Effect of steroidal and non-steroidal drugs on the microglia activation pattern and the course of degeneration in the retinal degeneration slow mouse.

BACKGROUND: In hereditary retinal degeneration, microglia cells become activated, migrate through the outer nuclear layer (ONL) and accumulate in the subretinal space. Although this inflammatory process is not likely to be responsible for the onset of photoreceptor apoptosis, cytotoxic substances secreted by activated microglia could potentially accelerate and perpetuate the degenerative process. Anti-inflammatory drugs have been shown to modulate the microglia response in neurodegenerative disorders and potentially ameliorate the disease progression in various animal model systems. In this study we wanted to test the impact of the most commonly used anti-inflammatory drugs (acetylsalicylate and prednisolone) on the microglia activation pattern, the rate of caspase-3-dependent photoreceptor apoptosis and the course of the degeneration in the retinal degeneration slow (rds) mouse retina. METHODS: 169 pigmented rds mice and 30 CBA wild-type mice were used for this study. The treatment groups were injected daily with either acetylsalicylate (200 mg/kg) or prednisolone (2 mg/kg) i.p. from day 0 up to 3 months. Animals were sacrificed at days 10, 14, 16, 18, 20, 30, 40, 60 and 90. Cryoprotected frozen sections were immunostained with F4/80 and cleaved caspase-3 antibodies. The main outcome measures were the total microglia count in the subretinal space, the total cleaved caspase-3-positive cells in the ONL and the averaged number of photoreceptor rows in the midperipheral retina. RESULTS: Neither acetylsalicylate nor prednisolone reduced subretinal microglia accumulation in the rds mouse degeneration model. Moreover, they aggravated migration and accumulation in the early time course. The apoptotic cascade started earlier and was more pronounced in both treatment groups compared to the control group. The pace of retinal degeneration was not reduced in the treatment groups compared to the untreated control. In contrast, acetylsalicylate did significantly accelerate the photoreceptor cell degeneration in comparison to the prednisolone (p < 0.001) and to the control group (p < 0.001). CONCLUSIONS: Acetylsalicylate and prednisolone do not decrease the microglia response in the rds mouse and are not neuroprotective. More research is needed to clarify the molecular mechanisms which lead to photoreceptor cell death and to elucidate the complex role of microglia in inherited retinal degeneration.

Long-term preservation of retinal function in the RCS rat model of retinitis pigmentosa following lentivirus-mediated gene therapy.

The Royal College of Surgeons (RCS) rat is a well-characterized model of autosomal recessive retinitis pigmentosa (RP) due to a defect in the retinal pigment epithelium (RPE). It is homozygous for a null mutation in the gene encoding , a receptor tyrosine kinase found in RPE cells, that is required for phagocytosis of shed photoreceptor outer segments. The absence of Mertk results in accumulation of outer segment debris. This subsequently leads to progressive loss of photoreceptor cells. In order to evaluate the efficacy of lentiviral-mediated gene replacement therapy in the RCS rat, we produced recombinant VSV-G pseudotyped HIV-1-based lentiviruses containing a murine Mertk cDNA driven by a spleen focus forming virus (SFFV) promoter. The vector was subretinally injected into the right eye of 10-day-old RCS rats; the left eye was left untreated as an internal control. Here, we present a detailed assessment of the duration and extent of the morphological rescue and the resulting functional benefits. We examined animals at various time points over a period of 7 months by light and electron microscopy, and electroretinography. We observed correction of the phagocytic defect, slowing of photoreceptor cell loss and preservation of retinal function for up to 7 months. This study demonstrates the potential of gene therapy approaches for the treatment of retinal degenerations caused by defects specific to the RPE and supports the use of lentiviral vectors for the treatment of such disorders.

Improvement of neuronal visual responses in the superior colliculus in Prph2(Rd2/Rd2) mice following gene therapy.

