Neurochemistry International 69 (2014) 14–19
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NADPH oxidase inhibition improves neurological outcome in experimental traumatic brain injury Xin-Yu Lu a,b, Han-Dong Wang a,⇑, Jian-Guo Xu a, Ke Ding a, Tao Li a a b
Department of Neurosurgery, Jinling Hospital, School of Medicine, Nanjing University, Nanjing, Jiangsu Province, China Department of Neurosurgery, People’s Hospital Affiliated to Jiangsu University, Zhenjiang, Jiangsu Province, China
a r t i c l e
i n f o
Article history: Received 3 November 2013 Received in revised form 13 January 2014 Accepted 25 February 2014 Available online 28 February 2014 Keywords: Traumatic brain injury NADPH oxidase NOX2 Apocynin
a b s t r a c t Purpose: Traumatic brain injury (TBI) is a worldwide health problem with oxidative stress recognized as a major pathogenetic factor. The present experimental study was designed to explore the neuroprotective effect of NADPH oxidase (NOX) inhibitor, apocynin, on mouse TBI. Methods: Moderately severe weight-drop impact head injury was induced in adult male mice, randomly divided into four groups: sham, TBI, TBI + vehicle and TBI + apocynin treatment. Apocynin (50 mg/kg) was injected intraperitoneally 30 min before TBI. The expression of NOX2 protein was investigated using immunoblotting techniques 1 and 24 h after TBI. Neurological score was evaluated 24 h after TBI. Blood–brain barrier disruption was detected by Evans blue extravasation and cortical apoptosis was analyzed by TUNEL assay. Additionally, we assessed tissue levels of malondialdehyde (MDA). Results: NOX2 expression increased rapidly following TBI in male mice, with an early peak at 1 h, followed by a second peak at 24 h. Pre-treatment with the NOX inhibitor, apocynin markedly inhibited NOX2 expression. Apocynin also attenuated MDA levels and TBI-induced blood–brain barrier dysfunction. In addition apocynin significantly attenuated TBI-induced neurological deficits and cortical apoptosis. Conclusion: Pre-treatment with apocynin effectively attenuates markers of cerebral oxidative stress after TBI, thus supporting the hypothesis that apocynin is a potential neuroprotectant and adjunct therapy for TBI patients. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Traumatic brain injury (TBI) is a major public health problem globally. While mortality rates have slightly improved since 1990, TBI incidence is increasing worldwide. The weighted average mortality for TBI is 39%, and that for unfavorable outcome on the Glasgow Outcome Scale is 60% (Rosenfeld et al., 2012). In spite of advances in research and care, the clinical outcome of patients with severe head injury is still poor, and its management poses enormous challenges to the medical team and burdens hospital resources. There is a compelling need for more effective therapeutic options to aid this critical population. The pathophysiological profile of TBI is heterogeneous and complex. Increasing evidence has shown that oxidative stress, which generates reactive oxygen species (ROS), plays an important ⇑ Corresponding author. Address: Department of Neurosurgery, Jinling Hospital, 305 East Zhongshan Road, Nanjing 210002, Jiangsu Province, China. Tel./fax: +86 25 51805396. E-mail address: [email protected] (H.-D. Wang). http://dx.doi.org/10.1016/j.neuint.2014.02.006 0197-0186/Ó 2014 Elsevier Ltd. All rights reserved.
role in TBI. Oxidative stress is implicated in the development of cerebral edema, breakdown of the blood–brain barrier (BBB), impairment of sensory–motor function and secondary neuronal injury. With the growing interest in NADPH oxidases (NOXs), the role of this family of enzymes in the physiological function and pathophysiological dysfunction of cerebral cells has attracted considerable interest. NADPH oxidase (NOX) is a multi-component enzyme comprising cytoplasmatic subunits (p47phox, p67phox, p40phox and Rac2), which upon phosphorylation, can form complexes that translocate to the plasma membrane and dock with the plasma membrane subunits p91phox and p22phox. Catalysis of NOX occurs at the p91phox subunit (NOX2). Numerous papers from several laboratories have reported that over-activated NOX2 significantly contributes to oxidative damage to neurons and other cell types in ischemic, traumatic and degenerative CNS conditions (Ano et al., 2010; Cairns et al., 2012; Kahles et al., 2007). Apocynin (4-hydroxy-3-methoxy-acetophenone) is a compound derived from the root extract of Picrorhiza kurroa, a Chinese medicinal herb that has been used for centuries to treat inflammatory
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diseases. Apocynin has been reported to protect against global cerebral ischemia–induced oxidative stress and neuronal injury in the hippocampus inhibiting superoxide production, to preserve the BBB, and to decrease ischemic areas and brain edema (Altintas et al., 2013). However, its role in the pathophysiology of traumatic brain injury is still obscure (Weston et al., 2013). In the present study we used a TBI mouse model to evaluate the neuroprotective effect of apocynin. Our hypothesis was that an intraperitoneal administration of apocynin prior to TBI in mice could down-regulate NOX2 expression following TBI, prevent TBI-induced BBB disruption, decrease the levels of lipid peroxidation product malondialdehyde (MDA), and thereby reduce tissue damage, thus ultimately leading to recovery of neurological function.
