A consistent, quantifiable, and graded rat lumbosacral spinal cord injury model

Abstract

The purpose of this study is to develop a rat lumbosacral spinal cord injury (SCI) model that causes consistent motoneuronal loss and behavior deficits. Most SCI models focus on the thoracic or cervical spinal cord. Lumbosacral SCI accounts for about one third of human SCI but no standardized lumbosacral model is available for evaluating therapies. Twenty-six adult female Sprague-Dawley rats were randomized to three groups: sham (n=9), 25 mm (n=8), and 50 mm (n=9). Sham rats had laminectomy only, while 25 mm and 50 mm rats were injured by dropping a 10 g rod from a height of 25 mm or 50 mm, respectively, onto the L4-5 spinal cord at the T13/L1 vertebral junction. We measured footprint length (FL), toe spreading (TS), intermediate toe spreading (ITS), and sciatic function index (SFI) from walking footprints, and static toe spreading (STS), static intermediate toe spreading (SITS), and static sciatic index (SSI) from standing footprints. At six weeks, we assessed neuronal and white matter loss, quantified axons, diameter, and myelin thickness in the peroneal and tibial nerves, and measured cross-sectional areas of tibialis anterior and gastrocnemius muscle fibers. The result shows that peroneal and tibial motoneurons were respectively distributed in 4.71 mm and 5.01 mm columns in the spinal cord. Dropping a 10-g weight from 25 mm or 50 mm caused 1.5 mm or 3.75 mm gaps in peroneal and tibial motoneuronal columns, respectively, and increased spinal cord white matter loss. Fifty millimeter contusions significantly increased FL and reduced TS, ITS, STS, SITS, SFI, and SSI more than 25 mm contusions, and resulted in smaller axon and myelinated axon diameters in tibial and peroneal nerves and greater atrophy of gastrocnemius and anterior tibialis muscles, than 25 mm contusions. This model of lumbosacral SCI produces consistent and graded loss of white matter, motoneuronal loss, peripheral nerve axonal changes, and anterior tibialis and gastrocnemius muscles atrophy in rats.

Keywords: immunohistochemistry; locomotor function; models of injury; rat; spinal cord injury.

FIG. 1.

Retrograde labeling of tibial and common peroneal nerve, and anatomy of rat's spine and spinal cord. (A) Anatomy of rat's spine and spinal cord, exposed by a L1 laminectomy. (B) Enlarged box area in A. The lower margin of T13 laminae is indicated by a dashed line. (C) Motoneurons labeled by fluororuby (red) and fluorogold (blue) backfilled respectively from the tibial nerve and common peroneal nerve by retrograde labeling. The spinal cord cut between T13 and L1. R13, rib 13; T13, vertebra T13; L1, L1 vertebra; L2, L2 vertebra. Scale bar, 500 μm.

FIG. 2.

Walking footprint and analysis. (A–C) Pictures of typical footprints obtained at one week after injury in the Sham, 25 mm, and 50 mm groups, respectively. Scale bar (A-C), 10 mm. Dash line: Footprint length (FL), maximum distance from the third toe to end of the print; Toe spread (TS), distance between the first and fifth toes; Intermediate toe spread (ITS), distance from the second to the fourth toe. (D) The increase in mean footprint length (FL) in the Sham (n=8), 25 mm (n=7), and 50 mm (n=8) groups. (E) and (F) show the decrease in toe spread (TS) and intermediate toe spread (ITS) in the three injury groups. (G) shows the decrease in mean sciatic function index (SFI) scores. The error bars indicate standard deviation. The three groups differed significantly from each other at all time-points after the first week. * indicates significant difference compared with the Sham group. # indicates significant difference between 25 mm and 50 mm groups. (H) shows a walking injured rat (2 weeks after injury) while (I) shows a normal rat. Both rats had just moved their left hindlimb forward. The heel of injured rat (H red arrow) touched the ground, while normal one did not. The toes of injured rat (H blue arrow) did not flatten and spread, while the normal one did. Neither of these behaviors would be rated in a BBB score.

