Nto slices of 2 mm in thickness. The fresh brain slices were
Nto slices of 2 mm in thickness. The fresh brain slices were immersed in 2 TTC solution in PBS for 20 min at 37 and their images were subsequently captured using an Epson Perfection 1260 desktop scanner (Seiko Epson Corporation, Nagano, Japan) at a resolution of 600 dpi. The area of the white infarct region was then analyzed using Adobe Photoshop CS4 Extended Version 11.0.1 (Adobe Systems Inc., San Jose, CA, USA). The infarct volume of a slice was calculated using the following equation:flexion (to the left limbs). NDS 2: Rats showing decreased resistance to lateral push to the left. NDS 3: Rats showing contralateral circling, i.e., circling to the left only. NDS 4: Rats showing spontaneous circling. The NDSs were measured at 1, 2, 3, 5 and 7 days after the MCAO.Beamwalking testInfarct volume = Infarct area of the slice ?thickness of the slice (2 mm)The total infarct volume was calculated by summing up the infarct volumes of the different slices.Cresyl violet stainingAt the end of the experiment at day 7, the animals were anesthetized and their brains were perfused transcardially with phosphate buffered saline, followed by 4 paraformaldehyde. The brains were subsequently excised, embedded with paraffin wax and cut coronally into slices of 6- thickness. These sections were then stained with 0.1 cresyl violet for 10 min.ImmunohistochemistryA beam-walking test was applied to assess the motor coordination and balance control Doravirine chemical information 28045099″ title=View Abstract(s)”>PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28045099 of the forelimbs and hind limbs of rats treated with the different extracts to evaluate their motor functional recovery after ischemia [33, 34]. The experimental animals were trained to complete a beam-walking test 3 days before undergoing MCAO to take baseline measurements, which involved them traversing an elevated narrow beam (2.5 ?120 cm, 80 cm above the floor) [35]. During the training days (2 days), the rats required less than 10 s to cross the beam. The rats were motivated to cross the beam by placing their home cage at the opposite end of the training stage. The rats were given 90 s to cross the walkway to their home cage. Performance was assessed by recording the mean latency of three trials to walk along the beam. This beam-walking test was performed at 1, 2, 3, 5 and 7 days after MCAO.Statistical analysisAll data have been presented as the mean values ? SD. Student’s t test and one way ANOVA, followed by Dunnett’s multiple comparison test (GraphPad Prism 5.0 software; San Diego, CA, USA), were used for the statistical analysis. Statistical significance was taken as P < 0.05.The expression levels of Nrf-2 and Bcl-2 at the penumbra of the rats were examined by immunohistochemistry. At 7 days after MCAO, the rats were euthanized and their brains were harvested and cut into slices of 6- thickness, as above mentioned. These sections were then blocked with FBS containing 10 goat serum and 1 BSA, and incubated with rabbit polyclonal anti-Nrf-2 (1:200; Abcam, Cambridge, UK) and rabbit polyclonal anti-Bcl-2 (1:400; Abcam) overnight at 4 . The slices were then washed with PBS and incubated with Alexa Fluor?555 Goat Anti-Rabbit IgG (H + L) (Invitrogen Life Technologies) for 1 h with dilutions of 1:400 and 1:800 for binding on anti-Nrf-2 and anti-Bcl-2, respectively. The slices were then washed with PBS and counterstained with DAPI. Fluorescence images were captured with ApoTome.2 system (Carl Zeiss AG, Jena, Germany).Neurological deficit scoreResultsCell viability measurements by MTT assayAn MTT.