Swimming exercise on spatial memory performance and hippocampal oxidative stress in colchicine-induced memory-impaired male Long-Evans rats
Authors
- Rokhsana Binte AminDepartment of Physiology, Bangabandhu Sheikh Mujib Medical University (currently Bangladesh Medical University), Dhaka, Bangladesh
- Puspita BasakDepartment of Physiology, Bangabandhu Sheikh Mujib Medical University (currently Bangladesh Medical University), Dhaka, Bangladesh
- Fhamida AkterDepartment of Physiology, Bangabandhu Sheikh Mujib Medical University (currently Bangladesh Medical University), Dhaka, Bangladesh
- Md. Saiful IslamDepartment of Physiology, Bangabandhu Sheikh Mujib Medical University (currently Bangladesh Medical University), Dhaka, Bangladesh
- Kazi Rafiqul IslamDepartment of Pharmacology, Bangladesh Agricultural University, Mymensingh, Bangladesh
- Taskina AliDepartment of Physiology, Bangabandhu Sheikh Mujib Medical University (currently Bangladesh Medical University), Dhaka, Bangladesh
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Published by Bangabandhu Sheikh Mujib Medical University (currently Bangladesh Medical University).
Methods: Thirty male aged 8 standard deviation (2) weeks; weight 225 (75) gm Long-Evans rats were assigned to normal control, sham control, colchicine control, pre colchicine swimming exercise and post colchicine swimming exercise groups. A memory-impaired rat model was established by administering colchicine intrahippocampally. Swimming exercise was performed before and after spatial memory impairment. For spatial reference and working memory performance evaluation, the Morris water maze test was used. Hippocampal malondialdehyde and glutathione peroxidase were estimated for oxidative stress assessment in all rats.
Results: Intrahippocampal colchicine administration significantly impaired spatial memory, and elevated malondialdehyde, decreased glutathione peroxidase level in the hippocampus of colchicine control rats. In contrast, both pre- and post-treatment with swimming exercise significantly improved learning and spatial memory retention and attenuated oxidative damage to nearly normal levels.
Conclusion: Swimming exercise prevents as well as alleviates colchicine-induced spatial memory impairment along with hippocampal oxidative stress in male Long-Evans rats. Moreover, this swimming exercise schedule is sufficient to reverse these alarming consequences to almost normal.
Currently, the long-term memory is known as reference memory (RM) [5]. Here, the information available for solving RM tasks remains constant throughout the trials and is reinforced by repeated training [6]. On the other hand, working memory (WM) is a form of short-term memory that retains information for a short period, usually while an individual plans an action based on it [7]. Both of these memories depend on the hippocampus, the most medial portion of the temporal lobe cortex. Neurodegeneration of the hippocampus can lead to memory impairment, and oxidative stress is one of the proposed mechanisms underlying this neurodegeneration [8]. Oxidative stress is caused by reactive oxygen species, which are part of a group of molecules called free radicals, and leads to cell injury [9]. Malondialdehyde (MDA), an end product of lipid peroxidation, is commonly measured as an indirect index of oxidative stress [10]. Superoxide dismutase, glutathione peroxidase (GPx) play a crucial role against oxidative injury as scavengers [9].
The beneficial effects of SwE on memory impairment, as well as oxidative stress, before or after hippocampal damage remained unresolved [18]. However, the volume of information regarding the effects of SwE on memory impairment and oxidative stress is not enough for reaching any final inference. The present study has been designed to examine the effects of SwE on memory performance, as well as hippocampal oxidative stress markers, before and after colchicine-induced spatial memory impairment in male Long-Evans rats.
This experimental study was conducted at the Department of Physiology, Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka, Bangladesh, from March 2023 to February 2024. The sampling technique was purposive. Here, the specified difference between the two means was compared to test the null hypothesis. So the formula, n = [(u + v)2 X (σ12 +σ22)] / (μ1 – μ2 )2, involving effect size was used to calculate the intended sample size for a group [19]. We used mean and standard deviation of ‘escape latency in reference memory’ version of the Morris Water Maze test, stated by Raghavendra et al. (2013) [20]. Thus, we used 30 rats.
A total of 30 male Long-Evans rats aged 8 (standard deviation 2) weeks; weight 225 (75) gm were collected from the central animal house of BSMMU and were housed in standard laboratory environments (temperature: 27°C, 12/12 hour light-dark cycle). The rats were provided with free access to standard laboratory food [21] and cooled boiled water ad libitum. All the experiments were conducted according to the ῾Guidelines for the Animal Experimentation Ethics Committee (AEEC, 15 May 2023) of the International Centre for Diarrhoeal Disease Research, Bangladesh and the advice of an expert veterinarian. All the experiments were performed during the daytime between 08:00 and 16:00 hours.
