Activation of caspase 9 in a rat model of experimental glaucoma

PURPOSE: We investigated retinal ganglion cell (RGC) death and activation of caspase 9 in rats with experimental glaucoma. METHODS: Elevated intraocular pressure (IOP) was induced in rats using the Morrison model. Surviving backlabeled RGC were counted and TUNEL staining detected apoptosis. Procaspase 9 expression and activated caspase 9 were studied by immunoblot and immunohistochemistry. RESULTS: IOP correlated with surviving RGC. TUNEL-positive RGC were observed in animals with elevated IOP. Procaspase 9 levels increased with IOP intensity. Cleaved caspase 9 was detected by immunoblot only in rats with peak IOP above 35 mm Hg for > or =6 days. Cleaved caspase 9 staining was seen only in the ganglion cell layer of retinas from rats with peak IOP > or =32 mm Hg. CONCLUSIONS: RGC loss is correlated with IOP in experimental glaucoma. These results support activation of caspase 9, the intrinsic caspase cascade, in RGC death in experimental glaucoma.     

Neuroprotective and intraocular pressure-lowering effects of (-)Delta9-tetrahydrocannabinol in a rat model of glaucoma

In glaucoma, retinal ganglion cell (RGC) death is induced by many risk factors, including ocular hypertension. It has been proposed that glutamate-mediated oxidative stress may also contribute to this RGC death. Cannabinoids are known to possess therapeutic properties including ocular hypotension and antioxidation. In this study, we test the hypothesis that (-)Delta(9)-tetrahydrocannabinol (THC) lowers intraocular pressure (IOP) and prevents RGC death in a rat model of glaucoma. Arat model of experimental glaucoma with chronic, moderately elevated IOP was produced unilaterally by cauterization of episcleral vessels. Rats received weekly injections of THC at a level of 5 mg/kg or vehicle for 20 weeks. IOP of both eyes was measured weekly on anesthetized animals immediately before THC treatment. RGCs were labeled in a retrograde fashion and counted in whole-mounted retinas. IOP was elevated in all operated eyes 1 day after the operation and remained elevated in the vehicle-treated rats throughout 20 weeks. In THC-treated rats, IOP elevation in operated eyes was diminished 2 weeks after operation and remained reduced. IOP in the contralateral control eyes was not affected by THC. In the operated eyes of vehicle-treated animals, there was a loss of approximately 50 and 40% of the RGCs in the peripheral and central retina, respectively. The RGC loss in the operated eyes of the THC-treated animals was reduced to 10-20%. These results demonstrate that THC is a neuroprotectant that preserves RGCs in an experimental model of glaucoma, possibly through a reduction in IOP.     

辅酶 Q10 对视网膜神经节细胞及高眼压动物模型氧化应激损伤保护作用的研究

目的探讨辅酶Q10对体外培养的人视网膜神经节细胞（RGC）及高眼压动物模型氧化应激损伤的保护所用，并对其作用机制进行分析。方法体外培养人RGC，辅酶Q10干预，干预后暴露于过氧化氢（H：0：）24h，通过光镜观察RGC形态改变；CCK-8检测细胞活性改变；用DCFH—DA荧光法检测细胞内活性氧（ROS）水平；流式细胞仪技术检测细胞凋亡情况。高眼压动物模型利用Wistar大鼠巩膜上静脉烧灼模型。辅酶Q10每日胃肠灌注喂养。Tonopen方法检测动物模型眼压水平；利用荧光金上丘逆行标记方法检测存活的RGC。用Western-印迹法检测与氧化应激及凋亡相关的caspase3，cytochromeC，BAX及BCL2蛋白的表达。结果辅酶Q10可改善暴露于氧化应激环境中的人RGC活力，并能在一定程度上抑制RGC的凋亡。DCFH—DA法结果表明辅酶Q10能够抑制RGC内ROS的产生。动物模型实验结果显示，巩膜上静脉烧灼术可以稳定地升高动物眼压。辅酶Q10可以保护RGC免受高眼压损伤。Western印迹法结果显示，经辅酶Q10预处理后的RGC及动物模型caspase3，cytochromeC及BAX表达量降低，而抑制凋亡的BCL2表达升高。结论辅酶Q10对体外培养的人RGC及高眼压动模型具有抗氧化损伤的保护作用，这种保护作用可能与抑制细胞凋亡有关。     

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.     

