All experimental procedures were approved by the University of Ilorin Ethical Review Committee (approval number UERC/ASN/2019/1949) and conducted in accordance with ARRIVE guidelines. Male Wistar rats weighing 150–200g, were used for the study and were housed in the Faculty of Basic Medical Sciences, University of Ilorin animal house. The rats were acclimatised for 2 weeks after which the experiments commenced. The animals were housed in cages made of wood, exposed to 12 h light/dark cycle and had unhindered access to water and food with exception to experimental procedure periods. All experimens were performed between 8am and 2pm to avoid possible diurnal variation in the behavioural tests. Pain tests or neurobehavioural test were carried out by experimenters blinded to the treatments. All efforts to minimise animal suffering and to use the minimum number of animals required to produce dependable results were employed. The animals were handled based on the laid down principle of the Guide for the Care and Use of Laboratory Animals published by National Institute of Health.
Drugs and Chemicals: Analytical grade glucosamine sulphate and chondroitin sulphate (Jiaxing Hengjie Biopharmaceutical Co., Ltd, China) were used. These drugs and their combinations were dissolved in 0.9% NaCl and orally administered using oral cannula. sodium pentobarbital (50 mg/kg i.p.) (Sigma-Aldrich, USA) was used as anesthetic agents. Other chemicals and materials were obtained from Bridge Biotech Ltd, Nigeria. 4.0 silk suture was used to induce CCI and suture the muscles, while 4.0 nylon suture was used to suture the skin.
Experimental Design: Animals were randomly grouped into seven (7) groups of six rats (n=6) per group with sample size determined based on G*power analysis  and the ethical concept of reduction . The grouping (Fig. 1A) consists of four (4) control groups.
The non-ligated control (NC) animals received normal saline (2.5 ml/kg oral) treatment without ligation. The sham control (SC) animals had the skin and muscle opened and sutured without nerve ligation. The sham animals were treated with 2.5 ml/kg orally administered normal saline. The CCI rats or the ligated control (LC) animals were induced with right sciatic nerve CCI and were treated with 2.5 ml/kg orally administered normal. GS treated animals underwent surgical procedure with chronic constriction of the right sciatic nerve and were treated with GS (240mg/kg oral). CS treated animals underwent surgical procedure with chronic constriction of the right sciatic nerve and were administered oral CS (900mg/kg oral.). GSCS treated animals underwent surgical procedure with CCI of the right sciatic nerve following which they were administered oral combination of GS (240mg/kg) and CS (900mg/kg). GB treated rats underwent surgical procedure with CCI of the right sciatic nerve and were treated with 60 mg/kg orally administered gabapentin (a reference control).
The treatments duration lasted for 21 days which commenced on the 3rd day post CCI intervention (Fig. 6).
Induction of chronic constriction injury: CCI model for neuropathic pain was applied to bring about neuropathy as portrayed by Bennett and Xie . Under sodium pentobarbital anesthesia, incision was made on the skin of the right hind limb and the sciatic nerves underneath the biceps femoris were exposed and freed of adhering tissue and three loose ligatures of silk 4.0 were tied around it with 3mm gap approximately. The sham control rats had the skin and femoris muscle opened and sutured without nerve ligation. To examine the synergistic effects, we administered a combination of GS (240mg/kg) and CS (900mg/kg) and investigated the effects on thermal hyperalgesia, mechanical allodynia, cold allodynia, sciatic nerve functional index and central proinflammatory markers.
Mechanical Allodynia (von Frey Test): To investigate the sensory functionality of the sciatic nerve, von Frey filament test (Ugo Basile, Italy) was used to measure paw withdrawal threshold to mechanical stimuli. The rats were housed in a transparent plastic with a mesh floor made of metal and before testing, were habituated to the environment for about 10 minutes. von Frey filaments of evaluator sizes 4.17, 4.31, 4.56, 4.74, 4.93, 5.07, 5.18, 5.46, 5.88 and 6.1 (corresponding to forces of 1.4g, 2g, 4g, 6g, 8g, 10, 15g, 26g, 60g, 100g respectively) were applied to the hind paw’s plantar surface. Each filament was used five times for a single paw and the mechanical threshold was calculated based on the method of Bonin et al. .
Mechanical allodynia was determined prior to CCI (baseline), then 3, 10, 17 and 24 days following CCI. Drugs treatment commenced on day 3 following CCI after post CCI tests.
Thermal Hyperalgesia (Hot-Plate Test): Thermal sensitivity of the hind paw was evaluated with a hot-plate apparatus (Hefe-joyce, China). The hot plate was preheated to 55o± 0.5o C and animals were placed on the preheated plate surface. The reaction time of paw withdrawal was recorded with a cut-of time set at 20s. Thermal analgesia was determined prior to CCI (baseline), then 3, 10, 17 and 24 days following CCI.
