Ischemic preconditioning recovers nerve growth factor, brain-derived neurotrophic factor and their high affinity receptors after forebrain ischemia

Background and aims: Preconditioning of gerbil brain with a sublethal forebrain ischemia is known protective to hippocampal CA1 neurons following a subsequent lethal ischemia (the second ischemia) which normally damages neurons (ischemic tolerance). The role of neurotrophic factor in the mechanism of ischemic tolerance has not been well discussed. Methods: Male Mongolian gerbils, 12-13 weeks old and weighing 65-85 g, were used for study. To induce ischemic tolerance, a 2-min period of occlusion of bilateral common carotid arteries (ischemic preconditioning) was followed by 3 days of reperfusion, and then a 3-min period of occlusion (the second ischemia) was induced. Animals were decapitated at 3 days of reperfusion after sham operation or 2-min ischemic preconditioning and at 4 hours, 1 day, 3 days and 7 days after the second ischemia (n= 5, in each time point). Neuronal apoptosis was detected by using an in situ apoptosis detection kit. Hematoxylin and eosin (H&E) staining and 4′,6′-diamidino-2-phenylindole hydrochloride (DAPI) staining were used to calculate the survived CA1 neurons. Immunofluorescent detection of NGF, TrkA, BDNF and TrkB was performed in brain sections. Confocal laser scanning microscopy of immunolabeled sections was carried out with use of a Leica TCS SP2 system. Western blot analysis was also studied. Results: Using confocal laser scanning microscope, we found that the immunofluoresence of NGF and BDNF and their high affinity receptors (TrkA and TrkB) was present in the hippocampal cells of sham-operation gerbils. A 2-min ischemic preconditioning caused little change of these proteins (ANOVA test, P> 0.05). After the second lethal ischemia, in the CA1 area with ischemic preconditioning, only BDNF but not NGF and their high affinity receptors showed a transient reduction at 4 hours (ANOVA test, P< 0.01) and improved from 1 day (ANOVA test, P> 0.05). In the CA1 area with no ischemic preconditioning, NGF and its high affinity receptor, TrkA, showed a consistent reduction from 4 hours to 7 days (ANOVA test, P< 0.05); BDNF and TrkB decreased transiently from 4 hours to 1 day (ANOVA test, P< 0.05) but were recovered in the survived neurons from 3 days. At 3 and 7 days after the second lethal ischemia, apoptotic cell injury could be seen in the CA1 area with no ischemic preconditioning, and was sparsely seen in the CA1 area with ischemic preconditioning. In the ischemia-resistant CA3 and dentate gyrus areas, only BDNF decreased significantly at 7 days in the CA3 area with no ischemic preconditioning. However, there was no significant change of NGF, TrkA and TrkB immunofluorescence from 4 hours to 7 days after the second lethal ischemia in the gerbils with or without ischemic preconditioning. Western blot study showed a similar change to the confocal immunofluorescent study. Conclusions: Our study showed that ischemic preconditioning recovers the initial decline of NGF and BDNF and their corresponding receptors in the vulnerable CA1 neurons after the second lethal ischemia, suggesting a possible role of growth factors in the protective mechanism of ischemic tolerance.