Pathophysiology of Ganglion Cell Death and Optic Nerve Degeneration Workshop
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November 12-13, 2002 On November 12-13, 2002, the National Eye Institute sponsored the Pathophysiology of Ganglion Cells and Optic Nerve Degeneration Workshop at the Airlie Conference Center, Warrenton, VA. The purpose of the workshop was to hold a dialogue on glaucoma-in the context of retinal ganglion cell compromise-in order to identify research needs and opportunities. The specific goals of the meeting were: to review the state of knowledge of neurodegeneration as it applies to glaucoma, to identify common mechanisms and technologies from other areas of neurodegeneration and neuroprotection that could be applied to research on glaucoma, and to summarize the workshop discussions and post the collective ideas on the NEI Web Site. Scientists and clinicians, representing a number of areas of medicine, were invited to speak on their area of expertise and brainstorm about the needs and opportunities as they pertain to retinal ganglion cell research. Even though the focus was glaucoma, it was recognized that this disease can productively be considered as part of the broad scope of other neurodegenerative diseases. This was emphasized in the open discussion session in which many of the remarks focused on the need for a broader, multi-disciplinary effort. The workshop is part of NEI's Phase II Program Planning. However, the workshop is not inclusive of all the important basic and clinical research being conducted on glaucoma. Future meetings will incorporate additional topics and will reflect the evolving research as the scientific community works to enhance understanding of a serious disease that affects over 3 million Americans and millions more world-wide. The report that follows summarizes presentations by the participants on: (1) retinal ganglion cell biology and physiology, (2) current hypotheses of glaucoma pathophysiology, including the role of vascular compromise, immune dysfunction, and glutamate excitotoxicity, (3) animal models of glaucoma and other chronic neurodegenerative disorders, (4) common mechanisms between glaucoma and other neurodegenerative conditions, and (5) endogenous and pharmacological molecules that promote axon survival and regeneration. The report contains the following sections: Background Information on Glaucoma Major Topics of Discussion by Workshop Participants A Summary of Needs and Opportunities Presentation Summaries Section I: Current Hypotheses of Glaucoma Pathophysiology What is Glaucoma? What is the Role of Intraocular Pressure in the Etiology of Glaucoma? Section II: Animal Models Contribute to the Study of Glaucoma Primate and Rat Models of Ocular Hypertension Section III: The Biology of the Inner Retina is affected in Glaucoma Retinal Ganglion Cell Interactions and Survival Section IV: Mechanisms of Chronic Neurodegeneration Relevant to Glaucoma Etiology Amyotrophic Lateral Sclerosis Section V: Degeneration and Regeneration Growth Factor Deprivation and Neuronal Cell Death Section VI: The Microenvironment in the Optic Nerve Head Role of Nitric Oxide in Retinal Ganglion Cell Degeneration Background Information on GlaucomaGlaucoma is a degenerative disorder of the optic nerve. It is characterized by a loss of ganglion cells and their axons. There are several types of glaucoma (e.g., open-angle, normal tension, and early onset-to name a few); however, all of these types have the same common feature, ganglion cell loss. Millions of people have glaucoma. In the United States, alone, there are approximately 3 million people with the disease, of which about 120,000 are now blind. Most of these cases are attributed to primary open angle glaucoma (POAG), an age-related form of the disease. In addition, glaucoma is the number one cause of blindness in African-Americans. Blindness from all forms of glaucoma is estimated in excess of $1.5 billion annually in U. S. Social Security benefits and health care expenditures. Elevated intraocular pressure (IOP) is frequently but not always associated with glaucoma. Because some people exhibit no association between increased IOP and glaucoma, it is thought that other mechanisms probably play a role in this blinding disease. For patients who present with high IOP, treatment may involve topical drugs or surgical intervention to lower pressure. New approaches to treatment are essential, because not all patients respond to current treatments, and vision that is lost cannot currently be restored. Since ganglion cell death is the common feature of glaucoma, it has become increasingly important to understand the fundamental pathophysiology of the retina. This report is intended to stimulate the development of new paradigms and neuroprotective methodologies for the clinical treatment of glaucoma. Major Topics of Discussion by Workshop ParticipantsNeurons exist in a highly complex relationship with other cells and with the extracellular environment. Studying the neurons of the normal retina and optic nerve will help identify key factors that are responsible for the health of CNS neurons. Trophic factor responsiveness, cell receptors, and influences from other cells like amacrine and Muller cells, are important areas to explore, as well as the effect of age on retinal ganglion cell numbers and robustness. Studying the pathophysiology of glaucoma requires a multidisciplinary approach, including research on cell and retinal tissue structure, vulnerability of specific retinal cells or cell components, trophic and mechanical factors and cell signaling, synaptic transmission, and apoptosis. The optic nerve head is unmyelinated and has different functional and mechanical properties from other regions of the optic nerve. Knowing the trophic factors, cell types, cell interactions, receptors, channels, and additional principles that govern the optic nerve head are undoubtedly important for understanding glaucoma. Studies on the optic nerve in rats, mice, and nonhuman primates are necessary, but it is important that these projects are not purely descriptive. Evidence suggests that axons of retinal ganglion cells destruct in a unique way, independent of the soma. Studying the vulnerability of the axon, especially in the brief unmyelinated section of the optic nerve head, will address whether glaucoma is a problem of the axon or soma. It would appear that injury to axons occurs first and that events in the soma follow. Experiments should look at the order of events in retinal ganglion cell death. Altered synaptic transmission and glutamate excitotoxicity are areas of intense study. There is debate on whether excess glutamate has a positive or negative influence on cells. It is possible that there are different sub-classes of retinal ganglion cells that