RGC death in mice after optic nerve crush injury: oxidative stress and neuroprotection

PURPOSE: To establish a method for morphometric analysis of retrogradely labeled retinal ganglion cells (RGCs) of the mouse retina, to be used for the study of molecular aspects of RGC survival and neuroprotection in this model; to evaluate the effect of overexpression of Cu-Zn-superoxide dismutase (CuZnSOD) on RGC survival after severe crush injury to the optic nerve, and to assess the effect of the alpha2-adrenoreceptor agonist brimonidine, recently shown to be neuroprotective, on RGC survival. METHODS: A severe crush injury was inflicted unilaterally in the orbital portion of the optic nerves of wild-type and transgenic (Tg-SOD) mice expressing three to four times more human CuZnSOD than the wild type. In each mouse all RGCs were labeled 72 hours before crush injury by stereotactic injection of the neurotracer dye FluoroGold (Fluorochrome, Denver, CO) into the superior colliculus. Survival of RGCs was then assessed morphometrically, with and without systemic injection of brimonidine. RESULTS: Two weeks after crush injury, the number of surviving RGCs was significantly lower in the Tg-SOD mice (596.6 +/- 71.9 cells/mm(2)) than in the wild-type control mice (863. 5 +/- 68 cells/mm(2)). There was no difference between the numbers of surviving RGCs in the uninjured retinas of the two strains (3708 +/- 231.3 cells/mm(2) and 3904 +/- 120 cells/mm(2), respectively). Systemic injections of brimonidine significantly reduced cell death in the Tg-SOD mice, but not in the wild type. CONCLUSIONS: Overexpression of CuZnSOD accelerates RGC death after optic nerve injury in mice. Activation of the alpha2-adrenoreceptor pathway by brimonidine enhances survival of RGCs in an in vivo transgenic model of excessive oxidative stress.     

Topographic and morphologic analyses of retinal ganglion cell loss in old DBA/2NNia mice

PURPOSE: To evaluate the relationship between retinal ganglion cell (RGC) size, density distribution, and survival in senescent DBA2/NNia mice that develop pigmentary glaucoma. To evaluate the validity of nearest neighbor distance (NND), a measure of focal density for surviving RGCs in the retina, as a method to quantify RGC loss in mice. METHODS: Fifteen-month-old DBA2/NNia mice were labeled retrogradely with fluorogold. Retinas were flat mounted and imaged in their entirety using an epifluorescence microscope with a motorized stage. Digital maps of the retinal wholemounts were constructed to automatically count and establish spatial coordinates for RGCs over the entire retina. RGC size and NND were determined from these maps. RESULTS: RGC counts in the group of 15-month-old DBA/2NNia animals ranged from 22,330 to 92,157 cells per retina. Mean RGC cell size per retina ranged from 22.35 to 35.64 microm2 and correlated linearly with total RGC counts. NND distribution histograms were compared for retinas with variable degrees of RGC loss. The distribution of NNDs in each retina was skewed toward larger distance values in more affected retinas. In partially damaged retinas, areas with severe pathology coincided with areas of maximal loss of large RGCs, and areas of preserved RGCs correlated with larger cell sizes. CONCLUSIONS: Damaged retinas have a smaller mean cell size, indicating preferential loss of larger RGCs or size reduction of surviving cells. NND analysis of the RGC population in a retina is a useful measure of glaucomatous RGC loss. The skewed NND distribution of surviving RGCs and the finding that RGC loss correlates with a shift/amplitude change in the mode of the histogram and its tail suggests two different patterns of RGC loss possibly attributable to different pathologic processes in glaucomatous DBA/2 mice.     

