64 research outputs found

    Comparison of different gene-therapy methods to treat Leber hereditary optic neuropathy in a mouse model

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    IntroductionTherapies for Leber hereditary optic neuropathy (LHON), in common with all disorders caused by mutated mitochondrial DNA, are inadequate. We have developed two gene therapy strategies for the disease: mitochondrial-targeted and allotopic expressed and compared them in a mouse model of LHON.MethodsA LHON mouse model was generated by intravitreal injection of a mitochondrialtargeted Adeno-associated virus (AAV) carrying mutant human NADH dehydrogenase 4 gene (hND4/m.11778G>A) to induce retinal ganglion cell (RGC) degeneration and axon loss, the hallmark of the human disease. We then attempted to rescue those mice using a second intravitreal injection of either mitochondrial-targeted or allotopic expressed wildtype human ND4. The rescue of RGCs and their axons were assessed using serial pattern electroretinogram (PERG) and transmission electron microscopy.ResultsCompared to non-rescued LHON controls where PERG amplitude was much reduced, both strategies significantly preserved PERG amplitude over 15 months. However, the rescue effect was more marked with mitochondrial-targeted therapy than with allotopic therapy (p = 0.0128). Post-mortem analysis showed that mitochondrial-targeted human ND4 better preserved small axons that are preferentially lost in human LHON.ConclusionsThese results in a pre-clinical mouse model of LHON suggest that mitochondrially-targeted AAV gene therapy, compared to allotopic AAV gene therapy, is more efficient in rescuing the LHON phenotype

    Modeling Retinal Ganglion Cell Dysfunction in Optic Neuropathies

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    As in glaucoma and other optic neuropathies cellular dysfunction often precedes cell death, the assessment of retinal ganglion cell (RGC) function represents a key outcome measure for neuroprotective strategies aimed at targeting distressed but still viable cells. RGC dysfunction can be assessed with the pattern electroretinogram (PERG), a sensitive measure of electrical activity of RGCs that is recorded non-invasively in human subjects and mouse models. Here, we offer a conceptual framework based on an intuitive state-transition model used for disease management in patients to identify progressive, potentially reversible stages of RGC dysfunction leading to cell death in mouse models of glaucoma and other optic neuropathies. We provide mathematical equations to describe state-transitions with a set of modifiable parameters that alter the time course and severity of state-transitions, which can be used for hypothesis testing and fitting experimental PERG data. PERG dynamics as a function of physiological stimuli are also used to differentiate phenotypic and altered RGC response dynamics, to assess susceptibility to stressors and to assess reversible dysfunction upon pharmacological treatment

    Using Noninvasive Electrophysiology to Determine Time Windows of Neuroprotection in Optic Neuropathies

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    The goal of neuroprotection in optic neuropathies is to prevent loss of retinal ganglion cells (RGCs) and spare their function. The ideal time window for initiating neuroprotective treatments should be the preclinical period at which RGCs start losing their functional integrity before dying. Noninvasive electrophysiological tests such as the Pattern Electroretinogram (PERG) can assess the ability of RGCs to generate electrical signals under a protracted degenerative process in both clinical conditions and experimental models, which may have both diagnostic and prognostic values and provide the rationale for early treatment. The PERG can be used to longitudinally monitor the acute and chronic effects of neuroprotective treatments. User-friendly versions of the PERG technology are now commercially available for both clinical and experimental use

    IOP-dependent retinal ganglion cell dysfunction in glaucomatous DBA/2J mice. Invest Ophthalmol Vis Sci.

