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N-acetylcysteine attenuates early induction of heme oxygenase-1 following traumatic brain injury
Brain Research 1033 (2005) 13 – 19 www.elsevier.com/locate/brainres
Research report
N-acetylcysteine attenuates early induction of heme oxygenase-1 following traumatic brain injury Jae-Hyuk Yi, Alan S. Hazell* Department of Medicine, Hoˆpital Saint-Luc (CHUM), University of Montreal, 1058 St-Denis St., Montreal, Quebec Canada H2X 3J4 Accepted 30 October 2004 Available online 7 January 2005
Abstract Traumatic brain injury (TBI) results in a cascade of events that includes the production of reactive oxygen species. Heme oxygenase-1 (HO1) is induced in glial cells following head trauma, suggestive of oxidative stress. We have studied the temporal and spatial effects of the antioxidant N-acetylcysteine (NAC) on HO-1 levels following lateral fluid-percussion injury by immunoblotting and immunohistochemistry. In the injured cerebral cortex, maximal HO-1 induction was seen 6 h post-TBI and was maintained for up to 24 h following the insult, while the ipsilateral hippocampus and thalamus showed marked induction at 24 h postinjury. In all three brain regions, little or no HO-1 immunoreactivity was observed on the contralateral side. Astrocytes exhibited positive immunoreactivity for HO-1 in the injured cerebral cortex, hippocampus, and thalamus, while some neurons and microglia were also immunoreactive in the injured cortex. The administration of NAC 5 min following TBI resulted in a marked reduction in this widespread induction of HO-1, concomitant with a decrease in the volume of injury in all three brain regions. Together, these findings indicate that HO-1 induction is related to both oxidative and injury characteristics of the affected tissue, suggesting that protein expression of this gene is a credible marker of oxidative damage in this model of TBI. D 2004 Elsevier B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Trauma Keywords: Heme oxygenase; Traumatic brain injury; Astrocyte; Oxidative stress; Fluid percussion; Antioxidant
1. Introduction Heme oxygenase-1 (HO-1, HSP32) is an inducible form of the heme oxygenases (HO-1, HO-2, and HO-3) that metabolizes heme molecules to carbon monoxide, iron, and biliverdin. While HO-1 is induced in response to numerous stimuli [27], HO-2 is constitutively expressed in most neurons in the brain [26], with HO-3 being highly expressed in the hippocampus and cerebellum [20]. In traumatic brain injury (TBI), HO-1 is induced in the glial cells of human brain as early as 6 h, and up to 6 months following the injury [4]. In rats, however, TBI has been reported to result in transient
* Corresponding author. Fax: +1 514 412 7314. E-mail address: [email protected] (A.S. Hazell). 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.10.055
HO-1 induction in astrocytes from 24 h postinjury, with a noticeable reduction of staining at 5 days postinjury [6]. Thus far, the precise mechanism(s) responsible for HO-1 induction in TBI remains unclear. N-acetylcysteine (NAC) is a cysteine analog commonly used to treat acetaminophen overdose [10]. While the precise beneficial mechanisms provided by NAC still require further elucidation, NAC can protect against reactive oxygen species through the restoration of intracellular glutathione [9,18]. In TBI, NAC has been effective in restoring cerebrovascular responsiveness to hyperventilation following fluid-percussion injury [5] and protection against mitochondrial dysfunction in cortical impact injury [28], both of which may greatly reduce the degree of oxidative stress resulting from the insult. In this study, the regional effects of NAC on HO-1 induction were studied using immunoblotting and immunohistochem-
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istry in a lateral fluid-percussion model of TBI of moderate severity.
