Growth-associated protein-43 expression in the lens of rats and primates.

Growth-associated protein-43 is a specific neuronal protein that regulates differentiation, growth and plasticity. In the present study, growth-associated protein-43 expression was studied in the lens of rats and primates (including man) at different postnatal ages by immunoblotting, immunohistochemistry and quantitative real-time polymerase chain reaction. Growth-associated protein-43 was expressed at all ages in primates and in developing, but not in adult rats. We demonstrate that the lens - a tissue that is devoid of nerves - expresses growth-associated protein-43 throughout life in primates, and in rats during development but not in adulthood. These results suggest that growth-associated protein-43 is involved in differentiation processes also outside the nervous system.

Transformation of adult retina from the regenerative to the axonogenesis state activates specific genes in various subsets of neurons and glial cells.

The purpose of this study was to identify the gene expression profile of the regenerating retina in vitro. To achieve this goal, three experimental groups were studied: (1) an injury control group (OC-LI group) that underwent open crush (OC) of the optic nerve and lens injury (LI) in vivo; (2) an experimental group (OC-LI-R group) that comprised animals treated like those in the OC-LI group except that retinal axons were allowed to regenerate (R) in vitro; and (3) an experimental group (OC-LI-NR group) that comprised animals treated as those in the OC-LI group, except that the retinas were cultured in vitro with the retinal ganglion cell (RGC) layer facing upwards to prevent axonal regeneration (NR). Gene expression in each treatment group was compared to that of untreated controls. Immunohistochemistry was used to examine whether expression of differentially regulated genes also occurred at the protein level and to localize these proteins to the respective retinal cells. Genes that were regulated belonged to different functional categories such as antioxidants, antiapoptotic molecules, transcription factors, secreted signaling molecules, inflammation-related genes, and others. Comparison of changes in gene expression among the various treatment groups revealed a relatively small cohort of genes that was expressed in different subsets of cells only in the OC-LI-R group; these genes can be considered to be regeneration-specific. Our findings demonstrate that axonal regeneration of RGC involves an orchestrated response of all retinal neurons and glia, and could provide a platform for the development of therapeutic strategies for the regeneration of injured ganglion cells.

Early electroretinographic features of streptozotocin-induced diabetic retinopathy.

BACKGROUND: This study set out to document the early electrophysiological and immunohistochemical changes that occur in the retina of experimentally induced diabetic rats. METHODS: Diabetes was induced in rats by intraperitoneal injection of 60 mg/kg of streptozotocin (STZ). Electroretinogram readings were taken monthly under either short-duration or long-duration stimuli for up to 3 months after STZ. Oscillatory potentials (OP) and the amplitudes and implicit times of a- and b-waves were analysed, and b-wave amplitudes were analysed using a Naka-Rushton fit. Scotopic a-waves were analysed with photoreceptor models, and Rmp3 (the maximum a-wave amplitude) and S (sensitivity) were calculated. Three months after STZ injection, immunohistochemistry for glial fibrillary acidic protein was performed on the retinas of the STZ-treated rats and age-matched controls. RESULTS: The implicit OP times were significantly longer in the diabetic rats as compared with the controls, and this difference was noted as early as 1 month following STZ treatment. Other electrophysiological parameters, such as OP amplitudes, a- and b-wave amplitude as well as the implicit times, did not differ from controls at this stage. The sacrificed STZ-treated rats also demonstrated marked enhancement of glial fibrillary acidic protein immunoreactivity, suggesting that at least in experimentally induced diabetic retinopathy there is increased Müller cell reactivity. CONCLUSION: The results of this study indicated that functional alterations in the retina develop rapidly after the onset of diabetes. Analysis of each electroretinogram component may be useful in further investigating the development mechanisms of diabetic retinopathy.

Retinal gene profiling in a hereditary rodent model of elevated intraocular pressure.

