Mice. Male Swiss mice (11-week old, 29-34 g and at least five times backcrossed) obtained from Central Animal Facility, University of Ilorin, Nigeria were used for this study. The mice were housed in the animals’ facility of Bioresearch Hub Laboratory where the experiment was carried out. Prior to any procedure, the mice were allowed to acclimatise for 1 week in a standard housing condition at a relatively constant temperature with regular light-dark cycle. Mouse pellet and water were accessible ad libitum before and during the experiment. All procedures here stated were as approved by the University Ethical Review Committee, University of Ilorin, Nigeria; and the proposal was issued with approval number – UERC/ASN/2019/1923.
Exposure to varied home-cage sizes. Mice were randomly divided into two, cage stationed (Control Group) and cage migrated (Test Group). For all the experiments performed, animals were housed in six (n=6) per group. Mice assigned to cage stationed group were kept in the same size cage of 45 x 25 x 15 cm (length x width x height) for 30-day duration of the experiment. These mice were transferred to clean same size cage within a period of 60-second at 8 am daily. The same toys and water bottle were returned into the cage after each transfer. Mice allotted to cage migrated were exposed to cage of diverse sizes. Exposure to new cage size was done daily (every 24 hours) at 8 am and all mice were moved into the new size cage within an average time of 60 seconds. The same toys and water bottle were returned to the new cage. From Days 1 to 15, mice were exposed to the following cage respectively - 45 x 25 x 15 cm, 35 x 20 x 15 cm, 60 x 30 x 20 cm, 45 x 15 x 15 cm, 50 x 25 x 20 cm, 30 x 20 x 14 cm, 45 x 20 x 20 cm, 40 x 25 x 14 cm, 60 x 15 x 30 cm, 35 x 30 x 15 cm, 30 x 30 x 14 cm, 40 x 24 x 20 cm, 50 x 30 x 25 cm, 35 x 15 x 8 cm, and 30 x 15 x 15 cm. On Days 16 to 30, mice were re-exposed to the above cages respectively starting from the earliest. All cages were transparent plastics with thick gauze-like galvanized metal lids.
Weight. The body weights were measured weekly with Kerro weighing scale (Kerro Scale BL3002, Taiwan) starting from the first day of the experiment. Body weights were monitored for four (4) weeks. At expiration of different cage size exposure (Day 30), mice were sacrificed under anesthesia and weights of vital organs were taken in relative to the body weight of each mouse (organosomatic ratio).
Fasting and Oral glucose tolerance test. Fasting blood glucose levels were determined in 12-hour fasted mice using blood from their tail veins. The glucose levels were quantified using ACCU-CHEK glucometer. Oral glucose tolerance test was carried out following a preceding oral glucose load of 50mg in 12-hour fasted mice and subsequence measurement of blood glucose levels at 0, 60 and 120 min using ACCU-CHEK glucometer machine. Values were presented in mg/dl.
Emotion-associated Behavioural Studies. Emotion-related behaviours were evaluated using paradigms such as open field maze, light and dark box, and tail suspension.
Open field maze was first described by Hall in 1934 and it provides means for simple assessment of activities, and general behaviours in rodents [10]. The open field maze used was 40 x 40 x 30 cm in dimensions with a dirty white tile floor. Using black paint, the floor was divided into a 16-cell of 10 cm2 each. Each mouse was placed at the centre of the maze with the animal facing forward away from its handler. Five (5) minutes of exploration was allowed and the entire maze was cleaned with 70% ethanol before another mouse was introduced. Total line crossing [ambulation], frequency of rearing, grooming time and time spent probing the central part of the maze were used in assessing emotional state of the mice.
Light/dark box test explores the inherent aversion to illuminated arena in rodents. The paradigm used was a two-compartment (a dark and a light) box with a total size of 83 x 42 x 30 cm. Each compartment was 40 x 40 x 30 cm in size and the two compartments were linked with a tunnel of 2 x 2 cm. Mice were individually placed in the brightly illuminated light compartment and allowed to explore the maze for 5 minutes. The total number of visits and time expended in the light or dark compartment were used to determine the emotional state of the animals as earlier described in the literature [11,12].
Tail suspension test was used to evaluate the behavioural and physiological aspect of emotional state in experimental mice [13]. Briefly, each mouse was suspended by its tail with paper tape on a fixed retort stand. Every mouse spent 5 minutes on tail suspension, total number of attempt to turn against the gravity (tail climbing) and time spent inactive were used to quantify mood of the animals.
Pain-related Behavioural Studies. Pain-linked behavioural responses were used to assess pain sensitivity in experimental mice subjected to noxious chemical, thermal and mechanical stimuli.
Formalin pain test was used to assess animals’ responses to noxious chemical. After 15 minutes habituation in a transparent glass arena, 20µl of 2.5% PBS-formalin solution was injected subcutaneously in the plantar surface of right hindpaw of each mouse. The total time spent on paw licking was monitored for 50 minutes and was used to evaluate pain perception as described in the literature [14]. The time spent were eventually grouped into 0-5 minutes for nociceptive [early phase] pain and 20-30 for inflammatory (late phase) pain.
