1122 Cellular molecular and developmental neuroscience
Hypoxia-inducible factor-1α upregulation in microglia following hypoxia protects against ischemia-induced cerebral infarction Tao Huanga,*, Weiyi Huangb,*, Zhiqiang Zhanga, Lei Yuc, Caijun Xiea, Dongan Zhua, Zizhuang Penga and Jiehan Chena Activated microglia were considered to be the toxic inflammatory mediators that induce neuron degeneration after brain ischemia. Hypoxia can enhance the expression of hypoxia-inducible factor-1α (HIF-1α) in microglia and cause microglial activation. However, intermittent hypoxia has been reported recently to be capable of protecting the body from myocardial ischemia. We established a high-altitude environment as the hypoxic condition in this study. The hypoxic condition displayed a neuroprotective effect after brain ischemia, and mice exposed to this condition presented better neurological performance and smaller infarct size. At the same time, a high level of HIF-1α, low level of isoform of nitric oxide synthase, and a reduction in microglial activation were also seen in ischemic focus of hypoxic mice. However, this neuroprotective effect could be blocked by 2-methoxyestradiol, the HIF-1α inhibitor. Our finding suggested that HIF-1α expression was involved in microglial activation in vitro and was regulated by oxygen supply. The microglia were inactivated by re-exposure to hypoxia, which might be due to overexpression of HIF-1α. These results indicated that hypoxic conditions can be exploited to achieve maximum neuroprotection after brain
Introduction Neuroinflammation caused by activated microglia was considered to be the vital factor of subsequent neurological function deficit after acute brain ischemia. Microglia constitute a type of resident cell in the central nervous system, which originates from monocyte–macrophage cell lines and constitute 20% of the total glial cells. When stimulated, microglia can transform into an activated state and exert their functions [1]. Activated microglia can recognize and phagocytose damaged neurons and promote healing of injured nerve tissue. Under pathological conditions, microglia are the dominant players in the process of nerve inflammation and eventually lead to nerve damage [2,3]. The underlying mechanism lies in the inflammatory cytokines released by microglia, such as arachidonic acid metabolites, interleukin-1, nitric oxide (NO), reactive oxygen species, and tumor necrosis factor-α. Although these factors are Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's website (www.neuroreport.com). 0959-4965 © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins
ischemia. This mechanism possibly lies in microglial inactivation through regulation of the expression of HIF-1α. NeuroReport 25:1122–1128 © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins. NeuroReport 2014, 25:1122–1128 Keywords: brain ischemia, hypoxia, hypoxia-inducible factor-1α, isoform of nitric oxide synthase, microglia, neuroprotection a Department of Neurosurgery, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine (Guangdong Provincial Hospital of Chinese Medicine), bThe National Key Clinic Specialty, The Neurosurgery Institute of Guangdong Province, Guangdong Provincial Key Laboratory on Brain Function Repair and Regeneration, Department of Neurosurgery, Zhujiang Hospital, Southern Medical University and cKey Laboratory of Construction and Detection of Guangdong Province, Department of Anatomy, Southern Medical University, Guangdong, China
Correspondence to Tao Huang, MD, PhD, Department of Neurosurgery, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine (Guangdong Provincial Hospital of Chinese Medicine), 111# Dade Road, Guangzhou, Guangdong Province 510120, China Tel/fax: + 86 20 81874903; e-mail: [email protected] *Tao Huang and Weiyi Huang contributed equally to the writing of this article. Received 10 June 2014 accepted 26 June 2014
essential components in normal nerve functioning, overexpression of these factors may lead to catastrophic neuronal apoptosis [4,5]. Thus, overactivation of microglia would cause irreversible damage to brain tissue and lead to neurodegenerative diseases by releasing excessive cytokines [3]. The emergence of a new phenotype and some specific proteins, such as the inducible isoform of nitric oxide synthase (iNOS), are the indicators of microglial activation [6,7]. In conclusion, we believed that the mechanism of brain injury and functional deficit caused by hypoxic ischemia may lie in the microglia activated by hypoxia, which express iNOS and secrete NO, reactive oxygen species, and proinflammatory cytokines. This iNOS-mediated NO production is considered vital in neuroinflammation after brain ischemia [8,9]. Research found that iNOS inhibitor and iNOS knockout mice displayed strong neuroprotection after cerebral ischemia through a neuron apoptosis-resisting effect [10]. Systemic infection and neuroinflammation are common syndromes after stroke in the clinic, and all these suggested that neuroinflammatory processes play a key role in neuron apoptosis after brain ischemia [11]. DOI: 10.1097/WNR.0000000000000236
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
HIF-1α upregulation in microglia Huang et al. 1123
Hypoxia-inducible factor-1 (HIF-1) consists of a basic helix–loop–helix–PAS domain, a heterodimeric transcription factor that contains the protein HIF-1α and an arylhydrocarbon receptor nuclear translocator (ARNT, HIF-1β) [12]. The availability of HIF-1 is determined primarily by HIF-1α, which is stably expressed. In contrast to HIF-1β, the amount of HIF-1α expressed is regulated in an oxygen-sensitive manner [13]. HIF-1α is efficiently degraded by the Von Hippel-Lindaudependent ubiquitin–proteasome pathway during normoxia [13]. Under hypoxic conditions, HIF-1α displays a remarkably high level of expression and heterodimerizes with HIF-1β to form HIF-1 after translocating into the nucleus. Gene transcription of vascular endothelial growth factor, erythropoietin, iNOS, and glycolytic enzymes is activated to enhance cellular adaptation to hypoxia [14]. In the brain, HIF-1α expression seems to be induced by hypoxia in neurons, astrocytes, ependymal cells, and possibly endothelial cells [14]. Neuroinflammation induced by microglia is crucial in neuron apoptosis after a stroke. The role of HIF-1α expression in microglia remained a controversial topic for the past two decades. In different neuroinflammatory diseases such as multiple sclerosis and Alzheimer’s disease, HIF-1α has different effects in the regulation of microglia [15]. In this study, short-term hypoxia was designed to investigate whether a high-altitude environment could be proposed as a therapeutic strategy for brain ischemia. The relation between microglial activation and HIF-1α expression after brain ischemia was also discussed in our study.
