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  • Progesterone Administration Modulates TLRs/NF-B Signaling Pathway in Rat Brain after Cortical Contusion

    Gang Chen,1 Jixin Shi,1 Wei Jin,1 Lin Wang,1 Weiying Xie,2 Jie Sun,2 and Chunhua Hang1

    Departments of 1Neurosurgery and 2Anesthesiology, Jinling Hospital, School of Medicine,Nanjing University, Nanjing, Jiangsu Province, China

    Abstract. This study investigated whether progesterone administration modulates toll-like receptors (TLRs) and the nuclear factor-kappa B (NF-B) signaling pathway in the injured rat brain following traumatic brain injury (TBI). Right parietal cortical contusion was made by a weight-dropping method. Male rats were given 0 or 16 mg/kg injections of progesterone at postinjury hr 1 and 6 and on days 1, 2, 3, 4, and 5. Brain samples were extracted at 5 days after trauma. We measured mRNA expression of TLR2 and TLR4 by reverse-transcriptase polymerase chain reaction (RT-PCR), NF-B binding activity by electrophoretic mobility shift assay (EMSA), concentrations of interleukin-1b (IL-1b), tumor necrosis factor-a (TNF-a), and interleukin-6 (IL-6) by enzyme-linked immunosorbent assay (ELISA), intercellular adhesion molecule-1 (ICAM-1) expression by immunohistochemistry, and brain damage by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL). The results showed that TBI induces strong up-regulation of TLR2, TLR4, NF-B, pro-inflammatory cytokines, and ICAM-1 in the pericontusional area. Administration of progesterone following TBI down-regulates the cortical levels of these agents related to the TLRs/NF-B signaling pathway. After progesterone administration, apoptotic TUNEL-positive cells in the injured brain were significantly decreased. In summary, post-TBI progesterone administration attenuates the TLRs/NF-B signaling pathway in injured rat brain, and this may be a mechanism whereby progesterone improves the outcome following TBI.

    Keywords: progesterone; traumatic brain injury; toll-like receptors; nuclear factor-kappaB

    Introduction

    Although traumatic brain injury (TBI) represents a significant public health problem in the world, there are currently no treatments that improve clinical outcome measures [1,2]. Recently, several clinical and experimental studies have shown that progesterone plays neuroprotective roles in TBI, which include reducing cerebral edema, preventing neuronal loss, and improving cerebral function [3,4]. Following the demonstration that proges-terone is effective in attenuating neurological abnormalities, the next step is to determine how

    progesterone mediates its neuroprotective effects. Because progesterone reduces edema after TBI and is considered an immune suppressant due to its role in maintaining pregnancy (eg, preventing immune-mediated rejection of the fetus) [5], we hypothesized that progesterone might help to promote recovery from TBI by modulating the pathophysiological pathways and signal transcription factors that are related to cerebral inflammation after TBI. Ten mammalian toll-like receptors (TRLs) have been identified by sequence analysis [6]. Among them, TRL2 and TRL4, which are widely expressed in brain, can detect endogenous agonists, such as the degradation products of macromolecules, heat shock protein 60 and 70, products of proteolytic cascades, intracellular components of ruptured cells, and products of genes that are activated by

    Address correspondence to Chunhua Hang, M.D., Dept. of Neurosurgery, Jinling Hospital, 305 East Zhongshan Road, Nanjing 210002, P.R.China; tel 86 25 8197 3916; fax 86 25 8481 7581; email nju_neurosurgery@163.com.

    0091-7370/08/0100-0065. $3.00. 2008 by the Association of Clinical Scientists, Inc.

    Available online at www.annclinlabsci.org

    Annals of Clinical & Laboratory Science, vol. 38, no. 1, 2008 65

  • inflammation [6]. Furthermore, TLR2 and TLR4 have been demonstrated to play an important role in initiating the cerebral inflammation related to stroke, Alzheimers disease, Huntingtons disease, and Parkinsons disease [7]. All TLRs activate a common signaling pathway that culminates in the activation of NF-B transcription factors and the mitogen-activated protein kinases (MAPKs). As one of the most important downstream molecules in the TRLs signaling pathway, NF-B is a trans-criptional factor required for the gene expression of many inflammatory mediators, such as interleukin-b (IL-1b), tumor necrosis factor-a (TNF-a), interleukin-6 (IL-6), and intercellular adhesion molecule-1 (ICAM-1) [8]. The aim of the current study was to see if progesterone attenuates the TBI induced activation of TRLs/NF-B signaling pathway in the peri-contusional area. We hypothesize that the effect of progesterone on modulating the TRLs/NF-B signaling pathway is a mechanism whereby proges-terone protects neurons, reduces cerebral edema, and promotes behavioral recovery after TBI.

