Activation of survival pathways in the degenerating retina of rd10 mice.

Blinding diseases of the retina are frequently characterized by loss of photoreceptor cells. The retinal degeneration 10 (rd10) mouse expresses a mutant form of rod phosphodiesterase leading to autosomal recessive photoreceptor degeneration. In contrast to rd1, rd10 mice have remaining rod function mimicking more closely most forms of human Retinitis Pigmentosa. Here we use morphology, biochemistry, retinal whole mounts, real-time PCR, Western blotting and immunofluorescence to compile a comprehensive report on progression of retinal degeneration in the rd10 retina up to one year of age. We show that retinal development, morphology, gene expression pattern and retinal vasculature was normal until postnatal day 15. Thereafter, a bi-phasic pattern of rod cell death emerged with a first rapid phase peaking around 3 weeks of age followed by a slower second phase. Death of cone cells followed with a delay and vessel dropout was prominent in the retinal periphery of 6 months old rd10 mice. At one year of age, RPE atrophy was evident. The degenerating retina rapidly induced expression of transcriptional regulators Atf3 and Cebpd. Induction of Atf3 was transient and lasted only for several days at the beginning of degeneration whereas levels of Cebpd remained elevated throughout the period of photoreceptor loss. Several protective genes such as Lif, Edn2 and Fgf2 which are implicated in a potent endogenous survival pathway, and Mt1 and Mt2 were strongly upregulated in the rd10 retina. In addition, increased expression of Casp1 and Il1b suggested an inflammatory response.

Retinal neuroprotection by hypoxic preconditioning is independent of hypoxia-inducible factor-1 alpha expression in photoreceptors.

Hypoxic preconditioning stabilizes hypoxia-inducible factor (HIF) 1 alpha in the retina and protects photoreceptors against light-induced cell death. HIF-1 alpha is one of the major transcription factors responding to low oxygen tension and can differentially regulate a large number of target genes. To analyse whether photoreceptor-specific expression of HIF-1 alpha is essential to protect photoreceptors by hypoxic preconditioning, we knocked down expression of HIF-1 alpha specifically in photoreceptor cells, using the cyclization recombinase (Cre)-lox system. The Cre-mediated knockdown caused a 20-fold reduced expression of Hif-1 alpha in the photoreceptor cell layer. In the total retina, RNA expression was reduced by 65%, and hypoxic preconditioning led to only a small increase in HIF-1 alpha protein levels. Accordingly, HIF-1 target gene expression after hypoxia was significantly diminished. Retinas of Hif-1 alpha knockdown animals did not show any pathological alterations, and tolerated hypoxic exposure in a comparable way to wild-type retinas. Importantly, the strong neuroprotective effect of hypoxic preconditioning against light-induced photoreceptor degeneration persisted in knockdown mice, suggesting that hypoxia-mediated survival of light exposure does not depend on an autocrine action of HIF-1 alpha in photoreceptor cells. Hypoxia-mediated stabilization of HIF-2 alpha and phosphorylation of signal transducer and activator of transcription 3 (STAT 3) were not affected in the retinas of Hif-1 alpha knockdown mice. Thus, these factors are candidates for regulating the resistance of photoreceptors to light damage after hypoxic preconditioning, along with several potentially neuroprotective genes that were similarly induced in hypoxic knockdown and control mice.

Physico-chemical Stability of MabThera Drug-product Solution for Subcutaneous Injection under in-use Conditions with Different Administration Materials.

MabThera is an essential component of the standard-of-care regimens in the treatment of non-Hodgkin lymphoma and Chronic Lymphatic Leukemia. MabThera for subcutaneous injection is a novel line extension that has been approved by the European Medicines Agency for the treatment of patients with follicular lymphoma and diffuse large B-cell lymphoma. This study aimed to evaluate in-use stability data of MabThera subcutaneous drug-product solution in single-use syringes for subcutaneous administration according to the European Medicines Agency guideline. The drug-product solution was exposed to material contact surfaces of five different administration setups commonly used in subcutaneous drug delivery. MabThera subcutaneous was transferred under aseptic conditions into polypropylene and polycarbonate syringes and stored for 1, 2, and 4 weeks at 2°C to 8°C followed by 24 hours at 30°C. After storage, subcutaneous administration was simulated and MabThera subcutaneous drug-product solution quality attributes were evaluated by using compendial physico-chemical tests, as well as suitable and validated molecule- and formulation-specific analytical methods. MabThera subcutaneous vials were treated and analyzed in parallel. The physico-chemical results of MabThera subcutaneous in the different setups were comparable to the control for all timepoints. No change in drug-product quality after storage and simulated administration was found compared to the control. However, since single-dose products do not contain preservatives, microbial contamination and growth needs to be avoided and product sterility needs to be ensured. The results showed that MabThera subcutaneous remains compatible and stable, from a physico-chemical perspective, for up to 4 weeks at 2°C to 8°C followed by 24 hours at 30°C with the contact materials tested in this study. In order to avoid and minimize microbial growth, MabThera subcutaneous should be used immediately after removal from the original packaging container and strict aseptic handling conditions need to be followed.

A mouse model for studying cone photoreceptor pathologies.

