Yoshioka T. A neurochemical^ distinct third channel in the macaque dorsal lateral geniculate nucleus. Science.1994;264(5158):575-577. Hevner RF, Wong-Riley MTT. Regulation of cytochrome oxidase protein levels by functional activity in the macaque monkey visual system. J Neurosci. 1990; 10(4): 1331 -1340. Higginbotham EJ. Clinical presentation of primary open-angle glaucoma, in Kaufman PL, Mittag TW (eds.): Textbook of Ophthalmology: Glaucoma, Vol . 7. London: Mosby, 1994a. 8.34. Higginbotham EJ. Tonometry and Tonography, in Kaufman PL, Mittag TW (eds.): Textbook of Ophthalmology: Glaucoma, Vol . 7. London: Mosby, 1994b. 3.1. Higginbotham EJ. Ophthalmoscopy: Examination Techniques, in Kaufman PL, Mittag TW (eds.): Textbook of Ophthalmology: Glaucoma, Vol . 7. London: Mosby, 1994c. 4.1. Hitchings RA, Spaeth GL. The optic disc in glaucoma. I: classification. Br J Ophthalmol. 1976;60:778. Holmin C, Storr-Paulsen A: The visual field after trabeculectomy. A follow up study using computerized perimetry. Act Ophthalmol 1984;62:230-234. Horton JC. Cytochrome oxidase patches: A new cytoarchitectonic feature of monkey visual cortex. 1984;304:199-253. Horton JC, Hocking DR. Intrinsic variability of ocular dominance column periodicity in normal macaque monkey. J Neurosci. 1996; 16(22)7228-7239. Horton JC, Hubel DH. Regular patchy distribution of cytochrome oxidase staining in primary visual cortex of macaque monkeys. Nature (Lond) 1981;292:762-764. Hoskins HD Jr, Kass M A . Angle-closure glaucoma with papillary block, in Hoskins HD Jr, Kass M A (eds): Becker-Shaffer's Diagnosis and Therapy of the Glaucomas, ed 6. St. Louis: C V Mosby, 1989, 208-237. Hubel DH, Wiesel TN. Receptive fields, binocular interaction and functional architecture in the cat's visual cortex. J. Physiol. (Lond.) 1962:160:106-154. 139 Hubel DH, Wiesel TN. Receptive fields and functional architecture in two non-striate visual areas (18 and 19) of the cat. J Neurophysiol 1965;28:229-289. Hubel DH, Wiesel TN. Laminar and columnar distribution of geniculocortical fibers in macaque monkeys. J Comp Neurol. 1972;146:421-450. Hubel DH, Wiesel TN. Ferrier Lecture: Functional architecture of macaque monkey visual cortex. Proc. R. Soc. Lond. (Biol.) 1977;198:1-59. Humphrey A L , Hendrickson AE. Background and stimulus-induced patterns of high metabolic activity in the visual cortex (area 17) of the squirrel and macaque monkey. J Neurosci. 1983;3(2)345-358. Johnson CA. Visual function measures in experimental glaucoma. J Glaucoma. 1997;6(6):351-352. Johnson L V . Tonographic survey. Am J Ophthalmol. 1966;61:680-689. Johnson H, Cowey A. Transneuronal retrograde degeneration of retinal ganglion cells following restricted lesions of striate cortex in the monkey. Exp Brain Res. 2000;132(2):269-75. Jonas JB, Gusek GC, Guggenmoos-Holzmann I, Naumann GOH. Variability of the real dimensions of normal human optic discs. Graefes Arch Clin Exp Ophthalmol. 1998; 226:332. Jonas JB, Gusek GC, Naumann GO. Optic disc morphometry in chronic primary open-angle glaucoma. I. Morphometric intrapapillary characteristics. Graefes Arch Clin Exp Ophthalmol. 1988;226(6):522-530. Jonas JB, Gusek GC, Naumann GOH. Optic disc, cup and neuroretinal rim size, configuration and correlation in normal eyes. Invest Ophthalmol Vis Sci. 1998;29:1151. Jonas JB, Fernandez M C , Sturmer J. Pattern of glaucomatous neuroretinal rim loss. Ophthalmology. 1993;100(l):63-68.. Kandel, ER. Processing of Form and Movement in the Visual System. In. Kandel ER, Schwartz J. Principles of Neural Science. 2nd ed. New York: Elsevier Science, 1985. Kanski J, McAllister JA, Salmon JF. Glaucoma: A colour manual of diagnosis and treatment 2 n d edition. Butterworth Heinemann, Oxford, Boston. 1996. Kass M A , Gordon MO, Hoff MR, Parkinson JM, Kolker A E , Hart W M Jr, Becker B. Topical timolol administration reduces the incidence of glaucomatous damage in ocular hypertensive individuals. Archives of Ophthalmology. 1989;107(11): 1590-1598. 140 Kaufman PL, Mittag TW. Medical Therapy of Glaucoma, in Kaufman PL, Mittag TW (eds.): Textbook of Ophthalmology: Glaucoma, Vol . 7. London: Mosby, 1994. 9.7-9.29. Kidd M , O'Connor. Progression of field ioss after trabeculectomy: A five-year follow-up. Br J Ophthalmol 1985;69:827-831. Kim D, Timothy Q. Duong TQ, Kim S. High-resolution mapping of isoorientation columns by fMRI. Nature Neuroscience. 2000;3(2)164-169. Kitazawa Y , Horie T, Aoki S, et al. Untreated ocular hypertension. Arch Ophthalmol. 1977;95:1180-1184. Klein BE, Klein R. Intraocular pressure and cardiovascular risk variables. Arch Ophthalmol. 1981;99(5):837-839. Kolker, A E . Visual prognosis in advanced glaucoma: A comparison of medical and surgical therapy for retention of vision in 101 eyes with advanced glaucoma. Trans A m Ophthalmol Soc. 1977;75:539-554. Krupin T. Setone in glaucoma surger, in Waltman ST, Keates RH, Hoyt CS et al (eds). Surgery of the Eye. New York. Churchill Livingstone, 1988, 377-385. Krupin T, Rosenberg LF, Ruderman JM. Drainage Implants, in Kaufman PL, Mittag TW (eds.): Textbook of Ophthalmology: Glaucoma, Vol . 7. London: Mosby, 1994. 9.62-9.63. Kuffler SW. Discharge patterns and functional organization of mammalian retina. J. Neurophysiol. 1953;16:37-68. Kupfer C. The distribution of cell size in the lateral geniculate nucleus of man. J Neuropathol Exp Neurol. 1965;24:653-661. Lam D Y , Kaufman PL, Gabelt BT, To EC, Matsubara JA. Neurochemical correlates of cortical plasticity after unilateral elevated intraocular pressure in a primate model of glaucoma. Investigative Ophthalmology & Visual Science. 2003;44(6):2573-2581. Lamping K, Bellows R, Hutchinson T, Afran S. Long term evaluation of initial filtration surgery. Ophthalmology. 1986;92:91-101. Lee PY, Podos SM, Howard-Williams JR, Severin C H , Rose A D , Siegel MJ . Pharmacological testing in the laser-induced monkey glaucoma model. Curr Eye Res. 1985;4(7):775-781. LeVay S, Wiesel TN, Hubel DH. The development of ocular dominance columns in normal and visually deprived monkeys. J Comp Neurol. 