Retinal degenerations in the dog II. Development of the retina in rod-cone dysplasia

The kinetics of rod outer segment production and turnover were studied in Irish setter dogs with a recessively inherited photoreceptor degeneration (rod-cone dysplasia). Affected and littermate control (heterozygote) dogs, 16–50 days of age, were injected intravitreally with [³H]leucine after electroretinographic studies. Normal age-matched mongrel dogs were also used as controls. Between 6 hr and 8 days following intravitreal injection, eyes were removed and processed for microscopic and radioautographic studies.In affected dogs, the rod components of the electroretinogram were not recordable. Cone components were recordable but failed to develop normally. Morphologically, rod inner segments were diminutive and the outer segments short and contained disorganized and disoriented disc material. Cone inner segments became broad and club-shaped. Radio-autography showed label accumulation within inner segments, but only sporadic and randomly distributed label in the narrow outer segment layer. These results suggest that in Irish setters with rod-cone dysplasia, there is a defect in the morphogenesis of the rod outer segments during postnatal retinal differentiation.     

Retinal histopathology of an XLRP carrier with a mutation in the RPGR exon ORF15

X-linked retinitis pigmentosa comprises the severe forms of RP, with early onset of night blindness, rapid constriction of visual fields and eventual loss of central acuity. Of the five distinct XLRP loci identified on the X chromosome, mutations have been found only in the RP2 and RPGR genes. Of these, mutations in RPGR are more common, particularly in a mutational hot spot that was identified in the newly discovered exon ORF15. We report on an extended family with a microdeletion in RPGR exon ORF15 and the retinal histopathology of a female carrier of this mutation. We found a 1bp deletion at position 632 in exon ORF15 in affected members of family XLRP-319. This mutation alters the reading frame of the predicted RPGR protein, resulting in a premature stop codon. The mutation segregated with disease in three generations of the family and was associated with severe early onset retinal disease in affected men. The retina from a 75 year old carrier female donor had slight photoreceptor loss in the less diseased areas. More severe atrophy with retinal pigment epithelium (RPE) migration was present in areas of the mid- and far periphery. By immunocytochemistry, loss of rhodopsin labelling in rods was found in the areas of focal atrophy and loss of uniform cone spacing was apparent even in well preserved regions. Small multifocal areas of outer retinal degeneration were present in the better preserved regions of the eye. In these foci, rod and cone loss did not coincide. The dissociation of rod and cone degeneration in areas of focal disease is consistent with random X-inactivation early in embryonic development and the occurrence of distinct patterns of radial (rod) and tangential (cone) dispersion during clonal expansion early in photoreceptor differentiation.     

Absence of voltage-dependent calcium channels delays photoreceptor degeneration in rd mice

Retinal degeneration results from the apoptotic cell death of photoreceptors. While mutations in a large number of genes give rise to retinal degeneration, the specific mechanisms are not well understood. One hypothesis involves mediation of apoptosis by high concentrations of intracellular Ca(2+). We used a mouse line that carries the rd mutation but also lacks the major L-type voltage-dependent Ca(2+) channel at the photoreceptor synapse to examine whether this route of Ca(2+) entry plays a role in photoreceptor degeneration. In both experimental and control mice, the photoreceptors degenerate. However, at postnatal days 16, 18, and 21 there is a delay in photoreceptor cell loss in the experimental mice, which lack L-type voltage-dependent Ca(2+) channels, compared to controls. These data indicate that Ca(2+) entry via the L-type voltage-dependent Ca(2+) channel contributes to the mechanisms responsible for photoreceptor cell death in this mouse model of retinitis pigmentosa.     

