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CHAPTER 3 of the 6 regions was marked on the animals’ back, and 10 consecutive innocuous “pokes” were delivered in each boundary at an inter-poke-interval of approximately 1-2 s, or until the animal recovered from movement evoked from the previous poke if longer than 2 seconds. Prior to testing, the operator practiced the stimulus procedure. This ensured that each poke was as brief as possible, that the filament landed normal (90°) to the skin surface, and that the final position of the filament handle was approximately half the distance to that of the distance at initial contact of the filament. This protocol ensured pokes of consistent duration and maximum force which was confirmed using a weighing balance (maximum bending force: 2.86 ± 0.09 g; n = 10 pokes). During sensitivity testing, animals were “semi-restrained” in a V-shaped plastic box. This restricted the animal’s ability to avoid the testing procedure and thereby facilitated the operator’s accuracy of each poke, but enabled sufficient movement for the animal to display behavioural responses of interest. Testing was recorded using a webcam (Logitec HD Pro C920). Videos were assessed blind to the observer in slow motion play back by evaluating the response to each innocuous poke that was graded into one of four categories as: I) no response; II) mild response characterized by acknowledgment of the stimulus, head turns, brief shuddering of the contacted skin, but no obvious pain avoidance behaviours; III) medium response, characterized by moderate signs of pain perception, including moderate avoidance attempts by moving away from the stimulus; IV) severe response, characterized by severe signs of pain perception, including attacking the stimulus and “desperate” avoidance attempts and escape behaviours including jumping, running, writhing or audible vocalization. The four categories, I-IV, were chosen because these behaviours are easily distinguishable. The frequency of each response category was multiplied by a weight; categories I-IV were multiplied by 0, 1, √2, and 2 respectively to provide greater separation between ordinal pain behaviours between non-painful and painful (Coderre et al., 1993), as well as to help minimize heteroscedasticity of the data. The sum of the 10 weighted responses provided a regional sensitivity score (RSS) for each region. This paradigm enables high resolution measures of sensitivity to 10 innocuous pokes with each possible RSS ranging between 0 and 20. Scores from ipsi- and contralateral regions were pooled to determine level sensitivity scores (LSS) above, at and below the level of injury. A cumulative sensitivity score (CSS) was derived for each animal by summing the RSS from all 6 regions; the maximum CSS possible is therefore 120. The hypersensitivity threshold was defined by the mean + 2 standard deviations (confidence interval of 95.5%) of CSSs calculated from uninjured intact rats (control group). 3.3.6 Somatosensory assessment Animals were anaesthetized with urethane (12.5% w/v; 1.4 g/kg; i.p.) and maintained at 37 °C on a heating mat. A tracheotomy was performed and animals were placed in a stereotaxic frame. The gracile nuclei were exposed through the foramen magnum by head flection and removal of overlying muscles and meninges. Both left and right sciatic and sural nerves were exposed by removal of the overlying skin followed by a splitting incision of the gluteus maximum and semimembranosus muscles respectively. The exposed nerves were isolated from adjacent 60PDF Image | Effects of Red Light Treatment on Spinal Cord Injury
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