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. Author manuscript; available in PMC: 2019 Jun 1.
Published in final edited form as: Neurochem Int. 2018 Mar 9;116:1–12. doi: 10.1016/j.neuint.2018.03.004

Tert-butylhydroquinone Post-treatment Attenuates Neonatal Hypoxic-ischemic Brain Damage in Rats

Juan Zhang 1,2, Lorelei Donovan Tucker 2, Dong Yan 2, Yujiao Lu 2, Luodan Yang 2, Chongyun Wu 2, Yong Li 2, Quanguang Zhang 2,*
PMCID: PMC5895521  NIHMSID: NIHMS953929  PMID: 29530758

Abstract

Hypoxic-ischemic (HI) encephalopathy is a leading cause of dire mortality and morbidity in neonates. Unfortunately, no effective therapies have been developed as of yet. Oxidative stress plays a critical role in pathogenesis and progression of neonatal HI. Previously, as a Nrf2 activator, tert-butylhydroquinone (TBHQ) has been demonstrated to exert neuroprotection on brain trauma and ischemic stroke models, as well as oxidative stress-induced cytotoxicity in neurons. It is, however, still unknown whether TBHQ administration can protect against oxidative stress in neonatal HI brain injury. This study was undertaken to determine the neuroprotective effects and mechanisms of TBHQ post-treatment on neonatal HI brain damage. Using a neonatal HI rat model, we demonstrated that TBHQ markedly abated oxidative stress compared to the HI group, as evidenced by decreased oxidative stress indexes, enhanced Nrf2 nuclear accumulation and DNA binding activity, and up-regulated expression of Nrf2 downstream antioxidative genes. Administration of TBHQ likewise significantly suppressed reactive gliosis and release of inflammatory cytokines, and inhibited apoptosis and neuronal degeneration in the neonatal rat cerebral cortex. In addition, infarct size and neuronal damage were attenuated distinctly. These beneficial effects were accompanied by improved neurological reflex and motor coordination as well as amelioration of spatial learning and memory deficits. Overall, our results provide the first documentation of the beneficial effects of TBHQ in neonatal HI model, in part conferred by activation of Nrf2 mediated antioxidative signaling pathways.

Keywords: Neonatal hypoxia-ischemia, Tert-butylhydroquinone, Nrf2, Oxidative stress, Neuroprotection

1. Introduction

Neonatal hypoxic-ischemic encephalopathy (HIE) is the most common perinatal brain disorder, and is associated with fearful morbidity and mortality (Fatemi et al., 2009; Zhu et al., 2015). HIE often leads to severe neurological sequelae, such as cerebral palsy, epilepsy and intellectual disabilities. Treatment and care for these neurological deficits imposes considerable financial and lifelong burdens to the affected individuals, their families, and society at large (Pazos et al., 2012). As a result, HIE is a significant global public health care problem. Many studies have been aimed at investigation of feasible and effective strategies for the treatment of HIE, such as therapeutic hypothermia (TH), hyperbaric oxygen therapy, stem cell therapy, anticonvulsant and neuroprotective drugs including erythropoietin, melatonin, desferrioxamine, resveratrol and sulforaphane (Buonocore et al., 2012; Zhu et al., 2015). Despite these efforts, however, there are limited effective therapy options currently available. Herein, there is an urgent need for more effective treatments to ameliorate the damage and disability associated with neonatal HIE.

The pathogenesis of HIE is complex and multifactorial, including energy failure, glutamatergic excitotoxicity, calcium overload, inflammation, reactive oxygen species (ROS)-mediated toxicity, endothelial cell dysfunction, etc., which culminates finally in neurocyte death by a mixture of necrosis and apoptosis (Northington et al., 2011). Recently, oxidative stress has been reported as a critical causative factor in HIE (Noor et al., 2007). The rich polyunsaturated fatty acid content, high rate of oxygen consumption, increased availability of free iron, and low antioxidant capabilities in the immature brain, compared to the adult brain, all contribute to sensitize the neonatal brain to oxidative stress after HIE (Esih et al., 2017). In pathological conditions, excessive free radical production can effectively overwhelm production of antioxidative enzymes (Birben et al., 2012). Free iron further contributes to cytotoxicity by promoting the generation of ROS (Abdal Dayem et al., 2017). Increased ROS output rapidly damages cell membranes, proteins, lipids, DNA, and results in a cascading inflammatory response. These derivatives contribute to a complex interplay of apoptosis, autophagy and necrosis resulting in brain injury (Zhao et al., 2016b). Previous investigations have indicated that, as a new therapeutic approach, exogenous antioxidant therapy exhibits effective neuroprotection against neonatal HI brain damage. Some of these exogenous antioxidants have demonstrated neuroprotective effects in human trials, i.e., the erythropoietin and melatonin (Arteaga et al., 2017). Before vital molecules are subject to insult, antioxidants can safely interact with oxygen free radicals and terminate that chain response to protect against oxidative damage (Lobo et al., 2010).

Notably, the nuclear factor erythroid 2-related factor-2 (Nrf2) pathway has pleiotropic actions in antioxidative stress, anti-apoptotic, anti-inflammatory, anti-atherosclerotic, and anti-tumorogenic signaling, and is known to regulate glutathione (GSH) synthesis and cerebrovascular reactivity, conferring neuroprotection against oxidative insult. In this manner, Nrf2 activators have become attractive neuroprotective candidates for cerebral ischemia (Jiang et al., 2017a; Lu et al., 2016). It has been shown that mice deficient of Nrf2 are especially sensitive to oxidative stress (Ma, 2013). Under normal conditions, Nrf2 is sequestered in the cytoplasm, but is translocated to the nucleus in response to oxidative stress. Next, it can activate a range of antioxidant downstream genes, such as heme oxygenase-1 (HO-1), NAD(P)H: quinone oxidoreductase-1 (NQO1) and superoxide dismutase2 (SOD2), to scavenge ROS and prevent damage by oxidative stress (Jiang et al., 2017b).

