Changes in Glial cell line-derived neurotrophic factor expression in the rostral and caudal stumps of the transected adult rat spinal cord

Abstract

Limited information is available regarding the role of endogenous Glial cell line-derived neurotrophic factor (GDNF) in the spinal cord following transection injury. The present study investigated the possible role of GDNF in injured spinal cords following transection injury (T(9)-T(10)) in adult rats. The locomotor function recovery of animals by the BBB (Basso, Beattie, Bresnahan) scale score showed that hindlimb support and stepping function increased gradually from 7 days post operation (dpo) to 21 dpo. However, the locomotion function in the hindlimbs decreased effectively in GDNF-antibody treated rats. GDNF immunoreactivty in neurons in the ventral horn of the rostral stump was stained strongly at 3 and 7 dpo, and in the caudal stump at 14 dpo, while immunostaining in astrocytes was also seen at all time-points after transection injury. Western blot showed that the level of GDNF protein underwent a rapid decrease at 7 dpo in both stumps, and was followed by a partial recovery at a later time-point, when compared with the sham-operated group. GDNF mRNA-positive signals were detected in neurons of the ventral horn, especially in lamina IX. No regenerative fibers from corticospinal tract can be seen in the caudal segment near the injury site using BDA tracing technique. No somatosensory evoked potentials (SEP) could be recorded throughout the experimental period as well. These findings suggested that intrinsic GDNF in the spinal cord could play an essential role in neuroplasticity. The mechanism may be that GDNF is involved in the regulation of local circuitry in transected spinal cords of adult rats.

Fig. 1

Somatosensory evoked potential was recorded in normal adult rats (P₁N₁P₂ type). The latency of SEP: P₁ (10.3 ± 0.26) ms, N₁ (8.81 ± 0.34) ms, P₂ (15.5 ± 0.43) ms, amplitude: P₁ (2.34 ± 0.02), N₁ (16.3 ± 0.14), P₂ (−5.06 ± 0.05)

Fig. 2

In the negative control group, no specific immunopositive staining was detected (a). GDNF positive profiles, simultaneously label-GFAP, were present in the white matter (b)

Fig. 3

Under higher magnification (400×), GDNF immunoreactivity (IR) was observed in neurons from the ventral horn of the gray matter in the normal spinal cord (a, arrow). GDNF (IR) in astrocytes was also seen in white matter (b, arrow). In the rostral stump, strong GDNF IR was detected in neurons from the ventral horn at 3 and 7 dpo (c, e, 400×, arrows). Moderate to intense immunostained neurons were labeled at 14 dpo (g, 400×, arrow). Intensely immunostained IR in astrocytes of the white matter were observed from 3 to 14 dpo (d, f, h, 400×, arrows)

Fig. 4

In the rostral stump, moderate to intense immunostained neurons were labeled at 21 dpo (a, 400×, arrow). Intensely immunostained IR in astrocytes of the white matter were also observed at 21 dpo (b, 400×, arrow). In the caudal stump, immunostained products were detected in neurons of the gray matter at 3 and 7 dpo (c, d, 400×, arrows). Strong immunostained IR for GDNF in neurons was observed at 14 dpo (e, 400×, arrow). Under higher magnification, immunoreactive structures of GDNF were localized in both cytoplasm and nuclei at 21 dpo (g, 400×, arrow), GDNF IR in astrocytes of the white matter was intensely immunostained at 14 and 21 dpo (f, h, 400×, arrows)

Fig. 5

In the spinal cord of adult rats, GDNF mRNA-positive product were markedly detected in neurons from the lamina III, IV and V and the central canal (a). Medium-sized neurons were seen in the field of intermediomedial nucleus and Nucleus dorsalis. In the ventral horn, GDNF mRNA-positive large neurons were also observed in lamina IX. Under higher magnification (200×), positive products with blue and purple staining were localized in the cytoplasm (b, arrows), also in white matter scattered glial cells were probe-labeled with light density (c, arrows)

Fig. 6

In the rostral stump, GDNF protein level increased at 3 dpo (P < 0.05), then reached its lowest point at 7 dpo which was below the control group (P < 0.05), then began to recover at 14 dpo, but it was less than that of normal level. This was followed by a continuous increase higher than the control group at 21 dpo (a). In the caudal stump, GDNF protein decreased at 3 dpo, reached its lowest point at 7 dpo (P < 0.05), then started to increase at 14 dpo, but it was lower than the normal level at 21 dpo (b)

Fig. 7

GDNF levels were significantly different from each other (P < 0.05), except for the comparison between the 21 dpo and normal group, as well as the 21 and 3 dpo group in the rostral stump. The groups were significantly different from each other (P < 0.05), except for the comparison between the 3 and 14 dpo group in the caudal stump

Fig. 8

BDA-positive axons staining as brown silkiness were detected in rostral cord sections near the injury site (a), but not in the field of scar tissue (b), and caudal cord sections near the injury site (c)