Sten Kjellström

Depleting Rac1 in mouse rod photoreceptors protects them from photo-oxidative stress without affecting their structure or function

In nonphagocytic cells, Rac1 is a component of NADPH oxidase that produces reactive oxygen species [Ushio-Fukai M (2006) Sci STKE 2006:re8]. Rac1 is expressed abundantly in mammalian retinal photoreceptors, where it is activated in response to light stimuli [Balasubramanian N, Slepak VZ (2003) Curr Biol 13:1306–1310]. We used Cre-LoxP conditional gene targeting to knock down Rac1 expression in mouse rod photoreceptors and found protection against light-induced photoreceptor death compared with WT litter-mates. We also found a similar protective effect on rods using apocynin, which inhibits NADPH oxidase activity. These results implicate both neuronal Rac1 and NADPH oxidase in cell death in this model of CNS degeneration. Studies in which dominant-mutants of Rac1 were expressed in transgenic Drosophila species demonstrated that Rac1 is a key regulator of photoreceptor morphogenesis and polarity [Chang HY, Ready DF (2000) Science 290:1978–1980]. However, we found that diminished Rac1 expression in mouse rods had no effect on retinal structure or function examined by light microscopy, electron microscopy, rhodopsin measurement, electroretinogram activity, and visual acuity, indicating rod outer segment morphogenesis proceeded normally in Rac1 conditional knockout mice. The lack of structural or functional effect of Rac1 depletion on photoreceptors, but protection under conditions of stress, indicate that the Rac1 pathway warrants exploration as a target for therapy in retinal neurodegenerative diseases.

Alterations in electroretinograms and retinal morphology in rabbits treated with vigabatrin

Purpose: To determine whether long-term treatment with the anti-epileptic drug vigabatrin causes damage to rabbit retina. Methods: Five rabbits were treated continuously with a daily dose of vigabatrin solution per orally during a period of 1–8 months. Two rabbits receiving water were used as controls. Repeated full-field electroretinograms (every two weeks) were assessed during this period. Vigabatrin serum concentration was repeatedly measured for securing successful drug administration. After termination of treatment the rabbits were sacrificed and the morphology of the sectioned retina was studied. Results: In all rabbits treated with vigabatrin the serum analyses repeatedly demonstrated elevated drug concentration. Full-field electroretinograms demonstrated normal rod function in all treated rabbits, but reduced cone function in two of the five treated rabbits verified by 30u2009Hz flicker stimulation. Morphologic studies of the sectioned retina demonstrated GFAP immunoactivity of the glial cells localized in the retinal periphery in all five treated rabbits, one of which had staining also in the centrally localized glial cells. The treated rabbits also demonstrated a weaker GAD staining in the IPL and less positive amacrine cells, compared to the controls. Only two treated rabbits had normal GABA staining while three had an enhanced GABA immunoreactivity and undistinguishable fibers in the IPL. In three out of five treated rabbits the Müller cells were short, stubby and fragmented, with swollen endfeet. Conclusion: This study demonstrates changes in histopathology caused by vigabatrin in an animal model, which has not been reported previously. We have found that vigabatrin orally administrated to rabbits does not affect rod function but may reduce cone function in the full-field electroretinogram, which is similar to the previously reported vigabatrin effect on the human ERG. The results indicate that vigabatrin may damage or influence, at least one cell type in the rabbit retina.

Long-term 12 year follow-up of X-linked congenital retinoschisis.

Purpose: To investigate the retinal structure and function during the progression of X-linked retinoschisis (XLRS) from childhood to adulthood. Methods: Ten patients clinically diagnosed with XLRS were investigated at 6–15 years of age (mean age 9 years) with a follow-up 8 to 14 years later (mean 12 years). The patients underwent regular ophthalmic examination as well as testing of best corrected visual acuity (BCVA), visual field (VF) and assessment of full-field electroretinography (ERG) during their first visit. During the follow-up, the same clinical protocols were repeated. In addition, macular structure and function was examined with multifocal electroretinography (mfERG) and optical coherence tomography (OCT). The patients were 18–25 years of age (mean age 21 years) at the follow-up examination. All exons and exon-intron boundaries of RS1-gene were sequenced for gene mutations in 9 out of the 10 patients. Results: Best corrected VA and VF were stable during this follow-up period. No significant progression in cone or rod function could be measured by full-field ERG. Multifocal electroretinography and OCT demonstrated a wide heterogeneity of macular changes in retinal structure and function at the time of follow-up visit. Three different mutations were detected in these nine patients, including a known nonsense mutation in exon 3, a novel insertion in exon 5 and an intronic mutation at 5′ splice site of intron 3. Conclusions: Clinical follow-up (mean 12 years) of ten young XLRS patients (mean age of 9 years) with a typical congenital retinoschisis phenotype revealed no significant decline in retinal function during this time period. MfERG and OCT demonstrated a wide variety of macular changes including structure and dysfunction. The XLRS disease was relatively stable during this period of observation and would afford opportunity for therapy studies to judge benefit against baseline and against the fellow eye.

