Expression of vascular endothelial growth factor receptors in rat spinal cord tissue

Chongyang FU; Guangxiang HONG; Fabin WANG

ABSTRACT

BACKGROUND:

Vascular endothelial growth factors (VEGF) can promote the division of endothelial cells and accelerate the growth of newborn vessels, whereas the expression and distribution of VEGF receptors (VEGFR) in spinal cord should be observed.

OBJECTIVE:

To observe the expression and distribution of VEGFR-1 and VEGFR-2 in rat spinal cord.

DESIGN:

A randomized controlled observation.

SETTING:

Department of Orthopaedic Surgery, the First Affiliated Hospital of Dalian Medical University; Department of Hand Surgery, Union Hospital affiliated to Tongji Medical College, Huazhong University of Science and Technology.MATERIALS Ten adult male Wistar rats of clean degree, weighing 180-200 g, were provided by the animal experimental center of Tongji Medical College, Huazhong University of Science and Technology. Immunohistochemical primary antibody was purchased from Santa Cruz Company; the second and third antibodies from Beijing Zhongshan Biotechnology Co., Ltd. Reverse transcription-polymerase chain reaction (RT-PCR) kit was the product of Promega,Trizol reagents were purchased from Invitrogen Company, and the VEGFR-1 and VEGFR-2 primers were designed by Beijing Aoke Company.

METHODS:

The experiment was carried out in the laboratory of Department of Hand Surgery of Wuhan Union Hospital from March to June in 2004. ① Detection of VEGFR expression in spinal cord anterior horn The rats were anesthetized by 100 g/L chloral hydrate, spinal cord of lumbar 4-6 (L4-6) was fixed in fixation solution at 4 ℃ for overnight, then routine dehydration, hyalinization and paraffin embedding were performed, and serial sections of about 5 μm were prepared for observing the VEGFR expressions using immuniohistochemical staining. ② Detection of VEGFR mRNA expression in spinal cord Five rats were selected, L4-6 spinal cord (50 mg) was removed and centrifugated, then the content of total RNA was determined with ultraviolet spectrophotometer. The synthetized cDNA was amplified with PCR, and the PCR products (10 μL) were treated with 20 g/L agarose gel electrophoresis, and stained with 0.1 mg/L ethidium bromide, the results were observed and recorded under ultraviolet lamp and photographed. The products were scanned and quantified with gel imaging analytical system to record the gray value of each band, the gray value of β-actin band was taken as 1,and those of the other objective fragments were compared with it to record the gray value ratio and analyze the expressions of the objective fragments.MAIN OUTCOME

MEASURES:

① Expression and distribution of VEGFR-1 and VEGFR-2 in rat spinal cord anterior horn;② Results of VEGFR mRNA expression in spinal cord.

RESULTS:

① Both VEGFR-1 and VEGFR-2 were expressed in the microvessels of normal rat spinal cord tissue.Besides, VEGFR-2 mainly expressed in motor neurons, glial cells and the nerve fibers in surrounding white matter. ②The results of gel imaging analytical system showed that the VEGFR-2 content in normal spinal cord was obviously higher than VEGFR-1 (0.874±0.222, 0.486±0.181, P ＜ 0.05).

CONCLUSION:

VEGF promote the formation of microvessels through the combined effects of VEGFR-1 and VEGFR-2,and it plays the neurotrophic and neuroprotective role through VEGFR-2.