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Single dose 3-nitropropionic acid (3-NPA) 24 hr before global ischemia improves neuronal survival in both, neocortex and hippocampus ('chemical preconditioning'). Neuronal survival after transient global ischemia requires new protein synthesis during recovery, especially of those with anti-apoptotic function. Bcl-2-protein is expressed in neurons that survive cerebral ischemia and may parallel the time course of tolerance after ischemic preconditioning. With this study we examined whether differences in bcl-2-protein expression compared to baseline may be involved in the induction of ischemic tolerance using 3-NPA. Male Wistar rats received either a single intraperitoneal (i.p.) dose of 3-NPA (20 mg/kg), and were observed for 3 (n = 4), 12 (n = 5) or 24 hours (n = 5) or the same amount of vehicle and were observed for 24 h (n = 8, controls). Immunohistochemistry allowed to compare the intensity of bcl-2 immunoreactivity at three subsequent time points in hippocampus, dentate gyrus and parietal neocortex with that of control animals. A single dose of 3-NPA caused a significant increase of bcl-2 protein immunoreactivity in hippocampal neurons, i.e. CA 1 (5 out of 5 animals, p = 0.003), CA 3 (5/5, p = 0.003), CA 4 (4/5, p = 0.025), and neocortex (5/5, p = 0.004), in a time dependent manner over a period of 24 hr after injection. Neuronal bcl-2 protein expression in CA 2 and dentate gyrus remained unchanged. The data suggest a possible role of bcl-2-protein in chemical induction of ischemic tolerance using a single subtoxic dose of 3-NPA. Bcl-2-protein expression may be initiated by increased levels of reactive oxygen species (ROS) after 3-NPA administration, as shown by others. Additional bcl-2 protein may then be available to (1) control postischemic ROS burst, (2) protect the mitochondrial membranes, and (3) inhibit pro-apoptotic mechanisms.

Many studies have reported ischemia protection using various preconditioning techniques, including single dose 3-nitropropionic acid (3-NPA), a mitochondrial 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 proapoptotic bax. 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 combination of both 3-NPA-pretreatment and subsequent global cerebral ischemia-reperfusion. Brain homogenates of adult Wistar rats (n = 25) were analyzed for bcl-2 and bax mRNA expression using a new highly sensitive and quantitative polymerase chain reaction (PCR) technique that allows real-time fluorescence measurements of the PCR product (LightCycler; Roche Diagnostics, Mannheim, Germany). Animals for mRNA analysis received 3-NPA (20 mg/kg, intraperitoneal; "chemical preconditioning") or vehicle (normal saline), and were 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 sections of additional rats (n = 68) to determine amount and localization of the respective Bcl-2 and Bax protein expression in various brain areas. One set of animals was injected with 3-NPA and observed for 3, 12, 24, and 96 hours; a second set was exposed to 15 minutes global cerebral ischemia, 3, 12, and 24 hours reperfusion; and a third set was pretreated with 3-NPA or saline 24 hours before the ischemic brain insult and observed for 96 hours of reperfusion. The authors found single dose 3-NPA treatment to be associated with an elevated bcl-2:bax ratio (increased bcl-2 expression, decreased bax expression), both on the transcriptional (mRNA) and the translational (protein) level. The differential influence of 3-NPA was maintained during early recovery from global cerebral ischemia (3 hours), when 3-NPA pretreated animals showed higher bcl-2 and lower bax mRNA levels compared with rats with saline treatment. Respective changes in protein expression were localized predominately in neurons vulnerable to ischemic damage. Compared with baseline, Bcl-2 protein was significantly higher in surviving neurons at 96 hours after the insult, whereas Bax protein remained unchanged. However, at this late time of postischemic recovery (96 hours), the protein expression pattern of surviving neurons was not different between animals with and without 3-NPA pretreatment. To the authors' knowledge, the current study is the first report on the differential expression of pro- and antiapoptotic genes after a single, nonlethal dose of 3-NPA. The current results suggest alterations in the balance between pro- and antiapoptotic proteins as a potential explanation for the reported protection provided by chemical preconditioning using 3-NPA in rats.

Temperature control during experimental ischemia continues to be of major interest. However, if exposure of brain tissue is necessary during the experiment, regional heat loss may occur even when the core temperature is maintained. Furthermore, valid non-invasive brain temperature monitoring is difficult in small rodents. This paper describes a method for both monitoring and maintenance of brain temperature during small animal preparations in a stereotaxic frame. The device used includes an ear-bar thermocouple probe and a small near-infrared radiator. The new equipment permitted to maintain peri-ischemic brain temperature at a desired level while carrying out non-invasive continuous recordings of cerebral blood flow (laser Doppler-flowmetry) and of electrical brain function (EEG). In contrast, without extracranial heat application, superficial and basal brain temperatures decreased during global cerebral ischemia by 4.1 +/- 0.1 and 4.6 +/- 0.4 degrees C (mean +/- SEM), respectively, returning to baseline values at 15-30 min of reperfusion while rectal (core) temperature remained stable at baseline values. The ear-bar thermocouple probe (tympanic membrane) reliably reflected basal brain temperature, and temperature in superficial brain areas correlated well with that in the temporal muscle. Our data show that the new system allows to exclude unwanted hypothermic neuroprotection, and does not interfere with optical and electrical measurement techniques.