Retinal degeneration mutants in the mouse

The Jackson Laboratory, having the world's largest collection of mouse mutant stocks and genetically diverse inbred strains, is an ideal place to look for genetically determined eye variations and disorders. Through ophthalmoscopy, electroretinography and histology, we have discovered disorders affecting all aspects of the eye including the lid, cornea, iris, lens and retina, resulting in corneal disorders, cataracts, glaucoma and retinal degenerations. Mouse models of retinal degeneration have been investigated for many years in the hope of understanding the causes of photoreceptor cell death. Sixteen naturally occurring mouse mutants that manifest degeneration of photoreceptors in the retina with preservation of all other retinal cell types have been found: retinal degeneration (formerly rd, identical with rodless retina, r, now Pde6b(rd1)); Purkinje cell degeneration (pcd); nervous (nr); retinal degeneration slow (rds, now Prph(Rd2)); retinal degeneration 3 (rd3); motor neuron degeneration (mnd); retinal degeneration 4 (Rd4); retinal degeneration 5 (rd5, now tub); vitiligo (vit, now Mitf(mi-vit)); retinal degeneration 6 (rd6); retinal degeneration 7 (rd7, now Nr2e3(rd7)); neuronal ceroid lipofuscinosis (nclf); retinal degeneration 8 (rd8); retinal degeneration 9 (Rd9); retinal degeneration 10 (rd10, now Pde6b(rd10)); and cone photoreceptor function loss (cpfl1). In this report, we first review the genotypes and phenotypes of these mutants and second, list the mouse strains that carry each mutation. We will also provide detailed information about the cpfl1 mutation. The phenotypic characteristics of cpfl1 mice are similar to those observed in patients with complete achromatopsia (ACHM2, OMIM 216900) and the cpfl1 mutation is the first naturally-arising mutation in mice to cause cone-specific photoreceptor function loss. cpfl1 mice may provide a model for congenital achromatopsia in humans.     

Retinal anatomy and function of the transthyretin null mouse.

Vitamin A (retinol) is vital for the normal development and function of many tissues in the body including the eye. The purpose of this project was to characterize the retinal anatomy and function of the transthyretin (TTR) null mouse. Mice lacking TTR have been constructed by homologous recombination. Immunocytochemistry was performed to localize short and mid-long wavelength cone opsins as well as morphological examination of the entire retina in wild-type and TTR null mice. Visual function was assessed using the electroretinogram (ERG) and resulting waveforms were analysed in terms of receptoral and postreceptoral components. Retinal morphology of the TTR null mouse was normal. In addition, short and mid-long wavelength cone opsins were localized normally in both TTR null and wild-type retinae. Consistent with these findings, TTR null mice show no anomalies of receptoral (P3) nor post-receptoral (b-wave) ERG components compared with wild-type mice. The results suggest that although circulating plasma levels of retinol and retinol binding protein (RBP) are extremely low, this reduction has little effect on the retinal structure or function of the TTR null mouse. These data are consistent with the existence of mechanisms for the transport of retinol to the retina independent of the classical retinol-RBP-TTR complex.     

Retinal degeneration in the nervous mutant mouse. IV. Inner retinal changes

We have recently noted that the inner nuclear layer (INL) and the inner plexiform layer (IPL) were significantly thinner in mice homozygous for the nervous defect (nr / nr) than in control (nr /+ or +/+) littermates. Here, we have carried out a series of anatomical studies to further understand these inner retinal changes. At postnatal day (P) 13, there was no difference in the inner retina between nervous and control mice, while a significant difference was observed at P30. Similar changes were not seen in other mouse models of photoreceptor degeneration. There was a significant reduction in the density of cells in the INL that were stained by antibodies against the inhibitory neurotransmitters GABA and glycine. These results indicate that the nervous defect causes a degeneration of one or more sub-types of amacrine cells, in addition to the loss of cerebellar Purkinje cells and retinal photoreceptors that is known to occur in these mutant animals. Finally, evidence is provided that photoreceptors die by an apoptotic pathway in nervous mice.     

