Functional recovery of stepping in rats after a complete neonatal spinal cord transection is not due to regrowth across the lesion site

Abstract

Rats receiving a complete spinal cord transection (ST) at a neonatal stage spontaneously can recover significant stepping ability, whereas minimal recovery is attained in rats transected as adults. In addition, neonatally spinal cord transected rats trained to step more readily improve their locomotor ability. We hypothesized that recovery of stepping in rats receiving a complete spinal cord transection at postnatal day 5 (P5) is attributable to changes in the lumbosacral neural circuitry and not to regeneration of axons across the lesion. As expected, stepping performance measured by several kinematics parameters was significantly better in ST (at P5) trained (treadmill stepping for 8 weeks) than age-matched non-trained spinal rats. Anterograde tracing with biotinylated dextran amine showed an absence of labeling of corticospinal or rubrospinal tract axons below the transection. Retrograde tracing with Fast Blue from the spinal cord below the transection showed no labeled neurons in the somatosensory motor cortex of the hindlimb area, red nucleus, spinal vestibular nucleus, and medullary reticular nucleus. Retrograde labeling transsynaptically via injection of pseudorabies virus (Bartha) into the soleus and tibialis anterior muscles showed no labeling in the same brain nuclei. Furthermore, re-transection of the spinal cord at or rostral to the original transection did not affect stepping ability. Combined, these results clearly indicate that there was no regeneration across the lesion after a complete spinal cord transection in neonatal rats and suggest that this is an important model to understand the higher level of locomotor recovery in rats attributable to lumbosacral mechanisms after receiving a complete ST at a neonatal compared to an adult stage.

Figure 1

Schematic of anatomical labeling techniques. A, biotinylated dextran amine (BDA) was bilaterally injected into the somatosensory motor cortex of the hindlimb area (SM HL) and red nucleus: the presence or absence of the anterograde tracer rostral and caudal to the transection site was examined In both intact and ST rats. B, ST animals were given a second spinal cord transection (re-transection) either rostral to, caudal to, or at the original transection site at 12 weeks after the initial transection. Gelfoam soaked in Fast Blue then was inserted into the transection site, and after 5 days the presence or absence of Fast Blue was examined in specific brain nuclei. C, intact and ST rats were given an injection of pseudorabies virus (PRV; Bartha strain) unilaterally into the tibialis anterior or soleus muscles: the presence or absence of the retrograde tracer in the brain was examined.

Figure 2

Step training improves hindlimb stepping in ST rats. The maximum number of steps performed by non-trained (black bars) and trained (gray bars) ST rats at 0 (30 days post-injury), 4, and 8 weeks after the initiation of step training is shown. The data are mean (±SEM) values from one min tests at a treadmill speed of 11 cm/s with 75% body weight support determined using video analysis. * and †, significant difference between non-trained (N = 14) and trained (N = 14) groups and between testing dates (in parentheses), respectively, at P< 0.05.

Figure 3

Re-transection rostral to the original transection site causes no apparent loss in stepping performance in ST trained rats. Stepping ability was determined by plotting the ankle position as recorded by the robotic arm attached to the rat’s ankle as it stepped on a treadmill. The stepping characteristics (X, step length; Y, step height) of a representative ST trained rat before and after a re-transection rostral to the original transection site are shown. Column A, graphs of X vs. Y of the left leg (step shape); column B, graphs of Y vs. time of left leg (step rhythm); and column C, graphs of right Y vs. left Y (interlimb coordination). Note that the rat exhibits nearly identical stepping performance under the two conditions. Chronological progression of stepping is indicated by the gradual change in color from blue (beginning) to red (end). All three graphs were plotted using the same set of ~15 steps as determined by left ankle position.

Figure 4

The kinematics of stepping in ST-trained rats are unaffected by a re-transection rostral to the original transection site. There was no significant difference in the PC1 X% (A) or Y% (B), and average step height (C), length (D), or duration (E) before and after the re-transection. Values are mean ± SEM for 5 rats.

Figure 5

BDA is not present in the spinal cord caudal to the transection site in both non-trained and trained ST rats. A–H, horizontal sections of the spinal cord from a representative intact (A, B, E, and F) and a representative ST trained (C, D, G, and H) rat injected with BDA in the somatosensory motor cortex of the hindlimb area. BDA staining is visible in sections rostral, but not caudal, to the transection site in the ST trained rat. I–P, transverse sections of the spinal cord from a representative intact (I, J, M, and N) and ST trained (K, L, O, and P) rat injected with BDA in the red nucleus. Again, staining is visible in sections rostral, but not caudal, to the transection site in the ST trained rat. E–H, M–P and M’–P’ are higher magnification images of the inset boxes in A–D, I–L and M–P, respectively. Scale bars in D=500 µm, H=100 µm, L=300 µm, P=30 µm and P’=5 µm and apply to A–D, E–H, I–L, M–P and M’–P’, respectively.

Figure 6

Fast Blue is present in brain nuclei of intact and ST non-trained rats re-transected rostral, but not caudal, to the original transection site. Coronal sections of the brain from a representative intact (A, E, I, and M), ST non-trained plus re-transection at a rostral level (B, F, J, and N), ST non-trained plus re-transected at the original transection site (C, G, K, and O), and ST non-trained plus re-transected at a caudal level (D, H, L, and P) rat are depicted. Staining for the somatosensory motor cortex of the hindlimb area (A–D), red nucleus (E–H), spinal vestibular nucleus (I–L), and medullary reticular nucleus (M–P) is shown. Labeled cells are observed in all regions in the intact and ST non-trained plus rostral re-transection, but not the ST non-trained plus re-transection at the original or caudal levels. Scale bar in N is 25 µm and applies to all panels.

Figure 7

No Fast Blue labeled neurons are observed in the brain of ST non-trained rats receiving a re-transection at the original transection site or at a level caudal to the transection site. Asterisks indicate a cell count of zero. Spinal rats that were re-transected either at or caudal to the original transection site (Tx Site) showed no Fast Blue stained neurons in any of the four nuclei examined. In contrast, intact and ST non-trained rats that were re-transected rostral to the original transection site showed a similar number of Fast Blue stained neurons in each of the four regions of the brain. Values are mean ± SEM for 7 intact and 15 ST non-trained rats.

Figure 8

PRV is present in four motor nuclei in the brain of intact, but not ST trained or non-trained rats. Coronal sections of the brain from a representative intact (A–D) and a representative ST trained (F–I) rat are depicted. Staining for the somatosensory motor cortex of the hindlimb area (A and F), red nucleus (B and G), spinal vestibular nucleus (C and H) and medullary reticular nucleus (D and I) is shown. Labeled cells are observed in all regions in the intact, but not the ST trained, rat. Transverse sections of the lumbar spinal cord from a representative intact (E) and ST trained (J) rat are shown. PRV-labeled cells are observed in both the intact and ST trained rats in the spinal cord below the lesion site. Scale bar in D is 50 µm and applies to A–I; scale bar in J is 200 µm and applies to E and J.