Materials and methods All mice were purchased from the Animal Experiment Center of Southern Medical University (Guangzhou, China). Animal experimental procedures were approved by the Southern Medical University Ethics Committee. All surgeries were performed under sodium pentobarbital anesthesia, and all efforts were made to minimize animal suffering. Animals
Twenty male mice (C57BL/6) weighing 20–22 g (12–14 weeks years old) were selected and anesthetized with intraperitoneal injection of 4% sodium chloral hydrate (100 mg/kg). The focal ischemic reperfusion model was prepared according to a previous study [16]. The mice were then treated with middle cerebral artery occlusion (MCAO) for 1.5 h. After that, the blood supply was restored. Twelve hours after the recovery of blood perfusion, all mice were randomly divided into two groups: a postoperative hypoxic group (hypoxia, n = 10) and a postoperative normoxic group (normal, n = 10). The hypoxic group was placed in the hypoxic box for 4 h, and the hypoxic conditions were set as a 5000 m altitude plateau environment. The normoxic group was placed in the normoxic environment as the
control group. Twelve hours after hypoxia, perfusion fixation was performed for both groups following a neurological assessment. Sodium chloral hydrate (4%) was used to anesthetize the mice, and cannulation was performed from the left ventricle into the ascending aorta. The vessels were rinsed using 100 ml saline (37°C, 140 mmHg) and perfusion fixation was performed for 30 min using 0.1 M phosphate buffer and 40 g/l paraformaldehyde. In the end, the mice were killed and their brain tissue was harvested (Supplement 1, Supplemental digital content 1, http://links. lww.com/WNR/A296). Another 20 male mice (C57BL/6) weighing 20–22 g (12–14 weeks old) were selected. Twelve hours after blood flow recovery, they were intraperitoneally injected with 2-methoxyestradiol (2-ME) (Sigma-Aldrich, St Louis, Missouri, USA), the HIF-1α inhibitor, at a dose of 100 mg/ kg. The mice were then randomly divided into two groups, a postoperative hypoxic group (hypoxia, n = 10) and a postoperative normoxic group (normal, n = 10), and then treated as described above (Supplement 1, Supplemental digital content 1, http://links.lww.com/WNR/A296). Hypoxia training protocol
Mice were housed in a thick-walled, high-pressure-resistantglass animal chamber that was commercially designed (76 × 50 × 50 cm3) and fitted with a brass lid and three brass outlets connected to the other components of the unit through vacuum tubes. Briefly, the first outlet was connected to a high-pressure vacuum pump with a pressure gauge through a copper tube. The second outlet was connected to a manometer that indicated the barometric pressure, whereas the third outlet was fitted to an adjustable knob to regulate the entry of air, which could develop pressure in the chamber. During the simulation, pressure was gradually decreased to a target level, equivalent to the pressure at an altitude of 3000 or 5000 m (i.e. PB 404 mmHg; PO2 84 mmHg, equivalent to an altitude of 5000 m). Four hours after exposing the animals to the decreased pressure, the pressure was gradually increased to a normal level over 15 min. For the normoxic control group, the animals were kept in the chamber (i.e. PB 760 mmHg; PO2 159.2 mmHg), which was circulated with room air for 4 h [17]. Neurological assessment score
Neurological evaluations were carried out 12 h after hypoxia according to a predetermined schedule, which excluded behavioral changes based on circadian rhythms. The examiners were not aware of the procedure the mice had undergone and the mice were scored according to the following criteria: score 0, no neurologic impairment; score 1, endoduction of the right anterior limbs and no complete tail stretch while walking to the left; score 2, circling toward the right during spontaneous walking; score 3, right-side lateriversion while walking; score 4, no spontaneous walking and some consciousness lost. We screened out mice with scores of 0 or 4 [18].
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
1124 NeuroReport 2014, Vol 25 No 14
Cerebral infarction size evaluation
The mice were killed by an overdose of anesthesia with 10% sodium chloral hydrate and decapitated immediately. Brain tissue was harvested and put into a − 70°C refrigerator for 10 min of quick freezing. Then tissue was obtained from the procerebrum and cut into five coronal brain slices of the same thickness (2 mm) from the frontal pole to the occipital pole. The slices were immediately put into 2% tetrazolium chloride (TTC)-PBS to shield from light before being incubated at 37°C for 30 min and fixed for 2 h with 4% paraformaldehyde. Normal brain tissue was dyed bright red, whereas the infarction focus presented pale white. The fixed brain slices were arranged and the areas of the prosencephalon and the cerebral infarction were measured by Image Pro Plus 5.0 (IPP, Media Cybernetics, Rockville, Maryland, USA) image processing software according to the formula V = t × (A1 + A2 + …An). In the formula, V represents the infarction volume or prosencephalon volume, t is the thickness of the slice, and A is the infarction size. Infarction volume and prosencephalon volume as well as the ratio of these two parameters (cerebral infarction volume/prosencephalon volume) were calculated. Tissue processing and immunohistochemistry
Mice were injected with sodium chloral hydrate (37°C, 140 mmHg) followed by 0.1 M phosphate buffer with 40 g/l paraformaldehyde over 30 min. Mice were then decapitated, and their brains were dissected and equilibrated for 24 h in 30% sucrose in PBS before being flashfrozen in an optimal cutting temperature compound. Brains were sectioned in the coronal plane at a thickness of 40 μm on a cryostat, blocked in PBS containing 0.3% Triton X-100 and 5% BSA (blocking solution), and incubated overnight at 4°C with the following primary antibodies: a mouse monoclonal antibody against ionized calcium-binding adapter molecule 1 (anti-Iba1, 019–19741; Wako Pure Chemical Industries Ltd, Tokyo, Japan) and anti-iNOS or anti-HIF-1α (SAB4502012, HPA001275; both from Sigma-Aldrich). The sections were then incubated with fluorescein isothiocyanate and tetramethylrhodamine B isothiocyanate-conjugated secondary antibodies (1 : 1000, H17101, A10532; Life Technologies, Carlsbad, California, USA) in blocking solution at room temperature for 2 h, washed with PBS, mounted on glass slides, and examined using a confocal microscope (Olympus, Tokyo, Japan). Microglia cultures
The BV-2 cell line is a stable microglia cell line derived from mice (gift from Department of Neurobiology of Southern Medical University). In this study, BV-2 cells were cultured in 75 cm2 flasks for 14 days in Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12; Gibco, Grand Island, New York, USA), supplemented with 10% fetal calf serum (Hyclone, Logan, Utah, USA), 100 U/ml penicillin, and 100 mg/ml streptomycin. Detached cells
were plated on 24-well plates at a density of 2 × 105 cells/ well. Cells were cultured for 2 days before treatment. Confluent cultures were passaged by trypsinization. Microglial activation and hypoxia
To activate microglia, BV-2 cells were placed in serum-free DMEM, gassed with 95% N2 and 5% CO2 (Anaerobic System PROOX model 110; BioSpherix, Redfield, New York,USA), and incubated at 37°C for 8 h. To reoxygenate hypoxic cultures, cells were transferred into a regular normoxic incubator (95% air, 5% CO2) and incubated for an additional 12 h. Then, the cultured cells were placed in serum-free DMEM and gassed with 95% N2 and 5% CO2 for 0, 4, and 8 h as a hypoxic condition (hypoxia); others were in normoxic condition (normal). Cell viability was only slightly reduced after exposure to hypoxia. 2-ME was dissolved in dimethyl sulfoxide at a concentration of 2.00 μM, and oxygen was restored after 8 h of hypoxia. The cells were incubated for the following 12 h, after which 2-ME (2.00 μM) was infused into the cultures. Western blotting