PURPOSE: Due to the low abundance of cone photoreceptors in the mouse retina and the scarcity of alternative animal models, little is known about mechanisms of cone degeneration. Nrl knockout mice develop exclusively the cone-type of photoreceptors. However, the cone photoreceptor layer in Nrl(-/-) mice displays an irregular morphology with severe rosette formation. Retinas of Rpe65(-/-);Nrl(-/-) mice have no rosettes due to the lack of 11-cis-retinal, but also are not functional. To develop a model with a functional all-cone retina that is morphologically well structured, we generated R91W;Nrl(-/-) double-mutant mice, which express a hypomorphic Rpe65 allele (R91W). METHODS: The following analyses were used to characterize the R91W;Nrl(-/-)mice: morphology by light and electron microscopy, protein distribution by immunofluorescence, cone function by electroretinography and optomotor response, RNA levels by RT-PCR, and chromophore levels by HPLC. Cone degeneration was assessed in R91W;Nrl(-/-) mice treated with MNU, and in triple R91W;Nrl(-/-);Cpfl1 and quadruple R91W;Nrl(-/-);Cpfl1;rd10 mutant mice. RESULTS: The all-cone retina of R91W;Nrl(-/-) mice is functional and relatively stable with only very slow age-related degeneration. Using triple and quadruple mutant mice, or a chemical treatment, we demonstrated that cone degeneration could be induced and analyzed in these mice. CONCLUSIONS: The reduced levels of visual chromophore prevented rosette formation and sustained function in the R91W;Nrl(-/-) retina. Thus, the R91W;Nrl(-/-) mouse allows study of the etiology of diseases related to cone degeneration in a "morphologically intact" and functional all-cone photoreceptor retina.

CDC42 is required for tissue lamination and cell survival in the mouse retina.

The small GTPase CDC42 has pleiotropic functions during development and in the adult. These functions include intra- as well as intercellular tasks such as organization of the cytoskeleton and, at least in epithelial cells, formation of adherens junctions. To investigate CDC42 in the neuronal retina, we generated retina-specific Cdc42-knockdown mice (Cdc42-KD) and analyzed the ensuing consequences for the developing and postnatal retina. Lack of CDC42 affected organization of the developing retina as early as E17.5, prevented correct tissue lamination, and resulted in progressive retinal degeneration and severely reduced retinal function of the postnatal retina. Despite the disorganization of the retina, formation of the primary vascular plexus was not strongly affected. However, both deeper vascular plexi developed abnormally with no clear layering of the vessels. Retinas of Cdc42-KD mice showed increased expression of pro-survival, but also of pro-apoptotic and pro-inflammatory genes and exhibited prolonged Müller glia hypertrophy. Thus, functional CDC42 is important for correct tissue organization already during retinal development. Its absence leads to severe destabilization of the postnatal retina with strong degeneration and loss of retinal function.

Normoxic activation of hypoxia-inducible factors in photoreceptors provides transient protection against light-induced retinal degeneration.

PURPOSE: Hypoxic preconditioning activates hypoxia-inducible transcription factors (HIFs) in the retina and protects photoreceptors from light-induced retinal degeneration. The authors tested whether photoreceptor-specific activation of HIFs in normoxia is sufficient for protection. METHODS: Rod-specific Vhl knockdown mice were generated using the Cre-lox system with the rod opsin promoter controlling expression of CRE recombinase to stabilize HIF transcription factors in normoxic rods. Cell death was induced by light exposure and quantified by ELISA. Rhodopsin was quantified by spectrophotometry. Gene expression was analyzed by real-time PCR, and levels of proteins were determined by Western blotting. Morphology was investigated by light microscopy and retinal function tested by ERG. RESULTS: The rod-specific Vhl knockdown stabilized HIF-α proteins and induced expression of HIF target genes in retinas of 10-week-old mice under normoxic conditions. Retinal morphology and function were normal. At 36 hours after exposure to excessive light, Vhl knockdowns showed significantly less photoreceptor cell death than did wild-type controls. Ten days after light exposure, however, photoreceptor degeneration in Vhl knockdowns was similar to that of control animals. Vhl knockdowns expressed Fgf2 at higher basal levels before light exposure. After light exposure, however, expression of Fgf2 was not significantly different from that of wild-type controls. CONCLUSIONS: Artificial activation of HIF transcription factors in normoxic photoreceptors results in an increased basal expression of Fgf2 that may contribute to a transient protection of rods against light damage. Full photoreceptor protection may require a hypoxia-like response in additional retinal cell types and/or the differential regulation of additional mechanisms.

Novel rodent models for macular research.

BACKGROUND: Many disabling human retinal disorders involve the central retina, particularly the macula. However, the commonly used rodent models in research, mouse and rat, do not possess a macula. The purpose of this study was to identify small laboratory rodents with a significant central region as potential new models for macular research. METHODOLOGY/PRINCIPAL FINDINGS: Gerbillus perpallidus, Meriones unguiculatus and Phodopus campbelli, laboratory rodents less commonly used in retinal research, were subjected to confocal scanning laser ophthalmoscopy (cSLO), fluorescein and indocyanine green angiography, and spectral-domain optical coherence tomography (SD-OCT) using standard equipment (Heidelberg Engineering HRA1 and Spectralis™) adapted to small rodent eyes. The existence of a visual streak-like pattern was assessed on the basis of vascular topography, retinal thickness, and the topography of retinal ganglion cells and cone photoreceptors. All three species examined showed evidence of a significant horizontal streak-like specialization. cSLO angiography and retinal wholemounts revealed that superficial retinal blood vessels typically ramify and narrow into a sparse capillary net at the border of the respective area located dorsal to the optic nerve. Similar to the macular region, there was an absence of larger blood vessels in the streak region. Furthermore, the thickness of the photoreceptor layer and the population density of neurons in the ganglion cell layer were markedly increased in the visual streak region. CONCLUSIONS/SIGNIFICANCE: The retinal specializations of Gerbillus perpallidus, Meriones unguiculatus and Phodopus campbelli resemble features of the primate macula. Hence, the rodents reported here may serve to study aspects of macular development and diseases like age-related macular degeneration and diabetic macular edema, and the preclinical assessment of therapeutic strategies.