Selective Brain Cooling in Rats Ameliorates Intracerebral Hemorrhage and Edema Caused by Penetrating Brain Injury: Possible Involvement of Heme Oxygenase-1 Expression

Abstract

Brain edema formation associated with trauma-induced intracerebral hemorrhage (ICH) is a clinical complication with high mortality. Studies have shown that heme oxygenase-1 (HO-1) plays an important role in ICH-induced brain edema. In order to understand the role of HO-1 in the protective effect of selective brain cooling (SBC), we investigated the time course of HO-1 changes following penetrating ballistic-like brain injury (PBBI) in rats. Samples were collected from injured and control animals at 6, 24, 48, and 72 h, and 7 days post-injury to evaluate HO-1 expression, heme concentration, brain water content, and immunohistochemistry (IHC). Following a 10% frontal PBBI, HO-1 mRNA and protein was increased at all time points studied, reaching maximum expression levels at 24–48 h post-injury. An increase in the heme concentration and the development of brain edema coincided with the upregulation of HO-1 mRNA and protein during the 7-day post-injury period. SBC significantly decreased PBBI-induced heme concentration, attenuated HO-1 upregulation, and concomitantly reduced brain water content. These results suggest that the neuroprotective effects of SBC may be partially mediated by reducing the heme accumulation, which reduced injury-mediated upregulation of HO-1, and in turn ameliorated edema formation. Collectively, these results suggest a potential value of HO-1 as a diagnostic and/or therapeutic biomarker in hemorrhagic brain injury.

Introduction

B rain edema is a serious, acute complication following brain trauma, causing elevated intracranial pressure (ICP) and potentially triggering a cascade of secondary injuries. In traumatic brain injury (TBI), when intracranial bleeding occurs such as with a penetrating brain injury, multiple mechanisms are involved in the formation of edema, among which hemoglobin and its breakdown products, such as heme, appear to be one of the important mediators (Chang et al., 2005; Huang et al., 2002; Xi et al., 1998). Hemoglobin and its breakdown products have been considered to be potent inducers of heme oxygenase-1 (HO-1) (Fukuda et al., 1995; Huang et al., 2002; Maines, 1988). HO-1 (or HSP 32) is the rate limiting enzyme in the degradation of the pro-oxidant hemin/heme from blood, and it is also a temperature-sensitive protein. HO-1 is barely detectable in the normal brain, but has been shown to be highly inducible by various injurious conditions, including TBI, intracerebral hemorrhage (ICH), heat shock, and oxidative stress (Fukuda et al., 1995; Huang et al., 2002; Maines, 1998; Ryter and Choi, 2002).

Although it is well accepted that HO-1 plays an important role in TBI, its contribution to injury/recovery mechanisms remain controversial. The major issue is whether or not induction of HO-1 reflects a protective response or is detrimental to the traumatized brain. Most observations support that HO-1 induction acts as an important cellular defense mechanism against oxidative stress, cellular injury, and neuronal apoptosis (Ahmad and Zhuang, 2006; Chang et al., 2005; Chen-Roetling et al., 2005; Clark et al., 2000; Fukuda et al., 1995; Ku et al., 2006; Orozco-Ibarra et al., 2009; Panahian et al., 1999). Whereas several other groups using non-selective HO-1 inhibitors found reversal or attenuation of the exacerbated HO-1 to be neuroprotective (Dennery et al., 2003;Huang et al., 2002; Koeppen et al., 2004; Pannizon et al., 1996; Vaya et al., 2007). Furthermore, studies using HO-1 knockout mice have reported detrimental effects of HO-1 expression after injury (Wang and Doré, 2007). Suttner and Dennery (1999) hypothesized that there is a beneficial threshold of HO-1 upregulation following TBI, and it is unlikely that a cytoprotective response is seen if HO-1 is abundantly expressed.