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in the supernatant by the bicinchoninic acid reagent (Solarbio, Beijing, China) method. Equal amounts (50 lg) of protein were subjected to 10% SDS–PAGE and electrotransferred onto a hydrophobic polyvinylidene difluoride membrane (Roche Diagnostics, Mannheim, Germany). After blocking, the membranes were incubated with primary antibodies against NOX2 (abcam 1:5000) and b-actin (Bioworld 1:5000) overnight at 4 °C. After washing, the membrane was probed with horseradish peroxidase (HRP)-conjugated secondary antibodies (Bioworld 1:3000). The protein bands were detected by chemiluminescence and exposed onto X-ray film. The films were scanned, and the signal densities were quantified using the UN-SCAN-IT gel analysis program (Silk Scientific, Orem, UT) and normalized to a loading control actin. 2.3. TUNEL assay
2. Materials and methods 2.1. Animals and TBI model All protocols including surgical procedures and animal use were approved by the Animal Care and Use Committee of Nanjing University and conformed to the Guide for the Care and Use of Laboratory Animals by the National Institutes of Health. Adult male ICR mice (28 g–32 g) were purchased from Animal Center of Nanjing Medical University, Nanjing China. The animals were housed in a temperature and humidity controlled environment and supplied with free diet and water. The mouse model of TBI was set up using a modified weightdrop device, with a minor modification (Flierl et al., 2009; Ling et al., 2013). Briefly, mice were anesthetized with sodium pentobarbital (50 mg/kg). Under anaesthesia, the animal was placed in a prone position and the scalp disinfected with alcohol pads. A midline incision was performed to expose the skull between bregma and lambda suture lines (2.0–2.5 cm) and the left lateral portion of the skull was exposed by reflecting the skin and surrounding soft tissue. Mice were placed on a foam mattress underneath a weight-drop device in which a 200 g weight falls freely through a vertical tube from 3 cm onto the left lateral skull. The scalp was then sutured. All mice were maintained at a core temperature of 36–37.5 °C during and after surgery, until recovery from anaesthesia. Sham-injured animals, undergoing identical treatment with the exception of the injury, served as a control group. Animals were sacrificed at designated times following the sham or injury procedure for sample collection. In our pilot study, we found that there was no statistical difference in all the detected variables in sham animals prepared at each time point (data not shown). Therefore, control animals were all sacrificed at 6 h after sham operation. All mice were randomly allocated into the following groups: Sham, TBI, TBI + vehicle (DMSO) and TBI treated with apocynin groups. Apocynin (Sigma–Aldrich Co., St. Louis, MO), at the dose of 50 mg/kg was administrated intraperitoneally 30 min before surgery (Kahles et al., 2007; Song et al., 2013). DMSO (Sigma–Aldrich Co., St. Louis, MO) was used as vehicle at final concentration of 1%. 2.2. Western blot analysis For Western blot analysis (Yoshioka et al., 2010), the left hemisphere was removed from decapitated animals at 0 (sham), 1, 3, 6, 12, 24, 72 h after TBI and immediately frozen on dry ice. These samples were then homogenized in radioimmunoprecipitation assay (RIPA) buffer and protease inhibitor (both Sigma–Aldrich Co., St. Louis, MO). The homogenate was then centrifuged at 12,000g for 15 min at 4 °C. Protein concentration was determined
Twenty-four hours after TBI, deeply anesthetized animals were transcardially perfused with 100 ml ice–cold 0.9% saline and then 60 ml 4% formaldehyde solution. The brains were quickly removed and frozen. Eight micron thick coronal sections containing the area of impact were collected at 500 lm intervals and processed for apoptotic cell detection by TUNEL assay (ISCDD, Boehringer Mannheim, Germany) according to the manufacturer’s instructions. The extent of brain damage was evaluated by apoptotic index (AI), calculated as the average percentage of TUNEL-positive cells in each section counted in 10 cortical microscopic fields at 200 magnification. 2.4. Neurological deficits Neurological deficits were assessed 24 h after TBI by an independent researcher blinded to the treatment using the scoring system reported by Garcia, with slight modifications (Garcia et al., 1995; Lo et al., 2007). The following sensorimotor tests were graded on a scale of 0–3: spontaneous activity, symmetry of walking, symmetry of movements, symmetry of forelimbs, climbing ability, vibrissae response, and side stroking response. Neurological deficit scores were assigned as follows: 0, complete deficit; 1, definite deficit with some function; 2, decreased response or mild deficit; 3, no evidence of deficit/symmetrical responses. The minimum neurological score was 3, and the maximum score was 18. 2.5. BBB permeability The integrity of the BBB was investigated using Evans blue (EB) extravasation, as described earlier (Dohi et al., 2010). Briefly, EB (2% in saline, 4 ml/kg) was injected intravenously 23 h post-TBI. Animals were then re-anesthetized after 1 h with sodium pentobarbital (50 mg/kg) and perfused using saline to remove blood from the intravascular compartment. The animals were then sacrificed and the brains quickly removed and weighed. Four milliliter of 99.5% formamide per gram of tissue was added and placed on a bain-marie at 37 °C for 72 h, the amount of EB dye extravasated into the brain was determined by spectrofluorophotometer. Measurements were conducted at excitation wavelength of 620 nm, and emission wave length of 680 nm. The tissue content of EB was quantified from a linear standard curve derived from known amounts of the dye and was expressed per gram of tissue. 2.6. Determination MDA levels To determine oxidative stress, the levels of the lipid peroxidation product, MDA were determined in brain samples collected at 24 h after TBI. Briefly, after weighing, the brains were homogenized in 9 volumes of g/L ice–cold saline for 10 min using a Dounce tissue grinder (Kimble and Kontes, Vineland, NJ, USA). Supernatant
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homogenate was collected after centrifugation at 2500 rpm for 10 min at 4 °C. MDA levels were measured using assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions. The results are expressed in nmol/g tissue.
apocynin 30 min before TBI and then brain tissue was harvested 1 and 24 h after TBI. Western blot analysis confirmed that NOX2 expression was markedly increased at 1 and 24 h compared with sham mice (P < 0.05). After apocynin administration, NOX2 expression was markedly down-regulated in TBI + apocynin group (P < 0.05; Fig. 2).