PURPOSE: To characterize the changes in retinal gene expression induced by elevated intraocular pressure (IOP) in a hereditary rodent model. METHODS: A rat model derived from the RCS-rdy- strain develops IOP elevation spontaneously without experimental manipulation. Retinal gene expression after IOP elevation was compared with age-matched RCS-rdy- retinas having normal IOP levels The MWG Rat 10k array, which comprises 9715 rat genes spotted onto one array was used. Quantitative real-time PCR (qRT-PCR) was used to verify the expression of heat shock protein-27 (Hsp-27), SA hypertension-associated gene, c-myc, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), vascular endothelial growth factor (VEGF), myocilin, interleukin-7 (IL-7), mitogen activated protein kinase 13 (MAPK-13) and crystallin beta-A1 (Cryba1). The cellular distribution of c-myc, glial fibrillary acidic protein (GFAP), VEGF, and SA was assessed using immunohistochemistry. RESULTS: Elevated IOP of 37.7+/-5.0 mmHg shifted the retina's program of gene expression, with 75 genes being upregulated (equal to or higher than 3.0 fold) and 45 genes being downregulated (equal to or lower than 0.3 fold). These genes mediate various cellular processes such as cell adhesion, cell structure, hypertension, immunity, protein sythesis, proteolysis, transcription, and signaling. The regulation pattern of SA, VEGF, c-myc, IL-7, and MAPK-13, which are uniquely regulated in our model were confirmed by qRT-PCR experiments. The regulation of Hsp-27, TIMP-1, myocilin, and Cryba1, which have previously been associated with elevated IOP were also confirmed with qRT-PCR. The protein products of c-myc, SA, and GFAP were localized to astrocytes and Müller cells. Neurons in the ganglion cell layer and inner nuclear layer were VEGF-immunopositive. CONCLUSIONS: This study identified some of the genes that are differentially regulated, probably in response to long-term IOP exposure, in this animal model. The expression pattern of many genes is common to experimental models of elevated IOP and other retinal disorders such as diabetic retinopathy. However many genes are uniquely expressed in the retina of our model. This suggests that the mode of IOP elevation be it experimental or spontaneous could be relevant in determining which genes are regulated. Müller glia acquire a reactive phenotype as indicated by the upregulation of GFAP, c-myc, SA, and other Müller cell markers, emphasizing their relevance in pressure related- and other types of retinal injury. These data provide further evidence that IOP-mediated retinal injury is multifactorial and depends upon the interaction of different neuronal, glial, extracellular matrix, and vasogenic components.

Correlation between retinal ganglion cell death and chronically developing inherited glaucoma in a new rat mutant.

Glaucoma is a progressive optic neuropathy with characteristic optic disc changes, retinal ganglion cell loss and progressive visual field defects. Elevated intraocular pressure is considered to be a major risk factor in glaucomatous neuropathy. This study aimed to characterize and document a new chronic glaucoma model in the rat with respect to the effect of elevated intraocular pressure on overall retinal dysfunction and retinal ganglion cell loss, and to elucidate the possible mechanisms underlying this cell loss. Intraocular pressure (IOP) was measured in rats using a Tonopen. RGCs were retrogradely labeled with the fluorescent dye, 4-[didecylaminostyryl]-N-methyl-pyridinium-iodide (4-Di-10 ASP) and quantified on retinal flat mounts using fluorescence microscopy. The optic nerve head was examined fundoscopically. Changes in the histological appearance of the whole eyes was studied in paraffin sections, and immunohistochemistry was carried out on cryostat sections. The levels of mRNA for several genes were compared between control and glaucomatous retinae using semi-quantitative RT-PCR. Mutant animals are affected with either a unilateral or bilateral enlargement of the globes having an IOP that ranged from 25 to 45 mmHg, as compared to control values of 12-16 mmHg. The IOP of glaucomatous eyes increased significantly with age to attain a value of 35+/-7.3 at 1.5 years. Concomitant with the rise in IOP, the number of labeled RGCs continued to decrease in number with age. A total of 1887+/-117RGC mm(-2) could be labeled in wild-type control and juvenile mutant pre-glaucomatous retinas, whereas this number dropped to 92+/-26RGC mm(-2) at 1.5 years. Ophthalmoscopy revealed atrophied optic nerve heads in the affected eyes. The pars plicata and the pars plana of the ciliary body of glaucomatous eyes were hypertrophied and elongated, respectively. The anterior chamber was narrow and the irido-corneal angle open in glaucoma eyes. The mRNA of glial-fibrillary-acidic protein, endothelin-1, STAT-3 and STAT-6 increased in the retinas correlating with the severity and duration of the disease. Changes in the expression of GFAP and endothelin-1 could be confirmed using immunohistochemistry. This model may help to address several fundamental issues in the pathogenesis of glaucoma and aid in the development of neuroprotective strategies.