- Normal control (NC): No stereotaxic surgery (SS) + no normal saline (NS) + no swimming exercise (no SwE).
- Sham control (SC): SS + hippocampal infusion of 1 µl NS + no SwE.
- Colchicine control (ColC): SS + hippocampal infusion of 15 µg of colchicine in 1µl NS + no SwE.
- Pre colchicine swimming exercise (Pre-SwE Exp): SwE for 28 days followed by SS + hippocampal infusion of 15 µg of colchicine in 1µl NS.
- Post colchicine swimming exercise (Post-SwE Exp): SS + hippocampal infusion of 15 µg of colchicine in 1µl NS followed by SwE for 28 days.
SwE was performed in a circular pool (150 cm X 50 cm) for 28 days in rats of Pre-SwE Exp and Post-SwE Exp groups. Treatment was conducted five days a week. During the initial two days, SwE was started at 10 minutes per day and gradually increased by 10 minutes every two days until reaching 1 hour per day by the 12th day. From that moment onward, training continued at a rate of one hour per day until day 26.
According to previous research, [22-24] colchicine (Incepta Pharmaceuticals Ltd, Bangladesh) was administered into the rat hippocampus by SS. Each rat was deeply anesthetised with thiopental sodium (45 mg/kg, i.p.) and positioned in a stereotaxic apparatus (BioMed Easy Technologies Co., Ltd.). The scalp was incised and retracted. The rat was infused through a Hamilton microsyringe with 15 µg of colchicine in 1 µL of NS in each hippocampus. Controls that received the vehicle injection were utilised. The coordinates were -3.6 mm anterior-posterior, ±2 mm lateral-medial, and -3.4 mm dorso-ventral relative to bregma. All intra-hippocampal applications of colchicine and/or NS were infused very slowly over 1 minute, and the micro syringe was kept in place for the next minute before being slowly withdrawn. Gentamicin (5 mg/kg, i.p.) was administered postoperatively to prevent sepsis.
Test tools and circumstances [25-27]
MWM was a large circular pool (150 cm x 50 cm) filled to a depth of 30 cm with water at 24 to 26 °C. This pool was arbitrarily divided into four quadrants: north-west (NW), north-east (NE), south-east (SE) and south-west (SW). A round platform (15 cm × 28 cm) was placed at the center of one quadrant, with its top 2 cm below the water's surface, and served as the only escape route for the rats from the water. To determine the start locations, eight points —north (N), south (S), east (E), west (W), northeast (NE), northwest (NW), southeast (SE), and southwest (SW) of MWM — were labelled. To prevent visual clues in the pool, the entire inner wall of the pool and platform was painted with a non-toxic black color. The pool was placed in a room containing extra maze cues that rats could use to assist them in navigating. Two distinct testing paradigms were sequentially employed to assess reference and working memory skills, as illustrated in the Supplementary file.
The rats swam without a platform for 3 minutes over three consecutive days to acclimatise and habituate themselves to the reference memory version. Then, during the acquisition phase, all rats underwent four trials each day for six consecutive days, with the platform consistently placed in the NE quadrant. In every trial, the rats were released from different starting points in a predetermined sequence each day and given 60 seconds to locate the platform and climb onto it. A 50-second inter-trial time (20 seconds on the platform and 30 seconds for self-drying) was allowed. The time taken for the rat to find the platform, also known as the mean escape latency (EL), was measured using a stopwatch to assess the rat's learning ability. On the first trial each day, the rats found the platform by chance, which served as the information stage. The subsequent trials necessitated matching to the new position each day, as the platform remained in the same place for 6 days. The average EL of the 5th and 6th acquisition days was measured to assess memory consolidation. Approximately 24 hours after the last trial on day 6, the platform was removed from the pool, and a final spatial probe trial was conducted to assess the strength of learning and retrieval. In this probe trial, rats swam freely for 60 sec during which target crossings (TC; number of passing the quadrant made by rats within 60 sec after the platform was removed) and time spent in target (TT; the time spent in the quadrant from where the platform was removed during the same 60 sec period) were measured [28].
