Induction Of Ocular Hypertension Research Articles

Macrogilal retinal cells show a bilateral early activation in a mouse model of unilateral laser‐induced experimental glaucoma

PurposeTo describe macroglial morphological features of activation after 24 h of unilateral laser‐induced ocular hypertension (OHT) in OHT‐eyes and their contralateral eyes.MethodsAlbino Swiss mice were divided into two groups, naïve (n = 6) and lasered (n = 6). Retinal whole‐mounts were immunolabeled with antibodies against GFAP and MHC‐II.ResultsWith respect to naïve, OHT eyes and contralateral eyes showed different features of macroglial activation in some retinal areas: i) Müller cells were reactive and expressed GFAP; ii) astrocytes were more robust and had more primary and secondary processes; and iii) MHCII was up‐regulated in astrocytes and in some Müller cells. In contralateral eyes, these signs were observed mainly in the periphery. In addition, in the OHT eyes: i) in some retinal vessels near the optic disc, the astroglial reactivity was so intense that the entire vascular walls were sheathed by astroglial cells; ii) in some areas of the retina, astrocytes and Müller cells formed glial scars. iii) MHC‐II immunoreaction in astrocytes varied depending on the retinal area; in those in which MHC‐II immunostaining was less intense, only astrocyte primary processes could be recognized;ConclusionsAt 24 h after unilateral OHT, macroglia showed features of activation not only in OHT eyes, but also in untreated contralateral eyes. Early up‐regulation of MHC‐II in macroglial cells in both OHT retinas and contralateral retinas, suggest an immune system‐related inflammation early after the induction of ocular hypertension.

Effect of Diltiazem on Intraocular Pressure in Normal and Ocular Hypertensive Rabbits

Background; Glaucoma is a multifactorial disease involving progressive optic neuropathy and altered intraocular hemodynamics; furthermore, glaucoma can cause blindness if it is left untreated.Aim of the study: To explore effects of topical diltiazem on intraocular pressure (IOP) in each of normal and ocular hypertensive rabbits.Materials and methods: A group of 54 males of the rabbits were included in the present study. Induction of ocular hypertension was achieved by injection of hydroxy propyl methylcellulose in the anterior chamber of rabbits right eye. In addition to distilled water (as negative control), each of timolol (as positive control) and diltiazem (the tested drug) eye drops were instilled 3 times daily to right eye prophylactically for 4 days and therapeuticallyResults: Part Ι: Diltiazem (0.5%) caused highly significant (P < 0.01) reduction in intraocular pressure of right eye .
 Part ΙΙ: diltiazem, at both concentrations (0.5% and 0.25%) caused highly significant reduction (P < 0.01) in IOP at 5th and 10th days post treatment.Conclusions: Diltiazem exerted a detectable ocular hypotensive effect on the eye of rabbits when applied at concentration (0.5%) or (0.25%) 3 times daily.

Effect of Topically – Applied Carvedilol on Intraocular Pressure in Normal and Ocular Hypertensive Rabbits

Background: In glaucoma, as optic neuropathy gradually proceeds unnoticed by the patient, early detection and treatment is of paramount importance in arresting or controlling the progress of damage.Aim of the study: To explore effects of topical carvedilol on intraocular pressure (IOP) in each of normotensive and ocular hypertensive eyes of rabbits, with assessment of drug safety.Material and methods: A group of 54 males rabbits were included in this study. Induction of ocular hypertension was achieved by injection of hydroxy propyl methylcellulose in the anterior chamber of rabbits right eye. The present study was designed to evaluate the possible beneficial therapeutic effect (part I) and both prophylactic as well as therapeutic effect (part II). The included rabbits were divided into distilled water group, timolol (0.25% and 0.5%) groups, and carvedilol (0.25% and 0.5%). Each of drug eye drops (including distilled water) were instilled into right eyes 3 times/day for 4 days prophylactically and for 10 days therapeutically. The rabbits had been examined for the IOP, pupil diameter, light reflex, corneal reflex, and conjunctival redness prior to instillation of drugs and along the trial period.Results: Ocular hypotensive effects of carvedilol (0.25%) and (0.5%) eye drops were more efficient than that of distilled water (P<0.01). Furthermore, carvedilol (0.25%) eye drop simulated timolol (0.25%) eye drop (P>0.05) in its ocular hypotensive effect along the trial period .Carvedilol eye drop required 4 days of instillation into normotensive eyes to achieve highly significant (P<0.01) ocular hypotensive effect whereas 10 days were needed to do so in ocular hypertensive eyes; such ocular hypotensive effect was more efficient than that of distilled water, but less efficient than that of timolol eye drop.In both parts of the present study and regarding each of mean pupil diameter, light reflex, corneal reflex and conjunctival redness, carvedilol (0.25% or 0.5%) eye drops had no significant adverse effect (P > 0.05).Conclusions: Carvedilol eye drops instilled 3 times / day had an obvious prophylactic role in normotensive and a beneficial, safe, and tolerable therapeutic ocular hypotensive effects on hydroxy propyl methyl cellulose - induced ocular hypertension in rabbits .

