Quantitative Real-Time RT—PCR Analysis of Inflammatory Gene Expression Associated with Ischemia—Reperfusion Brain Injury

Abstract

Ischemia-reperfusion brain injury initiates an inflammatory response involving the expression of adhesion molecules and cytokines, some of which are regulated by the nuclear transcription factor NF-kappaB. In this study the authors examined mRNA expression levels for several important genes associated with inflammation at five time points (3, 6, 12, 24, and 72 hours) after transient middle cerebral artery occlusion (MCAO) in Sprague-Dawley rats. A sensitive and quantitative technique (TaqMan real-time QRT-PCR) was used to simultaneously measure mRNA levels for key cell adhesion molecules and inflammatory cytokines. Gene expression increased significantly in the injured hemisphere for interleukin (IL)-1beta (12-fold increase at 24 hours), IL-6 (25-fold increase at 6 hours) and ICAM-1 (4-fold increase at 24 hours), and the interhemispheric differences for these genes were significant for every time point examined (P < 0.05 for all values). Tumor necrosis factor-alpha mRNA was upregulated in the injured versus uninjured hemisphere from 3 to 24 hours (5-fold increase at 6 hours), while E-selectin showed a significant increase in mRNA levels from 6 to 24 hours after MCAO (10-fold increase at 6 hours) (P < 0.05 for all values). VCAM-1 mRNA levels did not respond differentially to injury at any time point between the two brain hemispheres. At all time points examined, activated NF-kappaB immunoreactivity was observed in cells throughout the infarct-damaged tissue. These results are consistent with the proinflammatory properties of the induced molecules, which are involved in the initiation of the inflammatory cascade, and may thus contribute to secondary cellular responses that lead to further brain damage.

Identification of Cathepsin B as a Mediator of Neuronal Death Induced by Aβ-activated Microglial Cells Using a Functional Genomics Approach

“…shown to protect against ischemia-induced neuronal damage (55), which is at least in part due to inflammatory and immunological reactions (56,57). In the present study, CA-074, an irreversible cathepsin Bspecific inhibitor with very low membrane permeability, completely abolished the toxicity in the conditioned medium released by activated BV2 cells.…”

A Quantitative Study of Microglial—Macrophage Synthesis of Tumor Necrosis Factor during Acute and Late Focal Cerebral Ischemia in Mice

“…Regarding the size of the ischemia-induced increase in TNF mRNA expression, we in the present study observed a 58-fold increase in the level of TNF mRNA in mice with 12 hours postsurgical survival relative to unoperated control mice. This increase is larger than the 28-fold increase observed in rats at 12 hours after induction of pMCAO (Wang et al, 2001), and the 20-fold increase observed in rats 6 hours after a 2-hour episode of transient MCAO (Berti et al, 2002). The data on TNF protein is more sparse, the most thorough study being by Buttini et al (1996), who used Western blot and immunohistochemistry to demonstrate TNF from 8 hours to 1 day after induction of pMCAO in the rat.…”

“…In studies of transient MCAO in rats, TNF mRNA levels have been observed to peak at 3 hours (Wang et al, 1994) or 6 hours (Berti et al, 2002), whereas in pMCAO TNF mRNA levels were observed to peak either at 3 hours (Buttini et al, 1996), 6 hours (Kim et al, 2002), or 12 hours (Liu et al, 1994). Regarding the size of the ischemia-induced increase in TNF mRNA expression, we in the present study observed a 58-fold increase in the level of TNF mRNA in mice with 12 hours postsurgical survival relative to unoperated control mice.…”

Activation of Toll-like receptor 4 signaling contributes to hippocampal neuronal death following global cerebral ischemia/reperfusion

“…Inflammatory processes are known to be associated with the acute cerebral ischemic condition, including the activation of resident brain cells such as microglial cells localized within the ischemic region, and the rapid synthesis of cytokines and chemokines (Barone and Feuerstein, 1999). An increased level of pro-inflammatory cytokines is an early feature of acute brain injury induced by clinical and experimental cerebral ischemia (Berti et al, 2002;Zaremba and Losy, 2004). To investigate whether TLR4 deficiency will prevent the production of inflammatory cytokines following GCI/R, we examined IL-6, TNFα, Fas ligand and high mobility group box 1 (HMGB1) in HF tissue from TLR4 -/-and WT mice.…”