2. Materials and methods 2.1. Trauma model All procedures were undertaken with the approval of the Animal Ethics committee of Hoˆpital Saint-Luc and the University of Montreal and were conducted in accordance with the guidelines set out by the Canadian Council on Animal Care. A total of 64 male Sprague–Dawley rats (350– 400 g) was housed under constant conditions of temperature, humidity, and 12:12 h day/night cycle. The lateral (parasagittal) model of traumatic brain injury used in this study has previously been described in detail [13]. A 4.8-mm-diameter craniectomy, centered between the bregma and lambda and 2.5 mm lateral to the superior sagittal sinus, was made over the left hemisphere using a trephine drill, and a hollow female Luer-Lok fitting secured with dental cement. Rats were subsequently allowed to recover overnight and, the following day, reanesthetized with 2% isofluorane and exposed to lateral fluid-percussion injury of moderate severity (2.0–2.5 atm). Animals were studied at 6 (n = 6) and 24 h (n = 6) postTBI. Sham controls (n = 5) received identical treatment but without exposure to trauma. For the administration of NAC, rats received an injection of either drug (163 mg/kg, i.p.) or equal volume of saline 5 min following TBI. 2.2. Western blotting At the appropriate time, brains were removed and rapidly frozen in isopentane on dry ice, sectioned into appropriate regions corresponding to the injured cerebral cortex, hippocampus, and thalamus, and homogenized in ice-cold TE buffer with phenylmethylsulfonyl fluoride using a Teflon glass homogenizer on ice. Resulting homogenates were centrifuged for 30 min at 15,000 g, 4 8C. Pellets were washed with TE buffer, recentrifuged, the supernatant discarded, and solubilized in RIPA buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate, pH 8.0, and a protease inhibitor cocktail). Protein content was determined according to Lowry et al. [12]. Immunoblotting assays were performed as previously described [8]. Fifty micrograms of tissue was loaded on to 12% SDS polyacrylamide gel electrophoresis, separated overnight at 40 V, and transferred to PVDF membranes at 20 V constant volt overnight at 4 8C. Membranes were subsequently incubated in a blocking solution consisting of 5% dry nonfat milk in TTBS (10 mM Tris, 100 mM NaCl, and 0.1% Tween-20) and then probed with antisera against HO-1 (Stressgen Biotechnologies, Victoria, BC, Canada; 1:5,000) or h-actin (Sigma, St. Louis, MO, USA; 1:10,000) in blocking solution for 1 h, washed three times in TTBS, and then reblocked for 1 h. After further washes in TTBS,
membranes were incubated in HRP-coupled donkey antimouse or antirabbit secondary antiserum (1:10,000) in blocking solution for 1 h. Membranes were subsequently treated with enhanced chemiluminescence kit reagents (New England Nuclear, Boston, MA, USA), according to the manufacturer’s instructions, and exposed to Kodak X-OMAT film, followed by semiquantitation of the immunoband intensities using a computerized image analysis system (MCID, Imaging Research, London, ON, Canada). 2.3. Immunohistochemistry For immunohistochemistry and histology, rats (sham group, n = 2; 6 h post-TBI group, n = 3; 24 post-TBI group, n = 3; saline treated n = 8; NAC treated n = 8) were deeply anesthetized with pentobarbital (60 mg/kg) and perfused transcardially as described previously [8]. Briefly, brains were removed and postfixed overnight in 10% neutralbuffered formalin containing 4% formaldehyde, 0.5% sodium phosphate buffer, and 1.5% methanol, pH 7.0. Coronal sections (3.8 to 5.8 mm relative to the bregma) of 40-Am thickness were cut using a vibrotome based on the rat brain atlas of Paxinos and Watson [16]. Sections were used for HO-1 immunohistochemistry or stained with cresyl violet according to Hazell et al. [8]. Briefly, sections were incubated for 10 min in phosphate-buffered saline (PBS) containing 0.3% hydrogen peroxide to block endogenous peroxidase activity. Tissue sections were then washed in PBS (3 10 min) and blocked for 20 min in PBS containing 0.5% Triton X-100, 5% donkey serum, and 1% bovine serum albumin, followed by incubation of 24 h at 4 8C in a blocking solution with HO-1 antirabbit primary antibody (1:5000, Stressgen Biotechnologies). The following day, the sections were washed again in PBS (3 10 min) and incubated for 1 h with biotinylated donkey antirabbit IgG (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA) in PBS with 0.5% Triton X-100, followed by washing in PBS (3 10 min) and incubation for 1 h in streptavidin-HRP conjugate (1:100; Sigma) in PBS with 0.5% Triton X-100. The sections were then washed in PBS (310 min), followed by incubation with diaminobenzidine (0.05%) in PBS with 25 mg/ml nickel ammonium sulfate, for signal enhancement, and in the presence of H2O2 (0.03%) for 2–10 min to allow color development. Following subsequent washings in PBS, the sections were mounted on Superfrost Plus slides (Fisher Scientific, Ottawa, ON, Canada), dehydrated in ethanol (75%, 95%, and 100%), cleared in xylene, and coverslipped with Permount. Controls for the specificity of immunostaining consisted of the omission of the primary or secondary antibody, resulting in a subsequent loss of immunoreactivity. 2.4. 2,3,5-Triphenyltetrazolium chloride (TTC) staining Twenty four hours after TBI, rats (saline-treated group, n = 7; NAC-treated group, n = 7) were injected with sodium
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the saline + TBI and NAC + TBI groups was performed using the Mann–Whitney test.
3. Results 3.1. Regional changes in HO-1 levels with or without NAC treatment
Fig. 1. Representative bands for HO-1 induction in three different regions of the brain following TBI, with or without NAC administration. (A) HO-1 induction peaked as early as 6 h following the injury, and the level was sustained up to 24 h posttrauma in the cerebral cortex (Cx). In the hippocampus (Hc) and thalamus (Th), little or no expression was detected at 6 h. (B) HO-1 induction is attenuated following NAC administration (163 mg/kg, i.p.) in the three regions. A total of 34 rats was used (sham group, n = 5; TBI groups, n = 6; FNAC).
