Evaluation of Five Tests for Sensitivity to Functional Deficits following Cervical or Thoracic Dorsal Column Transection in the Rat.

The dorsal column lesion model of spinal cord injury targets sensory fibres which originate from the dorsal root ganglia and ascend in the dorsal funiculus. It has the advantages that fibres can be specifically traced from the sciatic nerve, verifiably complete lesions can be performed of the labelled fibres, and it can be used to study sprouting in the central nervous system from the conditioning lesion effect. However, functional deficits from this type of lesion are mild, making assessment of experimental treatment-induced functional recovery difficult. Here, five functional tests were compared for their sensitivity to functional deficits, and hence their suitability to reliably measure recovery of function after dorsal column injury. We assessed the tape removal test, the rope crossing test, CatWalk gait analysis, and the horizontal ladder, and introduce a new test, the inclined rolling ladder. Animals with dorsal column injuries at C4 or T7 level were compared to sham-operated animals for a duration of eight weeks. As well as comparing groups at individual timepoints we also compared the longitudinal data over the whole time course with linear mixed models (LMMs), and for tests where steps are scored as success/error, using generalized LMMs for binomial data. Although, generally, function recovered to sham levels within 2-6 weeks, in most tests we were able to detect significant deficits with whole time-course comparisons. On the horizontal ladder deficits were detected until 5-6 weeks. With the new inclined rolling ladder functional deficits were somewhat more consistent over the testing period and appeared to last for 6-7 weeks. Of the CatWalk parameters base of support was sensitive to cervical and thoracic lesions while hind-paw print-width was affected by cervical lesion only. The inclined rolling ladder test in combination with the horizontal ladder and the CatWalk may prove useful to monitor functional recovery after experimental treatment in this lesion model

Histological evaluation of lesion completeness.

<p>Animals received a dorsal column transection at T7 or C4 level or sham surgery and survived eight weeks after which ascending sensory axons were transganglionically labelled with cholera toxin-B (CTB). Sections were processed for immunohistochemistry for CTB. <b>(A)</b> Section of the brain stem of an animal that received a sham lesion. The higher magnification image shows that many CTB-positive axons are visible in the nucleus gracilis of the brain stem. <b>(B)</b> Section of the brain stem of a lesioned animal. Bar = 500μm. The higher magnification image shows that no CTB-positive fibres were detected in the brain stem. Bar = 50μm.</p

Rope crossing test.

<p>Sensorimotor dysfunction was assessed using the rope crossing test for eight weeks after cervical and thoracic dorsal column injury. Graphs show the mean ratios of errors to total steps taken. In animals that received either a <b>(A)</b> C4 or T7 <b>(B)</b> DC injury we observed an overall significant increase in the error rate compared to the sham groups. Whole time -course comparisons: # p < 0.05, ## p < 0.01 LESION † p < 0.05 for INT. *p < 0.05, ** p < 0.01 for single time-point comparisons. Error bars are SEM.</p

Comparisons of diferent tests and lesion levels.

<p>(A) Parameter estimates and 95% confidence intervals for the LESION parameter estimated by GLMM, for C4 and T7 lesions in the rope, horizontal ladder and inclined rolling ladder, on the log-odds scale (x-axis). The effect size and variability may be directly compared for these tests. (B) Power analysis for detection of the effect sizes in LESION seen in this study using n = 6, for all tests at C4 and T7 for which a significant effect was found. Power is shown for α = 0.05.</p

CatWalk gait analysis.

<p>Several gait parameters were assessed for eight weeks after cervical or thoracic DC lesion. <b>(A, B)</b> Base of support was increased in C4 lesioned animals but slowly returns to sham levels <b>(A)</b>, while in T7 lesioned animals the increase is sustained <b>(B). (C, D)</b> Hind paw print width was increased in C4 lesioned animals <b>(C)</b> but not in T7 lesioned animals <b>(D)</b>. <b>(E, F)</b> A minor sustained increase in swing time of the hind paws was seen in C4 lesioned animals <b>(E)</b> but in T7 lesioned animals a more marked but transient increase occurred <b>(F). (G, H)</b> Stride length increased over the time course in C4 injured animals compared to shams while in T7 injured animals stride length was actually decreased at several time points. <b>(I, J)</b> Mean pixel intensity was not different in C4 lesioned animals <b>(I)</b> and largely unchanged in T7 lesioned animals <b>(J)</b>. <b>(K, L)</b> Maximum contact area of the hind paws was unchanged after C4 lesion but decreased overall after T7 lesion.<b>(L)</b>. Whole time -course comparisons: # p < 0.05, ## p < 0.01, ### p< 0.001 for LESION; ††† p< 0.001 for INT. *p < 0.05, ** p < 0.01, *** p< 0.001 for single time-point comparisons. Error bars are SEM.</p

Histological evaluation of the spinal cord after injury.

<p>Animals received a dorsal column transection or a sham lesion at T7 or C4 level and survived eight weeks after which sensory axons were transganglionically labelled with CTB. Sections were processed for immunohistochemistry for CTB. <b>(A)</b> Parasagittal section of the lesion center in the spinal cord from an animal that received a cervical dorsal column injury. <b>(B)</b> Section of spinal cord from an animal that received a thoracic dorsal column injury. Bar = 100μm.</p

Horizontal ladder test.

<p>Animals were allowed to cross a horizontal grid with gaps of randomized size to assess dysfunction during eight weeks after cervical or thoracic dorsal column lesion. Graphs show the mean ratios of errors to total steps taken. For both <b>(A)</b> C4 and <b>(B)</b> T7 lesioned animals we observed an overall increase in the error rates compared to sham controls. Whole time -course comparisons: # p < 0.05, ### p < 0.001 for LESION; †† p < 0.01 for INT.*p<0.05, **p<0.01, ***p<0.001 for single time-point comparison. Error bars are SEM.</p

Tape removal test.

<p>The tape removal test was performed in order to evaluate sensory dysfunction of the left hind paw up to eight weeks after cervical and thoracic dorsal column injury. Graphs show the mean time to tape removal on a logarithmic scale. We observed overall deficits in the ability to sense the adhesive tape on the left hind paw in both <b>(A)</b> C4 and <b>(B)</b> T7 injured animals compared to the sham groups. Whole time -course comparisons: # p < 0.05 for LESION † p < 0.05 for INT. *p<0.05 for single time-point comparisons. Error bars are SEM.</p

Inclined rolling ladder test.

<p>Animals were tested on the inclined rolling ladder test to assess dysfunction related to proprioception and texture perception after cervical or thoracic dorsal column injury. <b>(A, B)</b> An overall significant decrease in the Good Smooth Steps measure (successfully executed steps off smooth rungs compared to total steps) was observed for animals that received a C4 <b>(A)</b> or T7 <b>(B)</b> dorsal column transection. <b>(C, D)</b> A significant increase in the Slips measure (slips compared to successful steps off smooth rungs) was seen in both C4 <b>(C)</b> and T7 <b>(D)</b> injured animals. Whole time -course comparisons: # p < 0.05, ## p < 0.01 for LESION. *p < 0.05, ** p < 0.01, *** p< 0.001 for single time-point comparisons. Error bars are SEM.</p

Schematic diagram of the inclined rolling ladder.

<p>The inclined rolling ladder consists of a ladder constructed at a 45 degrees angle with rungs that have an immobile, heavily textured half and a smooth, freely rolling half. Animals are required to walk up the ladder to the platform. After training, animals with intact sensation of texture should be able to discriminate between rough, fixed and smooth, rolling rungs, and either choose the textured sections or adapt their stepping action when they detected a smooth (and thus rolling) rung.</p