Microvascular changes of the human anterior optic nerve in glaucoma

George A. Cioffi and Da-You Zhao

Devers Eye Institute, Portland, OR, USA

Introduction

It is hypothesized that anomalies of the optic nerve circulation are involved in the development of glaucomatous optic neuropathy. Circulatory alterations could result from changes in either the vascular anatomy or physiology, or both. The hemodynamic status of the human optic nerve in glaucomatous eyes has been the subject of histological, clinical, and experimental studies. Many investigations have shown potential vascular contributions to the glaucomatous optic neuropathy.1-6 Moreover, the anatomy of the anterior optic nerve vasculature has been investigated using a variety of different techniques.2-11 Selective microvascular corrosion casting techniques have been established as a method for studying the three-di- mensional vascular patterns of the anterior optic nerve. This technique has been used to study the microvasculature of the human anterior optic nerve in normal and glaucomatous eyes, using modified methyl methacrylate media to permanently cast the vasculature.2,7 This allows detailed examination of the microvasculature using scanning electron microscopy. Using these techniques, comparisons can be made between the anterior optic nerve in glaucomatous and normal eyes of humans. In addition, the relationship between the microvasculature of the anterior optic nerve and the pre-mortem visual function can be described.

Techniques

Human eye-bank eyes with long retrobulbar optic nerve remnants (approximately 10 mm), allowing identification of the central retinal artery (CRA) and the posterior ciliary arteries (PCAs), are used in selective microvascular corrosion casting experiments. In the present investigation, 11 normal eyes were obtained from six Caucasian donors, ranging in age at the time of death from 63-83 years. Nine glaucomatous eyes were obtained from five patients, 70-88 years of age at the time

Address for correspondence: George A. Cioffi, MD, Devers Eye Institute, 1040 NW 22nd Avenue, Suite 200, Portland, OR 97210, USA

Glaucoma in the New Millennium, pp. 37–41

Proceedings of the 50th Annual Symposium of the New Orleans Academy of Ophthalmology, New Orleans, LA, USA, April 6-8, 2001

edited by Jonathan Nussdorf

© 2003 Kugler Publications, The Hague, The Netherlands

of death. The clinical data from the donors was obtained following appropriate informed consent, and all procedures followed the tenets of the Declaration of Helsinki. None of the donors had a history of known systemic vascular diseases, including diabetes mellitus. Previously described techniques were used to selectively cannulate the posterior ciliary arteries and/or central retinal arteries.2,7 Following multiple flushing with tissue plasminogen activator (Activase, Genetech, Inc., South San Francisco, CA), the optic nerve vasculature was filled with modified Batson’s No. 17 methyl methacrylate media12 (Polysciences, Inc., Warrington, PA), which has a viscosity of approximately 11 cPs. After complete polymerization, the tissues surrounding the vascular casts were corroded in 6-M potassium hydroxide. Under a binocular dissecting microscope, the castings were microdissected, mounted, and coated with gold-palladium for examination using electron microscopy.

Normal microvasculature of the anterior optic nerve

The anterior optic nerve can be divided into four anatomical regions: the superficial nerve fiber layer, prelaminar region, lamina cribrosa, and retrolaminar region. In the prelaminar and lamina cribrosa regions, most of the vasculature is composed of capillaries derived from the posterior ciliary circulation. It is generally believed that the laminar region is the site of insult in glaucomatous optic nerve disease. The central retinal artery has a minimal contribution to this region, and most of the capillary beds are oriented in a lamellar fashion, running parallel to the posterior sclera. In fact, most of the anterior optic nerve is supplied by either direct or secondary branches of the PCAs. Branches from either the circle of ZinnHaller (an anastomotic ring surrounding the optic nerve) or the short posterior ciliary arteries provide the arterial supply to the capillary beds within the prelaminar and laminar regions. In vascular castings from normal eyes (n = 5), the average number of feeding vessels supplying to the anterior optic nerve is 9.6 ± 1.5 and 10.8 ± 1.9 in the superior and inferior quadrant of anterior optic nerve, respectively.

