Time course of upregulation of inflammatory mediators in the hemorrhagic brain in rats: Correlation with brain edema

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Abstract

Intracerebral hemorrhage (ICH) can cause secondary brain damage through inflammation-related pathways. Thrombin and one of its receptors, protease activated receptor-1 (PAR-1); matrix metalloproteinase (MMP)-9; and aquaporin (AQP)-4 are stroke-related inflammatory mediators that have been implicated in ICH pathology. To further characterize the inflammatory response after ICH, we studied the temporal profile of the expression of these inflammatory mediators and assessed their potential correlation with brain edema formation after brain hemorrhage in rats. ICH was modeled by infusing autologous blood into the striatum. Then mRNA and protein expression was assessed over the course of 5 days. We found that the mRNA and/or protein expression of thrombin, PAR-1, AQP-4, and MMP-9 was upregulated between 2 h and 5 days after ICH. Each reached a maximal level at day 2, except for AQP-4 protein, which peaked at day 5. Brain water content after ICH presented a similar trend; it was increased at 2 h, peaked at day 2, and then decreased but remained elevated at day 5. Our data provide novel evidence that upregulation of these selected inflammatory mediators occurs very early and persists for several days after ICH, and that temporal patterns of expression of thrombin and AQP-4 are associated with brain edema formation. These findings have important implications for efforts to reduce secondary brain damage after ICH.

Introduction

Intracerebral hemorrhage (ICH) is a common and often devastating stroke subtype that frequently causes brain edema. Edema leads to an expansion of brain volume that has a severe negative impact on ICH outcomes. Inflammation has been implicated in ICH-induced brain edema formation (Wang and Doré, 2007b, Xi et al., 2006). Thrombin; one of its receptors, protease activated receptor-1 (PAR-1); matrix metalloproteinase (MMP)-9; and aquaporin (AQP)-4 are important stroke-related inflammatory mediators that might contribute to or modulate edema formation after ICH [reviewed in (Wang and Doré, 2007b, Wang and Tsirka, 2005a, Xi et al., 2003, Zador et al., 2009)].

Thrombin is released from blood clots, and through activation of PAR-1, stimulates astrocytes and microglia (Moller et al., 2000, Noorbakhsh et al., 2003) and causes neuronal death and brain edema (Lee et al., 1997, Xue and Del Bigio, 2001, Xue and Del Bigio, 2005). Inhibition of thrombin activity reduces ICH-induced brain edema and neuronal cell death (Kitaoka et al., 2002, Xue and Del Bigio, 2005, Xue et al., 2009b). Neurotoxicity of thrombin was shown to be partially blocked by a PAR-1 antagonist (Xue et al., 2006). We have observed an increase in thrombin expression in neurons and cerebral vasculature in human brain tissue after ICH (Wu et al., 2008). Upregulation of PAR-1 protein expression has been reported in rats only in a collagenase-induced model of ICH (Zheng et al., 2009).

In addition to thrombin, other proteases, particularly MMP-9, have been suggested to be involved in ICH-induced secondary brain injury and edema formation [reviewed in (Wang and Doré, 2007b)]. Increased MMP-9 activity detected by gel zymography has been observed in rodent and human brains after ICH (Rosell et al., 2006, Rosenberg and Navratil, 1997, Tejima et al., 2007, Wang and Tsirka, 2005b, Xue et al., 2009b). Inhibition of MMP-9 activity was able to reduce ICH-induced early brain injury, edema formation, and neurologic deficits (Rosenberg and Navratil, 1997, Wang and Tsirka, 2005b, Xue et al., 2009b). Furthermore, MMP-9 was reported to act synergistically with thrombin to exacerbate ICH injury (Xue et al., 2009a).

AQPs, which belong to a family of water channel proteins, help to regulate brain water balance. AQP-4 is the most abundant isoform in the brain (Zador et al., 2009). It has been reported that AQP-4 expression is upregulated in astrocytes after ICH in rodents (Qing et al., 2009) and humans (Wu et al., 2008).

Interestingly, although thrombin, PAR-1, MMP-9, and AQP-4 have been implicated in ICH pathology, a time course of their expression, particularly within the first day after ICH, has not been established. To further our understanding of the role of inflammation in ICH pathology, we characterized the temporal profile of the expression of thrombin, PAR-1, MMP-9, and AQP-4 from 2 h to 5 days after ICH and compared their expression patterns with brain edema formation in the autologous whole-blood model in rats. The advantage of this model is that only blood is introduced into the striatum of the rat brain.

Section snippets

Animals and ICH model

All experimental protocols and procedures conformed to the guidelines of the Chinese Council for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at the Harbin Medical University, China. Adequate measures were taken to ensure minimal pain or discomfort to rats. A total of 440 adult male Sprague–Dawley rats (200–250 g) were purchased from the Center for Experimental Animals, Harbin Medical University, China. Rats were allowed free access

Histopathologic findings

The blood model in rats produces hematoma mostly restricted to the caudate nucleus. We observed a few scattered neutrophils in the perihematomal area at 3 h. Tissue necrosis was not evident at this time. Brain swelling became visible at 24 h, with increased numbers of inflammatory cells that included microglia, astrocytes, and neutrophils. Brain swelling and tissue necrosis were more evident at 48 h. The hematoma started to dissolve with glial cell proliferation and new vessel formation at day 5

Discussion

To our knowledge, this is the first systematic study performed to characterize the temporal profile of the expression of thrombin, PAR-1, MMP-9, and AQP-4 at the translational and/or transcriptional levels in the hemorrhagic brains of rats and to assess their potential correlation with brain edema formation. The target mRNA and/or protein expression was upregulated between 2 h and 5 days after ICH; the peak level for each was observed at 2 days, except for that of AQP-4 protein, which reached

Acknowledgments

This work was supported by the Natural Science Foundation of Heilongjiang Province ZJY0705 and the Foundation of the First Clinical Hospital of Harbin Medical University Y08-009 (HW) and by AHA 09BGIA2080137 and NIH K01AG031926 (JW). We thank Claire Levine for assistance with this manuscript.

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    These authors contributed equally to this work.

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