Improvement of neurological recovery and stimulation of neural progenitor cell proliferation by intrathecal administration of Sonic hedgehog

Laboratory investigation

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Object

Sonic hedgehog (Shh) is a glycoprotein molecule that has been shown to be associated with the proliferative capacity of endogenous neural precursor cells during embryonic development. It has also been shown to regulate the proliferative capacity of neural stem cells in the adult subventricular zone (SVZ), which are also upregulated in animal models of ischemic stroke. In the present study, the effects of exogenous administration of intrathecal Shh protein were examined in the setting of a rodent model of ischemic stroke, with particular attention given to endogenous neural stem cell proliferation and migration as well as inducible differences in behavioral recovery.

Methods

A rodent model of ischemic stroke was created using the intraluminal suture method of reversible middle cerebral artery occlusion. Animals were treated with intrathecal administration of Shh protein at 24 hours after the onset of the stroke. Behavioral testing was performed, and the animals were killed for measurements of infarct volume 7 days after stroke. Immunohistochemical staining was performed and measurements of cellular proliferation were obtained, with a focus on the proportion and distribution of neural progenitor cells in the SVZ. These values were compared across experimental groups.

Results

Treatment with intrathecal Shh protein resulted in significant improvement in behavioral function compared with the control group, with a significant reduction of ischemic tissue in the cerebral hemisphere. An increase of nestin immunoreactive cells was observed along the SVZ.

Conclusions

Intrathecal Shh agonist at doses that upregulate spinal cord GLI1 transcription increases the population of neural precursor cells after spinal cord injury in adult rats. Intrathecal administration of Shh protein appears to have a neuroprotective effect in animal models of ischemic stroke and is associated with improved behavioral recovery, which may be related to its effects on neurogenesis in the SVZ and could be associated with improved functional recovery.

Abbreviations used in this paper:MCA = middle cerebral artery; Shh = Sonic hedgehog; SVZ = subventricular zone.

Article Information

Address correspondence to: Nicholas C. Bambakidis, M.D., 11100 Euclid Avenue, Cleveland, Ohio 44106. email: nicholas.bambakidis1@uhhospitals.org.

Please include this information when citing this paper: published online February 10, 2012; DOI: 10.3171/2012.1.JNS111285.

© AANS, except where prohibited by US copyright law.

Headings

Figures

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    Bar graph showing results of 2 behavioral tests: the inclined screen test and the adhesive removal test. Treatment with Shh resulted in decreased finish times for both tasks. If an animal failed to complete the task within 120 seconds, the task was ended and 120 seconds was entered for the animal's final time. Finish times were averaged for each group and are represented as mean ± SEM. MCAO = MCA occlusion.

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    The addition of Shh to stroke-induced rats resulted in a reduction in infarct volume. Left and Center: Images show representative thionin-stained sections from rats after MCA occlusion treated with Shh (left) and saline (center). The area of infarcted tissue is characteristically represented with vacuolization and cellular destruction (arrows). Original magnification × 40. Right: The graph shows the decreased stroke volume (% contralateral) after Shh treatment in rats after MCA occlusion presented as the individual data points and mean ± SEM of each group. Stroke volume is measured after calculating the infarct area in sections obtained serially throughout the brain using Bioquant computer software. The Shh-treated group (black triangles) was significantly different (*p < 0.05, Student t-test) from the no-injection group (open circles) and saline-treated group (gray triangles).

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    Upper: Bar graph of nestin-positive cells after MCA occlusion. Cell counts are presented as the mean ± SEM of nestin-positive cells in the SVZ and stroke-induced areas on the stroke side of the brain (n = 5), as well as the SVZ and area corresponding to a stroke on the contralateral (Contra) side of the brain (n = 5), calculated in a blinded fashion. Cells from the SVZ are counted in 3 separate fields, and cells from the stroke-induced or corresponding area are counted on 7 separate sections. Ipsi = ipsilateral. Lower: Sonic hedgehog enhances neurogenesis. Panels A–D and I–L depict nestin-positive (red) and DAPI-positive (blue) cells in an animal treated with saline after MCA occlusion. In these animals, there is a significant increase in the number of nestin-positive cells in the SVZ of the stroke-induced side of the brain (A, B, I, and J) compared with the contralateral side of the brain (C, D, K, and L). Sonic hedgehog administration 1 day after MCA occlusion enhances this increase in nestin-positive cells on the stroke-induced side of the brain (E, F, M, and N) but not on the contralateral side (G, H, O, and P). Bar = 100 μm (A–H), and 25 μm (I–P). LV = lateral ventricle; Str = striatum. Original magnification × 40.

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