respond differently to glutamate. Description of the death pathway for retinal ganglion cells will determine whether a truly apoptotic mechanism is involved. Nerve cell death may have both caspase-dependent and -independent components. Understanding this cascade will be important for developing strategies for identifying neuroprotective molecules. Animal models (genetic and experimentally-induced) help in understanding how retinal ganglion cells die and how insults, such as intraocular pressure changes, affect the retina. The number of axons of retinal ganglion cells in the optic nerve among rodent strains varies widely; special care must be taken to select controls and to use adequate numbers of animals. Therapeutic intervention relies heavily on fundamental observations that characterize the pathophysiology of the specific disease. However, there are general principles among similarly-related disorders that hold promise for a common therapeutic approach. Neuroprotective molecules being studied in amyotrophic lateral sclerosis, Parkinson's disease, and stroke are prime candidates for testing in glaucoma. Already, there are numerous drugs being screened for neurological disorders that could be tested in cell and animal models of glaucoma. A Summary of Needs and Opportunities
Section I: Current Hypotheses of Glaucoma PathophysiologyWhat is Glaucoma? What is the Role of Intraocular Pressure in the Etiology of Glaucoma? Glaucoma is not a single disease but rather a heterogenous group of disorders which share in common a slow progressive loss of ganglion cells and their axons of the optic nerve that resulting in a distinct pattern of visual loss. The major types of glaucoma are open-angle, "normal tension" glaucoma, glaucoma secondary to other medical conditions, and glaucoma with onset in early childhood. Clinically, increased intraocular pressure (IOP) has been at the center of diagnosis, and epidemiological data have supported the contention that IOP is important. IOP is controlled by a balance between the secretion and drainage of aqueous humor. The aqueous humor is secreted by the ciliary body and follows a course from behind the iris to in front of it (carrying nutrients to the iris, lens, and cornea) and then out of the eye into the venous circulation via a trabecular network and the canal of Schlemm. Traditionally, increased IOP has been thought to be due to excess fluid in the eye that was most likely caused by a decrease in its outflow. The relationship between IOP and a clinical diagnosis of glaucoma notwithstanding, many people have glaucomatous optic neuropathy with normal IOP, and conversely, many people who have elevated intraocular pressure do not necessarily develop optic nerve damage. For these reasons, it is recognized that other mechanisms must work in conjunction or in addition to IOP to cause optic nerve degeneration. The characteristic anatomical change in the optic nerve in glaucoma is a "cupping" of the optic disk where ganglion cell axons have been lost. The death of the axons is associated with a loss of ganglion cell bodies in the retina and ganglion cell axon terminals in the dorsal lateral geniculate body. Unfortunately, a problem with vision is often not detected until it is quite advanced. Thirty to fifty percent of ganglion cells must be lost before their absence is detected in a visual field test and, even then, a patient is unlikely to realize that a problem exists. Research into the pathophysiology of glaucoma has been based on the clinical manifestations of the disease and with the use of the limited animal models available. Clues have come from the specific pattern of optic nerve degeneration observed using visual fields. Results suggest that not all axons are equally susceptible to IOP, and there is evidence that increased IOP may affect even side-by-side ganglion cell fibers differentially. Prominent among the theories to explain the unique patterning observed clinically is one that invokes a mechanical compression due to increased IOP. Others include vascular compromise, neurotropic withdrawal, glutamate neurotoxicity, a nitric oxide effect, and immune dysfunction. To summarize, the belief until recently has been that the damage in glaucoma is at the level of the optic disk and that it is frequently caused by an increase in intraocular pressure. It is true that lowering IOP often slows the progress of optic nerve disease and that gross damage occurs at the level of the optic disk, but there is reason to believe that other mechanisms are at work and that by uncovering these mechanisms the eyesight of millions of people may be preserved. Facts about the optic nerve:
Current Hypotheses of Glaucoma PathophysiologyWhat is the Vascular Component of Glaucoma? The retina is dependent on its blood supply for meeting its high metabolic needs. The pathophysiology and loss of axons of ganglion cells in glaucoma may be related to a compromised blood supply of the eye. Evidence supporting a vascular role in glaucoma pathophysiology is the presence of hemorrhaging sometimes seen in patients with optic nerve atrophy, which can take the form of flame-shaped hemorrhages on or near the disc. In addition, anatomical constraints imposed on the optic nerve head suggest a contribution of the vasculature to optic nerve pathology. Two vascular systems contribute to the nutrition of the retina-the retinal artery and the ciliary arteries. The central retinal artery of the ophthalmic artery enters the optic nerve about 1.25 centimeters behind the eyeball and runs parallel to the nerve fibers. At the optic disc, it branches into superior and inferior branches, which subdivide into nasal and temporal arteries. These form the vascular network of the inner layers of the retina and supply portions of the nerve fiber layer of the optic nerve head. The small ciliary arteries, 10-20 of them, penetrate the sclera at the posterior pole of the eye to form the choroidal capillaries, a dense single-layered vascular network. The lamina cribrosa at the optic nerve head is supplied by branches of the short posterior ciliary arteries. The optic nerve head is subject to several distinctive pathologies that stem from its anatomy. Tightly fitted into a narrow scleral canal over most of its extent, the anterior-most part of the optic nerve has dual responsibilities: it must provide safe conduct from the eye for the unmyelinated axons of ganglion cells and simultaneously provide space for the central retinal blood vessels to enter the eye. To complicate matters, two distinct pressure domains exist, one to either side of a sieve-like membrane in structural continuity with the sclera, the lamina cribrosa. In separating two pressure domains, pore size of the lamina cribrosa is crucial. Pores must be large enough to permit unimpeded egress of groups