The Nrf2 activator, TBHQ is an effective phenolic antioxidant widely used in foods, as well as in medicines and cosmetics. Numerous previous studies reported that TBHQ has a hydroquinone-type electrophilic structure that contributes to activate Nrf2 transcription (Satoh et al., 2009), counteract oxidative damage and exhibit neuroprotective effects in different models of central nervous system injury (Jin et al., 2014; Lu et al., 2014; Shih et al., 2005). As the effects of TBHQ treatment against HIE has yet to be determined, the present study aims to examine whether TBHQ post-treatment confers neuroprotection in a neonatal HI rat model. As well, this study seeks uncover the underlying mechanisms involved to determine whether TBHQ may serve as a possible exogenous antioxidant therapeutic agent against HIE.

2. Materials and Methods

2.1. Animal model of HI brain injury

In a modified version of a previously described HI model (Yuan et al., 2014), unsexed 10-day-old Sprague-Dawley rats (Charles River Laboratories) were anesthetized, and the right common carotid artery (RCCA) was isolated and ligated permanently through a midline neck incision. Animals were returned to cages for 1.5 h after wound closure. Rats were then placed in a hypoxic environment (6% oxygen/94% nitrogen) for 2 h. The temperature was maintained at 37°C. All procedures were approved by the Animal Care and Use Committee of Augusta University and were in accordance with National Institutes of Health guidelines.

2.2. Experimental design and administration of drugs

Rats were randomly allocated into four groups: (a) Sham + vehicle control group, treated with 1% DMSO in PBS without RCCA ligation; (b) HI + vehicle group, treated with 1% DMSO in PBS; (c) HI + TBHQ group, treated with TBHQ (Acros Organics); and (d) HI + TBHQ + brusatol (Brus) group, treated with brusatol (Sigma-Aldrich), a unique inhibitor of the Nrf2 pathway. TBHQ was dissolved in 1% DMSO and injected intraperitoneally (i.p.) at a dose of 20 mg/kg after one hour of HI and repeated once daily for seven consecutive days. Fresh TBHQ solutions were prepared for each injection. To confirm the role of Nrf2 pathway in TBHQ neuroprotection, brusatol was dissolved in 1% DMSO and administrated at a dose of 1.0 mg/kg via i.p. in the combination with TBHQ. The experimental protocol was shown in Fig. 1A. HI brain injury was induced on rat postnatal day 10 (P10). From P11 to P14, the righting reflex test was applied to investigate the short-term behavioral outcomes. From P28 to P31, the long-term behavioral tests were performed, such as the beam-walking, cylinder and Barnes maze task. Rats were sacrificed under anesthesia at P17 and P31, respectively. The brains were collected for further analysis.

Fig. 1. TBHQ decreased infarct size at the late stage after HI.

Fig. 1

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A Schematic illustration of the experimental design. B, C Representative images for cresyl violet (CV) staining and analysis of brain infarct size at P31 from different experimental groups: (a, b) Sham + vehicle group (Sham); (c, d) HI + vehicle group (HI); (e, f) HI + TBHQ group (TBHQ); and (g, h) HI + TBHQ + brusatol (Brus) group. Infarct size was expressed as the percentage difference in area of CV staining. Scale bar = 500 μm. Data were presented as mean ± SE (n = 8). *P < 0.05 versus HI group, #P < 0.05 versus TBHQ group.

2.3. Histology examination and infarct measurement

Rats were anesthetized and perfused transcardially with cold PBS followed by 4% paraformaldehyde (PFA). Brains were extracted and postfixed overnight in 4% PFA, and were then submersed in 30% sucrose until they sank. Brains were then immersed in cryoprotectant and frozen overnight at −80°C. Coronal brain sections (25 μm) were prepared using a cryostat microtome. Histological examination was carried out using the methods as described previously by our laboratory (Lu et al., 2017a; Zhang et al., 2013). At least 3–5 representative sections in the coronal plane (>100 μm gap between each section, ~2.5–4.5 mm posterior from Bregma) of each animal were selected for analysis. Sections were stained with 0.01% (w/v) cresyl violet (CV) for 30 min, following graded ethanol dehydration as described previously by our laboratory (Ahmed et al., 2016). Stained sections were mounted and examined under light microscopy and the mean infarct area of each section was quantified with ImageJ software. Histological analysis was performed by investigators blinded to the staining groups. Infarct size was calculated according to the following formula: size = (area of contralateral hemisphere – area of intact ipsilateral hemisphere) / area of contralateral hemisphere × 100 %.

2.4. Brain homogenates and subcellular fractionations

The ipsilateral damaged cerebral cortex was microdissected quickly 7 d after HI (P17) and frozen in liquid nitrogen immediately. The cytosolic and nuclear extraction were performed, as previously reported by our laboratory (Zhang et al., 2008), with a few modifications. Briefly, tissue samples were homogenized in ice cold homogenization medium buffer A. Then, the samples were sharply vortexed and sonicated 2 times for 2s, then centrifuged at 800 × g for 10 min. Supernatants were centrifuged again at 15,000 × g for 30 min at 4°C, and were kept at −80°C as cytosolic fractions. The nuclear pellets were washed three times using buffer A, resuspended in buffer B, and then were vigorously rocked at 4°C for 30 min and sonicated 2 times for 2s. The samples were then centrifugated at 12,000 × g for 15 min at 4°C. The protein samples were aliquoted and frozen in liquid nitrogen for long-term storage at −80°C. Protein concentrations were detected using a modified Lowry protein assay.