Intravitreal injection of ciliary neurotrophic factor (CNTF) causes peripheral remodeling and does not prevent photoreceptor loss in canine RPGR mutant retina

Ciliary neurotrophic factor (CNTF) rescues photoreceptors in several animal models of retinal degeneration and is currently being evaluated as a potential treatment for retinitis pigmentosa in humans. This study was conducted to test whether CNTF prevents photoreceptor cell loss in XLPRA2, an early onset canine model of X-linked retinitis pigmentosa caused by a frameshift mutation in RPGR exon ORF15. Four different treatment regimens of CNTF were tested in XLPRA2 dogs. Under anesthesia, the animals received at different ages an intravitreal injection of 12 microg of CNTF in the left eye. The right eye served as a control and was injected with a similar volume of phosphate buffered saline (PBS). Ocular examinations were performed regularly during the treatment periods. At termination, the dogs were euthanatized, eyes collected and the retinas were processed for embedding in optimal cutting temperature (OCT) medium. The outer nuclear layer (ONL) thickness was evaluated on H&E sections and values in both CNTF- and PBS-treated eyes were compared. Morphologic alterations in the peripheral retina were characterized by immunohistochemistry using cell-specific markers. Cell proliferation in the retinas was examined on semi-thin plastic sections, and by BrdU pulse-labeling and Ki67 immunohistochemistry on cryosections. All CNTF-treated eyes showed early clinical signs of corneal epitheliopathy, subcapsular cataracts and uveitis. No statistically significant difference in ONL thickness was seen between the CNTF- and PBS-injected eyes. Prominent retinal remodeling that consisted in an abnormal increase in the number of rods, and in misplacement of some rods, cones, bipolar and Müller cells, was observed in the peripheral retina of CNTF-treated eyes. This was only seen when CNTF was in injected before the age at which the canine retina reaches full maturation. In XLPRA2 dogs, intravitreal injections of CNTF failed to prevent photoreceptors from undergoing cell death in the central and mid-peripheral retina. CNTF also caused ocular side-effects and morphologic alterations in the periphery that were consistent with cell dedifferentiation and proliferation. Our findings suggest that some inherited forms of retinal degeneration may not respond to CNTF's neuroprotective effects.     

Mutation c. 1142 del G in the <Emphasis Type="Italic">PRPF31</Emphasis> Gene in a Family with Autosomal Dominant Retinitis Pigmentosa (RP11) and Its Implications

Purpose

To identify a mutation in the PRPF31 gene in a family (Family K) with autosomal dominant retinitis pigmentosa (adRP) linked to 19q13.4 (RP11) and to find the frequency of mutations in the PRPF31 gene among Japanese families with adRP.

Methods

Genomic DNA specimens were prepared from five symptomatic and two asymptomatic members of Family K and an additional 39 patients of 39 unrelated families with adRP. Coding regions of the PRPF31 gene were amplified by polymerase chain reaction. The amplicons were analyzed by a direct sequencing method.

Results

All seven family members had a heterozygous c.1142delG mutation in the PRPF31 gene, which was identical to the mutation previously reported in a different Japanese family. No other mutation was found in the PRPF31 gene among the 39 additional patients with adRP.

Conclusion

Although the frequency of mutations in the PRPF31 gene is about 2.5% in Japanese families with adRP, it is possible that c.1142delG is a common mutation among Japanese patients with adRP associated with mutations in the PRPF31 gene.?Jpn J Ophthalmol 2007;51:45–48 © Japanese Ophthalmological Society 2007

     

Systemic aminoglycoside treatment in rodent models of retinitis pigmentosa

We studied the potential of systemically administered aminoglycosides as a therapy for retinal degeneration resulting from premature termination codon (PTC) mutations. Aminoglycosides were systemically delivered to two rodent models of retinal degeneration: a transgenic rat model of dominant disease caused by a PTC in rhodopsin (S334ter); and a mouse model of recessive disease (rd12) caused by a PTC in the retinoid isomerase Rpe65. Initial luciferase reporter assays were undertaken to measure the efficiency of gentamicin-induced read-through in vitro. These experiments indicated that gentamicin treatment induced on average a 5.3% extra read-through of the S334ter PTC in vitro, but did not affect the rd12 PTC. Beginning at postnatal day 5, animals received daily subcutaneous injections of gentamicin or geneticin at a range of doses. The effect of the treatment on retinal degeneration was examined by histopathology and electroretinography (ERG). Systemic treatment with aminoglycoside significantly increased the number of surviving photoreceptors in the S334ter rat model over several weeks of treatment, but was not effective in slowing the retinal degeneration in the rd12 mouse model. Similarly, ERG recordings indicated better preservation of retinal function in the treated S334ter rats, but no difference was observed in the rd12 mice. Daily subcutaneous injection of 12.5mug/g gentamicin was the only regimen that inhibited retinal degeneration without apparent adverse systemic side effects. Reduced effectiveness beyond postnatal day 50 correlated with reduced ocular penetration of drug as seen in gentamicin-Texas red (GTTR) conjugation experiments. We conclude that, in the rat model, an approximately 5% reduction of abnormal truncated protein is sufficient to enhance photoreceptor survival. Such a change in truncated protein is consistent with beneficial effects seen when aminoglycosides has been used in other, non-ocular animal models. In the rd12 mouse, lack of efficacy was seen despite this particular PTC being theoretically more sensitive to aminoglycoside modification. We conclude that aminoglycoside read-through of PTCs in vitro and in vivo cannot be predicted just from genomic context. Because there is considerable genetic heterogeneity amongst retinal degenerations, pharmacologic therapies that are not gene-specific have significant appeal. Our findings suggest that if adverse issues such as systemic toxicity and limited ocular penetration can be overcome, small molecule therapeutics, such as aminoglycosides, which target classes of mutation could hold considerable potential as therapies for retinal disease.     