The pig eye as a novel model of glaucoma

We validated the pig eye as a model of glaucoma, based on chronic elevation of intraocular pressure (IOP). IOP was elevated by cauterising three episcleral veins in each of the left eyes of five adult pigs. Right eyes were used as controls. Measurement of IOP was performed during the experiment with an applanation tonometer (Tono-Pen). Five months after episcleral vein occlusion, retinal ganglion cells (RGCs) from both cauterised and control eyes were retrogradely backfilled with Fluoro-Gold. Analysis of RGC loss and morphometric as characterization of surviving RGCs was performed using whole-mounted retinas. Elevation of IOP was apparent after three weeks of episcleral vein cauterisation and it remained elevated for at least 21 weeks (duration of the experiments). Analysis of RGC loss after chronic elevation of IOP revealed that RGC death was significant in the mid-peripheral and peripheral retina, mainly in the temporal quadrants of both retinal regions. Moreover the mean soma area of remaining RGCs was observed to increase and we found a greater loss of large RGCs in the mid-peripheral and peripheral retina. We conclude that the pattern of RGC death induced in the pig retina by episcleral vein cauterisation resembles that found in human glaucoma. On the basis of this study, the pig retina may be considered as a suitable model for glaucoma-related studies, based on its similarity with human and on its affordability.     

Retinal damage after 3 to 4 months of elevated intraocular pressure in a rat glaucoma model

PURPOSE: To characterize a long-term elevated intraocular pressure (IOP) glaucoma model in the rat with respect to electroretinographic (ERG) changes and the pattern and mechanism of retinal ganglion cell (RGC) death. METHODS; An approximate doubling of IOP was induced in one eye (G) of female Wistar rats (150-180 g) by cautery of 3 episcleral/limbal veins. At intervals over 3 to 4 months, measurements of IOP and ERG changes (contact-lens electrode) were made in both the G and contralateral normal (N) eyes. At the end of 3 to 4 months of elevated IOP, RGCs were fluorescently labeled with Fluorogold (retrogradely from the superior colliculus), or retinas were labeled by intravitreal injection of a mitochondrial potential indicator dye and stained for apoptotic nuclei with a DNA dye. Flatmounts of fixed, dye-labeled retinas were examined by epifluorescence, confocal, or interference contrast microscopy. RESULTS: Elevated IOP was consistently maintained for up to 4 months in G eyes, but ERG a- and b-waves showed a statistically significant decline, of 30% to 40% in amplitude, after 3 months. Loss of RGCs in G retinas was primarily focal with no statistically significant loss demonstrable outside of the focal areas when assessed by an area sampling method for counting RGCs, which totaled 2% to 3% of the entire retinal area. Mitochondrial membrane potential of cells in the RGC layer was reduced by 17.5% (P: < 0.05) in regions surrounding areas of focal loss compared with comparable locations in control N eyes. After 3.5 months' elevated IOP the G retinas showed cell nuclei at various stages of apoptosis, from initial DNA condensation to fragmentation. CONCLUSIONS: The three-vein episcleral/limbal vein occlusion model for inducing glaucomatous pathology in the rat eye gives a consistent long-term elevation of IOP. After 3 to 4 months of approximately 100% increased IOP, the ERG responses begin to decline, there is a variable focal loss of RGCs, and some of the remaining RGCs show characteristics of stress and apoptosis. These changes seem consistent with retinal damage in human glaucoma (focal field defects), and this rat model appears to mimic some features of primary open-angle glaucoma.     

Ocular hypotensive effects of topically administered agmatine in a chronic ocular hypertensive rat model

Agmatine, a primary polyamine and potential neuromodulator, exhibits a high affinity to the α₂-adrenergic receptor as well as imidazoline receptors. As α₂-adrenergic receptor agonists display positive ocular hypotensive effects, we assessed whether agmatine effectively lowers intraocular pressure (IOP) using a chronic ocular hypertensive rat model. We raised IOP in unilateral eyes of Sprague-Dawley rats by cauterizing three episcleral veins per eye. Four weeks later, we topically administered 10⁻³ M agmatine solution 4 times a day for 6 consecutive weeks. After confirming the recovery of IOP to pretreatment level at 13 weeks after cauterization, the retinal ganglion cells (RGCs) were retrogradely labeled and counted. Eyes subjected to episcleral vein cauterization (EVC) demonstrated significant increases in IOP (48.39% increase over baseline IOP), and the elevated IOP was well maintained until 12 weeks. Topically administered agmatine powerfully lowered IOP to 30.29% of its pretreatment level, and the associated washout period was about two weeks. EVC was associated with a 55.44% loss of RGCs in the control group, but agmatine appeared to attenuate this RGC loss to 18.65%. Overall, topically administered agmatine appeared to effectively lower IOP and rescue RGCs in a chronic ocular hypertensive rat model. Although the mechanism underlying these effects is not yet established, it is possible that agmatine offers a powerful new ocular hypotensive agent for eyes with chronic ocular hypertension and/or glaucoma.     