BV-2 cells were plated on six-well plates. After each condition, they were washed with cold PBS and lysed for 30 min on ice with radioimmunoprecipitation assay buffer. Protein samples containing 30 mg protein were separated on 8% SDS polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, Massachusetts, USA). The membranes were incubated for 1 h with 4% dry skim milk in PBS buffer to block nonspecific binding. The membranes were then incubated with rabbit antibodies against iNOS (1 : 1000, 610310; BD Transduction Lab, Lexington, Kentucky, USA) or mouse antibodies against HIF-1α (1 : 1000, NB100–105; Novus Biologicals, Littleton, Colorado, USA) and β-actin (1 : 1000, sc130065; Santa Cruz Biotechnology, Santa Cruz, California, USA). The membranes were then incubated with goat antirabbit or rabbit anti-mouse peroxidase-conjugated secondary antibody (1 : 1000, sc45101, sc358922; Santa Cruz Biotechnology) for 1 h. The blots were visualized by enhanced chemiluminescence (ECL; Santa Cruz Biotechnology) using Kodak X-OMAT LS film (Eastman Kodak, Rochester, New York, USA). All western blot experiments were repeated at least three times, with different sets of samples, throughout this study. Protein levels were quantified and normalized to β-actin levels. Quantification of western blots was performed by Image Pro Plus 6.0 (IPP, Media cybernetics, Rockville, Maryland, USA). Statistical analysis
SPSS software (SPSS Inc., Chicago, Illinois, USA) was used. Results were expressed as mean ± SE and comparisons were made using one-way analysis of variance with Bonferroni’s post-test. P value less than 0.05 was considered statistically significant.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
HIF-1α upregulation in microglia Huang et al. 1125
Results Neuroprotection was induced by hypoxia after middle cerebral artery occlusion and inactivated microglia associated with the enhancement of hypoxia-inducible factor-1α expression
At 1.5 h after treatment with MCAO, the blood supply was restored. Ten mice were then placed in hypoxic conditions for 4 h. Compared with the normoxic group, the neurological assessment score of the hypoxic group was lower and the size of cerebral infarction focus was significantly smaller (Fig. 1a and b). This indicated that
cerebral infarction and neurological damage caused by cerebral ischemia can be effectively attenuated by a short-term plateau hypoxic environment. The results of the fluorescent labeling experiment showed that microglial cells aggregated at the junction between the ischemic infarction focus and the normal tissues in the normoxic group. Besides, HIF-1α expression of microglia was significantly higher in the hypoxic group compared with the normoxic group. iNOS expression in the hypoxic group was significantly lower than that of the normoxic group (Fig. 1c and d). The results indicated
Fig. 1
(a)
Normal
(c)
Hypoxia
Normal
Hypoxia
HIF-1α
50 40 30
∗∗
20 10 0 Normal
Hypoxia
(d) 40
3
2
∗∗
1
Number of positive
The volume of infarct (%)
(b)
Neurological deficit score
iNOS
30
60 ∗∗ 40 ∗∗
20 20 10 0
0
0 Normal
Hypoxia
Normal Hypoxia HIF-1α
Normal
Hypoxia iNOS
(a) The TTC stain showed that the volume ratio of the cerebral infarction was significantly lower in the hypoxic group (n = 5) compared with the normal group (n = 5). (b) The neurological score of the hypoxic group (n = 10) and the normal group (n = 10). Data are expressed as mean ± SE. Significant differences between the hypoxic group and the normal group were seen in infarction volume as well as in neurological score (**P < 0.01). (c) Immunofluorescence of ischemic infarction focus and normal tissues was performed. Microglia was labeled with FITC (light grey). HIF-1α and iNOS were labeled with tetramethylrhodamine B (light grey). (d) Statistical analysis of HIF-1α-positive and iNOS-positive cells in ischemic infarction focus and normal tissues. Five visions of different areas were randomly chosen. FITC, fluorescein isothiocyanate; HIF-1α, hypoxia-inducible factor-1α; iNOS, isoform of nitric oxide synthase; TTC, tetrazolium chloride.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
1126 NeuroReport 2014, Vol 25 No 14
that HIF-1α expression in microglial cells, which was induced by hypoxia after cerebral ischemia, might be the reason for brain protection. Inhibition of hypoxia-inducible factor-1α in microglia blocked neuroprotection after hypoxic condition
There was no significant difference in infarction size and neurological assessment score between the hypoxic group and the control group in vivo after treatment with MCAO and injection with 2-ME, the HIF-1α antagonist (Fig. 2a and b).
This indicated that the neuroprotection after the hypoxic period following cerebral ischemia was closely related to HIF-1α activation. There was also no difference in HIF-1α and iNOS expression (Fig. 2c and d) in microglia that aggregated at the junction of the ischemic infarction focus and normal tissues. This indicated that the expression of iNOS in activated microglia was determined by the level of HIF-1α expression. That is to say, the brain protection effect may derive from the reduction of iNOS released, which resulted from overexpressed HIF-1α in hypoxia-treated microglia.
Fig. 2
(a)
Normal
(c)
Hypoxia
Normal
Hypoxia
HIF-1α
iNOS
40 30 20 10 0 Normal
Hypoxia
(d)
3
Number of positive
50
Neurological deficit score
The volume of infarct (%)
(b)
2
1
25
60
20 40
15 10
20
5 0
0 Normal
Hypoxia
0 Normal HIF-1α
Hypoxia
Normal
Hypoxia iNOS
(a) The TTC stain showed an identical volume ratio of the cerebral infarction in the hypoxic group (n = 5) and normal group (n = 5) after 2-methoxyestradiol injection. (b) There were no significant differences in the neurological score between the hypoxic group (n = 10) and normal group (n = 10) in vivo after injection of an HIF-1α antagonist. (c) Immunofluorescence was performed on the ischemic infarction focus and normal tissues. Microglia were labeled with FITC (light grey). HIF-1α and iNOS were labeled with tetramethylrhodamine B (light grey). (d) Statistical analysis of HIF-1α-positive and iNOS-positive cells in the ischemic infarction focus and normal tissues. Five visions of different areas were randomly chosen. FITC, fluorescein isothiocyanate; HIF-1α, hypoxia-inducible factor-1α; iNOS, isoform of nitric oxide synthase; TTC, tetrazolium chloride.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
HIF-1α upregulation in microglia Huang et al. 1127
Fig. 3
8h
0h
Hypoxia 4h
(b) 8h
HIF-1α iNOS
1.0
∗
4
1 0
0.0
iNOS
8h
0h
4h
8h
HIF-1α iNOS β-Actin
2-ME 0.20
2-ME 1.5
0.15
1.0
0.10 0.5
0.05
0.0
0.00
HIF-1α
N o H rma yp l o 0 N xia h or 0 h H ma yp l 4 o h N xia or 4 m h H al yp 8 ox h ia 8h
4h
Level of expression
0h
(d)
Hypoxia
N o H rma yp l o 0 N xia h or 0 h H ma yp l 4 o h N xia or 4 m h H al yp 8 ox h ia 8h
Normal
∗∗
3
HIF-1α (c)
∗∗
2 0.5
N o H rma yp l o 0 N xia h or 0 h H ma yp l 4 o h N xia or 4 m h H al yp 8 ox h ia 8h
β-Actin
∗∗
1.5
N o H rma yp l o 0 N xia h or 0 h H ma yp l 4 o h N xia or 4 m h H al yp 8 ox h ia 8h
0h
Normal 4h
Level of expression
(a)
iNOS
(a) Western blot results after oxygen was restored for 12 h. Results were obtained at 0, 4, and 8 h after hypoxia. iNOS expression was reduced in the hypoxic group compared with the normal group, which was associated with the high expression of HIF-1α. (b) Statistical analysis of HIF-1α and iNOS expression (n = 3; *P < 0.05, **P < 0.01). (c) There was no difference in iNOS or HIF-1α expression between the hypoxic and normal group following 2-methoxyestradiol treatment. (d) Statistical analysis of HIF-1α and iNOS expression after 2-methoxyestradiol treatment (n = 3). HIF-1α, hypoxiainducible factor-1α; iNOS, isoform of nitric oxide synthase; 2-ME, 2-methoxyestradiol.
Hypoxia-inducible factor-1α in microglia was activated by exposure to hypoxic conditions
Microglia were activated by a hypoxic condition in vitro and recovered in normoxia for 12 h. Western blot analysis showed no difference in the expressions of HIF-1α and iNOS in activated microglia for 0-h hypoxia and normoxia treatment. However, a higher level of HIF-1α expression and a lower level of iNOS expression in activated microglia were detected 4 and 8 h after exposure to hypoxic condition compared with normoxic condition (Fig. 3a and b). This result suggested that overexpression of HIF-1α through hypoxia may attenuate the microglial activation. After 2-ME was applied to activated microglia under different conditions such as rehypoxia and normoxia, no difference in iNOS expression between these two groups was seen (Fig. 3c and d). This indicated that the reduction in iNOS activation in microglia by hypoxia treatment may be regulated by HIF-1α activation.
