TAK1 inhibition attenuates early brain injury after SAH

Accumulating evidence suggests that activation of mitogen-activated protein kinases (MAPKs) and nuclear factor NF-κB exacerbates early brain injury (EBI) following subarachnoid hemorrhage (SAH) by provoking pro-apoptotic and pro-inflammatory cellular signaling. Here we evaluate the role of TGFβ-activated kinase 1 (TAK1), a critical regulator of the NF-κB and MAPK pathways, in the early brain injury following SAH. Although the expression level of TAK1 did not present significant alternation in the basal temporal lobe after SAH, the expression of phosphorylated TAK1 (Thr187, p-TAK1) showed substantial increase 24 h post SAH. Intracerebroventricular injection of a selective TAK1 inhibitor (10 min post-SAH), 5Z-7-oxozeaenol (OZ), significantly reduced the levels of TAK1 and p-TAK1 at 24 h post SAH. Involvement of MAPKs and NF-κB signaling pathways was revealed that OZ inhibited SAH-induced phosphorylation of p38 and JNK, the nuclear translocation of NF-κB p65, and degradation of IκBα. Further, OZ administration diminished the SAH-induced apoptosis and EBI. As a result, neurological deficits caused by SAH were reversed. Our findings suggest that TAK1 inhibition confers marked neuroprotection against EBI following SAH. Therefore, TAK1 might be a promising new molecular target for the treatment of SAH. Subarachnoid hemorrhage (SAH) is a devastating neurological injury associated with significant patient morbidity and mortality. Despite the severe impact of SAH on public health, no specific pharmacological therapy for SAH is available that would improve the outcome of these patients. A growing body of evidence suggests that early brain injury (EBI) may contribute to the poor outcome and early mortality following SAH (1-3); However, elucidation of the complex cellular mechanisms underlying EBI remains a major challenge. Recently, multiple signaling pathways that are activated within minutes after the initial bleeding, which leads to early brain injury, are identified (4). Among them, activation of mitogen-activated protein kinases (MAPKs), particularly c-jun-N-terminal kinases (JNK) and p38, exacerbates SAH injury by provoking pro-apoptotic and pro-inflammatory cellular signaling (5). Additionally, NF-κB signaling activation also raises SAH-induced inflammatory responses and leads to worse SAH outcomes (6). Since multiple mechanisms contribute to EBI pathophysiology, it is thought that a drug specific for a single target may not give rise to meaningful improvements in neurobehavioral outcome. Alternatively, inhibition of a target involved in multiple EBI pathways may be a successful strategy. For this reason, it is appreciated that those agents, which have the ability to intervene at more than one critical pathway in the early brain injury after SAH, will have greater advantage over other by gest on N ovem er 8, 2017 hp://w w w .jb.org/ D ow nladed from TAK1 inhibition attenuates early brain injury after SAH 3 single-target agents. TGFβ-activated kinase 1 (TAK1) may be such a target. TAK1 is a member of the mitogen-activated protein kinase (MAPK) kinase kinase family and was initially found to function in TGF-β-mediated MAPK activation (7). Previous study demonstrated that TGF-β is a fibrogenic factor involved in the etiology of post-SAH pathology and plays an important role in generating communicating hydrocephalus after SAH (8,9). Several other known stimuli of TAK1-signaling, including IL-1 β, TNF-α and TLR4 have also been shown to be activated and to play a detrimental role in SAH (10-12). The activated TAK1 in turn activates the NF-κB and MAPK pathways (13,14). Recent investigations also demonstrate that inhibition of TAK1 provides neuroprotection in ischemia and traumatic brain injury (15-17). Taking into account all this background, the present study aimed to evaluate whether TAK1 is activated after SAH. Moreover, the possible role of TAK1 in the regulation of SAH-induced neuronal apoptosis was also analyzed by means of TAK1 pharmacological modulation with its specific inhibitor 5Z-7-oxozeaenol (OZ). EXPERIMENTAL PROCEDURES Animals and subarachnoid hemorrhage modelThe male Sprague-Dawley rats weighing 300–350 g were used in this study. Rats were housed in a reversed 12-h light/12-h dark cycle controlled environment with free access to food and water. All procedures were approved by Nanjing University Animal Care and Use Committee and in accordance with the Guide for the Care and Use of Laboratory Animals by the National Institute of Health (NIH). Experimental SAH was performed as described previously in our laboratory and other laboratories (18-21). First, the rats were intraperitoneally anesthetized with 10% chloral