Eighty-four rats (6 weeks old) were randomly assigned into four groups (Siven male and seven female rats in each group). The obtained median approximate lethal dose (ALD) of BCP was 1,000 mg/kg b.w./day in rats. The four groups were divided into: high dose (1,000 mg/kg b.w./day), medium dose (500 mg/kg b.w./day and 250 mg/kg b.w./day), low dose (125 mg/kg b.w./day), vehicle control (4% ethanol solution in same volume), and control (not treat). Rats were administered once daily by gavage with BCP suspension at four doses as above (1,000, 500, 250, and 125 mg/kg b.w./day) at 9:00–10:00 a.m. throughout the experiment for 28 continuous days. The investigators held the animal with the left hand and the syringe with the right hand. Then, they kept the head and neck of the rat in a straight line, thereby enabling easy access to the mouth. In Baoding rats, the head must be fixed to avoid the head twisting at will and affecting the operation of gavage. The intragastric needle entered from the angulus oris of the animal to make the mouth and esophagus align. When the intragastric needle reached a depth of approximately 5 cm, the experimenters gently pushed the syringe slowly to administer the dose. If there was no excessive struggle, the drug was injected slowly. If the resistance was small, the full dose could be administered. In the case of rat struggle, the intragastric needle was pulled out, reserved and operated again. The animals’ general clinic observations, body weight, morbidity, and mortality were recorded daily during administration. Additionally, measurements of food consumption and water intake were recorded weekly.

Blood samples were collected and placed into tubes containing EDTA-K2 for the hematological analyses. At the end of the drug administration period, the hematological index of rats was analyzed. Standard hematological and biochemistry tests were used to determine the hematological parameters, enzymes, substrates, and products of metabolism. The indicators were white blood cell (WBC), red blood cell (RBC), platelet (PLT), neutrophil (NEUT), lymphocyte (LYM), monocyte (MONO), eosinophil (EOS), basophil (BASO), and reticulocyte (Retic) counts; hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC).

Blood samples were collected, centrifuged at 3,000 rpm for 10 min at 4°C, and stored at −20°C. At the end of the drug administration period, the biochemical blood index of rats in each group was analyzed. The primary biochemical indicators were alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT), blood urea nitrogen (BUN), creatinine (CREA), total protein (TP), albumin (ALB), cholesterol (T-CHOL), glucose (GLU), triglyceride (TG), total bilirubin (T-BIL), direct bilirubin (D-BIL), lactate dehydrogenase (LDH), creatine kinase (CK), uric acid (UA), calcium (Ca), phosphorus (IP), high-density lipoprotein (HDL), and low-density lipoprotein (LDL).

At the end of the 28-day toxicity test, SD rats were fasted overnight and underwent general anesthesia with CO2. Necropsies were conducted carefully on all the rats, which either died or survived during experiments. Tissues like liver, spleen, heart, kidney (both), adrenal gland, lung, brain, pituitary gland, thymus, urinary bladder, stomach, intestine, testis, ovary, epididymis, uterus, prostate, seminal vesicle, trachea, esophagus, thyroid gland, salivary gland, skin, femur, and Harderian gland nerves were examined for the macroscopic morphology, and then removed quickly, washed in saline, weighed, and kept in 10% neutral buffered formalin (#0151S, BBC Biochemical, Seattle, WA). In addition, the relative weight of each organ (viscera/body ratio) was calculated based on the animal’s body weight according to the following formula: organ weight/body weight on sacrifice day × 100.

Tissues were fixed in a 10% formalin solution (# 0151S, BBC Biochemical, Seattle, WA) containing neutral phosphate-buffered saline, trimmed, processed, and embedded in paraffin, and sectioned at 4-μm thickness; sections were stained with hematoxylin and eosin (H&E) according to routine histological techniques. The histopathological changes were then evaluated using light microscopy (Leica DM 3000, Wetzlar, Germany).

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