Guadalupe Ruiz

Vaccination for protection of retinal ganglion cells against death from glutamate cytotoxicity and ocular hypertension: Implications for glaucoma

Our group recently demonstrated that autoimmune T cells directed against central nervous system-associated myelin antigens protect neurons from secondary degeneration. We further showed that the synthetic peptide copolymer 1 (Cop-1), known to suppress experimental autoimmune encephalomyelitis, can be safely substituted for the natural myelin antigen in both passive and active immunization for neuroprotection of the injured optic nerve. Here we attempted to determine whether similar immunizations are protective from retinal ganglion cell loss resulting from a direct biochemical insult caused, for example, by glutamate (a major mediator of degeneration in acute and chronic optic nerve insults) and in a rat model of ocular hypertension. Passive immunization with T cells reactive to myelin basic protein or active immunization with myelin oligodendrocyte glycoprotein-derived peptide, although neuroprotective after optic nerve injury, was ineffective against glutamate toxicity in mice and rats. In contrast, the number of surviving retinal ganglion cells per square millimeter in glutamate-injected retinas was significantly larger in mice immunized 10 days previously with Cop-1 emulsified in complete Freunds adjuvant than in mice injected with PBS in the same adjuvant (2,133 ± 270 and 1,329 ± 121, respectively, mean ± SEM; P < 0.02). A similar pattern was observed when mice were immunized on the day of glutamate injection (1,777 ± 101 compared with 1,414 ± 36; P < 0.05), but not when they were immunized 48 h later. These findings suggest that protection from glutamate toxicity requires reinforcement of the immune system by antigens that are different from those associated with myelin. The use of Cop-1 apparently circumvents this antigen specificity barrier. In the rat ocular hypertension model, which simulates glaucoma, immunization with Cop-1 significantly reduced the retinal ganglion cell loss from 27.8% ± 6.8% to 4.3% ± 1.6%, without affecting the intraocular pressure. This study may point the way to a therapy for glaucoma, a neurodegenerative disease of the optic nerve often associated with increased intraocular pressure, as well as for acute and chronic degenerative disorders in which glutamate is a prominent participant.

Neuroprotective Effect of Memantine in Different Retinal Injury Models in Rats

PurposeTo evaluate the neuroprotective effect of memantine, an NMDA receptor channel blocker, in two retinal ganglion cell (RGC) injury models in rats. MethodsNeuroprotective effect of memantine was tested in partial optic nerve injury and chronic ocular hypertensive models. In the optic nerve injury model, memantine (0.1 – 30 mg/kg) was injected intraperitoneally immediately after injury. Two weeks later, optic nerve function was determined by measuring compound action potential and surviving RGC was determined by retrograde labeling with dextran tetramethyl rhodamine. Chronic ocular hypertension was attained by laser photocoagulation of episcleral and limbal veins. Memantine (5 or 10 mg/kg) was administered continuously each day with an osmotic pump, either immediately after or 10 days after first laser photocoagulation, for 3 weeks, after which RGC survival was determined. ResultsTwo weeks after partial optic nerve injury, there was ≈80% reduction in RGC number. Memantine (5 mg/kg) caused a twofold increase in compound action potential amplitude and a 1.7-fold increase in survival of RGCs, respectively. In the chronic ocular hypertension model there was 37% decrease in RGCs after 3 weeks of elevated intraocular pressure. Memantine (10 mg/kg daily) reduced ganglion cell loss to 12% when applied immediately after first laser photocoagulation, and prevented any further loss when applied 10 days after first laser photocoagulation. ConclusionThe protective effect of memantine suggests that excessive stimulation of NMDA receptors by glutamate is involved in causing cell damage in these RGC injury models.

Müller cell response to laser-induced increase in intraocular pressure in rats

The goal of this study was to investigate the reaction of the Müller cells to elevated intraocular pressure (IOP). Elevated IOP is one of the risk factors in glaucomatous retinal ganglion cell (RGC) degeneration. Müller cells play an important role in retinal homeostasis. The reaction of Müller cells was examined by evaluating temporal changes in glutamate aspartate transporter (GLAST), glutamine synthase (GS), glial fibrillary acidic protein (GFAP), and the B‐cell lymphoma (Bcl‐2) using immunoblotting and immunohistochemical techniques. After IOP was elevated for 4–60 days, there was a time‐related decrease in RGC ranging from 6% to 44%. There was also a time‐related increase in GLAST protein reaching maximum after 3 weeks of elevated IOP. On the other hand, there was very little change in the expression of GS during the first 2 weeks followed by some increase between 21 and 60 days. An increase in Bcl‐2 was biphasic with maximum increase after 4 days followed by decline after 15 and 21 days. GFAP, which is usually not expressed in normal Müller cells, was present at all time points. In all cases, the increase was most intense in the vicinity of the ganglion cells where the astrocytes and endfeet of the Müller cells are located. These results indicate that Müller cells react to the insult of elevated IOP by expressing GFAP and Bcl‐2, proteins that are expressed in reactive gliosis and other pathological conditions. The increase in GLAST along with minimum change in GS indicates a disturbance in glutamate homeostasis.