hydrate (400 mg/kg body weight) and placed in a stereotaxic head frame. An insulin injection needle (BD Science) was tilted 45° in the sagittal plane, and placed 8 mm anterior to bregma in the midline, with the hole facing the right side. It was lowered until the tip reached the base of the skull, 2-3 mm anterior to the chiasma (approximately 10-12 mm from the brain surface) and retracted 0.5 mm. Loss of cerebrospinal fluid and bleeding from the midline vessels were prevented by plugging the burr hole with bone wax before inserting the needle. A total of 300 μl non-heparinized fresh autologous arterial blood was slowly injected into the prechiasmatic cistern for 3 min under aseptic technique. The heart rate was monitored and the rectal temperature was kept at 37 ± 0.5 °C by using physical cooling (ice bag) when required throughout experiments. Arterial blood samples were analyzed intermittently to maintain pO2, pCO2, and pH, parameters within normal physiological ranges. To maintain fluid balance, all rats were supplemented with 2 ml of 0.9% NaCl administered subcutaneously. After recovering from anesthesia, rats were returned to their cages with free-access food and water provided adlibitum. In the present study we observed that the basal temporal lobe was always stained with blood as described before. Therefore, the brain tissue adjacent to the clotted blood was used for analysis in our study (Fig. 1D). Experimental design 1 A total of 64 rats were used in this experiment. Of them, six rats with SAH were excluded later from the study because of little blood in prechiasmatic cistern but lots of blood clot in the frontal lobe instead, and ten SAH rats died before the intended sacrifice. The animals were randomly assigned to six groups post SAH: animals surviving SAH for 2 h (n=6), 6 h (n=6), 12 h (n=6), 24 h (n=9), 48 h (n=6) and 72 h (n=6). Control (n=9) animals underwent the by gest on N ovem er 8, 2017 hp://w w w .jb.org/ D ow nladed from TAK1 inhibition attenuates early brain injury after SAH 4 exactly same procedure as described above with the exception that no blood was injected intracisternally. Tissue was harvested at the designed time points for the evaluation of TAK1 and p-TAK1 expression (n=6 each). Cellular distribution of TAK1 was also determined by double-staining immunefluorescence at 24 h post SAH and control groups (n=3 each). Experimental design 2 To determine the role of TAK1 in SAH, rats were randomly allocated into the following groups and survived 24 h: 1) Control group (n=18): Control animals were injected with 0.3 ml saline. 2) SAH group (n=12); 3) SAH+vehicle group (n=18): rats were subjected to SAH plus intracerbroventricular (ICV) administration of 5 μl DMSO; 4) SAH+OZ group (n=18): rats were subjected to SAH plus intracerbroventricular administration of the TAK1 inhibitor OZ (Tocris Bioscience;25 μg 10 min post SAH). This dose was based on our prior published work using OZ in traumatic brain injury model (15). Coordinates for the injection placement were 1.0 mm posterior to bregma, 1.4 mm lateral to midline, and 4.4 mm below the skull surface and the injection duration was 10 min. Following the behavioral test, brain tissue of half number of rats was harvested for biochemical and histopathological analysis at 24 h post SAH. A total of 85 rats were used in this experiment (Seven rats with SAH were excluded later from the study for the same reason mentioned in experiment 1, twelve SAH rats died before the intended sacrifice). The neurological scoring was performed with a separate cohort of rats at 24 h and 72 h after surgery. Finally, the rats were sacrificed for histological analysis. A total of 56 rats were used in this experiment (Four rats with SAH score of 0 were excluded from the study for low SAH grade, eight SAH rats died before the intended sacrificed). Experimental design 3 To determine whether administration of the TAK1 inhibitor OZ confers brain protection with a wide therapeutic window against SAH, OZ or vehicle was administered 4 h post SAH. Neurological scores were evaluated at 24 and 72 h post SAH, and histological analysis was performed at 72 h. A total of 30 rats were used in this experiment (Three rats with SAH score of 0 were excluded from the study for low SAH grade at 24 h post SAH, six SAH rats died before the intended sacrifice). Biochemical analysis The whole cell protein extraction and nuclear fractions were prepared as previously described (15). For western analysis, 35 μg of total protein were loaded in each lane of SDS-PAGE, electrophoresed, and transferred to a nitrocellulose membrane. The blot containing the transferred protein was blocked in blocking buffer for 1 h at room temperature followed by incubation with