2.1 Probiotic
The strain of Lactobacillus plantarum UBLP40 and Bacillus clausii UBBC07 was obtained from Unique Biotech Ltd, India. Certificate of Analysis of both strains included in supplementary file 1. It was inoculated in sterilized MRS broth solution and incubated at 37 °C in the bacteriological incubator. Bacterial species were pelleted by centrifugation at 2000-3000 rpm, organisms were re-suspended in 500 mL phosphate buffered saline (PBS, pH7.4) and administered in rats via oral gavage every day.
2.2 Acid resistance, Bile resistance and Bile salt hydrolase (BSH) activity:
Both the strain of probiotic Lactobacillus plantarum UBLP40 and Bacillus clausii UBBC07 subjected for evaluation of its acid resistance, bile resistance and BSH activity as described by Patel et al. (2020).
2.3 Animals and Housing
The SD rats weighing 170-200 gm were obtained from the Bombay Veterinary College, department of pharmacology and toxicology, Maharashtra, India. The study protocol was approved by the IAEC (No: SSR/IAEC/2019/05). The study was conducted as per the CPCSEA guide for the laboratory animals. Twenty-four rats were housed and maintained under the standard condition in the good laboratory practice (GLP) certified animal house facility at SSR College of Pharmacy, India.
2.4 Experimental Design
The SD rats were divided into four groups (n=6) and were acclimatized for a period of 1 week. After 7 days of acclimatization span,
- Group I was assigned as a negative control
- Group II served as induced control which was treated with thioacetamide (250 mg/kg/day i.p at an interval of 24 h for 3 days),
- Group III was treated with thioacetamide (250 mg/kg/day i.p, 3 days) + Lactobacillus plantarum UBLP40 (107 CFU/day, p.o for 14 days) + Bacillus clausii UBBC07 (107 CFU/day, p.o for 14 days)
- Group VI was given thioacetamide (250 mg/kg/day i.p at an interval of 24 h for 3 days) + Standard treatment i.e lactulose (2.5 ml per kg in 3 divided doses, p.o for 14 days).
On the 14thday, rats were tested for behavioural functions (after two hours of treatment). On the 15thday, rats were sacrificed, initially, blood was collected for the determination of biochemical parameters and then liver and brain samples were collected for determination of neurochemical parameters and histopathological studies (Fig 1).
2.5 Behavioural tests
2.5.1 Rotarod test
An accelerating rotarod instrument was employed to record motor coordination, as per Vijitruth et al. (2018) method. Rats were trained and the basal falling latency was recorded. After treatment, the final falling latency was recorded.
2.5.2 T-maze test
The cognitive ability of the rats was analysed using T- shaped maze. This test was performed according to the procedure stated by Hussein et al. (2018). Firstly, the animals are subjected to habituation period at intervals; this is a crucial stage where the animals will inculcate learning only through habituation. Secondly, they are rewarded properly based on how they act and what path they choose during the habituation process and then trained accordingly. Spontaneous alternation is the main working principle. After placing the rat at the entrance of t-maze for 10 s, the barrier was raised and the animal was permitted to travel the maze for 30 seconds. It was granted a free choice. Thus, when it entered one of the arms, it was blocked for 30 seconds, to explore this place. Afterwards, it was kept again at the starting point. The session was restarted if the rat didn’t choose either of the arms after the 30s. A correct response is “an alternating choice between the two arms”. The time of response (seconds) is also recorded. Is there any latency in entering the goal arms or not and if yes then how much?
2.5.3 Object recognition test
The design of test apparatus was as per Ennaceur and Delacour (1988). Two trials T1 and T2 were done 2-min each. In T1 (two identical objects) trial and T2 (one identical object was replaced with the unknown object), trial objects were placed in two opposite corners of the in which the rats were to be placed. After placing the rat into the ORT apparatus, it was allowed to explore the objects. Trial T2 was performed after 1-hour trial T1. Exploration was defined as follows: “directing the nose toward the object at a distance of not more than 2 cm and/or touching the object with the nose”.
2.6 Biochemical parameters
2.6.1 Serum Alanine aminotransferase (ALT), Aspartate aminotransferase (AST) and Alkaline Phosphatase (ALP)
The blood sample was centrifuged at 1500×g at 4 C° for 15 minutes. The serum was deliberately isolated and used for further testing (El-Marasy et al. 2019). Assay kits based on colorimetric method were utilized to detect ALT and AST. pNPP- AMP kinetic assay method kit was utilized to quantify the amount of ALP in serum.
2.6.2 Serum Ammonia
This technique is based on the formation of indophenol which is a blue color compound and should be this color is persistent. The testing procedure was performed as per Muramatsu (1967). This method comprises two steps: firstly, treatment of blood and second development of color. The optical density of the established color was recorded at 610 nm.
2.7 Neurochemical parameters
2.7.1 Brain tissue homogenization
In 20 % phosphate buffer saline (PBS), the brain tissue was homogenized, centrifuged for 15 minutes at 1500 g at 4 C° and the supernatant was obtained for direct measurement of oxidative stress parameters (El-Marasy et al. 2019).
2.7.2 Lipid peroxidation
TBARS method of Ruiz-Larrea et al. (1994) was used to determine lipid peroxides in the brain of rats. A pink colored component was formed after mixing the reactants and heating it for 30 minutes. The absorbance of this compound was measured at 532 nm.
2.7.3 Reduced glutathione
The content of reduced glutathione in brain tissue was measured using Ellman's reagent (DTNB) (Danduga et al. 2018) Centrifugation was done for 20 min to separate supernatant of the mixture [Equal volumes of tissue homogenate and TCA (10%)]. In 1ml of supernatant 3ml of phosphate buffer (0.2M, pH 8) and 0.5ml of DTNB reagent (0.6mM in 0.2M Phosphate buffer) were added and mixed after which absorbance was recorded at 412nm.
2.8 Histopathological study of brain and liver
After withdrawing blood samples, the rats were euthanized. Liver and brain from different group rats were isolated and fixed in 10% neutral buffered formalin. These samples were sent for histopathological slide and paraffin wax preparation to Sakshi Histopathology Works, India. The prepared slides were observed under the microscope, images were captured. The injury in liver cells, as well as brain cells injury and inflammation, were determined.
2.9 Statistical analysis
This analysis was done using Graph Pad Prism 8 (software, India). The results were indicated as mean±SEM. All the data were analyzed by a one-way ANOVA test with a significance level (P < 0.05).