of nerve fiber bundles, yet, be small enough to fulfill the task of containing the pressure of the eye. To the degree the neural structures within it exceed a critical diameter, the hydrostatic pressure differential across the lamina cribrosa could impose destructive shear forces on them. Moreover, the basket-like, perforated form of the lamina cribrosa dictates that it be more compliant than the surrounding sclera. Yet coaxial translation, again, risks injury to nerve fiber bundles in transit. Raised intraocular pressure, altered ocular perfusion, loss of autoregulation, and characteristics of the blood-brain barrier at the optic disc are also possible candidates in the pathogenesis of glaucoma. The vascular tone of the blood vessels of the retina and optic nerve head are autoregulated and depend on contraction of the smooth muscle cells of the vascular endothelium. The smooth muscle cells are controlled by a variety of influences including neurotransmitters, hormones, myogenic and metabolic factors (for example, PO2, PCO2), and endothelium-derived factors that include the powerful vasodilator nitric oxide. Retinal vessels typically contain tight junctions that form an impermeable seal between cells. An unusual feature of the optic disc is a small zone where there is no blood-brain barrier. When fluorescein is injected for angiographic studies, it is possible to detect its flow in the extracellular space of the optic nerve head. One of the impediments to identifying a role for vasculature in the pathophysiology of glaucoma is that the flow of blood in the eye is difficult to assess clinically and experimentally. Techniques include: scanning laser fluorescein angiography with image analysis, color doppler imaging, laser doppler velocimetry, scanning laser doppler flowmetry, and pulsatile ocular blood flow systems. Animal studies indicate that the blood flow of the optic nerve head responds to a change in IOP. Studies also indicate a response in oxidative metabolism to varied IOP and mean arterial blood pressure, and that low blood pressure leads to loss of autoregulation and ischemia of the optic nerve head. To conclude, anomalies or abnormalities in the blood flow of the retina and optic disc may be affecting the integrity of retinal ganglion cell axons. Blood vessel compression, loss of autoregulation, or leaking blood vessels may be involved. Just like their cell bodies, the axons of ganglion cells need a vigorous blood supply for normal metabolic activity. Any blood vessel dysfunction at the optic disc could cause axonal malfunction and atrophy. Current Hypotheses of Glaucoma PathophysiologyWhat Damages the Optic Nerve Head in Glaucoma? Primary open angle glaucoma is an optic neuropathy characterized by the loss of axons of the retinal ganglion cell fibers at the level of the lamina cribrosa in the optic nerve head resulting in the clinical manifestation of "cupping". Astrocytes are the major glial cell type in the nonmyelinated optic nerve head in most mammalian species providing cellular and metabolic support to the axons and forming the interface between connective tissue surfaces and surrounding blood vessels. As such, it is of great interest to determine if they play a role in the pathophysiology of the disease. In the lamina cribrosa, astrocytes line lamellar connective tissue plates that run horizontal and perpendicular to the axons of the retinal ganglion cells. Cellular components of the cribriform plates include: astrocytes, lamina cribrosa cells, microglia and smooth muscle cells, and endothelial cells of blood vessels. Components of the extracellular matrix include: elastic fibers, collagen fibers, microfibrils, proteoglycans and glycoproteins. The axons run through the lamina cribrosa in bundles. In humans and non-human primates there is a normal gradient in hydrostatic pressure between inside the eye and the retrolaminar optic nerve across the optic nerve head. When intraocular pressure increases above physiological levels, the pressure gradient also increases submitting the lamina cribrosa and the retinal ganglion cell axons to deformation and mechanical stress. In glaucoma, cupping of the optic disc and compression, stretching and rearrangement of the lamina cribrosa occurs in response to elevated IOP. Astrocytes react vigorously to injury and stress. In glaucoma as with other CNS injuries, reactive astrocytes change shape, migrate out into the extracellular matrix into the nerve bundle area, express new proteins, and are involved in massive destruction and remodeling of the extracellular matrix. The expression of various collagen mRNAs and glycoproteins by reactive astrocytes is increased and so is de novo expression of elastin mRNA. Induction of expression of elastin leads to the formation of pathological irregular shaped elastotic fibers. Changes in the extracellular matrix and in the elastic component of the lamina cribrosa leads to loss of compliance and resiliency needed to adapt to changes in intraocular pressure even within the normal range. Matrix metalloproteinases (MMPs) are selectively expressed in reactive astrocytes in glaucoma and may be key to the progress of the remodeling of the extracellular matrix. MMPs may play a role in the transition of a quiescent astrocyte to the reactive phenotype by activating growth factors and disrupting the attachment of the cells to their substrate allowing changes in shape and migration. Interestingly, expression of MMPs was not increased in reactive astrocytes of the optic nerve head of monkeys with loss of retinal ganglion cells after optic nerve transection compared to a monkey model of laser induced glaucoma. Despite the obvious involvement of astrocytes of the optic nerve in glaucoma, it is not clear what their role is in retinal ganglion cell death. Normal optic nerve astrocytes in culture respond to pressure-related stress adopting many features of the reactive phenotype. Reactive astrocytes cause large amounts of oxidative damage by synthesizing enzymes that lead to reactive oxygen species. The intermediate filament cytoskeleton reorganizes around the center of the cell to protect the nucleus and the cell's synthetic machinery, whereas the actin and microtubules prepare the cell to migrate. Mechanical stress increases astrocyte motility and decreases cell adhesion, both characteristic of reactive astrocytes. Recently, using oligonucleotide microarray technology, astrocytes grown from human glaucomatous optic nerve heads were compared with normal, age-matched cells. Many interesting genes and pathways appeared differentially expressed in glaucomatous astrocytes including enzymes involved in steroid metabolism such as 3-alpha-hydoxysteroid dehydrogenases and the synthesis of a variety of growth factors, cytokines, and receptors. Glaucomatous astrocytes exhibited