2.7. Statistical analysis 3.3. TBI-induced BBB dysfunction was attenuated by apocynin All data are expressed as mean + sd (standard deviation). Data were analyzed by one-way ANOVA. Differences between experimental groups were determined by Fisher’s LSD post-test. Significance was assigned at P < 0.05. 3. Results 3.1. NOX2 protein expression in the cerebral cortex following TBI We evaluated NADPH oxidase activation by assessing NOX2 protein expression. We first examined the temporal pattern of NOX2 activation in the adult male mouse cerebral cortex following TBI by Western blot analysis. As shown in Fig. 1, there was rapid, robust elevation of NOX2 expression in the cerebral cortex at 1 h after TBI. This rapid elevation was followed by a decrease to lower, but still elevated levels at 3–12 h after TBI. A second significant elevation was observed at 24 h after TBI with no significant difference in NOX2 expression in the treated 72 h group compared to the sham group. In the majority of the subsequent studies, we chose to use the 1 h time-point and 24 h time-point for further analysis, as this was the peak of NOX2 protein. 3.2. TBI-induced upregulation of NOX2 expression was reduced by apocynin treatment
BBB permeability was quantified with Evans blue extravasations test. In the control mice, the Evans blue extravasation was almost zero, indicating that BBB dysfunction had not occurred (Fig. 3). Extravasation was significantly higher compared to sham group 24 h following TBI. In contrast, in the TBI + apocynin group, extravasation was significantly down-regulated (P < 0.01). 3.4. TBI-induced neurological deficits are attenuated by apocynin Neurological deficits were assessed 24 h after TBI to investigate whether apocynin prevented TBI-associated functional deficits. As illustrated in Fig. 4, TBI caused significant neurological deficits as assessed 24 h after TBI compared with sham group. The neurological scores for the TBI + apocynin group were markedly increased when compared with the TBI group. There were no significant differences between the sham and the TBI + apocynin group. These data suggest a neuroprotective effect of apocynin when given as pre-treatment before TBI. 3.5. TBI-induced increase in MDA levels was attenuated by apocynin As a biomarker of oxidative stress, brain tissue MDA level were found to be significantly higher in the TBI group
To determine the influence of apocynin on NOX2 activity in the brains of mice subjected to TBI, Western blot analysis was performed to assess NOX2 expression. Mice were injected with
Fig. 1. Temporal pattern of changes in NOX2 expression in cerebral cortices of mice subjected to TBI at the indicated times. Typical Western blots are shown in the upper part of the panel. Sham animals, which did not undergo TBI, were used as controls. Data are expressed as fold changes vs. sham (mean + sd, n = 4). ⁄P < 0.05 vs. sham.
Fig. 2. Effect of apocynin treatment on NOX2 expression in cerebral cortices of mice subjected to TBI. Typical Western blots are shown in the upper part of the panel. TBI-induced upregulation of NOX2 at 1 and 24 h is reduced by apocynin treatment. Data are expressed as fold changes vs. sham and presented as mean + sd, n = 4. ⁄ P < 0.05 vs. sham; #P < 0.05 vs. TBI at respective times.
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Fig. 3. Apocynin attenuates BBB dysfunction 24 h after TBI. In the sham group Evans blue extravasation was almost zero while in injured mice it reached the highest levels. There was no difference between TBI and TBI + vehicle group (NS). Evans blue extravasation was decreased in mice treated with apocynin. Data are presented as mean + sd, n = 4. ⁄P < 0.05 vs. sham group; #P < 0.05 vs. TBI group.
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Fig. 5. Effect of apocynin on MDA levels. MDA levels, used to assess lipid peroxidation, were significantly increased in cerebral cortices of mice subjected to TBI. Apocynin pretreatment significantly decreased MDA. There was no difference between TBI and TBI + vehicle group (NS). Data are presented as mean + sd, n = 4. ⁄P < 0.05 vs. sham, #P < 0.05 vs. TBI.
Fig. 4. Apocynin improves neurological outcome 24 h after TBI. Neurological scores are significantly decreased in TBI as well as in TBI + vehicle group as compared to sham. Treatment with apocynin improved the neurological outcome compared with the TBI group. There was no difference between TBI and TBI + vehicle groups (NS). Data are presented as mean + sd, n = 4. ⁄P < 0.05 vs. sham; #P < 0.05 vs. TBI group.
(6.332 ± 0.753 nmol/mg protein) when compared to the sham group (4.243 ± 0.399 nmol/mg protein). In the treatment group, the elevated MDA levels were reduced to almost control levels (4.576 ± 0.884 nmol/mg protein) and this reduction was statistically significant compared to the TBI group. Thus, pre-treatment with apocynin significantly attenuated the TBI-induced MDA increase (Fig. 5). 3.6. Effect of apocynin on apoptosis in the cortex of mice subjected to TBI As shown in Fig. 6, a small, but quantifiable number of TUNELpositive cells were observed in sham mice. TBI caused a significant increase in TUNEL-positive cells compared to those in sham mice (P < 0.05). Notably, apocynin treatment significantly decreased the number of TUNEL-positive cells compared to TBI (P < 0.05). 4. Discussion The present study demonstrates that NOX2 expression was increased after TBI, with two peaks at 1 and 24 h. This effect was
Fig. 6. Effect of apocynin treatment on apoptotic cells in mouse brain tissue 24 h after TBI. Typical TUNEL staining in sham (A), TBI (B), TBI + vehicle (C) and TBI + apocynin (D) brain tissue. A high apoptotic index was found in the TBI group compared to sham. In the TBI + apocynin group, the apoptotic index was significantly decreased. There was no difference between TBI and TBI + vehicle groups (NS). Data are presented as mean + sd, n = 4. ⁄P < 0.05 vs. sham, #P < 0.05 vs. TBI group.
