A combined scoring method to assess behavioral recovery after mouse spinal cord injury

Abstract

Although the rat has been the predominant rodent used to investigate the pathophysiology and treatment of experimental spinal cord injury (SCI), the increasing availability of transgenic animals has led to greater use of mouse models. However, behavioral assessment after SCI in mice has been less extensively investigated than in rats and few studies have critically examined the correlation between behavioral tests and injury severity or tissue damage. The present study characterized hindlimb functional performance in C57Bl/6 mice after contusion SCI at T9 using the weight drop method. A number of behavioral tests were examined with regard to variability, inter-rater reliability, and correlation to injury severity and white matter sparing. Mice were subjected to sham, mild-moderate or moderate-severe SCI and evaluated at day 1 and weekly up to 42 days using the Basso mouse scale (BMS), ladder climb, grid walk, inclined plane, plantar test and tail flick tests. The ladder climb and grid walk tests proved sub-optimal for use in mice, but modifications enhanced their predictive value with regard to injury severity. The inclined plane, plantar test and tail flick test showed far too much variability to have meaningful predictive value. The BMS score proved reliable, as previously reported, but a combined score (BLG) using BMS, Ladder climb (modified), and Grip walk (modified grid walk) provided better separation across injury levels and less variability than the individual tests. These data provide support for use of a combined scoring method to follow motor recovery in mice after contusion SCI.

Figure 1

Schematic of the spinal cord injury device. A horizontal pin attached the impounder to the end of a hollow tube, which guided the weight and prevented it from bouncing after impact. The weight was supported by a removable pin, which would be located at 10 (mild-moderate) or 20mm (moderate-severe) above the impounder to produce mild-moderate or moderate-severe injuries, respectively.

Figure 2

BMS scores 7, 14, 21, and 28 days after injury (n=9/sham, n=11/mild-moderate, and n=9/moderate-severe). BMS scores were between 0–0.5 on day one (data not shown), increased on days 7 to 21 (A–C) and plateaued between 21 to 28 (C–D) days after injury.

Figure 3

Number of grips in the grip walk (GW) test at 7, 14, 21, and 28 days post injury. The number of grips were added for easy, medium, and hard test (n=9/sham, n=11/mild-moderate, and n=9/moderate-severe). The test began at day 7 after injury.

Figure 4

Number of grips in the ladder climb (LC) test at 7, 14, 21, and 28 days post injury (n=9/sham, n=11/mild-moderate, and n=9/moderate-severe). The test begins at day 7 after injury.

Figure 5

The highest angle in the inclined plane (A) and the latency in the plantar (B) test at 1, 7, 14, 21, and 28 days post injury (DPI). Tests were not carried to day 35 due to no significant differences between injury groups or high variability observed among sham animals (n=9/sham, n=11/mild-moderate, and n=9/moderate-severe).

Figure 6

The modified grip walk (A), ladder climb (B), the BMS alone (C), and the BLG score (D) are demonstrated at 28 days. The BMS score, and converted GW and LC were added to obtain the BLG score at indicated days post injury (DPI) and (n=9/sham, n=11/mild-moderate, and n=9/moderate-severe).

Figure 7

Sections from sham, mild-moderate, and moderate-severely injured mice were stained with eriochrome cyanine R at 28 (A) days after injury and white matter sparing was assessed. The spinal cord shrinks at the epicenter as shown in mild-moderate and moderate-severe injury. White matter sparing correlation with the injury severity (B) and a positive correlation with the BLG score (C). White matter sparing was significantly different in both groups from the control group (p<0.01). C shows a direct correlation between the BLG score and the white matter sparing (r²= 0.9055, p<0.0001) Scalebar = 500µm.