1980;191:1-51. 141 Leventhal A G , Rodieck RW, Dreher B. Retinal ganglion cell classes in the Old World monkey: morphology and central projections. Science. 1981 ;213(4512):1139-1142. Leydhecker W: Is glaucoma therapy useless? in Kriegelstein GK, Leydhecker W (eds): Glaucoma Update II. Berlin: Springer-Verlag, 1983, 95-102. Liu S, Wong-Riley M . Quantitative light- and electron- microscopic analysis of cytochrome-oxidase distribution in neurons of the lateral geniculate nucleus of the adult monkey. Vis. Neurosci. 1990;4(3):269-287. Livingstone MS, Hubel DH. Thalamic inputs to cytochrome oxidase-rich regions in monkey visual cortex. Proc Natl Acad Sci USA. 1982;79:6098-6101. Livingstone MS, Hubel DH. Anatomy and physiology of color system in the primate visual cortex. J. Neurosci. 1984;4:309-356. Lowe RF. Primary creeping angle-closure glaucoma. Br J Ophthalmol. 1964;48:544. Lowry OH. Energy metabolism in brain and its control. In. Ingvar D H , Lassen N A (eds.): Brain Work Benzon Symposium VIII. New York: Academic Press. 1975.48-64. Lowry OH, Roberts NR, Leiner K Y , Wu M L , Fair A L , Albers RW. The quantitative histochemisty of brain III. Ammon's horn. J Biol Chem. 1954;207:39-49. Luo X G , Hevner RF, Wong-Riley MTT. Double labeling of cytochrome oxidase and. gamma aminobutyric acid in central nervous system neurons of adult cats. J Neurosci. Methods. 1989;30:189-195. Lutjen-Drecoll E, Rohen JW. The Normal Anterior Segment: Section 1- Anatomy of Aqueous Humor Formation. In Kaufman PL, Mittag TW (eds.): Textbook of Ophthalmology: Glaucoma, Vol . 7. London: Mosby, 1994.1.3. Mao L K , Steward WC, Shields M B : Correlation between intraocular pressure control and progressive glaucomatous damage in primary open-angle glaucoma. Am. J. Ophthalmol. 1991;111:51-55. Matthews, MR, Cowan W M , Powell TPS. Transneuronal cell degeneration in the lateral geniculate nucleus of the macaque monkey. J Anat. 1960;94:145-169. Mawe G M , Gershon M D . Functional heterogeneity in the myenteric plexus: Demonstration using cytochrome oxidase as a verified cytochemical probe of the activity of individual enteric neurons. J Comp Neurol. 1986;249:381-391. Migdal C, Hitchings R. Control of chronic simple glaucoma with primary medical, surgical and laser treatment. Trans Ophthalmol Soc U K 1986;105:653-656. 142 Minckler DS. The organization of nerve fiber bundles in the primate optic nerve head. Arch Opthalmol. 1980;98(9): 1630-1636. Minckler DS, Bunt A H , Johanson GW. Orthograde and retrograde axoplasmic transport during acute ocular hypertension in the monkey. Invest Ophthalmol Vis Sci. 1977:16(5):426-441. Minckler DS, Heuer DK, Hasty B et al. Clinical experience with the singleplate Molteno implant in complicated glaucomas. Ophthalmology. 1988;95:1181-1188. Minckler DS, Spaeth GL. Optic nerve damage in glaucoma. Surv Ophthalmol. 1981;26:128-148. Mjaatveldt A E , Wong-Riley MTT. The relationship between synaptogenesis and cytochrome oxidase activity in Purkinje cells of the developing rat cerebellum. J Comp Neurol. 1988;277:155-182. Molteno A C B . Use of Molteno implants to treat secondary glaucoma in Cairns JE (ed): Glaucoma. Vol 1. New York: Grune & Stratton, 1986, 211-238. Moran J, Gordon B. Long-term visual deprivation in human. Vision Res. 1982;22:27-36. Morgan JE. Optic nerve head structure in glaucoma: astrocytes as mediators of axonal damage. Eye. 2000;14(Pt3b):437-444. Nickells RW. Retinal ganglion cell death in glaucoma: the how, the why. And the maybe. J Glaucoma. 1996;5:345-356. Nilsson SFE, Bi l l A. Physiology and Neurophysiology of Aqueous Humor Inflow and Outflow. . in Kaufman PL, Mittag TW (eds.): Textbook of Ophthalmology: Glaucoma, Vol. 7. London: Mosby, 1994. 1.19. Nouri-Mahdavi K, Brigatti L, Weitzman M , Caprioli J. Outcomes of trabeculectomy for primary open-angle glaucoma. Ophthalmology. 1995;102(12):1760-1769. O'Brien C, Schwartz B, Takamoto T, Wu DC. Intraocular pressure and the rate of visual field loss in chronic open-angle glaucoma. Am J Ophthalmol. 1991 ;111(4):491-500. Odberg T. Visual field prognosis in advanced glaucoma. Acta Opthalmologica 1987;65(suppl):27-29. Pearson HE. Stoffler DJ. Retinal ganglion cell degeneration following loss of postsynaptic target neurons in the dorsal lateral geniculate nucleus of the adult cat. Experimental Neurology. 1992;! 16(2): 163-71. 143 Peters, A. The Organization of the Primary Visual Cortex in the Macaque, in Peters A , Rockland KS (eds): Cerebral Cortex, New York:Plenum Press, 1994. Perkins ES. Hand-held applanation tonometer. Br J Opthalmol. 1965;49(11):591-593. Perkins TW. Treatment of Glaucoma by Lowering Intraocular Pressure, in Kaufman PL, Mittag TW (eds.): Textbook of Ophthalmology: Glaucoma, Vol . 7. London: Mosby, 1994. 9.1-9.6. Portney GL. Photogrammetric analysis of the three-dimensional geometry of normal and glaucomatous optic cups. Trans Am Acad Ophthalmol Otol. 1976;81:239. Quigley HA. Open-angle glaucoma. N Engl J Med. 1993;328:10797-1106. Quigley HA, Addicks E M . Chronic experimental glaucoma in primates. II. Effects of extended intraocular pressure on optic nerve head and axonal transport. Invest Ophthalmol Vis Sci. 1980;19(2): 137-152. Quigley HA, Dunkelberger GR, Green WR. Chronic human glaucoma causing selectively greater loss of large optic nerve fibers. Ophthalmology. 1988;95:357-363. Quigley HA, Hohman R M , Addicks E M , Green WR. Blood vessels of the glaucomatous optic disc in experimental primate and human eyes. Invest Ophthalmol Vis Sci. 1984;25(8):918-31. Quigley HA, Maumenee A E . Long-term follow-up of treated open-angle glaucoma. Am. J. Ophthalmol 1979;87:519-525. Quigley HA, Nickells RW, Kerrigan L A , Pease M E , Thibault DJ, Zack DJ. Retinal ganglion cell death in experimental glaucoma after axotomy occurs by apoptosis. Invest Ophthalmol Vis Sci. 1995;36:774-786. Quigley HA, Pease M E . Change in the optic disc and nerve fiber layer estimated with the glaucoma-scope in monkey eyes. Journal of glaucoma. 