Distribution of amyloid precursor protein and amyloid-beta immunoreactivity in DBA/2J glaucomatous mouse retinas

PURPOSE: Evidence suggests that altered metabolism of amyloid precursor protein (APP) may play a role in the pathophysiology of retinal ganglion cell (RGC) death in the etiology of glaucoma. The authors sought to determine the distribution of APP and amyloid-beta (Abeta) in DBA/2J glaucomatous mouse retinas. METHODS: The retinas of 3- and 15-month-old DBA/2J mice and C57/BL-6 mice (control group) were fixed with 4% paraformaldehyde and processed for immunohistochemistry. Antibodies used included a polyclonal antibody to the C terminus of Abeta 40 and a polyclonal antibody to the APP ectodomain. Immunohistochemically stained tissue was graded using light microscopy. Distribution and semiquantitative expression of APP and Abeta in young and old glaucomatous and normal retinas were determined and compared. RESULTS: Strong APP and Abeta immunoreactivity was found in the RGC layer, optic nerve, and pia/dura of old DBA/2J retinas, with considerably higher intensity found in the old compared with the young DBA/2J mice. In contrast to glaucomatous mice, the control group did not show any notable age-related difference. CONCLUSIONS: Disruption of the homeostatic properties of secreted APP with consecutive Abeta cytotoxicity might be a contributing factor of ganglion cell loss in glaucomatous mouse retinas.     

Neuroprotective effects of BDNF and GDNF in intravitreally transplanted mesenchymal stem cells after optic nerve crush in mice

AIM: To assess the neuro-protective effect of bone marrow mesenchymal stem cells (BMSCs) on retinal ganglion cells (RGCs) following optic nerve crush in mice. METHODS: C56BL/6J mice were treated with intravitreal injection of PBS, BMSCs, BDNF-interference BMSCs (BIM), and GDNF-interference BMSCs (GIM) following optic nerve crush, respectively. The number of surviving RGCs was determined by whole-mount retinas and frozen sections, while certain mRNA or protein was detected by q-PCR or ELISA, respectively. RESULTS: The density (cell number/mm2) of RGCs was 410.77±56.70 in the retina 21d after optic nerve crush without any treatment, compared to 1351.39±195.97 in the normal control (P<0.05). RGCs in BMSCs treated eyes was 625.07±89.64/mm2, significantly higher than that of no or PBS treatment (P<0.05). While RGCs was even less in the retina with intravitreal injection of BIM (354.07+39.77) and GIM (326.67+33.37) than that without treatment (P<0.05). BMSCs injection improved the internal BDNF expression in retinas. CONCLUSION: Optic nerve crush caused rust loss of RGCs and intravitreally transplanted BMSCs at some extent protected RGCs from death. The effect of BMSCs and level of BDNF in retinas are both related to BDNF and GDNF expression in BMSCs.     

Different responses of macrophages in retinal ganglion cell survival after acute ocular hypertension in rats with different autoimmune backgrounds

Recently macrophages were shown to play a protective role in retinal ganglion cells (RGCs) after optic nerve (ON) injury. In the present study, we investigated how macrophages responded after acute intraocular pressure (IOP) elevation in experimental autoimmune encephalomyelitis (EAE)-resistant Fischer 344 (F344) and Sprague Dawley (SD) rats and EAE-vulnerable Lewis rats. Acute IOP elevation was performed at 110mmHg for 2h to mimic acute glaucoma. Phagocytic cells in the eye were removed by intravitreal application of clodronate liposomes whereas macrophage activation was achieved by intravitreal injection of zymosan, a yeast wall preparation. Fluorescence dye, FluoroGold, was applied behind the eyeballs to retrogradely label surviving RGCs 40h before animal sacrifice. Macrophages in the retina were identified by ED1 immunostaining. Loss of 25% RGCs in F344 but over 90% in Lewis rats was seen 2 weeks after acute IOP elevation. Significant increase in the number of macrophages in the retina was seen to accompany the great RGC loss in Lewis rats; removal of these macrophages reduced the extent of RGC loss, suggesting the involvement of macrophages in RGC death in Lewis strain. Low numbers of macrophages were seen in F344 retinas after acute IOP elevation, and removal of macrophages did not show clear effect on RGC viability. Whereas macrophage activation by zymosan protected RGCs after ON axotomy in F344 rats, the same macrophage activation became detrimental to RGCs after acute IOP elevation. The extent of RGC loss 3 weeks after acute IOP elevation or after macrophage activation by zymosan in EAE-resistant SD rats was similar to that in F344 rats. We thus demonstrate that macrophages in rats with different autoimmune backgrounds react differently to acute IOP elevation and differentially modulate RGC loss, a phenomenon contrary to the protective action in RGCs after ON axotomy. These data suggest that autoimmune background plays a role in modulating vulnerability of RGCs to acute IOP elevation.     