2.5. Western blotting

Western blotting was performed as described previously by our laboratory (Zhang et al., 2008). The protein from cytosolic and nuclear extracts was separated on 4–20% SDS-polyacrylamide gels, and transferred onto PVDF membrane. The membranes were blocked and incubated with primary antibody Nrf2 (1:500), β-actin (1:2000) and Neuronal nuclear antigen (NeuN, 1:500, Proteintech) at 4°C overnight and HRP-conjugated secondary antibodies (1:1000, Cell signaling) for 1 h at room temperature (RT). Protein bands were visualized with digital imaging system. The protein levels of each sample were normalized as intensity ratio with β-actin or NeuN and were analyzed by ImageJ software.

2.6. Nrf2 DNA-binding activity assay

As previously described (Sukumari-Ramesh and Alleyne, 2016), Nrf2 DNA-binding activity was determined by a Nrf2 transcription factor assay kit obtained from Cayman Chemical (600594), using nuclear proteins from the ipsilateral damaged cerebral cortex collected at the early stage (P17). Briefly, protein samples (containing 30 μg of protein) were added to a 96-well plate which was pre-coated with oligonucleotide containing a consensus-binding site for Nrf2. After incubation, Nrf2 antibody (1:500) was added to each well and incubated for 2 h, followed by HRP-conjugated secondary antibody. Developing and stop solution were added and absorbance was read at 450 nm with a reference wavelength of 650 nm. Absorbance value revealed Nrf2 DNA-binding activity and was expressed as fold changes compared with sham group.

2.7. Immunofluorescent staining and confocal microscopy

As previously reported by our laboratory (Zhang et al., 2008), floating coronal brain sections were washed with PBS and then blocked with 10% normal donkey serum at RT for 1 h. After blocking, the sections were incubated overnight at 4°C with following primary antibodies: NeuN (1:200), Microtubule associated protein 2 (MAP2, 1:100), Ionized calcium-binding adapter 1 (Iba-1, 1:200), NQO1 (1:100), SOD2 (1:100), HO-1 (1:100), Glial fibrillary acidic protein (GFAP, 1:200, Proteintech), Cleaved Caspase-3/9 (1:100, Cell Signaling) and Malondialdehyde (MDA, 1:100, Novus Biologicals). After washes, sections were incubated with corresponding secondary antibodies and mounted with DAPI. Images were captured by a LSM700 Meta confocal laser microscope (Carl Zeiss).

2.8. Total antioxidant capacity assay

Total antioxidant capacity was determined by an antioxidant assay kit obtained from Cayman Chemical (709001). The procedures followed the assay protocol, as previously described by our laboratory (Lu et al., 2017b). Absorbance was measured at 750 nm. The ability of antioxidants in the sample to suppress radical cations was compared with that of Trolox. The standard curve was calculated by Trolox concentration and absorbance. Total antioxidant capacity in cytosolic extracts of the ipsilateral damaged cerebral cortex at P17 was determined using the standard curve. Samples were executed in triplicate.

2.9. Measurement of ROS levels

As previously described (Wang et al., 2011), ROS was detected by dihydroethidium (DHE) staining. Brain sections were incubated with 10 μM DHE (AnaSpec) in the dark for 10 min at RT. After washes, sections were mounted with PBS for visualization using a confocal microscope. The relative levels of ROS were further measured by a fluorescence spectrophotometer (PerkinElmer), using cytosolic proteins from the ipsilateral damaged cerebral cortex at P17. DHE was added to the homogenate to a final concentration of 10 μM. The mixture was incubated in the dark for 10 min at RT. The fluorescence intensity was measured at 485 nm excitation/590 nm emission.

2.10. Inflammatory cytokines assay

Enzyme-linked immunosorbent assay (ELISA) was performed to evaluate the expression levels of Interleukin (IL) 1β, IL-6, IL-18, Tumor necrosis factor-alpha (TNF-α), Intercellular adhesion molecule-1 (ICAM-1) and anti-inflammatory cytokines (IL-10) (1:500, Proteintech) as described in detail by our laboratory (Lu et al., 2017b). Briefly, samples were diluted with PBS to 50 μl containing the same amount of the cytosolic protein fractions taken from the ipsilateral damaged cerebral cortex at P17. The samples, in duplicate, were added to polyvinyl chloride ELISA microplate, blocked and probed with specific antibodies. Absorbance was read at 450 nm using a microplate reader.

2.11. Fluoro-Jade C and TUNEL Staining

Fluoro-Jade C staining was used to detect degenerating neurons in the brain (Dixon et al., 2016). Briefly, brain sections were rinsed in basic alcohol consisting of 1% sodium hydroxide in 80% ethanol, 70% ethanol, distilled water, 0.06% potassium permanganate, 0.0001% solution of Fluoro-Jade C (Histo-chem) dissolved in 0.1% acetic acid vehicle, distilled water and xylene. Then the slides were air-dried and coverslipped using mounting media. TUNEL staining was performed on cryosections using an apoptosis detection kit (Thermo Fisher Scientific) as previously described by our laboratory (Lu et al., 2017b).