Caspase-independent component of retinal ganglion cell death, in vitro

PURPOSE: Although in vitro and in vivo models demonstrate caspase activation in retinal ganglion cells (RGCs) undergoing apoptosis, the caspase-independent component of RGC death is unclear. Identification of the precise mechanisms of cell death in these distinct neurons is essential for the development of effective neuroprotective strategies in glaucoma. Because TNF-alpha and hypoxia have been implicated in RGC death during glaucomatous optic nerve degeneration, this study was conducted to determine whether RGCs survive exposure to TNF-alpha or hypoxia in the presence of caspase inhibitor treatment, and whether mitochondrial dysfunction is involved in RGC death induced by these glaucomatous stimuli. METHODS: Primary cultures of rat RGCs were exposed to TNF-alpha or hypoxia for up to 48 hours. The temporal relationship of RGC death with the loss of mitochondrial membrane potential and the release of cell death mediators, including cytochrome c and apoptosis-inducing factor (AIF), was studied in the absence and presence of specific inhibitors of caspases. In addition, treatment with a free-radical scavenger, 4-hydroxytetramethylpiperidine-1-oxyl (tempol; 5 mM), was used in some experiments. Cell viability was assessed using calcein assay, and annexin V binding combined with propidium iodide staining was used for the distinction of apoptotic and necrotic cells. Caspase-3-like protease activity was measured using a fluorometric assay, and for the in situ detection of caspase activity, immunocytochemistry was performed with a cleavage-site-specific antibody. The time course of alterations in the mitochondrial membrane potential and the release of cell death mediators in individual cells undergoing cell death were assessed with a fluorescent tracer and subsequent immunocytochemistry. In addition, a fluorescent dye, dihydroethidium was used to assess the generation of reactive oxygen species (ROS). RESULTS: Findings of this study revealed that the loss of mitochondrial membrane potential and the release of cell death mediators accompanied RGC death induced by TNF-alpha or hypoxia. Although caspase inhibitor treatment temporarily decreased the rate of apoptosis, caspase inhibition was not adequate to block RGC death if the mitochondrial membrane potential was lost and mitochondrial mediators were released. Despite the inhibited caspase activity, survival rate was less than 70% after a 48-hour incubation with death stimuli, and both apoptotic and necrotic cells were detectable in these cultures. When combined with caspase inhibition, tempol reduced the production of ROS and provided an additional 20% increase in RGC survival. CONCLUSIONS: Based on these novel findings, RGC death induced by TNF-alpha or hypoxia involves a caspase-independent component, and reducing the free-radical generation provides additional protection of RGCs temporarily saved by caspase inhibition. Therefore, neuroprotective strategies in glaucoma should include tools to improve the ability of these neurons to survive the cytotoxic consequences of mitochondrial dysfunction.     

Characterization of retinal damage in the episcleral vein cauterization rat glaucoma model