Anterior optic nerve microvasculature in glaucomatous optic neuropathy

A summary of the clinical visual field data of the glaucoma patients can be seen in Table 1. The time from the diagnosis of glaucoma to the time of death in this group of individuals ranged between seven and 19 years. The correlation between visual field changes and the arteriolar supply vessels to the anterior optic nerve is presented in Table 2. The six glaucomatous eyes with complete vascular filling were included in this analysis, in order to allow for quantification of the entire optic nerve. The total number of feeding vessels was diminished to between three and six in the superior and inferior quadrants, which correlated with the most severe visual function loss (Table 2). Capillary dropout mainly occurred within the prelaminar and laminar regions of the anterior optic nerve in the glaucomatous eyes.

Table 2. Comparison of the number of feeding vessels with average threshold deviation (dB) on visual field testing in the glaucomatous eyes

Using total threshold deviation except for * using pattern threshold deviation

Discussion

Selective microvascular casting studies in normal and glaucomatous anterior optic nerves demonstrate changes in the vasculature, consistent with a variety of previous reports.2-5,13,14,18 In addition, this technique allows precise anatomical description of the microvasculature and quantification of the arterial supply vessels to the anterior optic nerve. The arteriolar supply vessels to the anterior optic nerve can be quantified and compared to pre-mortem optic nerve function. As clinical observations support the theory that arterial insufficiency and decreased blood flow in the anterior portions of the optic nerve are involved in the production of visual field loss in glaucoma,1,15 this technique may allow identification of anatomical anomalies that correlate with functional deficits. In experimental glaucoma, François and Neetens reported that there was a significant reduction in the filling of the capillaries of the retina and choroid, and, in the optic nerve, the reduction was greater on the temporal side of the nerve.5 The results of experimental glaucoma studies are indicative of an alteration of blood flow pattern as one cause of the pathological changes in the optic nerve.5,16 Likewise, Hayreh and colleagues17 found that 70% of choriocapillaris and 67% of temporal peripapillary

choroids showed moderate to severe atrophy.

Using quantitative methods, Quigley and coworkers19 found that loss of nerve fibers leads to capillary loss, and a constant relationship is maintained between the tissue and capillaries in the optic nerve head following complete transection of the optic nerve. This finding was also confirmed in human glaucomatous eyes.13 Jonas and colleagues found that the diameter of the retrobulbar optic nerve decreases with optic nerve atrophy.20 It was suggested that greater retrobulbar optic nerve caliber may indicate a greater structural reserve capacity. Grunwald and associates21 showed that glaucoma patients have a reduced optic nerve blood flow that correlates with the degree of glaucomatous damage. As glaucoma damage is believed to occur at the level of the lamina cribrosa,22,23 alterations in the laminal arterial supply (either anatomical or physiological) may lead to structural laminal changes or direct axonal ischemia.

The correlation of a decreased number of arteriolar supply vessels in the quadrant of the optic nerve exhibiting the greatest amount of damage, as determined by visual function loss, suggests that anatomical alterations may proceed function loss. While we would expect loss of the capillaries within a region of neuronal atrophy, retrograde closure of larger supply vessels may represent pre-existing regions at risk for vascular insufficiency.

In conclusion, microvascular corrosion castings seem to be an ideal method for studying the three-dimensional microvasculature of the anterior optic nerve. Microvascular changes were found in the anterior optic nerve as well as in the juxtapapillary choroid and retina of glaucomatous eyes. It is possible that hemodynamic changes result from anomalous vascular anatomy and contribute to the pathogenesis of glaucomatous optic neuropathy.

Acknowledgments

This study was supported by grant No. EY 05231 from the NIH.

