Electrophysiological deficits in the retina of the DBA/2J mouse

The DBA/2J (D2J) is a genetic mouse model for glaucomatous neurodegeneration because the animals develop anatomical and functional retinal deficits that partially can be correlated with elevated intraocular pressure (IOP). The IOP starts to increase at an age of about 6 months as a result of morphological changes within the anterior eye segment, e.g., pigment dispersion and iris synechiae. The purpose of the present study was to investigate how ERG responses change in individuals at different ages in D2J mice and to compare these changes with normal aging effects in pigmented C57/B6 (B6) mice. IOP was measured in awake, non-sedated D2J and B6 mice with a rebound tonometer. At ages between 2–3 and 10 months, scotopic flash ERGs were measured five times with about 2 months’ intervals. In addition, light adapted flicker ERGs were recorded. Our data show that the D2J shows lower flicker ERG responses than the B6 mice already at an age of 2–3 months. Dark adapted flash ERG responses are not decreased at this age. In both mouse strains the ERG responses decrease as a function of age, but there is a stronger decrease in the D2J mice. The data of flicker ERGs suggest the presence of early functional deficits in the D2J retina that possibly have a post-receptoral origin. The scotopic flash ERG reveals a functional deficit that occurs at a later stage and that possibly is IOP dependent. But, the deficits appear at an age at which the IOP is still lower than in the B6 mouse, indicating that other factors play an additional role.     

Cochlin deposits in the trabecular meshwork of the glaucomatous DBA/2J mouse

Cochlin deposits were observed in the trabecular meshwork (TM) of 8-month-old glaucomatous DBA/2J mice, coincident with the reported onset of increased intraocular pressure and optic nerve damage. An age-dependent increase in cochlin was observed up to 10 months of age and was paralleled by a decrease in type II collagen. Similar expression patterns exist in the TM of humans with primary open-angle glaucoma. Cochlin deposits, absent in non-glaucomatous mouse and human TM, may disrupt the TM extracellular matrix and obstruct aqueous humor circulation. Studies of DBA/2J mice offer promise for understanding the role cochlin may play in glaucoma.     

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

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

Reduced retina microglial activation and improved optic nerve integrity with minocycline treatment in the DBA/2J mouse model of glaucoma

PURPOSE: In the context of the retinal ganglion cell (RGC) axon degeneration in the optic nerve that occurs in glaucoma, microglia become activated, then phagocytic, and redistribute in the optic nerve head. The authors investigated the potential contribution of retinal microglia activation to glaucoma progression in the DBA/2J chronic mouse glaucoma model. METHODS: The authors treated 6-week-old DBA/2J mice for 25 weeks with minocycline, a tetracycline derivative known to reduce microglia activation and to improve neuronal survival in other models of neurodegenerative disease. They quantified RGC numbers and characterized microglia activation, gliosis, and both axonal integrity and retrograde tracer transport by RGCs in mice systemically treated with minocycline or vehicle only. RESULTS: Minocycline reduced microglial activation and improved RGC axonal transport and integrity, yet it had no effect on the characteristic age-related ocular changes that lead to chronically elevated pressure and did not alter Müller or astrocyte gliosis. Specifically, minocycline increased the fraction of microglia with resting ramified morphology and reduced levels of Iba1 mRNA and protein, a microglia-specific calcium ligand linked to activation. The reduction in microglial activation was coupled to significant improvement in RGC axonal transport, as measured by neuronal retrograde tracing from the superior colliculus. Finally, minocycline treatment significantly decoupled RGC axon loss from increased intraocular pressure. CONCLUSIONS: These observations suggest that in glaucoma, retina and optic nerve head microglia activation may be a factor in the early decline in function of the optic nerve and its subsequent degeneration.     

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.     

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.     