Differential distribution of Mel(1a) and Mel(1c) melatonin receptors in Xenopus laevis retina

The hormone melatonin is an output signal of an endogenous circadian clock in retinal photoreceptors. Melatonin may act as a paracrine and/or intracrine neurohormone by binding to specific receptors in the eye. The distribution of Mel(1a) and Mel(1c) melatonin receptors in the Xenopus laevis retina was examined by immunocytochemistry, using antibodies prepared against specific sequences of the Xenopus receptor proteins. Antibodies that label dopaminergic and GABA-ergic amacrine cells were used in double-label experiments with the melatonin receptor antibodies. The distribution of Mel(1a) and Mel(1c) receptor immunoreactivity was similar insofar as the two receptors were localized in the inner plexiform layer. However, the Mel(1c) receptor displayed some immunoreactivity in the photoreceptor cells, whereas the Mel(1a) receptor displayed little if any photoreceptor labelling. The Mel(1c) antibody, but not the Mel(1a), labelled a population of ganglion cells. While both receptors were localized to the outer plexiform layer, they did not appear to localize to the identical cell types. These results demonstrate that the Mel(1a) and Mel(1c) receptor proteins are present in cells of the X. laevis retina, and their distribution in the photoreceptors and inner retina is very similar to that reported in the human retina. The differential pattern of expression of the melatonin receptors suggests that melatonin may convey differential effects on various target cells in the retina.     

Drusen are Cold Spots for Proteolysis: Expression of Matrix Metalloproteinases and Their Tissue Inhibitor Proteins in Age-related Macular Degeneration

Drusen are abnormal extracellular matrix deposits characteristic of age-related macular degeneration (AMD), a leading cause of blindness in the aging human population. The mechanisms underlying drusen formation are not well characterized. The purpose of this study was to examine the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in drusen, and in the surrounding cells and tissue. To assess the extent of MMP and TIMP expression by retinal pigment epithelial (RPE) cells, cDNA arrays were screened with probes generated from cultured human RPE cells. The distribution of MMP-1, -2 and -3 and TIMP-1, -2, -3 and -4 was determined using immunohistochemistry in human RPE choroid from donor eyes with and without a clinical history of AMD. Gelatinase activity was assessed in unfixed frozen sections using in situ zymography. In cultured RPE cells, expression of 10 MMP and all four known TIMP mRNAs was detected. MMP immunoreactivity was widespread in the RPE choroid, but was absent from the interior of drusen. TIMP-3, but not other TIMPs, was detected in the drusen interior. Likewise, metal ion dependent gelatinase activity could be detected in RPE choroid, but not in drusen. These results show that, while metalloproteinase activity is widespread throughout the RPE choroid, drusen are cold spots for proteolysis. The data lead to the speculation that high TIMP-3 concentrations within drusen could inhibit MMPs and as a result slow the proteolytic degradation of these deposits.     