appropriate primary antibody in blocking buffer for 2 h to overnight at 4°C. The primary antibodies waere against cleaved-caspase-3 (cat# 9661), p-ERK1/2 (cat# 4370P), p-JNK (cat# 4668P), p-c-Jun (cat# D47G9), p-P38 (cat# 4511), histone 3 (H3, cat# 9715), or β-actin (cat# 4967) (1:1000, from Cell Signaling Technology, Danvers, MA), interleukin (IL)-1β (cat# ab9722), occludin (cat# ab31721), p-TAK1(Ser439, cat# ab109404) and phospho-TAK1 (Thr187, cat# ab192443) ( 1:1000, Abcam, Cambridge, MA), TAK1 (cat# sc-7162), p65(cat# sc-372), IκBα (cat# sc-371), zonula occludens-1 (ZO-1, cat# sc-10804), and tumour necrosis factor (TNF)-α (cat# sc-52746), (1:200; from Santa Cruz Biotechnology, Santa Cruz, CA). Signal was detected with horseradish peroxidase-conjugated immunoglobulin G (IgG) using enhanced chemiluminescence detection reagents (Amersham International, Buckinghamshire, UK). Blot bands were quantified by densitometry with Image J software (Image J, NIH). by gest on N ovem er 8, 2017 hp://w w w .jb.org/ D ow nladed from TAK1 inhibition attenuates early brain injury after SAH 5 Histological examination Rats were anesthetized and transcardially perfused with saline and 4% paraformaldehyde. The brains were removed and postfixed in paraformaldehyde for 24 h. For immunofluorescence microscopy, the brains were frozen in O.C.T. media, sectioned (10 μm), and mounted onto slides. Immunofluorescence staining was performed as described previously (15). The specificity of immunofluorescence reaction was evaluated by replacement of the primary antibody with rabbit IgG. Terminal dUTP Nick-end Labeling (TUNEL) assay was conducted by using a TUNEL detection kit according to the manufacturer’s instructions (In situ Cell Death Detection Kit,Roche Applied Science, Indianapolis, IN). In brief, each section was incubated with 50 μl TUNEL reaction mixture (contains 5 μl Enzyme solution and 45 μl Label Solution) for 1 h at 37°C in the dark and then washed with PBS. Slides were then counter-stained with 4,6-diamidino-2phenylindole (DAPI), washed, cover slipped with a water-based mounting medium, and sealed with nail polish. For the negative control, the sections were only incubated with Label solution. The positive cells were identified, counted and analyzed under the light microscope by an investigator blinded to the grouping. The extent of brain damage was evaluated by the apoptotic index, defined as the average percentage of TUNEL-positive cells in each section counted in 10 cortical microscopic fields (at × 400 magnification). A total of four sections from each animal were used for quantification. The final average percentage of TUNEL-positive cells of the four sections was regarded as the data for each sample. Formalin-fixed brains (72 h post-SAH) were dehydrated, embedded in paraffin and sliced into 4 μm thick sections which were stained with Cresyl Violet. The sampled region for each subfield was demarcated in the inferior basal temporal lobe and cresyl-violet neuronal cell bodies were counted. To quantify the amount of Nissl staining, ten random high-power fields (400×) in each coronary section were chosen, and the mean number of intact neurons in the ten views was regarded as the data of each section.A total of four sections from each animal were used for quantification. The final average number of the four sections was regarded as the data for each sample. The averages of 10 different fields of view were calculated for each animal. A total of five sections (with a minimum of 100 μm from the next) from each animal were used for quantification. The final average number of the five sections was regarded as the data for each sample. Brain water content (Brain edema) Brains were removed 24 h after surgery in experiment 2 and weighed immediately (wet weight) and reweighed after drying in 100°C for 72 h (dry weight). The percentage of water content was calculated as [(wet weightdry weight)/wet weight] × 100%. Neurologic scoring Three behavioral activity examinations to record appetite, activity, and neurological deficits (Table 1) were performed by an investigator blinded to the study groups at 24 and 72 h after SAH using the scoring system reported previously (22,23). Statistical analyses Data are expressed as mean ± SEM. One-way ANOVA followed by Tukey test was used to analyze differences between groups except for the neurobehavioral scores, which were analyzed with nonparametric tests (Kruskal-Wallis, followed by Dunnˊs post-hoc test). SPSS 16.0 was used for the statistical analysis (SPSS, Inc., Chicago, IL). by gest on N ovem er 8, 2017 hp://w w w .jb.org/ D ow nladed from TAK1 inhibition attenuates early brain injury after SAH 6 Differences were determined to be significant with p< 0.05.