Discussion In our study, hypoxia was induced by a simulated highaltitude environment. Over the past decade, short-term and long-term intermittent hypoxic environments have been proposed to have a therapeutic effect for ischemia [17,19]. A potential neuroprotective effect conferred by short-term hypoxia after cerebral ischemic has not yet
been confirmed. Our results indicated that short-term hypoxic conditions could effectively protect neurons at the early stage of cerebral ischemia and present an anti-inflammatory effect in the central nervous system as well. According to recent research, neuroinflammation following microglial activation is considered one of the most crucial factors for neurological damage after cerebral ischemia [20]. While under cerebral hypoxia, the activation of HIF-1α is an important inductor for microglial activation [21]. Activated microglia may later induce neuronal apoptosis by releasing endogenous inflammatory cytokines. In the present study, a high level of HIF-1α expression was detected in activated microglia [22]. It was even higher when the microglial activation was induced by hypoxia after ischemia. In the junction of infarction area and normal tissue, accumulation of microglia and a high level of HIF-1α expression were both observed, which was identical to previous studies. In vivo, the activation of iNOS was positively regulated by HIF-1α. High expression of HIF-1α after hypoxia significantly inhibited the expression of iNOS. This suggested that overactivation of HIF-1α may suppress the activation of microglia. Thus, a decline of iNOS expression was observed in microglia after short-term hypoxia.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
1128 NeuroReport 2014, Vol 25 No 14
Microglia can be activated by hypoxic conditions in vitro, as well as by neuronal apoptosis. Meanwhile, a high level of HIF-1α expression was observed in cerebral resident cells after brain ischemia [23]. Our research indicated that, although microglia could be activated by hypoxia after brain ischemia, 12 h of hypoxia after MCAO treatment can attenuate the brain damage in mice, and the overexpression of HIF-1α can suppress microglial activation as well. After injection of 2-ME, no difference in HIF-1α expression was observed in the microglia at the infarction border zones. This was accompanied by the absence of the brain protection effect. Some studies have shown that antagonization of HIF-1α in early stage of brain ischemia presents a neuroprotective effect. The effect may be due to the absence of HIF-1α, which can reduce the activation of microglia [24]. 2-ME is a stable and nontoxic estradiol derivative and known as a widely used HIF-1α inhibitor. By inhibiting HIF-α activation, 2-ME can suppress tumor growth, lessen traumatic brain injury, and display an anti-immunoinflammatory effect [25]. In our study, hypoxia after cerebral ischemia had a certain brain protection effect, in which HIF-1α played an important regulatory role. The outcome of this study suggested that hypoxic conditions can be used to achieve the maximum brain protection effect. In our future work, we will continue to explore the underlying mechanisms of this transient hypoxia-induced brain protection effect in cerebral ischemia.
3 4 5
6 7
8
9
10
11
12
13
14
15
16
Conclusion The present results showed that hypoxia could enhance the expression of HIF-1α in microglia and cause microglial activation. Further, HIF-1α overexpression regulated microglia inactivated by re-exposure to hypoxia, leading to neuroprotection against ischemia-induced cerebral infarction. Thus, this study has identified that hypoxic conditions can be exploited to achieve maximum neuroprotection after brain ischemia through regulation of the expression of HIF-1α in microglial activation.
17
18
19
20
21
Acknowledgements This study was supported by the Natural Science Foundation of Guangdong Province (S2011010005403). Conflicts of interest
22
23
There are no conflicts of interest.
References 1 2
Marty S, Dusart I, Peschanski M. Glial changes following an excitotoxic lesion in the CNS-I. Microglia/macrophages. Neuroscience 1991; 45:529–539. Berta T, Park CK, Xu ZZ, Xie RG, Liu T, Lü N, et al. Extracellular caspase-6 drives murine inflammatory pain via microglial TNF-alpha secretion. J Clin Invest 2014; 124:1173–1186.
24
25
Rodriguez JI, Kern JK. Evidence of microglial activation in autism and its possible role in brain underconnectivity. Neuron Glia Biol 2011; 7:205–213. Loihl AK, Murphy S. Expression of nitric oxide synthase-2 in glia associated with CNS pathology. Prog Brain Res 1998; 118:253–267. Bal-Price A, Brown GC. Inflammatory neurodegeneration mediated by nitric oxide from activated glia-inhibiting neuronal respiration, causing glutamate release and excitotoxicity. J Neurosci 2001; 21:6480–6491. Chao CC, Hu S, Peterson PK. Modulation of human microglial cell superoxide production by cytokines. J Leukoc Biol 1995; 58:65–70. Regenhardt RW, Desland F, Mecca AP, Pioquinto DJ, Afzal A, Mocco J, Sumners C. Anti-inflammatory effects of angiotensin-(1–7) in ischemic stroke. Neuropharmacology 2013; 71:154–163. Kawase M, Kinouchi H, Kato I, Akabane A, Kondo T, Arai S, et al. Inducible nitric oxide synthase following hypoxia in rat cultured glial cells. Brain Res 1996; 738:319–322. Iadecola C, Ross ME. Molecular pathology of cerebral ischemia: delayed gene expression and strategies for neuroprotection. Ann N Y Acad Sci 1997; 835:203–217. Wang Y, Ahmad N, Kudo M, Ashraf M. Contribution of Akt and endothelial nitric oxide synthase to diazoxide-induced late preconditioning. Am J Physiol Heart Circ Physiol 2004; 287:H1125–H1131. Miura T, Hamawaki M, Hazama S, Hashizume K, Ariyoshi T, Sumi M, et al. Outcome of surgical management for active mitral native valve infective endocarditis: a collective review of 57 patients. Gen Thorac Cardiovasc Surg 2014 [Epub ahead of print]. Cao Y, Mao X, Sun C, Zheng P, Gao J, Wang X, et al. Baicalin attenuates global cerebral ischemia/reperfusion injury in gerbils via anti-oxidative and anti-apoptotic pathways. Brain Res Bull 2011; 85:396–402. Richards EM, Fiskum G, Rosenthal RE, Hopkins I, McKenna MC. Hyperoxic reperfusion after global ischemia decreases hippocampal energy metabolism. Stroke 2007; 38:1578–1584. Wang GL, Jiang BH, Rue EA, Semenza GL. Hypoxia-inducible factor 1 is a basic-helix–loop–helix–PAS heterodimer regulated by cellular O2 tension. Proc Natl Acad Sci USA 1995; 92:5510–5514. Zeis T, Graumann U, Reynolds R, Schaeren-Wiemers N. Normal-appearing white matter in multiple sclerosis is in a subtle balance between inflammation and neuroprotection. Brain 2008; 131 (Pt 1):288–303. Chiamulera C, Albertini P, Valerio E, Reggiani A. Activation of metabotropic receptors has a neuroprotective effect in a rodent model of focal ischaemia. Eur J Pharmacol 1992; 216:335–336. Zhu XH, Yan HC, Zhang J, Qu HD, Qiu XS, Chen L, et al. Intermittent hypoxia promotes hippocampal neurogenesis and produces antidepressant-like effects in adult rats. J Neurosci 2010; 30:12653–12663. Cao CX, Yang QW, Lv FL, Cui J, Fu HB, Wang JZ. Reduced cerebral ischemia-reperfusion injury in Toll-like receptor 4 deficient mice. Biochem Biophys Res Commun 2007; 353:509–514. Zhang G, Zhou SM, Yuan C, Tian HJ, Li P, Gao YQ. The effects of short-term and long-term exposure to a high altitude hypoxic environment on neurobehavioral function. High Alt Med Biol 2013; 14:338–341. Tang Z, Gan Y, Liu Q, Yin JX, Liu Q, Shi J, Shi FD. CX3CR1 deficiency suppresses activation and neurotoxicity of microglia/macrophage in experimental ischemic stroke. J Neuroinflammation 2014; 11:26. Kyotani Y, Ota H, Itaya-Hironaka A, Yamauchi A, Sakuramoto-Tsuchida S, Zhao J, et al. Intermittent hypoxia induces the proliferation of rat vascular smooth muscle cell with the increases in epidermal growth factor family and erbB2 receptor. Exp Cell Res 2013; 319:3042–3050. Lu DY, Liou HC, Tang CH, Fu WM. Hypoxia-induced iNOS expression in microglia is regulated by the PI3-kinase/Akt/mTOR signaling pathway and activation of hypoxia inducible factor-1alpha. Biochem Pharmacol 2006; 72:992–1000. Del Bigio MR, Becker LE. Microglial aggregation in the dentate gyrus: a marker of mild hypoxic-ischaemic brain insult in human infants. Neuropathol Appl Neurobiol 1994; 20:144–151. Chavez-Valdez R, Martin LJ, Flock DL, Northington FJ. Necrostatin-1 attenuates mitochondrial dysfunction in neurons and astrocytes following neonatal hypoxia-ischemia. Neuroscience 2012; 219:192–203. Schaible EV, Windschügl J, Bobkiewicz W, Kaburov Y, Dangel L, Krämer T, et al. 2-Methoxyestradiol confers neuroprotection and inhibits a maladaptive HIF-1alpha response after traumatic brain injury in mice. J Neurochem 2014; 129:940–954.