Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in retinopathies using non-viral and viral gene delivery to the retina and retinal pigment epithelium (RPE). Subretinal injection is the best approach to deliver viral vectors directly to RPE cells. Before the clinical trial of a gene therapy, it is inevitable to validate the efficacy of the therapy in animal models of various retinopathies. Thus, subretinal injection in mice becomes a fundamental technique for an ocular gene therapy. In this protocol, we provide the easy and replicable technique for subretinal injection of viral vectors to experimental mice. This technique is modified from the intravitreal injection, which is widely used technique in ophthalmology clinics. The representative results of RPE/choroid/scleral complex flat-mount will help to understand the efficacy of this technique and adjust the volume and titer of viral vectors for the extent of gene transduction.     

Performing Subretinal Injections in Rodents to Deliver Retinal Pigment Epithelium Cells in Suspension

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment epithelium (RPE) cells. RPE provide critical support for photoreceptors and death or dysfunction of RPE cells is characteristic of age-related macular degeneration (AMD), the leading cause of permanent vision loss in people age 55 and older. While no cure for AMD has been identified, implantation of healthy RPE in diseased eyes may prove to be an effective treatment, and large numbers of RPE cells can be readily generated from pluripotent stem cells. Several interesting questions regarding the safety and efficacy of RPE cell delivery can still be examined in animal models, and well-accepted protocols used to inject RPE have been developed. The technique described here has been used by multiple groups in various studies and involves first creating a hole in the eye with a sharp needle. Then a syringe with a blunt needle loaded with cells is inserted through the hole and passed through the vitreous until it gently touches the RPE. Using this injection method, which is relatively simple and requires minimal equipment, we achieve consistent and efficient integration of stem cell-derived RPE cells in between the host RPE that prevents significant amount of photoreceptor degeneration in animal models. While not part of the actual protocol, we also describe how to determine the extent of the trauma induced by the injection, and how to verify that the cells were injected into the subretinal space using in vivo imaging modalities. Finally, the use of this protocol is not limited to RPE cells; it may be used to inject any compound or cell into the subretinal space.     

眼细胞在实验性近视的研究进展

近视形成及发展过程中的机制研究一直是眼科领域的热点问题。在近视的实验研究中,越来越多的眼细胞被发现与近视有关,主要涉及的细胞有视网膜光感受器细胞、穆勒细胞、视网膜色素上皮细胞、脉络膜黑色素细胞、巩膜成纤维细胞等。它们通过分泌生长因子、内分泌激素、神经递质等活性物质和表达相关受体,在近视的发生发展中起着重要作用。本文将从眼细胞的角度对近视发病机理作一个综述,为进一步完善近视发病机制的研究提供理论依据。     

Decreased VEGF-A and sustained PEDF expression in a human retinal pigment epithelium cell line cultured under hypothermia

Background

Previous reports have described a decrease in retinal temperature and clinical improvement of wet age-related macular degeneration (AMD) after vitrectomy. We hypothesized that the retinal temperature decrease after vitrectomy plays a part in the suppression of wet AMD development. To test this hypothesis, we evaluated the temperature dependence of the expression of vascular endothelial growth factor-A (VEGF-A) and in vitro angiogenesis in retinal pigment epithelium (RPE).

Results

We cultured ARPE-19 cells at 37, 35, 33 and 31°C and measured the expression of VEGF-A, VEGF-A splicing variants, and pigment epithelium–derived factor (PEDF). We performed an in vitro tube formation assay. The dehydrogenase activity was also evaluated at each temperature. Expression of VEGF-A significantly decreased with decreased temperature while PEDF expression did not. VEGF165 expression and in vitro angiogenesis also were temperature dependent. The dehydrogenase activity significantly decreased as the culture temperature decreased.

Conclusions

RPE cultured under hypothermia that decreased cellular metabolism also had decreased VEGF-A and sustained PEDF expression, creating an anti-angiogenic environment. This mechanism may be associated with a beneficial effect after vitrectomy in patients with wet AMD.     

