Egr1 expression is induced following glatiramer acetate immunotherapy in rodent models of glaucoma and Alzheimer's disease.

PURPOSE: Immunization with glatiramer acetate (GA) alleviates the neuropathology associated with glaucoma and Alzheimer's disease (AD) in rodent models. This research was undertaken to screen for molecular factors underlying GA-induced neuroprotective mechanisms in these models of chronic neurodegeneration. METHODS: Gene expression profiles were analyzed in GA-immunized versus nonimmunized elevated-intraocular pressure (IOP) rat models of glaucoma by using whole genome cDNA microarrays and were further validated by quantitative real-time PCR analysis. A gene, prominently upregulated by GA in elevated IOP retina, was further studied in APP(SWE)/PS1(ΔE9)-transgenic (AD-Tg) mice after GA immunization. RESULTS: Seven days after treatment with GA, numerous genes were regulated in the retinas of rats with elevated IOP. Comprehensive functional classification and DAVID/KEGG enrichment analysis of GA-induced differentially expressed genes revealed annotation terms and pathways involved in neuroprotection, immune responses, cell communication, and regeneration. Specifically, increased mRNA levels of an early growth response (Egr) 1 gene were evident in GA-immunized retinas with elevated IOP. In AD-Tg mice, a significant increase in hippocampal EGR1 protein levels was also found in response to GA immunization. Nuclear EGR1 in the dentate gyrus colocalized more frequently with doublecortin-positive and Ki67 proliferating neural progenitors in GA-immunized as compared to nonimmunized AD-Tg mice. Further, EGR1 levels were negatively correlated with hippocampal amyloid-ß plaque burden. CONCLUSIONS: This study presents global gene expression profiles associated with GA immunization in a glaucoma rat model. Moreover, it identifies EGR1 transcription factor as a potential mediator for GA-induced neuroprotection in both glaucoma and AD.

Toll-like receptor 4 restricts retinal progenitor cell proliferation.

Retinal neurogenesis ceases by the early postnatal period, although retinal progenitor cells (RPCs) persist throughout life. In this study, we show that in the mammalian eye, the function of Toll-like receptor 4 (TLR4) extends beyond regulation of the innate immune response; it restricts RPC proliferation. In TLR4-deficient mice, enhanced proliferation of cells reminiscent of RPCs is evident during the early postnatal period. In vitro experiments demonstrate that TLR4 acts as an intrinsic regulator of RPC fate decision. Increased TLR4 expression in the eye correlates with the postnatal cessation of cell proliferation. However, deficient TLR4 expression is not sufficient to extend the proliferative period but rather contributes to resumption of proliferation in combination with growth factors. Proliferation in vivo is inhibited by both MyD88-dependent and -independent pathways, similar to the mechanisms activated by TLR4 in immune cells. Thus, our study attributes a novel role to TLR4 as a negative regulator of RPC proliferation.

Chondroitin sulfate-derived disaccharide protects retinal cells from elevated intraocular pressure in aged and immunocompromised rats.

PURPOSE: A disaccharide (DS) derived from the naturally occurring compound chondroitin sulfate proteoglycan (CSPG) was recently shown to have neuroprotective activity. The authors examined the ability of this compound (CSPG-DS) to protect retinal ganglion cells (RGCs) from death caused by elevated intraocular pressure (IOP). METHODS: With the use of chronic and acute models of elevated IOP, the authors examined the effects of CSPG-DS on RGC survival in adult ( approximately 2 months old), aged (10-12 months old), and immunocompromised Lewis rats. Systemic, topical, and oral routes of administration were examined. RESULTS: CSPG-DS protected RGCs from IOP-induced death. Treatment was effective in all three examined rat populations (normal adult, aged, and immunocompromised rats) and with all routes of administration, possibly in part through its control of microglial activity. CONCLUSIONS: Results point to the therapeutic potential of CSPG-DS for glaucoma, particularly in elderly populations for whom disease prevalence is high.