1996;5(2): 106-16. Quigley HA, Sanchez R M , Dunkelberger GR, L'Hernault N L , Baginski TA. Chronic glaucoma selectively damages large optic nerve fibers. Invest Ophthalmol Vis Sci. 1987;28:913-920. Radius RL, Anderson DR. The course of axons through the retina and optic nerve head. Arch Ophthalmol. 1979;97(6):1154-1158. Radius RL, Maumenee A E , Green WR. Pit-like changes of the optic nerve head in open-angle glaucoma. Br J Ophthalmol. 1978;62(6)389-393. 144 Rodieck RW, Binmoeller KF, Dineen J. Parasol and midget ganglion cells of the human retina. J Comp Neurol. 1985;233(1):115-132. Rohen JW, Lutjen-Drecoll E: Age changes of the trabecular meshwork in human and monkey eyes, in Bredt H, Rohen JW (eds): Ageing and Development, Vol I. Stuttgart, New York: Schattauer Verlag, 1971, 1-36. Rollins DF, Drance SM: Five-Year Follow-Up of Trabeculectomy in the Management of Chronic Open Angle Glaucoma. Symposium of Glaucoma. New Orleans Academy of Ophthalmology. St. Louis: CV Mosby, 1981, 295-230. Roth SM, Spaeth GL, Starita RJ, et al. The effects of postoperative corticosteroids on trabeculectomy and the clinical course of glaucoma: Five year follow-up study. Ophthalmic Surg 1991;22:724-729. Saini K D . Garey LJ . Morphology of neurons in the lateral geniculate nucleus of the monkey. A Golgi study. Experimental Brain Research. 1981;42(3-4):235-48. Segal P, Swiercynska J. Mass screening of adults for glaucoma. Ophthalmologica. 1967;153(5):336-348. Seligman A M , Karnovsky MJ, Wasserkrug HL, Hanker JS. Nondroplet ultrastructural demonstrations of cytochrome oxidase activity with a polymerizing osmiophilic reagent, diaminobenzidine (DAB). J Cell Biol. 1968;38:1-14. Shirakashi M , Abe H , Sawaguchi S, Iwata K. The relationship between deterioration and reversal of optic disc cupping in monkeys with chronic experimental high-pressure glaucoma. Graefes Archive for Clinical & Experimental Ophthalmology, 1998;236(7):546-552. Schumer RA, Podos SM. The nerve of glaucoma. Arch Ophthalmol. 1994; 112(l):37-44. Schwartz A L , Whitten M E , Bleiman B, Martin D. Argon laser trabeculoplasty in uncontrolled phakic open-angle glaucoma. Ophthalmology, 1981;88:203-212. Shields M B : Primary angle-closure glaucoma, in Shields M B (ed): Textbook of Glaucoma, ed 3. Baltimore: Williams & Wilkins, 1992, 198-219. Shields M B . Cyclodestructive procedures, in Kaufman PL, Mittag TW (eds.): Textbook of Ophthalmology: Glaucoma, Vol . 7. London: Mosby, 1994. 9.82-9.87. Shields M B , 1998.Textbook of Glaucoma (4 t h ed). USA: Williams & Wilkins, 1998. Shin DH, Kolker A E , Kass M A et al. Long-term epinephrine therapy of ocular hypertension. Arch Ophthalmol. 1976;94:2059-2060. 145 Shin DH, Lee M K , Briggs KS, Kim C, Zeiter JH, McCarty B. Intraocular pressure-related pattern of optic disc cupping in adult glaucoma patients. Graefes Arch Clin Exp Ophthalmol. 1992;230(6):542-546. Sillito A M . The contribution of inhibitory mechanism to the receptive field properties of neurons in the striate cortex of the cat. J Physiol (London). 1975;250:305-329. Sillito A M , Kemp JA, Wilson JA, Berardi N . A re-evaluation of the mechanisms of underlying simple cell orientation selectivity. Brain Res. 1980;194:517-520. Skuta GL. Laser iridotomy and iridoplasty. in Kaufman PL, Mittag TW (eds.): Textbook of Ophthalmology: Glaucoma, Vol . 7. London: Mosby, 1994. Smythe B A , Herschler J. Filtration Surgery, .in Kaufman PL, Mittag TW (eds.): Textbook of Ophthalmology: Glaucoma, Vol . 7. London: Mosby, 1994. Sommer A. Intraocular pressure and glaucoma. A m J Ophthalmol 1989;107:186-188. Sommer A , Tielsch JM, Katz J, Quigley HA, Gottsch JD, Javitt JC, Martone JF, Royall R M , Witt K A , Ezrine S. Racial differences in the cause-specific prevalence of blindness in east Baltimore. N Engl J Med. 1991a;325(20):1412-1417. Sommer A, Tielsch JM, Katz J, et al. Baltimore Eye Survey Research Group: Relationship between intraocular pressure and primary open angle glaucoma among white and black Americans. Arch Opthalmol. 1991b;109:1090-1095. Sommer A, Katz J, Quigley HA, et al. Clinically detectable nerve fiber atrophy precedes the onset of glaucomatous field loss. Arch Opthalmol. 1991;109:77-83. Spaeth GL. The effect of change in intraocular pressure on the natural history of glaucoma: Lowering intraocular pressure in glaucoma can result in improvement of visual fields. Trans Ophthalmol Soc UK. 1985;104:256-64. Spaeth GL. Control of glaucoma: A new definition. Ophthal Surg 1983;14:303-304. Spaeth GL, Hitchings RA, Sivalingam E. The optic disc in glaucoma. Pathogenetic correlation of five patterns of cupping in chronic open-angle glaucoma. Trans Am Ophthalmol Otol 1976;81:217. Tomlinson A , Philips CI. Applanation tension and axial length of the eyeball. Br J Opthalmol. 1970;54(8):548-553. Trusk TC, Kaboord WS, Wong-Riley MTT. Effects of monocular enucleation, tetrodotoxin and lid suture on cytochrome-oxidase reactivity in supragranular puffs of adult macaque striate cortex. Visual Neuroscience. 1990;4:185-204. 146 Trusk TC, Wong-Riley M , DeYoe EA. Changes in cytochrome oxidase and neuronal activity in V I induced by monocular TTX blockade in macaque monkeys. Soc. Neurosci. Abstr. 1992;18:298. Tuulonen A, Airaksinen PJ. Initial glaucomatous optic disk and retinal nerve fiber layer abnormalities and their progression. Am J Opthalmol. 1991 ;111:485-490. Tuulonen A, Airaksinen PJ. Fundoscopic and Photographic Characteristics of the Retinal Nerve Fiber Layer in Glaucoma, in Kaufman PL, Mittag TW (eds.): Textbook of Ophthalmology: Glaucoma, Vol . 7. London: Mosby, 1994. 6.12-6.21. Van Buskirk E M . Other Secondary Glaucomas, in Kaufman PL, Mittag TW (eds.): Textbook of Ophthalmology: Glaucoma, Vol . 7. London: Mosby, 1994. Varma R, Quigley HA, Pease M E . Changes in the optic disk characteristics and number of nerve fibers in experimental glaucoma. American Journal of Ophthalmology. 1992;114:554-559. Vickers JC, Hof PR, Schumer RA, Wang RF, Podos SM, Morrison JH. Magnocellular and parvocellular visual pathways are both affected in the monkey model of glaucoma. Australian and New Zealand Journal of Ophthalmology. 