Animals
Female Sprague-Dawley rats aged 6-7 weeks, weighing 150-180 g were used throughout the experimental work. Rats were obtained from the animal house of Pharos University in Alexandria, Egypt, and kept under observation for at least one week prior to study with free access to food and water. The Research Ethical Committee of the Medical Research Institute, Alexandria University, Egypt approved the study (No: AU0122021132). All procedures were performed according to ARRIVE guidelines and comply with the National Research Council’s guide for the care and use of laboratory animals. The study was conducted in accordance with the Basic & Clinical Pharmacology & Toxicology policy for experimental and clinical studies (19). The number of animals was kept to a minimum, the duration of experiment was as short as possible and the animals were killed immediately after cessation of the experiment. For evaluation of the analgesic effect algesiometric tests (tail clip and writhing tests) were performed. For evaluating the anti-inflammatory effects, rats have undergone acute and sub-chronic formalin test. In the algesiometric and acute formalin tests, each animal was used once and received only one dose of the test drug. All observations were performed by two observers in a randomized and blind manner.
Drugs
Acetic acid and formalin were purchased from El-Gomhouria for trading Pharmaceuticals, Chemicals and Medical Appliances, Alexandria, Egypt. Celecoxib capsules from Pfizer Pharmaceutical Company, Alexandria, Egypt, and neostigmine ampoules from Amriya Pharmaceutical industries, Alexandria, Egypt, and diclofenac sodium ampoules from NOVARTIS, Alexandria, Egypt were used in the experiment. All ELISA kits were purchased from MyBioSource, San Diego, USA. The activities of Cox-1 and Cox-2 were determined using colorimetric assay kit (Cayman Chemical Company; Ann Arbor, MI, USA).
Experimental Design
Rats were allocated into seven groups of 8 rats each as follows: Group I, normal rats receiving sterile saline, group II, positive control subjected either to tail clip or acetic acid or formalin and not treated, group III, receiving 0.2 mg/kg neostigmine (20), group IV, receiving 10 mg/kg diclofenac sodium (21), group V, receiving 2 mg/kg celecoxib (22), group VI, receiving a combination of neostigmine (0.2 mg/kg) and diclofenac sodium (10 mg/kg), group VII, receiving a combination of neostigmine (0.2 mg/kg) and celecoxib (2 mg/kg). All drugs were freshly prepared and dissolved in saline. Doses were chosen from previous studies and have been tried in a pilot phase before the start of the real experiment. Drugs were injected intraperitoneal 30 min once before performing the tail clip, writhing and acute formalin study. In the sub-chronic formalin test, drugs were given daily after induction of inflammation with formalin for 5 days (the first dose started 5 hours after formalin injection). All procedures were performed rapidly with a high degree of accuracy and reproducibility. At the end of the study, rats were anesthetized using a mixture of 80 mg/kg ketamine and 5 mg/kg xylazine and euthanized using cervical dislocation. Samples were then collected for further biochemical and histological studies. Rats were then kept frozen till incineration.
Evaluation of analgesic activity
HAFFNER’s tail clip method
An artery clip was applied to the root of the tail of rat (approximately 1cm from the body) to induce pain (23) and the reaction time was noted. The animal quickly responded to this noxious stimulus by biting the clip or the tail near the location of the clip. The time between stimulation onset and response was measured by a stopwatch in 1/10 seconds increments. In all groups, tail clip test was performed preceding drug administration, and at 15, 30, 45- and 60-minutes following drug administration. The reaction time (test latency) at each time interval was calculated. Percent analgesia was calculated as % analgesia = MPE = (TL – BL / ML – BL) *100 where, MPE = max possible effect, ML= max latency or cut off time, TL = test latency and BL = basal latency or control latency. Cut off time of ten seconds was obliged in all sets of experiments (24) .
Writhing test
Pain was induced by injection of irritant (0.7 % acetic acid) into the peritoneal cavity of rats (25). The animals reacted with a characteristic abdominal cramp, which is called writhing.
Only rats that showed a positive response to acetic acid were used for the test on the next day. Test animals were administered the drug or the standard 30 min prior to acetic acid administration, then rats were placed individually into glass cage, observed for a period of fifteen min and the number of writhes was recorded for each rat. Percent inhibition was calculated as following: % inhibition = (Wc –Wt)*100 / Wc, where, Wc = no. of writhes in control group and Wt = no. of writhes in test group. Drugs showing less than 70 % inhibition were considered to have minimal analgesic activity (24).