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associated to the appearance of neurological deficits, to an increase in MDA, a biomarker of oxidative stress, and to damage of the BBB. Importantly, the data revealed that apocynin injected intraperitoneally 30 min before TBI could decrease NOX2 and MDA expression. Lastly, the results showed that mice pre-treated with apocynin have better neurological outcome and decreased tunnel index. These results indicate that NOX activation is a key mediator of neuronal death and that apocynin may have neuroprotective effects. ROS generation and oxidative stress have been proposed to play a critical role in TBI. ROS are known to not only damage cellular membranes by inducing lipid peroxidation. They can also damage DNA, proteins and lipids. The most studied ROS are O2 — superoxide radical; H2 O2 — hydrogen peroxide; and OH — hydroxyl radical originating from one, two or three electron transfers to dioxygen (O2). It has been generally assumed that mitochondria are the major source of ROS following brain injury. Whereas, recent work has shown that NOX is the central enzyme in ROS cellular production (Kandasamy et al., 2013; Lambert and Brand, 2009). Nevertheless, despite a strong rational of ROS being a pharmacological target, all antioxidant interventions failed to improve functional outcome in human clinical trials. Antioxidants may interfere with physiological functions of ROS or may not reach the crucial target structures of ROS-induced injury effectively. As an evolution of this concept, recent studies have targeted NADPH – the sources of ROS – rather than ROS themselves (Cairns et al., 2012; Pendyala and Natarajan, 2010). NOX is a membrane enzyme that is composed of several subunits, which include NOX and phox subunits. There are at least five different cellular NOX isoforms, termed NOX1–5 (Bedard and Krause, 2007). Previous studies have shown that at acute time points (1 h) following TBI, NOX2 is located in neurons, whereas at later time-points (24–48 h) it is mainly located in the microglia (Dohi et al., 2010; Flierl et al., 2009). Accumulating evidence indicates that NOX2 activation is associated with an increased risk for neuronal damage following experimental TBI, stroke and other degenerative disease. De Silva and collaborators found that NOX2 / mice, subjected to middle cerebral artery occlusion (MCAO), had smaller infarct volumes than wild type mice, edema volume also tended to be smaller compared to wild type mice (De Silva et al., 2011). Liu and colleagues also found that the remarkable Evans blue extravasation, a measure of BBB damage, and significantly increased NOX2 expression in the ischemic brain present in wild type MCAO mice, were significantly attenuated in NOX2 knockout mice. The author confirmed that activation of NOX2 containing NADPH oxidase was implicated in the induction of MMP-9, in loss of occludin and BBB disruption following ischemic stroke (Liu et al., 2011). In the present study we demonstrated that NOX2 expression was upregulated after TBI and that two peaks were observed at 1 h and 24 h after TBI, associated with BBB disturbance and increase in MDA levels. These finding are in line with those by Zhang and collaborators, who also found two NADPH oxidase activity peaks and superoxide production in TBI mice (Zhang et al., 2012). These and other studies imply that NOX2 plays a significant role in TBI physiopathologic mechanism and identified NOX2 as a promising target for pharmacological therapy to treat the pathology and dysfunction caused by TBI. Apocynin is a highly selective, cell-permeable NOX inhibitor. It is thought to act by preventing the translocation of NOX cytosolic components, p47phox to p91phox (NOX2) (Peters et al., 2001; Ximenes et al., 2007). Since apocynin half-life is about 1 h, and since we have shown that there is a rapid and robust elevation in NOX2 within 1 h following TBI, we delivered a single dose of apocynin to prevent TBI-induced NOX activation 30 min prior to TBI. This dosing strategy is not expected to induce any toxicity and could have neuroprotective effects after brain injury (Van den Worm et al., 2001; Yong Choi et al., 2012). In this study we
found that apocynin decreases NOX2 and MDA activation, and protects the tissue against oxidative stress. Moreover apocynin has also been reported to reduce the ischemic area, enhance SOD antioxidant activity, attenuate the increased BBB permeability and further reduce brain edema following TBI (Yong Choi et al., 2012; Yoshioka et al., 2010). Accordingly, apocynin loses its neuroprotective effect in NOX2 / mice (Chen et al., 2011; Pepping et al., 2013). In a recent study, Ferreira and collaborators revealed that apocynin (5 mg/kg), when injected subcutaneously 30 min and 24 h after injury, provided protection against moderate fluid percussion injury (mlFPI)-induced object recognition memory impairment 7 days after neuronal injury. The same treatment also protected against mlFPI-induced IL-1, TNF-a and nitric oxide metabolite content (NOx) 3 and 24 h after neuronal injury (Ferreira et al., 2013). Apocynin also acts as a direct ROS scavenger in given experimental conditions (Heumüller et al., 2008). Taken together, this evidence demonstrates that apocynin has distinct neuroprotective effects achieved by inhibition of NOX2. The effect of inhibition of NOX on oxidative stress was assessed by measuring MDA, a product of lipoxygenase. MDA is a major biomarker of oxidative stress. It increases due to lipid peroxidation, which is one of the harmful consequences of TBI that rapidly causes cellular injury. In the present study, we reported that attenuation of NOX2 expression by apocynin markedly decreased TBI-induced MDA level in the brain. Thus attenuation of MDA activation may represent an effective strategy to prevent ROS-mediated brain damage (Altintas et al., 2013; Sahna et al., 2003). Several limitations of the present study should be considered when interpreting our results. The number of animals in each group was small, such that statistical power may have been compromised given the inherent variability in brain injury resulting from the weight-drop model. In addition, a ‘‘pretreatment’’ study is not really clinically applicable. It remains to be established if apocynin is useful as a therapeutic when administered after TBI. Moreover, our study design reveals associations, but is not indicative of causality, which might entail additional arms such as a nox2 knockout group, or use of direct nox2 blockers. Lastly, in the present study we omitted the sham + apocynin control group thus preventing a direct comparison with the sham and with the TBI + apocynin group. This may be potentially important in our pretreatment study, as the effects of pre-injury administration of the drug alone were not studied.
5. Conclusions In summary, the results of the present study demonstrate that NOX2 activation can contribute significantly to MDA generation, disturbance of BBB and neuronal cell death following TBI. Our data also suggest that administration of apocynin may be a viable neuroprotectant and a potential adjuvant therapy for patients with TBI. It remains to be established if apocynin administration following TBI is likewise effective in reducing the development of secondary damage.
Conflict of interest No competing financial interests exist.
Acknowledgment This study was supported by the Science and Technology Development Fund of Zhenjiang (No. SH20130037).
