Estrogen affects BDNF expression following chronic constriction nerve injury<?xml:namespace prefix = o ns = "urn:schemas-microsoft-com:office:office" />

Xin Zhao,^1,2 Jian Liu,² Rong Guan,³ Yi Shen,¹ Ping Xu⁴ and Jianguo Xu^2,CA

¹School of Life Science,Nanjing University,Nanjing 210093; ²Department of Anesthesiology, JinlingHospital,Nanjing 210002, PR China;

³Division of Cell and Molecular Biology,University of Texas Southwestern Medical Center at Dallas,Dallas,Texas 75390-9042,USA;

⁴Computer Science and Technology, Nanjing University, Nanjing 210093, PR China

^CACorresponding Author: zhmymy@hotmail.com

Received10 April 2003; accepted12 May 2003

DOI: 10.1097/01.wnr.0000086251.76504.38

INTRODUCTION<?xml:namespace prefix = o ns = "urn:schemas-microsoft-com:office:office" />

The impact of gender variability on nociceptive processing has received renewed attention. Gonadal hormones appear to have a role in noxious stimuli processing. Females report lower pain thresholds, higher pain ratings and less tolerance to noxious stimuli than males ^[1]. Studies in mice indicate that males and females use functionally distinct pain pathway, and the gonadal steroids, particularly estrogen, play a major role in regulating these pathways ^[2]. Estrogen modulates numerous molecular mechanisms that contribute to the pain process. It can regulate neurotransmitter and neuromodulator and exert its action via genomic or nongenomic mechanisms ^[3－5].

The rat with chronic constriction injury (CCI) ^[6] presents many of the characteristic signs and symptoms of neuropathic pain found in human, and provides a suitable model to study the mechanisms underlying this type of pain. Following peripheral nerve injury or inflammation, sensory information processing in the spinal dorsal horn appears to undergo significant changes and perhaps leads to the development of persistent pain ^[7,8].

A common gene product regulated by estrogen is brain derived neurotrophic factor (BDNF) ^[5]. BDNF, with a mol. mass of 27 kDa, is a member of the neurotrophin family of growth factors. It is located in specific neuronal populations at low levels in the peripheral nervous system and at much higher levels in the CNS ^[3－5]. Besides its role in the survival, differentiation, and outgrowth of select peripheral and central neurons during development and in adulthood ^[9,10], it also serves as a neurotransmitter or neuromodulator within the dorsal horn of the spinal cord ^[11].

Sciatic axotomy and inflammatory stimulus can upregulate BDNF expression in the dorsal root ganglion (DRG) neurons. Increased BDNF levels are transported anterogradely to the spinal dorsal horn ^[12－14]. Therefore, in order to investigate the effect of estrogen on its level in CCI rats, we detected BDNF mRNA in DRGs and its protein content in the spinal cord of different estrogen level rats by means of reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). We also used the hot-plate test as a sign of neuropathic pain.

MATERIALS AND METHODS<?xml:namespace prefix = o ns = "urn:schemas-microsoft-com:office:office" />

Female Sprague－Dawley rats (180～200 g) maintained under a 12:12 h light:dark cycle with lights on at 06.00 h, with food and water available ad lib, were used in this study. All rats were purchased between the age of 8 and 10 weeks and were housed for a minimum of 1 week prior to use. The hind paw withdrawal latency (PWL) of all animals was obtained. Those responded abnormal were excluded. Then the rats were randomly assigned to one of the following treatment groups (n=25 rats/condition) (1) ovariectomized (OVX): low estrogen level group, which received ovariectomy without injection of estradiol; (2) estrogen (E): high estrogen level group, which received injection of estradiol with gonadally intact; (3) normal (N): normal estrogen level group, gonadally intact without receiving injection of estradiol. All the three groups received CCI surgery. We made great efforts to minimize the discomfort of the animals. All animal experiments were performed in accordance with the national legislation, and with the National Institutes of Health Guide regarding the care and use of animals for experimental procedures.

Ovariectomy: On the first day of the research, rats of OVX group were anesthetized with a single injection of urethane (1 g/kg, i.p.). The ovaries were removed through bilateral incisions in the dorsal flank. The other two groups received sham operation in which ovaries were inspected without removal.

Hormone treatment: From day one rats of the E group received 17-β-estradiol (50μg, s.c., diluted in sesame oil) every 2 days. The other two groups received only sesame oil as a vehicle.

CCI surgery: Three weeks after the research began, the estrogen level of each rat reached a stable stage, as confirmed by the determination of estrogen in the plasma. CCI surgery was performed. Under urethane (1 g/kg, i.p.) anesthesia, the right sciatic nerve was exposed and proximal to the trifurcation, ～7mm of the common sciatic nerve was freed from adhering tissue. Four 4-0 chromic gut sutures were tied around the nerve at intervals of～1mm, and ligatures were tied loosely enough so that, on visual inspection, blood flow was not obstructed ^[15]. The left sciatic, used as control, was exposed but not ligated. The surgical incision was sutured and postsurgical recuperation was monitored daily. Special attention was paid to prevention of infection, thus minimizing the influence of inflammation.

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