Serum β-endorphin level
At the end of the tail clip test, after 60 min, rats were euthanized as mentioned earlier and blood was collected from aorta for measurement of the level of β-endorphin in all tested groups.
Evaluation of anti-inflammatory activity
Acute anti-inflammatory activity
For screening of anti-inflammatory drugs, 0.05 ml of 5 % solution of formalin was injected subcutaneously into the plantar side of the left hind paw of rats. The tested drugs were administered intraperitoneally 30 min before the challenge with formalin, to test the ability of such agents to prevent the edema produced in the hind paw of the rat after injection of the phlogistic agent. The paw diameter was measured by vernier caliper before the start of the experiment, then again after 3 h, and eventually 24 h after the challenge (26). Percent inhibition at different time interval was calculated as following: % inhibition = (paw volume in control group – paw volume in test group) divided by paw volume in control group and multiplied by hundred (24).
Sub-Chronic anti-inflammatory activity
Sub-Chronic phase of inflammation was enabled by subcutaneous injection of 0.05 ml 5 % solution of formalin into the plantar side of the right hind paw of rats. Tested drugs were administered intraperitoneally 5 hours after formalin injection and then daily for five days (26). The paw diameter was measured before the start of the experiment and then daily by vernier caliper. Percentage anti-inflammatory effect of drugs was calculated as following: % anti-inflammatory effect = (mean paw volume in control group – mean paw volume in test group) divided by mean paw volume in control group and multiplied by hundred.
At the end of experiment rats were euthanized using a mixture of 80 mg/kg ketamine and 5 mg/kg xylazine. Blood was collected from the posterior vena cava through a laparotomy incision to determine some serum parameters using ELISA. The anti-inflammatory parameters tumor necrosis factor- ɑ (TNF-ɑ), high sensitivity C-reactive protein (HS-CRP) and nuclear Factor-кB (NF-кB) were measured. The Cox activities (Cox-1, Cox-2 and Total Cox) assays were determined according to the procedures used by Kargman et al. (27). The Kit (Cayman Chemical Company; Ann Arbor, MI, USA) includes isozyme-specific inhibitors for distinguishing Cox-2 from Cox-1 activity. Protein was determined using a modified method of Lowry et al. (28). The specific activity of each enzyme was determined by dividing its activity by the protein concentration in the sample (U/mg protein) (27). Also, aspartate transaminase (AST) and alanine transaminase (ALT) as indicators of liver function were measured. Creatinine and urea were determined spectrophotometrically. Livers, inflamed hind paws and one kidney were removed, washed with ice-cold saline, kept in 10 % formalin for further histopathological examination.
Histopathology examination
For routine H&E staining (renal and liver tissue), paraffin blocks were prepared from tissue samples, fixed in 10% neutral buffered formalin solution after a routine tissue deparafinization process. From each tissue sample, 4 μm thick sections were obtained and stained with routine Hematoxylin-Eosin stain, and examined under light microscope. The liver activity index was scored as described by Ishak et al.(29), with the permission from authors and publisher for modification (Table 1) and according to NASH Clinical Research Network scoring system (30) as described in Table 2.
For sections of bone: samples were embedded in 100 ml of 5% nitric acid, the solution was changed once in 3 days. After ensuring complete decalcification, the tissues were washed using distilled water for 30 min, following which the specimens were subjected to manual tissue processing. After processing, the tissues were embedded in paraffin and were sectioned to a thickness of 7-8 μm using the soft tissue microtome. The sections were then stained by Hematoxylin and Eosin and examined under light microscope.
Quantification of inflammatory cells was done semiquantitative using the hot spot method where the area of highest inflammatory infiltrate was chosen in each section through screening of the slide on low power examination (x100). Then non overlapped high power fields (x400) were examined and the number of inflammatory cells was counted in each field. The mean number of inflammatory cells per high power field was calculated.
Statistical analysis
Values are presented as means ± SD (n=8). Data were analyzed using one-way analysis of variance (ANOVA), followed by Tukey multiple comparison post hoc test. For abnormally distributed data, Mann-Whitney Test was used to analyze two independent populations. The differences were considered to be significant at p<0.05. The graphs were drawn using Prism computer program (GraphPad software Inc. V5, San Diego, CA, USA).