Proteoglycans in the mouse interphotoreceptor matrix. IV. Retinal synthesis of chondroitin sulfate proteoglycan

To determine whether the mouse retina contributes chondroitin sulfate proteoglycan (CS-PG) to the interphotoreceptor matrix (IPM), 35SO4(2-) was used as a tracer for newly synthesized proteoglycan by retinas maintained in vitro in the absence of pigment epithelium. Following incubation with the tracer for 3 hr, the 35S-labeled proteoglycans present in the incubation medium and associated with isolated photoreceptor outer segments were analyzed separately. Proteoglycan was extracted with 4 M guanidine, and then separated on a G-25 column followed by DEAE ion exchange chromatography in the presence of 8 M urea. The proteoglycan fraction was eluted with a linear NaCl gradient of 0.15-1.0 M. Eluted 35S-labeled macromolecules were susceptible to chondroitinase AC and ABC degradation, indicating that virtually all the 35S-labeled proteoglycan synthesized by the mouse retina and secreted into the incubation media is of the chondroitin sulfate type. In parallel autoradiographic analysis of retinas following 35SO4(2-) incubation, silver grains were present over all retinal compartments, with 41-48% associated with the photoreceptor layer. Quantitative autoradiography of retinas following chondroitinase AC digestion of fixed retinas revealed significant (P less than 0.025) reduction in silver grains associated with the photoreceptor outer segment layer as compared to controls. These combined biochemical and autoradiographic studies indicate that the retina, possibly the photoreceptors, synthesize at least a portion of the CS-PG present in the IPM of the mouse.     

Retinal degeneration in the nervous mutant mouse. III. Electrophysiological studies of the visual pathway

The nervous (nr) mutation induces a progressive and severe degeneration of cerebellar Purkinje cells and retinal photoreceptors that is virtually complete within the first few months of life. Previous studies of the retina in nervous (nr/nr) mice have focused primarily on the structural abnormalities seen at the level of the photoreceptor cell bodies and outer segments. Here, we have carried out a series of functional studies of the visual pathway in nervous mice and have quantified the status of the inner retinal cell and plexiform layers. Affected animals were obtained by mating nr/+ heterozygotes and screening the offspring for the ataxia characteristic of nervous animals; phenotypically normal littermates (i.e. nr/+ or +/+) were used as controls. As described previously, there is a substantial loss of photoreceptors cells in the nervous retina and a marked shortening of the inner and outer segments. These changes are accompanied by a more modest decline in the thickness of the inner plexiform and inner nuclear layers. These anatomic abnormalities were accompanied by reproducible changes in visual function, as measured with the electroretinogram (ERG) and visual evoked potential (VEP). The dark-adapted ERGs of nervous and control mice had similar waveforms, although the nervous responses were substantially smaller in amplitude. The reductions in the amplitude of the ERG a-wave corresponded to the loss of photoreceptor cells and shortened outer segments seen histologically. Nevertheless, the kinetics of the leading edge of the a-wave did not differ between nervous and control mice, indicating that the rod outer segments of nervous mice continue to respond to light in a normal fashion. The amplitudes of cone ERGs were also reduced in nervous mice, although the extent of this reduction in any given animal was always less than that for rod-mediated ERG components. Overall, this result is consistent with cone involvement occurring only as a secondary effect of rod photoreceptor degeneration. The peak latencies of VEPs of nervous mice were slower than those of control littermates. These functional abnormalities correspond well to the structural changes induced by the nervous mutation, which does not appear to prevent visual signals from being transmitted centrally, beyond the limitations imposed by the degenerative process.     

Retinal pathway origins of the pattern ERG of the mouse

This study investigated contributions from the retinal On and Off pathways, and the spiking and nonspiking activity of neurons in those pathways to the pattern ERG of the mouse. Light-adapted pattern and ganzfeld ERGs were recorded from anesthetized C57BL/6 mice 3-4 months of age. Recordings were made before and after intravitreal injections of PDA (cis-2,3-piperidine-dicarboxylic acid) to block transmission to hyperpolarizing 2nd order and all 3rd order neurons, TTX (tetrodotoxin) to block Na⁺-dependent spiking, APB (2-amino-4-phosphonobutyric acid) to block synapses between photoreceptors and ON-bipolar cells, and APB + TTX and PDA + TTX cocktails. The pattern stimuli consisted of 0.05 cy/deg gratings reversing in contrast at 1 Hz, presented at various contrasts (50-90%) and a rod saturating mean luminance. For flash ERGs, brief green ganzfeld flashes were presented on a rod-suppressing green background. Recordings were made 39-42 days after unilateral optic nerve crush (ONC) in a subset of animals in which ganglion cell degeneration was subsequently confirmed in retinal sections. Pattern ERGs were similar in waveform for all contrasts, with a positive wave (P1) peak for 90% contrast around 60 ms on average and maximum trough for a negative wave (N2) around 132 ms after each contrast reversal; amplitudes were greatest for 90% contrast which became the standard stimulus. ONC eliminated or nearly eliminated the pattern ERG but did not affect the major waves of the flash ERG. PDA and TTX both delayed P1 and N2 waves of the pattern ERG, and reduced their amplitudes, with effects of PDA on N2 greater than those of TTX. In the flash ERG, PDA reduced a-wave amplitudes, removed OPs but hardly affected b-wave amplitudes. In contrast, TTX reduced b-wave amplitudes substantially, as previously observed in rat. APB removed P1 of the pattern ERG, but left a negative wave of similar timing and amplitude to N2. In the flash ERG, APB removed the b-wave, producing a negative ERG. Addition of TTX to the APB injection removed most of N2 of the pattern ERG, while other waves of the pattern and flash ERG resembled those after APB alone. Addition of TTX to the PDA injection had little effect on the pattern ERG beyond that of PDA alone, but it prolonged the b-wave of the flash ERG. In conclusion, this study confirmed that a selective lesion of ganglion cells will practically eliminate the pattern ERG. The study also showed that P1 of the mouse pattern ERG is dominated by contributions, mainly spiking, from ON pathway neurons, whereas N2 reflects substantial spiking activity from the OFF pathway as well as nonspiking contributions from both pathways.     

Interactions of chlorpromazine with alpha-, beta- and gamma-crystallins.

The binding parameters (binding affinity constant, K and number of binding sites, p) has been determined spectrofluorometrically for chlorpromazine (CPZ) binding to the lens proteins--alphaL-crystallin, betaL-crystallin and gamma-crystallin. The binding affinity constants for CPZ binding to alphaL- and gamma-crystallins are higher than the binding affinity constants for 3betaL-crystallin, although the number of CPZ binding sites for betaL-crystallin is comparatively higher than the number for the other two lens proteins. CPZ causes local conformational changes around the tryptophan moieties of the protein molecules but does not cause any gross conformational change within the protein moieties. Binding of CPZ to alphaL-crystallin does not significantly alter the anti-aggregation properties of the molecular chaperone, alphaL-crystallin against oxidation-induced aggregation of gamma-crystallin at 37 degrees C and thermal aggregation of alcohol dehydrogenase (ADH) at 48 degrees C. Therefore, CPZ induced alteration in chaperone activity of alphaL-crystallin is probably not associated with the formation of cataracts.     

Aging effects on the bovine lens gamma-crystallins

Sephadex gel filtration and SE-Sephadex chromatography of cattle and calf lens extracts revealed major differences in the protein patterns of the γ-crystallin fraction. With aging of the lens apparently a new form of γ-crystallin emerges. The older bovine lens contains two forms of γ-crystallin: one which is identical with calf lens γ is found in the nucleus; the second γ is found in the cortex and is of larger molecular weight than the calf lens γ. Amino acid analyses, N-group determinations and amide-N assays have further characterized differences and similarities between these two forms of γ-crystallins.     