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Hypoxia-inducible factor-1α upregulation in microglia following hypoxia protects against ischemia-induced cerebral infarction Tao Huanga,*, Weiyi Huangb,*, Zhiqiang Zhanga, Lei Yuc, Caijun Xiea, Dongan Zhua, Zizhuang Penga and Jiehan Chena Activated microglia were considered to be the toxic inflammatory mediators that induce neuron degeneration after brain ischemia. Hypoxia can enhance the expression of hypoxia-inducible factor-1α (HIF-1α) in microglia and cause microglial activation. However, intermittent hypoxia has been reported recently to be capable of protecting the body from myocardial ischemia. We established a high-altitude environment as the hypoxic condition in this study. The hypoxic condition displayed a neuroprotective effect after brain ischemia, and mice exposed to this condition presented better neurological performance and smaller infarct size. At the same time, a high level of HIF-1α, low level of isoform of nitric oxide synthase, and a reduction in microglial activation were also seen in ischemic focus of hypoxic mice. However, this neuroprotective effect could be blocked by 2-methoxyestradiol, the HIF-1α inhibitor. Our finding suggested that HIF-1α expression was involved in microglial activation in vitro and was regulated by oxygen supply. The microglia were inactivated by re-exposure to hypoxia, which might be due to overexpression of HIF-1α. These results indicated that hypoxic conditions can be exploited to achieve maximum neuroprotection after brain
Introduction Neuroinflammation caused by activated microglia was considered to be the vital factor of subsequent neurological function deficit after acute brain ischemia. Microglia constitute a type of resident cell in the central nervous system, which originates from monocyte–macrophage cell lines and constitute 20% of the total glial cells. When stimulated, microglia can transform into an activated state and exert their functions [1]. Activated microglia can recognize and phagocytose damaged neurons and promote healing of injured nerve tissue. Under pathological conditions, microglia are the dominant players in the process of nerve inflammation and eventually lead to nerve damage [2,3]. The underlying mechanism lies in the inflammatory cytokines released by microglia, such as arachidonic acid metabolites, interleukin-1, nitric oxide (NO), reactive oxygen species, and tumor necrosis factor-α. Although these factors are Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's website (www.neuroreport.com). 0959-4965 © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins
ischemia. This mechanism possibly lies in microglial inactivation through regulation of the expression of HIF-1α. NeuroReport 25:1122–1128 © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins. NeuroReport 2014, 25:1122–1128 Keywords: brain ischemia, hypoxia, hypoxia-inducible factor-1α, isoform of nitric oxide synthase, microglia, neuroprotection a Department of Neurosurgery, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine (Guangdong Provincial Hospital of Chinese Medicine), bThe National Key Clinic Specialty, The Neurosurgery Institute of Guangdong Province, Guangdong Provincial Key Laboratory on Brain Function Repair and Regeneration, Department of Neurosurgery, Zhujiang Hospital, Southern Medical University and cKey Laboratory of Construction and Detection of Guangdong Province, Department of Anatomy, Southern Medical University, Guangdong, China
Correspondence to Tao Huang, MD, PhD, Department of Neurosurgery, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine (Guangdong Provincial Hospital of Chinese Medicine), 111# Dade Road, Guangzhou, Guangdong Province 510120, China Tel/fax: + 86 20 81874903; e-mail: [email protected] *Tao Huang and Weiyi Huang contributed equally to the writing of this article. Received 10 June 2014 accepted 26 June 2014
essential components in normal nerve functioning, overexpression of these factors may lead to catastrophic neuronal apoptosis [4,5]. Thus, overactivation of microglia would cause irreversible damage to brain tissue and lead to neurodegenerative diseases by releasing excessive cytokines [3]. The emergence of a new phenotype and some specific proteins, such as the inducible isoform of nitric oxide synthase (iNOS), are the indicators of microglial activation [6,7]. In conclusion, we believed that the mechanism of brain injury and functional deficit caused by hypoxic ischemia may lie in the microglia activated by hypoxia, which express iNOS and secrete NO, reactive oxygen species, and proinflammatory cytokines. This iNOS-mediated NO production is considered vital in neuroinflammation after brain ischemia [8,9]. Research found that iNOS inhibitor and iNOS knockout mice displayed strong neuroprotection after cerebral ischemia through a neuron apoptosis-resisting effect [10]. Systemic infection and neuroinflammation are common syndromes after stroke in the clinic, and all these suggested that neuroinflammatory processes play a key role in neuron apoptosis after brain ischemia [11]. DOI: 10.1097/WNR.0000000000000236
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
HIF-1α upregulation in microglia Huang et al. 1123
Hypoxia-inducible factor-1 (HIF-1) consists of a basic helix–loop–helix–PAS domain, a heterodimeric transcription factor that contains the protein HIF-1α and an arylhydrocarbon receptor nuclear translocator (ARNT, HIF-1β) [12]. The availability of HIF-1 is determined primarily by HIF-1α, which is stably expressed. In contrast to HIF-1β, the amount of HIF-1α expressed is regulated in an oxygen-sensitive manner [13]. HIF-1α is efficiently degraded by the Von Hippel-Lindaudependent ubiquitin–proteasome pathway during normoxia [13]. Under hypoxic conditions, HIF-1α displays a remarkably high level of expression and heterodimerizes with HIF-1β to form HIF-1 after translocating into the nucleus. Gene transcription of vascular endothelial growth factor, erythropoietin, iNOS, and glycolytic enzymes is activated to enhance cellular adaptation to hypoxia [14]. In the brain, HIF-1α expression seems to be induced by hypoxia in neurons, astrocytes, ependymal cells, and possibly endothelial cells [14]. Neuroinflammation induced by microglia is crucial in neuron apoptosis after a stroke. The role of HIF-1α expression in microglia remained a controversial topic for the past two decades. In different neuroinflammatory diseases such as multiple sclerosis and Alzheimer’s disease, HIF-1α has different effects in the regulation of microglia [15]. In this study, short-term hypoxia was designed to investigate whether a high-altitude environment could be proposed as a therapeutic strategy for brain ischemia. The relation between microglial activation and HIF-1α expression after brain ischemia was also discussed in our study.
