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D.T. Nguyen, A.M. Hayden, F.N. Ross–Cisneros, A.A. Sadun; Activated Caspase 3 as a Marker for Apoptosis in Optic Nerves From HIV Positive Patients . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5626.
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Our laboratory has previously studied AIDS associated optic nerve axonal degeneration with various markers including TNF alpha, IL–1beta, and IL–6. Activated caspase 3 has been shown to be an early index for apoptosis. This study is to systematically evaluate activated caspase 3 as a marker for apoptosis in axons and surrounding cells of optic nerves in necropsy specimens obtained from chronically infected HIV patients.
Tissues were fixed in 10% neutral buffered formalin, processed, and embedded in paraffin. Cross sections of 6 control optic nerves, 10 retrobulbar optic nerves from HIV patients, and intraductal breast carcinoma positive controls were used. The sections were labeled using either primary antibody consisting of rabbit anti human activated caspase 3 or buffer solution without primary antibody. Secondary goat anti rabbit antibody conjugated to horseradish peroxidase was then bound. The primary–secondary antibody complex was then color developed with diaminobenzidine (DAB) chromogen and counterstained with hematoxylin. Apoptotic index was calculated as a ratio of the number of apoptotic cells to the total number of cells examined.
The HIV optic nerves stained positive for activated caspase 3, as did intraductal breast carcinoma tissue. Our negative control optic nerves also stained positive for caspase 3 although slightly less so. The apoptotic index for the HIV optic nerves and control optic nerves were 0.286 and 0.193 respectively. On low powered microscopy, the HIV optic nerves stained for caspase 3 in each cross section. At high magnification, there were cells with no DAB chromogen staining adjacent to cells with positive staining. The stained cells undergoing early stages of apoptosis appeared to be astrocytes. None of the slides treated with buffer solution followed by secondary antibody showed any nonspecific staining of tissues.
Activated caspase 3 is an enzyme that is upregulated in cells undergoing apoptosis and found in neuronal degeneration in HIV associated dementia. By labeling for activated caspase 3, we demonstrated that apoptotic events may also take place in optic nerves of patients chronically infected with the HIV virus. Understanding the signals for apoptosis may lead to new treatment modalities for HIV associated optic neuropathy.
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