many functional and biochemical characteristics of reactive astrocytes. Most of the work in the field of glaucoma has focused on the axons of the retinal ganglion cells. It is possible that the astrocytes (activated by elevated intraocular pressure or by other mechanisms) alter the environment of the axons and producing a milieu that may cause axonal degeneration or that may prevent survival of healthy retinal ganglion cells. Future work might suggest mechanisms for altering astrocytes or the environment to protect ganglion cells. Section II: Animal Models Contribute to the Study of GlaucomaPrimate and Rat Models of Ocular Hypertension Two compelling reasons exist for developing animal models in glaucoma. One is to understand the mechanism of pressure-induced optic nerve damage. The other is to develop therapies for protecting the optic nerve in glaucoma. An effective model should allow for the obstruction of aqueous humor outflow in order to raise IOP and for monitoring of intraocular pressure. Nonhuman primates have been the animal of choice for studying glaucoma. Monkeys have been shown to develop glaucomatous changes in the optic nerve when their intraocular pressure is elevated experimentally. On histological examination, ganglion cell nerve fiber degeneration and debris can be seen in the extracellular matrix of the optic nerve. Monkeys can be trained to perform visual field tests, and those with experimental glaucoma show deficits indicative of glaucoma. Because nonhuman primates are costly to acquire and raise, dangerous to handle for testing of IOP, and of uncertain age if they are caught in the wild, a rat model would be preferable. Validation of the rat model is facilitated by the following observations: 1) Frequent IOP measurements using a tonometer are easy to obtain in rats. 2) The composition of the rat optic nerve head is similar to that of the primate, including the connective tissue and cellular (astrocyte) support structures of the optic nerve axon bundles, and 3) Some etiological manifestations such as extracellular matrix material deposition, fluctuations in IOP, cupping, and ganglion cell axon and soma injury are similar to those seen in glaucoma patients. The fact that rats are inexpensive and easy to handle, yield ample tissue for analysis, and through breeding, exhibit less genetic variability are real advantages. In the near future, IOP monitoring of small mammals may be made easier through a transducer chip that would sit on the animal's sclera or through an indwelling catheter in the anterior chamber of the eye. A number of techniques for inducing increases in intraocular pressure in the rat are available. One that we have used successfully involves sclerosing the trabecular meshwork using hypertonic saline. By cannulating the veins and injecting the corrosive saline retrogradely into the canal of Schlemm, it is possible to scar and close up the trabecular meshwork. Intraocular pressure elevates in approximately a week and lesions begin to develop. The lesions seen in the rat optic nerve following this procedure includes a decrease in axon density and an increase in vesicular bodies and vacuoles that is most prominent in the superior region of the optic nerve. The patterning of damage seen in this model lends itself to developing hypotheses related to damage seen in human disease. Animal Models Contribute to the Study of GlaucomaMouse Models of Inherited Optic Nerve and Ganglion Cell Degeneration Inherited models of optic nerve and ganglion cell degeneration are another way to study glaucomatous changes to cell bodies and axons of the eye. Several mouse models-some in which the genetic mutation occurs spontaneously and others in which the mutation is induced-show progressive and gradual onset of disease. Two mouse models that develop spontaneous glaucoma characterized by progressive IOP elevation, RGC death, and optic nerve cupping (DBA/2J and AKXD-28/Ty) have been very valuable models for dissecting pathways of cell death in spontaneous glaucoma. The difference between the more progressive milder onset of pressure insult in these mice and the more sudden insult in experimentally-induced models may be very important. Since exposure to milder insults affects a cell's susceptibility to subsequent more severe insults, there may be meaningful differences in the relative importance of specific death mechanisms/pathways between spontaneous and induced diseases. Inbred mice allow researchers to study larger numbers of genotypically identical animals in highly controlled environments. The DBA/2J mouse has an iris disorder where pigment from the iris washes into the aqueous humor and plugs the drainage system of the eye. IOP rises and, after a few months, the animals' retinal ganglion cells and axons die. The optic disk in this mouse strain shows cupping and retinal ganglion cell bodies appear apoptotic. The phenotype is suppressed in a variant of the DBA/2J mouse, which makes no iris pigment. This variant develops no pressure increase or glaucomatous lesions, which argues strongly in favor of increased IOP as a cause of glaucoma in the DBA/2J model. The AKXD-28/Ty is so susceptible to pressure changes in the eye that its entire retina is affected by increased intraocular pressure. This mouse model could be useful for detecting modifier genes that regulate the influence of pressure. The ability to produce transgenic and gene-targeted mice, and the large number of existing mouse mutants, offers a unique opportunity to study the contribution of various factors to retinal ganglion cell degeneration. As unpublished data support involvement of an axonal degeneration pathway, mice with mutations in various genes that affect specific cell death pathways in the soma and axon will be invaluable in understanding glaucomatous neurodegeneration. In summary, genetic strategies have proven very effective when studying variable, asynchronous disease processes such as ganglion cell death in glaucoma. In fact, it is likely that genetic experiments will be essential for understanding the triggers of complex asynchronous neurodegenerative diseases like glaucoma. Section III: The Biology of the Inner Retina is affected in GlaucomaRetinal Ganglion Cell Interactions and Survival It is well known that shortly after development retinal ganglion cells and other central nervous system (CNS) neurons lose an ability to regenerate. Similarly, after an injury that separates neurons from their target tissue (e.g., other neurons) efforts to regrow CNS axons are uneventful. The cause of this inability to grow axons has eluded generations of researchers. One view is that the failure of axons to regenerate is related to the absence of neurotrophic factors from the target tissue or perhaps to the presence of inhibitory factors from glial and