Quantitative analysis of retinal ganglion cell (RGC) loss in aging DBA/2NNia glaucomatous mice: comparison with RGC loss in aging C57/BL6 mice

PURPOSE: To quantify the extent and pattern of retinal ganglion cell (RGC) loss in the DBA2/NNia glaucomatous mouse strain as a function of age and compare it with ganglion cell loss in a nonglaucomatous strain. METHODS: All the ganglion cells in retinas of DBA/2NNia and C57/BL6 mice of various ages (five eyes per age group in 3-month intervals from 3 to 18 months of age) were counted. A novel counting method that does not rely on sampling and that uses retrograde labeling of RGCs with Fluorogold (Fluorochrome; Englewood, CO) was used. RGC loss in the glaucomatous DBA/2NNia mouse strain was contrasted to RGC loss in C57 mice at the same ages. The total number of Fluorogold-labeled cells per retina was compared within and among the two strains as a function of age. In addition, RGC density maps were constructed for each retina, and the range of densities for each age group was compared within and among the two strains. IOP in awake, nonsedated DBA/2NNia mice was measured with a rebound tonometer. RESULTS: RGC loss started between 12 and 15 months of age in C57 mice and led to an approximate 46% reduction by 18 months of age. The rate of loss was best approximated by a second-order polynomial curve. In comparison, DBA/2NNia mice also began showing RGC loss at approximately 12 months of age, but it proceeded at a much faster rate, with approximately 64% of their RGCs dying by the 15th month of age but little additional loss thereafter. RGC loss in the DBA animals had a focal pattern that appeared more patchy and showed greater variability than the age-related loss in C57 mice, which was more diffuse. IOP and total retinal area in DBA/2NNia mice began to increase at approximately 6 months of age. IOP normalized after the 12th month of age. CONCLUSIONS: Age-related RGC loss of up to 50% can occur in the C57 mouse by 18 months of age. The loss does not proceed linearly with age, as is often assumed, and differs both in extent and locational pattern from pathologic RGC loss secondary to glaucoma in DBA/2NNia mouse retinas.     

Retinal ganglion cell death and optic nerve degeneration by genetic ablation in adult mice

Despite the magnitude of the problem, no effective treatments exist to prevent retinal ganglion cell (RGC) death and optic nerve degeneration from occurring in diseases affecting the human eye. Animal models currently available for developing treatment strategies suffer from cumbersome procedures required to induce RGC death or rely on mutations that induce defects in developing retinas rather than in mature retinas of adults. Our objective was to develop a robust genetically engineered adult mouse model for RGC loss and optic nerve degeneration based on genetic ablation. To achieve this, we took advantage of Pou4f2 (Brn3b), a gene activated immediately as RGCs begin to differentiate and expressed throughout life. We generated adult mice whose genomes harbored a conditional Pou4f2 allele containing a floxed-lacZ-stop-diphtheria toxin A cassette and a CAGG-Cre-ER™ transgene. In this bigenic model, Cre recombinase is fused to a modified estrogen nuclear receptor in which the estrogen-binding domain binds preferentially to the estrogen agonist tamoxifen rather than to endogenous estradiol. Upon binding to the estrogen-binding domain, tamoxifen derepresses Cre recombinase, leading to the efficient genomic deletion of the floxed-lacZ-stop DNA sequence and expression of diphtheria toxin A. Tamoxifen administered to adult mice at different ages by intraperitoneal injection led to rapid RGC loss, reactive gliosis, progressive degradation of the optic nerve over a period of several months, and visual impairment. Perhaps more reflective of human disease, partial loss of RGCs was achieved by modulating the tamoxifen treatment. Especially relevant for RGC death and optic nerve degeneration in human retinal pathologies, RGC-ablated retinas maintained their structural integrity, and other retinal neurons and their connections in the inner and outer plexiform layers appeared unaffected by RGC ablation. These events are hallmarks of progressive optic nerve degeneration observed in human retinal pathologies and demonstrate the validity of this model for use in developing stem cell therapies for replacing dead RGCs with healthy ones.     