Working memory test
A testing paradigm adapted from Sarihi et al. [25] was used to conduct the working memory version of the test approximately 48 hours following the probe trial. Here, the 6-day acquisition phase of the reference memory test was considered as the pre-training phase of the working memory test. Then, a training and test phase was conducted over four consecutive days, with four trials each day. Here, the platform position was altered daily but remained the same for the four trials. However, during the four daily trials, each rat was released from four distinct starting points, all of which were far from the platform position. Rats randomly arrived at the platform on the first trial of each day, which served as the information stage. The following trials needed to be matched to the new position for that day, as the platform was changed daily. The mean escape latency in the training and test phases was recorded as above to assess learning ability and savings (the difference in latency scores between trials 1 and 2, expressed as the percentage of savings increased from trial 1 to trial 2) was measured to assess learning efficiency [28, 29].
Rats were euthanised through decapitation under diethyl ether anesthesia, 24 hours following the final behavioral test [15]. Then the brain tissues were swiftly taken out, and the hippocampal tissues were promptly isolated. They were then carefully rinsed with ice-cold Phosphate-Buffered Saline (PBS) (0.1 M, pH 7.4) to ensure complete removal of excess blood. The tissue pieces were weighed and homogenised in PBS using a glass homogeniser on ice, maintaining a ratio of weight (g) to volume (mL) of 1:4. Next, the homogenate underwent centrifugation at 3500 rpm for 10 minutes to obtain the supernatant. The supernatant was collected for the estimation of MDA and GPx levels by ELISA according to the manufacturer’s protocol (Elab Science Biotechnology, USA). If any unintended delay occurred, the supernatant was stored in a laboratory at -20°C.
Data were expressed as mean (standard error) of the study variables and statistically analysed using ANOVA followed by the Bonferroni post hoc test (between groups) using SPSS (version 25.0), P <0.05 was considered statistically significant.
For reference memory performance
ColC rats showed significantly (P <0.01) higher mean EL than those of SC rats. However, SwE improved learning ability performance, as evidenced by statistically significant (P <0.01) differences in mean EL of our ColC and experimental (Pre-SwE Exp and Post-SwE Exp) rats. Notably, the differences in this variable between experimental and NC rats were found to be statistically non-significant on the last acquisition day. However, the differences of mean EL between Pre-SwE Exp vs Post-SwE Exp rats were found statistically non-significant in all acquisition days (Table 1). These data demonstrated that the learning ability of reference memory was impaired by colchicine, and this impairment was reversed almost to normal by SwE.
Mean escape latency of acquisition day | Groups | ||||
NC | SC | ColC | Pre-SwE Exp | Post-SwE Exp | |
Days |
|
|
|
|
|
1st | 26.2 (2.3) | 30.6 (2.4) | 60.0 (0.0)b | 43.2 (1.8)b | 43.8 (1.6)b |
2nd | 22.6 (1.0) | 25.4 (0.6) | 58.9 (0.5)b | 38.6 (2.0)b | 40.5 (1.2)b |
3rd | 14.5 (1.8) | 18.9 (0.4) | 56.5 (1.2)b | 34.2 (1.9)b | 33.8 (1.0)b |
4th | 13.1 (1.7) | 17.5 (0.8) | 53.9 (0.7)b | 35.0 (1.6)b | 34.9 (1.6)b |
5th | 13.0 (1.2) | 15.9 (0.7) | 51.7 (2.0)b | 25.9 (0.7)b | 27.7 (0.9)b |
6th | 12.2 (1.0) | 13.3 (0.4) | 49.5 (2.0)b | 16.8 (1.1)b | 16.8 (0.8)b |
Average of acquisition days | |||||
5th and 6th | 12.6 (0.2) | 14.6 (0.8) | 50.6 (0.7)b | 20.4 (2.1)a | 22.4 (3.2)a |
