INTRODUCTION

Neurologic injury caused by ischemia-reperfusion injury of the spinal cord has been a frequency between 3 % and 24 % (^{Cambria et al., 1997}). Neurologic damage developing after acute spinal cord injury is related with necrosis developing after secondary injury and primary mechanic injury and apoptosis which develops later (^{Anderson et al., 1982}). Oxygen-derived free radicals have been implicated in the pathogenesis of spinal cord neuronal injury after trauma. Decreasing the level of oxidative stress minimizes the secondary destruction effect after traumatic injury (^{Yazihan et al., 2008}). Numerous studies reported that increased oxidative stress due to ischemia-reperfusion injury is associated with decreased enzymatic and non-enzymatic antioxidant defense potential, such as glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) activities (^{Naik et al., 2006}; ^{Morsy et al., 2010}). Vascular endothelial growth factor (VEGF) is among the significantly upregulated genes found in SCI rats with allodynia (^{Nesic et al., 2005}).VEGF isoforms play important roles in the development of vascular network, it independently works on neuron to guide neurogenesis (^{Jin et al. (2002}), axon growth (^{Sondell et al., 1999}; Alitalo et al., 2011; ^{Erskine et al., 2011}; ^{Ruiz de Almodovar et al. 2011}) Nuclear Antigen (PCNA) is a significant cell-cycle regulated nuclear protein for DNApolymerase, the PCNA-labeled nuclei had been shown to identify cells in the late G1 and early S phases of the cell cycle, as well as cells undergoing DNA repair (^{Chen et al., 2005}). Oral administration of Ganoderma lucidum has been shown to significantly reduce both cerebral infarct area and neuronal apoptosis in the ischemic cortex (^{Zhou et al., 2012}). Recent studies have demonstrated the neuroprotective effect of Ganoderma lucidum to reduce oxidative stress in vitro (^{Zhao et al., 2005}), to induce neuronal differentiation (^{Cheung et al., 2000}), and to prevent the harmful effects of the exterminating toxin Ab in Alzheimer's disease in cultured rat neurons (^{Lai et al., 2008}). In this study. The protective effect of Ganoderma lucidum on neuronal and angiogenetic function after medulla spinalis injury was investigated.

MATERIAL AND METHOD

The investigation was conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by US National Institutes of Health (NIH Publication no. 85-23, revised 1996). All experimental protocols were approved by the Dicle University Animal Care and Use Committee. Male Sprague-Dawley rats (200- 230 g) were housed in an air-conditioned room with 12-h light and dark cycles, where the temperature (23±2 °C) and relative humidity (65-70 %) were kept constant.

Anesthetized (ip ketamine and chlorpromazine; 75 mg/kg and 1 mg/kg, respectively) rats were positioned on a heating pad in a prone position and a rectal probe was inserted. Under sterile conditions, following T5-12 midline skin incision and paravertebral muscle dissection, spinous processes and laminar arcs of T7-10 were removed. The dura was left intact. Weight-drop model was performed for SCI (^{Allen, 1911}). The animals were subjected to an impact of 100 g/cm to the dorsal surface of the spinal cord. The force was applied via a stainless steel rod (3 mm diameter tip weighing 10 g) that was rounded at the surface. The rod was dropped vertically through a 10-cm guide tube that was positioned perpendicular to the center of the spinal cord. Afterward, the muscles and the incision were sutured. Following surgical procedure, the rats were placed in warming chamber and their body temperatures were maintained at approximately 37 °C until they were completely awake. Thirty minutes after the spinal cord injury, rats were injected either saline or G. lucidum polysaccharides (GLPS) (400 mg/kg/day) via gastric gavage. The seventh day all 30 animals were killed after TBI. Thirty rats were divided into three groups as group 1 control group, 2 spinal cord injury group, 3 spinal cord injury + Gonoderma lucidum group (20 mL/kg per day via gastric gavage) Ganoderma lucidum was administered for 7 days after trauma. Each group consisted of 10 animals, and of these, six were used for biochemical (Table I) and the other four were used for the evaluation of histological analysis.