A rapid induction of HO-1 was detected by Western blotting following fluid-percussion injury of moderate severity (Fig. 1A). Little or no evidence of multiple bands was observed with this antibody. In the injured cerebral cortex, HO-1 induction was observed as early as 6 h
pentobarbital (60 mg/kg, i.p) and brains were quickly removed, transferred into Falcon tubes containing ice-cold saline solution, and stored at 4 8C for 10 min. A series of 2mm coronal sections were cut on a prechilled Rodent Brain Matrix (ASI instruments, MI, USA) and were rapidly transferred to a solution of 2% TTC (Sigma) in saline prewarmed to 37 8C in a water bath. Five sections from each brain were incubated for 15 min, then transferred to 10% neutral buffered formalin solution, and kept at 4 8C until scanned and processed for the determination of volume of injury (VOI). 2.5. Volume of injury Sections from each brain were scanned and saved as TIFF files. These files were processed using AdobeR PhotoshopR for the determination of the injury area. Brain lesion areas were determined in pixels and converted into mm2 (relative to a scale bar size of 0.01 mm scanned on the same image and a square area using this length), multiplied by the thickness of the section (2 mm), and then summed for the calculation of total VOI (mm3). For the consideration of brain edema, both ipsi- and contralateral sides of the brain were imaged, the relative swelling considered as previously described [2], and the final total VOI calculated using the following formula: Corrected injury volume ¼ contralateral hemisphere volume ðipsilateral hemisphere volume measured injury volumeÞ:
2.6. Statistical analysis Statistical analysis of immunoblotting data was performed using unpaired t test. Comparison of VOI between
Fig. 2. Quantitative immunoblot analysis of HO-1 expression following TBI alone or TBI with NAC treatment in the injured (A) cerebral cortex (Cx), (B) hippocampus (Hc), and (C) thalamus (Th). Asterisks indicate significant difference between TBI alone and TBI with NAC when same time point groups were compared (unpaired t test, p b 0.05).
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following TBI, and this level was sustained up to 24 h. In the injured hippocampus and thalamus, HO-1 induction occurred at 24 h, with little or no induction at 6 h post-TBI. These temporal and quantitative differences in HO-1 induction following TBI in the three regions were substantially modified following treatment with NAC, 5 min following the insult: in the injured cerebral cortex at 6 h, and in the hippocampus and thalamus regions at 24 h (Fig. 1B). Following NAC treatment, HO-1 induction in the injured cerebral cortex following NAC treatment was reduced to 16% of the value in the TBI alone group after 6 h (Fig. 2A). On the other hand, no significant effect of NAC on HO-1 levels was observed at 24 h post-TBI. In the hippocampus and thalamus, HO-1 levels remained almost undetectable at 6 h following NAC administration (Fig. 2B, C). After 24 h, however, HO-1 induction in the two brain regions was reduced to 37% and 46% of the values obtained with TBI alone, respectively.
induction in the ipsilateral (but not contralateral) Layer I of the cerebral cortex that was astrocytic in nature (Fig. 3A, inset). No obvious HO-1 immunoreactivity was observed in the hippocampus and thalamus of these control animals (Fig. 3D, G), with the exception of neurons in the dentate gyrus. TBI resulted in an early increase in HO-1 immunoreactivity in the injured cortex at 6 h postinjury (Fig. 3B). In the core of the injury, some microglia-like cells and neurons exhibited HO-1 immunoreactivity (Fig. 4A, black arrowhead), as well as astrocytes (Fig. 4A, white arrowhead). In other areas of the cortex, HO-1 immunoreactivity was localized predominantly to astrocytes. At 24 h post-TBI, immunostaining was also detected in the astrocytes of the ipsilateral hippocampus and thalamus (Fig. 3E, inset, and F) and in the cerebral cortex. Treatment with NAC led to a reduction in HO-1 immunoreactivity in all three regions (Fig. 4). 3.3. Effect of NAC on volume of injury
3.2. Regional and cell-type HO-1 profile Figs. 3 and 4 show the profile of HO-1 immunostaining. Sham treatment alone triggered some localized HO-1
The administration of NAC resulted in a reduction in the VOI (Mann–Whitney test, p = 0.0111) when the VOI of saline + TBI group was compared with that of the NAC +
Fig. 3. HO-1 immunoreactivity in the injured cerebral cortex (A–C), precedes hippocampus (D– F), and thalamus (G–I) following TBI. Panels A, D, and G: Sham. Panels B, E, and H: 6 h post-TBI. Panels C, F, and I: 24 h post-TBI. In Panel A, arrow points to cortex layer 1 where sham injury resulted in HO-1 induction (inset), but not in the deeper layers of the cortex. In Panel D, arrow points to neuronal staining in the dentate gyrus area of the sham brain. Abbreviations used: SRH, stratum radiatum of hippocampus; HF, hippocampal fissure; VPM, ventroposteriomedial thalamic nuclei. Bars represent 100 (low power) and 25 Am (high power inset).