应用眼底病小鼠模型须考虑的因素

随着眼科基础研究的发展,眼科疾病动物模型的应用越来越广泛,正确选择和使用合适的动物模型是合理解释眼科研究结果的保证.在眼底疾病的小鼠模型应用过程中,由于自发性和基因改造性眼底病小鼠模型来源于不同的种系,其中可能带有对所研究表型能产生干扰的基因突变.本文简单介绍最常见的4个基因的突变（变异）,以提出研究者在进行相关研究和选择动物模型时必须考虑的因素.Crb1基因、磷酸乙酰化酶6β（Pde6b）、Gnat2和RPE65基因的突变（变异）在实验小鼠中分布广泛,分别可产生rd8样视网膜变性、rd1样视网膜变性、视锥细胞功能不全和差别性光损伤敏感的表型.研究者在实验设计阶段要认真了解实验动物的遗传背景,排除有可能影响自己所研究的表型的种系.因各种条件所限确实无法避免时,也可通过设立合适的对照加以解决.     

Retinal degeneration in the pcd/pcd mutant mouse: accumulation of spherules in the interphotoreceptor space.

The Purkinje cell degeneration (pcd) mutant mouse rapidly loses cerebellar Purkinje cells and about 50% of its retinal photoreceptor cells at between 3 and 5 weeks of age, and thereafter slowly loses the remaining photoreceptor cells during the first year of life. An ultrastructural study of the developing photoreceptor cells of the pcd/pcd retina was undertaken using both transmission and scanning electron microscopy to characterize further the previously reported retinal vesicles associated with this mutation. Transmission electron microscopy (TEM) revealed an abundance of 'bead-like' vesicles or excrescences in the extracellular matrix surrounding the inner segment region at post-natal day (P) 25. The vesicles are membrane bound, amorphous in appearance and vary in size from 125 to 370 nm. Scanning electron microscopy suggests that the vesicles seen with TEM are actually spherules formed from outpocketing and pinching off of the plasma membrane in the mitochondria-rich region of the rod inner segment. At P25, the spherules are concentrated in the interphotoreceptor space at the level of rod inner segments; at P40, however, they are displaced from their origin and appear mostly at the level of rod outer segments and in the subretinal space.     

Retinal heparanase expression in streptozotocin-induced diabetic rats

Objective: Heparanase, an endoglycosidase, exhibits strong proangiogenic capacity that can induce vascular endothelial growth factor (VEGF) expression in tumour angiogenesis. The purpose of this study was to evaluate heparanase expression and its relationship with VEGF in streptozotocin (STZ)-induced diabetic rats' retinas.Design: Experimental study.Participants: STZ-induced rats and non-diabetic control rats.Methods: Heparanase expression was initially evaluated in cultured human retinal microvascular endothelial cells (HRECs) under high-glucose conditions by Western blot. Diabetes was induced in Sprague-Dawley rats by STZ intraperitoneal injection. Retinal heparanase expression was assayed in rats by immunohistochemistry. Heparanase inhibitor (phosphomannopentaose sulfate) was administrated to high-glucose-treated HRECs and diabetic rats. VEGF levels were evaluated in HRECs and retinas using enzyme-linked immunosorbent assay.Results: Heparanase expression was increased in HRECs under high-glucose conditions compared with controls (p < 0.01). Immunohistochemical studies indicated that heparanase signals were intense in the retinal vascular endothelia of diabetic rats, but faint in those of nondiabetic control rats. Quantitative analysis showed that heparanase protein expression was increased by 3.2-fold in diabetic rats' retinas compared with nondiabetic rats' retinas (p < 0.01). VEGF level was increased, as was heparanase expression, in high-glucose-treated HRECs and in the retinas of diabetic rats, and these increases were significantly decreased by phosphomannopentaose sulfate administration (p < 0.01).Conclusions: Heparanase expression was upregulated and associated with an increase of VEGF expression in STZ-induced diabetic rat retinas. The data suggest that heparanase may be involved in the development of diabetic retinopathy and represents a possible novel target for therapeutic intervention.     

