C57BL/6J, DBA/2J, and DBA/2J.Gpnmb+ mice have different visual signal processing in the inner retina

To characterize differences in retinal ganglion cell (RGC) function in mouse strains relevant to disease models. C57BL/6J (B6) and DBA/2J (D2) are the two most common mouse strains; D2 has two mutated genes, tyrosinase-related protein 1 (Tyrp1) and glycoprotein non-metastatic melanoma protein B (Gpnmb), causing iris disease and intraocular pressure (IOP) elevation after 6 months of age that results in RGC degeneration, and is the most widely used model of glaucoma. DBA/2J.Gpnmb(+) (D2.Gpnmb(+)) is the wild type for the Gpnmb mutation and does not develop IOP elevation and glaucoma. Young (2-4 months of age) B6, D2, and D2.Gpnmb(+) mice (n=6 for each group) were tested with pattern electroretinogram (PERG) in response to different contrasts and spatial frequencies. PERG amplitude and latency dependencies on stimulus parameters (transfer functions) were established for each mouse strain, together with corresponding thresholds for contrast and spatial resolution. PERG analysis showed that B6, D2, and D2.Gpnmb(+) mice had comparable contrast threshold and spatial resolution. Suprathreshold spatial contrast processing, however, had different characteristics in the three strains. PERG amplitude and latency changes with increasing contrast were different between B6 and D2 as well as between D2 and D2.Gpnmb(+). B6, D2, and D2.Gpnmb(+) mice have different characteristics of PERG spatial contrast processing consistent with different mechanisms of contrast gain control. This may imply differences in the activity of underlying PERG generators and synaptic circuitry in the inner retina

Mutant NADH dehydrogenase subunit 4 gene delivery to mitochondria by targeting sequence-modified adeno-associated virus induces visual loss and optic atrophy in mice

Although mutated G11778A NADH ubiquinone oxidoreductase subunit 4 (ND4) mitochondrial DNA (mtDNA) is firmly linked to the blindness of Leber hereditary optic neuropathy (LHON), a bona fide animal model system with mutated mtDNA complex I subunits that would enable probing the pathogenesis of optic neuropathy and testing potential avenues for therapy has yet to be developed. The mutant human ND4 gene with a guanine to adenine transition at position 11778 with an attached FLAG epitope under control of the mitochondrial heavy strand promoter (HSP) was inserted into a modified self-complementary (sc) adeno-associated virus (AAV) backbone. The HSP-ND4FLAG was directed toward the mitochondria by adding the 23 amino acid cytochrome oxidase subunit 8 (COX8) presequence fused in frame to the N-terminus of green fluorescent protein (GFP) into the AAV2 capsid open reading frame. The packaged scAAV-HSP mutant ND4 was injected into the vitreous cavity of normal mice (OD). Contralateral eyes received scAAV-GFP (OS). Translocation and integration of mutant human ND4 in mouse mitochondria were assessed with PCR, reverse transcription-polymerase chain reaction (RT-PCR), sequencing, immunoblotting, and immunohistochemistry. Visual function was monitored with serial pattern electroretinography (PERG) and in vivo structure with spectral domain optical coherence tomography (OCT). Animals were euthanized at 1 year and processed for light and transmission electron microscopy. The PCR products of the mitochondrial and nuclear DNA extracted from infected retinas and optic nerves gave the expected 500 base pair bands. RT-PCR confirmed transcription of the mutant human ND4 DNA in mice. DNA sequencing confirmed that the PCR and RT-PCR products were mutant human ND4 (OD only). Immunoblotting revealed the expression of mutant ND4FLAG (OD only). Pattern electroretinograms showed a significant decrement in retinal ganglion cell function OD relative to OS at 1 month and 6 months after AAV injections. Spectral domain optical coherence tomography showed optic disc edema starting at 1 month post injection followed by optic nerve head atrophy with marked thinning of the inner retina at 1 year. Histopathology of optic nerve cross sections revealed reductions in the optic nerve diameters of OD versus OS where transmission electron microscopy revealed significant loss of optic nerve axons in mutant ND4 injected eyes where some remaining axons were still in various stages of irreversible degeneration with electron dense aggregation. Electron lucent mitochondria accumulated in swollen axons where fusion of mitochondria was also evident. Due to the UGA codon at amino acid 16, mutant G11778A ND4 was translated only in the mitochondria where its expression led to significant loss of visual function, loss of retinal ganglion cells, and optic nerve degeneration recapitulating the hallmarks of human LHON