Potential role of Pax-2 in retinal axon navigation through the chick optic nerve stalk and optic chiasm.

The degree of fiber decussation at the optic chiasm differs between species, ranging from complete crossing in lower vertebrates to highly complex patterns of intermingling of the fibers from the two eyes seen in mammals and birds. Understanding the genetic control of fiber guidance through the chiasm is therefore important to unravel the developmental mechanisms within the visual system. Here we first report on early stages of chiasm formation, with pioneering axons from the left eye consistently arriving earlier than their counterparts from the right eye. This initial left-right asymmetry is transient and no functional significance is assigned to it yet. Secondly, we examined formation of the chiasm in relation with the expression of the transcription factor Pax-2 along the ventral eye cup and optic nerve stalk. Finally, in order to examine causal involvement of Pax-2 in chiasm formation, the gene was overexpressed along the neuraxis and in the eye cup at embryonic stages preceding the exit of axons from the eye, and hence arrival of axons at the chiasm. When studied with neuroanatomical tracing, Pax-2 overexpression resulted in visibly anomalous decussation of axons at the chiasm. A likely consequence of this perturbation was erroneous arrival of axons at the tectum, as observed by anterograde staining from the retina. These data suggest that balanced expression of Pax-2 results in the correct formation of the chick chiasm at early stages by imposing accurate pathfinding within the optic stalk and the midline.

Adeno-associated viruses containing bFGF or BDNF are neuroprotective against excitotoxicity.

PURPOSE: Brain-derived neurotrophic factor (BDNF) and basic fibroblast growth factor (bFGF) hold much promise for the protection of retinal ganglion cells against excitotoxic cell death. We tested the possibility of delivering these growth factors to retinal ganglion cells via an adeno-associated viral (AAV) vector and tested their efficacy in two models of excitotoxicity. METHODS: Rat retinas were infected with AAV vectors encoding bFGF or BDNF. A control vector containing green fluorescent protein (GFP) was injected in the contralateral eye. Eyes were subjected to either an intravitreal injection of N-methyl-D-aspartate (NMDA) or optic nerve crush, and ganglion cell survival was evaluated. RESULTS: AAV.CMV.bFGF and AAV.CBA.BDNF were neuroprotective against NMDA injection 1 month post-treatment. Additionally, AAV.CMV.bFGF was protective against optic nerve crush. CONCLUSION: AAV-mediated delivery of bFGF and BDNF can promote retinal cell survival following excitotoxic insult.

Retinal ganglion cells resistant to advanced glaucoma: a postmortem study of human retinas with the carbocyanine dye DiI.