Materials and methods All mice were purchased from the Animal Experiment Center of Southern Medical University (Guangzhou, China). Animal experimental procedures were approved by the Southern Medical University Ethics Committee. All surgeries were performed under sodium pentobarbital anesthesia, and all efforts were made to minimize animal suffering. Animals
Twenty male mice (C57BL/6) weighing 20–22 g (12–14 weeks years old) were selected and anesthetized with intraperitoneal injection of 4% sodium chloral hydrate (100 mg/kg). The focal ischemic reperfusion model was prepared according to a previous study [16]. The mice were then treated with middle cerebral artery occlusion (MCAO) for 1.5 h. After that, the blood supply was restored. Twelve hours after the recovery of blood perfusion, all mice were randomly divided into two groups: a postoperative hypoxic group (hypoxia, n = 10) and a postoperative normoxic group (normal, n = 10). The hypoxic group was placed in the hypoxic box for 4 h, and the hypoxic conditions were set as a 5000 m altitude plateau environment. The normoxic group was placed in the normoxic environment as the
control group. Twelve hours after hypoxia, perfusion fixation was performed for both groups following a neurological assessment. Sodium chloral hydrate (4%) was used to anesthetize the mice, and cannulation was performed from the left ventricle into the ascending aorta. The vessels were rinsed using 100 ml saline (37°C, 140 mmHg) and perfusion fixation was performed for 30 min using 0.1 M phosphate buffer and 40 g/l paraformaldehyde. In the end, the mice were killed and their brain tissue was harvested (Supplement 1, Supplemental digital content 1, http://links. lww.com/WNR/A296). Another 20 male mice (C57BL/6) weighing 20–22 g (12–14 weeks old) were selected. Twelve hours after blood flow recovery, they were intraperitoneally injected with 2-methoxyestradiol (2-ME) (Sigma-Aldrich, St Louis, Missouri, USA), the HIF-1α inhibitor, at a dose of 100 mg/ kg. The mice were then randomly divided into two groups, a postoperative hypoxic group (hypoxia, n = 10) and a postoperative normoxic group (normal, n = 10), and then treated as described above (Supplement 1, Supplemental digital content 1, http://links.lww.com/WNR/A296). Hypoxia training protocol
Mice were housed in a thick-walled, high-pressure-resistantglass animal chamber that was commercially designed (76 × 50 × 50 cm3) and fitted with a brass lid and three brass outlets connected to the other components of the unit through vacuum tubes. Briefly, the first outlet was connected to a high-pressure vacuum pump with a pressure gauge through a copper tube. The second outlet was connected to a manometer that indicated the barometric pressure, whereas the third outlet was fitted to an adjustable knob to regulate the entry of air, which could develop pressure in the chamber. During the simulation, pressure was gradually decreased to a target level, equivalent to the pressure at an altitude of 3000 or 5000 m (i.e. PB 404 mmHg; PO2 84 mmHg, equivalent to an altitude of 5000 m). Four hours after exposing the animals to the decreased pressure, the pressure was gradually increased to a normal level over 15 min. For the normoxic control group, the animals were kept in the chamber (i.e. PB 760 mmHg; PO2 159.2 mmHg), which was circulated with room air for 4 h [17]. Neurological assessment score
Neurological evaluations were carried out 12 h after hypoxia according to a predetermined schedule, which excluded behavioral changes based on circadian rhythms. The examiners were not aware of the procedure the mice had undergone and the mice were scored according to the following criteria: score 0, no neurologic impairment; score 1, endoduction of the right anterior limbs and no complete tail stretch while walking to the left; score 2, circling toward the right during spontaneous walking; score 3, right-side lateriversion while walking; score 4, no spontaneous walking and some consciousness lost. We screened out mice with scores of 0 or 4 [18].
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
1124 NeuroReport 2014, Vol 25 No 14
Cerebral infarction size evaluation
The mice were killed by an overdose of anesthesia with 10% sodium chloral hydrate and decapitated immediately. Brain tissue was harvested and put into a − 70°C refrigerator for 10 min of quick freezing. Then tissue was obtained from the procerebrum and cut into five coronal brain slices of the same thickness (2 mm) from the frontal pole to the occipital pole. The slices were immediately put into 2% tetrazolium chloride (TTC)-PBS to shield from light before being incubated at 37°C for 30 min and fixed for 2 h with 4% paraformaldehyde. Normal brain tissue was dyed bright red, whereas the infarction focus presented pale white. The fixed brain slices were arranged and the areas of the prosencephalon and the cerebral infarction were measured by Image Pro Plus 5.0 (IPP, Media Cybernetics, Rockville, Maryland, USA) image processing software according to the formula V = t × (A1 + A2 + …An). In the formula, V represents the infarction volume or prosencephalon volume, t is the thickness of the slice, and A is the infarction size. Infarction volume and prosencephalon volume as well as the ratio of these two parameters (cerebral infarction volume/prosencephalon volume) were calculated. Tissue processing and immunohistochemistry
Mice were injected with sodium chloral hydrate (37°C, 140 mmHg) followed by 0.1 M phosphate buffer with 40 g/l paraformaldehyde over 30 min. Mice were then decapitated, and their brains were dissected and equilibrated for 24 h in 30% sucrose in PBS before being flashfrozen in an optimal cutting temperature compound. Brains were sectioned in the coronal plane at a thickness of 40 μm on a cryostat, blocked in PBS containing 0.3% Triton X-100 and 5% BSA (blocking solution), and incubated overnight at 4°C with the following primary antibodies: a mouse monoclonal antibody against ionized calcium-binding adapter molecule 1 (anti-Iba1, 019–19741; Wako Pure Chemical Industries Ltd, Tokyo, Japan) and anti-iNOS or anti-HIF-1α (SAB4502012, HPA001275; both from Sigma-Aldrich). The sections were then incubated with fluorescein isothiocyanate and tetramethylrhodamine B isothiocyanate-conjugated secondary antibodies (1 : 1000, H17101, A10532; Life Technologies, Carlsbad, California, USA) in blocking solution at room temperature for 2 h, washed with PBS, mounted on glass slides, and examined using a confocal microscope (Olympus, Tokyo, Japan). Microglia cultures
The BV-2 cell line is a stable microglia cell line derived from mice (gift from Department of Neurobiology of Southern Medical University). In this study, BV-2 cells were cultured in 75 cm2 flasks for 14 days in Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12; Gibco, Grand Island, New York, USA), supplemented with 10% fetal calf serum (Hyclone, Logan, Utah, USA), 100 U/ml penicillin, and 100 mg/ml streptomycin. Detached cells
were plated on 24-well plates at a density of 2 × 105 cells/ well. Cells were cultured for 2 days before treatment. Confluent cultures were passaged by trypsinization. Microglial activation and hypoxia
To activate microglia, BV-2 cells were placed in serum-free DMEM, gassed with 95% N2 and 5% CO2 (Anaerobic System PROOX model 110; BioSpherix, Redfield, New York,USA), and incubated at 37°C for 8 h. To reoxygenate hypoxic cultures, cells were transferred into a regular normoxic incubator (95% air, 5% CO2) and incubated for an additional 12 h. Then, the cultured cells were placed in serum-free DMEM and gassed with 95% N2 and 5% CO2 for 0, 4, and 8 h as a hypoxic condition (hypoxia); others were in normoxic condition (normal). Cell viability was only slightly reduced after exposure to hypoxia. 2-ME was dissolved in dimethyl sulfoxide at a concentration of 2.00 μM, and oxygen was restored after 8 h of hypoxia. The cells were incubated for the following 12 h, after which 2-ME (2.00 μM) was infused into the cultures. Western blotting