Inherited retinal degenerations are a major cause of blindness for which there are currently no effective therapies. Significant progress concerning in vivo gene transfer has allowed retardation of degeneration or retinal functional improvement in different animal models. To date, there has been no evaluation of the impact of these treatments on higher visual function, a critical step for validating gene therapy treatment strategies. Here, we have used adeno-associated (AAV2)-mediated gene transfer of Prph2 in the Prph2(Rd2/Rd2) mouse model. We then assessed higher visual function by recording from central visually responsive neurons in the superior colliculus and improvements were correlated in individual animals with retinal function (ERG) and histological and biochemical changes. Although gene replacement therapy only partially restores photoreceptor morphology, it results in a 300% increase of the visual cycle protein rhodopsin, leading to retinal function improvement (250% increase of b-wave amplitude) and significantly higher central visual responses (166% increase at 24 cd/m(2)). These findings suggest that gene replacement therapy leading to even relatively modest structural improvement may result in improved central visual function.

Stable rAAV-mediated transduction of rod and cone photoreceptors in the canine retina.

Recombinant adeno-associated virus (rAAV) vectors are attractive candidates for the treatment of inherited and acquired retinal disease. Although rAAV vectors are well characterized in rodent models, a prerequisite to their clinical application in human patients is the thorough evaluation of their efficacy and safety in intermediate animal models. In this study, we describe rAAV-2-mediated expression of GFP reporter gene in retinal cells following local vector delivery in dogs. Subretinal delivery of rAAV.CMV.GFP was performed unilaterally in eight normal dogs from 6 weeks of age. The area of retinal transduction was maximized by the optimization of surgical techniques for subretinal vector delivery by pars-plana vitrectomy and the use of fine-gauge subretinal cannulae to create multiple retinotomies. rAAV-2 vectors mediated efficient stable reporter gene expression in photoreceptors and retinal pigment epithelial cells. We found efficient transduction of cone photoreceptors in addition to rods in both the canine retina and after subretinal vector delivery in another intermediate animal model, the feline retina. GFP expression in dogs was confined to the area of the retinal bleb and was sustained in cells at this site for at least 18 months. Electroretinography demonstrated a modest reduction in global rod-mediated retinal function following subretinal delivery of rAAV.CMV.GFP. Three of the eight animals developed delayed-onset intraocular inflammation, in two cases associated with a serum antibody response to GFP protein. We conclude that rAAV-2 vectors mediate efficient sustained transgene expression in rod and cone photoreceptors following subretinal delivery in this intermediate animal model. The possibility of adverse effects including intraocular immune responses and reduced retinal function requires further investigation prior to clinical applications in patients.

Intraocular gene delivery of ciliary neurotrophic factor results in significant loss of retinal function in normal mice and in the Prph2Rd2/Rd2 model of retinal degeneration.

Intraocular delivery of a variety of neurotrophic factors has been widely investigated as a potential treatment for retinal dystrophy (RD). The most commonly studied factor, ciliary neurotrophic factor (CNTF), has been shown to preserve retinal morphology and to promote cell survival in a variety of models of RD. In order to evaluate CNTF as a potential treatment for RD, we used the Prph2(Rd2/Rd2) mouse. CNTF was expressed intraocularly using AAV-mediated gene delivery either by itself or, in a second treatment group, combined with AAV-mediated gene replacement therapy of peripherin2, which we have previously shown to improve photoreceptor structure and function. We confirmed in both groups of animals that CNTF reduces the loss of photoreceptor cells. Visual function, however, as assessed over a time course by electroretinography (ERG), was significantly reduced compared with untreated controls. Furthermore, CNTF gene expression negated the effects on function of gene replacement therapy. In order to test whether this deleterious effect is only seen when degenerating retina is treated, we recorded ERGs from wild-type mice following intraocular injection of AAV expressing CNTF. Here a marked deleterious effect was noted, in which the b-wave amplitude was reduced by at least 50%. Our results demonstrate that intraocular CNTF gene delivery may have a deleterious effect on the retina and caution against its application in clinical trials.

[Progress in somatic gene therapy of retinal degeneration in the animal model]. / Fortschritte in der somatischen Gentherapie von Netzhautdegenerationen am Tiermodell.

More than 60 genes responsible for human retinal dystrophies have already been identified. Most of them are either expressed in the photoreceptor or in the retinal pigment epithelium (RPE). Therefore these cells have become the target of new therapeutical strategies on a molecular level. The most promising approach at present is somatic gene therapy, which has been developed over the last years and the principle has now been established in animal models. For gene therapy of inherited retinal degeneration, as for gene therapy in general, gene transfer has to be proven to be not only efficient but also safe. This has recently been achieved using the adeno-associated virus (AAV) as a vector to express a therapeutic gene within the photoreceptor cell. It could be demonstrated in mouse and dog models of retinal degeneration that expression of the therapeutic transgene leads to anatomical and functional restitution of degenerating photoreceptors. A significant immune response to AAV has not been detected so far. In this paper the recent success of gene therapy of retinal degeneration in animal models is reviewed.

Kinetics of transgene expression in mouse retina following sub-retinal injection of recombinant adeno-associated virus.

Using confocal microscopy we have examined in detail the temporal and spatial pattern of green fluorescent protein expression following sub-retinal injection of recombinant adeno-associated virus (rAAV) in the mouse and have determined the effect of viral titre on the number and type of cells transduced. Our results suggest that some transgene expression occurs as early as three days after injection, and that transgene expression occurs beyond the area of retinal detachment. Vector titre appears to have a substantial effect on both transduction efficiency and the speed of onset of photoreceptor cell transduction. Our data suggests that we have not yet reached the limits of photoreceptor transduction efficiency using AAV vectors. An increase in titre could still lead to an improved transduction efficiency and faster onset of photoreceptor transduction. We failed to detect transfected cones even in areas where nearly 100% of the rods were transduced, but we found efficient and sustained RPE transduction.

[Biochemical stress monitoring during cataract surgery; phenylephrine 10% shows no changes in serum-catecholamines in comparison with phenylephrine 5%]. / Biochemisches Stressmonitoring in der Kataraktchirurgie: Phenylephrin 10% zeigt im Vergleich mit Phenylephrin 5% keine Veränderung der Serum-Katecholamin-Werte.

BACKGROUND: There is a controversy about the concentration of topical phenylephrine recommended for diagnostic or therapeutic mydriasis. Phenylephrine 10% leads to a faster and more pronounced mydriasis but cardio-vascular side-effects like hypertension and arrhythmia have been reported. A maximal pupillary dilatation is a prerequisite for successful cataract surgery. The aim of this study was to evaluate the risk-benefit ratio of phenylephrine 10% in comparison to 5% in the daily practice of the cataract-surgery unit in our clinic by clinical assessment and monitoring of biochemical stress parameters. PATIENTS AND METHODS: 30 informed and consenting patients were randomly allocated to 2 groups of equal size. After a single application of 2 drops of phenylephrine 5% in group 1 and 10% in group 2 respectively and 1 drops of cyclopentolate 1% with neutral pupil (time 0), an ECG was recorded and blood pressure, pulse, oxygen-saturation and pupil size were measured. Simultaneously a blood-sample was taken and the serum-catecholamines adrenaline and noradrenaline were determined by HPLC (High Pressure Liquid Chromatography). These measurements were repeated after 5, 10 and 30 minutes. RESULTS: The mean pupil area after 30 minutes in group 1 was 31.97 (+/- 0.43) mm2 compared to 45.72 (+/- 0.39) mm2 in group 2. Our data showed no other significant variation between the groups: neither clinical monitoring nor catecholamine measurements showed concentration-dependent patterns in blood pressure development or serum levels. No systemic cardiovascular effects were observed. CONCLUSION: These results demonstrate that a controlled application of phenylephrine 10%--under observation of contraindications--yields no increased risk for the occurrence of cardio-vascular side-effects in comparison with phenylephrine 5%. Therefore, we recommend the use of phenylephrine 10% in the described dosage as routine medication for cataract surgery.