A TBI initiates metabolic processes that may exacerbate the injury (Marion et al., 1997; Sahuquillo et al., 2001; Stelmasiak et al., 2000). There is evidence that hypothermia, by reducing cerebral metabolic rate, edema, inflammatory responses, and apoptosis (Marion et al., 1997; Sahuquillo and Vilalta, 2007), is a promising therapy for TBI, and has the ability to influence the multiple biochemical/metabolic cascades that are set in motion after injury. Historically, hypothermia studies have used methods of systemic cooling to lower brain temperature. Although this method can provide neuroprotection, it may also present serious adverse effects, such as electrolyte imbalance, shivering, infections, and coagulopathy (Kronzon, 1997; Milhaud et al., 2005; Romlin et al., 2007; Tieu et al., 2007). Therefore, in order to make hypothermia a more efficient and practical therapy for TBI, we have developed a novel method of selective brain cooling (SBC), by the extraluminal cooling of bilateral common carotid arteries (CCA). Recently we have determined that SBC significantly reduces PBBI-induced ICH, elevations in ICP, and brain edema (Wei et al, 2010).

The penetrating ballistic-like brain injury (PBBI) model is characterized by significant ICH and swelling (Williams et al., 2005, 2006). Since SBC has been shown to reduce ICH, and HO-1 is a temperature-sensitive protein induced following ICH, the regulation of HO-1 expression may play a key role in the neuroprotective response of SBC. Therefore, the aims of this study were: (1) to further evaluate the effect of SBC therapy on brain edema and hemorrhage; (2) to investigate alterations in HO-1 expression following PBBI with or without SBC; and (3) to evaluate the potential value of HO-1 as a diagnostic biomarker as well as therapeutic target for neuroprotection.

Methods

Male Sprague-Dawley rats (250–300 g; Charles River Labs, Raleigh, VA) were used for this study. Anesthesia was induced by 5% isoflurane and maintained with 2% isoflurane during the surgery. All procedures were approved by the Institutional Animal Care and Use Committee at Walter Reed Army Institute of Research (WRAIR). Research was conducted in compliance with the Animal Welfare Act, the Guide for the Care and Use of Laboratory Animals (National Research Council), and other federal statutes involving animals. Animals were housed individually under a normal 12-h light/dark cycle (lights on at 6:00 am) in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International.

Experimental design

This study was defined by five experiments: (1) brain heme measurement; (2) brain water content measurement; (3) quantitative RT-PCR for measurement of HO-1 mRNA; (4) Western blot analysis of HO-1, and (5) IHC of HO-1. In each experiment animals were separated into two injury groups (PBBI alone and PBBI+SBC), and two control groups (sham alone and sham+SBC). In the injury groups, five time points were studied, 6, 24, 48, and 72 h, and 7 days post-PBBI, except for IHC assay (only at 48 h post-PBBI). Sham controls were designated at 48 h post-surgery. Six animals per group were used in each experiment except for IHC (n=4).

Penetrating ballistic-like brain injury model

The experimental PBBI model mimics the ballistic nature of a high-velocity bullet wound to produce temporary cavitation in the brain and has been previously characterized (Williams et al., 2005, 2006). Briefly, a 10% unilateral frontal PBBI was induced in rats by stereotaxic insertion of a specially designed probe into the right hemisphere of the brain. The probe was inserted through a cranial window over the frontal cortex and rapid inflation/deflation of a water-filled balloon was used to create a temporary cavity in the cerebrum. Sham rats received identical surgical procedures, but the described brain injuries were not induced.

Selective brain cooling procedure

SBC was achieved by circulating cool water through a custom-designed cooling cuff that was sheathed around a segment of each CCA (Wei et al., 2008). Briefly, each CCA was exposed and separated from the vagus nerve in anesthetized rats. Cooling cuffs were placed around a segment of the CCA and secured by a piece of suture. The cuff was connected to a roller pump via Silastic tubing, allowing cold water (withdrawn from an ice water bath) to be pumped through the cuff. SBC was initiated immediately following PBBI (within 1 min), and the brain temperature was reduced by 3°C for 2 h until re-warming was introduced by stopping the cold water circulation to allow the brain temperature to return to the 37°C spontaneously, which took approximately 30 min. Sham-alone animals received craniotomy surgery without PBBI, and received CCA cuff placements without the cold water circulation. Animals in the sham+SBC group received craniotomy surgery without PBBI, and received CCA cuff placements with the cold water circulation. During the entire period of SBC and re-warming, the body temperature was maintained at 37°C using a heating blanket.