References

1.Van Buskirk EM, Cioffi GA: Glaucomatous optic neuropathy. Am J Ophthalmol 113:447542, 1992

2.Zhao DY, Cioffi GA: Microvasculature of the human glaucomatous anterior optic nerve. Eye 14:445-449, 2000

3.Michelson G, Langhans MJ, Groh MJM: Perfusion of the juxtapapillary retina and neuroretinal rim area in primary open-angle glaucoma. J Glaucoma 5:91-98, 1996

4.Nicolela MT, Hnik P, Drance SM: Scanning laser Doppler flowmeter study of retinal and optic disk flow in glaucomatous patients. Am J Ophthalmol 122:775-783, 1996

5.François J, Neetens A: Vascularity of the eye and the optic nerve in glaucoma. Arch Ophthalmol 71:219-225, 1964

6.Wang L, Cioffi GA, Van Buskirk EM: The vascular pattern of the optic nerve and its potential relevance in glaucoma. Curr Opin Ophthalmol 9(2):24-29, 1998

7.Onda E, Cioffi GA, Bacon DR, Van Buskirk EM: Microvasculature of the human optic nerve. Am J Ophthalmol 120:92-102, 1995

8.Hayreh SS: The optic nerve head circulation in health and disease: the 1994 Von Sallman lecture. Exp Eye Res 61:259-272, 1995

9.Zhao Y, Li F: Microangioarchitecture of the optic papilla. Jpn J Ophthalmol 31:147-159, 1987

10.Olver JM, Spalton DJ, McCartney ACE: Quantitative morphology of human retrolaminar optic nerve vasculature. Invest Ophthalmol Vis Sci 35:3858-3866, 1994

11.Lieberman MF, Maumenee AE, Green WR: Histologic study of the vasculature of the anterior optic nerve. Am J Ophthalmol 82:405-423, 19876

12.Fahrenbach WH, Bacon DR, Morrison JC, Van Buskirk EM: Controlled vascular corrosion casting of the rabbit eye. J Electron Microsci Tech 10:15-26, 1988

13.Quigley HA, Hohman RM, Addicks EM, Green WR: Blood vessels of the glaucomatous optic disc in experimental primate and human eyes. Invest Ophthalmol Vis Sci 25:918-931, 1984

14.Hayreh SS: Blood supply of the optic nerve head and its role in optic nerve atrophy, glaucoma, and oedema of the optic disc. Br J Ophthalmol 53:721-748, 1969

15.Harrington DO: The pathogenesis of the glaucoma field: clinical evidence that circulatory insufficiency in the optic nerve is the primary cause of visual field loss in glaucoma. Am J Ophthalmol 47:177-185, 1959

16.Kalvin NH, Hamasaki DI, Gass JDM: Experimental glaucoma in monkeys. II. Studies of intraocular vascularity during glaucoma. Arch Ophthalmol 76:94-103, 1966

17.Hayreh SS, Pe’er J, Zimmerman MB: Morphologic changes in chronic high-pressure experimental glaucoma in rhesus monkeys. J Glaucoma 8:56-71, 1999

18.Radius RL, Anderson DR: Breakdown of the normal optic nerve head blood-brain barrier following acute elevation of intraocular pressure in experimental animals. Invest Ophthalmol Vis Sci 19:244-255, 1980

19.Quigley HA, Hohman RM, Addicks EM: Quantitative study of optic nerve head capillaries in experimental optic disk pallor. Am J Ophthalmol 93:689-699, 1982

20.Jonas JB, Schmidt AM, Müller-Bergh JA, Naumann GO: Optic nerve fiber count and diameter of the retrobulbar optic nerve in normal and glaucomatous eyes. Graefe’s Arch Clin Exp Ophthalmol 233(7):421-424, 1995

21.Grunwald JE, Piltz J, Hariprasad SM, Dupont J, Maguire MG: Optic nerve blood flow in glaucoma: effect of systemic hypertension. Am J Ophthalmol 127:516-522, 1999

22.Anderson DR, Hendrickson A: Effect of intraocular pressure on rapid axoplasmic transport in monkey optic nerve. Invest Ophthalmol Vis Sci 13:771-783, 1974

23.Quigley HA, Addicks EM, Green WR, Maumenee AE: Optic nerve damage in human glaucoma. II. The site of injury and susceptibility to damage. Arch Ophthalmol 99:635-649, 1981

Neuroprotection and glaucoma

How do I tell if a drug is neuroprotective?

