Design
A randomized, controlled, animal study.
The experiments were performed at the Key Laboratory of Xinjiang Medical University and Tumor Hospital of Xinjiang Medical University in 2012.
A total of 40 purebred male, adult Beagles, aged 1– 1.5 years, weighing 12.0 ± 1.5 kg, body length 60 ± 5 cm, were provided by the Laboratory Animal Institute of Sichuan Academy of Medical Sciences (license No. SCXK (Chuan) 2004-15). The dogs were separately housed at 20–23°C, with humidity of 40–60%, and allowed free access to food and water. The animal procedures were performed in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, issued by the Ministry of Science and Technology of China[15].
Methods
The 40 dogs were randomly assigned to groups, and T9-10 levels were confirmed as the target regions for radiation. The target region was accurately outlined using CT (CT Imaging System, Cleveland, OH, USA) assisted by computer and radiation procedures were formulated. The dogs were intravenously anesthetized and placed in the dog body membrane fixation mould, and subjected to IGRT and conventional radiation therapy (Figure 6), receiving 5 or 7 Gy (i.e. 50 or 70 Gy in total) each time, 300 cGy/min, once a day, 10 times over a 14 day period (Monday-Friday only)
Sampling and histological observation of T9-10 segments
Three months after radiation, the dogs were anesthetized and sacrificed, and T9-10 spinal cord was harvested under a microscope, immediately fixed in 10% formalin and 2.5% glutaraldehyde, stored in liquid nitrogen, paraffin embedded, and sectioned into 4 μm-thick sections, followed by routine hematoxylin- eosin staining. Spinal cord histological changes were observed by light microscopy (Leica, Wetzlar, Germany).
Transmission electron microscopy of injury spinal cord ultrastructure
Samples were washed with buffer solution, prefixed with 2.5% glutaraldehyde for 2 hours, post-fixed with 1% osmic acid for 2 hours, washed with PBS, dehydrated with an increasing gradient of ethanol and acetone, embedded, dried by baking overnight at 37°C, and sectioned at 50 nm thickness. The sections were observed and images were obtained by transmission electron microscopy (Hitachi, Hokkaido, Japan).
TUNEL detection for cell apoptosis
Spinal cord neuronal apoptosis was detected by the TUNEL method with an in situ apoptosis detection kit (Boehringer Mannheim, Berlin, Germany). The sections were dewaxed, hydrated, followed by in situ end labeling according to manufacturer’s instruction. The sections were visualized by diaminobenzidine (Boehringer Mannheim), and mounted with neutral gum. Ten fields of view with dense apoptotic cells from each section were selected using BT-2000 color pathological imaging analysis system (Hubei Botai Electronic Technology, Wuhan, Hubei, China) to calculate neuronal apoptotic index (%) = mean absorbance of TUNEL-positive cells × percentage of TUNEL-positive cells (percentage of positive cells in total cells) × 100%[14].
Immunohistochemistry for Fas and HSP70 expression
Streptavidin biotin peroxidase complex method was used to perform immunohistochemistry according to the kit. Briefly, paraffin sections were dewaxed, hydrated, incubated with the primary antibody, rabbit anti-dog Fas or HSP70 monoclonal antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C for 2 hours, followed by the secondary antibody, goat anti-rabbit IgG (1:100; Santa Cruz Biotechnology) at 37°C for 1 hour. Ten fields of view were randomly selected from two anterior horns of each section and the positively stained area was measured automatically using the BT-2000 color pathological imaging analysis system. The mean percentage of positive staining out of the total area was used as Fas and HSP70 expression.
