Gamma Knife Irradiation of Injured Sciatic Nerve Induces Histological and Behavioral Improvement in the Rat Neuropathic Pain Model

<div><p>We examined the effects of gamma knife (GK) irradiation on injured nerves using a rat partial sciatic nerve ligation (PSL) model. GK irradiation was performed at one week after ligation and nerve preparations were made three weeks after ligation. GK irradiation is known to induce immune responses such as glial cell activation in the central nervous system. Thus, we determined the effects of GK irradiation on macrophages using immunoblot and histochemical analyses. Expression of Iba-1 protein, a macrophage marker, was further increased in GK-treated injured nerves as compared with non-irradiated injured nerves. Immunohistochemical study of Iba-1 in GK-irradiated injured sciatic nerves demonstrated Iba-1 positive macrophage accumulation to be enhanced in areas distal to the ligation point. In the same area, myelin debris was also more efficiently removed by GK-irradiation. Myelin debris clearance by macrophages is thought to contribute to a permissive environment for axon growth. In the immunoblot study, GK irradiation significantly increased expressions of βIII-tubulin protein and myelin protein zero, which are markers of axon regeneration and re-myelination, respectively. Toluidine blue staining revealed the re-myelinated fiber diameter to be larger at proximal sites and that the re-myelinated fiber number was increased at distal sites in GK-irradiated injured nerves as compared with non-irradiated injured nerves. These results suggest that GK irradiation of injured nerves facilitates regeneration and re-myelination. In a behavior study, early alleviation of allodynia was observed with GK irradiation in PSL rats. When GK-induced alleviation of allodynia was initially detected, the expression of glial cell line-derived neurotrophic factor (GDNF), a potent analgesic factor, was significantly increased by GK irradiation. These results suggested that GK irradiation alleviates allodynia via increased GDNF. This study provides novel evidence that GK irradiation of injured peripheral nerves may have beneficial effects.</p></div

Further accumulation of Iba-1 positive macrophages in the ipsilateral sciatic nerves of PSL rats after GK-irradiation.

<p><b>A,</b> Effects of GK irradiation on Iba1 protein expression in the sciatic nerve. The top blots are examples of immunoreactive bands against anti-Iba-1 antibodies. The bottom blots are β-actin. <b>B,</b> Schematic figures of a ligation site and the areas analyzed. Histological photographs of injured nerves taken in four areas; proximal site, injured site, distal1 site and distal2 site. Red area; predicted 80% isodose irradiated area. Orange area; predicted 30% isodose irradiated area. <b>C,</b> Representative photographs of Iba-1 immunoreactivity on the ipsilateral sciatic nerve after GK-irradiation. <b>a;</b> sham-operated rats. <b>b;</b> ligation rats. <b>c;</b> GK-irradiated sham-operated rats. <b>d;</b> GK-irradiated ligation rats. Scale bar; 50 µm. <b>D,</b> The size of the Iba-1 positive area in each zone was measured using ImageJ. Data are presented as means ± SEM. **p<0.01 compared with Ligation group. <b>E,</b> The number of Iba-1 positive cells in each zone was determined using a cell counter with ImageJ. Data are presented as means ± SEM. **p<0.01 compared with Ligation group.</p

Morphological changes in GK-irradiated injured nerves analyzed by toluidine blue staining.

<p><b>A,</b> Representative photographs of toluidine blue stained cross sections of the ipsilateral sciatic nerve after GK-irradiation. Semi-thin cross sections (0.5 µm) of sciatic nerves 1 mm proximal to the injury site (<b>c</b> and <b>d</b>) or 1 mm distal to the injury site were stained (<b>e</b> and <b>f</b>) with 0.5% toluidine blue. <b>a;</b> sham-operated rats, <b>b;</b> GK-irradiated sham-operated rats, <b>c and e;</b> ligation rats, and <b>d and f;</b> GK-irradiated ligation rats. Scale bars; 20µm for each panel. Arrow; re-myelinated fiber. Arrowhead; immature re-myelinated axon. <b>B, a-c;</b> these graphs show the density of myelinated fibers per section from each rat. <b>a;</b> sham and sham+GK rats, <b>b</b>; proximal sections from ligation and ligation+GK rats, <b>c;</b> distal sections from ligation and ligation+GK rats. *p<0.05 compared with non-irradiated nerves from each group. <b>d and f,</b> Distribution of myelinated fiber diameters is shown as a histogram with 1µm bin. Data are presented as means ± SEM. <b>d;</b> sham and sham+GK rats. <b>f;</b> ligation and ligation+GK rats. <b>e and g,</b> Cumulative percentage distribution is plotted against the myelinated fiber diameter. <b>e;</b> sham (<i>n</i> = 3239 fibers) and sham+GK (<i>n</i> = 1972 fibers). <b>g;</b> ligation (<i>n</i> = 6179 fibers) and ligation+GK (<i>n</i> = 2386 fibers). *p<0.05 compared with Ligation group.</p

Immunoblot analyses of βIII-tubulin and P0 protein expressions two weeks after GK irradiation of the sciatic nerve.

<p><b>A,</b> The top blots are examples of immunoreactive bands against anti-βIII-tubulin antibodies. The middle blots are P0. The bottom blots are β-actin. <b>B,</b> One graph presents the optical density ratio of βIII-tubulin/β-actin, the other that of P0/β-actin. Data are presented as means ± SEM. **p<0.01 compared with the non-irradiated group.</p

Toluidine blue staining of injured nerves following partial sciatic nerve injury.

<p><b>A,</b> Time course analysis of morphological changes in the injured nerve. <b>a–g</b>; Semi-thin cross sections (0.5 µm) of sciatic nerves 1 mm proximal (b, c, and d) to the injury site and 1 mm distal (e, f, and g) to the injury site were stained with 0.5% toluidine blue. Representative photographs of cross sections of sciatic nerves. <b>a;</b> intact, <b>b and e;</b> one week after ligation, <b>c and f;</b> three weeks after ligation, <b>d and g;</b> seven weeks after ligation. Scale bars; 20µm for each panel. Arrow; myelin vacuolation and degenerating myelin. <b>h,</b> Density of myelinated fibers per section from each rat. **p<0.01, *p<0.05 compared with intact group. ††p<0.01, †p<0.05 compared with 3 weeks after ligation group. <b>i and j,</b> Distribution of myelinated fiber diameters is shown as a histogram with 1µm bin. <b>i;</b> Intact, <b>j;</b> Injured nerve. Data are presented as means ± SEM. <b>k,</b> Plot of the cumulative myelinated fiber diameter distribution. Black line; intact rat (<i>n</i> = 2264 fibers), gray line; 3 weeks after ligation (<i>n</i> = 6179 fibers), gray broken line; 7 weeks after ligation (<i>n</i> = 2510 fibers). **p<0.01 compared with intact group. ††p<0.01 compared with 3 weeks after ligation group. <b>B,</b> Immunoblot analysis of βIII-tubulin and P0 protein expressions after ligation. The top blots are examples of immunoreactive bands against anti-βIII-tubulin antibodies. The middle blots are P0. The bottom blots are β-actin. <b>a;</b> ipsilateral side, <b>b;</b> contralateral side. The graph presents the optical density ratios of βIII-tubulin/β-actin (open circle) and P0/β-actin (closed square). Data are presented as means ± SEM. **p<0.01, *p<0.05 compared with the intact group.</p