Automatic quantitative analysis of experimental primary and secondary retinal neurodegeneration: implications for optic neuropathies.

Secondary neurodegeneration is thought to play an important role in the pathology of neurodegenerative disease, which potential therapies may target. However, the quantitative assessment of the degree of secondary neurodegeneration is difficult. The present study describes a novel algorithm from which estimates of primary and secondary degeneration are computed using well-established rodent models of partial optic nerve transection (pONT) and ocular hypertension (OHT). Brn3-labelled retinal ganglion cells (RGCs) were identified in whole-retinal mounts from which RGC density, nearest neighbour distances and regularity indices were determined. The spatial distribution and rate of RGC loss were assessed and the percentage of primary and secondary degeneration in each non-overlapping segment was calculated. Mean RGC number (82 592±681) and RGC density (1695±23.3 RGC/mm2) in naïve eyes were comparable with previous studies, with an average decline in RGC density of 71±17 and 23±5% over the time course of pONT and OHT models, respectively. Spatial analysis revealed greatest RGC loss in the superior and central retina in pONT, but significant RGC loss in the inferior retina from 3 days post model induction. In comparison, there was no significant difference between superior and inferior retina after OHT induction, and RGC loss occurred mainly along the superior/inferior axis (~30%) versus the nasal–temporal axis (~15%). Intriguingly, a significant loss of RGCs was also observed in contralateral eyes in experimental OHT. In conclusion, a novel algorithm to automatically segment Brn3a-labelled retinal whole-mounts into non-overlapping segments is described, which enables automated spatial and temporal segmentation of RGCs, revealing heterogeneity in the spatial distribution of primary and secondary degenerative processes. This method provides an attractive means to rapidly determine the efficacy of neuroprotective therapies with implications for any neurodegenerative disorder affecting the retina.

Hypoxic Preconditioning Improved Neuroprotective Effect of Bone Marrow-Mesenchymal Stem Cells Transplantation in Acute Glaucoma Models

This study explored the novel strategy of hypoxic preconditioning of Bone Marrow Mesenchymal Stem Cells (BM-MSCs) before intra vitreal transplantation to improve neuroprotective effects of Retinal Ganglion Cells (RGCs) in Acute Glaucoma Models. The methods of this research were isolated mesenchymal stem cells from the bone marrow of adult wild-type Sprague-Dawley (SD) rats. BM-MSCs were cultured under normoxic or hypoxic (1% oxygen for 24 hours) conditions. Normoxic or hypoxic BM-MSCs were transplanted intravitreally 1 week after ocular hypertension induction by acutely increasing IOP to 100 - 120 mmHg for 60 minutes. Rats were killed 4 weeks after transplanted. Apoptosis was examined by tunnel assay and expression Brn3b (Brn3b = RGCs marker) by immunohistochemical analysis of the retina. Results showed that transplantation of hypoxic preconditioning BM-MSCs in acute glaucoma models resulted in a significant apoptosis decreasing (p < 0.05) and an significant increasing in RGCs (p < 0.05), as well as enhanced mor-phologic and functional benefits of stem cell therapy versus normoxic BM-MSCs transplantation. Conclusions: Hypoxic preconditioning enhances the capacity of BM-MSCs transplantation to improve neuroprotective effects of RGCs in Acute Glaucoma Models.