other cells. Neurotrophic factors are peptides that may be instrumental in instructing the cell body of the neuron to form a new axon. They are carried retrogradely from the target tissue to the cell body. A cut or crushed axon prevents trophic factors from reaching the soma. Not only do axons fail to regenerate but cell bodies deprived of neurotrophic factors undergo apoptotic changes and die. There are other possible reasons for the failure of neurons of the central nervous system to regenerate including signals from other cells or the loss of electrical activity in the cell. It is quite likely that a combination of events is at work. Amacrine cells of the retina, for example, have been shown to signal neonatal retinal ganglion cells to undergo a profound and irreversible loss of intrinsic axonal growth ability. Bipolar cells, hormones, superior collicular cells, astrocytes, oligodendrocytes, and microglia are among the possible influences on retinal ganglion cells. Our research into the mechanisms that promote the survival of rat retinal ganglion cells demonstrates that in contrast to PNS neurons, the survival of purified rat retinal ganglion cells in vitro is not promoted by peptide trophic factors unless their intracellular cAMP is increased pharmacologically or by depolarization. Long-term survival of most RGCs in culture can be promoted by a combination of trophic factors normally produced along the visual pathway, including BDNF, CNTF, IGF1, an oligodendrocyte-derived protein, and cAMP elevation. cAMP elevation enhances BDNF responsiveness by rapidly recruiting TrkB to the plasma membrane by translocation from intracellular stores. A crucial question is whether overcoming inhibitory cues will be sufficient to promote axonal regeneration by retinal ganglion cells and other neurons of the CNS. As death of RGCs in glaucoma is believed to be apoptotic, it is likely that RGCs die in glaucoma as their axons are severed at the optic nerve head either because their somas are cut off from target-derived trophic factors or because they lose trophic responsiveness. The survival of RGCs and regeneration of their axons will likely be promoted by exogenous stimulation of these trophic signaling pathways. The peptide trophic factors that normally signal RGCs to survive in vivo are presently unknown. By understanding the influences that affect developing and mature neurons, it may be possible to answer the question and to manipulate influences in order to achieve axonal regeneration. The Biology of the Inner Retina is affected in Glaucoma?Retinal Ganglion Cell Development and Neurodegenerative Events Connectivity of retinal ganglion cells (RGCs) in the rodent is different during development and later in life. Synaptic remodeling occurs as the animal matures. For example, during development, individual ganglion cells have many more connections with target cells than later on. The "synaptosis" (death of synapses) that gives rise to a new pattern of connectivity does not affect the survival of the RGCs. Effective remodeling, which involves the elimination of previously functional connections, occurs before rods and cones are mature. Remodeling requires ganglion cell signaling, since blocking action potentials prevents formation of the layers. These action potentials are endogenously generated in utero long before rods and cones are present; the ganglion cells fire spontaneously and synchronously, generating "waves" of activity that sweep across domains of the retina. This endogenous activity is also required for the regulation of gene expression by LGN neurons, as revealed by differential display comparing expression between control and activity-blocked LGN. A family of genes regulated by this activity is the Class I major histocompatibility complex (MHC I), which encode immunoglobulin superfamily members required for T-cell recognition in the mammalian immune system. These genes are also expressed by CNS neurons at times and regions of activity-dependent axonal rearrangements, in a manner that can be regulated by action potential activity. In mice genetically deficient for cell surface class I MHC, or for CD3 zeta, a required component of signaling for many receptors known to recognize class MHC I, the development of the retinogeniculate projection (known to undergo extensive activity-dependent refinement) is significantly altered from that of wildtype mice, suggesting that normal structural rearrangements have been disrupted. MHC I are also expressed by CNS neurons at times and regions of activity-dependent axonal rearrangements, in a manner that can be regulated by action potential activity. The contribution of the immune system in remodeling during retinal development and in the pathogenesis of glaucoma in the adult eye is an interesting question. A leaky blood-brain barrier at the lamina cribrosa in glaucoma may provide access of T- cells of the immune system to the retina and might lead to destruction of Class I MHC expressing neurons, such as retinal ganglion cells, as an unfortunate side effect of immune surveillance. Such interactions could underlie the mechanisms of many unexplained neurodegenerative disorders, including Parkinson's, Macular Degeneration, the degeneration of retinal ganglion cells associated with glaucoma, or perhaps even Alzheimers'. The Biology of the Inner Retina is affected in GlaucomaGlutamate and Synaptic Transmission Glutamate and calcium are involved in the transmission of signals from the photoreceptor cells to bipolar cells and ganglion cells. Light enters the eye, activates photoreceptor cells, and modulates their release of glutamate. This activates bipolar cells. Bipolar cells also release glutamate onto retinal ganglion cells and amacrine cells. It is important to realize that there is a difference between glutamate in the retina and glutamate elsewhere in the central nervous system (CNS). In the CNS, when a cell is stimulated glutamate is released briefly and quickly recycled back into the cell. In the retina, glutamate is present all the time, in the light and dark; it is released tonically from photoreceptor and bipolar cells. Ganglion cells are driven by bipolar and amacrine cells, receiving inhibitory influences from the GABAergic and glycinergic amacrine cells and excitatory input from the bipolar cells. The influence of bipolar cells on retinal ganglion cells is also affected by inhibitory feedback on them from amacrine cells. The synaptic terminal of the bipolar cell contains many glutamate-containing vesicles, many more than in cells in other parts of the CNS. Not much is known about how the vesicles are transported to their release site. It is known, however, that calcium is involved in the control of glutamate release into the synapse. Calcium enters photoreceptor and bipolar cells through