Cold Allodynia (Acetone Test): Latency of paw withdrawal to cold stimulus was investigated using acetone spray test as described by Yoon et al. . Cold allodynia was determined prior to CCI (baseline), then 3, 10, 17 and 24 days following CCI.
Histological Analysis: Five-ten hours after the last dose of drug treatment, rats were made unconscious and the sciatic nerve proximal to the bifurcation point was dissected. All animals were anaesthesized with sodium pentobarbital. Formalin (10%) was used to fix the DRG, included in paraffin and put through to 7µm thick transverse sections, followed by further hematoxylin and eosin (H&E) staining. The slides were examined by a person blinded to the treatments. The following parameters were analysed from the slides: nerve constituents, which include endoneurium, perineurium and epineurium, the nerve fiber, inflammatory cell infiltrates and Schwann cells.
Biochemical analysis of the DRG
Five to ten hours after the last dose of treatment, the spinal column was dissected from the base of the skull to the level of the femurs, it was cut down the mid-line and the spinal cord extruded and meninges removed as described by Sleigh et al. , L3&L4 DRG were extracted and rinsed and stored in ice-cold phosphate buffer solution (PBS) and frozen for biochemical analysis. DRG was homogenized with the aid of a glass homogenizer at 4oC in ice-cold saline (2ml) (11 mmol L−1 Tris buffer, pH 7.4). Homogenates were centrifuged at 5,000 RPM for ten minutes and supernatants were collected for the estimation of biochemical parameters using ELISA principle.
Evaluation of proinflammatory mediators
Prostaglandin E2 (PGE2), (Bioassay Technology Laboratory, Shanghai, China) Tumor necrotic factor alpha (TNF-α) (Elabscience Biotechnology Inc., Texas, USA), interleukin 6 (IL-6) (Elabscience Biotechnology Inc., Texas, USA), interleukin 1 beta (IL1-β) (Elabscience Biotechnology Inc., Texas, USA), nerve growth factor (NGF) (Bioassay Technology Laboratory, Shanghai, China) and nuclear factor kappa B-105 (NFκB105) (Elabscience Biotechnology Inc., Texas, USA) levels in the DRG homogenate were assessed using ELIZA techniques based on the manufacturer’s instructional manual.
DRG tissue (L5) was investigated using haematoxylin and eosin stain (H&E). Histological examination of slide was as described by Windsor  and Hopwood . The DRG tissues were fixed in 10% formalin for over 24 hours before dehydrating, clearing, embedding and staining.
Immunohistochemistry: Expression of nuclear factor kappa B (NFκB p65) was evaluated using the Thermo Scientific Pierce Peroxidase IHC Detection Kit (36000, Thermo Sciencific, USA) with modest procedure modification. Endogenous peroxidase activity was quenched by incubating tissue for 30 minutes in Peroxidase Suppressor, washed three times in Wash Buffer. Blocking buffer was added to the slides and incubated for 30 minutes. Excess buffer was blotted from the tissue sections, before addition of primary antibodies; NFkB p65 (Cat #14-6731-81, Thermo Sciencific, USA), at a dilution of 1:100 and left overnight in a humidified chamber at 4°C. Afterward, slides were washed two times for 3 minutes with Wash Buffer.
The tissue sections were treated with Biotinylated Secondary Antibody and incubated for 30 minutes. The slides washed three times for 3 minutes each with Wash Buffer, treated and incubated with the Avidin/Streptavidin-HRP for another 30 minutes, and washed three times for 3 minutes each with Wash Buffer. The tissues were incubated with Metal Enhanced DAB (3.3 diaminobenzidine) Substrate Working Solution for 5 minutes for desired staining to be achieved. The slides were rinsed with distilled water and drained. Adequate amount of Mayer’s hematoxylin stain was dropped on to the slide to cover the entire tissue surface and incubated for 1-2 minutes at room temperature. Drained off the hematoxylin and washed slide several times with distilled water. The slides were mounted and photomicrographed with Amscope MU900 digital camera attached to the microscope. The intensity of staining was examined with the open-source Fiji (ImageJ) software.
GraphPad Prism 8.0.1 software (USA) was used for statistical analyses. Values are expressed as Mean ± S.E.M. Behavioural data were analysed using one way and two-way analysis of variance (ANOVA) with Tukey’s post hoc multiple comparisons test. Biomarkers data were analysed with one-way ANOVA followed by Turkey’s post hoc multiple comparisons test. P<0.05 are considered significant.