Neural stem cells decrease neuronal loss induced by ischemia/reperfusion in mice

Abstract

We investigated the effect of neural stem cell (NSC) infusion on brain ischemia/reperfusion injury. Ischemia was achieved by middle cerebral artery occlusion in C57/Bl6 mice. A 6-0 monofilament nylon suture, blunted at the tip by heat and coated in poly-L-lysine was introduced into the common carotid artery and advanced so as to block middle cerebral artery. The filament was advanced until a >70% reduction of blood flow compared to preischemic baseline was observed. At the end of a 30 min ischemic period, blood flow was restored by removing the filament. To confirm the adequacy of the vascular occlusion, blood flow was measured by Laser Doppler flowmetry. Selected stem cells, isolated from newborn C57BL/6 mice, expressing b-galattosidase gene, were injected intracerebroventricularly in mice (5105/5 ml) at the end of ischemia. The distribution and migration of stem cells in ischemic and non-ischemic brains were evaluated by X-Gal staining at different time points. Two hours after ischemia, stem cells could be observed near the injection site, attached to the ependyma and beginning to move inside the brain parenchima. At 24 h stem cells were still present in the brain parenchima, but many of them could be found inside the vessels, both in the ipsi- and in the contralateral side. At day 5 and day 14 the majority of b-gal positive cells could be found inside the vessels, both in the ipsi- and in the contralateral side. At these two time-points in sham-operated mice no positive cell could be found in brain parenchyma or in vessels, indicating that if there was no lesion, cells were washed out. Fourteen days after ischemia neuronal loss was evaluated by cresyl violet staining. Neurons in the striatal region were counted by an image analysis software (AnalySIS). For each brain, three 20 mm sections, including the anterior, medial and posterior striatum, were analysed and on each section six fields were examined at 40' magnification. The number of neurons was estimated as percentage of neurons in the ipsilateral side versus those in the contralateral side. Mice receiving NSC showed a significant reduction of neuronal loss compared to those receiving saline (NCS: 4.93.5% saline: 26.27.1%) These data indicate that NSC are recruited by ischemic injury and that they are able to reduce the neuronal damage induced by ischemia/reperfusion.