青光眼动物模型DBA／2J小鼠的眼部特征及组织学观察

目的评价不同月龄DBA／2J小鼠的眼压、眼部特征及组织学变化。方法清洁级DBA／2J小鼠36只,3、5、7、9、11、14月龄各6只,3、9、14月龄的C57BL／6J小鼠各6只为对照。分别对实验鼠行眼前节照相,前房微管法眼压测量。用尼氏染色法对鼠视网膜切片进行染色并在光学显微镜下行视网膜神经节细胞（RGCs）计数,光学显微镜下对视网膜冰冻切片行视盘的形态学观察。结果鼠眼前节检查表明,DBA／2J小鼠从5月龄始逐渐发生虹膜色素播散、虹膜萎缩,虹膜透照可见瞳孔变形。眼压从7月龄开始升高,9月龄眼压升至高峰,14月龄降至对照组水平。各月龄DBA／2J小鼠眼压间的差异有统计学意义（F=27．600,P〈0．05）,各月龄C57BL／6J小鼠眼压间的差异无统计学意义（F=0．249,P=0．781）。DBA／2J鼠RGCs数量从7月龄开始减少,9～11月龄减少明显,各月龄DBA／2J鼠RGCs计数间的差异有统计学意义（F=23．594,P=0．000）,各月龄C57BL／6J小鼠RGCs计数的差异无统计学意义（F=1．816,P=0．211）。DBA／2J小鼠视盘凹陷于9～14月龄开始逐渐加深,而各月龄C57BL／6J小鼠的视盘形态无明显变化。结论随月龄的增长,DBA／2J小鼠眼前节病变逐渐加重,表现出继发性青光眼的相关形态学改变。DBA／2J小鼠是研究青光眼发病机制和视神经保护较好的动物模型。     

Time Course of Age-dependent Changes in Intraocular Pressure and Retinal Ganglion Cell Death in DBA/2J Mouse

Glaucoma is a major cause of irreversible blindness and is characterized by the death of retinal ganglion cells (RGCs) [1]. This death of RGCs is frequently associated with an elevation in intraocular pressure (IOP) [2].However, the understanding of how elevated IOP leads to cell death is hampered by the lack of an animal model that emulates the clinical time course for decades. Mouse studies have proven helpful for investigating human complex diseases. The DBA/2J mouse, which is inheri…     

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.     

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。     

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.     

Differential susceptibility to experimental glaucoma among 3 mouse strains using bead and viscoelastic injection

The purpose of this experiment was to test the susceptibility to retinal ganglion cell (RGC) axon loss and RGC layer cell loss from experimental glaucoma among 3 mouse strains, and between younger and older mice. We obstructed the mouse aqueous outflow channels by injecting 2 μL of 6 μm diameter, polystyrene beads followed by 3 μL of viscoelastic solution into the anterior chamber with a glass micropipette. We evaluated intraocular pressure (IOP) and damage to RGC as measured by optic nerve axon counts and RGC layer neuron counts in 3 strains of young mice (2 month old C57BL/6, DBA/2J, and CD1) and 10 month C57BL/6 mice. Bead and viscoelastic injection produced IOP elevation at ≥1 time point in 94.1% of eyes (112/119), with mean IOP difference from fellow eyes of 4.4 ± 3.0 mmHg. By 6-12 weeks, injected eyes were 10.8% longer and 7.6% wider (p < 0.0001). Young DBA/2J and C57BL/6 eyes increased axial length significantly more than young CD1 or older C57BL/6 (all p ≤ 0.02). RGC layer and axon loss was greatest in CD1 mice, significantly more than the other groups (p from 0.04 to <0.0001). Young C57BL/6 eyes elongated more and lost more RGC layer cells than older C57BL/6 mice (p = 0.02 and 0.01, respectively). With this mouse glaucoma model, there was differential susceptibility to ocular elongation and RGC layer and axon damage among mouse strains and by age. Factors that determine sensitivity to RGC injury can be studied using transgenic mouse strains with inducible models.     