George A. Cioffi

Devers Eye Institute, Portland, OR, USA

Defining neuroprotection

While a variety of complex definitions of neuroprotection exist, a simple diagram (Fig. 1) illustrates the concept best. Neuroprotection in glaucoma refers to the ability of a therapeutic modality to prevent the loss, or decrease the rate of loss, of retinal ganglion cells, thereby preventing subsequent loss of visual function. Many drugs have laid claim to being neuroprotective, however, other than lowering the intraocular pressure (IOP), no therapeutic interventions currently used in the treatment of glaucoma have been proven. Proof of neuroprotection requires three important scientific hurdles to be crossed before therapeutic application in humans, as follows: proof of concept; adequacy of drug delivery; and human clinical trials (Table 1). Until these investigative hurdles are overcome, the claim of neuroprotection in the treatment of glaucoma remains suspect.

Intrinsic and extrinsic factors

A multitude of factors, either intrinsic or extrinsic to the optic nerve, alone or in combination, may adversely affect the health and function of the optic nerve.1-8 Potential extrinsic factors that may contribute to the development of glaucomatous optic neuropathy include elevated IOP and aberrations of the systemic cardiovascular status. Factors intrinsic to the optic nerve that potentially contribute to axonal loss, include abnormalities of the composition of the support tissues of the optic nerve, or anomalies of the microcirculatory physiology within the nerve. Some individuals may have optic nerves with particular anomalies of these intrinsic factors, which result in heightened susceptibility to glaucomatous optic neuropathy. Other individuals may possess optic nerves that develop glaucomatous damage primarily as a result of extrinsic insult. Thus, a spectrum of factors should be considered in each individual case of glaucoma.

Address for correspondence: George A. Cioffi, MD, Devers Eye Institute, 1040 NW 22nd Avenue, Suite 200, Portland, OR 97210, USA

Glaucoma in the New Millennium, pp. 43–48

Proceedings of the 50th Annual Symposium of the New Orleans Academy of Ophthalmology, New Orleans, LA, USA, April 6-8, 2001

edited by Jonathan Nussdorf

© 2003 Kugler Publications, The Hague, The Netherlands

Fig. 1. Neuroprotection changes the slope of retinal ganglion cell (RGC) death. Drug X: any therapeutic modality.

Table 1. Three criteria for neuroprotection

1Proof of concept

cell death pathways and models of ‘glaucoma’

2Drug delivery

site of action and pharmacological doses

3Human clinical trials

endpoints, progression rate, study population

IOP plays a major role in the development of glaucomatous optic neuropathy in many subjects. One hypothesis of the etiology of glaucoma proposes that neuronal damage results from mechanical pressure on the optic nerve. This hypothesis focuses primarily on the potential deleterious effects that elevated IOP has on the extracellular matrix and glial support structures of the anterior optic nerve. It suggests that there is direct, mechanical insult, which results from IOP being increased above a tolerable threshold. The IOP causes backward bowing, stretching, and compression of the laminar plates within the lamina cribrosa. Misalignment of the fenestrations within the lamina cribrosa and compression of the connective tissue plates results in the inhibition of axoplasmic flow within the axons of the ganglion cells. Interruption of axoplasmic flow or direct mechanical compression of the neural axons causes death of the nerve cells and results in glaucomatous optic neuropathy.

This extrinsic, pressure-induced hypothesis of glaucomatous optic neuropathy is supported by both laboratory models of glaucoma and clinical observations. Increased IOP in non-human primate models causes obstruction of axoplasmic flow in the laminar region. Histology of such primate eyes demonstrates posterior bowing of the lamina cribrosa, with compression of the connective tissue plates and distortion of the laminar fenestrae. Transverse sections through the lamina cribrosa have documented the increased size of laminar fenestrae in the superior and inferior poles of the optic nerve. These regional differences of the lamina cribrosa may account for asymmetric mechanical damage to the axons within the various regions. In early glaucomatous optic neuropathy, damage to the ganglion cell axons primarily occurs in the superior and inferior regions of the optic nerve.