BV-2 cells were plated on six-well plates. After each condition, they were washed with cold PBS and lysed for 30 min on ice with radioimmunoprecipitation assay buffer. Protein samples containing 30 mg protein were separated on 8% SDS polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, Massachusetts, USA). The membranes were incubated for 1 h with 4% dry skim milk in PBS buffer to block nonspecific binding. The membranes were then incubated with rabbit antibodies against iNOS (1 : 1000, 610310; BD Transduction Lab, Lexington, Kentucky, USA) or mouse antibodies against HIF-1α (1 : 1000, NB100–105; Novus Biologicals, Littleton, Colorado, USA) and β-actin (1 : 1000, sc130065; Santa Cruz Biotechnology, Santa Cruz, California, USA). The membranes were then incubated with goat antirabbit or rabbit anti-mouse peroxidase-conjugated secondary antibody (1 : 1000, sc45101, sc358922; Santa Cruz Biotechnology) for 1 h. The blots were visualized by enhanced chemiluminescence (ECL; Santa Cruz Biotechnology) using Kodak X-OMAT LS film (Eastman Kodak, Rochester, New York, USA). All western blot experiments were repeated at least three times, with different sets of samples, throughout this study. Protein levels were quantified and normalized to β-actin levels. Quantification of western blots was performed by Image Pro Plus 6.0 (IPP, Media cybernetics, Rockville, Maryland, USA). Statistical analysis
SPSS software (SPSS Inc., Chicago, Illinois, USA) was used. Results were expressed as mean ± SE and comparisons were made using one-way analysis of variance with Bonferroni’s post-test. P value less than 0.05 was considered statistically significant.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
HIF-1α upregulation in microglia Huang et al. 1125
Results Neuroprotection was induced by hypoxia after middle cerebral artery occlusion and inactivated microglia associated with the enhancement of hypoxia-inducible factor-1α expression
At 1.5 h after treatment with MCAO, the blood supply was restored. Ten mice were then placed in hypoxic conditions for 4 h. Compared with the normoxic group, the neurological assessment score of the hypoxic group was lower and the size of cerebral infarction focus was significantly smaller (Fig. 1a and b). This indicated that
cerebral infarction and neurological damage caused by cerebral ischemia can be effectively attenuated by a short-term plateau hypoxic environment. The results of the fluorescent labeling experiment showed that microglial cells aggregated at the junction between the ischemic infarction focus and the normal tissues in the normoxic group. Besides, HIF-1α expression of microglia was significantly higher in the hypoxic group compared with the normoxic group. iNOS expression in the hypoxic group was significantly lower than that of the normoxic group (Fig. 1c and d). The results indicated
Fig. 1
(a)
Normal
(c)
Hypoxia
Normal
Hypoxia
HIF-1α
50 40 30
∗∗
20 10 0 Normal
Hypoxia
(d) 40
3
2
∗∗
1
Number of positive
The volume of infarct (%)
(b)
Neurological deficit score
iNOS
30
60 ∗∗ 40 ∗∗
20 20 10 0
0
0 Normal
Hypoxia
Normal Hypoxia HIF-1α
Normal
Hypoxia iNOS
(a) The TTC stain showed that the volume ratio of the cerebral infarction was significantly lower in the hypoxic group (n = 5) compared with the normal group (n = 5). (b) The neurological score of the hypoxic group (n = 10) and the normal group (n = 10). Data are expressed as mean ± SE. Significant differences between the hypoxic group and the normal group were seen in infarction volume as well as in neurological score (**P < 0.01). (c) Immunofluorescence of ischemic infarction focus and normal tissues was performed. Microglia was labeled with FITC (light grey). HIF-1α and iNOS were labeled with tetramethylrhodamine B (light grey). (d) Statistical analysis of HIF-1α-positive and iNOS-positive cells in ischemic infarction focus and normal tissues. Five visions of different areas were randomly chosen. FITC, fluorescein isothiocyanate; HIF-1α, hypoxia-inducible factor-1α; iNOS, isoform of nitric oxide synthase; TTC, tetrazolium chloride.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
1126 NeuroReport 2014, Vol 25 No 14
that HIF-1α expression in microglial cells, which was induced by hypoxia after cerebral ischemia, might be the reason for brain protection. Inhibition of hypoxia-inducible factor-1α in microglia blocked neuroprotection after hypoxic condition
There was no significant difference in infarction size and neurological assessment score between the hypoxic group and the control group in vivo after treatment with MCAO and injection with 2-ME, the HIF-1α antagonist (Fig. 2a and b).
This indicated that the neuroprotection after the hypoxic period following cerebral ischemia was closely related to HIF-1α activation. There was also no difference in HIF-1α and iNOS expression (Fig. 2c and d) in microglia that aggregated at the junction of the ischemic infarction focus and normal tissues. This indicated that the expression of iNOS in activated microglia was determined by the level of HIF-1α expression. That is to say, the brain protection effect may derive from the reduction of iNOS released, which resulted from overexpressed HIF-1α in hypoxia-treated microglia.
Fig. 2
(a)
Normal
(c)
Hypoxia
Normal
Hypoxia
HIF-1α
iNOS
40 30 20 10 0 Normal
Hypoxia
(d)
3
Number of positive
50
Neurological deficit score
The volume of infarct (%)
(b)
2
1
25
60
20 40
15 10
20
5 0
0 Normal
Hypoxia
0 Normal HIF-1α
Hypoxia
Normal
Hypoxia iNOS
(a) The TTC stain showed an identical volume ratio of the cerebral infarction in the hypoxic group (n = 5) and normal group (n = 5) after 2-methoxyestradiol injection. (b) There were no significant differences in the neurological score between the hypoxic group (n = 10) and normal group (n = 10) in vivo after injection of an HIF-1α antagonist. (c) Immunofluorescence was performed on the ischemic infarction focus and normal tissues. Microglia were labeled with FITC (light grey). HIF-1α and iNOS were labeled with tetramethylrhodamine B (light grey). (d) Statistical analysis of HIF-1α-positive and iNOS-positive cells in the ischemic infarction focus and normal tissues. Five visions of different areas were randomly chosen. FITC, fluorescein isothiocyanate; HIF-1α, hypoxia-inducible factor-1α; iNOS, isoform of nitric oxide synthase; TTC, tetrazolium chloride.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
HIF-1α upregulation in microglia Huang et al. 1127
Fig. 3
8h
0h
Hypoxia 4h
(b) 8h
HIF-1α iNOS
1.0
∗
4
1 0
0.0
iNOS
8h
0h
4h
8h
HIF-1α iNOS β-Actin
2-ME 0.20
2-ME 1.5
0.15
1.0
0.10 0.5
0.05
0.0
0.00
HIF-1α
N o H rma yp l o 0 N xia h or 0 h H ma yp l 4 o h N xia or 4 m h H al yp 8 ox h ia 8h
4h
Level of expression
0h
(d)
Hypoxia
N o H rma yp l o 0 N xia h or 0 h H ma yp l 4 o h N xia or 4 m h H al yp 8 ox h ia 8h
Normal
∗∗
3
HIF-1α (c)
∗∗
2 0.5
N o H rma yp l o 0 N xia h or 0 h H ma yp l 4 o h N xia or 4 m h H al yp 8 ox h ia 8h
β-Actin
∗∗
1.5
N o H rma yp l o 0 N xia h or 0 h H ma yp l 4 o h N xia or 4 m h H al yp 8 ox h ia 8h
0h
Normal 4h
Level of expression
(a)
iNOS
(a) Western blot results after oxygen was restored for 12 h. Results were obtained at 0, 4, and 8 h after hypoxia. iNOS expression was reduced in the hypoxic group compared with the normal group, which was associated with the high expression of HIF-1α. (b) Statistical analysis of HIF-1α and iNOS expression (n = 3; *P < 0.05, **P < 0.01). (c) There was no difference in iNOS or HIF-1α expression between the hypoxic and normal group following 2-methoxyestradiol treatment. (d) Statistical analysis of HIF-1α and iNOS expression after 2-methoxyestradiol treatment (n = 3). HIF-1α, hypoxiainducible factor-1α; iNOS, isoform of nitric oxide synthase; 2-ME, 2-methoxyestradiol.