Efficacy and safety of memantine, an NMDA-type open-channel blocker, for reduction of retinal injury associated with experimental glaucoma in rat and monkey.

Glutamatergic excitotoxicity has been implicated as a mechanism for injury in a variety of central nervous system pathologies, including glaucoma. Memantine, an NMDA-type glutamatergic open-channel blocker, has pharmacologic properties that make its efficacy greater under excitotoxic conditions, but lesser under normal conditions. Daily oral dosing for approximately 15 months with 4.0 mg/kg memantine in monkeys yielded plasma concentrations similar to those found in patients who received memantine treatment for Parkinsons disease. This same dose of memantine was not associated with any evidence of an effect on the normal function of the retina and central visual pathways, as indicated by measures of the electroretinogram (ERG) and visually-evoked cortical potential (VECP). Amplitude of the VECP response was reduced in eyes with experimentally induced glaucoma. When compared to vehicle-treated control animals, memantine-treated glaucoma eyes suffered significantly less reduction of VECP amplitude. Preliminary results in a rat model for experimental glaucoma also show that, when compared to control animals, systemic treatment with memantine (10 mg/kg/day) was associated with a significant reduction in glaucoma-induced loss of retinal ganglion cells.

Glaucoma Alters the Circadian Timing System

Glaucoma is a widespread ocular disease and major cause of blindness characterized by progressive, irreversible damage of the optic nerve. Although the degenerative loss of retinal ganglion cells (RGC) and visual deficits associated with glaucoma have been extensively studied, we hypothesize that glaucoma will also lead to alteration of the circadian timing system. Circadian and non-visual responses to light are mediated by a specialized subset of melanopsin expressing RGCs that provide photic input to mammalian endogenous clock in the suprachiasmatic nucleus (SCN). In order to explore the molecular, anatomical and functional consequences of glaucoma we used a rodent model of chronic ocular hypertension, a primary causal factor of the pathology. Quantitative analysis of retinal projections using sensitive anterograde tracing demonstrates a significant reduction (∼50–70%) of RGC axon terminals in all visual and non-visual structures and notably in the SCN. The capacity of glaucomatous rats to entrain to light was challenged by exposure to successive shifts of the light dark (LD) cycle associated with step-wise decreases in light intensity. Although glaucomatous rats are able to entrain their locomotor activity to the LD cycle at all light levels, they require more time to re-adjust to a shifted LD cycle and show significantly greater variability in activity onsets in comparison with normal rats. Quantitative PCR reveals the novel finding that melanopsin as well as rod and cone opsin mRNAs are significantly reduced in glaucomatous retinas. Our findings demonstrate that glaucoma impacts on all these aspects of the circadian timing system. In light of these results, the classical view of glaucoma as pathology unique to the visual system should be extended to include anatomical and functional alterations of the circadian timing system.

Assessment of the differences in pharmacokinetics and pharmacodynamics between four distinct formulations of triamcinolone acetonide.

Purpose: To compare the durability of Kenalog, Trivaris, Triesence, and compounding pharmacy preservative-free triamcinolone acetonide in pigmented rabbits with syneretic vitreous using direct visualization, pharmacodynamics, and pharmacokinetics. Methods: Twenty-five Dutch-belted rabbits were used. Pharmacokinetic experiment: Rabbits were intravitreally injected with one of four 4-mg triamcinolone acetonide formulations. Wide-field imaging was serially performed to document residual drug mass. Pharmacodynamics experiment: Four triamcinolone acetonide groups and one control group received intravitreal recombinant human vascular endothelial growth factor 165 every 2 weeks and were followed with fluorescein angiography to assess vascular endothelial growth factor retinal vasculopathy as a measure of residual steroid effect. Particle size of the formulations was measured with Mastersizer 2000. Results: Remaining triamcinolone acetonide mass after 19 weeks: 12,091 ± 2,512 pixels for the Kenalog group, 1,307.36 ± 695.57 for Trivaris, 5577 ± 1477 for Triesence, and 1,535 ± 329 for compounded preservative-free triamcinolone acetonide. Kenalog suppressed recombinant human vascular endothelial growth factor–induced retinopathy more effectively than the other triamcinolone acetonide groups at Week 39, the final time point assessed. Particle size (90th percentile) was 47 &mgr;m for Kenalog, 26 &mgr;m for Triesence, and 22 &mgr;m for both compounded preservative-free triamcinolone acetonide and Trivaris. Conclusion: Triamcinolone acetonide formulations do not have the same pharmacokinetics/pharmacodynamics. Kenalog has the longest vitreous visibility and durability. Particle size appears to correlate with efficacy and durability.

