Abstract: Primary vitreoretinal lymphoma (PVRL), as a subset of primary central nervous system lymphoma (PCNSL), is a rare and fatal ocular malignancy. Most PVRL masquerades as chronic posterior uveitis, which makes the clinical diagnosis challenging. Vitreous cells, subretinal lesions and imaging techniques are essential for clinical diagnosis. Importantly, cytopathology/histopathology identification of malignant cells is the gold standard for the diagnosis of PVRL. In addition, molecular detection of immunoglobulin heavy chain (IgH) or T cell receptor (TCR) gene rearrangements, immunophenotyping for cell markers, and cytokine analysis of interleukine-10 elevation are often used as adjunct procedures. Current management of PVRL involves local radiation, intravitreal chemotherapy (methotrexate and rituximab), with or without systemic chemotherapy depending on the involvement of non-ocular tissues. In cases with concomitant PCNSL, systemic high-dose methotrexate/rituximab based therapy in conjunction with local therapy, whole brain radiotherapy and/or autologous stem cell transplantation is considered. Although PVRL normally responds well to initial treatment, high rates of relapse and CNS involvement usually lead to poor prognosis and limited survival. A professional team of medical experts in ophthalmologists, ocular pathologists, neuro-oncologists and hemato-oncologists is essential for optimizing patient management.
Background: Congenital hereditary endothelial dystrophy (CHED) is characterized by blindness at birth or in early infancy resulting from bilateral corneal opacification, and is linked to mutation in the Slc4a11 gene. A Slc4a11 knockout (KO) mouse, generated by gene deletion (Vithana et al. Nat Genet 2006), was acquired in order to study this disease. To confirm the phenotype of this Slc4a11 KO mouse model as a function of age, using the wild type (WT) mouse as a control.
Methods: Genotyping was performed by PCR (REDExtract-N-AmpTM Tissue PCR Kit, Sigma-Aldrich, Oakville, ON). Slc4a11 WT and KO mice populations aged from 5 to 50 weeks were studied (n=5 animals per age group; 5-year age intervals). Slit lamp examination, anterior segment-ocular coherence tomography (OCT930SR; Thorlabs, Inc., Newton, NJ), corneal endothelial cell staining, and scanning (SEM) and transmission (TEM) electron microscopy were used to assess the morphological and cellular differences between the two groups. The expression of basolateral membrane transporter NaBC1 within the corneal endothelium was also assessed using immunohistochemistry.
Results: Diffuse and progressive corneal opacification was observed at the slit lamp in the Slc4a11 KO mice, starting at 10 weeks. The central corneal thickness (CCT) also increased progressively as a function of time. In comparison, Slc4a11 WT corneas remained clear over the entire study period. Early TEM results showed vacuole degeneration of the corneal endothelium in the 15-week KO mouse, which was not seen in the same age WT mouse.
Conclusions: The corneal phenotype of this Slc4a11 KO mouse is representative of the clinical manifestations of CHED in human subjects, confirming the usefulness of this model for studying pathophysiology and therapeutic alternatives for Slc4a11-associated corneal dystrophies.
Background: The expression, localization, and function of the endocannabinoid system has been well characterized in recent years in the monkey retina and in the primary thalamic relay, the lateral geniculate nucleus (dLGN). Few data are available on cortical recipients’ structures of the dLGN, namely the primary visual cortex (V1). The goal of this study is to characterize the expression and localization of the metabotropic cannabinoid receptor type 1 (CB1R), the synthesizing enzyme N-acyl phosphatidyl-ethanolamine phospholipase D (NAPE-PLD), and the degradation enzyme fatty acid amide hydrolase (FAAH) in the vervet monkey area V1.
Methods: Using Western blots and immunohistochemistry, we investigated the expression patterns of CB1R, NAPE-PLD, and FAAH in the vervet monkey primary visual cortex.
Results: CB1R, NAPE-PLD, and FAAH were expressed in the primary visual cortex throughout the rostro-caudal axis. CB1R showed very low levels of staining in cortical layer 4, with higher expressions in all other cortical layers, especially layer 1. NAPE-PLD and FAAH expressions were highest in layers 1, 2 and 3, and lowest in layer 4.
Conclusions: Interestingly enough, CB1R was very low in layer 4 of V1 in comparison to the other cortical layers. The visual information coming from the dLGN and entering layer 4Calpha (magno cells) and 4Cbeta (parvo cells) may be therefore modulated by the higher expression levels of CB1R in cortical layers 2 and 3 on the way to the dorsal and ventral visual streams. This is further supported by the higher expression of NAPE-PLD and FAAH in the outer cortical layers. These data indicate that CB1R system can influence the network of activity patterns in the visual stream after the visual information has reached area V1. These novel results provide insights for understanding the role of the endocannabinoids in the modulation of cortical visual inputs, and hence, visual perception.