Protective effects of a composition of Chinese herbs-Gurigumu-13 on retinal ganglion cell apoptosis in DBA/2J glaucoma mouse model

AIM: To explore the concrete mechanism of a Mongolian compound medicine-Gurigumu-13 (GRGM) for glaucoma treatment. METHODS: DBA/2J mice, as glaucoma models, were intragastric administrated with GRGM to study the effect of GRGM on retinal ganglion cells (RGCs). The loss of RGCs was evaluated with the number of RGCs and axons. The expression of the target protein of RGCs or mouse retinas was determined by Western blot. The relative content of malondialdehyde (MDA) was examined by ELISA assay. RESULTS: GRGM distinctly improved retina damage via increasing the number of neurons, RGCs and axons in a concentration dependent manner. Meanwhile, GRGM obviously decreased the high level of MDA and the expression of oxidative stress-related proteins in retinas of DBA/2J mice, but promoted the expression of antioxidant proteins. Additionally, GRGM also significantly inhibited the protein expression of Bip and Chop, which were markers of endoplasmic reticulum stress-induced apoptosis. CONCLUSION: GRGM have obvious protective effects on RGCs in DBA/2J mice, and increase the number of RGCs and axons via inhibiting oxidative stress and endoplasmic reticulum stress.     

A neurobehavioral analysis of the prevention of visual impairment in the DBA/2J mouse model of glaucoma

Purpose. Timoptic-XE treatment was used to examine the relationship between age-related changes in intraocular pressure (IOP), retinal cell loss, visual ability, and neuronal labeling in the superior colliculus in the DBA/2J mouse model of pigmentary glaucoma. Methods. Mice were administered Timoptic-XE (0.0%, 0.25%, or 0.50%) daily from 9 weeks to 12 months of age. Visual ability and IOP were evaluated at 3, 6, 9, and 12 months of age. Mice from each group were then given intraocular injections of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP), and estimates of the number of cells in the ganglion cell layer of the retina, WGA-HRP transneural labeling of cells, cell count, and cross-sectional area of Nissl-stained cells in the superior colliculus were obtained. Results. Mice treated with 0.50% and 0.25% Timoptic-XE maintained a high level of performance in behavioral vision tasks, while 12-month-old untreated mice (0.0% Timoptic-XE) exhibited impaired visual performance. Timoptic-XE therapy reduced IOP and cell loss in the ganglion cell layer of the retina and prevented somal shrinkage and the decrease in WGA-HRP transneural labeling in the superior colliculus that occurred in untreated mice at 12 months of age. Conclusions. This study provides a comprehensive assessment of the efficacy of Timoptic-XE in DBA/2J mice by correlating age-related visual system changes in the retina and brain with changes in IOP and visual ability. These results showed that reducing IOP not only rescued retinal ganglion cell atrophy but also restored visual function and altered patterns of neurodegeneration that occur with blindness.     

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.     

Alterations of amino acids and glutamate transport in the DBA/2J mouse retina; possible clues to degeneration

Background The DBA/2J mouse spontaneously develops ocular hypertension and time-dependent progressive retinal ganglion cell (RGC) loss. This study examines changes in amino acid levels in the vitreous, and changes in the expression of retinal glutamate transporters and receptors that occur during the progression of this pathology. Methods Retinas were obtained from DBA/2J mice at ages 3, 6 and 11 months. C57BL/6 mice were used as age-matched controls. Vitreal amino acid content was measured with HPLC. Western blotting and immunohistochemistry were performed using specific antibodies against the glutamate transporters (GLAST, GLT-1v, EAAC-1) and glutamate receptors, particularly NMDA (NR1, NR2A, NR2B) and AMPA (GluR1, GluR2/3, GluR4) receptors. Results HPLC showed retinal concentrations of glutamate, glutamine, glycine, alanine, lysine, serine, and arginine to be significantly higher in DBA/2J mice at 11 months of age compared to age-matched controls. Western Blots revealed a moderate decrease of GLAST and GLT-1v expression in DBA/2J mice at 6 and 11 months as compared to age-matched controls while there was no change in EAAC1. Immunohistochemically, no changes in expression of NMDA and AMPA receptors were seen. Conclusion Alterations of amino acid content and enhanced glutamate neurotransmission might be involved in the pathogenesis of retinal neurodegeneration in the DBA/2J mouse model of ocular hypertension. Moreover, these mice provide an animal model for studying excitotoxic retinal damage.     

