2.3. Transient Middle Cerebral Artery Occlusion (tMCAO)

Brain ischemic injury by tMCAO in TS and e-Cig exposed C57BL/6 J male mice was performed as previously reported with slight modifications. Surgery was performed using a Zeiss OPMI pico I surgical microscope (Carl Zeiss GmbH, Jena, Germany). Temperature was maintained at 37 °C, controlled by the thermostatic blanket (TC-1000 Temperature Controller, CWE, USA). Mice were anesthetized with 4% and maintained at 1–1.5% isoflurane with a facemask. The cerebral blood flow was continuously monitored throughout the surgery to confirm the occlusion and reperfusion of the brain by using non-invasive, real-time microcirculation imaging, Pericam PSI system (Perimed Inc., Marble Falls, TX) placed over the exposed skull in the territory of the left middle cerebral artery (MCA) perfusion area. A midline incision was made at the neck about 1.5 cm long. The left carotid bifurcation, external carotid artery (ECA) and common carotid artery (CCA) were isolated from the adjacent tissue. After occlusion of CCA using a micro clip, the left ECA was ligated, coagulated, and cut distally to the cranial thyroid artery. 6–0 nylon monofilament with a silicone coated tip (0.20–0.23 mm; Doccol Corporation) was gently introduced up to ~8.5–9 mm to block the origin of the MCA. IR injury was produced by tMCAO (30 min) according to established procedures in Dr. Abbruscato's laboratory [50]. After 30 min occlusion, the suture was withdrawn up to the left carotid bifurcation to restore blood flow, i.e. reperfusion. Animals that failed to recover at least 80% of baseline within 10 min after reperfusion were excluded from the experimental group. After reperfusion, neurological evaluation using several sensory-motor tests was carried out. These include neurologic deficit score on a four-point scale [50]. Once the brain tissue was resected, coronal sections (10 µm thick) from the contralateral and ipsilateral hemispheres were also analyzed by fluorescent microscopy to assess neuronal degeneration by using fluoro-Jade C (FJC) which is an anionic dye that specifically stains soma and neurites of degenerating neurons [51].