Background: Exposure to ethanol in utero leads to several brain development disorders including retinal abnormalities whose underlying cellular pathogenesis remains elusive. We have previously reported changes in electroretinogram recordings in moderate fetal alcohol exposure (MFAE) vervet monkeys. The goal of this study is to characterize the anatomical effects of moderate MFAE during the third trimester in the vervet monkey retina.
Methods: Using immunohistochemistry and Western blots, we analyzed changes in the expression of cell-type specific proteins that may occur in the MFAE retina compared to the normal retina. We also compared the basic retinal anatomy across groups by examining retinal layering and thickness.
Results: Our main result indicates that GFAP (a potent marker of astrocytes) immunoreactivity was increased in the MFAE retina indicating strong astrogliosis. There was no obvious change in the overall anatomy in the MFAE retina and no significant differences in the mean thickness of each retinal layer. Furthermore, no significant changes in the morphology of the photoreceptors, horizontal cells, bipolar cells, and amacrines cells was observed.
Conclusions: These data indicate that astrogliosis is a consequence of prenatal alcohol exposure and might explain the reported changes in the electroretinographic responses.
Background: Zellweger spectrum disorder (ZSD) is an autosomal recessive disease caused by mutations in any one of 13 PEX genes whose protein products are required for peroxisome assembly. Retinopathy leading to blindness is one of the major handicaps faced by affected individuals, but treatment for this is supportive only. To test whether we could improve visual function in ZSD, we performed a proof-of-concept trial for PEX1 gene augmentation therapy using the Pex1-G844D mouse model, which bears the equivalent to a common human mutation. This model exhibits a gradual decline in scotopic ffERG response, an always residual photopic ffERG response, diminished visual acuity, and cone and bipolar cell anomalies.
Methods: We administered subretinal injections of a PEX1-containing viral vector (AAV8.CMV.hPEX1.HA) to 2 mouse cohorts of 5 or 9 weeks of age. A GFP-containing vector was used as a control in the contralateral eye of each animal. Efficient expression of the virus was confirmed by retinal histology/immunohistochemistry, and its ability to recover peroxisome import was confirmed in vitro. Preliminary ffERG and optokinetic (OKN) analyses were performed on a subset of animals at 8, 16, and 20 weeks after gene delivery. Final ffERG and OKN measures were performed when each cohort reached 32 weeks of age (23 or 27 weeks post injection).
Results: Preliminary ffERG and OKN analyses at 8 weeks post injection showed mildly better retinal response and visual acuity, respectively, in the PEX1-injected eyes, as did ffERG analysis when each cohort reached 25 weeks of age (16 or 20 weeks after gene delivery). This effect was more pronounced in the cohort treated at 5 weeks of age, when ffERG response is highest in Pex1-G844D mice. At 32 weeks of age, the ffERG response in the PEX1-injected eyes was double that of GFP-injected eyes, on average, though there was no change in OKN. Furthermore, in PEX1-injected eyes the photopic ffERG response improved over time, and the decline in scotopic b-wave amplitude was ameliorated compared to un-injected eyes.
Conclusions: AAV8.CMV.hPEX1.HA was subretinally delivered into the left eye of 5- and 9-week-old Pex1-G844D retina. Successful expression of the protein with no gross histologic side effect was observed. Neither the injection, nor exposure to the AAV8 capsid or the transgenic protein negatively altered the ERG or OKN response. At 5–6 months after gene delivery, therapeutic vector-treated eyes showed improved ERG compared to control eyes, on average, in both the “prevention” and “recovery” cohorts. This implies clinical potential of gene delivery to improve vision in patients with ZSD. Retinal immunohistochemistry (to visualize retinal cell types) and biochemical analyses will be performed on treated and untreated retinas, and may inform the mechanism of ERG improvement.
Background: Retinal pigment epithelium (RPE) is vital for the homeostasis of the subretina including photoreceptors and choroid. Interestingly, our previous results suggested that the recently discovered lactate receptor GPR81 is abundantly expressed in RPE. To date, only one previous study has shown that activating GPR81 could enhance DNA repair by activating HDAC1. Consequently, we investigated whether GPR81 exhibits epigenetic modification in the subretina by using GPR81?/? mice.
Methods: GPR81?/? mice and wide type littermates were generated on a background of C57BL/6J mice. The thicknesses of their choroid were evaluated by immunohistochemistry. Meanwhile, Q-PCR, western blot and choroid sprout assay were performed. In vitro, primary retinal pigment epithelium (pRPE) cells were isolated from mice, and cultured for treatments.
Results: The thickness of choroid was reduced in GPR81?/? mice compared to GPR81+/+ mice, suggesting that GPR81 is important for the integrity of choroid. In the choroid sprout assay, lactate treated RPE/choroid complex showed a significant increase in angiogenesis compared to controls while lactate treated KO RPE/choroid complex showed no difference compared to their controls. For Q-PCR, most of the genes screened elevated their expression in GPR81?/? mice compared to WT mice, suggesting epigenetic modification may exist, which were confirmed by histone acetylation and HDACs activity assay.