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Fig. 4. Trauma-induced HO-1 immunoreactivity is substantially decreased in NAC-treated rats (B, D, F) compared with saline-treated rats (A, C, E). Panels A and B: injured cortex, 6 h post-TBI. Panels C and D: stratum radiatum of the hippocampus 24 h post-TBI. Panels E and F: ventroposteriomedial thalamus 24 h post-TBI. In Panel A, the box indicates the area depicted in the inset showing the glial staining of HO-1 (inset, white arrowhead), as well as the neuronal staining (inset, black arrowhead), in the injury core. Note the reduction in positively stained glial processes in Panels B, D, and F compared with Panels A, C, and E, respectively. A total of 16 rats was used (sham group, n = 2; TBI groups, n = 3). Bars represent 50 (low power) and 25 Am (high power inset).
Fig. 5. Comparison of volume of injury at 24 h post-TBI in animals treated with saline or NAC (163 mg/kg, i.p., 5 min post-TBI) from TTC staining. (A) Representative coronal TTC-stained sections of the saline- and NAC-treated TBI brains. (B) A significant difference was found in injury volumes between the two groups using the Mann–Whitney test ( p b 0.05). The values were 34.3 and 18.9 mm3 for saline- (n = 7) and NAC-treated (n = 7) TBI brains, respectively.
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TBI group (Fig. 5). The average VOI was 34.4 mm3 for the saline + TBI group, and 18.9 mm3 for the NAC + TBI group.
4. Discussion Pathophysiological changes in TBI often involve hemorrhage, with release of blood into the extracellular space that may partly account for the effects of secondary injury. This is particularly evident in the injured cortex, in which there is the development of a contusion, a consistent finding in this fluidpercussion model [13]. Hemorrhage associated with this contusion can induce focal vasospasm and cerebral ischemia, contributing to the injury process. Such a hemorrhage can also lead to neurotoxicity by producing a slow development of oxidative stress [19]. The present study demonstrates the regional and temporal induction of HO-1 protein and a beneficial effect of NAC administration following TBI. In two recent studies involving subarachnoid hemorrhage, HO1 induction was reported to occur [23,24], with antioxidant treatment having a preventative effect [24]. Our findings are consistent with the effects of NAC treatment in those studies. When TBI rats were administered NAC 5 min following injury, the induction of HO-1 was suppressed in all three brain regions examined. Previous studies have demonstrated that NAC treatment results in a rapid recovery of levels of reduced glutathione in brain and a reduction of oxidative stress following TBI [9,28]. The consequence of such a depletion may play an important role in the injury process. Thus, NAC may protect against neuronal death by improving the antioxidant status of the tissue. On the other hand, several reports indicate that HO-1 itself has a cytoprotective role to play due to its ability to break down heme (a prooxidant) to biliverdin and bilirubin, both of which are powerful antioxidants. This has been demonstrated under both in vitro [1,25] and in vivo conditions [14,15]. The suppression of HO-1 induction by NAC treatment would therefore seem inconsistent with an antioxidant role ascribed to HO-1. To further investigate this apparent inconsistency, we have assessed how NAC affects VOI. The use of TTC staining is often employed for determining the VOI in stroke. In TBI, it has proven to be a rapid and reproducible method for quantifying brain lesion volume in fluidpercussion injury [17], as well as cortical impact injury [2], where the estimation of brain edema enabled the determination of absolute injury volume [2]. Our findings indicate that NAC decreases the VOI in TBI. Such an effect of this drug has also been reported in focal ischemia [21]. In addition, the present results are consistent with previous findings that HO-1 induction, on its own, decreases ROS levels [11]. Decreased VOI by NAC suggests a neuroprotective role for this drug under these conditions, concomitant with a reduction of HO-1 levels. Together, these findings suggest that the induction of HO-1 is a marker of oxidative stress-related damage, with the expres-
sion of this gene being designed to antagonize this effect. The suppression of HO-1 induction in the present study appears to be a consequence of the increased antioxidant status of the tissue brought about by NAC treatment, making it less necessary to mount a further protective response of this nature to the TBI insult. In addition, other factors have been reported to participate in the regulation of HO-1 in the brain. Recent studies indicate that the gene repressor protein Bach-1 down-regulates HO-1 and that the silencing of this protein using RNA interference results in a dramatic up-regulation of HO-1 [22], thus suggesting that certain inhibitory factors are responsible for preventing the expression of this gene. In addition, hypoxia has been reported to lead to the induction of HO-1 in the cerebral medulla [3,7]. In summary, the expression of HO-1 occurred in the injured cerebral cortex at 6 h following TBI and was sustained for up to 24 h postinjury. On the other hand, in the ipsilateral hippocampus and thalamus, little or no HO-1 protein was detected at 6 h, with HO-1 induction occurring at 24 h following TBI. HO-1 immunoreactivity was localized primarily to the astrocytes, although in the core of the injured cortex, some neuronal and microglial immunostaining was also detected, consistent with previous findings [6]. These changes were regionally delayed or suppressed and were associated with a reduction in the VOI following treatment with the antioxidant NAC. Our findings suggest that HO-1 induction is a credible marker of oxidative damage following brain trauma.