Erythropoietin promotes survival of retinal ganglion cells in DBA/2J glaucoma mice

PURPOSE: Retinal ganglion cell (RGC) loss occurs in response to increased intraocular pressure (IOP) and/or retinal ischemia in glaucoma and leads to impairment of vision. This study was undertaken to test the efficacy of erythropoietin (EPO) in providing neuroprotection to RGCs in vivo. METHODS: The neuroprotective effects of EPO were studied in the DBA/2J mouse model of glaucoma. Mice were intraperitoneally injected with control substances or various doses of EPO, starting at the age of 6 months and continuing for an additional 2, 4, or 6 months. RGCs were labeled retrogradely by a gold tracer. IOP was measured with a microelectric-mechanical system, and EPO receptor (EPOR) expression was detected by immunohistochemistry. Axonal death in the optic nerve was quantified by para-phenylenediamine staining, and a complete blood count system was used to measure the number of erythrocytes. RESULTS: In DBA/2J mice, the average number of viable RGCs significantly decreased from 4 months to 10 months, with an inverse correlation between the number of dead optic nerve axons and viable RGCs. Treatment with EPO at doses of 3000, 6000, and 12,000 U/kg body weight per week all prevented significant RGC loss, compared with untreated DBA/2J control animals. EPO effects were similar to those of memantine, a known neuroprotective agent. IOP, in contrast, was unchanged by both EPO and memantine. Finally, EPOR was expressed in the RGC layer in both DBA/2J and C57BL/6J mice. CONCLUSIONS: EPO promoted RGC survival in DBA/2J glaucomatous mice without affecting IOP. These results suggest that EPO may be a potential therapeutic neuroprotectant in glaucoma.     

Diabetes Exacerbates the Intraocular Pressure-Independent Retinal Ganglion Cells Degeneration in the DBA/2J Model of Glaucoma

PurposeGlaucoma is a multifactorial disease, causing retinal ganglion cells (RGCs) and optic nerve degeneration. The role of diabetes as a risk factor for glaucoma has been postulated but still not unequivocally demonstrated. The purpose of this study is to clarify the effect of diabetes in the early progression of glaucomatous RGC dysfunction preceding intraocular pressure (IOP) elevation, using the DBA/2J mouse (D2) model of glaucoma.MethodsD2 mice were injected with streptozotocin (STZ) obtaining a combined model of diabetes and glaucoma (D2 + STZ). D2 and D2 + STZ mice were monitored for weight, glycemia, and IOP from 3.5 to 6 months of age. In addition, the activity of RGC and outer retina were assessed using pattern electroretinogram (PERG) and flash electroretinogram (FERG), respectively. At the end point, RGC density and astrogliosis were evaluated in flat mounted retinas. In addition, Müller cell reactivity was evaluated in retinal cross-sections. Finally, the expression of inflammation and oxidative stress markers were analyzed.ResultsIOP was not influenced by time or diabetes. In contrast, RGC activity resulted progressively decreased in the D2 group independently from IOP elevation and outer retinal dysfunction. Diabetes exacerbated RGC dysfunction, which resulted independent from variation in IOP and outer retinal activity. Diabetic retinas displayed decreased RGC density and increased glial reactivity given by an increment in oxidative stress and inflammation.ConclusionsDiabetes can act as an IOP-independent risk factor for the early progression of glaucoma promoting oxidative stress and inflammation-mediated RGC dysfunction, glial reactivity, and cellular death.     