1997;25:239-243. Vickers JC, Schumer RA, Podos SM, Wang RF, Riederer B M , Morrison JH. Differential vulnerability of neurochemically identified subpopulations of retinal ganglion neurons in monkey model of glaucoma. Brain Res. 1995;680:23-35. Vinet J, Bernier PJ, Parent A. Bcl-2 expression in thalamus, brainstem, cerebellum and visual cortex of adult primate. Neurosci Res. 2002;42:269-277. Vnek N , Ramsden B M , Hung CP, Goldman-Rakic PS, Roe A W . Optical imaging of functional domains in the cortex of the awake and behaving monkey. Proc Natl Acad Sci USA. 1999;96:4057-4060. Wald, G. Molecular basis of visual excitation. Science. 1968;162:230-239. Weber A J , Chen H, Hubbard WC, Kaufman PL. Experimental glaucoma and cell size, density, and number in the primate lateral geniculate nucleus. Invest Ophthalmol Vis Sci. 2000;41:1370-1379. Weber AJ , Kaufman PL, Hubbard WC. Morphology of single ganglion cells in the glaucomatous primate retina. Invest Ophthalmol Vis Sci. 1998;39:2304-2320. Werner EB, Drance SM, Schulzer M . Trabeculectomy and the progression of glaucomatous visual field loss. Arch Ophthalmol 1977;95:1374-1377. 147 Wikstrom M , Krab K, Saraste M . Proton-translocating cytochrome complexes. Annu Rev Biochem. 1981;50:623-655. Wilensky JT, Jampol L M . Laser therapy for open-angle glaucoma. Ophthalmology. 1981;88:203-212. Wise JB, Witter SL: Argon laser therapy for open-angle glaucoma; a pilot study. Arch Ophthalmol 1979;97:319-322. Wiesel TN, Hubel D H , Lam D M K . Autoradiographic demonstration of ocular-dominance columns in the monkey striate cortex by means of transneuronal transport. Brain Res. 1974;79:273-279. Wong-Riley MTT Columnar cortico-cortical interconnections within the visual system of the squirrel and macaque monkeys. Brain Res. 1979a;162:201-217. Wong-Riley MTT. Changes in the visual system of monocularly sutured or enucleated cats demonstrable with cytochrome oxidase histochemistry. Brain Research. 1979b;171:ll-28. Wong-Riley MTT. Cytochrome oxidase: An endogenous metabolic marker for neuronal activity. Trends Neurosci. 1989;12:94-101. Wong-Riley MTT. Primate Visual Cortex: Dynamic Metabolic Organization and Plasticity Revealed by Cytochrome Oxidase, in Peters A, Rockland KS (eds.): Cerebral Cortex, Vol.10. New York: Plenum Press, 1994. Wong-Riley MTT, Carroll EW. The effect of impulse blockage on cytochrome oxidase activity in the monkey visual system. Nature. 1984;307:262-264. Wong-Riley MT, Norton TT. Histochemical localization of cytochrome oxidase activity in the visual system of the tree shrew: normal patterns and the effect of retinal impulse blockage. J Comp Neurol 1988;278(4):633-634. Wong-Riley MTT, Kageyama GH. Localization of cytochrome oxidase in the spinal cord and dorsal root ganglia, with quantitative analysis of ventral horn cells in the monkey. J Comp Neurol. 1986;245:41 -61. Wong-Riley M , Riley DA. The effect of impulse blockage on cytochrome oxidase activity in the cat visual system. Brain Res. 1983;261:185-193. Wong-Riley M , Merzenich M M , Leake PA. Changes in endogenous enzymatic reactivity to D A B induced by neuronal inactivity. Brain Research. 1978;141:185-192. Wong-Riley MTT, Tripathi SC, Trusk TC, Hoppe DA. Effect of retinal impulse 148 blockage on cytochrome oxidase-rich zones in the macaque striate cortex: I. Quantitative electron microscopic (EM) analysis of neurons. Vis Neurosci. 1989a;2(5):483-497. Wong-Riley MTT, Tripathi SC, Trusk TC, Hoppe DA. Effect of retinal impulse blockage on cytochrome oxidase-rich zones in the macaque striate cortex: II. Quantitative electron microscopic (EM) analysis of neuropil. Vis Neurosci. 1989b;2(5):499-514. Wong-Riley MTT, Trusk TC, Kaboord W, Huang Z. Interpuffs in the macaque striate cortex: Quantitative E M analysis of neurons before and after unilateral retinal impulse blockade. Soc Neurosci Abstr. 1992;18:299. Wong-Riley MTT, Trusk T, Huang Z. Interpuffs in the macaque striate cortex: Quantiative E M analysis of neuropil before and after unilateral retinal impulse blockade, Soc Neurosci Abst. 1993;19:334. Wong-Riley M , Anderson B, Liebl W, Huang Z. Neurochemical organization of the macaque striate cortex: correlation of cytochrome oxidase with Na+K+ATPase, N A D P H -diaphorase, nitric oxide synthase, and N-methyl-D-aspartate receptor subunit 1. Neuroscience. 1998;83(4):1025-1045. Yucel Y H , Gupta N , Kalichman M W , Mizisin AP, Hare W, deSouza L M , Zangwill L, Weinreb RN. Relationship of optic disc topography to optic nerve fiber number in glaucoma. Arch Ophthalmol. 1998;166(4):493-497. Yucel Y H , Kalichman M W , Mizisin AP, Powell HC, Weinreb RN. Histomorphometric analysis of optic nerve changes in experimental glaucoma. J Glaucoma. 1999;8(l):38-45. Yucel Y H , Zhang Q, Gupta N , Kaufman PL, Weinreb RN. Loss of neurons in magnocellular and parvocellular layers of the lateral geniculate nucleus in glaucoma. Arch Ophthalmol. 2000; 118(3):378-384. Yucel Y H , Zhang Q, Weinreb RN, Kaufman PL, Gupta N . Atrophy of relay neurons in magno- and parvocellular layers in the lateral geniculate nucleus in experimental glaucoma. Investigative Ophthalmology & Visual Science. 2001 ;42(13):3216-3222. Zalta A H . Specific Types of Glaucoma, in. Kaufman PL, Mittag TW (eds.): Glaucoma, Vol. 7. London: Mosby, 1994. Zalta A H . Gonioscopy. in. Kaufman PL, Mittag TW (eds.): Glaucoma, Vol . 7. London: Mosby, 1994b. 3.11. 