2.1. Chemical and reagents
Progesterone was obtained as a gift sample from Symbiotec Pharmalab Pvt. Limited, Indore. Noscapine was purchased from government opium and alkaloid works undertaking, Neeemuch, M.P. (Drug license no.20/75) as suggested by the chief controller, Government opium and alkaloid works, New Delhi. All other chemicals and reagents used in this work were of analytical grade and commercially availed from regular drug and reagents suppliers.
2.2. Animals and ethical approval
Wistar rats (250–280 g) were obtained from the Central Animal Facility of the institute and used for the experiment. Animals were housed in polypropylene cages and maintained under standard laboratory environmental conditions: temperature 25 ± 2°C, 12-h light and 12-h dark cycle with free access to food and water ad libitum. The experimental protocol (Protocol No. PH/IAEC/2K16/010) of the study was approved by the Institutional Animal Ethics Committee.
2.3. Bilateral common carotid artery occlusion model
The bilateral common carotid artery occlusion (BCCAO), the procedure was adapted from the method of (Iwasaki et al. 1989). After 7 days of pretreatment with PG and NOS, rats were anesthetized with chloral hydrate (350 mg/ kg ip). Subsequently, a ventral midline cervical incision was performed to expose both common carotid arteries (CCAs), and they were freed from their sheaths and carefully separated from the adjacent vagus nerves. Occlusion of CCAs reduced cerebral blood flow to about 70% (Li et al. 2016). After 30 min of CCAs occlusion, reperfusion began by removing the carotid clips and was continued up to 24h at which time measurements of the different parameters were made. It has been reported that BCCAO causes significant cerebral ischemia in rats (Yanpallewar et al. 2004).
2.4. Study design for In-vivo studies
Male Wistar rats weighing 250–280 g were randomly distributed into five groups containing 30 rats in each group and administered with respective treatment. Group 1, sham-operated control group, these animals received saline 2 ml /kg, PO as a vehicle; group 2, ischemia-reperfusion (I-R) ( i.e, BCCAO rats received saline vehicle); group 3, received progesterone (PG) 8mg/kg PO; group 4, received noscapine (NOS) 10mg/kg PO ; group 5, PG + NOS (8mg/kg + 10 mg/kg) in combination., 7 days pre-treatment and on the day after surgery (8th day). Experiments were performed on five sets of animals (n = 6) within each group. The post-ischemic behaviors were recorded 3 days after reperfusion in the Morris water maze and an open field was also performed to assess spatial learni.0ng memory and general locomotor activities. The first set was used for observation of physiological parameters and determination of infarct volume. The second set was used for the estimation of behavioral parameters. The third set was used for the estimation of oxidative stress parameters: superoxide dismutase, glutathione reductase (SOD, GSH), lipid peroxidation, and myeloperoxidase (MPO) activity. The BBB permeability was evaluated in the fourth set, while a fifth set was for observation of histological changes (Fig. 1).
2.4.1. Infarct analysis
For infarct, analysis was performed in coronal slices (2 mm) of the promptly isolated brain and stained with 2% 2, 3, 5-triphenyltetrazoliumchloride (TTC) for 30 min at room temperature. A 10% formalin solution was used to fix the slices for the overnight period and evaluate the infarct area using Image J software 1.30V (http://www.rsb.into.nih/ ij). The infarct area was added together, for all sections, to obtain the total infarct area, which was multiplied with the thickness of the brain slice to obtain infarct volume per brain (mm3) and corrected for edema (Thiyagarajan and Sharma 2004).
2.4.2. Brain water content
After the measurement of infarct volume, brain tissue was dried in an oven at 100°C for 24 h and reweighed to obtain the dry weight. The brain water content indicating brain swelling volume was expressed as (Schwab et al. 1997). The tissue water content is expressed as the percentage of the wet tissue weights as follows:
% of water content in brain = Wet weight - Dry weight x 100/ Wet weight
2.4.3. Evaluation of motor function
The Rotarod is one of the most frequently used tests of motor function after 24 h of injury in the rats. The speed selector was set so that the roller rod makes 15 rpm. Before the test, each animal was given 1 min exposure to the moving rod. The animals were placed on the roller for 3 min. Latency to fall from the rolling rod was observed. A normal animal could maintain its equilibrium for an indefinite period (Rogers et al. 1997).
2.4.4. Morris water maze test
The cognitive function of rats was assessed by using the Morris water maze test (MWM) as described earlier (Morris et al. 1982; Tiwari et al. 2009; Tuzcu and Baydas 2006). The test apparatus was a circular water tank (180 cm in diameter and 60 cm high) that was partially filled with water (24 ± 1°C). Full cream milk (1.5 l) was used to render the water opaque. The pool was divided virtually into four equal quadrants, labeled A–B–C–D. A platform (12.5 cm in diameter and 38 cm high) was placed in one of the four maze quadrants (the target quadrant) and submerged 2.0 cm below the water surface. The platform remained in the same position during training days (reference memory procedure). All animals followed this sequence for that session. Each rat was placed in the water facing the wall at the start location and was allowed 120 sec to find the hidden platform. The animal was allowed a 20 sec rest on the platform. The latency to reach the platform was recorded. If the rat was unable to locate the hidden platform, it was lifted out and placed on the platform for 20 sec. The procedure was repeated for all four start locations. Two sessions of four trials each were conducted on each day of testing separated by 4 h. After that, the platform was removed and a probe trial (without platform) was conducted. Each rat was placed in the pool at the same randomly selected starting pole and the swimming path was observed and time spent in the quadrant of the pool which initially contained the platform was measured. On completion of the probe trial, a black platform that extended 1 cm above the surface of the water was placed in a quadrant other than that chosen for the submerged platform. Each rat was then given four trials of 120 sec to locate it. The latency to reach the platform was recorded (working memory procedure) (Patil et al. 2015; Yanpallewar et al. 2004; Wang et al. 2017).
2.4.5. The Open Field Test
Locomotor function and anxiety were assessed using the open field test, as described previously (Lu et al. 2016). A square wooden open field (44 × 44 × 32 cm) was subdivided into 16 even squares with thin white stripes. Each mouse was placed next to the wall of the arena, facing away from the experimenter. Behavior was recorded for 10 min. outcome measures were it was observed for the ambulations (number of squares crossed), the total period of immobility (in seconds), number of rearings, and groomings at the end of each trial, the arena was cleaned using 70% ethanol to prevent olfactory cue bias (Yanpallewar et al. 2004; Almahozi et al. 2019).
2.4.6. Assessment of oxidative stress
The brain tissue (1g) was homogenized in ice-cold 10% trichloroacetic acid (TCA) using a tissue homogenizer. Malondialdehyde (MDA) levels were assayed as an index of lipid peroxidation by monitoring the formation of thiobarbituric acid-reactive substances at 532 nm and expressed as malondialdehyde (MDA) content, mmol MDA⁄mg of tissue protein (Slater and Sawyer 1971). Superoxide dismutase (SOD) activity was measured by the inhibition of pyrogallol autoxidation at 420 nm for 5 min (Marklund and Marklund 1974) one unit activity was determined as the amount of enzyme that inhibited the oxidation of pyrogallol by 50%. Similarly, reduced glutathione (GSH) was determined using 5, 5’-dithiobis (2-nitrobenzoic acid) (DTNB) reagent at 412 nm and expressed as µg of GSH/mg of protein (Moron et al. 1979). Tissue protein was estimated in each sample with the method reported by Lowery et al. (1951).
2.4.7. Evaluation of BBB dysfunction
The integrity of BBB was investigated by Evans blue (EB) extravasation due to leakage in the brain. Permeability of the BBB was quantified as µg of EB/ hemisphere and considered an index of vascular permeability. At the commencement of reperfusion, EB (0.1 ml of a 4% solution) was injected into the tail vein. The rat was anesthetized and perfused transcardially with 100 ml heparinized saline solution (10 IU/ml) after reperfusion. Once sacrificed, the rat brain was removed. Rat Brain was homogenized in 1 ml of 0.1 mol/l PBS and then centrifuged at 1000 g for 15 min. Trichloroacetic acid (0.7 ml of a 100% solution) was added to the 0.7 ml supernatant. The mixture was allowed to incubate at 14°C for 18 h and then centrifuged again at 1000 g for 30 min. The amount of EB in the supernatant was determined spectrophotometrically at 610 nm by comparison against readings obtained from standard solutions (Gursoy-ozdemir et al. 2000).
2.4.8. Estimation of myeloperoxidase activity
The myeloperoxidase enzyme was extracted and its activity was measured using a method described by Bradley et al. Briefly, plasma samples were homogenized in 50 mmol/l potassium phosphate buffer, pH 6, containing 0.5% hexadecyltrimethyl ammonium bromide. The homogenate was freeze-thawed three times and then centrifuged for 20 min at 11000 g and 4˚C. The supernatant (34 ml) was mixed with the same phosphate buffer (986 ml) containing 0.167 mg/ml ortho-dianisidine dihydrochloride and 0.0005% hydrogen peroxide. The change in absorbance at 460 nm was recorded using a spectrophotometer. One unit of MPO activity was defined as that consuming 1 nmol peroxide/min at 22˚C. Results were expressed as unit/mg of tissue protein (Bradley et al. 1982).