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

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

Electrophysiological assessment of retinal ganglion cell function

The function of retinal ganglion cells (RGCs) can be non-invasively assessed in experimental and genetic models of glaucoma by means of variants of the ERG technique that emphasize the activity of inner retina neurons. The best understood technique is the Pattern Electroretinogram (PERG) in response to contrast-reversing gratings or checkerboards, which selectively depends on the presence of functional RGCs. In glaucoma models, the PERG can be altered before histological loss of RGCs; PERG alterations may be either reversed with moderate IOP lowering or exacerbated with moderate IOP elevation. Under particular luminance-stimulus conditions, the Flash-ERG displays components that may reflect electrical activity originating in the proximal retina and be altered in some experimental glaucoma models (positive Scotopic Threshold response, pSTR; negative Scotopic Threshold Response, nSTR; Photopic Negative Response, PhNR; Oscillatory Potentials, OPs; multifocal ERG, mfERG). It is not yet known which of these components is most sensitive to glaucomatous damage. Electrophysiological assessment of RGC function appears to be a necessary outcome measure in experimental glaucoma models, which complements structural assessment and may even predict it. Neuroprotective strategies could be tested based on enhancement of baseline electrophysiological function that results in improved RGC survival. The use of electrophysiology in glaucoma models may be facilitated by specifically designed instruments that allow high throughput, robust assessment of electrophysiological function. •Retinal ganglion cell function is measurable with electrophysiology in in-vivo glaucoma models.•PERG is the most specific and sensitive method.•Retinal ganglion cell function is a necessary outcome measure

The mouse pattern electroretinogram

Mouse models of optic nerve disease such as glaucoma, optic neuritis, ischemic optic neuropathy, and mitochondrial optic neuropathy are being developed at increasing rate to investigate specific pathophysiological mechanisms and the effect of neuroprotective treatments. The use of these models may be greatly enhanced by the availability of non-invasive methods able to monitor retinal ganglion cell (RGC) function longitudinally such as the Pattern Electroretinogram (PERG). While the use of the PERG as a tool to probe inner retina function in mammals is known since 25 years, relatively less information is available for the mouse. Here, the PERG technique and the main applications in the mouse are reviewed

Electrophysiological testing in glaucoma

Glaucoma is a complex disease with multifactorial etiology; at present, the intraocular pressure is the only treatable risk factor. If detected early, disease progression can frequently be arrested or slowed with medical and surgical treatment. Therefore, key aspects of glaucoma management are early detection of disease and monitoring of its progression, including evaluation of treatment effectiveness. In this context, electrophysiological testing may provide specific and unique information. The past few years have seen an increasing interest in electrophysiological testing in glaucoma. This article will provide a review of recent developments in the field

FOLLOW-UP-STUDY WITH PATTERN ERG IN OCULAR HYPERTENSION AND GLAUCOMA PATIENTS UNDER TIMOLOL MALEATE TREATMENT

1. Pattern-reversal (8 Hz) electroretinograms (PERGs) to sinusoidal gratings of variable spatial frequency (0.6-4.8 c/deg) have been recorded in 28 normal subjects and in 20 patients with ocular hypertension (OHT; n = 11) or early open-angle glaucoma (EOAG, n = 9) who were under timolol maleate treatment during a 24-month clinical follow-up. 2. Visual fields and optic disk appearances did not vary significantly in all patients over the follow-up period. Under treatment intraocular pressure (IOP) ranged 15-25 mmHg in both OHT and EOAG patients. 3. Two PERG recording were obtained for each patient: the first after 12 months and the second after 24 months of treatment and clinical follow-up. At the first recording, the mean PERG amplitude was significantly reduced, as compared to age-matched controls, on both OHT (1 and 1.4 c/deg) and EOAG (0.6-1.4 c/deg) patients. PERG amplitudes tended to be mom reduced in EOAG than OHT patients and did not correlate with the corresponding IOP values before or under treatment. 4. At the second PERG recording the average PERG amplitude was not significantly different from the first control at any of the tested spatial frequencies. However, some patients showed amplitude decreases (2 OHT and 2 EOAG) or increases (3 OHT and 3 EOAG) which exceeded the range of normal test-retest variability. 5. In individual OHT and EOAG patients, amplitude differences between the 1st and 2nd PERG recording (2nd - 1st test) were negatively correlated (P < 0.05) with the corresponding IOP values recorded during the follow-up period. This relationship was statistically significant only at intermediate spatial frequencies (1-1.4 c/deg). The amount of PERG reductions with increasing IOP tended to be larger in EOAG than in OHT patients. 6. These results indicate that there are selective PERG changes associated with IOP control in treated OHT and EOAG patients