Acknowledgments This study was funded by the Marie Robert Head Trauma Foundation (Quebec) and by a grant from the Canadian Institutes of Health Research (MOP-53110).
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Research report
N-acetylcysteine attenuates early induction of heme oxygenase-1 following traumatic brain injury Jae-Hyuk Yi, Alan S. Hazell* Department of Medicine, Hoˆpital Saint-Luc (CHUM), University of Montreal, 1058 St-Denis St., Montreal, Quebec Canada H2X 3J4 Accepted 30 October 2004 Available online 7 January 2005
Abstract Traumatic brain injury (TBI) results in a cascade of events that includes the production of reactive oxygen species. Heme oxygenase-1 (HO1) is induced in glial cells following head trauma, suggestive of oxidative stress. We have studied the temporal and spatial effects of the antioxidant N-acetylcysteine (NAC) on HO-1 levels following lateral fluid-percussion injury by immunoblotting and immunohistochemistry. In the injured cerebral cortex, maximal HO-1 induction was seen 6 h post-TBI and was maintained for up to 24 h following the insult, while the ipsilateral hippocampus and thalamus showed marked induction at 24 h postinjury. In all three brain regions, little or no HO-1 immunoreactivity was observed on the contralateral side. Astrocytes exhibited positive immunoreactivity for HO-1 in the injured cerebral cortex, hippocampus, and thalamus, while some neurons and microglia were also immunoreactive in the injured cortex. The administration of NAC 5 min following TBI resulted in a marked reduction in this widespread induction of HO-1, concomitant with a decrease in the volume of injury in all three brain regions. Together, these findings indicate that HO-1 induction is related to both oxidative and injury characteristics of the affected tissue, suggesting that protein expression of this gene is a credible marker of oxidative damage in this model of TBI. D 2004 Elsevier B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Trauma Keywords: Heme oxygenase; Traumatic brain injury; Astrocyte; Oxidative stress; Fluid percussion; Antioxidant
1. Introduction Heme oxygenase-1 (HO-1, HSP32) is an inducible form of the heme oxygenases (HO-1, HO-2, and HO-3) that metabolizes heme molecules to carbon monoxide, iron, and biliverdin. While HO-1 is induced in response to numerous stimuli [27], HO-2 is constitutively expressed in most neurons in the brain [26], with HO-3 being highly expressed in the hippocampus and cerebellum [20]. In traumatic brain injury (TBI), HO-1 is induced in the glial cells of human brain as early as 6 h, and up to 6 months following the injury [4]. In rats, however, TBI has been reported to result in transient
* Corresponding author. Fax: +1 514 412 7314. E-mail address: [email protected] (A.S. Hazell). 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.10.055
HO-1 induction in astrocytes from 24 h postinjury, with a noticeable reduction of staining at 5 days postinjury [6]. Thus far, the precise mechanism(s) responsible for HO-1 induction in TBI remains unclear. N-acetylcysteine (NAC) is a cysteine analog commonly used to treat acetaminophen overdose [10]. While the precise beneficial mechanisms provided by NAC still require further elucidation, NAC can protect against reactive oxygen species through the restoration of intracellular glutathione [9,18]. In TBI, NAC has been effective in restoring cerebrovascular responsiveness to hyperventilation following fluid-percussion injury [5] and protection against mitochondrial dysfunction in cortical impact injury [28], both of which may greatly reduce the degree of oxidative stress resulting from the insult. In this study, the regional effects of NAC on HO-1 induction were studied using immunoblotting and immunohistochem-
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istry in a lateral fluid-percussion model of TBI of moderate severity.