149 5. Appendix A. Central visual pathway i. Visual neuroanatomy a. The Retina Light enters through the pupil of the eye and is focused through the lens onto the retina at the back of the eye (Figure 27). The retina consists of five major cell classes: receptor cells, bipolar cells, horizontal cells, amacrine cells and ganglion cells. The receptor cells (rods and cones) make direct synaptic contact with bipolar cells (interneurons), which connect to the ganglion cells. The ganglion cells project to the lateral geniculate nucleus and the superior colliculus as well as to the brainstem. Horizontal cells and amacrine cells modulate the flow of information from receptor to bipolar to ganglion cells. The cell bodies of these five classes of neurons reside in three layers: 1) the outer nuclear layer (receptors) 2) the inner nuclear layer (bipolar, horizontal and amacrine), and 3) the ganglion cell layer (ganglion). The processes of these cells interact in two distinct synaptic layers: the outer plexiform layer and the inner plexiform layer. The outer plexiform layer connects the processes of receptor, bipolar and horizontal cells, whereas the inner plexiform layer contains the processes of bipolar, amacrine and ganglion cells. Visual information flows from receptor to ganglion cells in two major pathways. The simplest pathway is a direct route from receptor cells to bipolar cells to ganglion cells. The second pathway involves the integration of horizontal cells to 150 Figure 27: Pathway of light through the retina. Light enters through the pupil of the eye and is focused through the lens onto the retina at the back of the eye. The receptor cells (rods and cones) make direct synaptic contact with bipolar cells (interneurons), which connect to ganglion cells, which project to the L G N . Except for at the foveola (the centre of the fovea), light must first pass through these overlying layers of nerve cells before reaching the photoreceptors. (Figure adapted from Bailey and Gouras, 1985). 151 transfer information from distant receptor cells to the bipolar ganglion cell pathway. Ultimately, the absorption of light is transduced into chemical signals, which leave the eye via the optic nerve. The axons of the retinal ganglion cell travel through the nerve fiber layer to the optic disc head where it becomes bundles of axons that exit the eye (Kuffler, 1953; Wald, 1968). b. The LGN Axons form the optic nerve, and project to the dorsal lateral geniculate nucleus (LGN) on the inferior side of the thalamus. However, prior to reaching the thalamus some axons within the optic nerve cross at the optic chiasm and project to the L G N contralateral to the eye. Axons of retinal ganglion cells from nasal retina cross over at the optic chiasm to the other hemisphere and continue to the contralateral L G N . Axons of the retinal ganglion cells of the temporal retina remain uncrossed and terminate in the ipsilateral L G N . The L G N is therefore a bilateral thalamic structure. It is composed of 6 layers of neurons separated by intervening layers of axons and dendrites. Each layer receives input from one eye only. Layers 1, 4, and 6 receive visual input from the contralateral nasal retinal fibers and layers 2, 3, and 5 receive visual input from the ipsilateral temporal retinal fibers. Despite the segregated input from the two eyes into different layers, the topographical arrangement of ganglion receptive fields are conserved in each layer of the L G N . Together, the layers form six maps of the contralateral hemifield in vertical register. However, not all parts of the retina are represented.equally. Proportionally, more of the nucleus is devoted to the representation of the central area than to the periphery of the retina. The central area contains a higher number of ganglion 153 cells and therefore projects more axons to the L G N . The specific segregation of input within the L G N allows for evaluation on the consequences of monocular retinal damage caused by elevated IOP at this level. c. The visual cortex Projection cells from the L G N send their axons to the visual cortex. The visual cortex is about 3 mm thick and is composed of alternating layers of fibers and cells dispersed from the pial surface to white matter (Kandel and Schwartz, 1985). Each layer of the visual cortex performs specific tasks. Each hemisphere of the brain receives information from the opposite visual field. For example, information from the left visual field is received in the right hemisphere and information from the right visual field is received in the left hemisphere. Area V I (Brodmann's area 17) of the primary visual cortex receives the strongest input from the L G N . Axons from the L G N terminate mostly in layer IV. This layer is divided into three major sub layers: IVA, IVB and IVC. Cells in layer IVC receive input from either one eye or the other via the separate layers of the L G N (Figure 28). The output from layer IVC goes to the layers above or below so that the signals from both eyes can converge on individual cells. i i . The Retinotopic map The L G N is mapped onto the primary visual cortex in a point-to-point manner. Each geniculate axon terminates in and contacts only a small part of layer IV. In primates, the primary visual cortex contains an orderly map of the visual field. The 154 Figure 28: Termination of L G N afferents in the visual cortex. Projections from the magnocellular layers (M) of the L G N terminate in layer IVCa and to a lesser degree layer VI. Projections from the parvocellular layers (P) terminate in Layer IVCp and to a lesser degree to layer IVA and VI. Input representing one eye is segregated from the other eye into separate ocular dominance columns. (Figure adapted from Berne and Levy, 1983). 155 I ocular dominance columns IVa IVb IVC-alpha IVC-beta VI white matter r 1 p i MS ( A 1 from from from from LGN layer LGN layer LGN layer LGN layer 1 (M) 4,6 (P) 2 (M) 3,5 (P) contralateral eye column ipsilateral eye column Figure 28 | 5£ cortex is located on the medial surface at the posterior pole of the cerebral hemisphere. Various parts of the cortex are devoted to the representation of specific areas of the visual field. For example, each half of the visual field is mapped to the contralateral hemisphere. Further, the upper fields are mapped below the fundus of the calcarine fissure and the lower fields above it. As mentioned before, more retinal ganglion cells are devoted to the central region of the visual field, and therefore this region of the cortex is magnified compare to the areas representing peripheral regions (Figure 29) (Berne and Levy, 1983). iii. Organization of the primary visual cortex The connections of the cells in each cortical