Biphasic bisperoxovanadium administration and Schwann cell transplantation for repair after cervical contusive spinal cord injury

Abstract

Schwann cells (SCs) hold promise for spinal cord injury (SCI) repair; however, there are limitations for its use as a lone treatment. We showed that acute inhibition of the phosphatase and tensin homolog deleted on chromosome ten (PTEN) by bisperoxovanadium (bpV) was neuroprotective and enhanced function following cervical hemicontusion SCI. We hypothesized that combining acute bpV therapy and delayed SC engraftment would further improve neuroprotection and recovery after cervical SCI. Adult female Sprague-Dawley (SD) rats were randomly sorted into 5 groups: sham, vehicle, bpV, SC transplantation, and bpV+SC transplantation. SCs were isolated from adult green fluorescent protein (GFP)-expressing SD rats (GFP-SCs). 200 μg/kg bpV(pic) was administered intraperitoneally (IP) twice daily for 7 days post-SCI in bpV-treated groups. GFP-SCs (1×10(6) in 5 μl medium) were transplanted into the lesion epicenter at the 8th day post-SCI. Forelimb function was tested for 10 weeks and histology was assessed. bpV alone significantly reduced lesion (by 40%, p<0.05) and cavitation (by 65%, p<0.05) and improved functional recovery (p<0.05) compared to injury alone. The combination promoted similar neuroprotection (p<0.01 vs. injury); however, GFP-SCs alone did not. Both SC-transplanted groups exhibited remarkable long-term SC survival, SMI-31(+) axon ingrowth and RECA-1(+) vasculature presence in the SC graft; however, bpV+SCs promoted an 89% greater axon-to-lesion ratio than SCs only. We concluded that bpV likely contributed largely to the neuroprotective and functional benefits while SCs facilitated considerable host-tissue interaction and modification. The combination of the two shows promise as an attractive strategy to enhance recovery after SCI.

Keywords: Neuroprotection; PTEN; Schwann cell transplantation; Spinal cord injury; bpV.

Figure 1. Experimental design and treatments for the bpV(pic)/GFP-SC combination study

(A) Time course and milestones of the experiment. (B) The structure of bpV(pic). (C) A photomicrograph of confluent pure GFP-SCs used for transplantation. Scale bar = 200 μm.

Figure 2. Forelimb sensorimotor assessment scores

Rats were tested on coordinated forelimb movement and manipulation of cereal rings during eating. Post-SCI behavior was performed 1 week after SCI and just before the 2^nd surgery. At the end of the study bpV + SCs and bpV-only group scores were significantly higher than the Vehicle group. bpV + SCs, *, p< 0.05 vs. Veh; ** p< 0.01 vs. Veh; *** p< 0.001 vs. Veh. bpV, #, p< 0.05 vs. Veh; ## p< 0.01 vs. Veh; ### p< 0.001 vs. Veh. SCs, ^$, p< 0.05 vs. Veh; ^$$, p< 0.01 vs. Veh; ^$$$ p< 0.001 vs. Veh.

Figure 3. bpV and bpV + SCs reduced lesion and enhanced spared tissue

(A) Schematic drawing illustrated how lesion and spared issue are defined. (B–E) GFAP immunostaining clearly defined astrocytic glial boarder around the lesion in all groups. Marked reduction of lesion was found in bpV and bpV + SCs treatment groups. (F and G) Significant reduction of percent lesion area (F) and increase in percent spared tissue (G) were found as compared to Vehicle-treated group. Animals treated with SCs alone exhibited a trend in neuroprotective measures, but showed no statistical significance compared to the Vehicle group. (H) Lesion reduction closely correlated with the mean functional scores of each group (R² = 0.93). *, p < 0.05. Scale bar = 1 mm. Bars = mean ± SEM.

Figure 4. bpV, SCs, used alone or in combination, reduced lesion cavity

(A–D) Cresyl violet/eosin (CVE) staining shows cavities at the lesion epicenter in different groups. All three bpV and/or SC treatment groups (B–D) showed marked reduction of lesion cavities as compared to the Vehicle-treated control (A). (E) The three treatment groups showed statistically significant reduction of lesion cavitation as compared to the Vehicle group, although no difference was found between these treatment groups. *, p< 0.05; **, p< 0.01. Scale bar = 1 mm.

Figure 5. bpV and bpV + SCs increased the number of ventral horn neurons adjacent to the epicenter of injury

(A) Cresyl violet/eosin-stained cross sections cut at 2mm rostral, epicenter, and 2 mm caudal to the injury in groups received different treatments. Ventral horn neurons were clearly seen (right column). (B) Only bpV and bpV + SCs groups significantly increased the number of ventral horn neurons at 2 mm caudal to the lesion. *, p< 0.05. Scale bar: 2 mm rostral & epicenter = 1mm; 2mm caudal = 250 μm.

Figure 6. Comparison of GFP-SC graft area between SCs and bpV + SCs groups

(A–D) SCs in both transplantation groups (C and D) but not in the Vehicle (A) or bpV (B) group showed GFP labeling in the lesion, indicating the survival of the grafted SCs. (E) Areas occupied by grafted GFP-SCs was similar between the SCs and bpV + SCs groups. (F) bpV + SCs exhibited greater GFP-SC/Lesion Area ratio than animals receiving SC transplantation alone although the difference was not statistically significant. Scale bar = 1 mm.

Figure 7. SC transplantation promoted axonal growth within the lesion

(A–L) High power cross-section view of the ipsilateral lesion epicenter in the Vehicle, SCs, and bpV-SCs groups. (A–D) In the Vehicle control group receiving no SC transplantation, no axonal growth into the lesion was found. (E–L) In groups that receiving SCs (E–H) or bpV-SC (I–L), considerable axonal growth, immunolabeled by SMI-31, into the lesion site was found (G, K) and they were closely associated with grafted GFP-SCs (F–J). (M–O) There was no statistically significance difference between the total SMI-31⁺ area (M), nor SMI-31⁺ as a fraction of SC-graft area (N). However, axonal growth into the graft as an index of lesion size was significantly greater in the bpV-SC treatment group as compared with the SCs only group (O). *, p < 0.05. Scale bar = 750 μm.

Figure 8. SCs promoted vascular growth into the graft

(A–A′) In the Vehicle control group receiving no SC transplantation, no RECA-1⁺ vasculature was found within the lesion. (B–C′) RECA-1⁺ vascular growth into the GFP-SC grafts was clearly seen in groups receiving SCs (B–B′) and bpV + SCs (C–C′). White arrowheads indicate RECA-1⁺ blood vessels. Scale bar = 1 mm (A), 500 μm (B & B′), 50 μm (C & C′).