Pilotstudie zu musterelektroretinographischen Veränderungen bei der DBA/2NNia-Maus Tiermodell eines angeborenen Engwinkelglaukoms

PURPOSE: The aim of this study was to document the time-course of retinal dysfunction by pattern-electroretinography (PERG) in eyes of the DBA/2NNia substrain of mouse that develop an inherited angle-closure glaucoma. METHODS: Twelve DBA/2NNia mice and 12 control C57BL/6 J mice were studied by PERG recordings from 2 to 10 months of age. PERGs were recorded using different spatial and temporal frequencies. RESULTS: PERGs recorded with a temporal frequency of 7.5 Hz and a spatial frequency of 0.4 cycles/degree performed best to discriminate between DBA/2NNia mice and C57BL/6 J mice. When compared with normal C57BL/6 J mice, significant amplitude reductions of the PERG (Student's t-test; p < 0.01) were found in DBA/2NNia mice by 5 months of age and continued to decline as the animals aged. At beginning of follow-up, the mean PERG amplitude in DBA/2NNia mice was 2.3 +/- 0.5 microV. At 5 months of age, the mean PERG amplitude was reduced by 0.9 +/- 0.45 microV (paired t-test; p < 0.0001). CONCLUSION: Previously, a significant loss of retinal ganglion cells was found in the DBA/2NNia mouse substrain at 6-7 months of age. In the present study, we found decreases in PERG amplitudes, occurring from the age of 5 months onward. Similarities with the findings in human glaucoma indicate the relevance of this animal model for further glaucoma research.     

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.     

Neuronal nitric oxide synthase (nNOS) positive retinal amacrine cells are altered in the DBA/2NNia mouse, a murine model for angle-closure glaucoma

PURPOSE: To characterize retinal amacrine cell changes in eyes of DBA/2NNia (DBA) mice that develop an inherited angle-closure glaucoma. METHODS: DBA and non-glaucomatous C57BL/6J mice of different age groups (2 to 23 months of age) were studied and compared. Morphologic investigations included NADPH-diaphorase staining of retinal whole mounts and fluorescence immunohistochemistry of cryosections with antibodies against neuronal nitric oxide synthase (nNOS), tyrosin hydroxylase (TH), gamma aminobutyric acid (GABA), and vesicular acetylcholine transporter (VAChT). RESULTS: Immunohistochemistry of amacrine cell subpopulations in the retinae of DBA mice revealed no significant staining differences in the two mouse strains at all ages using antibodies against TH, GABA, and VAChT. However, staining with nNOS and NADPH diaphorase revealed significant differences between the DBA strain and the C57BL/6J mice. With the onset of elevated IOP and glaucoma beginning at around 6 months in the DBA mice, the total number of NOS positive amacrine cells continuously decreased from 1000 cells at 6 months of age down to 480 cells in animals older than 20 months of age, but did not decline in age-matched C57 mouse retinas. CONCLUSION: We previously described a parafoveal loss of nNOS positive amacrine cells in the monkey glaucoma model. The fact that there is also a significant decrease of nNOS amacrine cells in the glaucomatous mouse eye indicates a specific response of nNOS positive amacrine cells in glaucomatous retinopathy.     

Dependency of intraocular pressure elevation and glaucomatous changes in DBA/2J and DBA/2J-Rj mice