Episcleral vein cauterization (EVC) is used in rats to generate a glaucoma model with high intraocular pressure (IOP). The long-term retinal damage in this glaucoma model, however, has not been accurately quantified. We report the location and amount of retinal ganglion cell (RGC) damage caused by (EVC) induced IOP elevation in two rat strains. IOP was raised in one eye of Wistar (N = 5) and Brown-Norway(B-N)(N = 7) rats by EVC and monitored monthly until IOP in contralateral eyes equalized at 5 months post-surgery. Animals were maintained for 3.5-4.5 additional months. B-N rats (N = 7) that had no EVC served as controls for this strain. Scotopic flash ERGs were recorded at baseline and just prior to euthanasia. Automated counts of all retrogradely labeled RGCs in retinal flat-mounts were determined and compared between contralateral eyes. RGC density maps were constructed and RGC size distribution was determined. Oscillatory potentials in the group of eyes which had elevated IOP were decreased at the time of euthanasia, when IOP had returned to normal. The group of normal B-N rats had similar RGC counts between contralateral eyes. In the experimental group the mean number of RGCs was not significantly different between control and experimental eyes, but 1 of 5 Wistar and 2 of 7 B-N experimental eyes had at least 30% fewer RGCs than contralateral control eyes. Total retinal area in B-N experimental eyes was higher compared to contralateral eyes. Cumulative IOP exposure of the experimental eyes was modestly correlated with RGC loss while oscillatory potentials appeared to be inversely related to RGC loss. In retinas with extensive (> 30% RGC loss) but not complete damage, smaller cells were preserved better than larger ones. The above results indicate that RGC loss in both Wistar and B-N strains is variable after a prolonged elevation of IOP via EVC. Such variability despite equivalent IOP levels and ERG abnormalities, suggests unknown factors that can protect IOP-stressed RGCs. Identification and enhancement of such factors could prove useful for glaucoma therapy.     

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.     

内源性睫状神经营养因子对青光眼鼠视网膜的保护作用

目的 探讨睫状神经营养因子(CNTF)蛋白在慢性高眼压性青光眼大鼠视网膜中的定位及表达情况.方法采用水下电凝巩膜静脉建立大鼠慢性高眼压性青光眼模型,在1、3、7、14、28 d分别摘取眼球,运用免疫组织化学和Western blot法检测大鼠视网膜CNTF蛋白的定位及表达变化.结果对照组大鼠视网膜神经节细胞(RGCs)层有少量CNTF蛋白表达,青光眼大鼠视网膜CNTF蛋白的表达显著增加,建立模型后第7～14 d表达量达到高峰,此时除了神经节细胞层外,内外核层亦发现有CNTF蛋白的表达,之后表达减少.结论青光眼大鼠视网膜内源性CNTF表达增加,可能与促进损伤的RGCs的存活和轴突再生密切相关.     

Obstructed axonal transport of BDNF and its receptor TrkB in experimental glaucoma

PURPOSE: In both animal model system and in human glaucoma, retinal ganglion cells (RGCs) die by apoptosis. To understand how RGC apoptosis is initiated in these systems, the authors studied RGC neurotrophin transport in experimental glaucoma using acute intraocular pressure (IOP) elevations in rats and chronic IOP elevation and unilateral optic nerve transections in monkeys. METHODS: Eyes were studied in masked fashion by light and electron microscopy and by immunohistochemistry with antibodies directed against the tyrosine kinase receptors (TrkA, B, and C) and against brain-derived neurotrophic factor (BDNF), as well as by autoradiography to identify retrograde axonal transport of 125I-BDNF injected into the superior colliculus. RESULTS: With acute glaucoma in the rat, RGC axons became abnormally dilated, accumulating vesicles presumed to be moving in axonal transport at the optic nerve head. Label for TrkB, but not TrkA, was relatively increased at and behind the optic nerve head with IOP elevation. Abnormal, focal labeling for TrkB and BDNF was identified in axons of monkey optic nerve heads with chronic glaucoma. With acute IOP elevation in rats, radiolabeled BDNF arrived at cells in the RGC layer at less than half the level of control eyes. CONCLUSIONS: Interruption of BDNF retrograde transport and accumulation of TrkB at the optic nerve head in acute and chronic glaucoma models suggest a role for neurotrophin deprivation in the pathogenesis of RGC death in glaucoma.     

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.     