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Neurochemistry International journal homepage: www.elsevier.com/locate/nci
NADPH oxidase inhibition improves neurological outcome in experimental traumatic brain injury Xin-Yu Lu a,b, Han-Dong Wang a,⇑, Jian-Guo Xu a, Ke Ding a, Tao Li a a b
Department of Neurosurgery, Jinling Hospital, School of Medicine, Nanjing University, Nanjing, Jiangsu Province, China Department of Neurosurgery, People’s Hospital Affiliated to Jiangsu University, Zhenjiang, Jiangsu Province, China
a r t i c l e
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Article history: Received 3 November 2013 Received in revised form 13 January 2014 Accepted 25 February 2014 Available online 28 February 2014 Keywords: Traumatic brain injury NADPH oxidase NOX2 Apocynin
a b s t r a c t Purpose: Traumatic brain injury (TBI) is a worldwide health problem with oxidative stress recognized as a major pathogenetic factor. The present experimental study was designed to explore the neuroprotective effect of NADPH oxidase (NOX) inhibitor, apocynin, on mouse TBI. Methods: Moderately severe weight-drop impact head injury was induced in adult male mice, randomly divided into four groups: sham, TBI, TBI + vehicle and TBI + apocynin treatment. Apocynin (50 mg/kg) was injected intraperitoneally 30 min before TBI. The expression of NOX2 protein was investigated using immunoblotting techniques 1 and 24 h after TBI. Neurological score was evaluated 24 h after TBI. Blood–brain barrier disruption was detected by Evans blue extravasation and cortical apoptosis was analyzed by TUNEL assay. Additionally, we assessed tissue levels of malondialdehyde (MDA). Results: NOX2 expression increased rapidly following TBI in male mice, with an early peak at 1 h, followed by a second peak at 24 h. Pre-treatment with the NOX inhibitor, apocynin markedly inhibited NOX2 expression. Apocynin also attenuated MDA levels and TBI-induced blood–brain barrier dysfunction. In addition apocynin significantly attenuated TBI-induced neurological deficits and cortical apoptosis. Conclusion: Pre-treatment with apocynin effectively attenuates markers of cerebral oxidative stress after TBI, thus supporting the hypothesis that apocynin is a potential neuroprotectant and adjunct therapy for TBI patients. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Traumatic brain injury (TBI) is a major public health problem globally. While mortality rates have slightly improved since 1990, TBI incidence is increasing worldwide. The weighted average mortality for TBI is 39%, and that for unfavorable outcome on the Glasgow Outcome Scale is 60% (Rosenfeld et al., 2012). In spite of advances in research and care, the clinical outcome of patients with severe head injury is still poor, and its management poses enormous challenges to the medical team and burdens hospital resources. There is a compelling need for more effective therapeutic options to aid this critical population. The pathophysiological profile of TBI is heterogeneous and complex. Increasing evidence has shown that oxidative stress, which generates reactive oxygen species (ROS), plays an important ⇑ Corresponding author. Address: Department of Neurosurgery, Jinling Hospital, 305 East Zhongshan Road, Nanjing 210002, Jiangsu Province, China. Tel./fax: +86 25 51805396. E-mail address: [email protected] (H.-D. Wang). http://dx.doi.org/10.1016/j.neuint.2014.02.006 0197-0186/Ó 2014 Elsevier Ltd. All rights reserved.
role in TBI. Oxidative stress is implicated in the development of cerebral edema, breakdown of the blood–brain barrier (BBB), impairment of sensory–motor function and secondary neuronal injury. With the growing interest in NADPH oxidases (NOXs), the role of this family of enzymes in the physiological function and pathophysiological dysfunction of cerebral cells has attracted considerable interest. NADPH oxidase (NOX) is a multi-component enzyme comprising cytoplasmatic subunits (p47phox, p67phox, p40phox and Rac2), which upon phosphorylation, can form complexes that translocate to the plasma membrane and dock with the plasma membrane subunits p91phox and p22phox. Catalysis of NOX occurs at the p91phox subunit (NOX2). Numerous papers from several laboratories have reported that over-activated NOX2 significantly contributes to oxidative damage to neurons and other cell types in ischemic, traumatic and degenerative CNS conditions (Ano et al., 2010; Cairns et al., 2012; Kahles et al., 2007). Apocynin (4-hydroxy-3-methoxy-acetophenone) is a compound derived from the root extract of Picrorhiza kurroa, a Chinese medicinal herb that has been used for centuries to treat inflammatory
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diseases. Apocynin has been reported to protect against global cerebral ischemia–induced oxidative stress and neuronal injury in the hippocampus inhibiting superoxide production, to preserve the BBB, and to decrease ischemic areas and brain edema (Altintas et al., 2013). However, its role in the pathophysiology of traumatic brain injury is still obscure (Weston et al., 2013). In the present study we used a TBI mouse model to evaluate the neuroprotective effect of apocynin. Our hypothesis was that an intraperitoneal administration of apocynin prior to TBI in mice could down-regulate NOX2 expression following TBI, prevent TBI-induced BBB disruption, decrease the levels of lipid peroxidation product malondialdehyde (MDA), and thereby reduce tissue damage, thus ultimately leading to recovery of neurological function.
