The anxiety status is changed along with memory impairments in intracerebroventricular colchicine injected rat model of Alzheimer Disease (cAD) due to neurodegeneration, which has been indicated to be mediated by inflammation. Inducible cox-2, involved in inflammation, may have important role in the colchicine induced alteration of anxiety status. Therefore, the present study was designed to investigate the role of cox-2 on the anxiety behavior (response to novelty in an elevated open field space) of cAD by inhibiting it with three different doses (10, 20, and 30 mg) of etoricoxib (a cox-2 blocker) in two time points (14 and 21 days). The results showed anxiolytic behavior in cAD along with lower serum corticosterone level, both of which were recovered at all the doses of etoricoxib on day 21. On day 14 all of the anxiety parameters showed similar results to that of day 21 at high doses but not at 10 mg/kg body weight. Results indicate that the parameters of anxiety were dependent on neuronal circuitries that were probably sensitive to etoricoxib induced blocking of neurodegeneration. The present study showed that anxiolytic behavior in cADr is predominantly due to cox-2 mediated neuroinflammation induced neurodegeneration in the brain.
Colchicine is a plant alkaloid and has neurotoxic properties [
Sil et al. [
Therefore, the present study was designed to investigate the role of cox-2 on the different attributes of anxiety like behavior in icv colchicine injected AD rats by blocking its expression and activity by etoricoxib (a cox-2 blocker) in 14- and 21-day study.
144 healthy, adult male albino rats (Charles-Foster strain) weighing 200–250 g (6–8 weeks of age) were used in this study. Animals were housed individually in polypropylene animal cages with food pellet and water ad libitum. The animal room was maintained at the temperature of
144 rats were used in this study and were divided in the following way.
Etoricoxib (Ranbaxy, India) was dissolved in distilled water and it was administered orally through a gastric cannula attached to a 1 mL syringe. The daily dose of etoricoxib was divided equally into two parts given at 6-hour intervals each. Three doses of etoricoxib (10, 20, and 30 mg/kg body wt.) were given po to different groups of rats for 14 and 21 days each starting from 4 days prior to colchicine (icv) injection (for AD rats) and 4 days prior to vehicle (icv) injection (for sham operated rats). Control rats were also treated with etoricoxib for the same time period.
In the lateral ventricle of rat, 7.5
The anxiety behavior was measured by the method described by Ennaceur et al. [
The apparatus consists of a wooden platform (width 80 cm × length 80 cm × height 50 cm) elevated by 120 cm from the ground. The objects (width 8 cm × length 8 cm × height 13 cm) which were to be explored were in triplicate and were alternated between animals. They were made of white ceramic, a biologically neutral material. The objects were of heavy weight so that the animals could not move them around in the arena. These objects had never been associated with a reinforcer and are not known to have any ethological significance for the rats. The field of the open space was divided in outer area, inner area, and object area. The outer area was 15 cm wide from the edge of the field. The inner area was 10 cm × 10 cm wide located in the middle of the field. The object area is defined by a circle drawn in the centre, which is 2 cm larger than the bottom surface of the objects that was used in the experiment.
The experiments were carried out in open space with an object in the centre. These required testing animals in single session on 14th and 21st day after colchicine or vehicle injection along with control rats for all the groups. In each session, all the rats in each group were tested for 10 min. The surface of the platform of the apparatus was cleaned with 90% ethanol after testing of each rat and left to dry before the introduction of the next rat to minimize the effects of lingering olfactory cues. Rats were released into an open space from any part of the outer area (randomly predefined) facing away from the inner area. They were released for 10 min to explore the area of the open space. Rats were observed on a screen monitor connected to a video camera suspended above the test arena. The latency of first entry to the inner area, frequency of entry to the inner area, total time spent in the inner area, total time spent in the outer area, latency of first approach to the object area, and frequency of approach to the object area were noted in each session.
Blood was collected (1.5 mL, between 11:00 a.m. and 11:30 a.m.) from the heart of anesthetized rat (Diethyl Ether, Kabra Drugs, India) by a syringe and was kept for serum collection without any anticoagulant. With this serum corticosterone level was measured in different groups of rats in 14- and 21-day study.
Serum corticosterone (CORT) concentration was determined by radioimmunoassay using a commercially available kit (125I Rat Corticosterone (MP Biomedicals, LIC, Diagnostics Division, Ohio)) and gamma counter. The antisera used for the assay were highly specific for the Rat Corticosterone and it had 1.58% cross-reactivity with 11-deoxycorticosterone. The assay sensitivity is approximately 10 ng/mL and intra-assay-interassay coefficient variation was less than 10%. Quality control serum was used for the assay and all the experimental samples were run in duplicate.
