Granzyme-b is involved in mediating post-ischemic neuronal death during focal cerebral ischemia in rat model

• Published in: Neuroscience 2010-02, Vol.165 (4), p.1203-1216
• Main Authors: Chaitanya, G.V, Schwaninger, M, Alexander, J.S, Babu, P. Prakash
• Format: Article
• Language: English
• Subjects: Animals; Apoptosis - physiology; Biological and medical sciences; Brain - enzymology; Brain - physiopathology; Brain Ischemia - enzymology; Brain Ischemia - physiopathology; CD8 Antigens - metabolism; cell death; Cell Death - physiology; cerebral ischemia; cytotoxic T lymphocytes; Disease Models, Animal; Fundamental and applied biological sciences. Psychology; granzyme-b; Granzymes - metabolism; Infarction, Middle Cerebral Artery - enzymology; Infarction, Middle Cerebral Artery - physiopathology; Male; Medical sciences; Necrosis - enzymology; Necrosis - physiopathology; Nerve Degeneration - enzymology; Nerve Degeneration - physiopathology; Neurology; neurons; Neurons - enzymology; Neurons - physiology; PC12 Cells; Random Allocation; Rats; Rats, Wistar; T-Lymphocytes, Cytotoxic - enzymology; T-Lymphocytes, Cytotoxic - physiology; Time Factors; TUNEL; Vascular diseases and vascular malformations of the nervous system; Vertebrates: nervous system and sense organs
• ISSN: 0306-4522; 1873-7544
• DOI: 10.1016/j.neuroscience.2009.10.067

Abstract Although peripheral immune cells infiltrate ischemic infarct tissue and elicit immune injury, the role of Cytotoxic T Lymphocytes (CTLs) and the toxins they release in mediating neuronal death is not well understood. Granzyme-b (Gra-b), a serine protease found in the cytoplasmic granules of CTLs and natural killer cells, plays an important role in inducing target cell death by activating several caspases and by initiating caspase-independent pathways that contribute to target cell death. To determine if CTLs and Gra-b are involved in post-ischemic cerebral cell death; we investigated the role of CD8+ CTLs and Gra-b in ischemic rat brain infarct after transient middle cerebral artery occlusion (tMCAO) and in sham-operated animals. We observed that CTLs infiltrate the ischemic infarct within 1 h of reperfusion. There was a significant increase in Gra-b levels in the ischemic region starting from 1 h until 3 day which correlated with increased levels of chemokines (IP-10/CXCL10, IL-2) and TNF-α. Co-immunoprecipitation experiments show that Gra-b interacts with Bid, PARP, and caspase-3 in ischemic samples. Immunofluorescence analysis of Gra-b and TUNEL showed that Gra-b is present both in apoptotic and necrotic cells. Triple immunostaining further confirmed that the Gra-b positive degenerating cells were neurons. CTLs in close spatial proximity to degenerating neurons, increased levels of Gra-b, localization in neurons positive for TUNEL, and interaction with other pro-apoptotic proteins indicate that Gra-b and CTLs play a significant role in neuronal death following cerebral ischemia in the rat brain after tMCAO. Based on the above findings we support our hypothesis that Gra-b secreted from activated CTLs might be involved in aggravating post-ischemic damage by mediating neuronal death.