A rat model for acute rise in intraocular pressure: immune modulation as a therapeutic strategy.

PURPOSE: To establish a rat model of acute increase in intraocular pressure (IOP) and to investigate the therapeutic window for protection against death of retinal ganglion cells (RGCs) by vaccination with glatiramer acetate (Cop-1) or by treatment with brimonidine or MK-801. DESIGN: Animal study, laboratory investigation. METHODS: IOP was transiently increased in anesthetized Lewis rats by infusing normal saline (0.9%) into the anterior chamber of the eye for one hour. RGC survival was assessed one week and two weeks later by counting the RGCs retrogradely labeled with rhodamine dextran. MAIN OUTCOME MEASURES: RGC survival. RESULTS: IOP rose to 100 cm H(2)O (76 mm Hg) and returned to baseline after 24 hours. The RGC count decreased by 23% a week after the insult and by a further 7% after the second week. Vaccination with Cop-1 on the day of the insult prevented 50% of the IOP-induced RGC loss. Similar neuroprotection was achieved by daily intraperitoneal injections of brimonidine, but not with MK-801. CONCLUSIONS: A transient increase in IOP to 100 cm H(2)O causes death of RGCs in rats. A single immunization with Cop-1 or daily injections of brimonidine protected up to 50% of potentially doomed RGCs from IOP-induced death, suggesting that not all of the cell death in the untreated model results from the IOP insult directly, but that some of it is caused by insult-induced environmental cytotoxicity, which is unrelated to glutamate toxicity or at least to NMDA receptors. These findings can be applied immediately as a basis for acute glaucoma therapy.

A sulfated disaccharide derived from chondroitin sulfate proteoglycan protects against inflammation-associated neurodegeneration.

Chondroitin sulfate proteoglycan (CSPG), a matrix protein that occurs naturally in the central nervous system (CNS), is considered to be a major inhibitor of axonal regeneration and is known to participate in activation of the inflammatory response. The degradation of CSPG by a specific enzyme, chondroitinase ABC, promotes repair. We postulated that a disaccharidic degradation product of this glycoprotein (CSPG-DS), generated following such degradation, participates in the modulation of the inflammatory responses and can, therefore, promote recovery in immune-induced neuropathologies of the CNS, such as experimental autoimmune encephalomyelitis (EAE) and experimental autoimmune uveitis (EAU). In these pathologies, the dramatic increase in T cells infiltrating the CNS is far in excess of the numbers needed for regular maintenance. Here, we show that CSPG-DS markedly alleviated the clinical symptoms of EAE and protected against the neuronal loss in EAU. The last effect was associated with a reduction in the numbers of infiltrating T cells and marked microglia activation. This is further supported by our in vitro results indicating that CSPG-DS attenuated T cell motility and decreased secretion of the cytokines interferon-gamma and tumor necrosis factor-alpha. Mechanistically, these effects are associated with an increase in SOCS-3 levels and a decrease in NF-kappaB. Our results point to a potential therapeutic modality, in which a compound derived from an endogenous CNS-resident molecule, known for its destructive role in CNS recovery, might be helpful in overcoming inflammation-induced neurodegenerative conditions.

Pattern electroretinography in a rat model of ocular hypertension: functional evidence for early detection of inner retinal damage.

With the increasing use of the rat as an animal model for glaucoma and for the evaluation of neuroprotective treatments, there is a need for a sensitive test of retinal ganglion cell (RGC) function in this species. The aims of this study were to detect functional abnormalities of the inner retina in a rat model of high intraocular pressure (IOP) using the pattern electroretinogram (PERG), and to correlate them with morphometric analysis of RGC survival and the functional integrity of the inner retina. Unilateral ocular hypertension was induced in 17 Lewis rats through laser photocoagulation. Pattern ERGs were recorded prior to lasering and 3 weeks later, using a series of shifting patterns of decreasing spatial frequency projected directly onto the animals' fundus. IOP was measured at the same intervals, and the number of surviving RGCs estimated. Low amplitude PERG signals could be recorded in response to a narrow grating of 0.368 cycles per degree (cpd), and increased with stimulus size. Lasering caused mean (+/-s.d.) IOP to increase significantly from 18.3+/-4.5 (baseline) to 29.8+/-8.8 mmHg within 3 weeks (p<0.0001). At this time, PERG amplitudes were significantly reduced (p<0.05), declining an average of 45% compared to the normotensive, control eyes. No outer retinal damage was observed, but the mean number of RGCs decreased significantly (p<0.001), from 2 525.0+/-372.4 to 1 542.8+/-333.8 cells per mm2. This decrease in RGC number was significantly (p=0.03) correlated the decrease in PERG amplitude. The correlation between functional integrity of the inner retina and the rat PERG was further demonstrated by intravitreal tetrodotoxin injections, which temporarily abolished the PERG but did not affect outer retinal activity, reflected in the flash ERG. The evidence for early functional deficits, combined with tonometry and documentation of correlated ganglion cells loss, confirms the sensitivity of this diagnostic tool and the validity and importance of this animal model in glaucoma research.

T-cell-based vaccination for morphological and functional neuroprotection in a rat model of chronically elevated intraocular pressure.

Acute or chronic glaucoma is often associated with an increase in intraocular pressure (IOP). In many patients, however, therapeutic pressure reduction does not halt disease progression. Neuroprotection has been proposed as a complementary therapeutic approach. We previously demonstrated effective T-cell-based neuroprotection in experimental animals vaccinated with the synthetic copolymer glatiramer acetate (copolymer-1, Cop-1), a weak agonist of self-antigens. This study was undertaken to test different routes and modes of vaccination with Cop-1 as treatment modalities for protection against retinal ganglion cell (RGC) death caused by chronic elevation of IOP in rats, and to determine whether anatomical neuroprotection is accompanied by functional neuroprotection. In a chronic model of unilaterally high IOP, Cop-1 vaccination, with or without an adjuvant, protected rats against IOP-induced loss of RGCs by eliciting a systemic T-cell-mediated response capable of cross-reacting with self-antigens residing in the eye. In rats deprived of T cells, Cop-1 (unlike treatment with alpha2-adrenoreceptor agonists) was not protective of RGCs, substantiating the contention that its beneficial effect is not conferred directly but is T-cell-mediated. Pattern electroretinography provided evidence of functional protection. Thus, vaccination with adjuvant-free Cop-1 can protect RGCs from the consequences of elevated IOP in rats. This protection is manifested both morphologically and functionally. These findings can be readily implemented for the development of a therapeutic vaccination to arrest the progression of glaucoma.

A disaccharide derived from chondroitin sulphate proteoglycan promotes central nervous system repair in rats and mice.

Chondroitin sulphate proteoglycan (CSPG) inhibits axonal regeneration in the central nervous system (CNS) and its local degradation promotes repair. We postulated that the enzymatic degradation of CSPG generates reparative products. Here we show that an enzymatic degradation product of CSPG, a specific disaccharide (CSPG-DS), promoted CNS recovery by modulating both neuronal and microglial behaviour. In neurons, acting via a mechanism that involves the PKCalpha and PYK2 intracellular signalling pathways, CSPG-DS induced neurite outgrowth and protected against neuronal toxicity and axonal collapse in vitro. In microglia, via a mechanism that involves ERK1/2 and PYK2, CSPG-DS evoked a response that allowed these cells to manifest a neuroprotective phenotype ex vivo. In vivo, systemically or locally injected CSPG-DS protected neurons in mice subjected to glutamate or aggregated beta-amyloid intoxication. Our results suggest that treatment with CSPG-DS might provide a way to promote post-traumatic recovery, via multiple cellular targets.

Antigenic specificity of immunoprotective therapeutic vaccination for glaucoma.