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in the supernatant by the bicinchoninic acid reagent (Solarbio, Beijing, China) method. Equal amounts (50 lg) of protein were subjected to 10% SDS–PAGE and electrotransferred onto a hydrophobic polyvinylidene difluoride membrane (Roche Diagnostics, Mannheim, Germany). After blocking, the membranes were incubated with primary antibodies against NOX2 (abcam 1:5000) and b-actin (Bioworld 1:5000) overnight at 4 °C. After washing, the membrane was probed with horseradish peroxidase (HRP)-conjugated secondary antibodies (Bioworld 1:3000). The protein bands were detected by chemiluminescence and exposed onto X-ray film. The films were scanned, and the signal densities were quantified using the UN-SCAN-IT gel analysis program (Silk Scientific, Orem, UT) and normalized to a loading control actin. 2.3. TUNEL assay
2. Materials and methods 2.1. Animals and TBI model All protocols including surgical procedures and animal use were approved by the Animal Care and Use Committee of Nanjing University and conformed to the Guide for the Care and Use of Laboratory Animals by the National Institutes of Health. Adult male ICR mice (28 g–32 g) were purchased from Animal Center of Nanjing Medical University, Nanjing China. The animals were housed in a temperature and humidity controlled environment and supplied with free diet and water. The mouse model of TBI was set up using a modified weightdrop device, with a minor modification (Flierl et al., 2009; Ling et al., 2013). Briefly, mice were anesthetized with sodium pentobarbital (50 mg/kg). Under anaesthesia, the animal was placed in a prone position and the scalp disinfected with alcohol pads. A midline incision was performed to expose the skull between bregma and lambda suture lines (2.0–2.5 cm) and the left lateral portion of the skull was exposed by reflecting the skin and surrounding soft tissue. Mice were placed on a foam mattress underneath a weight-drop device in which a 200 g weight falls freely through a vertical tube from 3 cm onto the left lateral skull. The scalp was then sutured. All mice were maintained at a core temperature of 36–37.5 °C during and after surgery, until recovery from anaesthesia. Sham-injured animals, undergoing identical treatment with the exception of the injury, served as a control group. Animals were sacrificed at designated times following the sham or injury procedure for sample collection. In our pilot study, we found that there was no statistical difference in all the detected variables in sham animals prepared at each time point (data not shown). Therefore, control animals were all sacrificed at 6 h after sham operation. All mice were randomly allocated into the following groups: Sham, TBI, TBI + vehicle (DMSO) and TBI treated with apocynin groups. Apocynin (Sigma–Aldrich Co., St. Louis, MO), at the dose of 50 mg/kg was administrated intraperitoneally 30 min before surgery (Kahles et al., 2007; Song et al., 2013). DMSO (Sigma–Aldrich Co., St. Louis, MO) was used as vehicle at final concentration of 1%. 2.2. Western blot analysis For Western blot analysis (Yoshioka et al., 2010), the left hemisphere was removed from decapitated animals at 0 (sham), 1, 3, 6, 12, 24, 72 h after TBI and immediately frozen on dry ice. These samples were then homogenized in radioimmunoprecipitation assay (RIPA) buffer and protease inhibitor (both Sigma–Aldrich Co., St. Louis, MO). The homogenate was then centrifuged at 12,000g for 15 min at 4 °C. Protein concentration was determined
Twenty-four hours after TBI, deeply anesthetized animals were transcardially perfused with 100 ml ice–cold 0.9% saline and then 60 ml 4% formaldehyde solution. The brains were quickly removed and frozen. Eight micron thick coronal sections containing the area of impact were collected at 500 lm intervals and processed for apoptotic cell detection by TUNEL assay (ISCDD, Boehringer Mannheim, Germany) according to the manufacturer’s instructions. The extent of brain damage was evaluated by apoptotic index (AI), calculated as the average percentage of TUNEL-positive cells in each section counted in 10 cortical microscopic fields at 200 magnification. 2.4. Neurological deficits Neurological deficits were assessed 24 h after TBI by an independent researcher blinded to the treatment using the scoring system reported by Garcia, with slight modifications (Garcia et al., 1995; Lo et al., 2007). The following sensorimotor tests were graded on a scale of 0–3: spontaneous activity, symmetry of walking, symmetry of movements, symmetry of forelimbs, climbing ability, vibrissae response, and side stroking response. Neurological deficit scores were assigned as follows: 0, complete deficit; 1, definite deficit with some function; 2, decreased response or mild deficit; 3, no evidence of deficit/symmetrical responses. The minimum neurological score was 3, and the maximum score was 18. 2.5. BBB permeability The integrity of the BBB was investigated using Evans blue (EB) extravasation, as described earlier (Dohi et al., 2010). Briefly, EB (2% in saline, 4 ml/kg) was injected intravenously 23 h post-TBI. Animals were then re-anesthetized after 1 h with sodium pentobarbital (50 mg/kg) and perfused using saline to remove blood from the intravascular compartment. The animals were then sacrificed and the brains quickly removed and weighed. Four milliliter of 99.5% formamide per gram of tissue was added and placed on a bain-marie at 37 °C for 72 h, the amount of EB dye extravasated into the brain was determined by spectrofluorophotometer. Measurements were conducted at excitation wavelength of 620 nm, and emission wave length of 680 nm. The tissue content of EB was quantified from a linear standard curve derived from known amounts of the dye and was expressed per gram of tissue. 2.6. Determination MDA levels To determine oxidative stress, the levels of the lipid peroxidation product, MDA were determined in brain samples collected at 24 h after TBI. Briefly, after weighing, the brains were homogenized in 9 volumes of g/L ice–cold saline for 10 min using a Dounce tissue grinder (Kimble and Kontes, Vineland, NJ, USA). Supernatant
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homogenate was collected after centrifugation at 2500 rpm for 10 min at 4 °C. MDA levels were measured using assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions. The results are expressed in nmol/g tissue.
apocynin 30 min before TBI and then brain tissue was harvested 1 and 24 h after TBI. Western blot analysis confirmed that NOX2 expression was markedly increased at 1 and 24 h compared with sham mice (P < 0.05). After apocynin administration, NOX2 expression was markedly down-regulated in TBI + apocynin group (P < 0.05; Fig. 2).