L-type channels. A defect in calcium channels on photoreceptor cells has been demonstrated in a form of congenital night blindness. Perhaps by manipulating calcium channels it would be possible to circumvent photoreceptor degeneration. Glutamate released from the photoreceptor and bipolar cell activates calcium-sensitive receptors [NMDA- and AMPA-containing receptors] on post-synaptic ganglion or amacrine cells. Blocking receptors with antagonists can sometimes prevent calcium from entering the cells and may confer some neural protection. Calcium is extruded from cells through a calcium-sodium exchange or plasma-membrane calcium ATPase. It may be affected by availability of ATPase or by a calcium-sodium exchange that could change acidity in the cell. Glutamate released into the synaptic cleft is taken up by glial cells through glutamate transporters that depend on a potassium and sodium gradient. This is a high affinity transporter system that keeps glutamate at very low levels. Glutamate inside the glial (Muller) cells is converted by glutamine synthetase to glutamine. What may be happening to glutamine is uncertain but it may be recycled back into the synaptic cleft and converted back into glutamate. In experimental glaucoma in a monkey model, more glutamine gathers in the glial cells. This raises the possibility that poorly functioning cellular pumps and transporters may be involved in the pathogenesis of glaucoma. It is also important to note that there is a presumption that glutamate in the cleft and high intracellular calcium are excitotoxic and bad. It is also conceivable that glutamate and NMDA can be good and even be neuroprotective. Raising calcium to a certain level is protective; raising it too high, though, can be neurotoxic. Section IV: Mechanisms of Chronic Neurodegeneration Relevant to Glaucoma EtiologyAmyotrophic Lateral Sclerosis In amyotrophic lateral sclerosis (ALS), there is a selective loss of upper motor neurons in the cerebral cortex and lower motor neurons in the brainstem and spinal cord. Theories of pathobiology in ALS include astrocyte dysfunction, glutamate excitotoxicity, chaperone protein dysfunction, and glutamate transport abnormalities. Transgenic mice have been developed that manifest clinicopathology similar to what is seen in patients with ALS. One with mutations in the gene encoding for superoxide dismutase (SOD1), a protein responsible for causing one form of familial amyotrophic lateral sclerosis, exhibits changes that include pathology of glial and motor neuron cells. Recent work has begun to investigate mechanisms underlying this effect. However, because only a subset of cases of human ALS can be attributed to the mutation of SOD1, it is likely that the cause of ALS is a multifactorial disease. A second transgenic mouse for studying ALS is the SUV-1 mutant. The SUV (small unilamellar vesicle) protein is abundant in degenerating neuronal tissue. It is believed that astrocytes may play a role in the cascade of events that kill neurons in ALS. Degenerating motor neurons of the spinal cord are typically surrounded by reactive astrocytes. The glial cells exhibit aberrant RNA metabolism, excess production of glutamate as evidenced by an increased presence in cerebrospinal fluid, and increased metabolism of glial fibrillary acidic protein (GFAP), the main component of astroglial intermediate filaments. Further evidence of the role of glial cells in ALS is that glial-restricted precursors (GRP) protect against glutamate toxicity and that overexpression of the glutamate transporter EAAT2 prolongs life of neurons. These transporters are believed to be critical in reducing potentially toxic concentrations of glutamate through rapid uptake into nerve terminals and glial cells. A screening of about a thousand compounds revealed a subset of about 20 that increase synthesis of glutamate transport proteins, up to 7-fold. Some of these are FDA-approved drugs that people take all the time. Numerous drugs are being screened for amyotrophic lateral sclerosis. The hope is that the drugs will treat patients effectively and improve the understanding of basic mechanisms of neural degeneration. Some drugs in the pre-clinical research stage, including bioassays of existing drugs, are being applied in animal models, and should be available for clinical trials soon. Additionally, a number of drugs are in development in collaborations between universities and pharmaceutical and biotechnology firms. Mechanisms of Chronic Neurodegeneration Relevant to Glaucoma EtiologyParkinson's Disease and Stroke Neuronal death in neurological disorders may be related to selective vulnerability and resistance of nerve cells. Studying and finding a cure for neurological disease may require knowledge in five areas: cause of injury, cell death cascade, cell survival pathways, regeneration of surviving cells, and replacement of lost cells. Caspase independent cell death has been implicated in experimental models of ischemic injury and Parkinson's disease. Depending on the region of the brain, type of cells, and the cause of ischemia, cells die in different ways. Nerve cell degeneration following stroke, for example, is thought to stem in part from overexcitation of N-methyl-D-aspartate (NMDA) receptors by glutamate. This causes intracellular activation of nitric oxide synthase and an increase in production of nitric oxide (NO). A scaffolding protein is involved in the functioning of the synthase molecule and there is evidence that by disrupting the scaffolding that NO production and cytotoxicity can be eliminated. NO is a membrane-permeable molecule that defuses out of the cell to surrounding neural tissue. Glutamate induces a simultaneous production in mitochondria of superoxide anion and the formation of peroxynitrite. Peroxynitrite has a very specific and damaging effect on DNA leading to strand breaks and activation of poly (ADP-ribose) polymerase (PARP). PARP activation plays a role in NMDA excitotoxicity, focal cerebral ischemia, and death of dopamine producing cells in an animal model of Parkinson's disease. To look at how PARP activation causes neuronal cell death, the researchers compared a mitochondrial associated protein called apoptosis-inducing factor (AIF) in normal and PARP knockout mice. They looked at toxically-exposed nerve cells starting at 10 minutes after exposure, up to 24 hours and found that the course of AIF translocation is concomitant with PARP activation. AIF, under the influence of the excitotoxic PARP stimulus, translocates from the mitochondria to the nucleus (which becomes apoptotic). This does not occur in PARP knockout mice; the cells survive both in vitro and in vivo despite excitotoxicity. The nerve cell death cascade appears to be as follows: Ischemia or another injury causes DNA to be damaged. This activates