Time-dependent retinal ganglion cell loss, microglial activation and blood-retina-barrier tightness in an acute model of ocular hypertension

Glaucoma is a group of neurodegenerative diseases characterized by the progressive loss of retinal ganglion cells (RGCs) and their axons, and is the second leading cause of blindness worldwide. Elevated intraocular pressure is a well known risk factor for the development of glaucomatous optic neuropathy and pharmacological or surgical lowering of intraocular pressure represents a standard procedure in glaucoma treatment. However, the treatment options are limited and although lowering of intraocular pressure impedes disease progression, glaucoma cannot be cured by the currently available therapy concepts. In an acute short-term ocular hypertension model in rat, we characterize RGC loss, but also microglial cell activation and vascular alterations of the retina at certain time points. The combination of these three parameters might facilitate a better evaluation of the disease progression, and could further serve as a new model to test novel treatment strategies at certain time points. Acute ocular hypertension (OHT) was induced by the injection of magnetic microbeads into the rat anterior chamber angle (n = 22) with magnetic position control, leading to constant elevation of IOP. At certain time points post injection (4d, 7d, 10d, 14d and 21d), RGC loss, microglial activation, and microvascular pericyte (PC) coverage was analyzed using immunohistochemistry with corresponding specific markers (Brn3a, Iba1, NG2). Additionally, the tightness of the retinal vasculature was determined via injections of Texas Red labeled dextran (10 kDa) and subsequently analyzed for vascular leakage. For documentation, confocal laser-scanning microscopy was used, followed by cell counts, capillary length measurements and morphological and statistical analysis. The injection of magnetic microbeads led to a progressive loss of RGCs at the five time points investigated (20.07%, 29.52%, 41.80%, 61.40% and 76.57%). Microglial cells increased in number and displayed an activated morphology, as revealed by Iba1-positive cell number (150.23%, 175%, 429.25%,486.72% and 544.78%) and particle size analysis (205.49%, 203.37%, 412.84%, 333.37% and 299.77%) compared to contralateral control eyes. Pericyte coverage (NG2-positive PC/mm) displayed a significant reduction after 7d of OHT in central, and after 7d and 10d in peripheral retina. Despite these alterations, the tightness of the retinal vasculature remained unaltered at 14 and 21 days after OHT induction. While vascular tightness was unchanged in the course of OHT, a progressive loss of RGCs and activation of microglial cells was detected. Since a significant loss in RGCs was observed already at day 4 of experimental glaucoma, and since activated microglia peaked at day 10, we determined a time frame of 7–14 days after MB injection as potential optimum to study glaucoma mechanisms in this model.

Neuroprotective effects of intravitreally transplanted adipose tissue and bone marrow–derived mesenchymal stem cells in an experimental ocular hypertension model

Background aimsThe purpose of this study was to investigate the neuroprotective effects of bone marrow bone marrow–derived and adipose tissue–derived mesenchymal stromal cells (MSCs) that were intravitreally transplanted in an experimental ocular hypertension (OHT) model. MethodsAn OHT rat model was generated by means of intracameral injection of hyaluronic acid into the anterior chamber. MSCs labeled with green fluorescence protein were transplanted intravitreally 1 week after OHT induction. At the end of the second and fourth weeks, retinal ganglion cells were visualized with the use of a flat-mount retina method and were evaluated by means of immunofluorescence staining against green fluorescence protein, vimentin, CD105, and cytokines (interleukin [IL]-1Ra, prostaglandin E2 receptor, IL-6, transforming growth factor-β1, interferon-γ and tumor necrosis factor-α). ResultsThe retinal ganglion cell numbers per area were significantly improved in stem cell–treated OHT groups compared with that in the non-treated OHT group (P < 0.05). The results of immunohistochemical analyses indicated that a limited number of stem cells had integrated into the ganglion cell layer and the inner nuclear layer. The number of cells expressing proinflammatory cytokines (interferon-γ and tumor necrosis factor-α) decreased in the MSC-transferred group compared with that in the OHT group after 4 weeks (P < 0.01). On the other hand, IL-1Ra and prostaglandin E2 receptor expressions were increased in the rat bone marrow–derived MSC group but were more significant in the rat adipose tissue–derived MSC group (P < 0.01). ConclusionsAfter intravitreal transplantation, MSCs showed a neuroprotective effect in the rat OHT model. Therefore, MSCs promise an alternative therapy approach for functional recovery in the treatment of glaucoma.