PURPOSE: The present study was conducted to examine whether the morphology of the retinal ganglion cells is altered in advanced glaucoma. Perikaryal, axonal, and dendritic alterations were monitored in glaucoma-resistant retinal ganglion cells by postvitam application of the fluorescent dye DiI. METHODS: The retinas of four amaurotic glaucomatous eyes and four normal eyes enucleated after death were used in this study. The retinas were freed from surrounding tissue, prepared as flatmounts on a nitrocellulose filter, and fixed overnight in 4% paraformaldehyde. The retinal ganglion cells were labeled by introducing crystals of the fluorescent carbocyanine dye DiI, into the optic fiber layer. This dye diffuses along membranes of ganglion cell axons, completely labeling them and their cell bodies and dendrites. Further characterization of the retinas and optic nerves included hematoxylin-eosin and van Gieson histochemical staining as well as immunohistochemistry against glial fibrillary acidic protein. RESULTS: Because of the advanced stage of the disease, the retinas were almost completely depleted of ganglion cells, which had degenerated and therefore could not be stained. The few remaining ganglion cells were considered to be resistant to glaucoma. They showed drastic morphologic alterations, such as abnormal axonal beading, the cell bodies were normal in size but had irregular silhouettes or swellings, and there were fewer dendritic bifurcations. The size of the dendritic trees was smaller, implicating pruning of smaller dendritic branches. Glial cells were also detected immunocytochemically indicating their involvement in the pathologic course of glaucoma. CONCLUSIONS: The data suggest that the few ganglion cells that survive the elevated intraocular pressure associated with loss of visual function display morphologic changes that are manifested both on the cell body and on their intraretinal processes, including axons and dendrites.

Benzodiazepine and kainate receptor binding sites in the RCS rat retina.

BACKGROUND: The effect of age and photoreceptor degeneration on the kainate subtype of glutamate receptors and on the benzodiazepine-sensitive gamma-aminobutyric acid-A receptors (GABA(A)) in normal and RCS (Royal College of Surgeons) rats were investigated. METHODS: [(3)H]Kainate and [(3)H]flunitrazepam were used as radioligands for kainate and GABA(A)/benzodiazepine()receptors, respectively, using the quantitative receptor autoradiography technique. RESULTS: In both normal and RCS rat retina we observed that [(3)Eta]flunitrazepam and [(3)Eta]kainate binding levels were several times higher in inner plexiform layer (IPL) than in outer plexiform layer (OPL) at all four ages studied (P17, P35, P60 and P180). Age-related changes in receptor binding were observed in normal rat retina: [(3)Eta]flunitrazepam binding showed a significant decrease of 25% between P17 and P60 in IPL,and [(3)Eta]kainate binding showed significant decreases between P17 and P35 in both synaptic layers (71% in IPL and 63% in OPL). Degeneration-related changes in benzodiazepine and kainate receptor binding were observed in RCS rat retina. In IPL, [(3)Eta]flunitrazepam and [(3)Eta]kainate binding levels were higher than in normal retina at P35 (by 24% and 86%, respectively). In OPL, [(3)Eta]flunitrazepam binding was higher in RCS than in normal retina on P35 (74%) and also on P60 (62%). CONCLUSIONS: The results indicate that postnatal changes occur in kainate and benzodiazepine receptor binding sites in OPL and IPL of the rat retina up to 6 months of age. The data also suggest that the receptor binding changes observed in the RCS retina could be a consequence of the primary photoreceptor degeneration.

Effects of anti-glaucoma medications on ganglion cell survival: the DBA/2J mouse model.

We studied whether several agents, approved or undergoing trials in human glaucoma, were effective in preventing ganglion cell loss in the DBA/2J mouse. Adult DBA/2J mice were treated with timolol, pilocarpine, brimonidine, dorzolamide, or NMDA-receptor antagonist memantine. Surviving retinal ganglion cells of treated and control mice were retrogradely labeled with fluorogold and counted after whole mount preparation. In treated mice, only memantine and timolol had significant effects on retinal ganglion cell survival (P<0.0001, analysis of variance). Brimonidine was lethal to these mice, and these retinae were not analyzed further. The DBA/2J mouse represents a promising candidate for further experimentation in ocular hypertension.

Detection of early neuron degeneration and accompanying microglial responses in the retina of a rat model of glaucoma.