Data are expressed as mean ± SEM. One way ANOVA was employed to compare the data of the control, sham operated, and AD experimental groups followed by Tukey’s multiple comparison test using the Statistical Package for Social science Software (SPSS software: 20.0.0, USA).
The latency of first entry to the inner area was significantly increased [
The latency of first entry to the inner area in an elevated open space with a novel object (for anxiety status) of different experimental groups of rats.
Groups | Day 14 | Day 21 |
---|---|---|
Control | 0.09 |
0.02 |
Control + E (10 mg/kg) | 0.07 |
0.06 |
Control + E (20 mg/kg) | 0.06 |
0.08 |
Control + E (30 mg/kg) | 0.07 |
0.06 |
Sham | 0.08 |
0.09 |
Sham + E (10 mg/kg) | 0.08 |
0.04 |
Sham + E (20 mg/kg) | 0.07 |
0.08 |
Sham + E (30 mg/kg) | 0.09 |
0.06 |
AD | 3.73 |
6.11 |
AD + E (10 mg/kg) | 0.98 |
0.26 |
AD + E (20 mg/kg) | 0.72 |
0.13 |
AD + E (30 mg/kg) | 0.33 |
0.11 |
Frequency of entry to the inner area significantly decreased [
The frequency of entry to the inner area in an elevated open space with a novel object (for anxiety status) of different experimental groups of rats.
The total time spent in the inner area significantly decreased [
The total time spent in the inner area in an elevated open space with a novel object (for anxiety status) of different experimental groups of rats.
The total time spent in the outer area significantly increased [
The total time spent in the outer area in an elevated open space with a novel object (for anxiety status) of different experimental groups of rats.
The latency of first approach to the object area significantly increased [
The latency of first approach to the object area in an elevated open space with a novel object (for anxiety status) of different experimental groups of rats.
The frequency of approach to the object area significantly decreased [
The frequency of approach to the object area in an elevated open space with a novel object (for anxiety status) of different experimental groups of rats.
The serum corticosterone level was significantly decreased [
Serum corticosterone level in different experimental groups of rats.
Though any animal model may not always exhibit all the characteristic features of a disease but some salient features of the disease are found in the animal models [
Anxiety along with dementia in AD patients has been reported for a long time. Though memory impairment is exhibited in most of the animal models of AD, the anxiety behavior is variable to some extent. The anxiety behavior depends not only on the animal models of AD but also on the methods of investigation [
It appears from these results that the parameters of anxiety (e.g., latency of first entry to inner area, frequency of entry to inner area, total time spent in the inner area, and latency of first approach to the object area) which were reduced completely to the level of control at the dose of 10 mg/kg body weight of etoricoxib in cAD rats on day 14 were dependent on the neuronal circuitry that were probably more sensitive to etoricoxib induced blocking of neurodegeneration than that of the other parameters of anxiety (total time spent in the outer area and frequency of approach to the object area) which did not reduce to the level of control at the dose of 10 mg/kg body weight of etoricoxib in cAD rats at the same time point. The anxiolytic behavior in the cAD rats may be the sum total effect of the impaired function of different neural areas which were affected by colchicine induced neurodegeneration. The protection from neurodegeneration of these brain areas after administration of etoricoxib may be the cause behind recovery from anxiolytic behavior observed in the present study. Etoricoxib can cross the blood brain barrier [
The lower serum corticosterone level was observed in the cAD rats and the administration of etoricoxib in these animals resulted in complete recovery of corticosterone levels at the lowest observed dose (10 mg/kg body wt.) on both the days 14 and 21 and continued to remain so at all the other observed doses in this study. This recovery of corticosterone in etoricoxib treated cAD rats can be corroborated with the recovery of anxiolytic behavior in these rats. The serum corticosterone level in cAD rats may be related with the anxiety status of the animals. It has been reported that the subcutaneous injection of corticosterone in rats [
The present study showed that anxiolytic behavior in cAD rats is predominantly due to cox-2 mediated neuroinflammation induced neurodegeneration in the brain.
The authors declare no conflict of interests.
This research work has been supported by UGC Major Research Project (F. no. 42-532/2013 (SR) dt. 22 March 2013). Dr. Pratip Chakraborty, IICB, Dr. S. N. Kabir, IICB, and Bidisha Bhaduri (M.S. student, CU 2013) are acknowledged for their assistance in some parts of the work.