2.7. Statistical analysis 3.3. TBI-induced BBB dysfunction was attenuated by apocynin All data are expressed as mean + sd (standard deviation). Data were analyzed by one-way ANOVA. Differences between experimental groups were determined by Fisher’s LSD post-test. Significance was assigned at P < 0.05. 3. Results 3.1. NOX2 protein expression in the cerebral cortex following TBI We evaluated NADPH oxidase activation by assessing NOX2 protein expression. We first examined the temporal pattern of NOX2 activation in the adult male mouse cerebral cortex following TBI by Western blot analysis. As shown in Fig. 1, there was rapid, robust elevation of NOX2 expression in the cerebral cortex at 1 h after TBI. This rapid elevation was followed by a decrease to lower, but still elevated levels at 3–12 h after TBI. A second significant elevation was observed at 24 h after TBI with no significant difference in NOX2 expression in the treated 72 h group compared to the sham group. In the majority of the subsequent studies, we chose to use the 1 h time-point and 24 h time-point for further analysis, as this was the peak of NOX2 protein. 3.2. TBI-induced upregulation of NOX2 expression was reduced by apocynin treatment
BBB permeability was quantified with Evans blue extravasations test. In the control mice, the Evans blue extravasation was almost zero, indicating that BBB dysfunction had not occurred (Fig. 3). Extravasation was significantly higher compared to sham group 24 h following TBI. In contrast, in the TBI + apocynin group, extravasation was significantly down-regulated (P < 0.01). 3.4. TBI-induced neurological deficits are attenuated by apocynin Neurological deficits were assessed 24 h after TBI to investigate whether apocynin prevented TBI-associated functional deficits. As illustrated in Fig. 4, TBI caused significant neurological deficits as assessed 24 h after TBI compared with sham group. The neurological scores for the TBI + apocynin group were markedly increased when compared with the TBI group. There were no significant differences between the sham and the TBI + apocynin group. These data suggest a neuroprotective effect of apocynin when given as pre-treatment before TBI. 3.5. TBI-induced increase in MDA levels was attenuated by apocynin As a biomarker of oxidative stress, brain tissue MDA level were found to be significantly higher in the TBI group
To determine the influence of apocynin on NOX2 activity in the brains of mice subjected to TBI, Western blot analysis was performed to assess NOX2 expression. Mice were injected with
Fig. 1. Temporal pattern of changes in NOX2 expression in cerebral cortices of mice subjected to TBI at the indicated times. Typical Western blots are shown in the upper part of the panel. Sham animals, which did not undergo TBI, were used as controls. Data are expressed as fold changes vs. sham (mean + sd, n = 4). ⁄P < 0.05 vs. sham.
Fig. 2. Effect of apocynin treatment on NOX2 expression in cerebral cortices of mice subjected to TBI. Typical Western blots are shown in the upper part of the panel. TBI-induced upregulation of NOX2 at 1 and 24 h is reduced by apocynin treatment. Data are expressed as fold changes vs. sham and presented as mean + sd, n = 4. ⁄ P < 0.05 vs. sham; #P < 0.05 vs. TBI at respective times.
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Fig. 3. Apocynin attenuates BBB dysfunction 24 h after TBI. In the sham group Evans blue extravasation was almost zero while in injured mice it reached the highest levels. There was no difference between TBI and TBI + vehicle group (NS). Evans blue extravasation was decreased in mice treated with apocynin. Data are presented as mean + sd, n = 4. ⁄P < 0.05 vs. sham group; #P < 0.05 vs. TBI group.
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Fig. 5. Effect of apocynin on MDA levels. MDA levels, used to assess lipid peroxidation, were significantly increased in cerebral cortices of mice subjected to TBI. Apocynin pretreatment significantly decreased MDA. There was no difference between TBI and TBI + vehicle group (NS). Data are presented as mean + sd, n = 4. ⁄P < 0.05 vs. sham, #P < 0.05 vs. TBI.
Fig. 4. Apocynin improves neurological outcome 24 h after TBI. Neurological scores are significantly decreased in TBI as well as in TBI + vehicle group as compared to sham. Treatment with apocynin improved the neurological outcome compared with the TBI group. There was no difference between TBI and TBI + vehicle groups (NS). Data are presented as mean + sd, n = 4. ⁄P < 0.05 vs. sham; #P < 0.05 vs. TBI group.
(6.332 ± 0.753 nmol/mg protein) when compared to the sham group (4.243 ± 0.399 nmol/mg protein). In the treatment group, the elevated MDA levels were reduced to almost control levels (4.576 ± 0.884 nmol/mg protein) and this reduction was statistically significant compared to the TBI group. Thus, pre-treatment with apocynin significantly attenuated the TBI-induced MDA increase (Fig. 5). 3.6. Effect of apocynin on apoptosis in the cortex of mice subjected to TBI As shown in Fig. 6, a small, but quantifiable number of TUNELpositive cells were observed in sham mice. TBI caused a significant increase in TUNEL-positive cells compared to those in sham mice (P < 0.05). Notably, apocynin treatment significantly decreased the number of TUNEL-positive cells compared to TBI (P < 0.05). 4. Discussion The present study demonstrates that NOX2 expression was increased after TBI, with two peaks at 1 and 24 h. This effect was
Fig. 6. Effect of apocynin treatment on apoptotic cells in mouse brain tissue 24 h after TBI. Typical TUNEL staining in sham (A), TBI (B), TBI + vehicle (C) and TBI + apocynin (D) brain tissue. A high apoptotic index was found in the TBI group compared to sham. In the TBI + apocynin group, the apoptotic index was significantly decreased. There was no difference between TBI and TBI + vehicle groups (NS). Data are presented as mean + sd, n = 4. ⁄P < 0.05 vs. sham, #P < 0.05 vs. TBI group.