2.5. Tissue and plasma distribution study
Animals (Wistar rats 200-250g) were divided into three different groups containing 12 rats in each group and administered with respective treatment. PG (8 mg/kg, i.p) treated group; NOS (10 mg/kg PO) treated group; and combination treated PG (8mg/kg) + NOS (10mg/kg) groups, 7 days pre-treatment and on the day after surgery (8th day). The blood sample was collected at different time points from the time of administration as 0, 0.5, 1, 2, 4, 8, 16, 24, 48, and 72 h. For drug distribution study in the brain after 24 h of reperfusion, rats were sacrificed and the brain was collected for further analysis. The levels of drugs in rat plasma and brain were analyzed using HPLC (Singh and Pai 2013; Xu et al. 2014). Pharmacokinetics parameter evaluation of PG and NOS including Cmax, Tmax, t1/2, and AUC0 − t were calculated by non-compartmental analysis using the excel add-in PK Solver (Version 2.0) (Zhang et al. 2010) (Fig. 1).
2.5.1. Simultaneous RP-HPLC bio-analytical method development
A series of the trail has been done to obtain good peak shape and resolution between analytes at Agilent 1260 HPLC system consist of the analytical column Kromasil C8 (150*4.6mm, 5um). HPLC grade methanol, acetonitrile, and water were used as mobile phase and diluent at different ratios. The elute was monitored through UV based detector set at 241nm (Karlsson et al.
1990; Zhang et al. 2004; Aneja et al.
2007; Liu et al.
2008; Yan et al.
2014).
2.5.2. Plasma/brain sample preparation
Plasma samples were obtained after centrifugation of blood samples at 10,000 rpm; 2–4°C and supernatant was collected. To 100 µl of rat plasma collected at each study point, 300 µl of methanol was added and centrifuged at 10000 rpm; 2–4°C for 10 min. The supernatant was collected and stored at -20°C for HPLC analysis.
The brain tissue was homogenated with isotonic buffer solution (pH 7.4) and centrifuge at 10,000 rpm2-4°C, the supernatant was collected in an Eppendorf tube. 100 µl of brain homogenate was mixed with 300 µl of methanol and further centrifuged at 10,000 rpm; 2–4°C, the supernatant was collected and stored in -20°C for HPLC analysis (Wang et al. 2011; Feng et al. 2017; Liao et al. 2018).
2.5.3. Standard and quality control sample preparation:
Noscapine and Progesterone standard solution was prepared in HPLC grade methanol similarly a combination of both drug and a quality control sample was also prepared in HPLC grade methanol. Appropriate dilutions of the standard were prepared in HPLC grade methanol to produce 2, 4, 6, 8, and, 10 µg/ml.
Plasma and brain homogenate calibration samples and were prepared by spiking 100 µl of standard solution with 100 µl of rat plasma and 100 µl of brain homogenate respectively. This mixture was further vortexed for 2 min and 300 µl of methanol was added in this mixture and vortexed for 2 min and finally centrifuged at 10000 rpm for 10 min under cold conditions. The supernatant was collected and injected into HPLC. Samples for the determination of recovery, precision, and accuracy were prepared by spiking control rat plasma and brain homogenate in at appropriate concentrations and were stored at − 80°C until analysis (Karlsson et al.1990; Zhang et al. 2004; Aneja et al. 2007). Bioanalytical method validation parameters were also performed (Supplemental Data-II).
2.6. Histological examination
The rat was decapitated; the brain was rapidly dissected out, washed immediately with saline, and fixed in 10% buffered formalin. Cerebral hemispheres were embedded in paraffin and sections were cut and stained using hematoxylin and eosin to observe under a microscope for histological change (Margaritescu et al. 2009).
2.7. Statistical analysis
The results were calculated as mean ± standard error of the mean (SEM); the analysis of variance (ANOVA) was applied to calculate the statistical difference. The changes in the oxidative biomarker infarct area and vascular permeability were compared with the sham-operated group by using one-way ANOVA followed by Tukey’s test. The data were presented as mean ± SEM; p < 0.05 was considered for statistical significance using Prism GraphPad software (GraphPad Software, San Diego, CA, 409 USA).