Background
Neuronal damage occurs in the ischemic core but also in ‘the ischemic penumbra’. The core grows rapidly at the expense of the penumbra and thus, neuropathological investigations of the cell death proves to be complicated in arterial occlusion models. In our model we perform a critical reduction of regional cerebral blood flow, mimicking the metabolic conditions in the penumbra in order to study the temporal course of cell death and potential apoptotic mechanism involved.
Material and Methods
Male Wistar rats were anaesthetized, intubated and ventilated throughout the entire experiment. The left carotid artery was cannulized for blood pressure monitoring and blood gaze control. Local cerebral blood flow (lCBF) was bilaterally assessed by laser Doppler. Tissue impedance as a parameter for cell swelling was measured in the ipsilateral (penumbra) hemisphere. After stable baseline (30 min) penumbra conditions were provoked by hypobaric hypotension causing a critical CBF reduction in the ipsilateral hemisphere for 30 minutes. In addition cortical spreading depression (SD) was elicited four times during ‘penumbra-period’ by intracortical injection of 150 mM KCl as metabolic challenge. To study the time course of cell death rats were allowed to survive for either one (n=3;Gr-A), two (n=6;Gr-B) or seven days (n=6;Gr-C) before histological evaluation. In order to investigate whether apoptosis-related mechanisms are involved animals received an intracerebroventricular injection of a pan-caspase-inhibitor (zVADfmk 160 ng, n=6;Gr-D) or placebo (0.2% DMSO, n=6;Gr-E) 20 minutes after end of hypotension. These animals survived 7 days for histological evaluation.
Results
Hypobaric hypotension induced a significant 53% lCBF reduction in the ipsilateral hemisphere in groups A, B, C. Accordingly, cerebral perfusion declined by 55% in the caspase-inhibitor experiment (Gr-D and Gr-E). After induced cortical spreading depression during hypotension the tissue impedance increased by 51% indicating massive cell swelling triggered by SD. Cell swelling recovered only after normalization of blood pressure. The histological evaluation showed a late cell loss in the ipsilateral hemisphere (364.2±74 /mm2, Gr-A; 262.0±69 /mm2, Gr-B; 217.0±69/mm2, Gr-C). Compared to native brains (mean density 369.0±71 /mm2), a significant cell deficit was in group C. That delayed cell death was prevented by a zVADfmk bolus (Gr-D: 376.4±58 neurons/mm2) as compared to the placebo group (Gr-E: 264.21±84.59 /mm2). There were no differences in neuron density in the contralateral hemispheres neither of the ‘time-course’-groups (413.8±63/mm2, Gr-A; 335.2±74 /mm2, Gr-B; 319.7±37 /mm2, Gr-C) nor of the placebo (385.9±61 /mm2) and zVADfmk (398.9±62 /mm2) group.
Conclusion
zVADfmk reduces neuronal damage and thus, caspase-dependent mechanisms are involved in the delayed neuronal cell death occurring in tissue with critically reduced CBF.