Cytokine chemokine expression in contused rat spinal cord

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Abstract

Spinal cord injury within the first few hours, is complicated by inflammatory mechanisms, including the influx of monocyte/macrophages as well as the activation of resident spinal microglia and astrocytes. Numerous studies have suggested that the initial infiltration of the hematogenous cells may be due to the secretion of cytokines and chemokines in the injured CNS. In order to elucidate which chemotactic factors may be expressed following traumatic spinal cord contusion, the presence of mRNA for a number of cytokines, chemokines and growth factors was examined in contused rat spinal cord by reverse transcriptase-polymerase chain reaction and immunohistochemistry. Spinal injury was accompanied by an increase in glial fibrillary acidic protein mRNA suggesting astrocyte activation and astrogliosis. TNFα message levels were upregulated as early as 1 h post injury and returned to baseline levels by 3 days post injury (DPI). By immunocytochemistry, staining for TNFα increased at 1 and 3 dpi and was predominantly diffuse in the necrotic tissue. The chemokines IP-10, MCP-1, and MIP-1α were also detected in the injured spinal cord. mRNA levels of IP-10 peaked around 6 h post injury and were upregulated up to 7 dpi. MCP-1 mRNA was detected at 1 h post injury and its levels returned to baseline by 14 dpi. An increase in MCP-1 staining was observed from 1 to 7 dpi. The staining was also diffuse in the necrotic tissue and also localized to cells near the site of injury. The presence of aFGF and bFGF was also detected in the injured spinal cord. mRNA for aFGF was detected at 0 time, increased at 6 h post injury, peaked at 3 days, and remained elevated up to 21 days. bFGF mRNA was initially detected at 1 h post injury, increased between 6 h and 3 days, declined thereafter and returned to baseline levels by 21 days.

Introduction

Following mechanical injury to the central nervous system (CNS), breakdown of the blood–brain barrier results in recruitment of hematogenous cells at the site of injury. As in numerous other tissues, the initial infiltration of these cells in the injured CNS has been attributed to chemokines. The production of these factors in the CNS has not only been attributed to the activated lymphoid and mononuclear cells that infiltrate the CNS, but also to the resident glial cells, especially reactive astrocytes and microglia. Chemokines are functionally related cytokines that induce specific actions on the immune system and are released in response to infection, inflammation and trauma. Studies conducted in many laboratories have shown that chemokines play a central role in the process of leukocyte recruitment to tissues. The α family chemokines contain in their structure a conserved CXC motif and are known to recruit polymorphonuclear cells. The β family chemokines contain a CC motif and provide priming signals for macrophages, lymphocytes, eosinophils and basophils. Among the chemokines from the β family, monocyte chemoattractant protein (MCP-1), human homolog of the mouse JE gene product, is the most well characterized. Increased MCP-1 expression was detected in astrocytes and in perivascular mononuclear cells in experimental allergic encephalomyelitis (EAE), an inflammatory disease of the CNS. MCP-1 protein levels have been shown to parallel development of clinical disease and macrophage infiltration (Hulkower et al., 1993, Ransohoff et al., 1993, Brosnan et al., 1993). Besides immune mediated inflammation, MCP-1 expression has been found in rat/mouse subjected to brain trauma (Berman et al., 1996). Some studies have shown a close correlation between macrophage infiltration and elevated levels of MCP-1 (Berman et al., 1996). Following a penetrating cortical injury, MCP-1 has been localized to astrocytes, endothelial cells, and macrophages (Glabinski et al., 1996). MCP-1 expression in human endothelial cells has been shown to increase adhesion of monocytes to endothelial cells (Shyy et al., 1993). Mononuclear infiltrate composed mainly of monocytes and macrophages was detected in transgenic mice overexpressing MCP-1 in the brain. A few studies have shown that MCP-1 expression in astrocytes can be modulated by cytokines such as interleukin 1 (IL-1), interferon gamma (IFN-γ), and transforming growth factor beta (TGFβ) (Barna et al., 1994, Hurwitz et al., 1995, Majumder et al., 1996). Macrophage inflammatory protein — 1 alpha (MIP-1α) and macrophage inflammatory protein — 1 beta (MIP-1β) expression has been shown predominantly in myeloiod and lymphoid cells. These factors act as chemotactic and activating agents for macrophages (Wilson et al., 1990). MIP-1α and MIP-1β mRNAs have been demonstrated in EAE and MIP-1α expression has been correlated with development of severe clinical disease (Karpus et al., 1995, Godiska et al., 1995).