layer are complex, but it is possible to recognize certain consistent patterns (Figure 30). In general, neurons in layer IVC receive input from the L G N and send their axons superficially to layers II and III. These layers project to layer V , which have connections to layer VI. Layer VI completes the loop with connections to layer IV. Moreover, each of these layers has strong connections to other areas of the brain, therefore allowing information to leave this loop from any layer. There are two main types of cells involved in this loop, stellate cells and pyramidal cells. Stellate cells are exclusively involved with local integration of cortical activity. In contrast, pyramidal cells have axons that leave Brodmann's area 17 to project to adjacent areas of the cortex or brainstem (Gilbert and Wiesel, 1979). The primary visual cortex is further organized into narrow columns that extend from the pial surface to the white matter. Each column contains cells in layer IVC with concentric receptive fields. Simple cells with almost identical retinal positions and 157 Figure 29: Visual field representation on the visual cortex. The various parts of the visual field (top) are numbered to match the corresponding areas in the primary visual cortex (bottom). This cortex is located at the posterior pole of the cerebral hemisphere on the medial surface. Each half of the visual field is mapped to the contralateral hemisphere. Moreover, the upper visual field is mapped below the calcarine fissure while the lower visual field is mapped above the fissure. It is also important to note that greater regions of the cortex is devoted to the central region of the visual field compared to representation of the peripheral regions. (Figure adapted from Berne and Levy, 1983). 158 visual field primary visual fissure cortex Figure 29 ISi Figure 30: Modular organization of the primary visual cortex. Within one hypercolumn of Area 17 of the visual cortex, a complete set of orientation columns representing 360° exist. A set of ocular dominance columns separating input from left and right eye is another module of organization. In addition, several cortical pegs exist within a hypercolumn. These are concerned with color rather than an axis of orientation. (Figure adapted from Kandel, 1985). 160 o c u l a r c o r t i c a l pegs p r i m a r y v i s u a l cor tex (area 1 7) Figure 30 identical axes of orientation are situated above and below the cells in layer IVC. Further, complex cells exist in each column, which receive input from the simple cells. The organization of the cells enables processing of orientation selectivity and color. Cells with the same axis of orientation tend to be grouped together in columns (Hubel and Wiesel, 1965). In contrast, cells arranged in vertical pegs through the six layers of the cortex are not concerned with orientation, but with color (Livingstone and Hubel, 1984). In addition to this, alternating columns exist devoted to input from the left or the right eye that are independent of the other two organizing modes of colour and orientation. These columns are concerned with ocular dominance properties that are important for binocular interaction and depth perception. The axons from cells in the L G N terminate on stellate cells within layer IVC of the primary visual cortex systematically. Fibers from layers 1, 4, 6 of the L G N alternate with fibers from layers 2, 3, 5, to terminate on columns of stellate cells beside each other. These alternating columns receive visual inputs from the contralateral eye (layer 1, 4, 6) and the ipsilateral eye (layer 2, 3, 5) (Wiesel et al., 1974; Hubel and Wiesel, 1977). The overall separation of input of one eye from the other is the basis for ocular dominance columns observed under models for monocular deprivation. While the retinogeniculocortical pathway is the primary visual pathway, the retinal axons also project to the superior colliculus, the pulvinar nucleus, the pregeniculate nucleus, the olivary pretectal nucleus, the nucleus optic tract, the dorsal, lateral and medial terminal accessory optic nucleus and the suprachiasmatic nucleus (Cowey and Stoerig, 1993). These pathways are responsible for driving ocular motility, circadian rhythms, and governing pupillary light reflex. 162 B . Types of glaucoma i . Open-angle glaucoma By definition, chronic, or primary open-angle glaucoma (POAG), is an optic neuropathy that has no apparent obstruction of the trabecular meshwork by structures such as the iris, on gonioscopic examination. The angle of the anterior chamber can be wide or narrow, but it remains open under all circumstances. P O A G can occur through a wide range of IOPs (Sommer et al., 1991a), where patients with a low IOP or normal IOP may develop clinical symptoms, while patient with a high IOP may not (Graham, 1972). IOP is only slightly elevated in the early stages of the disease. As it advances, the IOP becomes progressively higher. The development of persistent elevated IOP is a significant risk factor for glaucoma, but it is not a causative one because without the accompanying optic nerve head and visual field defect, the patient may not develop glaucoma (Chauhan and Drance, 1992; O'Brien et al., 1991; Weber et al., 1998). Blockage of aqueous outflow in primary open-angle patients are due to clusters of extracellular material known as "sheath-derived plaques" derived from elastic-like fibers located within the cribriform layer of the trabecular meshwork. They also develop around connecting fibrils, which then thicken and merge (Figure 31). Plaque formation is a generalized, continuous and age dependent process. However, its formation becomes deleterious when the aqueous pathway situated in the inner wall of Schlemm's canal or in the cribriform layer, is narrowed or obstructed. Blockage can also occur when extracellular material other than sheath-derived plaques is deposited by, within or around the cribiform pathways. Further, loss of trabecular cells accompanied with the thickening 163 Figure 31: Blockage of aqueous outflow in primary open-angle glaucoma. The inner wall of Schlemm's canal and the subendothelially located elastic fiber network is depicted in normal eyes (A) and glaucomatous eyes (B). Thickening of the sheaths by sheath-derived plaques (type I, II, and III) cause narrowing of outflow pathways. As a result, the giant vacuole disappears and aqueous outflow is blocked. (Figure adapted from Lutjen-Drecoll, 1994). 