Acetylation preserves retinal ganglion cell structure and function in a chronic model of ocular hypertension.

The current studies investigate if the histone deacetylase (HDAC) inhibitor, valproic acid (VPA), can limit retinal ganglion cell (RGC) degeneration in an ocular-hypertensive rat model. Intraocular pressure (IOP) was elevated unilaterally in Brown Norway rats by hypertonic saline injection. Rats received either vehicle or VPA (100 mg/kg) treatment for 28 days. Retinal ganglion cell function and number were assessed by pattern electroretinogram (pERG) and retrograde FluoroGold labeling. Western blotting and a fluorescence assay were used for determination of histone H3 acetylation and HDAC activity, respectively, at 3-day, 1-week, and 2-week time points. Hypertonic saline injections increased IOPs by 7 to 14 mm Hg. In vehicle-treated animals, ocular hypertension resulted in a 29.1% and 39.4% decrease in pERG amplitudes at 2 and 4 weeks, respectively, and a 42.9% decrease in mean RGC density at 4 weeks. In comparison, VPA treatment yielded significant amplitude preservation at 2 and 4 weeks and showed significant RGC density preservation at 4 weeks. No significant difference in RGC densities or IOPs was measured between control eyes of vehicle- and VPA-treated rats. In ocular-hypertensive eyes, class I HDAC activity was significantly elevated within 1 week (13.3 ± 2.2%) and histone H3 acetylation was significantly reduced within 2 weeks following the induction of ocular hypertension. Increase in HDAC activity is a relatively early retinal event induced by elevated IOP, and suppressing HDAC activity can protect RGCs from ocular-hypertensive stress. Together these data provide a basis for developing HDAC inhibitors for the treatment of optic neuropathies.

Complement activation after induction of ocular hypertension in an animal model

Although an elevated intraocular pressure (IOP) is known as the main risk factor for glaucoma, many studies also showed an involvement of the immune system in this disease. In this study we investigated if a moderate increase in IOP leads to activation of the complement system. The IOP was elevated experimentally in the left eye of rats, whereas the fellow eye served as the control. The IOP was measured at regular intervals. The number of retinal ganglion cells (RGC) was quantified via NeuN staining. To evaluate the activation of the complement system staining for C3, membrane attack complex (MAC), and mannose-binding lectin (MBL) was performed. Furthermore, we investigated possible glia activation (GFAP and vimentin) and apoptosis (Bax). A moderate elevation of the IOP was noted from day 11 after induction of ocular hypertension (OHT) until the end of the study (28 days, p = 0.0005). In the OHT-group significantly fewer RGCs (p = 0.02) were detected. Additionally, we noted significant C3 and MAC activation in the ganglion cell layer (C3, p = 0.001 and MAC, p = 0.02) as well as in the total retina (C3, p = 0.002 and MAC, p = 0.012). An activation via the lectin pathway by MBL staining could not be detected (p = 0.40). At this point in time no alterations with regard to glia cells were noted (GFAP, p = 0.97 and vimentin, p = 0.99). No apoptosis via Bax pathway could be observed (p = 0.90). The results suggest that the complement system is involved in the loss of RGCs even by a moderate IOP elevation which was indicated by significantly more C3 and MAC depositions in the OHT group.

Electroretinogram and Visual-Evoked Potential Assessment of Retinal and Central Visual Function in a Rat Ocular Hypertension Model of Glaucoma