Hot- and cold- plate test were used to quantify pain sensitivity of mice to noxious thermal stimuli of 55 oC and 0 oC respectively. Each mouse was placed within the plexiglass walls positioned on a preheated iron plate surface maintained at 55 oC and the time taken for the mouse to jump was detailed as nociception threshold point. The cold plate used was a thin glass surface stand with attached perforated restriction glass chambers on it. The restriction boxes were blinded to prevent mice from seeing one another. Following 15 minutes acclimatisation, ice was applied to the plantar surface of the right hindpaw till animal demonstrated nocifensive behaviour such as lifting the affected limb or moving away from the stimulus. Time taken to exhibit these behaviours was accepted as nociceptive end-point and the average of three trials within 3 minutes apart was recorded for each mouse.
Von Frey Test. Pain sensitivity to mechanical noxious stimulus was assessed via quantifying withdrawal thresholds of the right hind paw to calibrated filament [15]. The filament was applied with enough pressure to cause bucking against the ventral surface of hind paw and was allowed to remain for 5 seconds before its removal for an unresponsive mouse. From the least filaments of 0.008 g to 6 g, the twelve von Frey filaments were used to probe for pain sensitivity in a continuous ascending order starting from the smallest calibration. The withdrawal threshold was accepted for a calibrated von Frey filaments if animal withdrew its hind paw at least five times from seven applied stimuli.
Note – Every maze was cleaned with 70% ethanol solution before introducing another mouse. All behavioural studies were video-recorded and extraction of data was done by three trained persons blinded to the experiment.
Biochemical assays. Mice were sacrificed at experimental endpoint (Day 30) under continuous inhalation of anesthetic drug, isoflurane. Blood was collected with 1 ml syringe via cardiac puncture and was emptied into lithium-heparinized Eppendorf bottle. This was then centrifuged at 3000 rpm for 10 minutes under 4 oC. The plasma extracted from all samples were used for chemical analysis. Immediately after blood collection, mouse was transcardially perfused with 20 ml of cold PBS, the brain was carefully removed, homogenised, centrifuged (3000 rpm, 10 minutes and at 4 oC) and the supernatant was collected into plane bottle.
Protein assay. Bradford protein assay was used to evaluate the total protein content of the plasma, harvested organs and brain homogenates. The principle of this assay borders on capacity of protein molecule to bind on Coomassie dye. The reaction gives best result under acidic conditions and it is characterised with a colour change from brown to blue. In this study, the homogenate was diluted to obtain 50μg protein/30μl. 30μl of the sample or 30μl of the standard solution was added to separate test tubes. 30μl of water was added to two other test tubes for the standard curve. 30μl of buffer was added to the test tube containing the sample, followed by the addition 1.5ml of Bradford reagent to each test tube. The test tubes were incubated at room temperature for 5minutes after which the absorbance was read at 595nm.
Inflammatory mediators and neurotransmitters assays. NGF, 1L-10, 1L-6, 1L-1β, TNF-α, NF-Kb, serotonin, noradrenaline and GABA were assessed in the brain in duplicate using ELISA kits manufactured by Elabscience, China. The samples were appropriately added to the varied kit plates and the assays were performed in accordance to the manufacturer instructions.
Malondialdehye. This assay is based on the reaction of a chromogenic reagent, 2-thiobarbituric acid with MDA at 25o C. One molecule of MDA reacts with 2 molecules of 2-thiobarbituric acid via knoevenagel-type condensation to yield a chromophore with absorbance maximum at 532 nm. Standard and samples solutions were prepared and 300µl of each was added into microwell, followed by the addition of 300µl of indicator to the sample and standard wells which were mixed. The mixtures were incubated for 45 minutes at room temperature. The absorbance of the resulting solution was taken at 532 nm using microplate reader.
Histological Studies. On the last day of the experiment, blood was collected from anaesthetised mice, perfused with cold phosphate-buffered saline and then followed by perfusion with 4% phosphate-buffered paraformaldehyde via cardiac puncture. Sections of the brain showing motor and anterior cingulate cortices were exercised, fixed in 4% phosphate-buffered paraformaldehyde, stored under 4oC for 24 hours, processed in ascending grades of ethanol, cleared in xylene and embedded in paraffin. Sections (5µm; MK 1110 rotary microtome) of the cortices were stained with hematoxylin and eosin (H&E), and Cresyl fast violet (CFV). Immunohistochemistry was also performed on similar coronal sections of paraffin-embedded cortices tissues using anti-mouse-Ki-67 (1:100, BIO-RAD, UK) and GfaP (1:100, BIO-RAD, UK). Sections were deparaffinised, subjected to antigen retrieval and immunostained with the Ki-67 and Gfab antibodies. Photomicrographs of the processed tissues were captured under 100X objective len using the Zeiss Axiostar plus light microscope.
Statistical Analysis. The present data were analyzed with 7.0 version of Graph Pad Prism. The specific tool used include unpaired Student’s t-test and EC50 shift. Data were reported in mean ± SEM and statistical significance was accepted at p < 0.05.