    Materials and Methods

    Animals. Male Wistar rats (250 to 300 g) were purchased from the Animal Center of the Chinese Academy of Sciences, Shanghai, China. The rats were housed in temperature- and humidity-controlled animal quarters with a 12-hr light/dark cycle. All experimental protocols complied with the Laboratory Animal Care and Use Guidelines of the Medical School of Nanjing University.

    Cortical contusion trauma. Following anesthesia with urethane (1000 mg/kg, ip), the rats head was fixed in the stereotactic frame. A right parietal craniotomy (diameter 5 mm) was drilled under aseptic conditions 1 mm posterior and 2 mm lateral to the bregma. Trauma was induced by a modification of the Feeneys weight-drop model [9] in which a freefalling weight drops onto the exposed intact cranial dura to produce a standardized parietal contusion. A steel rod (weight 40 g) with a flat end diameter of 4 mm was allowed to fall from a height of 25 cm onto a piston resting on the dura. The piston was allowed to compress the brain tissue a maximum of 5 mm. After this procedure, the rats were returned to their cages and the room temperature kept at 231C. Heart rate, arterial blood pressure, and rectal temperature were monitored, and the rectal temperature was kept at 370.5C by physical cooling (ice bag) when required throughout the experiment. Sham-operated control rats were anesthetized and mounted in the stereotaxic apparatus, and their scalps were cut and sutured, but were not trephinated.

    Experimental protocol. The experimental groups consisted of sham+vehicle (SV; n = 6), lesion+vehicle (LV; n = 6), and lesion+progesterone (LP; n = 6). Progesterone (4-pregnene-3, 20-dione, Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 2-hydroxypropyl-b-cyclodextrin (Sigma-Aldrich); the pro-portion was 20 mg (progesterone):4 ml (2-hydroxypropyl-b-cyclodextrin). Rats of the LP group received injections of 16 mg/kg progesterone (dose volume 3.2 ml/kg) at 1 and 6 hr and 1, 2, 3, 4, and 5 days after the surgery (ip for the first injection and sc for the remaining six). Rats of the SV and LV groups received equal volumes (3.2 ml/kg) of 2-hydroxypropyl-b-cyclodextrin [10,11]. The rats were decapitated 5 days after injury for tissue assays. The surrounding brain tissue of the injured cortex (Fig. 1) was dissected on ice as described in our previous study [12]; a portion of the tissue was fixed in 10% buffered formalin, the remainder was immediately stored in liquid nitrogen until analysis.

    RNA extraction and RT-PCR. The levels of TLR2 and TLR4 mRNA expression were determined by reverse-transcriptase polymerase chain reaction (RT-PCR). Total RNA was extracted with TriPure Reagent (Roche Diagnostics, Indiana-polis, IN, USA) according to the manufacturers instructions. The cDNA synthesis from the isolated RNA was performed using a reverse transcriptional system. Briefly, 4 g of total brain RNA was reversely transcribed using 0.5 g oligo(dT)15 and incubated with 15 U Avian Myeloblastosis Virus Reverse Transcriptase (AMV RT) (all from Promega, Madison, WI, USA). The cDNA was amplified by PCR using oligonucleotide primers (Table 1), which amplified rat TLR2, TLR4, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA in preliminary experiments. Thereafter, the amplified frag-ments were detected by agarose gel electrophoresis and

    Fig. 1. Schematic representation of the cortical contusion area induced by weight-dropping trauma and the studied region surrounding the injured brain.

    Annals of Clinical & Laboratory Science, vol. 38, no. 1, 200866

  • visualized by ethidium bromide staining. The intensity of the bands was quantified using Glyko Bandscan software. As a control, GAPDH mRNA was detected in all samples, and TLR2/GAPDH and TLR4/GAPDH product ratios were used as indices of TLR2 and TLR4 mRNA expression.

    Nuclear protein extract and electrophoretic mobility shift assay (EMSA). Nuclear protein was extracted and quantified as described previously [13,14]. EMSA was performed using a gel shift assay system (Promega, Madison, WI). NF-B oligo-nucleotide probe (5-AGTTGAGGGGACTTTCCCAGGC-3) was end-labeled with [-32P]ATP (Free Biotech, Beijing, China). EMSA was performed as in previous studies [13,14].

    Enzyme-linked immunosorbent assay (ELISA). The levels of inflammatory mediators were quantified using specific ELISA kits for rats according to the manufacturers instructions (TNF-a from Diaclone Research, France; IL-1b and IL-6 from Biosource Europe SA, Belgium) and previous studies [13,14]. Results were expressed as ng/g protein.

    Immunohistochemical studies were conducted on formalin-fixed, paraffin-embedded sections. Rabbit-anti-rat monoclonal antibody to ICAM-1 (diluted 1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used. Immunohistochemical assays were performed according to our previous study [12]. The number of positive microvessels in each section was counted in 10 microscopic fields (at 100 magnificatio