Lack of evidence for cell death among avian spinal cord interneurons during normal development and following removal of targets and afferents

Chick embryos and posthatched chicks were examined at several ages for the presence of pyknotic interneurons in the lumbar spinal cord. Because no pyknotic interneurons were found, direct cell counts of healthy interneurons were carried out and a comparison made between early-and late-stage embryos and hatchlings. There was no decrease in the number of interneurons in the ventral intermediate gray matter of the spinal cord between embryonic day (E) 8 and 2 weeks posthatching (PH) or in the dorsal horn between E10 and 2 weeks PH. To study whether interneuron survival is regulated by targets or afferents, a situation known to exist in other developing neural populations, early embryos were subjected to (1) removal of one limb, resulting in the loss of lateral motor column motoneurons and dorsal root ganglion sensory afferents; (2) transection of the thoracic spinal cord, thereby removing both descending afferents and rostral targets of spinal interneurons, or (3) a combination of the two operations. No reductions in interneuron numbers were found as a result of these operations. Furthermore, morphometric analysis also revaled no change in neuronal size following these experimental manipulations. By contrast, there was a slight decrease in the total area of spinal gray matter that was most prominent in the dorsal region following limb bud removal. Our results indicate (1) that spinal interneurons fail to exhibit the massive naturally occurring death of postmitotic neurons that has been observed for several other populations of spinal neurons, and (2) spinal interneurons appear to be relatively resistant to induced cell death following the removal of substantial numbers of afferent inputs and targets.     

Thresholds for masking responses to light in three strains of retinally degenerate mice

Mutant mice with retinal degeneration (rd/rd) were given 1-h pulses of light of varying brightness at times of the night when they would normally be active. The mutant mice showed a significantly greater inhibition of locomotor activity to light (negative masking) than wildtype controls. Lack of impairment, or even enhancement of negative masking suggests that this response may depend on sparing in retinally degenerate mice of the same receptor type that mediates clock resetting, because synchronization of the circadian system is known to be unimpaired in these mutants. With very dim light pulses, mutants did not change their activity, but wildtypes actually became more active (positive masking). Positive and negative masking appear to depend on different sensory and central processes. Accepted: 11 May 1998     

雷珠单抗玻璃体内注射对早产儿视网膜病变患儿临床疗效及视网膜功能发育的影响

目的:探讨雷珠单抗玻璃体内注射对早产儿视网膜病变(ROP)患儿临床疗效及视网膜功能发育的影响。方法:收集2014年6月～2018年6月我院收治的80例ROP患儿,随机分为对照组和观察组各40例,对照组患儿采用激光治疗方案,观察组患儿采用雷珠单抗玻璃体内注射治疗方案。比较治疗后两组疗效和复发情况;随访6个月,采用闪光视网膜电图(F-ERG)检查治疗后视网膜功能的发育状况。结果:治疗后,观察组病变控制率明显高于对照组,病变进展率和复发率明显低于对照组(P0.05);F-ERG检查结果显示,对照组视杆细胞系统反应振幅较观察组明显降低,潜伏期较观察组明显延长,最大混合反应a、b波振幅明显低于观察组(P0.05);两组间最大混合反应波振幅比值b/a、潜伏期比较差异无统计学意义(P0.05)。两组间视锥细胞反应a波潜伏期及a、b波振幅比较差异无统计学意义(P0.05);对照组视锥细胞反应b波潜伏期明显比观察组延长,震荡电位(Ops)比观察组明显降低(P0.05)。结论:雷珠单抗玻璃体内注射治疗ROP患儿,疗效确切,操作简单且快速,视网膜功能的发育比激光治疗更趋向正常,适用于ROP患儿。     

Dopaminergic Regulation of Cone Retinomotor Movement in Isolated Teleost Retinas: I. Induction of Cone Contraction Is Mediated by D2 Receptors