经房角镜光凝小梁网法建立慢性高眼压大鼠模型及其与经角膜缘光凝法的比较

背景 慢性高眼压动物模型的建立是青光眼发病机制研究的基础,以往激光光凝建立慢性高眼压动物模型的方法存在模型眼压波动大,需要重复光凝和并发症多的问题,造模方法的改良对于顺利开展相关的实验研究具有重要意义. 目的 用经房角镜光凝小梁网法建立大鼠慢性高眼模型,并与以往的经角膜缘光凝法进行比较. 方法 选取8 ～12周龄清洁级Fischer344大鼠36只,将动物分为正常对照组、经角膜缘光凝组和经房角镜光凝组,每组12只,经角膜缘光凝组采用532 nm YAG激光经角膜缘光凝大鼠右眼小梁网建立慢性高眼压模型,激光能量为440 ～ 500 mW,激射光斑40 ～ 60个;经房角镜光凝组激光能量为800 ～850 mW,激射光斑100～120个.光凝后用Tonolab眼压计测量并观察各组大鼠眼压变化;于光凝后第3周每组处死5只大鼠,分离视网膜,采用免疫荧光技术检测并比较各组大鼠视网膜中Tuj-1阳性的视网膜神经节细胞(RGCs)数目.实验动物的使用及喂养遵循ARVO声明.结果 造模后3周各组大鼠一般情况可,眼表无明显损伤.经角膜缘光凝组和房角镜光凝组慢性高眼压模型的成模率分别为75％和100％.正常对照组、经角膜缘光凝组和经房角镜光凝组大鼠模型眼造模后3周的平均眼压分别为(11.0±1.3)、(23.4±12.6)和(25.3±4.9)mmHg(1 mmHg=0.133 kPa),峰眼压分别为(12.3±1.0)、(50.5±7.3)和(44.3±12.3)mmHg,组间总体比较差异均有统计学意义(F=25.496、80.762,均P＜0.001),其中经角膜缘光凝组和经房角镜光凝组大鼠模型眼平均眼压均明显高于正常对照组,差异均有统计学意义(均P＜0.001),而2个组间平均眼压和峰眼压差异均无统计学意义(P=1.000、0.195).正常对照组、经角膜缘光凝组和经房角镜光凝组大鼠视网膜中Tuj-1阳性RGCs数目分别为(2 048.2±148.5)、(645.2±177.1)及(1 223.7±148.6)/mm2,总体比较差异有统计学意义(F=98.767,P＜0.001),其中经角膜缘光凝组和经房角镜光凝组大鼠视网膜中Tuj-1阳性RGCs数目均明显少于正常对照组,且经角膜缘光凝组大鼠视网膜中Tuj-1阳性RGCs数目明显少于经房角镜光凝组,差异均有统计学意义(均P＜0.01). 结论 经房角镜光凝小梁网能够诱导大鼠慢性高眼压并导致RGCs损害,但眼压升高模式及RGCs损害程度与经角膜缘光凝法有所不同,经房角镜光凝法建立慢性高眼压模型成模率更高.     

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.     

Rat chronic glaucoma model induced by intracameral injection of microbeads suspended in sodium sulfate–sodium hyaluronate

Purpose

To establish a rat glaucoma model with chronic intraocular pressure (IOP) elevation induced by microbeads suspended in sodium sulfate–sodium hyaluronate.

Methods

Chronic elevation of IOP was induced unilaterally by injecting polystyrene microbeads, suspended in 4 % sodium sulfate and 3 % sodium hyaluronate, into the anterior chamber. The microbead suspension was injected through either the clear corneal (CC) or sclerocorneal (SC) tunnel. IOP changes were monitored up to 8 weeks after injection. The loss of retinal ganglion cells (RGCs) was assessed using fluorogold retrograde labeling of RGCs. RGC axons were evaluated by immunohistochemistry and immunoblotting.

Results

The resulting IOP elevation was maintained up to 3 weeks after the intracameral injection of microbeads through the CC route and up to 4 weeks after injection through the SC route. The density of RGCs was significantly reduced at 4 weeks after injection, with the SC route leading to more RGC loss than the CC route (p = 0.037). The neurofilament immunoreactivity and protein levels in the optic nerve were also significantly reduced at 4 weeks after injection. Some eyes in the SC route cohort received re-injection of the microbead suspension at 4 weeks after the initial injection, which led to an elevated IOP more than 8 weeks after the initial injection, and eventually a 27.5 % loss of RGC density compared with the control eyes.

Conclusion

The intracameral injection of microbeads suspended in hyaluronate effectively produced chronic IOP elevation and subsequent RGC degeneration in rat eyes. The sclerocorneal tunnel approach yielded a longer period of IOP elevation than the clear corneal approach. Our modified microbead injection offers a reliable high-pressure glaucoma model.     