Brain and blood collection

Samples of brain tissue and blood (1 mL) were collected at 6, 24, 48, and 72 h, and 7 days post-injury for all experiments except for the IHC assay, which was terminated at 48 h post-injury. All collection procedures were performed on animals anesthetized with 70 mg/kg ketamine and 6 mg/kg xylazine. Blood was obtained by cardiac puncture into 1.5-mL heparin-coated tubes and centrifuged at 2000g for 10 min at 4°C to collect the plasma. After blood collection, brain tissue was collected depending on the experimental design. For water content measurement, the whole brain was collected. For RNA and protein analysis a 2-mm coronal section of brain tissue starting 5 mm from the frontal pole of the brain was dissected and separated from the injured, ipsilateral hemisphere. For heme measurement, at each time point animals were perfused with 0.01 M phosphate-buffered saline (PBS; pH 7.4), and ipsilateral brain tissues were suspended in lysis buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 5 mM EDTA, and 1% protease inhibitor mix; GE Healthcare, Piscataway, NJ) and homogenized. For IHC, animals at 48 h post-injury were perfused with 0.01 M PBS (pH 7.4) followed by 4% paraformaldehyde. The brains were extracted, immersed in 4% paraformaldehyde for 6 h, and then transferred to 0.1 M phosphate buffer containing 20% sucrose (pH 7.4, 4°C), and sent to FD Neurotechnologies (Baltimore, MD) for histopathological and immunostaining procedures.

Heme concentration measurements

The heme content of brains subjected to PBBI was quantified with pyridine reagent (Sigma-Aldrich, St Louis, MO; Sinclair et al., 1999). Brain homogenates (0.84 mL) were mixed with 0.2 mL pyridine and 0.1 mL 1 M NaOH. The concentration of heme was measured at 500–600 nm by a SpectraMax M5 spectrophotometer (MDS Analytical Technologies, Sunnyvale, CA) at 25°C. Heme concentration was calculated based on the millimolar extinction coefficient of 20.7 for the difference in absorption between the peak at 557 nm and the trough at 541 nm.

Brain water content measurement

Brain edema was determined based on brain wet-to-dry weight ratio (Xi et al., 1998). Briefly, the brain was removed from the skull and divided into two hemispheres along the midline. The brain samples were immediately weighed on an electric analytic balance (model AE 100; Mettler Instrument Corp., Hightstown, NJ) to obtain the wet weight (WW), and were then dried at 100°C for 2 days to obtain the dry weight (DW). The percentage of water content was calculated as follows: \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*}{\rm Water \ content} \ (\%) = ({\rm WW} - {\rm DW}) / {\rm WW} \times 100 \% \end{align*} \end{document}

HO-1 mRNA analysis

HO-1 mRNA was analyzed by quantitative real-time RT-PCR. Brain samples were homogenized in TRIzol Reagent (Life Technologies, Gaithersburg, MD). Total RNA was extracted from the tissue according to the manufacturer's suggested protocol (Yao et al, 2002). The total RNA concentration was determined spectrophotometrically (absorbency 260 and 280 nm). The primers and probe for HO-1 were synthesized by AB Applied Biosystems (Foster City, CA).