Hypoxia-inducible factor-1α in microglia was activated by exposure to hypoxic conditions
Microglia were activated by a hypoxic condition in vitro and recovered in normoxia for 12 h. Western blot analysis showed no difference in the expressions of HIF-1α and iNOS in activated microglia for 0-h hypoxia and normoxia treatment. However, a higher level of HIF-1α expression and a lower level of iNOS expression in activated microglia were detected 4 and 8 h after exposure to hypoxic condition compared with normoxic condition (Fig. 3a and b). This result suggested that overexpression of HIF-1α through hypoxia may attenuate the microglial activation. After 2-ME was applied to activated microglia under different conditions such as rehypoxia and normoxia, no difference in iNOS expression between these two groups was seen (Fig. 3c and d). This indicated that the reduction in iNOS activation in microglia by hypoxia treatment may be regulated by HIF-1α activation.
Discussion In our study, hypoxia was induced by a simulated highaltitude environment. Over the past decade, short-term and long-term intermittent hypoxic environments have been proposed to have a therapeutic effect for ischemia [17,19]. A potential neuroprotective effect conferred by short-term hypoxia after cerebral ischemic has not yet
been confirmed. Our results indicated that short-term hypoxic conditions could effectively protect neurons at the early stage of cerebral ischemia and present an anti-inflammatory effect in the central nervous system as well. According to recent research, neuroinflammation following microglial activation is considered one of the most crucial factors for neurological damage after cerebral ischemia [20]. While under cerebral hypoxia, the activation of HIF-1α is an important inductor for microglial activation [21]. Activated microglia may later induce neuronal apoptosis by releasing endogenous inflammatory cytokines. In the present study, a high level of HIF-1α expression was detected in activated microglia [22]. It was even higher when the microglial activation was induced by hypoxia after ischemia. In the junction of infarction area and normal tissue, accumulation of microglia and a high level of HIF-1α expression were both observed, which was identical to previous studies. In vivo, the activation of iNOS was positively regulated by HIF-1α. High expression of HIF-1α after hypoxia significantly inhibited the expression of iNOS. This suggested that overactivation of HIF-1α may suppress the activation of microglia. Thus, a decline of iNOS expression was observed in microglia after short-term hypoxia.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
1128 NeuroReport 2014, Vol 25 No 14
Microglia can be activated by hypoxic conditions in vitro, as well as by neuronal apoptosis. Meanwhile, a high level of HIF-1α expression was observed in cerebral resident cells after brain ischemia [23]. Our research indicated that, although microglia could be activated by hypoxia after brain ischemia, 12 h of hypoxia after MCAO treatment can attenuate the brain damage in mice, and the overexpression of HIF-1α can suppress microglial activation as well. After injection of 2-ME, no difference in HIF-1α expression was observed in the microglia at the infarction border zones. This was accompanied by the absence of the brain protection effect. Some studies have shown that antagonization of HIF-1α in early stage of brain ischemia presents a neuroprotective effect. The effect may be due to the absence of HIF-1α, which can reduce the activation of microglia [24]. 2-ME is a stable and nontoxic estradiol derivative and known as a widely used HIF-1α inhibitor. By inhibiting HIF-α activation, 2-ME can suppress tumor growth, lessen traumatic brain injury, and display an anti-immunoinflammatory effect [25]. In our study, hypoxia after cerebral ischemia had a certain brain protection effect, in which HIF-1α played an important regulatory role. The outcome of this study suggested that hypoxic conditions can be used to achieve the maximum brain protection effect. In our future work, we will continue to explore the underlying mechanisms of this transient hypoxia-induced brain protection effect in cerebral ischemia.
3 4 5
6 7
8
9
10
11
12
13
14
15
16
Conclusion The present results showed that hypoxia could enhance the expression of HIF-1α in microglia and cause microglial activation. Further, HIF-1α overexpression regulated microglia inactivated by re-exposure to hypoxia, leading to neuroprotection against ischemia-induced cerebral infarction. Thus, this study has identified that hypoxic conditions can be exploited to achieve maximum neuroprotection after brain ischemia through regulation of the expression of HIF-1α in microglial activation.
17
18
19
20
21
Acknowledgements This study was supported by the Natural Science Foundation of Guangdong Province (S2011010005403). Conflicts of interest
22
23
There are no conflicts of interest.
References 1 2
Marty S, Dusart I, Peschanski M. Glial changes following an excitotoxic lesion in the CNS-I. Microglia/macrophages. Neuroscience 1991; 45:529–539. Berta T, Park CK, Xu ZZ, Xie RG, Liu T, Lü N, et al. Extracellular caspase-6 drives murine inflammatory pain via microglial TNF-alpha secretion. J Clin Invest 2014; 124:1173–1186.
24
25
Rodriguez JI, Kern JK. Evidence of microglial activation in autism and its possible role in brain underconnectivity. Neuron Glia Biol 2011; 7:205–213. Loihl AK, Murphy S. Expression of nitric oxide synthase-2 in glia associated with CNS pathology. Prog Brain Res 1998; 118:253–267. Bal-Price A, Brown GC. Inflammatory neurodegeneration mediated by nitric oxide from activated glia-inhibiting neuronal respiration, causing glutamate release and excitotoxicity. J Neurosci 2001; 21:6480–6491. Chao CC, Hu S, Peterson PK. Modulation of human microglial cell superoxide production by cytokines. J Leukoc Biol 1995; 58:65–70. Regenhardt RW, Desland F, Mecca AP, Pioquinto DJ, Afzal A, Mocco J, Sumners C. Anti-inflammatory effects of angiotensin-(1–7) in ischemic stroke. Neuropharmacology 2013; 71:154–163. Kawase M, Kinouchi H, Kato I, Akabane A, Kondo T, Arai S, et al. Inducible nitric oxide synthase following hypoxia in rat cultured glial cells. Brain Res 1996; 738:319–322. Iadecola C, Ross ME. Molecular pathology of cerebral ischemia: delayed gene expression and strategies for neuroprotection. