Refinement of the motorised laminectomy-assisted rat spinal cord injury model by analgesic treatment

Abstract

Usage and reporting of analgesia in animal models of spinal cord injury (SCI) have been sparse and requires proper attention. The majority of experimental SCI research uses rats as an animal model. This study aimed to probe into the effects of some commonly used regimens with NSAIDs and opioids on well-being of the rats as well as on the functional outcome of the model. This eight-week study used forty-two female Wistar rats (Crl: WI), randomly and equally divided into 6 treatment groups, viz. I) tramadol (5mg/kg) and buprenorphine (0.05mg/kg); II) carprofen (5mg/kg) and buprenorphine (0.05mg/kg); III) carprofen (5mg/kg); IV) meloxicam (1mg/kg) and buprenorphine (0.05mg/kg); V) meloxicam (1mg/kg); and VI) no analgesia (0.5 ml sterile saline). Buprenorphine was administered twice daily whereas other treatments were given once daily for five days post-operatively. Injections were given subcutaneously. All animals underwent dental burr-assisted laminectomy at the T10-T11 vertebra level. A custom-built calibrated spring-loaded 200 kilodynes force deliverer was used to induce severe SCI. Weekly body weight scores, Rat Grimace Scale (RGS), and dark-phase home cage activity were used as markers for well-being. Weekly Basso Beattie and Bresnahan (BBB) scores served as markers for functionality together with Novel Object Recognition test (NOR) at week 8 and terminal histopathology using area of vacuolisation and live neuronal count from the ventral horns of spinal cord. It was concluded that the usage of analgesia improved animal wellbeing while having no effects on the functional aspects of the animal model in comparison to the animals that received no analgesics.

Fig 1. Body weight comparison on days 7(a), 14 (b), 21 (c) and 28 (d) showed differences between groups.

* = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** P <0.0001.

Fig 2. Rat Grimace Scale showed changes between groups with respect to the baseline on days 7 (a) and 14 (b).

* = P < 0.05, ** = P < 0.01, *** = P < 0.001.

Fig 3. Dark phase activity in home cages showed differences between groups on days 1 (a), 7 (b) and 14 (c).

* = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** P <0.0001.

Fig 4. Basso beattie and bresnahan score (BBB).

There existed no differences between groups on days 1 (a), 7 (b), 14 (c), 21 (d), 28 (e), and 56 (f) in the BBB scores which is a measure of motor activity functionality in spinal cord injured rats.

Fig 5. Novel object recognition test (NOR).

The time spent (in seconds) to explore the novel object (T_N) in comparison to familiarised object (T_F) differed in all the groups before surgery at (Day 0) compared to the end of the study (Day 56). * = P < 0.05, **** P <0.0001.

Fig 6

a. Cross section of the spinal cord an intact spinal cord with inset showing the site of analysis, the ventral horn region bilaterally, performed for the entire study. b. Cross section of the spinal cord with spinal cord injury at lesion epicentre with inset showing the site of analysis for area of vacuolation and live neuronal count.

Fig 7. Percentage of vacuolation in ventral horns of spinal cord in rats.

All the groups differed significantly in vacuolation area in comparison to the non-SCI control group. * = P < 0.05.

Fig 8. Vacuolation in ventral horns in Hematoxylin and Eosin staining of spinal cord in rats.

Representative photographs from each group as analysed for comparison (Fig-8a to 8f represents Groups I to VI respectively, serially in order). The scale-bar used in the picture is 50μm.

Fig 9. Live neuronal count- All the groups differed significantly in number of live neurons in comparison to the non-SCI control.

* = P < 0.05.

Fig 10. Live neuronal count- Representative pictures of live neurons counted in ventral horns in Nissl’s staining of spinal cord in rats.

Live neurons from each group as analysed for comparison is shown (Fig- 10a to 10f represents Groups I to VI respectively, serially in order). The scale-bar used in the picture is 50μm.