PURPOSE: In this study parameters relevant for glaucoma in DBA/2J (D2J) mice were compared with those in age-matched DBA/2J-Rj (D2Rj) mice, to challenge the postulated role of D2J mice as a model for secondary high-tension glaucoma. METHODS: Genotyping for three known short nucleotide polymorphisms (SNPs) in the Tyrp1 gene and the Gpnmb gene by MALDI-TOF-MS and immunohistochemical staining for Gpnmb was performed in D2J and D2Rj mice. Twelve C57Bl/6 (B6), 8 D2Rj, and 11 D2J mice between 1 and 4 months of age were screened qualitatively and quantitatively for morphologic differences within the anterior eye segment. The IOP progression of 25 D2Rj and 18 D2J mice were investigated between 4 to 10.5 months after birth. At the end of this study, in 10 randomly selected individuals of each D2J and D2Rj cohort, correlation of IOP progression and optic nerve damage were determined in each eye. RESULTS: D2J and D2Rj strains were homozygous for both Tyrp 1 amino acid substitutions, so far only described in D2J mice. The Gpnmb(R150X) point mutation present in D2J mice was not detected in D2Rj. Accordingly, immunoreactivity (IR) for Gpnmb was present only in D2Rj and B6 eyes, but not in D2J. Compared with B6, both DBA/2 mice (D2) showed a significantly narrowed chamber angle caused by an anteriorly displaced ciliary body. IOP measurements showed an average IOP of approximately 14 mm Hg between age 4 and 7 months in D2Rj, which decreased to approximately 11 mm Hg in the period from 8 to 10.5 months. In D2J the average IOP showed a steady increase in the observed period from 4 to 10.5 months (from 8.65 to 15.58 mm Hg). Individuals with IOP peaks up to 30 mm Hg were detected in D2Rj, but none of these mice showed signs of an optic neuropathy after 10.5 months. In contrast, 30% of the investigated D2J mice at the age of 10.5 months showed a severe optic neuropathy. Individual data analyses, however, showed no significant correlation between elevated IOP and glaucomatous changes within the D2J population. CONCLUSIONS: Individual correlations of IOP course with axon loss in the single eyes confirmed that in D2J mice, hypertension is not the only causative factor in glaucomatous optic neuropathy. For further investigations on the pathogenesis of glaucoma in D2J mice, the D2Rj strain without a Gpnmb(R150X) mutation and without glaucomatous changes, but with individual IOP elevation, can be used as an interstrain control for D2J.     

Noninvasive assessment of aqueous humor turnover in the mouse eye

PURPOSE: To develop a noninvasive test for monitoring changes in aqueous humor turnover in the mouse eye. METHODS: After topical instillation of fluorescein, the rate of decay of fluorescence from aqueous humor and cornea was monitored in Black Swiss, C57 Bl6, and DBA 2J mice with a microscope equipped with epifluorescence and a charge-coupled device (CCD) camera. RESULTS: The rate of decay of fluorescence was identical in right and left eyes over an approximately 70-minute measurement period. The rate of decay was similar in normal mice aged 2 and 18 months. Pilocarpine and latanoprost, known to enhance aqueous humor outflow in humans, accelerated the decay of fluorescence. Levobunolol, known to inhibit aqueous humor inflow in humans, slowed decay. Dimethylamiloride, an inhibitor of the Na(+),H(+) exchanger that is known to act on cultured cells of both the ciliary epithelium and trabecular meshwork and to lower mouse intraocular pressure (IOP), enhanced decay. DBA 2J mice, in which secondary glaucoma develops, displayed a slower decay of fluorescence at 18 months of age than age-matched unaffected animals. CONCLUSIONS: Monitoring decay of fluorescence provides a noninvasive index of aqueous humor dynamics in the mouse eye that facilitates study of ocular hypotensive drugs and mouse models of glaucoma. Coupled with knowledge of IOP, it permits semiquantitative conclusions about the relative roles of aqueous humor inflow and outflow in conditions with altered IOP. Based on this approach, dimethylamiloride appears to lower mouse IOP primarily by enhancing outflow of aqueous humor.     