IOP-dependent retinal ganglion cell dysfunction in glaucomatous DBA/2J mice

PURPOSE: To characterize the effect of postural IOP elevation and pharmacological IOP lowering on retinal ganglion cell (RGC) function in the DBA/2J mouse model of glaucoma. METHODS: Four groups of DBA/2J mice (3 months old, n = 7; 5 months old, n = 7; 10 months old, n = 7; and 11 months old, n = 8) were anesthetized by intraperitoneal injection (0.6 mL/kg) of a mixture of ketamine (42.8 mg/mL), xylazine (8.5 mg/mL), and acepromazine (1.4 mg/mL). IOP and pattern electroretinogram (PERG) were sequentially measured with mice at 0 degrees (horizontal), 60 degrees head-down, and again at 0 degrees . IOP and PERG were also measured before and after intraperitoneal mannitol 25% (2.5 g/kg) administration with mice in a horizontal position. RESULTS: The head-down position induced reversible IOP elevations of 32% to 38% in all age groups (P < 0.01), and age-dependent reductions of PERG amplitude (3 months: +3%; 5 months: -47%, P < 0.01; and 10 months: -65%, P < 0.01). Administration of mannitol to 11-month-old mice resulted in a reduction in IOP of approximately 38% (P < 0.01) and a PERG amplitude improvement of approximately 83% (P < 0.001). IOP and PERG amplitude changes were inversely correlated (10 months head-down r(2) = 0.58, P < 0.001; 10-month-old mannitol r(2) = 0.41, P < 0.001). For all conditions, the light-adapted flash ERG was unaltered. CONCLUSIONS: In the DBA/2J mouse, RGC susceptibility to artificial IOP elevation increases with age. Abnormal RGC function in older mice may be improved with IOP lowering. Evaluation of PERG changes in response to artificial IOP modulation may represent a powerful tool to assess noninvasively RGCs' susceptibility to IOP insult in genetically distinct mouse models of glaucoma.     

TonoLab眼压计在遗传性小鼠高眼压模型中眼压测量的可重复性

背景 准确的眼压测量是青光眼实验研究的基础.TonoLab是一种新型的动物眼压计,其测量眼压的可重复性尚有待验证. 目的 评估新型眼压计TonoLab在遗传性高眼压模型中的准确性. 方法 采用新型眼压计TonoLab对麻醉状态下的63只7月龄遗传性高眼压DBA/2J小鼠进行眼压测量,测量时由两位技术娴熟的操作者分别、连续测量同一只眼的眼压3次,测量不分先后顺序,采用操作者盲法获取测量数据.计算测量结果的组内标准差（Sw）、组内相关系数（ICC）及组内变异系数（CVw）,以评估每位操作者测量结果的可重复性,采用Bland-Altman一致性检验评估两位操作者之间测量结果的可重复性. 结果 共测量63只小鼠126只眼,其中正常眼压者105只眼,操作者1测得的平均眼压为（12.98±1.05） mmHg（1 mmHg=0.133 kPa）,操作者2为（12.41±0.92） mmHg;高眼压者21只眼,操作者1和操作者2测得的平均眼压分别为（27.76±1.95） mmHg和（27.49±1.28） mmHg.正常眼压组两位操作者测量结果的Sw、ICC及CVw分别为1.34/1.11 mmHg、0.90/0.92和8.24％/7.42％;高眼压组两位操作者测量结果的Sw、ICC及CVw分别为2.21/1.49 mmHg、0.89/0.95和6.97％/4.82％.正常眼压组两位操作者测量结果之间的平均偏差为0.60 mmHg[95％可信区间（CI）：-5.20 ～6.30 mmHg],高眼压组为0.30 mmHg（95％ CI：-7.30～ 7.80 mmHg）.结论 TonoLab眼压计在正常眼压及高眼压小鼠中单个操作者测量结果的组内可重复性高,但两位操作者测量结果之间的可重复性较低,因此单个操作者用TonoLab眼压计测得的眼压结果可靠、准确.     

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.     

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.