The alpha chemokine family includes gamma-interferon-inducible protein (IP-10), platelet factor 4, IL-1, and melanoma growth stimulatory activity (MGSA/gro/KC) (Stoeckle and Barker, 1990). IL-8 and KC are potent chemoattractants and activators of neutrophils (Baggiolini et al., 1989, Oppenheim et al., 1991, Baggiolini and Clark-Lewis, 1992). Recombinant IP-10 is chemotactic for monocytes and activated lymphocytes and potentiates activated T-cell adhesion to endothelial cells in vitro (Taub et al., 1993, Amichay et al., 1996). In the CNS, IP-10 expression has been shown to occur at the onset of CNS inflammation and neurological signs in murine EAE (Ransohoff et al., 1993, Godiska et al., 1995). These studies revealed that astrocytes surrounding the perivascular inflammatory cuff were the source of IP-10 (Ransohoff et al., 1993, Tani and Ransohoff, 1994). Klein et al., (1996) have reported the presence of IP-10 message following injection of lipopolysaccharide into mouse cerebral cortex. In contrast, IP-10 mRNA was not detected after nitrocellulose membrane stab injury in mouse brain (Glabinski et al., 1996). In vitro, IP-10 mRNA expression has been reported in human astrocytoma cells (CRT) (Majumder et al., 1996) and in cultured primary rat astrocytes and microglia (Vanguri, 1995, Vanguri and Farber, 1994).

Current studies on a penetration (stab) wound in the rat brain which immediately breaks the blood–brain barrier has shown a rapid increase in MCP-1α, MIP-1β, regulated on activation, normal T cell expressed and secreted (RANTES), IP-10, TNFα, bFGF, and TGFβ. The present study reports a combined RT-PCR and immunocytochemical analysis of rat spinal cord after a contusion injury. A select group of factors was chosen based on previous brain and spinal cord injury studies (Ghirnikar et al., 1996; 1998). A preliminary report of this study has already been presented (Lee et al., 1997).

Section snippets

Spinal cord contusion injury

Male Sprague–Dawley rats weighing 200–250 g were maintained under standard conditions at the PAVAMC. The spinal cord contusion injury was performed as described previously (Wrathall et al., 1985). Briefly, animals were anesthetized with sodium pentobarbitol (50 mg/kg body weight) and a laminectomy performed at T8. Spinous processes and lamina were removed to expose a circular region of dura approximately 2.8 mm in diameter. The spinal column was stabilized by clamping the spinous processes of

The RT-PCR data are summarized in Fig. 1

MCP-1 mRNA was present at baseline in normal spinal cord, increased at 1 h, peaked at 24 h, and returned to low level by 14 days. MIP-1 α mRNA was present in normal spinal cord, increased at 1 h, peak by 3–6 h, decreased by day 1, remained unchanged to day 7, and returned to a low level by day 14. TNFα mRNA, not detected in normal spinal cord, had a high level by 1 h; remained high to day 1, returned to a low level by day 3, and not detected by day 5. bFGF mRNA was not detected in normal spinal

Discussion

The induction of TNFα mRNA which was not detected in normal spinal cord, reach a high level by 1 h, remained high to day 1, back to low level by day 3, and not detected by day 5 is in general agreement with the report of Wang et al. (1995). The present study shows that the responses of MCP-1, IP-10, bFGF, and MIP-1α, β span 1–7 days after contusion injury while GFAP and aFGF remained elevated throughout the experimental period of 21 days. We have recently found that IP-10 is rapidly induced

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