Microarray analysis of retinal gene expression in the DBA/2J model of glaucoma

PURPOSE: The DBA/2J mouse is a model for secondary angle-closure glaucoma, due to iris atrophy and pigment dispersion, which ultimately lead to increased intraocular pressure (IOP). The study was undertaken to correlate changes in retinal gene expression with IOP elevation by performing microarray analysis of retinal RNA from DBA/2J mice at 3 months before disease onset and at 8 months after IOP elevation. METHODS: IOP was monitored monthly in DBA/2J animals, and animals with normal (3 months) or elevated IOP (8 months) were identified. RNA was prepared from three individual retinas at each age, and the RNA was amplified and used to generate biotin-labeled probe for high-density mouse gene microarrays (U430.2; Affymetrix, Santa Clara, CA). A subset of genes was selected for confirmation by quantitative RT-PCR, by using independent retina samples from DBA/2J animals at 3, 5, and 8 months of age and compared to retinas from C57BL/6J control animals at 3 and 8 months. RESULTS: There were changes in expression of 68 genes, with 32 genes increasing and 36 genes decreasing at 8 months versus 3 months. Upregulated genes were associated with immune response, glial activation, signaling, and gene expression, whereas downregulated genes included multiple crystallin genes. Significant changes in nine upregulated genes and two downregulated genes were confirmed by quantitative RT-PCR, with some showing changes in expression by 5 months. CONCLUSIONS: DBA/2J retina shows evidence of glial activation and an immune-related response after IOP elevation, similar to what has been reported after acute elevation of IOP in other models.     

CYP1B1基因缺失小鼠在高糖皮质激素环境下对青光眼的易感性

目的观察高浓度糖皮质激素对CYP1B1^-/-小鼠青光眼易感性的影响。方法采用成年CYP1B1^-/-小鼠作为实验模型,以同龄C57BL／6J小鼠作为对照。每3天结膜下注射0．04mL倍他米松,用Tonopen眼压（IOP）笔每周定期测定小鼠IOP,于第一次给药前,给药后4、8、12周分别摘除眼球,制备石蜡切片,光学显微镜下观察小鼠视网膜形态和厚度,采用TUNEL法检测视网膜神经节细胞（RGCs）的凋亡。结果与给药前相比,2组小鼠给药后4、8、12周的IOP均较前升高,差异有统计学意义（P〈0．05）;CYP1B1^-/-小鼠的IOP在给药后8周、12周较C57BL／6J小鼠显著升高,差异有统计学意义（P〈0．05）。随着倍他米松注射时间的推移,2组小鼠视网膜纤维层均变薄,各时间组的总体差异有统计学意义（P〈0．05）;且CYP1B1^-/-小鼠视网膜纤维层厚度在给药后8周、12周较C57BL／6J小鼠显著变薄,差异有统计学意义（P〈0．05）。2组小鼠RGCs凋亡的速度与给药前相比均显著增加,差异有统计学意义（P〈0．05）;且CYP1B1^-/-小鼠与C57BL／6J小鼠相比结果更为显著（P〈0．05）。结论在高浓度糖皮质激素的诱导下,CYP1B1^-/-小鼠对青光眼的易感性增加。     

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.     

Cochlin and glaucoma: a mini-review

Primary open angle glaucoma (POAG) is a leading cause of late onset, progressive, irreversible blindness and, although its etiology is poorly understood, elevated intraocular pressure (IOP) often appears to be a contributory factor. Proteomic and Western analyses of trabecular meshwork (TM) from patients with POAG and age-matched controls originally implicated cochlin as possibly contributing to glaucoma pathogenesis. Cochlin deposits were subsequently detected in glaucomatous but not in control TM and older glaucomatous TM was found to contain higher levels of cochlin and significantly lower amounts of collagen type II. More recently, similar results were reported in DBA/2J mice, which at older ages develop elevated IOP, retinal ganglion cell degeneration, and optic nerve damage. Notably, cochlin was absent in TM from C57BL/6J, CD1, and BALBc/ByJ mice, which do not exhibit elevated IOP or glaucoma. Cochlin was found in the TM of very young DBA/2J mice, prior to elevated IOP, suggesting that over time the protein may contribute to the events leading to increased IOP and optic nerve damage. Here we review these findings and describe how future studies in DBA/2J mice can help resolve whether cochlin plays a causal role in mechanisms of POAG and elevated IOP.     