RT and PCR were carried out using a GeneAmp RNA PCR Core Kit and TaqMan Universal PCR Master Mix kit (Perkin-Elmer, Waltham, MA), according to the manufacturer's specifications. A two-step RT/PCR was performed. The RT reaction used 4 μg total RNA in a total volume of 50 μL containing 1×PCR Buffer II, 5 mmol/L MgCl₂, 1 mmol/L each of dNTP, 2.5 mol/L random hexamers, 1 U/μL RNase inhibitor, and MultiScribe Reverse Transcriptase. The RT reaction was carried out at 42°C for 15 min, then at 99°C for 5 min. Quantitative PCR was performed at 50°C for 2 min, at 95°C for 10 min, and then run for 40 cycles at 95°C for 15 sec, and again at 60°C for 1 min on the ABI PRISM 7000 Detection System (Foster City, CA).

Western blot analysis

Western blot analysis was performed on brain and plasma samples. Protein concentration was determined by using a BCA protein assay kit (Pierce Protein Research Products, Rockford, IL). From each sample, 20 μg protein was separated by 4–20% SDS-polyacrylamide gradient gel electrophoresis and then transferred to an Immobilon-P membrane. The blots were then blocked for 1 h in PBST (1×PBS and 0.1% Tween 20) containing 5% nonfat dry milk, and incubated overnight at 4°C with primary antibodies against HO-1 (Assay Designs, Ann Arbor, MI), and anti-β-actin (Sigma-Aldrich) (as the internal control) in PBST containing 3% nonfat milk. The blots were washed four times in PBST and incubated for 1 h with horseradish peroxidase-conjugated secondary antibody in PBST containing 3% nonfat dry milk. Immunoreactivity of the protein bands was detected by an enhanced chemiluminescence autoradiography (ECL kit; Amersham Pharmacia Biotech, Piscataway, NJ) per the manufacturer's instructions.

Immunohistochemistry

Immunohistochemistry was performed on brain samples collected at 48 h post-injury using methods similar to those previously described (Yao et al., 2009). Briefly, 40-μm cryostat sections were cut coronally through the cerebral cortex containing the striatum from approximately 3.7 to −8.0 mm relative to the bregma. Every first and second section of each series of 12 sections was separately mounted on a Superfrost Plus microscope slide. The first series of sections were stained with hematoxylin and eosin (H&E), and the rest of the series was processed for HO-1 IHC, or double-labeling of HO-1 and OX-42 (activated microglia marker) to verify that immunoreactivity of HO-1 occurred in microglia cells (FD NeuroTechnologies, Inc., Catonsville, MD). For the double-labeling, the separated optical images of the HO-1 and OX-42 immunoreactivity were captured from the same sections and were pseudo-colored red (HO-1) or green (OX-42). A digital overlay was generated by superimposing companion images such that areas of co-localization appeared in yellow double staining.

Data analysis

Values are presented as mean±standard error of the mean (SEM). Statistical comparisons were performed using analysis of variance (ANOVA), followed by post-hoc t-test analysis of evaluation between groups. Values were considered significant at p<0.05.

Results

SBC reduces heme accumulation and alleviates brain edema produced by PBBI

As shown in Figure 1, the heme concentrations in the injury-alone group were increased between two- and threefold from 6 h to 7 days after injury compared to sham controls. Heme concentrations in the PBBI+SBC group were significantly lower during the first 48 h post-injury compared to PBBI alone.

FIG. 1.

Heme concentration. Values are mean±standard error of the mean (n=6/group; **p<0.05 at each time point compared with sham controls; *p<0.05 for comparison between PBBI alone and PBBI+SBC; PBBI, penetrating ballistic-like brain injury; SBC, selective brain cooling).

As shown in Figure 2, following PBBI the brain water content was significantly increased in both the PBBI alone and PBBI+SBC groups from 6 h to 7 days post-injury at each time point compared to the sham groups. However, the percent brain water content in the PBBI+SBC groups was significantly lower at 24 h (79.85±0.55% versus 81.02±0.56%), and 48 h (80.31±0.39% versus 81.56±0.38%) post-injury compared to PBBI alone (p<0.05). The water content for the sham alone and sham+SBC groups showed no difference, and compared to sham controls the water content in the contralateral hemisphere in all injured animals was unchanged (data not shown).

FIG. 2.

