Axonal sprouting into the denervated spinal cord and synaptic and postsynaptic protein expression in the spinal cord after transplantation of bone marrow stromal cell in stroke rats

Abstract

We investigated whether compensatory reinnervation in the corticospinal tract (CST) and the corticorubral tract (CRT) is enhanced by the administration of bone marrow stromal cells (BMSCs) after experimental stroke. Adult male Wistar rats were subjected to permanent right middle cerebral artery occlusion (MCAo). Phosphate-buffered saline (PBS, control, n=7) or 3x10(6) BMSCs in PBS (n=8) were injected into a tail vein at 1 day postischemia. The CST of the left sensorimotor cortices was labeled with DiI 2 days prior to MCAo. Functional recovery was measured. Rats were sacrificed at 28 days after MCAo. The brain and spinal cord were removed and processed for vibratome sections for laser-scanning confocal analysis and paraffin sections for immunohistochemistry. Normal rats (n=4) exhibited a predominantly unilateral pattern of innervation of CST and CRT axons. After stroke, bilateral innervation occurred through axonal sprouting of the uninjured CRT and CST. Administration of BMSCs significantly increased the axonal restructuring on the de-afferented red nucleus and the denervated spinal motoneurons (p<0.05). BMSC treatment also significantly increased synaptic proteins in the denervated motoneurons. These results were highly correlated with improved functional outcome after stroke (r>0.81, p<0.01). We conclude that the transplantation of BMSCs enhances axonal sprouting and rewiring into the denervated spinal cord which may facilitate functional recovery after focal cerebral ischemia.

Figure 1. Neurological functional test and lesion volume

Compared to the control animals (n=7), BMSC treatment (n=8) significantly improved the functional recovery measured by the adhesive-removal test after stroke (A, *p<0.05). The lesion volume showed no difference between the control and BMSC treatment groups at four weeks after stroke (B).

Figure 2. CRT and CST tracing with DiI cortical injections

The CRT and CST arising from the left intact hemisphere were labeled by DiI cortical injections as shown in the schematic diagram (A). In normal rats, the CRT innervation was unilateral (B). Some CRT fibers changed their pathway to the lesioned red nucleus after stroke (C). Rats with BMSC treatment after stroke had an increased density of DiI-labeled fibers in the contralateral de-afferented red nucleus (D). Quantitation shows a significant CRT restructuring in BMSC treated animals as compared with controls (E, *p<0.05). In normal rats, no obvious axons crossed the midline into the opposite part of dorsal funiculus in a representative cervical longitudinal section (F). Four weeks after stroke, some DiI-labeled CST fibers were observed in the denervated side of the dorsal funiculus extending toward to the gray matter (G). The sprouting of branched CST axons from the intact side was enhanced by BMSC treatment postischemia (arrows in H). Compared with controls, the length of CST fibers was significantly increased in BMSC treated animals both in the cervical and lumbar cord (I, *p<0.05). WM: white matter, GM: gray matter. Scale Bar=250 μm in B–D, =25 μm in F–H.

Figure 3. Transverse confocal images showing the axonal sprouting into the spinal gray matter of the cervical cord

In normal rats, the CST showed a unilateral innervation manner (A). In rats after MCAo, DiI-labeled intact CST axons extended into the denervated side of the gray matter (B). BMSC treatment significantly increased the intact CST axonal sprouting into the denervated side in both the cervical and lumbar levels (C and D, *p<0.05). Insert of A, schematic drawing of the spinal gray matter showing the position of the photomicrograph appearing in A, B and C. cc: central canal. Scale Bar=25 μm in A–C.

Figure 4. Immunohistochemical staining of synaptophysin and PSD-95 on spinal motoneurons in the denervated side of the spinal cord

In normal rats, synaptophysin positive staining encircled spinal motoneurons, including their large cell bodies and dendritic trees (A). After stroke, the synaptophysin density was reduced (B). Synaptophysin staining was increased in the motor pool of the denervated ventral horn after BMSC treatment (C). Quantitative data show that the expression of synaptophysin was significantly enhanced in the cervical and lumbar cord by BMSC administration compared to controls (D, *p<0.05). PSD-95 staining was observed in the cytoplasm with thick proximal dendrites of spinal motoneurons in normal rats (E). PSD-95 expression was reduced postischemia (F). BMSC treatment significantly increased the PSD-95 level in the denervated spinal motoneurons (G and H, *p<0.05). Insert of A, schematic drawing of the spinal gray matter showing the position of the photomicrograph appearing in A–C and E–G. Scale Bar=25 μm in A–C and E–G.