Effects of elevated intraocular pressure on mouse retinal ganglion cells

We developed and characterized a mouse model of elevated intraocular pressure (IOP) to investigate the underlying cellular and genetic mechanisms of retinal ganglion cell (RGC) death. IOP was unilaterally increased in C57BL/6J mice by photocoagulation of the episcleral and limbal veins. IOP was measured using an indentation tonometer. RGC survival was measured by retrograde labeling using DiI applied to the superior colliculous. The mechanism of RGC death was investigated using TUNEL staining, immunostaining for cleaved caspase-3, and Western blot for Bcl-2 and Bax expression. RT-PCR was used to measure changes in Bcl-2, Bax, Bad, Bak, P53, ICE and Fas. Mean IOP was increased in the treated eyes from 13+/-1.8 to 20.0+/-2.8 mmHg at four weeks and 17+/-2.2 mmHg at eight weeks. RGC loss was 15.6+/-3.4% at two weeks and 27.3+/-4.5% at four weeks after laser photocoagulation. TUNEL staining and caspase-3 positive cells were increased in the ganglion cell layer (GCL) in the treated eyes and seldom found in the control eyes. Bcl-2 expression in control group was higher than in the experimental group, while Bax expression in the control group was less than in experimental group. This mouse model resulted in a consistent, sustained increase in IOP with a reduction in the number of RGCs in the treated eye. The RGCs in eyes with elevated IOP were TUNEL-positive, with increased caspase-3 and decreased Bcl-2, consistent with apoptosis as the mechanism of neuronal cell death.     

Neuroprotective effects of acteoside in a glaucoma mouse model by targeting Serta domain-containing protein 4

AIM: To explore the therapeutic effect and main molecular mechanisms of acteoside in a glaucoma model in DBA/2J mice. METHODS: Proteomics was used to compare the differentially expressed proteins of C57 and DBA/2J mice. After acteoside administration in DBA/2J mice, anterior segment observation, intraocular pressure (IOP) monitoring, electrophysiology examination, and hematoxylin and eosin staining were used to analyze any potential effects. Immunohistochemistry (IHC) assays were used to verify the proteomics results. Furthermore, retinal ganglion cell 5 (RGC5) cell proliferation was assessed with cell counting kit-8 (CCK-8) assays. Serta domain-containing protein 4 (Sertad4) mRNA and protein expression levels were measured by qRT-PCR and Western blot analysis, respectively. RESULTS: Proteomics analysis suggested that Sertad4 was the most significantly differentially expressed protein. Compared with the saline group, the acteoside treatment group showed decreased IOP, improved N1-P1 wave amplitudes, thicker retina, and larger numbers of cells in the ganglion cell layer (GCL). The IHC results showed that Sertad4 expression levels in DBA/2J mice treated with acteoside were significantly lower than in the saline group. Acteoside treatment could improve RGC5 cell survival and reduce the Sertad4 mRNA and protein expression levels after glutamate injury. CONCLUSION: Sertad4 is differentially expressed in DBA/2J mice. Acteoside can protect RGCs from damage, possibly through the downregulation of Sertad4, and has a potential use in glaucoma treatment.     

Longitudinal evaluation of retinal ganglion cell function and IOP in the DBA/2J mouse model of glaucoma

PURPOSE: To characterize progressive changes of retinal ganglion cell (RGC) function and intraocular pressure (IOP) in the DBA/2J mouse model of spontaneous glaucoma. METHODS: Serial pattern electroretinograms (PERGs) and IOPs measures were obtained from both eyes of 32 anesthetized DBA/2J mice over an age range of 2 to 12 months at 1-month intervals. Cone-driven flash-ERGs (FERGs) were also recorded. The endpoint was defined as the age at which the PERG amplitude reached the noise level in at least one eye. At that point, both eyes were histologically processed to evaluate the thickness of the retinal fiber layer (RNFL). RESULTS: IOP increased moderately between 2 and 6 months ( approximately 14-17 mm Hg) and then more steeply, until it leveled off at approximately 28 mm Hg by 9 to 11 months. The mean PERG amplitude decreased progressively after 3 months of age to reach the noise level (85% reduction of normal amplitude) at approximately 9 to 12 months in different animals. When the PERG was at noise level, the RNFL showed a relatively smaller reduction (40%) in normal thickness. The FERG displayed minor changes throughout the observation period. IOP and PERG changes were highly correlated (r(2) = 0.51, P < 0.001). CONCLUSIONS: Results indicate that inner retina function in DBA/2J mice progressively decreases after 3 months of age, and it is nearly abolished by 10 to 11 months, whereas outer retina function shows little change and the RNFL thickness is relatively spared. This result suggests that surviving RGCs may not be functional. Progression of inner retinal dysfunction is strongly associated with increased IOP.     