Characteristics of Optic Nerve Damage Induced by Chronic Intraocular Hypertension in Rat

Purpose:To set up the Sharma‘s chronic intraocular hypertension model and investigate the intraocular pressure (IOP) as well as the optic nerve damage of this model in rat.Methods :The operations of the chronic intraocular hypertension model were performed as described by Sharma in 60 male Lewis albino rats. IOP was measured using the TonoPen XL immediately after surgery and then at 5 day, 2 week or 4 week intervals. Cresyl violet staining of whole-mounted retinas was used to label retinal ganglion cells (RGCs),then RGCs were counted. Paraphenylenediamine (PPD) staining was performed in the semi-thin cross sections of optic nerve of rat, in order to know whether the axons of optic nerve were degenerated or not.Results:There were 47 rats with higher IOP after the episcleral veins cauterized in 60 rats. The ratio of elevated IOP was 78.3%. The IOPs were stable in 4 weeks. After cresyl violet staining, the RGCs loss was 11.0% and 11.3% was found in the central and peripheral retina respectively after 2 weeks of increased IOP. After 4 weeks of increased IOP, the loss of RGCs was 17% for the central retina and 24.6% for the peripheral retina. In the retinas without higher IOP, there was no loss of RGCs. PPD staining showed that optic nerve of rat with about 5.3% damage of axons located at the superior temporal region. Region of affected optic nerve 1 mm posterior to the globe by light microscope showed evidence of damaged axons with axonal swelling and myelin debris.Conclusion:Sharma‘s chronic intraocular hypertension model is a reproducible and effective glaucoma model, which mimics human glaucoma with chronically elevation IOP and induced RGCs loss and damage of optic nerve.     

Age and intraocular pressure in murine experimental glaucoma

Age and intraocular pressure (IOP) are the two most important risk factors for the development and progression of open-angle glaucoma. While IOP is commonly considered in models of experimental glaucoma (EG), most studies use juvenile or adult animals and seldom older animals which are representative of the human disease. This paper provides a concise review of how retinal ganglion cell (RGC) loss, the hallmark of glaucoma, can be evaluated in EG with a special emphasis on serial in vivo imaging, a parallel approach used in clinical practice. It appraises the suitability of EG models for the purpose of in vivo imaging and argues for the use of models that provide a sustained elevation of IOP, without compromise of the ocular media. In a study with parallel cohorts of adult (3-month-old, equivalent to 20 human years) and old (2-year-old, equivalent to 70 human years) mice, we compare the effects of elevated IOP on serial ganglion cell complex thickness and individual RGC dendritic morphology changes obtained in vivo. We also evaluate how age modulates the impact of elevated IOP on RGC somal and axonal density in histological analysis as well the density of melanopsin RGCs. We discuss the challenges of using old animals and emphasize the potential of single RGC imaging for understanding the pathobiology of RGC loss and evaluating new therapeutic avenues.     

Melanopsin-expressing retinal ganglion cells are more injury-resistant in a chronic ocular hypertension model

PURPOSE: To investigate the survival of melanopsin-expressing retinal ganglion cells (mRGCs) after the induction of chronic ocular hypertension. METHODS: Intraocular pressure (IOP) was elevated in adult Sprague-Dawley rats using an argon laser to photocoagulate the episcleral and limbal veins. IOP was measured with a calibrated tonometer and monitored for a period. Seven days before the animals were killed, a piece of sterile foam soaked with gold fluorescent dye was placed onto the superior colliculus (SC) to label the SC-projecting retinal ganglion cells (scRGCs) retrogradely. mRGCs were visualized by free floating immunohistochemistry on whole-mounted retinas. The number of surviving scRGCs and mRGCs were counted on flatmounted retinas. The branching pattern of dendrites and soma size of mRGCs were examined. RESULTS: An approximately 1.7-fold increase of IOP and a significant loss of scRGCs were found in experimental eyes after laser photocoagulation. However, no significant cell loss or morphologic changes on mRGCs and their dendrites after the induction of chronic ocular hypertension are noticed over a 12-week period. CONCLUSIONS: Although the degeneration of retinal ganglion cells (RGCs) is a major concern in glaucomatous damage, the findings show that mRGCs are less susceptible to death after the induction of chronic ocular hypertension. This result indicates that mRGCs carry some unique properties that are different from those of other subpopulations of RGCs. The immunohistochemistry approach can be used to distinguish easily these mRGCs from other subtypes. This method provides a useful tool to investigate their injury-resistant properties that are informative for the development of effective neuroprotective treatment for glaucoma.     

β-Ⅲ-Tubulin:a reliable marker for retinal ganglion cell labeling in experimental models of glaucoma

AIM:To evaluate the reliability of β-III-Tubulin protein as a retinal ganglion cell (RGC) marker in the experimental glaucoma model.