Conclusions: Taking together, the lactate receptor GPR81 in RPE is very important for maintaining homeostasis of the subretina. This novel discovery sheds new light on the relationship between metabolism and epigenetic modification.
Background: The neovascular aged-related macular degeneration (AMD) is the leading cause of legal blindness in the elderly. It is presently treated by anti-VEGF intravitreal injection in order to stop the neovascularization. In seeking of more efficient treatments to prevent retinal damage, it has been proposed that the kinin-kallikrein system (KKS), a key player in inflammation, could be involved in AMD etiology. However, the role of kinin receptors and their interaction with VEGF in AMD is poorly understood.
Methods: In order to address this question, choroidal neovascularization (CNV) was induced in the left eye of Long-Evans rat. After laser induction, anti-VEGF or IgG control were injected into the vitreal cavity. Gene expression was measured by qRT-PCR, retinal adherent leukocytes were labelled with FITC-Concanavalin A lectin, vascular leakage by the method of Evans blue and cellular localisation by immunohistochemistry.
Results: The number of labelled adherent leucocytes was significantly increased in laser-induced CNV compared to the control eye. This was significantly reversed by one single injection of anti-VEGF. Extravasation of Evans blue dye was significantly increased in laser-induced CNV eyes compared to control eyes and partially reversed by one single injection of anti-VEGF or by R954 treatment. The mRNA expression of inflammatory mediators was significantly increased in the retina of CNV rats. Immunodetection of B1R was significantly increased in CNV eyes. B1R immunolabeling was detected on endothelial and ganglion cells.
Conclusions: This study is the first to highlight an effect of the kinin/kallikrein system in a model of CNV that could be reduced by both anti-VEGF therapy and topically administered B1R antagonist R-954.
Abstract: Since the 21st century, the development of corneal tissue engineering technology has been developing rapidly. With the progress of biomaterials, cell culture and tissue engineering technology, tissue engineering cornea has gained great development in both basic scientific research and clinical application. In particular, tissue engineered corneal scaffolds are the core components of tissue engineered corneas. It is the focus of current research on tissue engineering cornea to search for scaffolds with good biocompatibility, high safety and good biomechanical properties. In this paper, the recent research progress of tissue engineering corneal materials is reviewed.
Abstract: Autoimmune retinopathy (AIR) refers to both paraneoplastic and non-paraneoplastic forms of a rare, acquired retinal degeneration thought to be mediated by the production of antiretinal antibodies. However, the mechanisms underlying AIR pathogenesis are incompletely understood, and it remains a diagnosis of exclusion given the lack of definitive testing as well as its protean clinical presentation. This review summarizes the current literature on the epidemiology, diagnosis, and management of AIR, with a focus on non-paraneoplastic disease and the potential role of immunomodulatory therapy. A recent expert consensus statement on diagnosis and management of non-paraneoplastic AIR served as a framework for interpreting the limited data available, a process that was complicated by the small sample sizes, heterogeneity, and retrospective nature of these studies. Additional work is needed to characterize AIR patients on the basis of cytokine and immunogenetic profiling; to establish the pathogenicity of antiretinal antibodies; and to standardize treatment regimens as well as assessment of clinical outcomes.
Abstract: In the early days of deciphering the injured neuronal tissues led to the realization that contrast is necessary to discern the parts of the recovering tissues from the damaged ones. Early attempts relied on available (and often naturally occurring) staining substances. Incidentally, the active ingredients of most of them were small molecules. With the advent of time, the knowledge of chemistry helped identify compounds and conditions for staining. The staining reagents were even found to enhance the visibility of the organelles. Silver impregnation identification of Golgi bodies was discovered in owl optic nerve. Staining reagents since the late 1800s were widely used across all disciplines and for nerve tissue and became a key contributor to advancement in nerve-related research. The use of these reagents provided insight into the organization of the neuronal tissues and helped distinguish nerve degeneration from regeneration. The neuronal staining reagents have played a fundamental role in the clinical research facilitating the identification of biological mechanisms underlying eye and neuropsychiatric diseases. We found a lack of systematic description of all staining reagents, whether they had been used historically or currently used. There is a lack of readily available information for optimal staining of different neuronal tissues for a given purpose. We present here a grouping of the reagents based on their target location: (I) the central nervous system (CNS), (II) the peripheral nervous system (PNS), or (III) both. The biochemical reactions of most of the staining reagents is based on acidic or basic pH and specific reaction partners such as organelle or biomolecules that exists within the given tissue type. We present here a summary of the chemical composition, optimal staining condition, use for given neuronal tissue and, where possible, historic usage. Several biomolecules such as lipids and metabolites lack specific antibodies. Despite being non-specific the reagents enhance contrast and provide corroboration about the microenvironment. In future, these reagents in combination with emerging techniques such as imaging mass spectrometry and kinetic histochemistry will validate or expand our understanding of localization of molecules within tissues or cells that are important for ophthalmology and vision science.