Purpose/aim: The aim of the study was to investigate the long-term functional changes that may occur in the retina and visual cortex in a rat ocular hypertension (OHT) model of glaucoma, used in our lab for treatment studies, using electroretinogram (ERG) and visual-evoked potential (VEP) cortical recordings in order to test the hypothesis that experimental glaucoma has differential retinal and central effects.Materials and methods: Experimental glaucoma was induced unilaterally in Dark Agouti rats using hypertonic saline injection into the episcleral veins. After 3, 8, 16 and 26 weeks, ERGs and VEPs were recorded under scotopic conditions using brief full-field white flashes (10 μcd s m−2 to 10.4 cd s m−2) and under photopic conditions using a rod-adapting background and white light flashes (0.13–10.4 cd s m−2).Results: At 16 and 26 weeks after OHT induction, there was a significant reduction in the amplitudes of the a- (50% and 30% of unoperated eye values, respectively) and b-waves (55% and 40%, respectively) of the scotopic ERG and the b-waves of the photopic ERG (55% and 45%, respectively) in the glaucomatous eyes. However, no significant changes in the VEPs simultaneously recorded over the visual cortex were seen at any of the time points.Conclusions: The reductions in ERG amplitudes suggest that this model of glaucoma not only causes retinal ganglion cell (RGC) degeneration but also degeneration of the outer retinal cells, and this was confirmed by histology showing a reduction in the outer retinal layers in the glaucomatous eyes. Cortical VEPs did not show detrimental effects suggesting that the retinal damage in this model was not extensive enough to be detected with the VEP methods used or that there could be central compensation in this model of glaucoma.

Acetylcholinesterase Inhibition Promotes Retinal Vasoprotection and Increases Ocular Blood Flow in Experimental Glaucoma

A clear correlation between vascular deficits and retinal ganglion cell (RGC) loss in glaucoma has not yet been established. The question arose as to whether there is loss of inner retinal vessels following intraocular pressure (IOP) increase and, if so, whether it occurs prior to, concomitantly with, or after RGC death. We also sought to establish whether galantamine, an acetylcholinesterase inhibitor that promotes RGC survival, can protect the retinal microvasculature and enhance blood flow in experimental glaucoma. Ocular hypertension was induced in Brown Norway rats by injection of hypertonic saline into an episcleral vein. Retinas were processed for simultaneous visualization of the retinal microvasculature and RGCs in glaucomatous and control eyes. Retinal blood flow was examined by quantitative autoradiography using N-isopropyl-p-[(14)C]-iodoamphetamine. Vascular reactivity was further assessed using an in vitro retinal microvasculature preparation. Substantial loss of retinal capillaries was observed after induction of ocular hypertension. The onset of both microvasculature and RGC loss occurred early and proceeded at a similar rate for at least 5 weeks after the initial damage. Systemic administration of galantamine preserved microvasculature density and improved retinal blood flow in glaucomatous retinas. The vasoactive effects of galantamine on retinal microvessels occurred through activation of muscarinic acetylcholine receptors both in vitro and in vivo. The onset and progression of microvessel and RGC loss are concomitant in experimental glaucoma, suggesting a tight codependence between these cellular compartments. Early interventions aimed to protect the retinal microvasculature and improve blood supply are likely to be beneficial for the treatment of glaucoma.

Degenerative effects in rat eyes after experimental ocular hypertension

This study was used to evaluate the degenerative effects on the retina and eye-cup sections after experimental induction of acute ocular hypertension on animal models. In particular, vascular events were directly focused in this research in order to assess the vascular remodeling after transient ocular hypertension on rat models. After local anaesthesia by administration of eye drops of 0.4% oxibuprocaine, 16 male adult Wistar rats were injected in the anterior chamber of the right eye with 15 µL of methylcellulose (MTC) 2% in physiological solution. The morphology and the vessels of the retina and eye-cup sections were examined in animals sacrificed 72 h after induction of ocular hypertension. In retinal fluorescein angiographies (FAGs), by means of fluorescein isothiocyanate-coniugated dextran (FITC), the radial venules showed enlargements and increased branching, while the arterioles appeared focally thickened. The length and size of actually perfused vessels appeared increased in the whole superficial plexus. In eye-cup sections of MTC-injected animals, in deep plexus and connecting layer there was a bigger increase of vessels than in controls. Moreover, the immunolocalization of astrocytic marker glial fibrillary acidic protein (GFAP) revealed its increased expression in internal limiting membrane and ganglion cell layer, as well as its presence in Müller cells. Finally, the pro-angiogenic factor vascular endothelial growth factor (VEGF) was found to be especially expressed by neurones of ganglion cell layer, both in control and in MTC-injected eyes. The data obtained in this experimental model on the interactions among glia, vessels and neurons should be useful to evaluate if also in glaucomatous patients the activation of vessel-adjacent glial cells might play key roles in following neuronal dysfunction.