164 Figure 31 of trabecular lamellae may also lead to narrowing of the intertrabecular spaces. Finally the complete fusion of trabecular beams may also be responsible for obstruction of the trabecular meshwork. This type of glaucoma develops slowly, sometimes without noticeable sight loss for years. It usually responds well to medication i f caught early and treated. Primary open-angle glaucoma is often painless in nature and therefore symptoms may not be reported until later, after gradual loss of peripheral vision (Higginbotham, 1994). ii. Normal-tension glaucoma Normal-tension glaucoma, is a form of open-angle glaucoma that is characterized by glaucomatous optic disc damage and visual field loss (Kanski et a l , 1996). Unlike POAG, normal tension glaucoma has IOP within the normal range. However, its range is still higher than those of the normal population. This slight increase in IOP is suspected to be a slight causative factor for this type of glaucoma. Patients are asymptomatic and are diagnosed based on the optic nerve appearance (Cartwright and Anderson, 1988; Crichton et al., 1989; Araie et a l , 1994). iii. Chronic narrow-angle glaucoma Chronic narrow-angle glaucoma, like open-angle glaucoma, can be symptom-less until vision loss is experienced. It occurs in less than 10% of glaucoma patients. In this form of the disease, aqueous humor cannot drain out of the eye due to very narrow drainage angles that are usually blocked by the iris. This condition can occur slowly and progressively, or very quickly (Lowe, 1964). 166 iv. Closed-angle glaucoma In contrast, patients with primary closed-angle glaucoma experience sudden unilateral onset of pain and decreased vision (Shields, 1992). The angle-closure glaucomas include a variety of acute and chronic conditions where temporary and/or permanent obstruction of the trabecular meshwork by the iris occurs. This causes a decreased aqueous outflow and subsequent elevated IOP. Primary angle-closure glaucoma is less common than primary open-angle glaucoma, with some studies suggesting a 1:4 to 1:10 ratios (primary-angle closure glaucoma: primary open-angle glaucoma for white patients) (Hoskins and Kass, 1989). Primary-angle closure occurs as a result of posterior mechanisms in which the peripheral iris is pushed into the angle. Aqueous humour is trapped within the posterior chamber due to a block between the pupillary iris and the anterior lens surface. As a result, the peripheral iris is shifted forward, which closes the anterior chamber angle (Zalta, 1994). This type of glaucoma is prone to occur in eyes with shallow anterior chambers (Congdon et al., 1992). v. Congenital glaucoma Congenital glaucoma is a rare form of glaucoma, affecting babies. Eighty percent of the cases are diagnosed by age one. These children are born with narrow anterior chamber angles or with some other defect in the trabecular meshwork. It is the most common type of glaucoma in infants and is a significant cause of childhood blindness (Shields, 1992). 167 vi . Secondary glaucomas Secondary glaucoma is usually acquired due to ocular or systemic disorders that are unrelated to glaucoma. These also include congenital, open-angle or closed-angle glaucoma as well. Situations in which glaucoma may result include: ocular trauma, chemical burns, drug use such as steroids, uveitis, increased venous pressure, intraocular tumors, cataract surgery, and intraocular hemmorhage (Van Buskirk, 1994). C. Method of diagnosis for glaucoma There are four main tests for glaucoma: tonometry, ophthalmoscopy, perimetry and gonioscopy. The tonometry test measures the inner pressure of the eye. The pressure is derived from applying a known force to a known area on the ocular surface. Various instruments are available for this technique (Higginbotham, 1994b). Ophthalmoscopy is used to examine the inside of the eye, especially the optic nerve. Its shape and color is observed through an opthalmoscope (Higginbotham, 1994c). If the eye pressure is not within the normal range, or if the optic nerve is unusual, perimetry or gonioscopy tests that are specific for glaucoma are performed. The perimetry or visual field test is used to map vision. Quantitative perimetry (sensitivity) rather than quantitative (confrontation and color saturation) perimetry is used due to its greater suitability for early detection of glaucoma. Actual measurements of the sensitivity are performed at various locations of the visual field (Heijl, 1994). Gonioscopy is a biomicroscopic technique used to examine the anterior angle of the eye. This technique is an important tool for examining iridocorneal relationships that can be altered through 168 time, surgery and pharmacological agents. It is very useful for differentiating between open-angle and angle closure glaucoma (Zalta, 1994b). D. Current therapies for glaucoma i. Medications Medications presently used for glaucoma therapy alter cellular functions in the eye by direct interaction with receptors or with certain specific enzymes. They are most often applied through eye drops. The first stage of glaucoma treatment is through the use of beta blockers, which lowers fluid production in the eye. Carbonic anhydrase inhibitors also decrease pressure by lowering fluid production. Other medications lower pressure by increasing drainage in the eye. These include adrenergic, cholinergic, cholinesterase inhibitors, docosanoid, prostaglandin, and prostamide drugs. Finally, alpha agonists lower IOP by both increasing fluid drainage and decreasing fluid production (Kaufman and Mittag, 1994). ii. Lasering Lasering is the next line of treatment