Autophagy in axonal degeneration in glaucomatous optic neuropathy

The role of autophagy in retinal ganglion cell (RGC) death is still controversial. Several studies focused on RGC body death, although the axonal degeneration pathway in the optic nerve has not been well documented in spite of evidence that the mechanisms of degeneration of neuronal cell bodies and their axons differ. Axonal degeneration of RGCs is a hallmark of glaucoma, and a pattern of localized retinal nerve fiber layer defects in glaucoma patients indicates that axonal degeneration may precede RGC body death in this condition. As models of preceding axonal degeneration, both the tumor necrosis factor (TNF) injection model and hypertensive glaucoma model may be useful in understanding the mechanism of axonal degeneration of RGCs, and the concept of axonal protection can be an attractive approach to the prevention of neurodegenerative optic nerve disease. Since mitochondria play crucial roles in glaucomatous optic neuropathy and can themselves serve as a part of the autophagosome, it seems that mitochondrial function may alter autophagy machinery. Like other neurodegenerative diseases, optic nerve degeneration may exhibit autophagic flux impairment resulting from elevated intraocular pressure, TNF, traumatic injury, ischemia, oxidative stress, and aging. As a model of aging, we used senescence-accelerated mice to provide new insights. In this review, we attempt to describe the relationship between autophagy and recently reported noteworthy factors including Nmnat, ROCK, and SIRT1 in the degeneration of RGCs and their axons and propose possible mechanisms of axonal protection via modulation of autophagy machinery.     

Inflammatory demyelination induces axonal injury and retinal ganglion cell apoptosis in experimental optic neuritis

Optic neuritis is an inflammatory disease of the optic nerve that often occurs in patients with multiple sclerosis and leads to permanent visual loss mediated by retinal ganglion cell (RGC) damage. Optic neuritis occurs with high frequency in relapsing-remitting experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, with significant loss of RGCs. In the current study, mechanisms of RGC loss in this model were examined to determine whether inflammation-induced axonal injury mediates apoptotic death of RGCs. RGCs were retrogradely labeled by injection of fluorogold into superior colliculi of 6-7 week old female SJL/J mice. EAE was induced one week later by immunization with proteolipid protein peptide. Optic neuritis was detected by inflammatory cell infiltration on histological examination as early as 9 days after immunization, with peak incidence by day 12. Demyelination occurred 1-2 days after inflammation began. Loss of RGC axons was detected following demyelination, with significant axonal loss occurring by day 13 post-immunization. Axonal loss occurred prior to loss of RGC bodies at day 14. Apoptotic cells were also observed at day 14 in the ganglion cell layer of eyes with optic neuritis, but not in control eyes. Together these results suggest that inflammatory cell infiltration mediates demyelination and leads to direct axonal injury in this model of experimental optic neuritis. RGCs die by an apoptotic mechanism triggered by axonal injury. Potential neuroprotective therapies to prevent permanent RGC loss from optic neuritis will likely need to be initiated prior to axonal injury to preserve neuronal function.     