Optimization of laser-induced choroidal neovascularization in African green monkeys

We developed and validated a new nonhuman primate model of laser-induced choroidal neovascularization (CNV) that addresses study design limitations prevalent in laser-induced CNV-based efficacy studies. Laser-induced Bruch’s membrane disruption triggers CNV and has been widely utilized in animals to model neovascular (“wet”) age-related macular degeneration (AMD). Despite widespread use of the approach, detailed assessment of experimental parameters and their influence on pathophysiological endpoints critical for disease modeling has been extremely limited and largely based on anecdotal observations. We evaluated laser power parameters and endpoint measures to optimize methods for CNV formation and quantification to facilitate drug efficacy screening in African green monkeys. Six laser spots of 350, 550, 750, 950 or 1500 mW laser power were positioned bilaterally 1.5 disc diameters from the fovea, within the macula. Fluorescein angiograms were collected 3–5 weeks later and scored by trained masked investigators using graded (I–IV) and densitometric methods. Histopathology assessments were also performed, including determination of CNV area. Test system sensitivity to angiogenesis inhibition was subsequently assessed by evaluating the effect of intravitreal bevacizumab (Avastin) pretreatment (one day prior to laser photocoagulation) on incidence of CNV. Grade III and grade IV lesions were considered clinically relevant, demonstrating early hyperfluorescence and late leakage within or beyond the lesion borders. By 4 weeks post-laser all treatment groups demonstrated evidence of grade III lesions with greatest incidence observed in lesions induced by 750 and 950 mW laser power (72.9% and 69.4% respectively). Grade IV lesions were confined to eyes receiving 550 mW laser power or higher, with highest incidence of grade IV lesions observed in eyes receiving 950 (19.4%) and 1500 mW (31%) laser spots, incidence peaking 4 weeks post-laser photocoagulation. Densitometric analyses of angiograms corroborated visual scoring. Bevacizumab completely abolished grade IV lesion development and significantly lowered lesion fluorescein signal intensity (P < 0.0001) and CNV area (P = 0.038) compared to vehicle-treated controls. Our studies demonstrate that laser power of 950–1500 mW and angiography analysis 4 weeks post-laser are optimal parameters to evaluate treatment effects on CNV induction following laser photocoagulation. Bevacizumab significantly attenuated CNV development, as determined by fluorescein angiography and histopathology assessments in this model, supporting the application of African green monkeys in preclinical modeling of CNV. Laser parameters and time points for therapeutic dosing and angiography endpoints are critical factors to the laser-induced CNV model and must be validated for robust assessment of efficacy. The newly optimized nonhuman primate model described will facilitate preclinical efficacy assessments of novel therapeutics for CNV.     

Comprehensive screening of the USH2A gene in Usher syndrome type II and non-syndromic recessive retinitis pigmentosa

A screen of the entire coding region of the USH2A gene in 129 unrelated patients with Usher syndrome type II (USH2) and in 146 unrelated patients with non-syndromic autosomal recessive retinitis pigmentosa (ARRP) uncovered 54 different sequence variations, including 18 likely pathogenic mutations (13 frameshift, three nonsense, and two missense), 12 changes of uncertain pathogenicity (11 missense changes and one in-frame deletion), and 24 non-pathogenic rare variants or polymorphisms. Of the 18 likely pathogenic mutations, nine were novel. Among the USH2 patients, 50 (39%) had one or two likely pathogenic mutations. The most common mutant allele in USH2 patients was E767fs, which was found in 29 patients, including one homozygote. Among the ARRP patients, we found 17 (12%) with one or two likely pathogenic mutations. The most common mutant allele in ARRP patients was C759F and it was found in 10 patients. The C759F allele was also found in two USH2 patients; in neither of them was a change in the other allele found. The second most common mutant allele in both patient groups was L1447fs (found in 6/50 USH2 patients and 6/17 ARRP patients). Of the 50+17=67 patients with identified USH2A mutations, only one mutation in one allele was found in 41+12=53 (79%); the reason for the high proportion of patients with only one identified mutation is obscure. Our results indicate that USH2A mutations are found in about 7% of all cases of RP in North America, a frequency similar to the RPGR gene (8%) and the rhodopsin gene (10%).     