Interleukin-6 is an efficacious marker of axonal transport disruption during experimental glaucoma and stimulates neuritogenesis in cultured retinal ganglion cells

It is increasingly recognised that chronically activated glia contribute to the pathology of various neurodegenerative diseases, including glaucoma. One means by which this can occur is through the release of neurotoxic, proinflammatory factors. In the current study, we therefore investigated the spatio-temporal patterns of expression of three such cytokines, IL-1β, TNFα and IL-6, in a validated rat model of experimental glaucoma.First, only weak evidence was found for increased expression of IL-1β and TNFα following induction of ocular hypertension. Second, and much more striking, was that robust evidence was uncovered showing IL-6 to be synthesised by injured retinal ganglion cells following elevation of intraocular pressure and transported in an orthograde fashion along the nerve, accumulating at sites of axonal disruption in the optic nerve head. Verification that IL-6 represents a novel marker of disrupted axonal transport in this model was obtained by performing double labelling immunofluorescence with recognised markers of fast axonal transport. The stimulus for IL-6 synthesis and axonal transport during experimental glaucoma arose from axonal injury rather than ocular hypertension, as the response was identical after optic nerve crush and bilateral occlusion of the carotid arteries, each of which is independent of elevated intraocular pressure. Moreover, the response of IL-6 was not a generalised feature of the gp130 family of cytokines, as it was not mimicked by another family member, ciliary neurotrophic factor. Finally, further study suggested that IL-6 may be an early part of the endogenous regenerative response as the cytokine colocalised with growth-associated membrane phosphoprotein-43 in some putative regenerating axons, and potently stimulated neuritogenesis in retinal ganglion cells in culture, an effect that was additive to that of ciliary neurotrophic factor. These data comprise clear evidence that IL-6 is actively involved in the attempt of injured retinal ganglion cells to regenerate their axons.

Soluble Nogo-66 Receptor Prevents Synaptic Dysfunction and Rescues Retinal Ganglion Cell Loss in Chronic Glaucoma

Myelin inhibitory proteins inhibit axon growth and synaptic function by binding to the Nogo-66 receptor (NgR)1 in the central nervous system. Glaucoma is a progressive neuropathy characterized by loss of vision as a result of retinal ganglion cell (RGC) death. Synaptic degeneration is thought to be an early pathology of neurodegeneration in glaucoma and precedes RGC loss. The authors aimed to examine whether the NgR1 antagonist promotes synaptic recovery and RGC survival in glaucoma. Experimental ocular hypertension model was induced in adult rats with laser coagulation of the episcleral and limbal veins. NgR1 antagonist, soluble NgR1 (sNgR-Fc) was administrated to examine their effect on synaptic recovery and RGC survival. Expression of c-Fos, a neuronal connectivity marker, in the retinas was investigated using immunohistochemistry. NgR1 was expressed in RGCs and upregulated after intraocular pressure elevation. Treatment with sNgR-Fc significantly reduced RGC loss at 2 and 4 weeks after the induction of ocular hypertension and also promoted RGC survival after optic nerve transection. There was no RGC loss at 5 days but there was significant synaptic degeneration as measured by c-Fos. Administration of sNgR-Fc attenuated synaptic degeneration at 5 days, and at 2 and 4 weeks. These data suggest that synaptic degeneration may be an initial molecular mechanism for neurodegeneration in glaucoma and appropriate NgR1 antagonism may delay the progression of the disease.

Protection of Retinal Ganglion Cells and the Optic Nerve During Short-term Hyperglycemia in Experimental Glaucoma

To evaluate the neuroprotective effect of short-term hyperglycemia on the retinal ganglion cell body and axon in a rat model of experimental glaucoma. Using a well-described limbal laser technique, unilateral ocular hypertension was induced in 2 groups (26 per group) of Sprague-Dawley rats. One group remained normoglycemic; the other was rendered hyperglycemic by means of an intraperitoneal injection of streptozocin. After 2 weeks of elevated intraocular pressure, axonal and retinal damage profiles were compared using several histological techniques. Immunohistochemical changes in the retina and optic nerve were also assessed. We found convincing evidence of delayed axonal degeneration and retinal ganglion cell death in hyperglycemic rats. Axon loss was reduced by about 50% 2 weeks after induction of ocular hypertension. Survival of retinal ganglion cell perikarya increased to a similar extent in hyperglycemic rats. The optic nerve and retinal ganglion cells are partially protected by short-term hyperglycemia in this rat model of experimental glaucoma. Energy substrate availability may therefore play a role in glaucomatous optic neuropathy. Our findings, to some extent, support the claims of the Ocular Hypertension Treatment Study, in which diabetes appeared to protect against the conversion to glaucoma. Targeted manipulation of neuronal energy metabolism may delay optic nerve degeneration and may represent a novel neuroprotective strategy for neurodegenerative diseases of the visual system such as glaucoma.