Percent brain water content. Values are mean±standard error of the mean (n=6/group; **p<0.05 for comparison between injured hemisphere and sham animals; *p<0.05 for comparison between PBBI alone and PBBI+SBC; PBBI, penetrating ballistic-like brain injury; SBC, selective brain cooling).

SBC reverses overexpression of HO-1 in rat brain and plasma

Figure 3 shows that HO-1 mRNA was increased three- to fourfold from 6 h to 7 days after PBBI compared to sham controls, reaching peak levels at 24 h. However, SBC attenuated the PBBI induced upregulation of HO-1 by 23–45% at all time intervals studied, without affecting HO-1 gene expression in the sham alone and sham+SBC controls.

FIG. 3.

Time course of heme oxygenase-1 (HO-1) gene expression following penetrating ballistic-like brain injury (PBBI) with or without selective brain cooling (SBC). Values are mean±standard error of the mean (n=6/group; *p<0.05 for comparison of PBBI alone versus PBBI+SBC).

The alteration of HO-1 in both brain (Fig. 4A) and plasma (Fig. 4B) showed a protein expression pattern similar to the gene expression at all time points studied. Quantitative measurement of Western blot density indicated that HO-1 was significantly upregulated from 6 h to 7 days post-PBBI, reaching peak levels at 48 h in both brain and in plasma (approximately a fourfold increase) compared to their respective shams (Fig. 4B). SBC attenuated the upregulation of HO-1 protein significantly at 6, 24, 48, and 72 h post-PBBI in both brain and plasma. No significant differences in HO-1 proteins were detected at 7 days post PBBI between the SBC and normothermic groups. Similarly SBC did not affect HO-1 in brain and plasma samples from sham groups.

FIG. 4.

Western blot (WB) analysis of heme oxygenase-1 (HO-1) protein levels and quantitative measurement of HO-1 WB optical density in brain (A) and in plasma (B) n=6/group at 6, 24, 48, and 72 h and 7 days post-PBBI with or without selective brain cooling (SBC). Values are mean±standard error of the mean (**p<0.05 for comparison between injured hemisphere and sham animals; *p<0.05 for comparison between PBBI alone and PBBI+SBC; S, sham alone; S-C, sham+SBC; P, PBBI alone; P-C, PBBI+SBC; PBBI, penetrating ballistic-like brain injury).

To examine the localization of HO-1 expression in different cell types, IHC was performed in the brain at 48 h post-PBBI. As shown in Figure 5 (panel A; H&E staining) ipsilateral hemispheres were enlarged with an obvious midline shift following PBBI, and hemorrhage and necrosis were observed surrounding the PBBI probe track in the injured hemispheres. The distribution of the HO-1-immunostained cells was adjacent to the site of injury, and somewhat correlated with the pattern of ICH (panel A, HO-1). A reduction in hemorrhage and HO-1-immunoreactive signal in the lesion region were observed in the SBC-treated group compared to the untreated normothermic group (Fig. 5A). Results of double-labeling indicated that the HO-1 staining was predominant in activated microglia populations that also exhibited OX-42 staining. The microglia HO-1 signal was decreased in SBC brain sections compared to sections from untreated animals (Fig. 5B).

FIG. 5.

(A) Representative coronal sections of rat brains at 48 h following penetrating ballistic-like brain injury (PBBI). Purple-colored slides indicate hematoxylin and eosin (H&E) staining, and light yellow slides indicate heme oxygenase-1 (HO-1) by immunohistochemistry (IHC; scale bar=1.0 mm). (B). Double-immunofluorescence labeling with HO-1 and OX-42 from the primary motor cortex region at 48 h following PBBI (HO-1-labeled cells=red; OX-42-labeled cells=green; HO-1 plus OX-42 labeling=yellow; scale bar=50 μm; SBC, selective brain cooling).