2. Materials and methods 2.1. Trauma model All procedures were undertaken with the approval of the Animal Ethics committee of Hoˆpital Saint-Luc and the University of Montreal and were conducted in accordance with the guidelines set out by the Canadian Council on Animal Care. A total of 64 male Sprague–Dawley rats (350– 400 g) was housed under constant conditions of temperature, humidity, and 12:12 h day/night cycle. The lateral (parasagittal) model of traumatic brain injury used in this study has previously been described in detail [13]. A 4.8-mm-diameter craniectomy, centered between the bregma and lambda and 2.5 mm lateral to the superior sagittal sinus, was made over the left hemisphere using a trephine drill, and a hollow female Luer-Lok fitting secured with dental cement. Rats were subsequently allowed to recover overnight and, the following day, reanesthetized with 2% isofluorane and exposed to lateral fluid-percussion injury of moderate severity (2.0–2.5 atm). Animals were studied at 6 (n = 6) and 24 h (n = 6) postTBI. Sham controls (n = 5) received identical treatment but without exposure to trauma. For the administration of NAC, rats received an injection of either drug (163 mg/kg, i.p.) or equal volume of saline 5 min following TBI. 2.2. Western blotting At the appropriate time, brains were removed and rapidly frozen in isopentane on dry ice, sectioned into appropriate regions corresponding to the injured cerebral cortex, hippocampus, and thalamus, and homogenized in ice-cold TE buffer with phenylmethylsulfonyl fluoride using a Teflon glass homogenizer on ice. Resulting homogenates were centrifuged for 30 min at 15,000 g, 4 8C. Pellets were washed with TE buffer, recentrifuged, the supernatant discarded, and solubilized in RIPA buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate, pH 8.0, and a protease inhibitor cocktail). Protein content was determined according to Lowry et al. [12]. Immunoblotting assays were performed as previously described [8]. Fifty micrograms of tissue was loaded on to 12% SDS polyacrylamide gel electrophoresis, separated overnight at 40 V, and transferred to PVDF membranes at 20 V constant volt overnight at 4 8C. Membranes were subsequently incubated in a blocking solution consisting of 5% dry nonfat milk in TTBS (10 mM Tris, 100 mM NaCl, and 0.1% Tween-20) and then probed with antisera against HO-1 (Stressgen Biotechnologies, Victoria, BC, Canada; 1:5,000) or h-actin (Sigma, St. Louis, MO, USA; 1:10,000) in blocking solution for 1 h, washed three times in TTBS, and then reblocked for 1 h. After further washes in TTBS,
membranes were incubated in HRP-coupled donkey antimouse or antirabbit secondary antiserum (1:10,000) in blocking solution for 1 h. Membranes were subsequently treated with enhanced chemiluminescence kit reagents (New England Nuclear, Boston, MA, USA), according to the manufacturer’s instructions, and exposed to Kodak X-OMAT film, followed by semiquantitation of the immunoband intensities using a computerized image analysis system (MCID, Imaging Research, London, ON, Canada). 2.3. Immunohistochemistry For immunohistochemistry and histology, rats (sham group, n = 2; 6 h post-TBI group, n = 3; 24 post-TBI group, n = 3; saline treated n = 8; NAC treated n = 8) were deeply anesthetized with pentobarbital (60 mg/kg) and perfused transcardially as described previously [8]. Briefly, brains were removed and postfixed overnight in 10% neutralbuffered formalin containing 4% formaldehyde, 0.5% sodium phosphate buffer, and 1.5% methanol, pH 7.0. Coronal sections (3.8 to 5.8 mm relative to the bregma) of 40-Am thickness were cut using a vibrotome based on the rat brain atlas of Paxinos and Watson [16]. Sections were used for HO-1 immunohistochemistry or stained with cresyl violet according to Hazell et al. [8]. Briefly, sections were incubated for 10 min in phosphate-buffered saline (PBS) containing 0.3% hydrogen peroxide to block endogenous peroxidase activity. Tissue sections were then washed in PBS (3 10 min) and blocked for 20 min in PBS containing 0.5% Triton X-100, 5% donkey serum, and 1% bovine serum albumin, followed by incubation of 24 h at 4 8C in a blocking solution with HO-1 antirabbit primary antibody (1:5000, Stressgen Biotechnologies). The following day, the sections were washed again in PBS (3 10 min) and incubated for 1 h with biotinylated donkey antirabbit IgG (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA) in PBS with 0.5% Triton X-100, followed by washing in PBS (3 10 min) and incubation for 1 h in streptavidin-HRP conjugate (1:100; Sigma) in PBS with 0.5% Triton X-100. The sections were then washed in PBS (310 min), followed by incubation with diaminobenzidine (0.05%) in PBS with 25 mg/ml nickel ammonium sulfate, for signal enhancement, and in the presence of H2O2 (0.03%) for 2–10 min to allow color development. Following subsequent washings in PBS, the sections were mounted on Superfrost Plus slides (Fisher Scientific, Ottawa, ON, Canada), dehydrated in ethanol (75%, 95%, and 100%), cleared in xylene, and coverslipped with Permount. Controls for the specificity of immunostaining consisted of the omission of the primary or secondary antibody, resulting in a subsequent loss of immunoreactivity. 2.4. 2,3,5-Triphenyltetrazolium chloride (TTC) staining Twenty four hours after TBI, rats (saline-treated group, n = 7; NAC-treated group, n = 7) were injected with sodium
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the saline + TBI and NAC + TBI groups was performed using the Mann–Whitney test.
3. Results 3.1. Regional changes in HO-1 levels with or without NAC treatment
Fig. 1. Representative bands for HO-1 induction in three different regions of the brain following TBI, with or without NAC administration. (A) HO-1 induction peaked as early as 6 h following the injury, and the level was sustained up to 24 h posttrauma in the cerebral cortex (Cx). In the hippocampus (Hc) and thalamus (Th), little or no expression was detected at 6 h. (B) HO-1 induction is attenuated following NAC administration (163 mg/kg, i.p.) in the three regions. A total of 34 rats was used (sham group, n = 5; TBI groups, n = 6; FNAC).