Retinal function and histopathology in rabbits treated with Topiramate.

PurposeTo evaluate retinal function and histopathology in rabbits treated orally with the anti-epileptic drug topiramate.MethodsSix rabbits were treated with a daily oral dose of topiramate during a period of eight months. Six rabbits receiving water served as controls. Blood samples were analyzed for determination of topiramate serum levels in order to ensure successful drug exposition. Standardized full-field electroretinograms (ERGs) were performed before treatment and then at 2, 3 and 8xa0months during the treatment period. After terminating treatment the rabbits were sacrificed and the morphology of the sectioned retina was studied.ResultsAfter eight months of treatment the full-field ERG demonstrated normal rod function in treated and control rabbits, but the light adapted 30xa0Hz flicker b-wave amplitude was significantly reduced in the treated rabbits. This was the case for both the light adapted (Wilcoxon signed ranks test, Pxa0=xa00.046) and the dark adapted (Wilcoxon signed ranks test, Pxa0=xa00.028) 30xa0Hz flicker response from the treated rabbits. Retinal immunohistology revealed a severe accumulation of GABA in amacrine cells and in the inner plexiform layer in 4 of 6 treated rabbits compared to the controls.ConclusionsTopiramate, orally administrated to rabbits, may cause a significant reduction of the retinal function demonstrated by the reduced b-wave amplitude in the full-field ERG, as well as changes in immunohistology characterized by a severe accumulation of GABA in the inner retina. The retinal dysfunction and the morphological changes indicate that topiramat may damage the retina, similarly to vigabatrin (another anti-epileptic drug).

Retinal function in rabbits does not improve 4-5 months after terminating treatment with vigabatrin.

Purpose: We have previously reported changes in retinal function and histopathology in rabbits treated with vigabatrin. The purpose of the present study was to evaluate retinal function and histopathology of retina in rabbits 4–5xa0months after terminating vigabatrin medication. Methods: Five rabbits were treated with a daily per oral dose of vigabatrin during 12–13xa0months. After terminating treatment an observation period of 4–5xa0months followed. Six rabbits receiving water served as controls. Standardized full-field electroretinograms were performed every 6–8xa0weeks, using a Burian–Allen bipolar contact lens. After 18xa0months the rabbits were sacrificed and the morphology of the sectioned retina was studied. The antibodies used for staining were GABA, GFAP, GAD, and vimentin. Results: After 12–13xa0months of treatment the full-field ERG was reduced in all rabbits treated with vigabatrin. There was a statistically significant difference in the dark adapted cone b-wave amplitude between treated animals and controls (Wilcoxon signed-rank test, p = 0.043). This difference was consistent also 4–5xa0months after terminating treatment. Immunohistology of the sectioned retina demonstrated no significant difference in immunoreactivity between treated animals and controls. All treated rabbits demonstrated elevated serum concentration of the drug during medication. Conclusion: Four to five months after terminating treatment with vigabatrin the rabbit full-field ERG remains reduced in isolated cone b-wave amplitude indicating that vigabatrin induced retinal dysfunction may be irreversible. However, immunohistology is normal after a period without treatment, implying that the previously described changes in retinal morphology and glial cell activity are reversible, and probably exist only during treatment.

Rifabutin accumulates in the lens and reduces retinal function in the rabbit eye

Purpose: To study the toxicology of rifabutin in the rabbit eye with emphasis on retinal function and histopathology. Methods: Seven rabbits received a daily dose of rifabutin during 15 months. Six rabbits receiving only the vehicle were used as controls. Repeated standardized full-field electroretinograms (ERG) were assessed. After discontinuing treatment, the rabbits were killed and the cornea, the lens, and the sectioned retina was studied. Immunhistochemistry directed against vimentin, glial fibrillaryacidic protein (GFAP), protein kinase C (PKC), and peanut agglutinin (PNA) was performed. Results: Rifabutin was detected in serum of the treated rabbits. During treatment, the full-field ERG demonstrated significantly reduced b-wave amplitudes in the total rod-cone response as well as in the isolated rod and cone response compared with the recordings before treatment. The control rabbits did not demonstrate a reduction of the ERG amplitudes. The treated rabbits developed a discoloration of the lens, not seen in the control group. No retinal pathology was demonstrated using immunohistochemical methods. Conclusion: Rifabutin causes a discoloration of the lens and reduces both rod and cone function in rabbits, but does not alter retinal morphology. Previous reports on ocular side effects caused by rifabutin are supported by the results of this study.