2.12. Caspase activity assay

Caspase-9 and caspase-3 activities in the cytosolic protein samples, taken from the ipsilateral damaged cerebral cortex at P17, were quantified as previously reported by our laboratory (Lu et al., 2017b). The values were expressed as fold change in fluorescent units compared with sham group.

2.13. Neurobehavioral analysis

2.13.1. Righting reflex test

As previously described (Schuch et al., 2016), righting reflex is related to subcortical maturation. As neonatal rats mature, the coordination of their movements and speed of righting developed quickly. Rats were put on their backs, and the time taken to turn over to the prone position was measured. The righting reflex time was measured from P11 to P14.

2.13.2. Beam-walking test

As previously reported by our laboratory (Ahmed et al., 2016), the beam-walking test was conducted to observe motor coordination at P30. A wooden beam 100 cm long and 7 cm wide was elevated 100 cm above the ground. The rat was placed at one end of the beam with home cage at the opposite end to motivate the animal to cross the beam and return to its cage. Rats were trained for 2 days before P30. The time taken to traverse the beam (completion time) was measured by an ANY-maze video tracking system.

2.13.3. Cylinder test

The cylinder test was applied to evaluate the asymmetry in use of the contralateral (left) forepaw indicated the deficit of ipsilateral (right) neurological function at the late stage (P31) as previously described by our laboratory (Ahmed et al., 2016). The animal was placed in a transparent glass cylinder, 10 cm in diameter and 15 cm high. The testing session was recorded for 2 min. The experimenter counted the numbers of independent uses of the left or right forepaws to contact with the side of the cylinder. The percentage of initial forepaw preference was scored by dividing contralateral forepaw use by total forepaw use.

2.13.4. Barnes maze test

Spatial learning and memory were assessed by Barnes maze test, as previously reported (Lu et al., 2017b). The maze was a circular platform with 18 equally spaced holes. A black escape box (target box) was located under one of the holes. A darkened opaque curtain surrounded the maze and bright flood incandescent light shining down on the maze center to ensure a uniform surrounding visual field. When the test started, rats were placed in the middle of the Barnes maze. Aversive noise was used to impel rats to find and enter the target box. The behavior tests were divided into training trials and probe test. The rat was given three minutes to find the target box in 3 days of training trials. If after three minutes the rat had not found the box, it was gently directed to it. Upon entering the escape box, the rat was allowed to remain in it with the hole covered for 30 seconds to habituate. On each training trial day, latency to find the escape hole was recorded by an ANY-maze video tracking system. The probe test was carried out at P31. The escape box was removed and the hole was covered. Time spent in the target quadrant where the target box had been located was recorded with probe duration of 90s. The quadrant occupancy was quantified by ANY-maze software.

2.14. Data analysis

Data were presented as mean ± standard error (SE). Our preliminary work and extensive past experience using the ischemic model, as well as power analysis, assure the sample size was sufficient to observe statistical differences between treatment groups. Data analysis was performed using one-way analysis of variance (ANOVA) followed by Student-Newman Keul’s or Dunnett’s post-hoc tests to determine group differences, unless otherwise noted. Significance was accepted at p < 0.05.

3. Results

3.1. Effects of TBHQ post-treatment on infarct size and neuronal damage

To determine the effects of TBHQ administration on neonatal HI brain damage at P31, whole-brain sections were stained with CV. As shown in Fig. 1B and C, the result revealed that no damage was observed in the either cerebral hemisphere of sham animals. In contrast, a large lesion developed in the ipsilateral side featured with the shrinkage of the right hemisphere and significant tissue loss after HI. Remarkably, animals treated with TBHQ displayed an infarct size that was significantly lower than that of HI group (p < 0.05). Intriguingly, the protective effect of TBHQ against HI injury was remarkably attenuated by brusatol, a selective inhibitor of the Nrf2 pathway (Fig. 1B and C).

Furthermore, NeuN, MAP2 and CV staining were used to identify the effects of TBHQ treatment in the ipsilateral damaged cerebral cortex at P17 after HI insult. As shown in Fig. 2A(j–l), the HI group revealed a significant loss of NeuN positive neuronal cells in the ipsilateral cortex compared to sham that was reduced with TBHQ treatment. MAP2 is largely accepted as a sensitive and early indicator for the assessment of neuronal injury. The results depicted in Fig. 2A(m–o) indicated less MAP2 fluorescent staining intensity and increased dispersion in the HI group compared to sham, an effect reversed distinctly by TBHQ treatment (Fig. 2B and C). Representative images of CV staining for neuropathological injury at P17 were also shown in Fig. 2A(p–r). The sham group had abundant cytoplasm and Nissl bodies present with a clear, intact cell outline. Substantial neuronal loss and dead cells were present in the damaged cortex of HI animals, with blurred cell outlines and sparse arrangement. The number of cells displaying eumorphism was significantly increased by TBHQ treatment in contrast with HI group, demonstrating robust neuroprotection conferred by TBHQ.

Fig. 2. TBHQ increased Nrf2 nuclear accumulation and mitigated neuronal damage.