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    PURPOSE. To characterize the effect of postural IOP elevation and pharmacological IOP lowering on retinal ganglion cell (RGC) function in the DBA/2J mouse model of glaucoma. METHODS. Four groups of DBA/2J mice (3 months old, n ϭ 7; 5 months old, n ϭ 7; 10 months old, n ϭ 7; and 11 months old, n ϭ 8) were anesthetized by intraperitoneal injection (0.6 mL/kg) of a mixture of ketamine (42.8 mg/mL), xylazine (8.5 mg/mL), and acepromazine (1.4 mg/mL). IOP and pattern electroretinogram (PERG) were sequentially measured with mice at 0°(horizontal), 60°head-down, and again at 0°. IOP and PERG were also measured before and after intraperitoneal mannitol 25% (2.5 g/kg) administration with mice in a horizontal position. RESULTS. The head-down position induced reversible IOP elevations of 32% to 38% in all age groups (P Ͻ 0.01), and age-dependent reductions of PERG amplitude (3 months: ϩ3%; 5 months: Ϫ47%, P Ͻ 0.01; and 10 months: Ϫ65%, P Ͻ 0.01). Administration of mannitol to 11-month-old mice resulted in a reduction in IOP of approximately 38% (P Ͻ 0.01) and a PERG amplitude improvement of approximately 83% (P Ͻ 0.001). IOP and PERG amplitude changes were inversely correlated (10 months head-down r 2 ϭ 0.58, P Ͻ 0.001; 10-month-old mannitol r 2 ϭ 0.41, P Ͻ 0.001). For all conditions, the lightadapted flash ERG was unaltered. CONCLUSIONS. In the DBA/2J mouse, RGC susceptibility to artificial IOP elevation increases with age. Abnormal RGC function in older mice may be improved with IOP lowering. Evaluation of PERG changes in response to artificial IOP modulation may represent a powerful tool to assess noninvasively RGCs' susceptibility to IOP insult in genetically distinct mouse models of glaucoma. (Invest Ophthalmol Vis Sci. 2007;48:4573-4579) DOI:10.1167/iovs.07-0582 M ouse models with genetic alterations relevant to glaucoma are receiving increasing attention in an effort to gain better understanding of the complex nature of the disease and to design specific treatments to prevent death of retinal ganglion cells (RGCs) and their axons. 9 -12 The noninvasive nature of the PERG allows serial recordings as a function of changing conditions (e.g., age, IOP levels). Using PERG, we have been able to characterize the natural history of RGC dysfunction and its association with IOP in a 12-month longitudinal study of BDA/2J mice (Saleh M et al. IOVS 2007;48:ARVO E-Abstract 210). On average, the IOP increased from 14 to 18 mm Hg between 2 and 6 months and then more steeply, leveling off by 11 months at approximately 28 mm Hg. After 3 months, the PERG amplitude decreased progressively with age to approach the noise level at approximately 10 to 11 months. The time-course of IOP elevation and PERG amplitude reduction were closely associated. Histologic analysis of eyes with abolished PERG showed that the retinal nerve fiber layer (RNFL) had lost approximately 40% of its normal thickness and the cone-flash ERG did not significantly change (Saleh M et al. IOVS 2007;48:ARVO E-Abstract 210). Taken together, these results indicate that DBA/2J mice have progressive functional damage in the inner retina (abnormal PERG) but not in the outer retina (normal flash-ERG) that seems to precede anatomic damage of the optic nerve (relatively spared RNFL). Recently, Aihara et al. 15 Preliminary results of this study have been presented earlier in abstract form (Nagaraju M et al. IOVS 2007;48:ARVO E-Abstract 212). 16 METHODS Animals and Husbandry A total of 29 DBA/2J mice of different ages-3 months, n ϭ 7 (7 eyes); 5 months, n ϭ7 (7 eyes); 10 months, n ϭ 7 (7 eyes); and 11 months, n ϭ 8, (16 eyes)-obtained from Jackson Laboratories (Bar Harbor, ME) were tested. The mice were maintained in a cyclic light environment (12-hour 50 lux, 12-hour dark) and fed ad libitum. For both IOP and PERG measurements, the mice were weighed and anesthetized with intraperitoneal injection (0.5-0.7 mL/kg) of a mixture of ketFrom the Bascom Palme

    The visual physiology of the wild type mouse determined with pattern VEPs

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    AbstractGenetically manipulated mice are important tools for studies on plasticity and degeneration/regeneration in the visual system. However, a description of the basic properties of the visual performance of the wild type mouse is still lacking. To characterize the visual physiology of the wild type (C57BL/6J) mouse we recorded Visual Evoked Potentials (VEPs) from the primary visual cortex. As compared to behavioral methods, VEPs may have the advantage that different aspects of vision can be screened readily and simultaneously in the same animals, including those with poor visual behavior due to motor or learning deficits. Local VEP responses to patterned visual stimuli have been recorded from the binocular visual cortex of anesthetized mice. Spatial (visual acuity, contrast threshold) and temporal (temporal function, response latency, motion sensitivity) aspects of VEPs were evaluated. The mouse VEP acuity was 0.6 c/deg, which is comparable to the behavioral visual acuity. The VEP peak contrast threshold was 5% (no behavioral data are available). Cortical representation of visual coordinates and cortical magnification factor corresponded to those previously reported using single cell recordings. Laminar analysis of VEPs indicated a dipole source in the supragranular layers of the visual cortex as a major response generator. VEPs showed contribution from both eyes, although biased strongly towards the eye contralateral to the recorded cortex. Results provide a comprehensive framework for characterizing visual phenotypes of a variety of transgenic mice

    Longitudinal evaluation of retinal ganglion cell function and

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    PURPOSE. To characterize progressive changes of retinal ganglion cell (RGC) function and intraocular pressure (IOP) in the DBA/2J mouse model of spontaneous glaucoma. METHODS. Serial pattern electroretinograms (PERGs) and IOPs measures were obtained from both eyes of 32 anesthetized DBA/2J mice over an age range of 2 to 12 months at 1-month intervals. Cone-driven flash-ERGs (FERGs) were also recorded. The endpoint was defined as the age at which the PERG amplitude reached the noise level in at least one eye. At that point, both eyes were histologically processed to evaluate the thickness of the retinal fiber layer (RNFL). RESULTS. IOP increased moderately between 2 and 6 months (ϳ14 -17 mm Hg) and then more steeply, until it leveled off at approximately 28 mm Hg by 9 to 11 months. The mean PERG amplitude decreased progressively after 3 months of age to reach the noise level (85% reduction of normal amplitude) at approximately 9 to 12 months in different animals. When the PERG was at noise level, the RNFL showed a relatively smaller reduction (40%) in normal thickness. The FERG displayed minor changes throughout the observation period. IOP and PERG changes were highly correlated (r 2 ϭ 0.51, P Ͻ 0.001). CONCLUSIONS. Results indicate that inner retina function in DBA/2J mice progressively decreases after 3 months of age, and it is nearly abolished by 10 to 11 months, whereas outer retina function shows little change and the RNFL thickness is relatively spared. This result suggests that surviving RGCs may not be functional. Progression of inner retinal dysfunction is strongly associated with increased IOP. (Invest Ophthalmol Vis Sci. 2007;48:4564 -4572