A rapid induction of HO-1 was detected by Western blotting following fluid-percussion injury of moderate severity (Fig. 1A). Little or no evidence of multiple bands was observed with this antibody. In the injured cerebral cortex, HO-1 induction was observed as early as 6 h
pentobarbital (60 mg/kg, i.p) and brains were quickly removed, transferred into Falcon tubes containing ice-cold saline solution, and stored at 4 8C for 10 min. A series of 2mm coronal sections were cut on a prechilled Rodent Brain Matrix (ASI instruments, MI, USA) and were rapidly transferred to a solution of 2% TTC (Sigma) in saline prewarmed to 37 8C in a water bath. Five sections from each brain were incubated for 15 min, then transferred to 10% neutral buffered formalin solution, and kept at 4 8C until scanned and processed for the determination of volume of injury (VOI). 2.5. Volume of injury Sections from each brain were scanned and saved as TIFF files. These files were processed using AdobeR PhotoshopR for the determination of the injury area. Brain lesion areas were determined in pixels and converted into mm2 (relative to a scale bar size of 0.01 mm scanned on the same image and a square area using this length), multiplied by the thickness of the section (2 mm), and then summed for the calculation of total VOI (mm3). For the consideration of brain edema, both ipsi- and contralateral sides of the brain were imaged, the relative swelling considered as previously described [2], and the final total VOI calculated using the following formula: Corrected injury volume ¼ contralateral hemisphere volume ðipsilateral hemisphere volume measured injury volumeÞ:
2.6. Statistical analysis Statistical analysis of immunoblotting data was performed using unpaired t test. Comparison of VOI between
Fig. 2. Quantitative immunoblot analysis of HO-1 expression following TBI alone or TBI with NAC treatment in the injured (A) cerebral cortex (Cx), (B) hippocampus (Hc), and (C) thalamus (Th). Asterisks indicate significant difference between TBI alone and TBI with NAC when same time point groups were compared (unpaired t test, p b 0.05).
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following TBI, and this level was sustained up to 24 h. In the injured hippocampus and thalamus, HO-1 induction occurred at 24 h, with little or no induction at 6 h post-TBI. These temporal and quantitative differences in HO-1 induction following TBI in the three regions were substantially modified following treatment with NAC, 5 min following the insult: in the injured cerebral cortex at 6 h, and in the hippocampus and thalamus regions at 24 h (Fig. 1B). Following NAC treatment, HO-1 induction in the injured cerebral cortex following NAC treatment was reduced to 16% of the value in the TBI alone group after 6 h (Fig. 2A). On the other hand, no significant effect of NAC on HO-1 levels was observed at 24 h post-TBI. In the hippocampus and thalamus, HO-1 levels remained almost undetectable at 6 h following NAC administration (Fig. 2B, C). After 24 h, however, HO-1 induction in the two brain regions was reduced to 37% and 46% of the values obtained with TBI alone, respectively.
induction in the ipsilateral (but not contralateral) Layer I of the cerebral cortex that was astrocytic in nature (Fig. 3A, inset). No obvious HO-1 immunoreactivity was observed in the hippocampus and thalamus of these control animals (Fig. 3D, G), with the exception of neurons in the dentate gyrus. TBI resulted in an early increase in HO-1 immunoreactivity in the injured cortex at 6 h postinjury (Fig. 3B). In the core of the injury, some microglia-like cells and neurons exhibited HO-1 immunoreactivity (Fig. 4A, black arrowhead), as well as astrocytes (Fig. 4A, white arrowhead). In other areas of the cortex, HO-1 immunoreactivity was localized predominantly to astrocytes. At 24 h post-TBI, immunostaining was also detected in the astrocytes of the ipsilateral hippocampus and thalamus (Fig. 3E, inset, and F) and in the cerebral cortex. Treatment with NAC led to a reduction in HO-1 immunoreactivity in all three regions (Fig. 4). 3.3. Effect of NAC on volume of injury
3.2. Regional and cell-type HO-1 profile Figs. 3 and 4 show the profile of HO-1 immunostaining. Sham treatment alone triggered some localized HO-1
The administration of NAC resulted in a reduction in the VOI (Mann–Whitney test, p = 0.0111) when the VOI of saline + TBI group was compared with that of the NAC +
Fig. 3. HO-1 immunoreactivity in the injured cerebral cortex (A–C), precedes hippocampus (D– F), and thalamus (G–I) following TBI. Panels A, D, and G: Sham. Panels B, E, and H: 6 h post-TBI. Panels C, F, and I: 24 h post-TBI. In Panel A, arrow points to cortex layer 1 where sham injury resulted in HO-1 induction (inset), but not in the deeper layers of the cortex. In Panel D, arrow points to neuronal staining in the dentate gyrus area of the sham brain. Abbreviations used: SRH, stratum radiatum of hippocampus; HF, hippocampal fissure; VPM, ventroposteriomedial thalamic nuclei. Bars represent 100 (low power) and 25 Am (high power inset).
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Fig. 4. Trauma-induced HO-1 immunoreactivity is substantially decreased in NAC-treated rats (B, D, F) compared with saline-treated rats (A, C, E). Panels A and B: injured cortex, 6 h post-TBI. Panels C and D: stratum radiatum of the hippocampus 24 h post-TBI. Panels E and F: ventroposteriomedial thalamus 24 h post-TBI. In Panel A, the box indicates the area depicted in the inset showing the glial staining of HO-1 (inset, white arrowhead), as well as the neuronal staining (inset, black arrowhead), in the injury core. Note the reduction in positively stained glial processes in Panels B, D, and F compared with Panels A, C, and E, respectively. A total of 16 rats was used (sham group, n = 2; TBI groups, n = 3). Bars represent 50 (low power) and 25 Am (high power inset).
Fig. 5. Comparison of volume of injury at 24 h post-TBI in animals treated with saline or NAC (163 mg/kg, i.p., 5 min post-TBI) from TTC staining. (A) Representative coronal TTC-stained sections of the saline- and NAC-treated TBI brains. (B) A significant difference was found in injury volumes between the two groups using the Mann–Whitney test ( p b 0.05). The values were 34.3 and 18.9 mm3 for saline- (n = 7) and NAC-treated (n = 7) TBI brains, respectively.
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TBI group (Fig. 5). The average VOI was 34.4 mm3 for the saline + TBI group, and 18.9 mm3 for the NAC + TBI group.
4. Discussion Pathophysiological changes in TBI often involve hemorrhage, with release of blood into the extracellular space that may partly account for the effects of secondary injury. This is particularly evident in the injured cortex, in which there is the development of a contusion, a consistent finding in this fluidpercussion model [13]. Hemorrhage associated with this contusion can induce focal vasospasm and cerebral ischemia, contributing to the injury process. Such a hemorrhage can also lead to neurotoxicity by producing a slow development of oxidative stress [19]. The present study demonstrates the regional and temporal induction of HO-1 protein and a beneficial effect of NAC administration following TBI. In two recent studies involving subarachnoid hemorrhage, HO1 induction was reported to occur [23,24], with antioxidant treatment having a preventative effect [24]. Our findings are consistent with the effects of NAC treatment in those studies. When TBI rats were administered NAC 5 min following injury, the induction of HO-1 was suppressed in all three brain regions examined. Previous studies have demonstrated that NAC treatment results in a rapid recovery of levels of reduced glutathione in brain and a reduction of oxidative stress following TBI [9,28]. The consequence of such a depletion may play an important role in the injury process. Thus, NAC may protect against neuronal death by improving the antioxidant status of the tissue. On the other hand, several reports indicate that HO-1 itself has a cytoprotective role to play due to its ability to break down heme (a prooxidant) to biliverdin and bilirubin, both of which are powerful antioxidants. This has been demonstrated under both in vitro [1,25] and in vivo conditions [14,15]. The suppression of HO-1 induction by NAC treatment would therefore seem inconsistent with an antioxidant role ascribed to HO-1. To further investigate this apparent inconsistency, we have assessed how NAC affects VOI. The use of TTC staining is often employed for determining the VOI in stroke. In TBI, it has proven to be a rapid and reproducible method for quantifying brain lesion volume in fluidpercussion injury [17], as well as cortical impact injury [2], where the estimation of brain edema enabled the determination of absolute injury volume [2]. Our findings indicate that NAC decreases the VOI in TBI. Such an effect of this drug has also been reported in focal ischemia [21]. In addition, the present results are consistent with previous findings that HO-1 induction, on its own, decreases ROS levels [11]. Decreased VOI by NAC suggests a neuroprotective role for this drug under these conditions, concomitant with a reduction of HO-1 levels. Together, these findings suggest that the induction of HO-1 is a marker of oxidative stress-related damage, with the expres-
sion of this gene being designed to antagonize this effect. The suppression of HO-1 induction in the present study appears to be a consequence of the increased antioxidant status of the tissue brought about by NAC treatment, making it less necessary to mount a further protective response of this nature to the TBI insult. In addition, other factors have been reported to participate in the regulation of HO-1 in the brain. Recent studies indicate that the gene repressor protein Bach-1 down-regulates HO-1 and that the silencing of this protein using RNA interference results in a dramatic up-regulation of HO-1 [22], thus suggesting that certain inhibitory factors are responsible for preventing the expression of this gene. In addition, hypoxia has been reported to lead to the induction of HO-1 in the cerebral medulla [3,7]. In summary, the expression of HO-1 occurred in the injured cerebral cortex at 6 h following TBI and was sustained for up to 24 h postinjury. On the other hand, in the ipsilateral hippocampus and thalamus, little or no HO-1 protein was detected at 6 h, with HO-1 induction occurring at 24 h following TBI. HO-1 immunoreactivity was localized primarily to the astrocytes, although in the core of the injured cortex, some neuronal and microglial immunostaining was also detected, consistent with previous findings [6]. These changes were regionally delayed or suppressed and were associated with a reduction in the VOI following treatment with the antioxidant NAC. Our findings suggest that HO-1 induction is a credible marker of oxidative damage following brain trauma.
Acknowledgments This study was funded by the Marie Robert Head Trauma Foundation (Quebec) and by a grant from the Canadian Institutes of Health Research (MOP-53110).
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