The Expression Patterns of Nogo-A, Myelin Associated Glycoprotein and Oligodendrocyte Myelin Glycoprotein in the Retina After Ocular Hypertension

Nogo-A, a major myelin inhibitory protein, inhibits axon growth and synaptic function in the central nervous system. Glaucoma is a progressive neuropathy as a result of retinal ganglion cell (RGC) death. Synaptic degeneration is thought to be an early pathology of neurodegeneration in glaucoma and precedes RGC loss. Here experimental ocular hypertension model was induced in adult rats with laser coagulation of the episcleral and limbal veins. The expression of Nogo-A, myelin-associated glycoprotein (MAG) and oligodendrocyte myelin glycoprotein (OMgp) in the retina was investigated using immunohistochemistry and Western blotting. We found that Nogo-A was expressed in the RGCs and upregulated after the induction of ocular hypertension. OMgp was only expressed in the inner plexiform layer. There was no MAG expression in the retina. Our data provided, for the first time, the expression patterns of three myelin proteins in the adult retina and suggested an important role of Nogo-A in the RGC death and synaptic degeneration in glaucoma.

Neuroprotective Effects of Intravitreal Mesenchymal Stem Cell Transplantation in Experimental Glaucoma

Purpose. Retrograde neurotrophic factor transport blockade has been implicated in the pathophysiology of glaucoma. Stem cell transplantation appears to ameliorate some neurodegenerative conditions in the brain and spinal cord, in part by neurotrophic factor secretion. The present study was conducted to determine whether local or systemic bone marrow-derived mesenchymal stem cell (MSC) transplantation can confer neuroprotection in a rat model of laser-induced ocular hypertensive glaucoma. Methods. MSCs were isolated from the bone marrow of adult wild-type and transgenic rats that ubiquitously express green fluorescent protein. MSCs were transplanted intravitreally 1 week before, or intravenously on the day of, ocular hypertension induction by laser photocoagulation of the trabecular meshwork. Ocular MSC localization and integration were determined by immunohistochemistry. Optic nerve damage was quantified by counting axons within optic nerve cross-sections 4 weeks after laser treatment. Results. After intravitreal transplantation, MSCs survived for at least 5 weeks. Cells were found mainly in the vitreous cavity, though a small proportion of discrete cells migrated into the host retina. Intravitreal MSC transplantation resulted in a statistically significant increase in overall RGC axon survival and a significant decrease in the rate of RGC axon loss normalized to cumulative intraocular pressure exposure. After intravenous transplantation, MSCs did not migrate to the injured eye. Intravenous transplantation had no effect on optic nerve damage. Conclusions. Local, but not systemic, transplantation of MSCs was neuroprotective in a rat glaucoma model. Autologous intravitreal transplantation of MSCs should be investigated further as a potential neuroprotective therapy for glaucoma.

Combined effect of brain-derived neurotrophic factor and LINGO-1 fusion protein on long-term survival of retinal ganglion cells in chronic glaucoma

Glaucoma is a progressive neuropathy characterized by loss of vision as a result of retinal ganglion cell (RGC) death. There are no effective neuroprotectants to treat this disorder. Brain-derived neurotrophic factor (BDNF) is well known to transiently delay RGC death in ocular hypertensive eyes. The CNS-specific leucine-rich repeat protein LINGO-1 contributes to the negative regulation to some trophic pathways. We thereby examined whether BDNF combined with LINGO-1 antagonists can promote long-term RGC survival after ocular hypertension. In this study, intraocular pressure was elevated in adult rats using an argon laser to photocoagulate the episcleral and limbal veins. BDNF alone shows slight neuroprotection to RGCs after a long-term progress of 4 weeks following the induction of ocular hypertension. However, combination of BDNF and LINGO-1-Fc prevents RGC death in the same condition. We further identified that (1) LINGO-1 was co-expressed with BDNF receptor, TrkB in the RGCs, and (2) BDNF combined with LINGO-1-Fc activated more TrkB in the injured retina compared to BDNF alone. These results indicate that the combination of BDNF with LINGO-1 antagonist can provide long-term protection for RGCs in a chronic ocular hypertension model. TrkB may be the predominant mediator of this neuroprotection.