In the retinas of lower vertebrates, retinal photoreceptors and melanin pigment granules of the retinal pigment epithelium (RPE) undergo characteristic movements in response to changes in light intensity and to signals from an endogenous circadian clock. To identify agents responsible for mediating light and/or circadian regulation of these retinomotor movements, we investigated the effects of hormones and neurotransmitters on cone, rod, and RPE movements in the green sunfish, Lepomis cyanellus. We report here that 3,4-dihydroxyphenylethylamine (dopamine) mimics the effect of light by inducing light-adaptive retinomotor movements in all three cell types. In isolated dark-cultured retinas, dopamine induced light-adaptive cone contraction with a half-maximal effect at 10(-8) M. This effect of dopamine was inhibited by antagonists with a potency order characteristic of D2 receptor mediation. The dopamine uptake blocker benztropine also induced light-adaptive cone contraction in isolated dark-cultured retinas, suggesting that there is continuous dopamine release in the dark but that concomitant uptake normally prevents activation of cone contraction. That dopamine plays a role in light regulation of cone movement is further suggested by the observation that light-induced cone contraction was partially inhibited by sulpiride, a selective D2 dopamine antagonist, or by Co2+, a blocker of synaptic transmission. Sulpiride also promoted dark-adaptive cone elongation in isolated light-adapted retinas, suggesting that continuous dopamine action is required in the light to maintain the light-adapted cone position. Dopamine can act directly on D2 receptors located on rod and cone inner/outer segments: dopamine induced light-adaptive retinomotor movements in isolated distal fragments of dark-adapted photoreceptors cultured in the dark. Together our results indicate that dopamine induces light-adaptive retinomotor movements in cones, rods, and RPE cells by activating D2 receptors. We suggest that, in vivo, dopamine plays a role in both light and circadian regulation of retinomotor movements.     

Retina Maturation Following Administration of Thyroxine in Developing Rats: Effects on Polyamine Metabolism and Glutamate Decarboxylase

Abstract: The effects of subcutaneous daily treatment with thyroxine on cell proliferation, differentiation, polyamines, and γ-aminobutyric acid metabolism in the rat retina were studied during the first 20 postnatal days. The retinal layers of the treated rats displayed an enhanced cell differentiation which reached its maximum 9–12 days from birth; but this effect stopped very quickly and was finished by the 20th postnatal day. Primarily there was an increase in ornithine decarboxylase activity which was accompanied by an increase in putrescine, spermidine, and spermine levels. S -Adenosylmethionine decarboxylase was induced later than ODC; corresponding with the enhanced synaptogenesis, glutamate decarboxylase increased 15-fold between the fourth and 15th days. Our data are consistent with the hypothesis that thyroxine may exert some of its effects by inducing the enzymes which regulate polyamine metabolism and synaptogenesis.     

A cytochemical study of myeloid bodies in the retinal pigment epithelium of the newt Notophthalmus viridescens

Summary It has been suggested (Yorke and Dickson 1984) that myeloid bodies (MBs) in the retinal pigment epithelium (RPE) of the newt, Notophthalmus viridescens, may represent areas of endoplasmic reticulum where lipids, such as 11-cis retinal derived from phagocytized outer segment tips, accumulate prior to esterification. Experiments in which an artificial ester substrate was added during in-vitro incubations have shown that esterase activity is represented in all areas of the newt RPE endoplasmic reticulum, including sites adjacent to all MBs. In related tests in which the localization of enzyme activity was restricted to areas of the cell where there had been accumulations of naturally-occurring (endogenous) esters, the products of ester hydrolysis were restricted to profiles of endoplasmic reticulum associated with lipid droplets, and with the interior of about 20% of those MBs that appeared completely circular in sections. This enzyme activity was not associated with other MB configurations. Results from endogenous-ester hydrolysis were identical to those obtained after staining with ZIO. This ZIO-reactivity was not affected by pre-incubation with agents that blocked or protected sulphydryl groups, and ZIO-reactive sites associated with MBs did not form complexes with digitonin. These observations suggest that MBs are a site of lipid-ester formation, but that they do not represent unique intracellular areas for this activity.