Endogenous stem cell proliferation induced by intravenous hedgehog agonist administration after contusion in the adult rat spinal cord

Laboratory investigation

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Object

Sonic hedgehog (Shh) is a glycoprotein molecule that upregulates the transcription factor Gli1. The Shh protein plays a critical role in the proliferation of endogenous neural precursor cells when directly injected into the spinal cord after a spinal cord injury in adult rodents. Small-molecule agonists of the hedgehog (Hh) pathway were used in an attempt to reproduce these findings through intravenous administration.

Methods

The expression of Gli1 was measured in rat spinal cord after the intravenous administration of an Hh agonist. Ten adult rats received a moderate contusion and were treated with either an Hh agonist (10 mg/kg, intravenously) or vehicle (5 rodents per group) 1 hour and 4 days after injury. The rats were killed 5 days postinjury. Tissue samples were immediately placed in fixative. Samples were immunohistochemically stained for neural precursor cells, and these cells were counted.

Results

Systemic dosing with an Hh agonist significantly upregulated Gli1 expression in the spinal cord (p < 0.005). After spinal contusion, animals treated with the Hh agonist had significantly more nestin-positive neural precursor cells around the rim of the lesion cavity than in vehicle-treated controls (means ± SDs, 46.9 ± 12.9 vs 20.9 ± 8.3 cells/hpf, respectively, p < 0.005). There was no significant difference in the area of white matter injury between the groups.

Conclusions

An intravenous Hh agonist at doses that upregulate spinal cord Gli1 transcription also increases the population of neural precursor cells after spinal cord injury in adult rats. These data support previous findings based on injections of Shh protein directly into the spinal cord.

Abbreviations used in this paper: GAPDH = glyceraldehyde-3-phosphate-dehydrogenase; Hh = hedgehog; NPC = neural progenitor cell; PCR = polymerase chain reaction; SCI = spinal cord injury; Shh = sonic hedgehog.

Article Information

Address correspondence to: Nicholas C. Bambakidis, M.D., University Hospitals Case Medical Center, 11100 Euclid Avenue, Cleveland, Ohio 44106. email: Nicholas.Bambakidis@uhhospitals.org.

© AANS, except where prohibited by US copyright law.

Headings

Figures

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    Bar graph demonstrating significantly higher activation of Gli1 transcription factor in uninjured rats that received an intravenously delivered Hh agonist (10 mg/kg) on Days 1 and 4 after injection (tissue harvesting occurred on Day 5). A comparison was made with tissue from the lung parenchyma (controls) and with animals treated with an intraperitoneal Hh agonist (20 mg/kg). *p < 0.005. IP = intraperitoneal; IV = intravenous.

  • View in gallery

    A: Photomicrograph of sagittal midthoracic spinal cord section of a rat with a moderate spinal cord contusion induced from a height of 12.5 mm. Weil stains show a spared rim of white matter surrounding a central cavity (arrowheads). B: Photomicrograph of surrounding area of white matter staining positively for glial fibrillary acidic protein while the central lesioned area (asterisks) does not. Areas around the rim of the lesion cavity were targeted for further quantification of nestin-positive cells at a higher power (square). C: High-power photomicrograph of the region bounded by the square in panel B. Nestin-positive cells are diffusely present and appear as primitive cells with 1 or 2 cellular processes (arrows). These cells represent NPCs.

  • View in gallery

    Graph representing the number of nestin-positive cells in rats treated with an Hh agonist versus those in untreated rats. Cell counts are presented as the means ±SDs of nestin-positive cells in a given microscopic field. Cell counts were made in a blinded fashion in 5 separate fields on 10 different sections. Rats were lesioned with a moderate spinal cord contusion. Treated rats intravenously received an Hh agonist at a dose of 10 mg/kg on Days 1 and 4 after injury. Data were analyzed with repeated-measures analysis of variance, followed by the Student-Newman-Keuls post hoc t-test. *p < 0.005.

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