NC indicates normal control; SC, Sham control; ColC, colchicine control; SwE, swimming exercise exposure. aP <0.05; bP <0.01. |
Categories | Number (%) |
Sex |
|
Male | 36 (60.0) |
Female | 24 (40.0) |
Age in yearsa | 8.8 (4.2) |
Education |
|
Pre-school | 20 (33.3) |
Elementary school | 24 (40.0) |
Junior high school | 16 (26.7) |
Cancer diagnoses |
|
Acute lymphoblastic leukemia | 33 (55) |
Retinoblastoma | 5 (8.3) |
Acute myeloid leukemia | 4 (6.7) |
Non-Hodgkins lymphoma | 4 (6.7) |
Osteosarcoma | 3 (5) |
Hepatoblastoma | 2 (3.3) |
Lymphoma | 2 (3.3) |
Neuroblastoma | 2 (3.3) |
Medulloblastoma | 1 (1.7) |
Neurofibroma | 1 (1.7) |
Ovarian tumour | 1 (1.7) |
Pancreatic cancer | 1 (1.7) |
Rhabdomyosarcoma | 1 (1.7) |
aMean (standard deviation) |
Variables in probe trial day | Groups | ||||
NC | SC | ColC | Pre-SwE Exp | Post-SwE Exp | |
Target crossings | 8.0 (0.3) | 7.3 (0.3) | 1.7 (0.2)a | 6.0 (0.3)a | 5.8 (0.4)a |
Time spent in target | 18.0 (0.4) | 16.2 (0.7) | 5.8 (0.8)a | 15.3 (0.7)a | 15.2 (0.9)a |
NC indicates normal control; SC, Sham control; ColC, colchicine control; SwE, swimming exercise exposure. aP <0.01. |
Pain level | Number (%) | P | ||
Pre | Post 1 | Post 2 | ||
Mean (SD)a pain score | 4.7 (1.9) | 2.7 (1.6) | 0.8 (1.1) | <0.001 |
Pain categories | ||||
No pain (0) | - | 1 (1.7) | 31 (51.7) | <0.001 |
Mild pain (1-3) | 15 (25.0) | 43 (70.0) | 27 (45.0) | |
Moderete pain (4-6) | 37 (61.7) | 15 (25.0) | 2 (3.3) | |
Severe pain (7-10) | 8 (13.3) | 2 (3.3) | - | |
aPain scores according to the visual analogue scale ranging from 0 to 10; SD indicates standard deviation |
Here, colchicine caused significantly higher mean EL (P <0.01) and lower savings (P <0.01) in the training and test phases in ColC rats compared to those in SC rats. However, both of our experimental rats showed significantly lower mean EL [Pre-SwE Exp (P <0.01 in trial 1; P <0.01 in trial 2, 3 and 4) and Post-SwE Exp (P<0.05in trial 1; P <0.01 in trial 2; P <0.01 in trial 3 and 4)], but not savings, when compared to those of ColC rats. Moreover, when we compared these variables between our experimental and NC rats, mean EL was found to be statistically non-significant in trial 4, except for savings. Furthermore, no statistically significant difference was observed in any of these variables between Pre-SwE Exp and Post-SwE Exp rats (Table 3). Therefore, based on these findings, it is evident that our 28-day SwE could improve the learning disability of colchicine-induced WM impairment in our rats, although not entirely and efficiently.
Trials | Groups | ||||
NC | SC | ColC | Pre-SwE Exp | Post-SwE Exp | |
1 | 20.8 (0.6) | 22.1 (1.8) | 41.1 (1.3)b | 31.9 (1.9)b | 32.9 (1.8)a, b |
2 | 10.9 (0.6) | 14.9 (1.7) | 37.4 (1.1)b | 24.9 (2.0)b | 26.8 (2.5)b |
3 | 8.4 (0.5) | 9.9 (2.0) | 32.8 (1.2)b | 22.0 (1.4)b | 21.0 (1.4)b |
4 | 7.8 (0.5) | 10.4 (1.3) | 27.6(1.1)b | 12.8 (1.2)b | 13.0 (1.4)b |
Savings (%)c | 47.7 (3.0) | 33.0 (3.0) | 10.0 (0.9)b | 23.6 (2.7)b | 18.9 (5.3)b |
NC indicates normal control; SC, Sham control; ColC, colchicine control; SwE, swimming exercise exposure. aP <0.05; bP <0.01. cThe difference in latency scores between trials 1 and 2, expressed as the percentage of savings increased from trial 1 to trial 2 |
Hippocampal oxidative stress markers | Groups | ||||
NC | SC | ColC | Pre-SwE Exp | Post-SwE Exp | |
Malondialdehyde (ng/mg protein) | 8.0 (1.4) | 8.3 (0.6) | 17.3 (2.0)b | 9.7 (1.4)b | 11.7 (2.0)a |
Glutathione peroxidase (pg/mg protein) | 316.2 (28.9) | 258.3 (14.6) | 121.8 (6.4)b | 308.8 (22.2)b | 296.0 (26.2)b |
NC indicates normal control; SC, Sham control; ColC, colchicine control; SwE, swimming exercise exposure. aP <0.01; bP <0.001. |