Histological Preparation and Analysis. At the end of the experiment, all animals were anesthetized via the intraperitoneal administration of ketamine HCL (0.15 ml/ 100 g body weight). The cerebellum were dissected. For the histological examination, cerebellum tissues were fixed in 10 % formaldehyde solution, post fixed in 70 % alcohol, and embedded in paraffin wax. The sections were stained with Hematoxylin-Eosin

Immunohistochemical staining. An antigen-retrieval process was performed in citrate buffer solution (pH 6.0) two times: first for 7 min, and then for 5 min in a microwave oven at 700 W. They were allowed to cool to room temperature for 30 min and washed in distilled water for 5 min twice. Endogenous peroxidase activity was blocked in 0.1 % hydrogen peroxide for 15 min. Ultra V block (Histostain-Plus Kit, Invitrogen, Carlsbad, CA) was applied for 10 min prior to the application of the primary antibodies (VEGF antibody, mouse monoclonal, 1/200, Santa Cruz Biotechnology) overnight.

The secondary antibody (Histostain-Plus Kit, Invitrogen, Carlsbad, CA) was applied for 20 min. Then the slides were exposed to streptavidin-peroxidase for 20 min. Diaminobenzidine (DAB, Invitrogen, Carlsbad) was used as a chromogen. Control slides were prepared as mentioned above but omitting the primary antibodies. After counterstaining with Hematoxylene, washing in tap water for 5 min, and in distilled water for 2 ¥ 5 min, the slides were mounted.

Tunnel technique. Anti-proliferating cell nuclear antigen (PCNA) antibody was employed for detection of the proliferation rate of enterocytes. Sections taken to distilled water were then washed in PBS 2x5 min sections were kept in freshly prepared permeabilization solution (0.1 % Triton X-100 in 0.1 % sodium citrate) 8 min on ice. then washed in PBS 2x5 min and Added TUNEL Mixture (diluted 1:1 with TUNEL Dilution Buffer) on sections in the dark in humidified chamber for 1 at 37 ºC. They were subsequently washed in PBS 2x5 min, As a substrate solution, Fast Red (Roche) was applied on sections. Sections were counterstained with Hematoxyline, Mounted with Kaiser's Glycerol Gelatine (Roche).

Measurement of MPO Activity. MPO activity in tissues was measured by a procedure similar to that described by ^{Hillegass et al. (1990}).Spinal cord tissue samples were homogenized in 50 mM potassium phosphate buffer with a pH of 6.0, and centrifuged at 41,400 g for 10 min. The pellets were then suspended in 50 mM PB containing 0.5 % hexa-decyl trimethyl-ammonium bromide (HETAB). After three freeze and thaw cycles, with sonication between cycles, the samples were centrifuged at 41,400 g for 10 min. Aliquots (0.3 ml) were added to 2.3 ml of reaction mixture containing 50 mM PB, o-dianisidine, and 20 mM H2O2 solution. One unit of enzyme activity was defined as the amount of MPO present that caused a change in absorbance, measured at 460 nm for 3 min. MPO activity was expressed as U/g tissue.

Malondialdehyde (MDA) and Glutathione (GSH) Assays. Spinal cord tissue samples were homogenized with ice-cold 150 mMKCl for the determination of MDA and GSH levels. The MDA levels were assayed for the products of lipid peroxidation and results are expressed as nmol MDA/g tissue (^{Buege & Aust, 1978}), GSH was determined by a spectrophotometric method based on the use of Ellman's reagent and results are expressed as mmol GSH/ g tissue (^{Beutler et al., 1963}).

Statistical Analysis. All data are expressed as means ± S.D. Groups of data were compared with an analysis of variance (ANOVA) followed by Tukey's multiple comparison tests. Values of p <0.05 were considered as significant.

RESULTS AND DISCUSSION

MPO activity, which is accepted as an indicator of neutrophil infiltration, was significantly higher in the spinal cord tissues of traumatic rats than those of the control group (p<0.001). Ganoderma treatment significantly decreased spinal cord tissue MPO levels (p<0.01). (Tabla I and Fig. 1).