PARP in the cell nucleus. PARP activation triggers crosstalk between nuclear and mitochondrial proteins that results in AIF release from mitochondria. The nucleus shrinks and neuronal cell death ensues. Evidence for this series of events includes the finding that neutralizing AIF with an anti-AIF antibody is protective against cell death. It may be possible to protect neuronal cells against death. Different strategies for identifying neuroprotective molecules include screening DNA primary libraries and using retroviral vectors to express preconditioning libraries. Thus far the strategies have yielded 40 neuroprotective molecules, five of which show promise against apoptosis. Yet to be screened are 20 additional molecules. The phenomenon of preconditioning occurs in the retina. Survival molecules identified in screens from preconditioned tissue might direct attention to new ways to spare the ganglion cell and optic nerve from degeneration or possibly slow degeneration. Molecules identified in the CNS might be protective. Additionally, the strategies to identify survival genes in the retina could be employed to find eye specific survival molecules. Section V: Degeneration and RegenerationGrowth Factor Deprivation and Neuronal Cell Death Sympathetic neurons in the developing animal require nerve growth factor (NGF) for survival. Without NGF, they undergo cell death (apoptosis). Studying rodent sympathetic neurons in culture containing NGF, or without NGF, can help reveal the process of apoptosis. Apoptosis is an active process that demands the expression of new proteins. In 1994, hundreds of genes that might cause neurons to die were studied. The expression of some increased during apoptosis, including c-Jun, which presages the involvement of the c-Jun N-terminal kinase (JNK) pathway. Studies on the JNK pathway demonstrate that JNK is required for apoptosis to occur. Apoptosis occurs through intrinsic and extrinsic pathways. Growth factor withdrawal involves the intrinsic pathway and includes damage to mitochondria. The extrinsic pathway involves death receptors (Fas, TNF, etc.) or cytotoxic T-cells and activation of proteolytic enzymes. The apoptotic pathways are modified by anti-apoptotic and pro-apoptotic members of the BCL-2 family of proteins. NGF-deprived sympathetic neurons in cell culture from animals lacking the BAX gene of the Bcl-2 family, for example, never die. BAX, it shows, is pro-apoptotic and essential in the intrinsic apoptotic pathway. BAX is contained in the cell at all times, but after an insult, it becomes translocated from the cytosol to the mitochondria where it triggers events that activate a caspase cascade and destruction of the cell. (Expression of anti-apoptotic members of the Bcl-2 family that block BAX can prevent or retard translocation.) If BAX is not induced, other genes might be induced to drive the pathway. One is a pro-apoptotic gene called BIM. Another is DP-5. In other words, there are critical molecules involved in apoptosis including transcription factors, pro- and anti-apoptotic members of the Bcl-2 family, and kinase cascades. An interesting molecule called CEP1347 is being studied as a possible therapeutic agent to prevent apoptosis. It has neurotropic-like properties and is a selective inhibitor of a family of kinases in the JNK pathway. NGF-deprived neurons in the presence of CEP1347 continue to grow. It acts upstream in the apoptosis pathway by blocking the development of "competence to die" in response to cytosolic cytochrome c. Caspase inhibitors, in contrast, act later in the process. Briefly, inhibition of the JNK pathway prevents the catabolic and atrophic effect on nerve cells of NGF-deprivation. Remarkably, even in the absence of growth factor, inhibition of the JNK pathway reverses the impact on the mitochondria. JNK pathway inhibition may have advantages over caspase inhibition in saving neurons in that it appears to preserve the functioning of the cell by acting at an earlier point in the process of apoptosis. Degeneration and RegenerationFactors Promoting Retinal Ganglion Cell Survival and Axon Regeneration Surprising researchers and perhaps turning a dogma on its ear is the finding that retinal ganglion cells exposed in vivo or in vitro to excess levels of the excitatory neurotransmitter glutamate survive. Traditionally, high levels of glutamate are considered neurotoxic and-by increasing damage to mitochondria and other cell components-responsible for cell death in certain neurological disorders. New evidence suggests that excess glutamate may even enhance some properties of retinal ganglion cells, like their responsiveness to trophic factors. We have recently reexamined the effects of glutamate and NMDA on rat RGCs in vitro and in vivo. We have found that highly purified RGCs express NR1 and NR2 receptor proteins by Western blotting and immunostaining, and functional NMDA receptors by whole-cell patch clamp recording. Nonetheless, high concentrations of glutamate and NMDA fail to induce death of any RGCs, even with or prolonged exposure, or in mixed retinal cultures. To determine whether RGCs in an intact retina are vulnerable to excitotoxicity, we retrogradely labeled RGCs using fluorogold and exposed intact acutely-isolated retinas to glutamate and NMDA. This produced substantial loss of amacrine cells, as measured by propridium iodide uptake, including most displaced amacrine cells in the RGC layer, but RGCs were not damaged. These results reveal that RGCs in vitro and in vivo are relatively invulnerable to glutamate excitotoxicity and suggest that neuroprotection with glutamate antagonists may not be an effective treatment for glaucoma or retinal ischemia. It may be difficult to kill retinal ganglion cells with glutamate but not so for all neuronal cells. Hippocampal cells, for example, are easily poisoned by glutamate. This suggests a differential susceptibility to neurotoxicity and injury in different parts of the brain. It is already known that neurons in different regions of the brain die at different rates following a generalized ischemic insult, indicating that neuronal cells do not all share a common pathway for cell death. Degeneration/RegenerationThe Optic Nerve as a Model of CNS White Matter Damage Signals generated by cell bodies of neurons are transmitted through the axons that compose the white matter (WM) of the brain. Also contained within the white matter are astrocytes and oligodendrocytes. The oligodendrocytes produce myelin to speed conduction of signals. Astrocytes play a part in modulating the extracellular environment. Injury to gray matter-cell bodies-from stroke, for example, receives the lion's share of attention from researchers and clinicians seeking strategies to rescue neurons from death. But the white matter, which composes approximately 