available. Argon laser trabeculoplasty (ALT) involves the scattering of approximately 100 argon laser burns over the whole 360° of the trabecular meshwork (Wise and Witter, 1979). The burns are not strong enough to create penetrating holes through the trabecular meshwork, but can cause a local area of coagulation and a decrease in IOP. There are several theories attempting to explain the mechanism of action of this technique. Overall, observations agree that A L T increases tonographic facility of aqueous outflow from the anterior chamber, thus 169 lowering IOP (Schwartz et al., 198.1; Wilensky and Jampol, 1981). A L T is used mainly for patients with primary open-angle glaucoma. Laser iridotomy and iridoplasty are other procedures available for angle-closure glaucoma. A laser is applied to the iris to form a hole or holes, allowing it to fall back from the fluid channel and helping the fluid drain (Skuta, 1994). iii. Filtration surgery When standard medical and laser therapy is inadequate for controlling satisfactory levels of IOP, filtration surgery is the treatment of choice. The goal in filtration surgery is to create an opening between the inside of the eye (usually the anterior chamber) and the subconjunctival space. Two main forms of filtration surgery are performed, partial-thickness (trabeculectomy) and full-thickness (sclerotomy). In these procedures, generally a tiny drainage hole is made in the sclera. The new drainage hole allows fluid to flow out of the eye and helps lower eye pressure. Trabeculectectomy offers the advantage of having a loosely sutured scleral flap overlying the internal ostium to provide some resistance to outflow of fluid. After a sclerotomy, the aqueous drains directly into the subconjunctival space without resistance (Grant, 1958; Lamping et al., 1986). Typical postoperative IOP readings after partial-thickness surgery are in the mid to high teens. In contrast, IOP readings range in the mid to low teens following full-thickness surgery (Lamping et al., 1986). Therefore, modifications such as making smaller scleral flaps, or releasing the sutures holding the scleral flap in place are used to mimic the results of a full-thickness procedure. Trabeculectomy is presently the most popular form of filtration surgery (Smythe and Herschler, 1994). 170 iv. Drainage implants When filtration surgery does not result with prolonged IOP control, implantation of a drainage device is the next option for therapy. Foreign materials are implanted into the eye to facilitate drainage of aqueous humor. Early devices were categorized as paracentesis drains, cyclodialysis implants, or sclerostomy implants. A variety of materials including horse hairs, silk loops, silicone strips, silk threads, platinum wires, magnesium strips, tantalum foil, inert plastics have all been traditionally used as drainage devices. However, the long-term results of these implants were poor and complications were great due to excessive postoperative inflammation or foreign body reaction that lead to bleb scarring (Krupin et al., 1994). Today, Molteno implants, a type of posterior tube shunt, are commonly used for patients with elevated IOP that had prior failure to filtration surgery. Posterior tube shunts all consist of an open tube that is placed into the anterior chamber to shunt aqueous humor posteriorly into an area of encapsulation around an episcleral explant. These implants are used for primary open-angle, closed-angle, congenital, and a variety of secondary glaucomas. While some complications of earlier glaucoma-implant procedures have been addressed since the invention of Molteno implants, operative and postoperative complications remain (Krupin, 1988). Moreover, IOPs after a successful Molteno implant unfortunately tends to be higher than after a successful trabeculectomy (Minckler et al., 1988, Krupin et al., 1994). v. Cyclodestructive procedures: Under certain high-risk situations such as glaucoma associated with active inflammation or glaucoma after multiple failed filtering procedures, medications, 171 lasering, filtration surgery and implants have a low probability of success. In these cases, cyclodestructive procedures for reducing aqueous production may be beneficial. Portions of ciliary processes are destroyed by cyclophotocoagulation. This procedure is noninvasive and relatively quick and easy. However, unpredictable results and frequent complications arise due to the significant disadvantages of the inability to see the processes being treated, as well as damage to adjacent tissue (Shield, 1994). 172 F i g u r e 32: Example of procedure for calculating IOP Ratio. A) Sample IOP data table from animal SI. B) Sample calculation for determining the area under the curve between two data points (yellow) of a graph recording IOP over the number of days post lasering. Total IOP area under the curve is the sum of these individual values. C) IOP Ratio was calculated by comparing the total area under the curve of the control eye to the treated (or glaucomatous) eye. 173 Date Days elapsed post laser O S (treated) IOP area under the curve Days elapsed post laser OD (control) IOP area under the curve 13-Sep 1 23 1 22 17-Sep 5 20 86 5 10 64 23-Sep[| 11 17 111 11 15 75 29-Sep 17 24 123 17 21 108 11-Oct 29 16 240 29 18 234 21-Oct 39 23 195 39 19 185 26-Oct 44 19 105 44 20 98 28-Oct 46 19 38 46 19 39 1-Nov 50 17 72 50 20 78 3-Nov 52 24 41 52 23 43 15-Nov 64 18 252 64 20 258 22-Nov 71 18 126 71 21 144 29-Nov 78 24 147 78 23 154 6-Dec 85 25 172 85 22 158 13-Dec 92 18 151 92 18 140 20-Dec 99 19 130 99 24 147 27-Dec 106 26 158 106 23 165 4-Jan 114 20 184 114 18 164 19-Jan 129 25 338 129 26 330 Total IC under th )P area e curve 2667 Total IOP area under the curve 2582 Area under the curve = Mean IOP x # days elapsed Area under the curve = [(23 + 20)/2] x (5-1) Area under the curve = 86 IOP Ratio = Control Eye (total IOP area under the curve) Treated Eye (total IOP area under the curve) IOP Ratio = 2667 IOP Ratio = 0.97 Figure 32 174 

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