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An Optic Nerve Crush Injury Murine Model to Study Retinal Ganglion Cell Survival
Published on: April 25, 2011
[Retinal neuronal cell death: molecular mechanism and neuroprotection]
1Department of Ophthalmology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621, Japan.
Abstract:
In retinitis pigmentosa, retinal detachment, age-related macular degeneration, and glaucoma, retinal neuronal cells are damaged by a common mechanism, apoptosis. Because apoptosis is an active process that requires de novo expression of a "death message", this process can be controlled by inhibiting the expression of the "death message". We first studied whether a retinal ischemia-reperfusion model can be used as a model for retinal neuronal apoptosis. In the retinal ischemia-reperfusion injuries, typical features of apoptosis, including TUNEL-positive cells, DNA ladder formation, and ultrastructural features of apoptosis were found. Using the model, systematic research to identify the "death message" was done by DNA microarray analysis. About 200 messages were found to be up- or down-regulated during the process of retinal ischemia-reperfusion. These genes were divided into four groups: (1) transcription factor genes, (2) cell cycle-related genes, (3) reactive oxygen scavenger genes and (4) molecular chaperon genes. The possible roles of such genes in neuronal apoptosis following retinal ischemia-reperfusion injury were studied. In the model, reactive oxygen species produced by reperfusion was found to generate lipid peroxides and induced up-regulation of a transcription factor, c-Jun, that further induced aberrant expression of cell cycle-related genes such as cyclin D1 in amacrine cells. However, because no controlled expression of cell cycle-related genes takes place in retinal neurons, amacrine cells died by a G1 arrest mechanism. On the other hand, horizontal cells never expressed cyclin D1 and the cells were found to die by necrosis. The study revealed a possible mechanism of retinal neuronal apoptosis and it also became apparent that different types of neurons use different "death messages". Furthermore, the possibility that inhibition of a "death message" sometimes induces necrosis rather than apoptosis was shown. This means that we need to try inhibition of the death mechanism upstream rather than downstream. Administration of thioredoxin, an endogenous reactive oxygen species that blocks generation of lipid peroxides and thus inhibits the death process upstream, was found to be neuroprotective against retinal ischemia-reperfusion injury. Aberrant expression of c-Jun and cyclin D1 was down-regulated by the treatment. Possible roles of caspases were also studied by using the ischemia-reperfusion injury, RCS rat, and excessive light exposure damage in wild type and caspase-1 deficient mice. Also, application of adeno-associated virus that carries Bcl-xL was tested to find possible neuroprotective effects on RCS rats. Our studies showed that caspase-1 played a more important role in the retinal photoreceptors and caspase-3 was important in neurons in the inner nuclear layer. Caspase-2 was found to be a major caspase in the retinal ganglion cell layer. In agreement with the findings, caspase-1 deficient mice showed less prominent light damage than wild type mice. Gene therapy by Bcl-xL was effective to protect retinal photoreceptor damage in RCS rats.
Insights
Retinal neuronal cell death in diseases like glaucoma involves apoptosis, an active process. This study identified key "death messages" and found upstream inhibition, like using thioredoxin, offers neuroprotection against retinal injury.
Area of Science:
- Ophthalmology
- Neuroscience
- Molecular Biology
Context:
- Retinal neuronal cell death underlies conditions such as retinitis pigmentosa, retinal detachment, age-related macular degeneration, and glaucoma.
- Apoptosis, a programmed cell death pathway, is a common mechanism in these retinal diseases.
- Understanding the molecular triggers of apoptosis is crucial for developing neuroprotective strategies.
Purpose:
- To investigate retinal ischemia-reperfusion injury as a model for studying retinal neuronal apoptosis.
- To identify the specific molecular "death messages" involved in retinal neuronal apoptosis using DNA microarray analysis.
- To explore upstream inhibition strategies for neuroprotection and investigate the roles of specific caspases in retinal cell death.
Summary:
- Retinal ischemia-reperfusion injury exhibits typical apoptotic features, allowing for the identification of approximately 200 differentially expressed genes.
- Key findings indicate reactive oxygen species induce c-Jun and cyclin D1, leading to amacrine cell apoptosis via G1 arrest, while horizontal cells undergo necrosis.
- Upstream inhibition of lipid peroxide generation with thioredoxin demonstrated neuroprotective effects, down-regulating c-Jun and cyclin D1 expression.
Impact:
- This research elucidates a potential mechanism of retinal neuronal apoptosis and highlights that different neuron types may utilize distinct "death messages".
- The study suggests that inhibiting apoptosis upstream, rather than downstream, may be a more effective neuroprotective strategy.
- Findings on caspase roles (caspase-1 in photoreceptors, caspase-3 in inner nuclear layer, caspase-2 in ganglion cell layer) and successful Bcl-xL gene therapy in rats provide avenues for future therapeutic interventions.