50 percent of the brain tissue and has metabolic requirements independent of the cell bodies, is also jeopardized by ischemia. In fact, about 20 percent of ischemic strokes involve predominantly WM. Clinically, damage to WM can result in serious disability, as seen in stroke, spinal cord and traumatic brain injury, and some forms of vascular dementia. Axons contain their own mitochondria and make most of their own ATP. Cell bodies provide for axons by producing proteins to replace axonal proteins that wear out, such as ion channels and transport proteins. Models of ischemia, where blood supply is compromised and nutrients such as oxygen and glucose are reduced, can help define mechanisms of loss of neuronal cell function and irreversible injury in the white matter. One such model is the rat optic nerve, a representative white matter tract that is particularly accessible. Experimentally, a stable compound action potential (CAP) can be generated by a supramaximal electrical stimulus to the optic nerve; the CAP can be quantitatively assessed and is altered by anoxia and other toxic stimuli. Ischemia causes ionic disruption in the optic nerve. ATP drops quickly. Intracellular sodium and extracellular potassium become elevated. Acidosis occurs. Function of the optic nerve is lost after extended periods of anoxia or ischemia. As in other tissues, it has been established that ischemia-induced white matter injury is dependent on the presence of extracellular calcium. The pathophysiology of WM damage can be studied at a molecular level using the isolated rodent optic nerve as a model. Optic nerve axons are damaged during ischemia by an uncontrolled influx of extracellular Ca2+ due to reverse Na+/Ca2+ exchange and activation of L-type Ca2+ channels on axons. Reverse Na+/Ca2+ exchange develops because of Na+ loading via non-inactivating Na+ channels and membrane depolarization. The sequence of events activated by elevated [Ca2+]i in axons is poorly understood and may include free radical formation, nitric oxide, and activation of destructive enzymes such as the calpains. Current evidence indicates that Ca2+ enters axons directly to produce axon injury. Nodes of the axon enlarge, mitochondria burst, and the cytoskeleton becomes disorganized when ischemia takes place in the presence of Ca2+. In contrast, without calcium, the axon is totally preserved. Calcium appears to be entering via reverse sodium-calcium exchanger and L-type Ca2+ channels. Blocking the exchanger and L-type Ca2+ channels during ischemia/anoxia blocks Ca2+ entry and protects the axon. Glutamate release during ischemia also appears to influence survival of axons. Staining axons and cells with a variety of markers in a normal white matter preparation to which glutamate has been applied shows breakdown of myelin but no effect on axons proper. Hence, glutamate may be seeking out glial elements. More evidence of glutamate involvement is that AMPA-kainate receptor blockade during ischemia protects the mouse optic nerve. Glutamate may be driven out of axons or astrocytes by reverse sodium-glutamate transport, or perhaps by other mechanisms including opening of hemichannels in astrocytes. Emerging evidence suggests that different white matter areas of the brain respond uniquely to ischemia or anoxia and have somewhat different mechanisms of injury. It is not known what the risk factors (e.g., aging, diabetes) are for axons, how injured glial cells and neurons interact, and what the sequence of events is in chronic white matter injury. Therapies can be developed as these questions are answered. Knowledge of the molecular pathophysiology of ischemic optic nerve (i.e. WM) injury points to several target systems with protective potential, including use-dependent Na+ channel blockers (i.e. anticonvulsant drugs like dilantin), blockers of reverse Na+/Ca2+ exchange, Ca2+ channel blockers, non-NMDA glutamate antagonists, and blockade of glutamate release. Section VI: The Microenvironment in the Optic Nerve HeadRole of Nitric Oxide in Retinal Ganglion Cell Degeneration Age Age has been shown to be an important factor for survival of retinal ganglion cells in rats, mice, monkeys, and humans. All lose about 30-40 percent of their retinal ganglion cells over a lifetime. When ischemia is induced in the eye of a rat model by raising intraocular pressure, retinal ganglion cells in older animals are more susceptible to injury. Caloric restriction, which prolongs life in rats, decreases their loss of retinal ganglion cells. Ischemic injury in young and old animals has revealed the presence of reactive retinal ganglion cells and of reactive astrocytes. Furthermore, ischemia in rats that already have glaucoma increases the vulnerability of their remaining ganglion cells. Nitric oxide Nitric oxide is a potent vasodilator and possible neurotransmitter. There is evidence that excess nitric oxide is neurotoxic and may have a role in the degeneration of the axons of the retinal ganglion cells in glaucoma. The affects of intraocular pressure on optic nerve head astrocytes appears to be important in the pathogenesis of glaucoma. Under in vitro conditions where astrocytes are grown under increased hydrostatic pressure, the cells produce nitric oxide as revealed by the presence of nitric oxide synthase (NOS-2). Glaucomatous optic nerve from the human eye reveals NOS-2 staining and suggests that nitric oxide from astrocytes could be killing retinal ganglion cells. Can astrocytes and retinal ganglion cells be protected from nitric oxide? The answer appears to be yes. This was shown by adding aminoguanidine, an inhibitor of NOS-2, to drinking water of rats with experimentally-induced glaucoma. The optic disc of control animals that received no aminoguanidine showed optic disc cupping, a sign of retinal ganglion cell loss; whereas, treated animals did not. Untreated animals had significant retinal ganglion cell degeneration. Aminoguanidine significantly protected against the loss of retinal ganglion cells in the glaucoma model. SC-51, a Pharmacia compound and a more specific blocker of NOS-2, has a similar effect. Some NOS-2 is necessary for normal immunological function. If research can separate NOS-2 induced by pressure from NOS-2 activated to fight inflammation, it may be possible to create a therapy to prevent nitric oxide neurotoxicity in glaucoma. Early results indicate that by using specific agents that stimulate discrete receptors (e.g., pressure-sensitive EGF receptors on astrocytes), these pathways can be distinguished and perhaps manipulated pharmacologically. In vivo, in the normal eye, there are few EGF receptors. The opposite is found in the glaucomatous eye. An inhibitor of EGF-tyrosine kinase may be useful as a pharmacological neuroprotective agent to prevent the induction of NOS-2 in glaucoma. |