Histology shows non-uniform distribution with increased numbers of large ganglion cell axons in the inferior and superior poles of the optic nerve. Large ganglion cell axons appear to be preferentially damaged early in glaucomatous optic neuropathy. This apparent increased susceptibility of the large cell axons may be due to the relative distribution of these axons within the regions of the optic nerve, which are primarily distorted by mechanical forces. Other glial support tissues within the optic nerve also show changes in glaucomatous optic neuropathy. Extracellular matrix components, including elastin fibers of the lamina cribrosa, are altered in glaucomatous eyes and in experimental models of glaucoma. These changes may be the result of increased IOP with concomitant mechanical stress, and may be associated with axonal death in glaucoma.

However, accumulating epidemiological and laboratory evidence demonstrates that elevated IOP is not the sole cause of optic nerve injury in glaucoma. As many as 20-30% of individuals with glaucoma never exhibit statistically elevated IOP, and among individuals treated for glaucoma, approximately 20-30% continue to lose vision despite maximal IOP-lowering therapy. These findings highlight the need for neuroprotection and have stimulated the investigation of alternative hypotheses. A commonly cited hypothesis proposes that intraneural ischemia leads to the development of glaucomatous optic neuropathy. This hypothesis proposes that vascular perfusion of the neural tissue within the optic nerve is deficient in glaucoma. Vascular perfusion is dependent upon arterial blood pressure, venous outflow, tissue pressure surrounding the vasculature, autoregulation, and local and regional vasomodulators. Insufficiency or abnormality of any or all of these components may result in regional ischemia. In laboratory studies, axoplasmic flow of the ganglion cell axons is obstructed by interrupting the short posterior ciliary artery circulation to the optic nerve. This may lead to ganglion cell death similar to that found with mechanical compression. Autoregulatory mechanisms within the regional vascular beds are believed to accommodate changes in arterial blood pressure in normal individuals. However, deficient autoregulation of the optic nerve vasculature would result in decreased optic nerve perfusion. The vascular hypothesis suggests that elevated IOP results in increased tissue pressure within the optic nerve tissue surrounding the vasculature. This causes vascular collapse and decreased neural perfusion, resulting in glaucomatous optic neuropathy. Increased tissue pressure from elevated IOP is only one possible cause of decreased vascular perfusion of the optic nerve. Individuals with glaucoma are more likely to have systemic vascular disorders, including diabetes mellitus, systemic hypertension, peripheral vascular disease, and vasospastic syndromes. These associations between extrinsic vascular factors and glaucoma support a vascular etiology in the development of glaucomatous optic neuropathy.

Glaucomatous optic neuropathy likely derives from many potential insults working independently or in concert. A combination of the various intrinsic and extrinsic factors may contribute to varying degrees in each individual. Many possible scenarios can be considered. Increases in IOP with mechanical distortion of the optic nerve may result in increased tissue pressure on the microvasculature of the optic nerve and decreased perfusion of the neuronal tissue. Mechanical distortion of the lamina cribrosa fenestrae could directly compromise ganglion cell axons, rendering them more susceptible to damage from an insufficient vascular supply. Further studies examining changes in the compliance and structure of the optic nerve resulting from increased IOP should increase our understanding of the

mechanical component of the development of glaucomatous optic neuropathy. Beyond these studies, investigations into potential neuroprotective therapies offer the promise of halting progressive vision loss in glaucoma. Perhaps the most important contribution of these types of investigations is that novel targets for therapeutic intervention can be developed and investigated. These potential therapies must then meet each of the three criteria (Table 1) in order to prove their therapeutic benefit.