FIG. 3.

Static foot positions and analysis. (A–C) Images of typical foot positions at two weeks after injury in the Sham, 25 mm, and 50 mm groups, respectively. (D) and (E) show the declines in mean static toe spreading (STS) and static intermediate toe spreading (SITS) in the three injury groups. (F) shows the declines of Static Sciatic Index scores in the three injury groups. The error bars indicate standard deviation. Animal numbers are the same as in Figure 2. All changes of foot positioning scores were statistically different among the three injury groups after the third week. * indicates significant difference compared with the Sham group. # indicates significant difference between the 25 mm and 50 mm groups.

FIG. 4.

Counts of spared NeuN-stained motoneurons. (A) Normal spinal cord. Large motoneurons are located in the ventral horn. (B) Injured spinal cord in Group 50 mm about 3 mm proximal to injury epicenter. (C) Injury epicenter in Group 50 mm. (D) Injured spinal cord in Group 50 mm about 3 mm distal to injury epicenter. Scale bar (A-D), 200 μm. (E) Graph of the numbers of spare motoneurons of±5 mm of the injury epicenter in the Sham (n=4), 25 mm (n=3), and 50 mm (n=4) injury groups. The error bars indicate standard deviation.

FIG. 5.

Retrograde labeling of spared motoneurons. (A–C) are exemplary images of contused spinal cords at T13/L1 from the Sham, 25 mm and 50 mm injury groups, respectively. (D–F) are camera lucida drawings corresponding to A-C, indicating counted motoneurons. Scale bar (A-F), 500 μm. (G) shows a graph of the number of counted spared motoneurons back-labeled from the tibial and peroneal nerves in the Sham (n=4), 25 mm (n=4), and 50 mm (n=4) injury groups. The error bars (D) indicate standard deviation. * indicates p<0.05 versus the Sham group, # indicates p<0.05 versus the 25 mm group.

FIG. 6.

Fast blue staining of white matter. (A) Cross-section in Group Sham. (B) Epicenter of Group 25 mm. (C) Epicenter of Group 50 mm. (D) Percentage of spared white matter compared with Group Sham. Animal numbers are the same as in Figure 4. Scale bar (A–C), 200 μm. The error bars (D) indicate standard deviation.

FIG. 7.

Measurements of myelinated axons after surgery. (A–C) shows exemplary images of combined silver and fast blue staining of tibial nerve in the Sham, 25 mm, and 50 mm injury groups, respectively. Axons were stained black and surrounding myelin sheathes were stained blue. (D) shows the numbers of myelinated axon in tibial and peroneal nerves of the three injury groups. (E–G) are higher magnification images of tibial nerve in the Sham, 25 mm, and 50 mm, groups, respectively. Arrows indicate densely silver-stained axon bulbs. (H) shows mean axon diameters in the three injury groups. (I) shows myelin sheath thickness in the three injury groups. Scale bar (A–C), 100 μm; (E–G), 10 μm. The error bars (D, H, I) indicate standard deviation. * indicates p<0.05 versus Sham; # indicates p<0.05 versus 25 mm. The data represent means and standard errors of means from 3 rats from the Sham group and 4 rats each from the 25 mm and 50 mm groups.

FIG. 8.

Histologic changes of muscles. (A–C) show exemplary images of hematoxylin and eosin (H & E) stained gastrocnemius (GA) muscles in the Sham (A), 25 mm (B), and 50 mm (C) groups at six weeks after surgery. Scale bar (A–C), 100 μm. (D) Mean muscle fiber cross-sectional areas (CSA) of tibialis anterior (TA) and GA muscle in the Sham (n=3), 25 mm (n=4), and 50 mm (n=4) groups. The error bars indicate standard deviation. Mean muscle fiber CSA in the 25 mm and 50 mm groups were significantly smaller than in the Sham group and from each other. * indicates p<0.05 versus the Sham group; # indicates p<0.05 versus 25 mm group.