Discussion

In the present study we demonstrated (1) changes in intracerebral hemorrhage and brain edema in the PBBI model as measured by changes in heme concentration and brain water content, (2) significant upregulation of HO-1 mRNA and protein in the brain and plasma of PBBI animals, (3) that SBC alleviated brain edema, probably by reducing heme accumulation and reversing the upregulation of HO-1 expression post-PBBI, and (4) the possibility of using HO-1 plasma levels as a diagnostic biomarker of hemorrhagic TBI.

Brain edema is a serious acute complication following brain trauma. Although there are multiple mechanisms involved in the induction of perihematomal edema formation, hemoglobin appears to be one of the important mediators (Fukuda et al., 1995; Wu et al., 2006). Hemoglobin is a putative pro-oxidant due to the iron content of its heme groups, and has been shown to exacerbate excitotoxicity in cortical cell cultures and produce widespread concentration-dependent neuronal death in cortical cell cultures (Regan and Panter, 1996). Heme is generated as a breakdown product of hemoglobin and other heme proteins resulting from traumatic subarachnoid hemorrhages, intraparenchymal contusions, or hematomas (Chang et al., 2005). The accumulation of hemorrhaged blood is highly toxic to the brain because it leads to high levels of heme metabolites that can cause oxidative stress, excitoxocity, and inflammation, and induce secondary brain edema formation and cause cell death (Clark et al., 2000, 2008; Dennery et al., 2003; Gong et al., 2004; Huang et al., 2002; Huffman et al., 2000, Ostrow et al., 2003; Regan and Panter, 1996; Thiex and Tsirka, 2007; Xi et al., 1998). Our previous studies have demonstrated the presence of ICH and brain edema associated with experimental PBBI (Williams et al., 2005, 2006). As such the results of the current study also confirmed similar pathological changes and support our hypothesis that overexpression of HO-1 may have a role in this PBBI-induced pathophysiology.

One of the most widely studied mechanisms of heme toxicity is focused on its degradation, which is catalyzed by HO-1, resulting in the release of free iron, biliverdin, and carbon monoxide (Fukuda et al., 1995; Huang et al., 2002; Maines, 1998). The functions of these heme by-products appear to be disparate with regard to their pro- (iron) and/or anti- (carbon monoxide and biliverdin) oxidative properties (Elbrit and Bonkovsky 1999; Frankel et al., 2000, Schipper, 2004; Suttner and Dennery, 1999; Matsumoto et al., 2006). This disparity may be partially responsible for the putative paradox of HO-1 for its protective and toxic functions observed under different types of injuries. Numerous studies have confirmed that HO-1 is markedly upregulated in different types of TBI, and for the most part this upregulation is beneficial (Anderson et al., 2009; Cousar et al., 2006; Fukuda et al., 1995; Koeppen et al., 2004; Song et al., 2007; Wang and Doré, 2007). For example, HO-1 protects astrocytes from oxidative toxicity caused by hemoglobin (Regan et al., 2002), and deficiency of HO-1 in humans and in HO-1-knockout mice leads to iron deposition and vulnerability to oxidative stress and inflammation (Koeppen et al., 2004; True et al., 2007). Another interesting finding from our study was that HO-1 expression was detected primarily in activated microglial cells, and SBC also decreased the enhanced HO-1 immunoreactivity in these cells. Activated microglial cells have been considered as major sources of inflammatory responses after brain injury, and have also been implicated in the worsening of ICH (Wang and Doré, 2007). These findings, along with our present results, suggest that HO-1 upregulation in hemorrhagic brain injury may be important in heme degradation, and thus in the regulation of brain edema.