PURPOSE: To characterize the early reaction of retinal ganglion cells (RGCs) in a rat model of glaucoma using in vivo imaging and to examine the involvement of retinal microglia in glaucomatous neuropathy. METHODS: Glaucoma was induced in adult female Sprague-Dawley rats by cauterizing two episcleral veins, which resulted in a 1.6-fold increase in intraocular pressure (IOP). Retinal ganglion cells were retrogradely labeled with the fluorescent dye, 4-[didecylaminostyryl]-N-methyl-pyridinium-iodide (4-Di-10ASP) and monitored in vivo after elevation of IOP using fluorescence microscopy imaging. The number of RGCs was quantified on retinal flatmounts. Dying RGCs were surrounded by activated microglia that became visible after taking up the fluorescent debris. Immunocytochemistry was conducted to characterize further the ganglion cells and microglia. RESULTS: Cauterizing two of the four episcleral veins resulted in a consistent increase of IOP to 25.3 +/- 2.0 mm Hg, as measured with a handheld tonometer. IOP remained high for at least 3 months in glaucomatous eyes. The earliest sign of RGC death was detected in anesthetized animals 20 hours after induction of glaucoma. RGCs continued to decrease in number over time, with 40% of RGCs having degenerated after 2.5 months. Fundoscopic examination of the optic nerve head revealed cupping 2 months after induction of glaucoma. In addition, microglia were detected on retinal flatmounts as early as 72 hours after induction. Activated microglia and RGCs were also identified immunocytochemically, with an antibody against ionized calcium-binding adaptor molecule (Iba)-1 and an antibody specific to the 200-kDa subunit of the neurofilament protein, respectively. CONCLUSIONS: Occlusion of episcleral veins is a reproducible method that mimics human glaucoma, with chronically elevated IOP-induced RGC loss. This study shows that in vivo imaging permits the detection of ganglion cells in the living animal in the early stages of the disease and highlights the importance of in vivo imaging in understanding ophthalmic disorders such as glaucoma. Secondly, activation of intraretinal microglia coincides with degeneration of RGCs in glaucoma.

In vivo FM: using conventional fluorescence microscopy to monitor retinal neuronal death in vivo.

Post-traumatic death of mature retinal neurons occurs in glaucoma and after optic nerve injury. The death is a dynamic process that can be fully analyzed with methods that monitor changes over time. We have coupled the development of retrogradely transportable fluorescent dyes with modification of conventional epifluorescence microscopy to manipulate and visualize rat retinal neurons in vivo. The method is a relatively new concept and has potential for the monitoring of retinal conditions, such as glaucoma or optic nerve transection, and for evaluation of neuroprotective strategies in the near future.

Phenytoin blocks retinal ganglion cell death after partial optic nerve crush.

Phenytoin is a well-characterized sodium channel blocker in widespread use as an anticonvulsant. In 1972, Becker and co-workers reported that phenytoin could reverse visual field loss from glaucoma. The authors therefore explored whether phenytoin could protect retinal ganglion cells from optic nerve crush. The optic nerve of Long-Evans rats was partially crushed; animals were given a single dose of either intraperitoneal phenytoin or vehicle. A third group underwent sham optic nerve crush. In a second set of experiments, the effect of phenytoin was compared to the N -methyl- D -receptor antagonist, memantine. Retinal ganglion survival was evaluated 1 week later. In addition, the effect of memantine and phenytoin on glutamate-induced intracellular calcium fluxes was evaluated.Phenytoin and memantine significantly reduced ganglion cell loss after optic nerve crush, and blunted the rise in intracellular calcium seen after administration of glutamate. Co-administration of the two agents, however, did not increase ganglion cell survival, and had no effect on ganglion cell calcium fluxes. Phenytoin can preserve retinal ganglion cells after partial optic nerve crush. This effect was not additive with a glutamate antagonist, suggesting that both agents alone are equally protective at saving the same population of ganglion cells at risk. In fact, the neuroprotective effect of the combined administration of phenytoin and memantine was significantly less than either of the two drugs alone. Phenytoin is known to decrease neuronal firing and neurotransmitter release; this may underlie its ability to serve as a neuro-protectant in this experimental paradigm.