Fig. 2

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A Immunofluorescent staining of Nrf2 (a–c; 40×), NeuN (d–f; 40×), and MAP2 (m–o; 40×) as well as the CV staining (p–r; 20×) in the ipsilateral damaged cerebral cortex region. a–i and m–o are magnified in the boxed regions (j–l) showing the damaged cerebral cortex. Boxed areas in (g–i) shown in merged images indicated co-localization of Nfr2 and NeuN at a higher magnification. Scale bars: a–i and m–r = 50 μm, j–l = 500 μm. B, C Quantification of MAP2 fluorescent intensity and dispersion were analyzed by using ImageJ software. D Representative Western blotting and quantification of Nrf2 levels were shown. Protein levels of Nrf2 from the cytosolic (a) and nuclear extracts (b) were expressed as ratio to β-actin and NeuN, respectively. Data were expressed as fold changes versus sham and the analysis was represented from 4–5 independent animals. E DNA binding activity of Nrf2 was detected as described in methods. Data were expressed as percentage changes versus sham group (mean ± SE, n = 6). *P < 0.05 versus sham group, #P < 0.05 versus HI group, $P < 0.05 versus TBHQ group.

3.2. Effects of TBHQ post-treatment on Nrf2 translocation and DNA binding activity

As previously reported and mentioned above, TBHQ affords cellular protection and defense against oxidative stress by inducing Nrf2 nuclear translocation. Herein, Nrf2 translocation and DNA binding activity were investigated after TBHQ treatment in the damaged cortex region at P17. As depicted in Fig. 2A(a–i), TBHQ was demonstrated to induce activation of the Nrf2 pathway, as evidenced by increased the levels of Nrf2 nuclear localization. In contrast, neuronal Nrf2 expression of sham and HI groups was mainly cytoplasmic. Further, semiquantitative immunoblotting was performed to determine the expression of Nrf2 levels in the cytosolic and nuclear extracts, an indicated in Figure 2D(a, b). The results agreed well, demonstrating significantly increased nuclear accumulation of Nrf2 observed in the TBHQ group as evidenced by fluorescent staining (p <0.05, Fig. 2B). In addition, we analyzed the efficacy of TBHQ in inducing Nrf2 DNA binding activity and the effect of brusatol in inhibiting the Nrf2 pathway, 1 h after administration to show responsiveness to the drug in the brain. As shown in Fig. 2E, rats treated with TBHQ displayed significant increases in Nrf2 DNA-binding activity compared to HI and sham animals, an effect could be effectively reversed in combination treatment with brusatol.

3.3. Effects of TBHQ post-treatment on oxidative stress status

Immunofluorescent staining was used to determine protein expression of three known downstream antioxidative Nrf2 transcriptional targets: NQO1, SOD2 and HO-1. As shown in Fig. 3A(a–i) and B–D, TBHQ treatment increased the levels of NQO1, SOD2 and HO-1 expression in cortical samples at P17 significantly, compared with HI group. To determine the immunoreactive level of the oxidative stress marker MDA, immunofluorescent staining was performed. Sham group presented very weak fluorescent intensity indicating an absence of oxidative stress. In contrast, great increases in immunostaining intensity for MDA were found in the HI group (Fig. 3A(j–l)), which was reduced dramatically by TBHQ treatment (p <0.05, Fig. 3E). Additionally, measurement of ROS level by DHE staining and fluorescence signal under spectrophotometer revealed that the HI rats exhibited a markedly higher ROS levels compared to sham, which was mitigated significantly by TBHQ administration (Fig. 3A(m–o), F and G). Consistent with these previous findings, total antioxidant capacity was markedly higher in the TBHQ group than that of the HI group (Fig. 3H).

Fig. 3. TBHQ prevented oxidative stress and attenuated oxidative damage.

Fig. 3

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A Representative microscopy images of the damaged cerebral cortex showing NQO1, SOD2, HO-1, MDA and DHE staining at P17. B–F Fluorescent intensity was calculated by ImageJ analysis software and expressed as percentage changes versus respective sham group. G The relative level of ROS from protein samples was quantified and detected by a fluorescence spectrophotometer. H Quantitative analysis of total antioxidant capacity was analyzed by an antioxidant assay kit. Scale bar = 20 μm, magnification: 40×. Data were expressed as mean ± SE (n = 6). *P < 0.05 versus sham, #P < 0.05 versus HI.

3.4. Effects of TBHQ post-treatment on glial activation and inflammatory cytokine expression

To examine the effects of TBHQ treatment on reactive gliosis and pro-inflammatory cytokine levels following HI injury, brain sections taken 7 days after HI injury were selected for GFAP and Iba1 immunofluorescent staining (Fig. 4A). Activated astrocytes and microglia had larger cell bodies and thicker processes. As expected, quantitative analyses indicated astrocytic marker GFAP and microglial marker Iba-1 intensity were both significantly alleviated in the damaged cerebral cortex by TBHQ administration compared with HI group at P17 (p <0.05, Fig. 4B, C). In addition, our results demonstrated that treatment with TBHQ in HI rats significantly reduced the expression of pro-inflammatory cytokines such as IL-1β and ICAM-1. There was, however, no difference in the levels of IL-6, IL-18 and TNF-α ιn TBHQ group compared with the HI group (data not shown). The expression of anti-inflammatory cytokine IL-10 in TBHQ rats was remarkably increased in comparison to the HI group (Fig. 4D–F).

Fig. 4. TBHQ inhibited glial activation and pro-inflammatory cytokine release.

Fig. 4

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A Representative photographs for GFAP (red) and Iba-1 (green) were shown. Nuclei were counterstained with DAPI (blue). B, C Fluorescent intensity of GFAP and Iba1 in each group were further quantified and shown as percentage changes versus sham group. D–F Quantitative analyses of pro-inflammatory and anti-inflammatory cytokine levels were shown. Scale bar = 20 μm, magnification: 40×. Data were expressed as mean ± SE (n = 6). *P < 0.05 versus sham, #P < 0.05 versus HI.