Head-up tilt lowers IOP and improves RGC dysfunction in glaucomatous DBA/2J mice

The inbred DBA/2J (D2) mouse strain is a well established model of spontaneously elevated intraocular pressure (IOP), progressive glaucomatous loss of retinal ganglion cells (RGCs), and early damage of RGC axons at the level of optic nerve head. Pattern electroretinogram (PERG) studies have shown that surviving RGCs in mice 6-12-month-old may be dysfunctional. RGC dysfunction seems to be IOP-dependent, since it may be exacerbated by means of acute IOP elevation with head-down body tilt. Here we test the hypothesis that head-up body posture lowers IOP, resulting in improvement of PERG amplitude in aged D2 mice with glaucoma. We show that head-up body tilt induces age-independent IOP lowering whose magnitude increases with the angle of tilt. For a fixed angle (−60°) of head-up tilt, IOP progressively decreases with a time constant of about 5 min and stabilizes at a value lower by about 5-6 mm Hg compared to the baseline. Head-up tilt also results in an improvement of PERG amplitude in older D2 mice with glaucoma but not in younger D2 mice without glaucoma. Improvement of PERG amplitude in aged D2 mice upon head-up-induced IOP lowering is consistent with the idea that RGCs undergo a stage of IOP-dependent, reversible dysfunction before death. The head-up IOP/PERG protocol may represent a non-invasive way to probe the potential for recovery of RGC dysfunction in D2 mice.     

腺相关病毒介导的脑源性神经营养因子对DBA／2J小鼠视网膜神经节细胞的保护作用

背景神经营养因子的缺乏与青光眼的视神经损害密切相关。外源性神经营养因子的补充具有短暂的保护作用。腺相关病毒（AAV）介导的神经营养因子可在眼内长期表达,但是否对青光眼动物的视神经具有持久的保护作用有待研究。目的评估AAV介导的脑源性神经营养因子（BDNF）基因在DBA／2J小鼠眼内的表达及其对视网膜神经节细胞（RGCs）的保护作用。方法健康清洁级DBA／2J小鼠10只从4月龄起,每月使用Tonolab眼压计测量眼压。6月龄时左眼玻璃体腔内注射AAV介导的BDNF和绿色荧光蛋白（GFP）基因（AAV—BDNF—GFP）1μ1,右眼注射等量的生理盐水作为对照。注射后3个月心脏灌流后取出视网膜,荧光显微镜下观察GFP在视网膜中的表达,免疫组织化学法计算存活的RGCs数目并进行比较。结果DBA／2J小鼠4月龄时AAV—BDNF—GFP眼眼压平均为11．90mmHg,对照眼眼压为11．40mmHg。实验眼与对照眼眼压5月龄时均开始升高,8月龄时达到高峰。从4月龄到9月龄,实验组和对照组眼压比较差异无统计学意义（t=-1．78～0．61,P=0．11—0．90）。玻璃体腔注射AAV—BDNF—GFP3月龄后视网膜可以观察到GFP阳性细胞,转染率为46．33％±8．08％。AAV—BDNF—GFP组实验眼的平均RGCs的密度为（3168．13±1319．33）mm^2,对照眼为（2024．81±796．38）mm^2,差异有统计学意义（t=2．75,P=0．02）。结论AAV介导的BDNF对DBA／2J小鼠RGCs有保护作用。     

Three experimental glaucoma models in rats: comparison of the effects of intraocular pressure elevation on retinal ganglion cell size and death

Glaucoma is a chronic and progressive optic nerve neuropathy involving the death of retinal ganglion cells (RGCs). Elevated intraocular pressure (IOP) is considered to be the major risk factor associated with the development of this neuropathy. The objective of the present study was to compare the effects on RGC survival of three different experimental methods to induce chronic elevation of IOP in rats. These methods were: (i) injections of latex microspheres into the eye anterior chamber; (ii) injections into the anterior chamber of a mixture of microspheres plus hydroxypropylmethylcellulose (HPM) and (iii) cauterization of three episcleral veins. The IOP of right (control) and left (glaucomatous) eyes was measured with an applanation tonometer in awake animals. Thirteen to 30 weeks later, RGCs were retrogradely labeled with 3% fluorogold. Subsequently, we analyzed the density of RGCs, as well as the major axis length and area of RGC soma resulting from the application of each method. A significant increase in IOP was found following application of each of the three methods. Cell death was evident in the glaucomatous eyes as compared to controls. However, no statistical differences were found between the extent of cell death associated with each of the three methods. IOP increase also induced a significant increase in the size of the soma of the remaining RGCs. In conclusion, the three methods used to increase IOP induce a similar degree of RGC death. Moreover, the extent of cell death was similar when the retinas were maintained under conditions of elevated IOP for 24 weeks in comparison to 13 weeks.     