T和B细胞缺陷对真菌性角膜炎发病过程的影响

目的：探讨特异性免疫应答是否参与真菌性角膜炎(fungal keratitis, FK)发病早期的病理过程。

方法：通过角膜基质内注射1×10⁵白色念珠菌孢子或烟曲霉菌孢子的方法在相同免疫遗传背景的Balb/c小鼠和重症联合免疫缺陷小鼠(severe combined T/B-null immuno- deficiency, SCID)诱导FK。用裂隙灯监测疾病临床变化特征并依据角膜病灶面积和病变深度以及角膜表面规则性进行临床评分。在病变显著的时间点,摘除小鼠眼球用HE和PAS染色观察小鼠角膜组织的病理变化特征。用菌落形成实验检测角膜组织中活菌数量的变化,用ELISA检测小鼠血浆和角膜组织匀浆液中细胞因子IL17、IFNγ和IL10的动态变化。

结果：Balb/c小鼠和SCID小鼠都可诱导出典型的FK。在白念菌性角膜炎或烟曲霉菌性角膜炎中,无论是大体临床表现和疾病分数,抑或组织病理变化和真菌负荷分析,在两种小鼠之间均无显著性差异; 两种小鼠间在血浆或角膜组织中IL17及IFNγ的水平等方面也没有显着差异。但诱导两种FK的SCID小鼠中,无论角膜匀浆液抑或外周血浆,在任何时间点均检测不到IL10的表达,而同样发生角膜炎的Balb/c小鼠则在FK发病早期检测到IL10的表达。

结论：在免疫遗传背景相同的条件下,小鼠中获得性免疫组分的存在与否并不影响FK的发病过程,提示小鼠中初次FK的发病与固有性免疫组分相关。     

Targeted inactivation of synaptic HRG4 (UNC119) causes dysfunction in the distal photoreceptor and slow retinal degeneration, revealing a new function

HRG4 (UNC119) is a photoreceptor protein predominantly localized to the photoreceptor synapses and to the inner segments to a lesser degree. A heterozygous truncation mutation in HRG4 was found in a patient with late onset cone-rod dystrophy, and a transgenic (TG) mouse expressing the identical mutant protein developed late onset retinal degeneration, confirming the pathogenic potential of HRG4. Recently, the dominant negative pathogenic mechanism in the TG model was shown to involve increased affinity of the truncated mutant HRG4 for its target, ARL2, which leads to a delayed decrease in its downstream target, mitochondrial ANT1, mitochondrial stress, synaptic degeneration, trans-synaptic degeneration, and whole photoreceptor degeneration by apoptosis. In this study, the mouse HRG4 (MRG4) gene was cloned and targeted to construct a knock-out (KO) mouse model of HRG4 in order to study the effects of completely inactivating this protein. The KO model was examined by genomic Southern blotting, Western blotting, immunofluorescence, funduscopy, LM and EM histopathology, ERG, and TUNEL analyses. The KO model developed a slowly progressive retinal degeneration, characterized by mottling in the fundus, mild thinning of the photoreceptor layer, and increase in apoptosis as early as 6 months, dramatic acceleration at approximately 17 months, and virtual obliteration of the photoreceptors by 20 months. When compared to retinal degeneration in the TG model, significant differences existed in the KO consisting of more severe and early photoreceptor death without evidence of early synaptic and trans-synaptic degeneration as seen in the TG, confirmed by LM and EM histopathology, ERG, and Western blotting of synaptic proteins. The results indicated a dysfunction in the KO outside the synapses in the distal end of photoreceptors where MRG4 is also localized. Differences in the phenotypes of retinal degeneration in the KO and TG models reflect a dysfunction in the two opposite ends of photoreceptors, i.e., the distal inner/outer segments and proximal synapses, respectively, indicating a second function of MRG4 in the distal photoreceptor and dual functionality of MRG4. Thus, inactivation of MRG4 by gene targeting resulted in a retinal degeneration phenotype quite different from that previously seen in the TG, attesting to the multiplicity of MRG4 function, in addition to the importance of this protein for normal retinal function. These models will be useful in elucidating the functions of HRG4/MRG4 and the mechanism of slow retinal degeneration.     

Association of LOC387715/ARMS2 (rs10490924) Gene Polymorphism with Age-Related Macular Degeneration in the Brazilian Population

Background: An association between LOC387715/ARMS2 (rs10490924) gene polymorphism and AMD has been reported. The aim of this study was to evaluate whether this polymorphism is associated with AMD in a Brazilian cohort.