3.5. Effects of TBHQ post-treatment on apoptotic pathway and neuronal degeneration

To investigate the efficacy of TBHQ treatment on suppressing HI-induced neuronal apoptosis and degeneration, TUNEL, Fluoro-Jade C and cleaved caspase-3 staining were performed as previously described. As shown in Fig. 5A(a–f), the results showed that the number of TUNEL and Fluoro-Jade C positive cells in HI group increased markedly when compared with sham group, but this increase was substantially reduced in the TBHQ group at the late stage P31 (Fig. 5B, C), indicating TBHQ treatment repressed apoptosis and reduced neuronal death after HI injury. Furthermore, immunofluorescent staining for cleaved caspase-3 demonstrated a predominant activation of caspase-3 in the peripheries of the infarcted cortex of the HI animals (Fig. 5A(g–i)), while caspase-3 was significantly down-regulated by post-treatment of TBHQ (Fig. 5D). As shown in Fig. 5E and F, further statistical analyses revealed that the HI-induced levels of caspase-9 and caspase-3 activities were significantly reduced at p17 by administration of TBHQ (p <0.05), in accordance with results above. Therefore, TBHQ treatment could attenuate HI-induced neuronal deficits and regulate apoptosis, in part via inhibition of the caspase-9 and caspase-3 apoptotic pathway.

Fig. 5. TBHQ repressed HI-induced apoptosis and neuronal degeneration.

Fig. 5

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A Typical images of TUNEL, Fluoro-Jade C and cleaved caspase-3 staining were taken from the peri-infarct cortex region at P31. B–D Quantitative analyses were performed by counting the number of TUNEL, Fluoro-Jade C and caspase-3 positive neurons. E, F Activities of caspase-9 and caspase-3 were detected by a chromogenic substrate assay using cytosolic proteins from ipsilateral damaged cerebral cortex at P17. Scale bar = 20 μm, magnification: 40×. Data were expressed as mean ± SE (n = 6). *P < 0.05 versus sham, #P < 0.05 versus HI.

3.6. Effects of TBHQ post-treatment on neurobehavioral deficits in HI rats

To examine the impact of TBHQ on HI-induced neurological reflex and motor coordination disturbances, three neurobehavioral tests (righting reflex, beam-walking and cylinder test) were carried out at different timepoints after HI. As noted in Fig. 6A, HI delayed the development of righting reflex. TBHQ treated rats displayed a significantly shorter righting reflex time compared to HI group at P11, as shown by improved the correct response. On the beam-walking test, completion time in the TBHQ group was lower than that of the HI group (p <0.05, Fig. 6B). The HI injury also resulted in the motor deficit in the contralateral forepaw. Fig. 6C indicated that there was a significant decrease in use of the contralateral (left) forepaw exhibited by the HI rats in the cylinder test. In contrast, relative contralateral forepaw usage in the TBHQ group was markedly improved. In addition, spatial learning and memory were impaired due to the HI injury. On the Barnes maze, a well-established behavioral test examining spatial learning and memory, TBHQ treated rats spent less time to find the escape box (Fig. 6D, a&b) and more time in the target quadrant than HI rats (Fig. 6D, c&d). In addition, the performance in the TBHQ treated rats after HI injury was similar to sham. These results suggested that neurobehavioral deficits were ameliorated by TBHQ administration.

Fig. 6. TBHQ treatment ameliorated neurobehavioral deficits after HI insult.

Fig. 6

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A Daily performance of the righting reflex was shown from P11 to P14. B Completion time (sec) spent in the beam-walking test at P30. C Relative contralateral forepaw usage was recorded in the cylinder test at P31. D The trial and probe test results of sham, HI and TBHQ in Barnes maze from P28 to P31. The escape latency was shown in (a). Representative tracking plots on the third trial day (P30) were presented in (b). (c) The occupancy time of probe test in target quadrant (blue color) was recorded at P31. (d) Tracking plots from the probe test indicated typical tracking for sham, HI and TBHQ groups. Values are expressed as mean ± SE (n = 8–11). *P < 0.05 versus sham, #P < 0.05 versus HI.

In light of previous studies (Shih et al., 2005), fresh TBHQ solutions were prepared for each injection. We hypothesized that, in this manner, the adverse effects of TBHQ could be potentially avoided or reduced. Rats administered fresh TBHQ solutions displayed only transient ataxia that presented within a very short time after injection, and no other adverse effect was observed afterwards.

4. Discussion

There are several main findings in the present study. TBHQ significantly ameliorated neurological deficits and neuropathological damage in the cerebral cortex after HI. When post-treated with TBHQ, the Nrf2 signaling pathway was activated and accompanied with up-regulated expression of NQO1, SOD2 and HO-1. TBHQ also significantly repressed oxidative stress, reactive gliosis, neuroinflammation, apoptosis and neuronal degeneration following HI. These findings indicated, for the first time, that TBHQ post-treatment can effectively modulate the activation of the Nrf2 signaling pathway and protect against oxidative damage, neuroinflammation and neuronal cell death in the neonatal HI rat model. Additionally, application of brusatol, a unique inhibitor of the Nrf2 pathway (Ren et al., 2011), abolished both Nrf2 DNA-binding activity and brain protection induced by TBHQ. Taken together, this investigation highlights the potential neuroprotective qualities of TBHQ as a novel therapeutic strategy for HI brain damage via upregulation of the Nrf2 signaling pathway.