PURPOSE: To investigate the antigenic specificity of the immune neuroprotective mechanism that can protect retinal ganglion cells (RGCs) against death caused by high intraocular pressure (IOP). METHODS: A unilateral increase in IOP was induced in rats by argon laser photocoagulation of the episcleral veins and limbal plexus. Rats with high IOP were immunized with glatiramer acetate (Cop-1, a synthetic copolymer) or with myelin-derived or uveitogenic peptides. When the steroid drug methylprednisolone was used, it was administered intraperitoneally every other day for 12 days. RESULTS: Vaccination with myelin-derived peptides that reside in the axons failed to protect RGCs from death caused by high IOP. In contrast, IOP-induced RGC loss was reduced by vaccination with R16, a peptide derived from interphotoreceptor retinoid-binding protein, an immunodominant antigen residing in the eye. The benefit of protection against IOP-induced RGC loss outweighed the cost of the monophasic experimental autoimmune uveitis (EAU) that transiently developed in a susceptible rat strain. Treatment with methylprednisolone alleviated the disease symptoms, but caused further loss of RGCs. Cop-1 vaccination was effective in both EAU-resistant and EAU-susceptible strains. CONCLUSIONS: To benefit damaged neurons, immune neuroprotection should be directed against immunodominant antigens that reside in the site of damage. In a rat model of high IOP, RGCs can benefit from vaccination with peptides derived from proteins that are immunodominant in the eye but not from myelin-associated proteins. This suggests that the site of primary degeneration in IOP-induced RGC loss is in the eye. Cop-1 vaccination apparently circumvents the site-specificity barrier and provides protection without risk of inducing autoimmune disease.

Resistance of retinal ganglion cells to an increase in intraocular pressure is immune-dependent.

PURPOSE: Glaucoma is widely accepted as a neurodegenerative disease in which retinal ganglion cell (RGC) loss is initiated by a primary insult to the optic nerve head, caused, for example, by increased intraocular pressure (IOP). In some cases, the surviving RGCs, despite adequate IOP control, may continue to degenerate as a result of their heightened susceptibility to self-destructive processes evoked by the initial damage. In animal models of mechanical or biochemical injury to the optic nerve or retina, a T-cell-mediated immune response evoked by the insult helps to reduce this ongoing loss. The current study was conducted to find out whether the ability to resist the IOP-induced loss of RGCs in a rat model is affected by the immune system. METHODS: The ocular veins and limbal plexus of rats of two strains differing in their resistance to experimental autoimmune encephalomyelitis (EAE) and in their ability to manifest a beneficial autoimmune response were laser irradiated twice to induce an increase in IOP. The pressure was measured weekly, and RGC losses were assessed 3 and 6 weeks after the first irradiation. To verify the existence of a relationship between the immune system and RGC survival, we assessed neuronal survival in Sprague-Dawley (SPD) rats devoid of mature T cells as well as after transferring splenocytes from Fisher rats, an EAE-resistant rat strain capable of manifesting T-cell-mediated neuroprotection, to rats of a major histocompatibility complex (MHC)-matched EAE-susceptible strain (Lewis), in which the ability to manifest such protective immunity is limited. RESULTS: Both 3 and 6 weeks after the increase in IOP was initiated, the number of surviving RGCs in SPD rats, a strain in which a beneficial autoimmune response can be evoked spontaneously, was significantly higher than in Lewis rats. Moreover, in SPD rats that were thymectomized at birth, the number of surviving RGCs after an increase in IOP as adults was significantly diminished. Passive transfer of splenocytes from Fisher rats to Lewis rats significantly reduced the IOP-induced loss of RGCs in the latter. CONCLUSIONS: In rats of different strains, a similar increase in IOP results in differing amounts of RGC loss. This disparity was found to correlate with immune potency. These findings may explain why patients with glaucoma experience different degrees of visual loss after pressure reduction, even when the severity of the disease at the time of diagnosis is similar. The results have far-reaching prognostic and therapeutic implications.