The upregulated expression of sonic hedgehog in motor neurons after rat facial nerve axotomy

Abstract

Nerve injury leads to the induction of a large number of genes to repair the damage and to restore synaptic transmission. We have attempted to identify molecules whose mRNA expression is altered in response to facial nerve axotomy. Here we report that facial nerve axotomy upregulates Sonic hedgehog (Shh) and its receptor Smoothened (Smo) in facial motor neurons of adult rats, whereas facial nerve axotomy does not upregulate mRNA of Shh or Smo in neonatal rats. We tested whether overexpression of Shh in facial motor neurons of axotomized neonatal rats may promote neuronal survival. Adenovirus-mediated overexpression of Shh, but not that of beta-galactosidase, transiently rescues axotomy-induced neuronal cell death for 3-5 d after axotomy. Finally, the pharmacological inhibitor of Shh signaling, cyclopamine, induces motor neuron death in adult rats after axotomy. These results suggest that Shh plays a regulatory role in nerve injury.

Figure 1.

a, Upregulated expression of Shh after axotomy in adult rats: in situ hybridization of Shh antisense riboprobes in a coronal section at 3, 6, and 24 hr and 5 d after the axotomy. The relatively large size of cells corresponding to motor neurons in facial nucleus shows the expression of Shh mRNAs. Compared with the control side (left), the signal intensity was severalfold higher in the axotomized facial nucleus (right). Scale bar, 500 μm. b, After capturing the in situ hybridization image, the section was stained with Nissl to calculate the ratio of Shh-upregulated cells. The number of motor neurons that display upregulation of Shh increases to 83 ± 1.4% at 18 hr after the operation. c, d, Quantitative analyses of Shh expression after axotomy by Northern blot (c) and Western blot (d) analyses. Each lane contains 30 μg of total RNA (c) and 10 μg of total protein (d) purified from pooled facial nuclei. Expression of Shh mRNA and 19 kDa polypeptides increased at 24 hr after axotomy and was sustained up to 4 weeks. The control blot using radiolabeled G3PDH for Northern blot and anti-PLP antibody for Western blot revealed that relatively equal amounts of RNA and protein were loaded in each lane.

Figure 2.

Expression of the Shh receptor Smo and Ptc in the facial nucleus of control and axotomized adult rats at 24 hr after axotomy. a, The total RNA (30 μg) purified from pooled facial nuclei was subjected to RNase protection analyses. For the expression of Smo, a 384 bp antisense riboprobe was hybridized, and a 302 bp band was protected after RNase digestion. For the expression of Ptc1, a 442 bp antisense riboprobe yielded a 300 bp protected band after RNase digestion. Control experiments using a G3PDH antisense riboprobe showed that equal amounts of total RNA were used in these experiments. b, In situ hybridization histochemistry showing the cellular localization of Smo transcripts in facial nucleus. The axotomy of adult rat facial nerve induced upregulated expression of Smo mRNA in the cell bodies of motor neurons. Scale bar, 500 μm.

Figure 3.

a, In situ hybridization histochemistry of Shh in neonatal rats at 24 hr after axotomy. The control side of facial motor neurons expresses Shh mRNA at a low level, whereas axotomy of facial nerves rather suppresses the Shh expression. Scale bar, 100 μm. b, c, Quantitative analyses of Shh expression after axotomy by Northern blot (b) and Western blot (c) analyses. Each lane contains 30 μg of total RNA (b) and 10 μg of total protein lysates (c) purified from pooled facial nuclei corresponding to representative time course. No expression of Shh mRNA and protein was detected in the facial nuclei after 5 d in axotomized neonatal rats because the axotomy of neonatal rats induces motor neuron death. Control blots were performed using radiolabeled G3PDH or anti-PLP antibody. d, The RNase protection analysis revealed that axotomy of neonatal rat facial nerves did not induce the expression of Smo and Ptc1 mRNA.

Figure 6.

a, Efficacy of adenovirus-mediated overexpression of lacZ after axotomy in P1 and adult rats in the lesioned motor neurons. The pups were treated for 4 d after axotomy after the implantation of gelfoams containing AdV-lacZ. The number of lacZ-positive neurons was counted. In the neonatal rats, more lacZ-positive neurons were observed than in the adult rats by the adenovirus gene transfer. The number of Nissl-positive survived neurons was counted from the sections covering bilateral facial nuclei. Results of three independent experiments were subjected to statistical analysis (Student's t test, ^*p < 0.01; ^**p < 0.001). Shh-adenovirus significantly potentiates the survival of axotomized motor neurons from 3 to 5 d after axotomy and adenovirus application (a). An application of adenovirus after axotomy did not influence the number of survived neurons compared with PBS application (b).

Figure 4.

We tested the effect of Shh overexpression in neonatal rats by the implantation of Gelfoams containing AdV-lacZ in the left operation side and Gelfoams containing AdV-Shh in the right operation side. At 3 d after the operation, the cryosections were stained with rabbit polyclonal anti-Shh (c, d) and mouse monoclonal anti-lacZ (e, f) antibodies visualized with Alexa-Fluor 488-conjugated (green) and Alexa-Fluor 594-conjugated (red) secondary antibodies. After capturing the confocal images, sections were stained with Nissl to visualize the neuronal nuclear staining (a, b). Five independent experiments were performed, and a representative result is shown. Scale bar, 500 μm.

Figure 5.

Cellular localization of adenovirus-mediated Shh expression in the lesioned facial nuclei of neonatal rats. The immunoreactivities of the anti-Shh monoclonal antibody were visualized with Alexa-Fluor 488 (green, left). The neuronal (p75 and GAP-43) and non-neuronal (m, Iba1 for microglia and macrophage; a, GFAP for astrocyte) markers were visualized with Alexa-Fluor 594 (red, middle). Shh immunoreactivities (arrows) were observed in the cell bodies of p75-positive or GAP-43-positive neurons but not detected in the cell bodies of Iba1-positive (m) or GFAP-positive (a) cells. Scale bar, 40 μm.

Figure 7.

Inhibition of Shh signaling in axotomized adult rats. The cyclopamine application resulted in the loss of motor neurons in vivo. Bilateral facial nerves were axotomized and a Gelfoam soaked with cyclopamine (1.0 or 5.0 μg/ml), with tomatidine (5.0 μg/ml), or with vehicle (45% w/v HBC) was implanted in each side. The total number of Nissl-positive cells was counted and compared between the implanted side and the control side. Five independent animals were operated on and statistically analyzed by Student's t test (^*p < 0.05; ^**p < 0.01).