Several previous studies have reported that TBHQ is neuroprotective in different pathological conditions. Shih et al. determined that TBHQ has neuroprotective effects in the middle cerebral artery occlusion (MCAO) and endothelin-1 vasoconstriction ischemia models (Shih et al., 2005) wherein Intracerebroventricular or i.p. pre-treatment with TBHQ reduced cortical damage and sensorimotor deficits. In addition, loss of Nrf2 function abrogated TBHQ mediated neuroprotection. Other studies have reported that, TBHQ can exert neuroprotective effects against experimental subarachnoid hemorrhage, traumatic brain injury and spinal cord injury (Jin et al., 2014; Lu et al., 2014; Wang et al., 2014). More recently, it was demonstrated that post-injury administration of TBHQ increased DNA-binding activity of Nrf2 and reduced oxidative and inflammatory brain damage after intracerebral hemorrhage (ICH) (Sukumari-Ramesh and Alleyne, 2016).

In this current study, the results were consistent with previous findings performed in other brain injury models. Our data indicated that TBHQ exerts not only neuroprotection but also improves critically important functional recovery. Further mechanistic investigation was performed in our study. The brain is uniquely vulnerable to oxidative damage and as such, ROS generation and resultant oxidative stress are crucial factors in the pathogenesis of HIE. Previous work by others has indicated that administration of TBHQ can activate Nrf2 and regulate antioxidant enzymes, subsequently alleviating damage resulting from oxidative stress (Liu et al., 2008; Lu et al., 2014). Moreover, Nrf2 is a major antioxidative stress signaling pathway in the neonatal HIE (Ping et al., 2010; Zhao et al., 2016a). TBHQ was previously found to prevent oxidative stress-induced cytotoxicity and cell death in neurons by up-regulating the expression of NQO1, SOD2 and HO-1 (Li et al., 2012; Sun et al., 2015; Zhang et al., 2017).

In our present study, TBHQ treatment increased translocation of Nrf2 into the nuclei in comparison with non-treated HI group animals. Subsequently, TBHQ was demonstrated to facilitate the expression of NQO1, SOD2 and HO-1. Furthermore, TBHQ administration attenuated oxidative damage, as evidenced by significantly decreased MDA and ROS levels significantly and improved total antioxidant capacity. These results demonstrate that the Nrf2 pathway was activated via TBHQ administration after HI injury.

Neuroinflammation is widely accepted to be a major characteristic feature in HIE (Li et al., 2016). After the initiation of HI, oxidative stress plays a key role in neuroinflammatory reactions. Initially, microglia and astrocytes are activated in response to HI attack, and produce excess inflammatory cytokines including TNF-α, IL-1β and IL-6, etc. Accumulation of these pro-inflammatory cytokines enhances expression of cell adhesion molecules and neutrophil infiltration. These are thought to provoke the loss of endothelial cell integrity in the brain resulting in permanent brain cell damage (Zhao et al., 2016b). Upregulation of the Nrf2 antioxidant pathway can contribute to abating microglial activation and pro-inflammatory cytokine expression (Jiang et al., 2017b; Li et al., 2008). TBHQ was demonstrated to reduce microglial activation and mitigated the expression of the pro-inflammatory cytokine IL-1β after ICH (Sukumari-Ramesh and Alleyne, 2016). Moreover, HO-1 can inhibit the upregulation of adhesion molecules in the HI-induced brain injury by removing free heme (Ping et al., 2010). In accordance with these findings, results in the current study showed that reactive gliosis and the pro-inflammatory cytokine IL-1β were reduced, and the anti-inflammatory cytokine IL-10 was significantly enhanced by TBHQ administration significantly. TBHQ administration also decreased expression of ICAM-1 via up-regulating levels of Nrf2 and HO-1 to remove free heme.

Notably, numerous evidences suggest that oxidative stress is also an important step on the path towards neuronal cell death after neonatal HI injury (Northington et al., 2011). Oxidative stress produces excessive ROS that then damages cellular macromolecules, leading to apoptosis and necrosis (Rodrigo et al., 2013). Previous studies indicated that TBHQ could protect against lead neurotoxicity and inhibit oxidative stress and apoptosis via the Nrf2 pathway (Ye et al., 2016). Intriguingly, Nrf2 activation or overexpression in astrocytes has been shown to be neuroprotective in multiple neurodegenerative animal models, in part due to activation of antioxidative gene expression (Chen-Roetling et al., 2017; Chen et al., 2009; Gan et al., 2012; Haskew-Layton et al., 2013). In line with this evidence, the current study verified that TBHQ post-treatment suppressed the number of TUNEL and Fluoro-Jade C positive cells, and inhibited neuronal cell death after HI in neonatal rats. We further examined the mechanisms of TBHQ treatment against apoptosis. As a crucial cell death component that is integral for apoptosis, caspase-9 and caspase-3 play a pivotal role in affecting the cell death process (Abraham and Shaham, 2004). Activated caspase-3 is expressed at higher levels in the neonatal brain after HI insult, which can activate endonucleases to cleave nucleic acids and lead, ultimately, to cell death (Fan et al., 2010). Our data indicated that TBHQ post-treatment play a pivotal role in inhibiting the activation of the caspase-9 and caspase-3 apoptosis pathway, and promoting the survival of cells maybe by up-regulating Nrf2 Pathway. In this study, Nrf2 signaling upregulation was predominately observed in neurons. However, we cannot rule out the role of astroglial Nrf2 activation in contributing to TBHQ neuroprotection