Three important considerations

Proof of concept, drug delivery and clinical trials

With advances in molecular and cellular biology, understanding of the mechanisms that may lead to neural damage has expanded enormously. Apoptosis (programmed cell death without necrosis) has been cited as a potential pathway of retinal ganglion cell death in both human glaucoma and experimental primate glaucoma associated with elevated IOP.9-11 Apoptosis is a cell death pathway in which a complex cascade of cellular events occurs (including nuclear clumping, DNA condensation, cellular shrinkage and involution, and finally macrophage engulfment without inflammation).12-15 As the genetic information necessary for apoptosis exists in neural cells, the question arises as to what is the initiating signal that leads to this terminal cascade of events. Potential initiating signals of apoptosis in central nervous system diseases include growth factor deprivation (Fig. 2), excitotoxicity (Fig. 3), oxygen-free radical production, nitric oxide synthesis, and abnormalities of calcium metabolism. In the eye, growth factor deprivation of retinal ganglion cells (the ‘neurotrophic hypothesis’) results from blockade of retrograde transport at the lamina cribrosa preventing growth factors from reaching their site of action in the cell body.16,17 Calcium and oxygen-free radicals influence biological pathways that can damage the optic nerve. Increased levels of intracellular calcium may damage neurons by stimulating catabolic enzymes or oxygenfree radical production.18 Oxygen-free radicals, such as superoxide ion and hydroperoxyl radical, have unpaired electrons that make them highly reactive.19 Following transient ischemia, these molecules are commonly liberated resulting in ‘re-perfusion injury’. Oxygen-free radicals preferentially damage the mostly unsaturated lipid cell membranes of neural tissues. Aberrant nitric oxide synthesis has also been demonstrated within the glaucomatous anterior optic nerve.20 Neuroscience has provided us with a variety of novel targets to aim our potential therapeutic agents at. Claims of neuroprotection that do not focus on one of the known pathways of neuronal cell death should be viewed with skepticism. “Does the therapy make sense, given our current knowledge of neuronal cell death pathways?” should be the first question when determining whether a new claim of neuroprotection is valid. Caution should be used when extrapolating from in vitro or animal experiments and clinically applying the findings to human disease. Proof of concept allows selection of the most promising drugs for further investigation and human clinical trials.

The adequacy of drug delivery is the next important issue that should be considered when determining whether a drug is neuroprotective. Does the drug reach the target tissue? It is commonly assumed that topically applied medication reaches the posterior segment of the eye and exerts effects (either beneficial or detrimental)

Fig. 2. Neurotrophic hypothesis. RGC: retinal ganglion cells; GF: growth factor.

Fig. 3. Glutamate excitotoxicity.

on the retina and optic nerve. However, evidence that topical medications reach the posterior segment in pharmacologically appropriate dosages is lacking and, at times, conflicting. Most available evidence indicates that topically applied medications have highly variable penetration to the posterior pole, while many systemic medications reach the optic nerve and retina. However, the integrity of the bloodretinal barrier and the blood-brain barrier may limit the amount of drug present in the target tissues, even following systemic administration. Drug concentrations sufficient for the proposed mechanism of action at the site of action must be demonstrated.

Human clinical trials are the final hurdle that must be cleared in order to establish proof of neuroprotection. Due to high costs, long duration, large sample sizes, difficulties of study protocols, and ill-defined endpoints, this hurdle is by far the most difficult to overcome. While traditional pharmaceutical trials in glaucoma

have used the surrogate marker of IOP to assess the adequacy of therapy, definitions of optic nerve structural and functional endpoints have lagged behind. Regulatory agencies and pharmaceutical companies alike have struggled to establish appropriate and meaningful guidelines for these trials. In addition, glaucoma is a very slowly progressive disease. This slow rate of progression mandates large study populations and lengthy study durations in order to test potential neuroprotectants. This translates into very expensive investigations, and limits the number of agents that can be examined. While basic science proof of concept and adequate drug delivery are important considerations in determining the potential of a neuroprotective agent, human clinical trials remain the gold standard for determining efficacy.

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