Retinal AAV8-RS1 Gene Therapy for X-Linked Retinoschisis: Initial Findings from a Phase I/IIa Trial by Intravitreal Delivery

This study evaluated the safety and tolerability of ocular RS1 adeno-associated virus (AAV8-RS1) gene augmentation therapy to the retina of participants with X-linked retinoschisis (XLRS). XLRS is a monogenic trait affecting only males, caused by mutations in the RS1 gene. Retinoschisin protein is secreted principally in the outer retina, and its absence results in retinal cavities, synaptic dysfunction, reduced visual acuity, and susceptibility to retinal detachment. This phase I/IIa single-center, prospective, open-label, three-dose-escalation clinical trial administered vector to nine participants with pathogenic RS1 mutations. The eye of each participant with worse acuity (≤63 letters; Snellen 20/63) received the AAV8-RS1 gene vector by intravitreal injection. Three participants were assigned to each of three dosage groups: 1e9 vector genomes (vg)/eye, 1e10 vg/eye, and 1e11 vg/eye. The investigational product was generally well tolerated in all but one individual. Ocular events included dose-related inflammation that resolved with topical and oral corticosteroids. Systemic antibodies against AAV8 increased in a dose-related fashion, but no antibodies against RS1 were observed. Retinal cavities closed transiently in one participant. Additional doses and immunosuppressive regimens are being explored to pursue evidence of safety and efficacy (ClinicalTrials.gov: NCT02317887).

Comparison of a new digital KM screen test with conventional Hess and Lees screen tests in the mapping of ocular deviations

PURPOSEnTo evaluate the digital KM screen computerized ocular motility test and to compare it with conventional nondigital techniques using the Hess and Lees screens.nnnMETHODSnPatients with known ocular deviations and a visual acuity of at least 20/100 underwent testing using the digital KM screen and the Hess and Lees screen tests. The examination duration, the subjectively perceived difficulty, and the patients method of choice were compared for the three tests. The accuracy of test results was compared using Bland-Altman plots between testing methods.nnnRESULTSnA total of 19 patients were included. Examination with the digital KM screen test was less time-consuming than tests with the Hess and Lees screens (Pxa0<xa00.001 and Pxa0=xa00.003, resp., compared with the digital KM screen). Patients found the test with the digital KM screen easier to perform than the Lees screen test (Pxa0=xa00.009) but of similar difficulty to the Hess screen test (Pxa0=xa00.203). The majority of the patients (83%) preferred the digital KM screen test to both of the other screen methods (Pxa0=xa00.008). Bland-Altman plots showed that the results obtained with all three tests were similar.nnnCONCLUSIONSnThe digital KM screen is accurate and time saving and provides similar results to Lees andxa0Hess screen testing. It also has the advantage of a digital data analysis and registration.

Alteration of Vitreal Retinoschisin Level in Human Primary Retinal Detachment

Alteration of Vitreal Retinoschisin Level in Human Primary Retinal Detachment Retinoschisin (RS1), the product of RS1 located on the X chromosome, is expressed mainly in retina.1 The 24-kDa RS1 encodes a highly conserved sequence motif termed the discoidin domain, which is a critical determinant of the structural and functional integrity of the retina.2 Mutations in RS1 that lead to either complete loss of RS1 expression or its accumulation as a nonfunctional misfolded form are the underlying causes of X-linked retinoschisis (XLRS).3 This disorder, seen exclusively in young males, is characterized by splitting, or schisis, affecting all retinal layers. The precise molecular mechanism by which RS1 functions is still undefined. During development, there is a wave of RS1 expression beginning at the retinal surface and spreading progressively in more distal retinal layers as they differentiate.4 In adult mice, RS1 is expressed in all retinal neurons except horizontal cells,4 with a predominance in photoreceptor inner segments and bipolar cells. In patients with XLRS, the retina is more prone to retinal detachment (RD) compared with the general population (10% vs 0.01%, respectively).5 These detachments are difficult to surgically reattach, making the postoperative outcome unfavorable.6,7 To assess the potential role of RS1 in retinal response to detachment, we analyzed RS1 levels in vitreous samples derived from patients’ eyes with or without RD.