Hypothermia is clinically designed to combat the deleterious metabolic responses that may exacerbate an injury (Marion et al., 1997). Numerous pre-clinical studies support the concept that hypothermia applied after brain injury reduces cerebral edema, mortality, and cerebrospinal fluid pressure (Barone et al., 1997; Chatzipanteli et al., 2000; Ehrlich et al., 2002). Recently, it has been reported that SBC is a safe and effective therapy for experimental brain injury, in part by reducing intracranial pressure, edema, and inflammation, with fewer systemic adverse effects (Sarkar et al., 2009; Wei et al., 2008, 2010). The optimal benefits of hypothermia therapy can be impacted by the cooling and re-warming conditions. Normally, fast post-hypothermic re-warming is considered problematic because it may exacerbate mitochondrial pathology and accelerate various radical-mediated processes (Povlishock and Wei, 2009). Our method of spontaneous re-warming allowed restoration to 37°C of the brain temperature within 30 min after the termination of the cold water circulation, and in previous studies it has been proven to be benign (Wei et al., 2008, 2010). The safety of our re-warming method is probably attributable to the fact that it was spontaneous, directly affecting the brain without systemic complications. However, the precise mechanisms by which SBC confers therapeutic benefits remain unclear. The mechanism of protection underlying hypothermia treatments is strongly correlated to downregulation of cellular and tissue metabolism (Yenari et al., 2008). In the present study, SBC lowered PBBI-induced increases in heme level, brain water content, mRNA, and protein levels of HO-1, in a time-related manner compared to PBBI-normothermic groups, which is suggestive of a neuroprotective effect of SBC in TBI. It is interesting to note that although SBC lowered PBBI-induced increases in heme concentration and brain water content only up to the first 48 h following injury, HO-1 mRNA and protein levels were significantly attenuated up to 72 h post-injury. These differences may be related to either the differential effects of SBC on HO-1 gene transcription and heme/water content, or to the limited duration of the SBC therapy used herein (only 2 h post-injury), thereby producing a transient early beneficial effect on heme and water content that lasts only up to 48 h post-injury. Studies are ongoing in our laboratory to investigate if extending the duration of SBC therapy could prolong the duration of protection. Although direct evidence that SBC inhibited heme accumulation and negated HO-1 expression was not readily apparent in this study without specific manipulations of heme or HO-1, our results are supported by similar findings showing that hypothermia significantly decreases post-traumatic elevation of hemoglobin concentrations after fluid percussion injury in rats (Kinoshita et al., 2002), and attenuates brain edema and oxidative/nitrosative stress by limiting upregulation of HO-1 and inducible nitric oxide synthase (iNOS) in the rat brain after acute liver failure (Jiang et al., 2009). The results of the current study demonstrating that injury-induced upregulation of HO-1 was attenuated, and brain hemorrhage/edema were alleviated by SBC, support our hypothesis that SBC is an effective neuroprotective therapy, and the alteration of the HO-1 expression level closely relates to TBI-induced hemorrhage and brain edema.

As mentioned earlier, the role that HO-1 plays in TBI and other pathophysiological conditions remains disputable. The major conflict is whether or not induction of HO-1 reflects a protective response or may indeed be detrimental to TBI. Based on our findings, we consider that in the PBBI model overexpression of HO-1 is detrimental to cell survival. We hypothesize that SBC provides neuroprotection not only by reducing the general metabolic state of the brain post-injury, but also by targeting several destructive mechanisms inclusive of the upregulation of HO-1, leading to a decrease in heme accumulation, a reduction of microglial activation, and subsequent alleviation of the brain edema. Clearly, further investigations are needed to confirm these observations. Finally, regardless of whether the overexpression of HO-1 in the brain augments or attenuates cellular injury, our demonstration that HO-1 expression in plasma mirrors the brain expression (including its attenuation following SBC), suggests that HO-1 may be predictive of ongoing metabolic compromise, and therefore has potential value as a diagnostic and therapeutic biomarker of TBI and/or TBI treatment.

Footnotes

Acknowledgments

The authors thank MAJ Kara Schmid for her review and comments on the manuscript, and Mr. Zhilin Liao for his excellent technical assistance in these studies.

Author Disclosure Statement

This material has been reviewed by the Walter Reed Army Institute of Research. There is no objection to its presentation and/or publication. The opinions or assertions contained herein are the private views of the authors, and are not to be construed as official, or as reflecting true views of the Department of the Army or the Department of Defense.

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