Retinal morphology and ERG response in the DBA/2NNia mouse model of angle-closure glaucoma

PURPOSE. To document the time course of retinal dysfunction by scotopic electroretinography (ERG) and by quantitative morphology in eyes of the DBA/2NNia substrain of mouse (DBA) with inherited angle-closure glaucoma. METHODS. DBA and control C57BL/6J (C57) mice were studied by ERG recordings from 5 to 15 months of age, and by morphology from 1 to 14 months of age. Scotopic ERGs were simultaneously recorded from both eyes of dark-adapted anesthetized mice. Changes in the central neuronal retina were evaluated by quantitative morphometry performed on serial semithin sections of Epon-embedded eyes. RESULTS. When compared with normal C57 mice, DBA mice showed significant reductions of the a-wave and b-wave amplitudes by 7 to 8 months, and the decline continued as the animals aged. The b-wave implicit time in DBA mice showed a gradual prolongation beginning at 8 months of age, when compared with C57 mice. Logistic regression analyses revealed significant correlations in a- and b-wave amplitude reductions between ipsilateral and contralateral eyes of DBA mice at ages when ERG parameters were greatly altered. Morphologically, thinning of the whole retina was already evident in DBA mice at 4 months of age, but loss of ganglion cells and thinning of the outer plexiform layer were first seen in 7- to 8-month-old animals. These changes progressed to the end of the 13-month period studied. CONCLUSIONS. Progressive thinning of the outer retinal layers in DBA mice was found to correlate with decreases in ERG a- and b-wave amplitudes, both occurring from the age of 7 to 8 months onward. Similarities with the findings in human late-stage glaucomatous retinopathy indicate the relevance of this animal model in further glaucoma research.     

Apoptotic retinal ganglion cell death in the DBA/2 mouse model of glaucoma

The DBA/2 mouse has been used as a model for spontaneous secondary glaucoma. We attempted to determine the in vivo time course and spatial distribution of retinal ganglion cells (RGCs) undergoing apoptotic death in DBA/2 mice. Female DBA/2 mice, 3, 9-10, 12, 15, and 18 months of age, received intravitreal injections of Annexin-V conjugated to AlexaFluor 1h prior to euthanasia. Retinas were fixed and flat-mounted. Annexin-V-positive RGCs in the hemiretina opposite the site of injection were counted, and their locations were recorded. Positive controls for detection of apoptotic RGCs by Annexin-V labeling included rats subjected to optic nerve ligation, and C57BL/6 mice subjected to either optic nerve ligation or intravitreal injection of NMDA. To verify that Annexin-V-labeled cells were RGCs, intravitreal Annexin-V injections were also performed on retinas pre-labeled retrogradely with FluoroGold or with DiI. Annexin-V-positive RGC locations were analyzed to determine possible clustering and areas of preferential loss. Annexin-V labeled apoptotic RGCs in eyes after optic nerve ligation, intravitreal NMDA injection, as well as in aged DBA/2 animals. In glaucomatous DBA/2 mice 95-100% of cells labeled with Annexin-V were also FluoroGold- and DiI-positive. This confirms that Annexin-V can be used to specifically detect apoptotic RGCs in rodent retinas. In DBA/2 mice, apoptotic RGC death is maximal from the 12th to the 15th month of age (ANOVA, p<0.001, Fisher's post hoc test) and occurs in clusters. These clusters are initially located in the midperipheral retina and progressively occur closer to the optic nerve head with increasing age. Retrograde axonal transport of FluoroGold in the glaucomatous mouse retina is functional until at least 2-3days prior to initiation of apoptotic RGC death.