Materials and Methods: In total, 126 unrelated AMD patients (mean age 74.17?±?7.64) were compared with 86 healthy controls (mean age 71.82?±?7.12). Study subjects were classified according to the International ARM Epidemiological Study Group definition for early and late-stage AMD. LOC387715/ARMS2 rs10490924 polymorphism was evaluated through polymerase chain reaction and direct sequencing.

Results: The T allele frequency was significantly higher in AMD patients than in controls (39.6% compared to 20.3%). The odds ratio (OR) for AMD was 2.05 (95% CI 1.13–3.71) for heterozygotes (TG) and 8.32 (95% CI 2.30–45.99) for homozygotes (TT).

Conclusions: These results suggest that there is a contribution of the rs10490924 SNP of the LOC387715/ARMS2 gene to AMD susceptibility in this sample of the Brazilian population.     

Nicotinamide prevents N-methyl-N-nitrosourea-induced photoreceptor cell apoptosis in Sprague-Dawley rats and C57BL mice

In previous studies, it was found that a single systemic administration of N-methyl-N-nitrosourea (MNU) to rats and mice resulted in the retinal degeneration in all treated animals over a 7 day period. Retinal degeneration was due to photoreceptor cell apoptosis that was identical to the apoptosis seen in human retinitis pigmentosa (RP). In the present study, nicotinamide (NAM), a water-soluble B-group vitamin (vitamin B(3)), suppressed photoreceptor cell loss in a dose-dependent manner when administered immediately after MNU treatment. In rats, a dose of NAM >or=25 mg kg(-1) completely suppressed photoreceptor cell loss, and 10 mg kg(-1) partially suppressed photoreceptor cell loss. In mice, doses of 1000 and >or=100 mg kg(-1) were needed for complete and partial suppression, respectively. Thus, rats were more responsive to NAM than mice. The retinoprotective effect of 1000 mg kg(-1) NAM lasted throughout the long-term (35 days) observation period, with no apparent toxicity. Also, in rats, 1000 mg kg(-1) NAM completely suppressed photoreceptor cell loss when administered up to 4 hr after MNU treatment, and partially suppressed photoreceptor cell loss when administered 6 hr after MNU treatment. In mice, administration of NAM 2-6 hr after MNU resulted in partial suppression. NAM did not reduce levels of 7-methyldeoxyguanosine DNA adduct, but did reduce photoreceptor cell apoptosis. Although the mechanism of action underlying this retinoprotection remains to be clarified, NAM may be a potential therapeutic agent for the treatment of retinal degeneration.     

Expression patterns of cytochrome P4501B1 (Cyp1b1) in FVB/N mouse eyes

Homozygous and compound heterozygous mutations in cytochrome P4501B1 (CYP1B1) cause primary congenital glaucoma (PCG) in humans. It is hypothesized that developmental anomalies of the trabecular meshwork prevent appropriate drainage of the aqueous humor and cause PCG in human patients. In this report, we studied the expression patterns of Cyp1b1 in the eye of albino FVB/N mouse at different developmental stages. We isolated a cDNA fragment corresponding to the 3'untranslated region (3'UTR) of the Cyp1b1 gene by PCR and used it to make an (35)S-labelled riboprobe for in situ hybridization. We found that Cyp1b1 is expressed in both anterior and posterior segments of the eye. Anteriorly, the expression is confined to the ciliary body, most likely in the outer/pigmented ciliary epithelial cells. Cyp1b1 mRNA can be detected in these cells at postnatal day 4 (P4) and the expression continues into adulthood. Surprisingly, no above-background levels of Cyp1b1 mRNA were found at or around the trabecular meshwork at all the stages we examined. In the posterior region of the embryonic day 15 (E15) eye, Cyp1b1 is expressed in the retinal neuroepithelium and in the tissues surrounding the optic nerve, but not in the optic nerve itself. In the P7 retina, Cyp1b1 mRNA is found in the inner nuclear layer. Based on our finding that Cyp1b1 is expressed in the developing and mature ciliary body of the mouse eye, we speculate that mutation in this gene can directly contribute to the abnormal elevation of the intraocular pressure (IOP) in the PCG patients or indirectly affect the aqueous outflow by disrupting the proper development of the trabecular meshwork in these patients.