Immunological properties of rat lens gamma-crystallins. III. Changes during development and ageing

The rat lens contains three immunologically different gamma-crystallins, gamma 1-, gamma 2-, and gamma 3-crystallins. With the aid of specific antisera, it could be established that partial identity exists between gamma 1- and gamma 2-crystallins, and non-identity between gamma 1- and gamma 3-, and between gamma 2- and gamma 3-crystallins. The occurrence of these three crystallins has been investigated in rat lenses, aged 5-1,162 days, by isoelectric focusing, immunofocusing and immunodiffusion. The gamma 1- and gamma 2-crystallins are already present in 5-day-old lenses, but diminish during ageing and finally disappear. In contrast, gamma 3-crystallin develops first in 10-day-old lenses, it remains present during progressed ageing.     

Retinal hemorrhages in the newborn.

There were 100 babies examined within 24 hours of birth. Retinal hemorrhages were found in 35% of newborns. Birth trauma from forceps delivery and prolonged labor were thought to be significant. Babies born by cesarean section were less likely to have retinal hemorrhages. Asphyxia was also found to be significant. The role of low plasma prothrombin levels, the vacuum extractor and increased intracranial pressure were discussed as etiologic agents. According to Von Noorden in 1973 there appears to be no positive correlation between hemorrhage of the macula and the occurrence of strabismus.     

Free isoelectric focusing of bovine lens gamma-crystallins

A method is described for separation of γ-crystallin components from the major bulk of crystallin residues of the bovine lens cortex. Carrier-free isoelectric focusing in a pH gradient from 3 to 10 of bovine γ-crystallins in a coil of polyethylene tubing is used to purify a cow lens cortex γ-crystallin fraction obtained from Sepharose 6B chromatography. Purity of the γ-crystallin preparation thus obtained is checked by polyacrylamide thin layer isoelectric focusing, immunoelectrophoresis, and two-dimensional antigen/antibody crossed electrophoresis against a polyvalent and a specific antiserum.Free isofocusing appears to be an excellent method by which to separate γ-crystallin components in purest form from a preparation containing 75% β-crystallins. Isoelectric points of protein components are determined by polyacrylamide thin layer isoelectric focusing on an analytical scale. It is defined that β-crystallins have isoelectric points of 7·00 and lower, and that γ-crystallins have isoelectric points over 7·00. The proteins from the cortex of the adult bovine lens contain about 1% of a crystallin which is identical to the γ-crystallins of the nucleus of the lens, which were originally of embryonic origin.     

视网膜损伤与c-fos基因表达

c-fos基因可能是最具特征性的即早基因(immediate early gene,IEG)。c-fos基因在调控视网膜功能上有重要作用。视网膜损伤,如光、缺血、穿通伤、视网膜脱离等,可诱导视网膜c-fos基因表达的改变。损伤性刺激可能是通过第一信使和第二信使的激活使c-fos基因在核内表达;c-fos基因调控着后期效应基因的表达,在对外界刺激-转录耦联的信息传递过程中起着第三信使的重要作用。本文就视网膜损伤所致c-fos基因表达的变化及其有关机理作一综述。     

Retinal pathology in the Kearns-Sayre syndrome.

Examination of the retinal tissues obtained at necropsy from a 14-year-old boy with Kearns-Sayre syndrome showed marked photoreceptor and pigment epithelial cell loss in the retinal periphery and around the optic nerve head. Electron microscopy of surviving retinal pigment epithelial (RPE) cells indicated a loss of apical microvilli and basal infoldings. The RPE was unusually devoid of melanosomes and showed no evidence of phagocytosis of photoreceptor debris. The cytoplasm of the RPE contained numerous, often enlarged, mitochondria. These structural changes suggested that a breakdown in the energy dependent interrelationships between the RPE and the photoreceptor layer was responsible for the outer retinal degeneration. The finding of numerous macrophages in the subretinal space suggests a secondary inflammatory component in the retinal degeneration.