Expression of matrix metalloproteinases and inhibitors on the scleral tissue of lamina cribrosa in rat with experimental chronic ocular hypertension

To investigate the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in the sclera of lamina cribrosa in rat chronic elevated intraocular pressure (IOP) and its the relationship with the pathogenesis of glaucoma. It was an experimental study. Chronic ocular hypertension (OHT) model was induced in the left eyes of 43 Wistar rats by cauterizing episcleral venous while the right eyes were used as control. The rats were divided into three groups: 13 for immunohistology, 15 for Western blotting, and 15 for RT-PCR by randomly computerized assignment and sacrificed at week 12 after OHT induction. The expression of MMP-2, MMP-9, TIMP-1, and TIMP-2 in the sclera lamina cribrosa was detected using immunohistology, Western blotting, and RT-PCR technique, respectively. Repeated measures ANOVA of two-factor Studies in SAS statistic software were used to analyze the data. After 12 weeks, compared with control eyes, the IOP of rat OHT eyes was significantly (F = 1519.67, P < 0.01) elevated. The expression of MMP-2 and MMP-9 was increased in cell nucleus and cytoplasm in sclera lamina cribrosa in rat OHT when compared to control eyes with negative expression. No expressions of TIMP-1, TIMP-2 in sclera lamina cribrosa were found in both OHT and control eyes. The mean gray-scale values of MMP-2, MMP-9, TIMP-1, and TIMP-2 in OHT were significantly (F = 405.55, F = 436.11, F = 1167.77, and F = 4629.64, P < 0.01) higher than control eye by Western blotting (193.88 +/- 8.84 vs 117.38 +/- 10.76, 202.65 +/- 8.37 vs 134.13 +/- 5.06, 283.63 +/- 5.65 vs 186.88 +/- 7.14, and 284.75 +/- 5.50 vs 183.0 +/- 5.58), while by RT-PCR (200.50 +/- 3.25 vs 181.88 +/- 9.36, 200.13 +/- 2.95 vs 181.75 +/- 5.85, 201.88 +/- 3.14 vs 179.25 +/- 9.21, and 195.50 +/- 3.55 vs 179.75 +/- 7.12), respectively. The increased expression of MMP-2, MMP-9, TIMP-1, and TIMP-2 in lamina cribrosa in rat OHT eye indicates that MMPs and TIMPs may be involved in the pathogenesis of glaucoma.

Synaptic degeneration of retinal ganglion cells in a rat ocular hypertension glaucoma model.

Glaucoma is a common neurodegenerative disease that affects retinal ganglion cells (RGCs) and their axons. Little is known of the synaptic degeneration involved in the pathophysiology of glaucoma. Here we used an experimental ocular hypertension model in rats to investigate this issue. Elevated intraocular pressure (IOP) was induced by laser coagulation of the episcleral and limbal veins. RGCs were retrogradely labeled with Fluoro-Gold (FG). The c-fos protein was used as a neuronal connectivity marker. Expression of c-fos in the retinas was investigated by immunohistochemistry at 5 days and 2 weeks after the induction of ocular hypertension. Both surviving RGCs as revealed by retrograde FG-labeled and c-fos-labeled RGCs were counted. The c-fos protein was mainly expressed in the nuclei and nucleoli of cells in the ganglion cell layer and inner nuclear layer in the normal retina. We also confirmed that c-fos was also expressed in the nuclei and nucleoli of RGCs retrogradely labeled with FG. There was no significant RGC loss at 5 days but about 13% RGC loss at 2 weeks after the induction of ocular hypertension. The number of RGCs expressing c-fos was significantly lower in the experimental animals at both 5 days and 2 weeks than normal. Our study suggests that there is synaptic disconnection for RGCs after ocular hypertension and it may precede the cell death in the early stage. It may provide insight into novel therapeutic strategies to slow the progress of glaucoma.