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associated to the appearance of neurological deficits, to an increase in MDA, a biomarker of oxidative stress, and to damage of the BBB. Importantly, the data revealed that apocynin injected intraperitoneally 30 min before TBI could decrease NOX2 and MDA expression. Lastly, the results showed that mice pre-treated with apocynin have better neurological outcome and decreased tunnel index. These results indicate that NOX activation is a key mediator of neuronal death and that apocynin may have neuroprotective effects. ROS generation and oxidative stress have been proposed to play a critical role in TBI. ROS are known to not only damage cellular membranes by inducing lipid peroxidation. They can also damage DNA, proteins and lipids. The most studied ROS are O2 — superoxide radical; H2 O2 — hydrogen peroxide; and OH — hydroxyl radical originating from one, two or three electron transfers to dioxygen (O2). It has been generally assumed that mitochondria are the major source of ROS following brain injury. Whereas, recent work has shown that NOX is the central enzyme in ROS cellular production (Kandasamy et al., 2013; Lambert and Brand, 2009). Nevertheless, despite a strong rational of ROS being a pharmacological target, all antioxidant interventions failed to improve functional outcome in human clinical trials. Antioxidants may interfere with physiological functions of ROS or may not reach the crucial target structures of ROS-induced injury effectively. As an evolution of this concept, recent studies have targeted NADPH – the sources of ROS – rather than ROS themselves (Cairns et al., 2012; Pendyala and Natarajan, 2010). NOX is a membrane enzyme that is composed of several subunits, which include NOX and phox subunits. There are at least five different cellular NOX isoforms, termed NOX1–5 (Bedard and Krause, 2007). Previous studies have shown that at acute time points (1 h) following TBI, NOX2 is located in neurons, whereas at later time-points (24–48 h) it is mainly located in the microglia (Dohi et al., 2010; Flierl et al., 2009). Accumulating evidence indicates that NOX2 activation is associated with an increased risk for neuronal damage following experimental TBI, stroke and other degenerative disease. De Silva and collaborators found that NOX2 / mice, subjected to middle cerebral artery occlusion (MCAO), had smaller infarct volumes than wild type mice, edema volume also tended to be smaller compared to wild type mice (De Silva et al., 2011). Liu and colleagues also found that the remarkable Evans blue extravasation, a measure of BBB damage, and significantly increased NOX2 expression in the ischemic brain present in wild type MCAO mice, were significantly attenuated in NOX2 knockout mice. The author confirmed that activation of NOX2 containing NADPH oxidase was implicated in the induction of MMP-9, in loss of occludin and BBB disruption following ischemic stroke (Liu et al., 2011). In the present study we demonstrated that NOX2 expression was upregulated after TBI and that two peaks were observed at 1 h and 24 h after TBI, associated with BBB disturbance and increase in MDA levels. These finding are in line with those by Zhang and collaborators, who also found two NADPH oxidase activity peaks and superoxide production in TBI mice (Zhang et al., 2012). These and other studies imply that NOX2 plays a significant role in TBI physiopathologic mechanism and identified NOX2 as a promising target for pharmacological therapy to treat the pathology and dysfunction caused by TBI. Apocynin is a highly selective, cell-permeable NOX inhibitor. It is thought to act by preventing the translocation of NOX cytosolic components, p47phox to p91phox (NOX2) (Peters et al., 2001; Ximenes et al., 2007). Since apocynin half-life is about 1 h, and since we have shown that there is a rapid and robust elevation in NOX2 within 1 h following TBI, we delivered a single dose of apocynin to prevent TBI-induced NOX activation 30 min prior to TBI. This dosing strategy is not expected to induce any toxicity and could have neuroprotective effects after brain injury (Van den Worm et al., 2001; Yong Choi et al., 2012). In this study we
found that apocynin decreases NOX2 and MDA activation, and protects the tissue against oxidative stress. Moreover apocynin has also been reported to reduce the ischemic area, enhance SOD antioxidant activity, attenuate the increased BBB permeability and further reduce brain edema following TBI (Yong Choi et al., 2012; Yoshioka et al., 2010). Accordingly, apocynin loses its neuroprotective effect in NOX2 / mice (Chen et al., 2011; Pepping et al., 2013). In a recent study, Ferreira and collaborators revealed that apocynin (5 mg/kg), when injected subcutaneously 30 min and 24 h after injury, provided protection against moderate fluid percussion injury (mlFPI)-induced object recognition memory impairment 7 days after neuronal injury. The same treatment also protected against mlFPI-induced IL-1, TNF-a and nitric oxide metabolite content (NOx) 3 and 24 h after neuronal injury (Ferreira et al., 2013). Apocynin also acts as a direct ROS scavenger in given experimental conditions (Heumüller et al., 2008). Taken together, this evidence demonstrates that apocynin has distinct neuroprotective effects achieved by inhibition of NOX2. The effect of inhibition of NOX on oxidative stress was assessed by measuring MDA, a product of lipoxygenase. MDA is a major biomarker of oxidative stress. It increases due to lipid peroxidation, which is one of the harmful consequences of TBI that rapidly causes cellular injury. In the present study, we reported that attenuation of NOX2 expression by apocynin markedly decreased TBI-induced MDA level in the brain. Thus attenuation of MDA activation may represent an effective strategy to prevent ROS-mediated brain damage (Altintas et al., 2013; Sahna et al., 2003). Several limitations of the present study should be considered when interpreting our results. The number of animals in each group was small, such that statistical power may have been compromised given the inherent variability in brain injury resulting from the weight-drop model. In addition, a ‘‘pretreatment’’ study is not really clinically applicable. It remains to be established if apocynin is useful as a therapeutic when administered after TBI. Moreover, our study design reveals associations, but is not indicative of causality, which might entail additional arms such as a nox2 knockout group, or use of direct nox2 blockers. Lastly, in the present study we omitted the sham + apocynin control group thus preventing a direct comparison with the sham and with the TBI + apocynin group. This may be potentially important in our pretreatment study, as the effects of pre-injury administration of the drug alone were not studied.
5. Conclusions In summary, the results of the present study demonstrate that NOX2 activation can contribute significantly to MDA generation, disturbance of BBB and neuronal cell death following TBI. Our data also suggest that administration of apocynin may be a viable neuroprotectant and a potential adjuvant therapy for patients with TBI. It remains to be established if apocynin administration following TBI is likewise effective in reducing the development of secondary damage.
Conflict of interest No competing financial interests exist.
Acknowledgment This study was supported by the Science and Technology Development Fund of Zhenjiang (No. SH20130037).
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