Intriguingly, published studies have shown that TBHQ has both chemoprotective and detrimental effects (Gharavi et al., 2007). The beneficial effects of TBHQ include its contribution to Nrf2 pathway activation. On the contrary, the reported carcinogenicity or toxicity of TBHQ was likely due to a reaction mediated by GSH-conjugates (Peters et al., 1996). According to the literature (Shih et al., 2005), i.p. injection of fresh TBHQ solution (16.7 mg/kg; 3 times) was performed in 8 h intervals, starting at 24h before MCAO and protected the brain from cerebral ischemia in vivo. A mild and transient ataxia was observed within the first 5 min of injection without other distinct negative side effects; nevertheless, it should be noted that TBHQ stock solutions were prepared fresh for each injection. This method of preparation was also mentioned in a previous study (Saykally et al., 2012). In addition, a 7-day or longer time course of TBHQ treatment confers neuroprotection against amyloid β-protein and traumatic brain injury induced apoptosis, as well as 3-nitropropionic acid toxicity in rats and mice, using two different methods of administration (i.p. and oral) (Nouhi et al., 2011; Saykally et al., 2012; Silva-Palacios et al., 2017). Both experimental and clinical observations have demonstrated that HI brain injury is an evolving process (Gunn, 2000). The progression of lesion size will be extensive in the first few days after initial insult, and many neurons may be rescued during this “window of opportunity” (Fatemi et al., 2009). Moreover, previous studies suggested multiple doses of antioxidant treatments are required to achieve optimal protective effects (Arteaga et al., 2017). Therefore, we hypothesized that the application of multiple-dose TBHQ must be sustained for a prolonged period to delay the evolution of HI damage processes. As is well known, a pretreatment therapy is not clinically applicable, so in the current study i.p. injection of TBHQ (20 mg/kg) was performed once daily for seven days. Consistent with previous studies (Shih et al., 2005), only rapid and transient adverse behaviors were observed in a short period of time after the injection of TBHQ but recovered very soon therefter. Herein, we hypothesized fresh TBHQ solutions may be prepared for each injection to avoid detrimental side effects.

According to the work presented above, we believe that the beneficial effects of TBHQ appear to be mediated by up-regulating the Nrf2 antioxidative signaling pathway. However, investigating methods to avoid or decrease the detrimental effects of TBHQ while maximizing the targeted neuroprotective actions is the next challenging goal in developing TBHQ as a viable therapeutic strategy for HI injury.

To best serve the purpose making the beneficial effects of TBHQ more clinically relevant and translational, further studies will be needed to discern whether TBHQ provides long-term neuroprotective effects without severe side effects. Drug trials for HIE have essentially been nonexistent in the newborn infant except for TH (Yager, 2004). TH is the most effective approach for treatment of HIE in humans currently, but it has a narrow therapeutic time window and its utility is limited to mild cases. Because of complex pathogenesis and clinical features, a single treatment is difficult to exert the more effective therapeutic action in neonatal HIE. With respect to the literature, combinational therapies with TH may be more effective to reach more targets of HI insult, including increased therapeutic time window and enhanced prevention of acute brain injury in the long term (Dixon et al., 2015). For some of antioxidants, beneficial effects against HIE have been shown when they are used as an adjunct therapy in combination with TH for HIE (Arteaga et al., 2017). Thus, whether combination of TBHQ and TH can enhance the neuroprotection against HIE merits further research. Likewise, more detailed work will be needed to explore the appropriate therapeutic window of TBHQ post-treatment effectiveness. In the present study, TBHQ was administered by i.p. injection one hour after HI insult and continued once daily for seven days. It remains to be defined whether the initial TBHQ therapeutic window can be delayed and whether the course of TBHQ treatment may be decreased to minimize potential adverse effects.

5. Conclusion

In conclusion, the current study found that HI-induced early brain injury, long-term lesion size, behavioral and cognitive dysfunction were ameliorated after administration of TBHQ, suggesting for the first time that TBHQ has protective effects on HI brain injury in neonatal rats. In addition, TBHQ post-treatment could mitigate oxidative damage, and suppress reactive gliosis and inflammation, as well as inhibit activation of the caspase-3/9 apoptosis pathway and neuronal cell death, which indicated that a possible mechanism of the beneficial effects of TBHQ lies in upregulation of the Nrf2 antioxidative pathway. Taken together, the present study demonstrated that TBHQ may be a new antioxidative therapeutic option for HIE in neonatal rats. With further work delineating the most effective treatment strategy, TBHQ may serve as a potent tool in the treatment of neonatal HIE greatly improving the lives of children and their families.

Highlights.

  • Tert-butylhydroquinone (TBHQ) reduces infarct size in hypoxic-ischemic (HI) rats.

  • TBHQ ameliorates neurobehavioral deficits after HI brain damage.

  • TBHQ promotes Nrf2 nuclear activation and attenuates oxidative damage after HI.

  • TBHQ suppresses neuroinflammation in the cerebral cortex after HI brain damage.

  • TBHQ inhibits HI-induced neuronal apoptosis and reduces neuronal cell death.

Acknowledgments

This study was supported in part by Research Grant NS086929 from the National Institute of Neurological Disorders and Stroke, National Institutes of Health, USA; an American Heart Association Grant-in-Aid 15GRNT25240004; a Natural Science Foundation of China (81403140); and a Natural Science Foundation of Shaanxi Province (2014JQ4150).

Footnotes

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