The neurobiology of cell death in glaucoma

Glaucoma is an optic neuropathy in which the optic nerve axons are damaged, resulting in death of retinal ganglion cells (RGCs). The primary region of damage is thought to be the optic nerve head (ONH), with the lateral geniculate nucleus (LGN) and optic radiations to the visual cortex being secondarily affected. Neurotrophin deprivation resulting from optic nerve injury is thought to cause RGCs to die by apoptosis by inhibition of cell survival pathways. However, disruption of retrograde axonal transport is not the only mechanism associated with optic nerve damage and RGC death, and thus, an additional mechanism of injury is likely to be involved in glaucomatous optic neuropathy.     

Effect of glatiramer acetate on primary and secondary degeneration of retinal ganglion cells in the rat

PURPOSE: After crush injury to the optic nerve, elevated intraocular pressure, and glutamate toxicity, the immune modulator glatiramer acetate (GA, Cop-1; Copaxone; Teva Pharmaceutical Industries, Pitach Tikva, Israel) has been shown to reduce the delayed cell death of retinal ganglion cells (RGCs). This study was undertaken to confirm the protective effect of GA on secondary degeneration of RGCs in the rat, by using a spatial, rather than temporal, model. METHODS: A total of 131 Wistar rats divided into 10 groups underwent bilateral stereotactic injection of fluorescent tracer (Fluorogold; Fluorochrome, Denver, CO) into the superior colliculus to label RGCs. They received a concurrent subcutaneously injection of (1) GA mixed with complete Freund's adjuvant (CFA), (2) CFA alone, or (3) saline. One week later, the superior one third of the left optic nerve was transected in animals in the six partial transection groups. Optic nerves in four additional groups underwent full transection. Rats were killed and retinas harvested from both eyes 1 or 4 weeks after partial transection and 1 or 2 weeks after full transection. RGC densities were calculated from retinal wholemounts, and differences between right (control) and left (transected) eyes were compared across treatment groups. RESULTS: Among the partial transection groups, differences in the mean percentage of RGC loss in the inferior retinas were not significant at 1 or 4 weeks (ANOVA; P = 0.20, P = 0.12, respectively). After full transection, there was significantly more RGC loss in the GA group than in the CFA group when comparing whole retinas at 1 week, but not at 2 weeks (two-tailed t-test; P = 0.04, P = 0.36, respectively). CONCLUSIONS: There is no evidence that GA has a neuroprotective effect after optic nerve transection, either for primarily injured or secondarily involved RGC.     

Intravitreal administration of erythropoietin and preservation of retinal ganglion cells in an experimental rat model of glaucoma

PURPOSE: The aim of this pilot study was to evaluate the potential neuroprotective effect of an intravitreal injection of erythropoietin (EPO) on retinal ganglion cell (RGC) preservation in an episcleral vessel cautery-induced rat model of glaucoma. METHODS: The animals were randomly assigned into an unoperated control group (n = 11) and three experimental groups: episcleral vessel cautery only (EVC: n = 4), episcleral vessel cautery with intravitreal normal saline injection (EVC-NS; n = 5), and episcleral vessel cautery with intravitreal EPO treatment (EVC-EPO; n = 9). The intravitreal injections were limited to 5 mul containing either normal saline alone or 200 ng of EPO in normal saline administered immediately after the cautery procedure. RGCs were labeled retrogradely by FluoroGold neuron tracer 5 to 7 days prior to the collection of eyes at day 21 and counted in whole flat-mounted retinas with fluorescence microscopy. RESULTS: Compared to the RGC counts in retinal specimens from unoperated control rats (12,619 +/- 310), the corresponding RGC counts were significantly decreased in both the EVC (9116 +/- 273; p < 0.005) and EVC-NS (9489 +/- 293; p < 0.005) groups but not significantly decreased in the EVC-EPO (11,212 +/- 414; p = 0.051) treated retinas. CONCLUSIONS: A single intravitreal 200 ng dose of EPO appears to have a protective effect on RGC viability in an in vivo rat model of glaucoma. Further experimental studies are needed to confirm these preliminary results and to optimize the appropriate dose and frequency of EPO delivery in animal models of glaucoma.     

Characterization of intraocular pressure pattern and changes of retinal ganglion cells in DBA2J glaucoma mice

AIM: To characterize the pattern of intraocular pressure (IOP) change and the deficit of retinal ganglion cells (RGCs) in DBA2J, which is most wellcharacterized chronic glaucoma mouse model and wild type (WT) C57bl/6 mice, and to study the relationship between IOP change and RGCs deficit. METHODS: IOP was monitored with a rebound tonometer in WT C57bl/6 and DBA2J mice from 3 to 15-monthold. Retinal function was evaluated by dark-adapted electroretinogram (ERG) in DBA2J and WT mice of 15monthold. A dye (Neurobiotin) was applied to optic nerve stump to retrograde label RGCs. TO-PRO-3 visualized all nuclei of cells in the RGC layer. RESULTS: The IOP in WT mice was 9.03±0.6 mm Hg on average and did not increase significantly as aging. The IOP in DBA2J mice, arranging from 7.2 to 28 mm Hg, was increasing significantly as aging, and it was normal at 3monthold compared with WT mice, slightly increased from 7-monthold and increased in 50% animals at 11monthold and in 38% animals at 15-monthold. The RGCs density in DBA2J mice started reducing by 7month-old, continuously decreased until reached about 20% of RGC in WT retina by 15monthold. RGC density was not linearly correlated with IOP in 15-monthold DBA2J mice. The amplitude of positive scotopic threshold response, and negative scotopic threshold response of ERG were significantly reduced in DBA2J mice of 15-monthold than that in agepaired WT mice. CONCLUSION: The present study found that DBA2J mice display pathological and functional deficits of the retina that was not linearly correlated with IOP.     

Anti-apoptotic effects of human granulocyte colony-stimulating factor (G-CSF) on retinal ganglion cells after optic nerve crush are PI3K/AKT-dependent

The purpose of present study is to dissect the role of PI3K/AKT signaling in the anti-apoptotic effects of human granulocyte colony-stimulating factor (G-CSF) on rat retinal ganglion cells (RGCs) after optic nerve (ON) crush. The ONs of seventy-two adult male Wistar rats were crushed by a standardized method. Control eyes received a sham operation. G-CSF or phosphate-buffered saline (PBS) was immediately administrated after the ON event for 5 days. Twelve rats were used to investigate the signaling pathways using western blot analysis. In other sixty rats, each eye also received intravitreal injections of PI3K/AKT inhibitor (LY294002) or PBS immediately after the experiments. Rats were euthanized at 1 or 2 weeks after the experiment. RGC density was counted by retrograde labeling with Fluorogold. Western blot analysis of p-AKT, TUNEL assays, and immunohistochemistry of the retinas were conducted. Two weeks after ON injury, RGC densities in the central and mid-peripheral retinas of ON-crushed, G-CSF treated rats were significantly higher than those of corresponding ON-crushed, G-CSF-treated and LY294002-injected rats (survival rates of 60% vs. 39% and 43% vs. 33%, respectively; p < 0.01). Decreased TUNEL staining and the up-regulations of p-AKT signaling in retinas of ON-crushed, G-CSF-treated rats were blocked by intravitreal injections of LY294002. The double staining showed that p-AKT expression co-localized with RGCs in the ON crushed, G-CSF treated retinas. In conclusion, the anti-apoptotic effects of G-CSF on RGCs are PI3K/AKT signaling dependent in the retinas to rescue RGCs after ON crush injury.