Pharmacological characterization of β-adrenoceptor subtype involvement in the ocular hypotensive response to β-adrenergic stimulation

The decrease in intraocular pressure elicited by isoproterenol in ocular normotensive animals is widely recognized. The participation of the β-adrenoceptor subtypes in mediating this ocular hypotensive response has, however, remained unclear, because previous studies have been limited to monitoring the activity of single, supramaximal doses of relatively selective β2-adrenoceptor agonists. The studies herein report a relatively extensive pharmacological characterization of β1-and β2-adrenoceptor involvement in ocular hypotension associated with β-adrenergic stimulation in the pigmented rabbit. A β2-adrenoceptor mechanism was indicated by the following evidence: (1) isoproterenol and relatively selective β2-adrenoceptor agonists produced ocular hypotension over a similar dose range (0·001–0·1%; (2) β1-adrenoceptor agonists, at doses likely to confer β1-adrenoceptor specificity, did not cause a similar decrease in intraocular pressure; (3) the ocular hypotensive response to isoproterenol was abolished by topical timolol and pindolol and the relatively selective β2-adrenoceptor antagonist ICI 118551, whereas the relatively selective β1-adrenoceptor antagonists metoprolol and betaxolol were topically inactive; (4) intravenous injection of β1-adrenoceptor-specific doses of metoprolol and betaxolol had little effect on isoproterenol-induced ocular hypotension, whereas the response was antagonized by a β2-adrenoceptor-specific i.v. dose of ICI 118551. These pharmacological results are consistent with radioligand binding and β-adrenoceptor-linked adenylate cyclase studies which indicate a predominantly β2-adrenoceptor population associated with the ocular ciliary processes. None of the β-blockers themselves altered normal intraocular pressure in the pigmented rabbit.

Elevated albumin in retinas of monkeys with experimental glaucoma.

PURPOSE To establish the identity of a prominent protein, approximately 70 kDa, that is markedly increased in the retina of monkeys with experimental glaucoma compared with the fellow control retina, the relationship to glaucoma severity, and its localization in the retina. METHODS Retinal extracts were subjected to 2-D gel electrophoresis to identify differentially expressed proteins. Purified peptides from the abundant 70 kDa protein were analyzed and identified by liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) separation, and collision-induced dissociation sequencing. Protein identity was performed on MASCOT (Matrix Science, Boston, MA) and confirmed by Western blot. The relationship between the increase in this protein and glaucoma severity was investigated by regression analyses. Protein localization in retina was evaluated by immunohistochemistry with confocal imaging. RESULTS The abundant protein was identified as Macaca mulatta serum albumin precursor (67 kDa) from eight non-overlapping proteolytic fragments, and the identity was confirmed by Western blot. The average increase in retinal albumin content was 2.3 fold (P = 0.015). In glaucoma eyes, albumin was localized to some neurons of the inner nuclear layer, in the inner plexiform layer, and along the vitreal surface, but it was only found in blood vessels in control retinas. CONCLUSIONS Albumin is the abundant protein found in the glaucomatous monkey retinas. The increased albumin is primarily localized to the inner retina where oxidative damage associated with experimental glaucoma is known to be prominent. Since albumin is a major antioxidant, the increase of albumin in the retinas of eyes with experimental glaucoma may serve to protect the retina against oxidative damage.

Effect of histamine on signal transduction in cultured human trabecular meshwork cells

Stimulation of cultured human trabecular meshwork cells by histamine caused time and dose related increases in inositol phosphates and intracellular free calcium. The increase in inositol trisphosphate (IP3) was immediate and calcium independent while that of inositol monophosphate (IP1) was gradual and calcium dependent. The rise in intracellular calcium was also rapid and occurred as a result of mobilization from intracellular stores and influx from external medium. Histamine also caused time and concentration related de novo synthesis of inositol phospholipids. Mepyramine but not cimetidine inhibited the action of histamine. These results indicate that histamine, via H1 receptor, evokes an early hydrolysis of inositol phospholipids and increase in intracellular free calcium, signals which may be involved with the function of the trabecular meshwork cells.