Tolerance-inducing dose of 3-nitropropionic acid modulates bcl-2 and bax balance in the rat brain: A potential mechanism of chemical preconditioning

Many studies have reported ischemia protection using various preconditionin g techniques, including single dose 3-nitropropionic acid (3-NPA), a mitoch ondrial toxin. However, the cellular signal transduction cascades resulting in ischemic tolerance and the mechanisms involved in neuronal survival in the tolerant state still remain unclear. The current study investigated the mRNA and protein expression of the antiapoptotic bcl-2 and the proapoptoti c bar, two antagonistic members of the bcl-2 gene family, in response to a single dose of 3-NPA, to global cerebral ischemia-reperfusion, and to the c ombination of both 3-NPA-pretreatment and subsequent global cerebral ischem ia-reperfusion. Brain homogenates of adult Wistar rats (n = 25) were analyz ed for bcl-2 and bar mRNA expression using a new highly sensitive and quant itative polymerase chain reaction (PCR) technique that allows real-time flu orescence measurements of the PCR product (LightCycler; Roche Diagnostics, Mannheim, Germany). Animals for mRNA analysis received 3-NPA (20 mg/kg, int raperitoneal; "chemical preconditioning") or vehicle (normal saline), and w ere either observed for 24 plus 3 hours or were subjected to 15 minutes of global cerebral ischemia 24 hours after the pretreatment and observed for 3 hours of reperfusion. Immunohistochemistry was applied to serial brain sec tions of additional rats (n = 68) to determine amount and localization of t he respective Bcl-2 and Bar protein expression in various brain areas. One set of animals was injected with 3-NPA and observed for 3, 12, 24, and 96 h ours; a second set was exposed to 15 minutes global cerebral ischemia, 3, 1 2, and 24 hours reperfusion; and a third set was pretreated with 3-NPA or s aline 24 hours before the ischemic brain insult and observed for 96 hours o f reperfusion. The authors found single dose 3-NPA treatment to be associat ed with an elevated bcl-2:bax ratio (increased bcl-2 expression, decreased bar expression), both on the transcriptional (mRNA) and the translational ( protein) level. The differential influence of 3-NPA was maintained during e arly recovery from global cerebral ischemia (3 hours), when 3-NPA pretreate d animals showed higher bcl-2 and lower bar mRNA levels compared with rats with saline treatment. Respective changes in protein expression were locali zed predominately in neurons vulnerable to ischemic damage. Compared with b aseline, Bcl-2 protein was significantly higher in surviving neurons at 96 hours after the insult, whereas Bar protein remained unchanged. However, at this late time of postischemic recovery (96 hours), the protein expression pat tern of surviving neurons was not different between animals with and w ithout 3-NPA pretreatment. To the authors' knowledge, the current study is the first report on the differential expression of pro- and antiapoptotic g enes after a single, nonlethal dose of 3-NPA. The current results suggest a lterations in the balance between pro- and antiapoptotic proteins as a pote ntial explanation for the reported protection provided by chemical precondi tioning using 3-NPA in rats.