Fig. 1 A. Control group: Canalis ependimalis covered with oval nucleus cubic-shaped epithelium, multipolar and bipolar neuron structures and glial cells in the Cornu anterior region were regularly observed. H-E staining Bar 100 mm. B. Spinal cord injury group: Degeneration and loss in Kanalis ependimalis cells, deterioration in some radical cells, hyperplasia in funicular cells, dilation in small capillary vessels, vacuolar structures in the area of substantia grisea were observed.HE staining Bar 100 mm. C. Spinal cord injury group+Ganoderma lucidum group: Mild degeneration in radicular cells, hyperplasia in funicular cells, vascular dilatation and reduction of vacuolar structures in the substantia grisea region. H-E staining Bar 100 mm. 

Trauma caused a significant increase in the MDA levels (p<0.001) with a concomitant decrease in GSH levels (p<0.001). Ganoderma treatment significantly reduced the increase in MDA levels and restored GSH content at seventh day.

Spinal cord trauma results in a rapid and extensive oxidative stress. It has long been established that oxidative stress plays a critical role in the pathophysiology of SCI (^{Christie et al., 2008}; ^{DeRuisseau et al., 2009}; ^{Savas et al., 2009}).

G. lucidum extract reduced the expressions of proinflammatory and cytotoxic factors from the activated microglia, and effectively protected the dopaminergic neurons against inflammatory and oxidative damage (^{Huang et al., 2010}). Some studies have shown that administration of VEGF to the injured spinal cord has neuroprotective effects, induces angiogenesis and improves neurobehavioral results (^{Widenfalk et al., 2003}; ^{Patel et al., 2009}; ^{Liu et al., 2010}). Our data show that VEGF expression is increased in spinal cord injury, but VEGF expression in neuron and endothelial cells is decreased in spinal cord injury of Ganoderma lucidum treatment.Vascular endothelial growth factor (VEGF), angiogenesis and vascularization of spinal cord injury (SCI) are important contributors. VEGF is thought to be an important neurotrophic for the continuity of spinal cord neurons and endothelial cells. (Fig. 2).

Fig. 2 A.Control group: Negative VEGF expression in radicular and funicular cells, VEGF expression in glia cells and vascular endothelium was weakly observed.VEGF immunostaining staining Bar 100 µm; B. Spinal cord injury group:Positive VEGF expression was observed in Glia cells and vascular endothelial cells.VEGF immunostaining staining Bar 100µm; C. Spinal cord injury group+Ganoderma lucidum group: In multipolar and bipolar neurons, weak VEGF expression was seen in glial cells and vascular endothelial cells.VEGF immunostaining staining Bar 100 µm; D. Control group: Negative PCNA expression in the radicular and funicular cells and glial cells. PCNA immunostaining Bar 50 µm; E. Spinal cord injury group: In radicular and funicular cells, the expression of PCNA in the nucleus of the glial cells and astrocytes in white matter was increased. PCNA immunostaining staining Bar 50µm; F. Spinal cord injury group+Ganoderma lucidum group: In the radicular and funicular cells, the PCNA expressions in the nucleus structure of the glial cells were weakened PCNA immunostaining staining Bar 50µm. 

Reactive astrocytes also play a crucial role in wound healing and functional recovery after SCI. At the subacute phase, astrocytes migrate to compact the lesion, presumably secluding the inflammatory cells to prevent them from spreading into the parenchyma of the spinal cord (^{Okada et al. 2006}). Xiangdong et al. observed that PCNA expression in astrocytes increased in white matter after spinal cord injury. In our study, the expression of astrocytes in PCNA and white matter increased in the nuclei of glial cells in radicular and funicular cells. In spinal cord injury, it was observed that the DNA structure changed and caused apoptotic changes. PCNA expression in the ganoderma group showed a weak reaction in the gray matter and white matter, and DNA and apoptotic changes in the nucleus structure decreased. Ganoderma lucidum, an antioxidant effective dose and duration dependent inhibitor of cell apoptosis and altered angiogenetic activity, has been implicated in changes in neurons and glial cells in spinal cord injury and in angiogenesis, impairing its dose and duration.