Progesterone and allopregnanolone reduce inflammatory cytokines after traumatic brain injury
Introduction
Recent studies demonstrate that progesterone can significantly reduce cerebral edema and enhance functional recovery from traumatic brain injury (TBI) and stroke in several animal models Asbury et al., 1998, Chen et al., 1999, Roof et al., 1994, Roof et al., 1996. Allopregnanolone, a GABAA-potentiating metabolite of progesterone, has been shown to have similar effects Bender and Norenberg, 1998, He et al., 2004, Limmroth et al., 1996. Since interleukin-1β (IL-1β) and tumor necrosis factor α (TNF-α) are major proinflammatory cytokines that mediate cerebral edema formation and secondary neuronal loss Patel et al., 2003, Raivich et al., 2002, Stover et al., 2000, Tehranian et al., 2002, we hypothesized that progesterone or allopregnanolone exerts their neuroprotective actions by suppressing the expression of these cytokines. Evidence from several studies has supported this theory.
Elevated IL-1β and TNF-α have been detected in the cerebrospinal fluid (CSF) and the brain parenchyma within hours after brain trauma in humans and rodents, respectively McClain et al., 1987, Ross et al., 1994, Woodroofe et al., 1991. This enhancement of proinflammatory cytokines markedly exacerbates ischemic, traumatic, or excitotoxic brain injury Lawrence et al., 1998, Loddick and Rothwell, 1996, Yamasaki et al., 1995. In particular, intracerebral administration of IL-1β results in breakdown of the blood–brain barrier (BBB), leading to cerebral edema and secondary cell death Holmin and Mathiesen, 2000, Touzani et al., 1999, whereas overexpression of the IL-1 receptor antagonist (IL-1ra), or the blockade of IL-1β-converting enzyme (caspase-1, ICE) activity, inhibits excitotoxic neuronal damage and reduces lesion size and early edema formation in a variety of brain injury models Berkenbosch, 1992, Fan et al., 1995, Holmin and Mathiesen, 2000, Touzani et al., 1999, Yamasaki et al., 1995. Similarly, TNF-α directly disrupts the BBB and causes vasogenic brain edema Knoblach and Faden, 2000, Megyeri et al., 1992, Stolpen et al., 1986. Neutralizing TNF-α activity lessens edema formation and facilitates recovery of motor function after TBI (Shohami et al., 1996). These data can be taken to suggest that elevated levels of IL-1β and TNF-α after TBI exacerbate brain damage (DeVries et al., 1996) and contribute to neurological impairment by increasing cerebral edema and by causing apoptotic cell death Allan et al., 2000, Arvin et al., 1996, Knoblach et al., 1999, Ross et al., 1994, Trembovler et al., 1999.
This elevation of inflammatory cytokines occurs very early post-injury Knoblach and Faden, 2000, Knoblach et al., 1999, before the invasion of peripheral neutrophils. Therefore, it is likely that the early production of these cytokines is due to injury-triggered local synthesis (Hopkins and Rothwell, 1995) in the resident CNS cells, most likely microglia Bartholdi and Schwab, 1997, Uno et al., 1997 and possibly neurons (Knoblach et al., 1999).
Most importantly, several in vitro studies have shown that progesterone and allopregnanolone suppress the production and the activity of proinflammatory cytokines including IL-1β and TNF-α Drew and Chavis, 2000, Polan et al., 1989, Salzman et al., 2000. For example, progesterone suppressed microglial activation and reduced the production and expression of TNF-α, IL-1β, and IL-1β-induced nitric oxide in various cell cultures Drew and Chavis, 2000, Polan et al., 1989, Salzman et al., 2000. Progesterone and allopregnanolone, but not estrogen, inhibits LPS-induced TNF-α transcription and intracellular TNF-α protein synthesis in culture Ghezzi et al., 2000, Miller and Hunt, 1998. They also protect cells against apoptotic cell death both in vitro (Vegeto et al., 1999) and in vivo (Djebaili et al., 2004). Additionally, progesterone administration stimulates peripheral nerve remyelination via the intracellular receptor (Koenig et al., 1995) and enhances neurite growth in the peripheral nerve system (PNS) via its metabolism to allopregnanolone (Koenig et al., 2000).
The aim of the current study was to determine whether progesterone and/or allopregnanolone could reduce the TBI-induced elevation of proinflammatory cytokines, in particular IL-1β and TNF-α, following severe cortical injury in the adult laboratory rat. We hypothesized that the regulation of post-TBI inflammation could be a mechanism by which progesterone and allopregnanolone protect neurons, reduce cerebral edema, and promote behavioral recovery after TBI. We sought to detect changes in both gene expression and protein concentrations of the cytokines in brain-injured rats treated with progesterone or allopregnanolone using reverse transcripted polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively.
Section snippets
Subjects and experimental design
Male Sprague–Dawley rats (weighing 300–350 g, approximately 90 days of age) were used (Emory University IACUC protocol #101-99). All animals were maintained in a light-, temperature-, and humidity-controlled environment and were housed individually in suspended stainless steel cages with food and water provided ad libitum.
One hundred and twenty rats were randomly assigned to groups indicated in Table 1. IL-1β and TNF-α gene expression was measured by assessing mRNA levels for these cytokines.
Reduced IL-1β mRNA expression after progesterone or allopregnanolone treatment
Two-way ANOVA revealed significant overall treatment (F5,115 = 11.486, P < 0.05) and post-injury time (F3,115 = 20.585, P < 0.05) effects on the amount of IL-1β mRNA expression. There was also a significant interaction between the treatment effects and the post-injury time points (F15,115 = 5.973, P < 0.05) Fig. 1, Fig. 3.
The subsequent pairwise comparisons on the main effects of each treatment group showed the following: First, there were no differences among the three sham groups
Discussion
Our results demonstrated that progesterone and allopregnanolone transiently attenuate the TBI-induced elevations of proinflammatory cytokines, in particular, IL-1β and TNF-α, at 3 h post-injury despite continued administration of these steroids. TBI resulted in the substantial upregulation of IL-1β and TNF-α mRNA during the acute phases of injury (i.e. 3, 8, and 12 h post-injury). Both the administration of progesterone (at 8 mg/kg) and that of allopregnanolone (at 4 mg/kg) reduced the
Acknowledgements
This research is supported by NIH Grant RO1 NS38664 (D.G.S.), a gift from GeneralCologne Reinsurance Corporation, the Emory University Research Committee (S.W.H.), and the Emory University Graduate School of Arts and Sciences (J.H.).
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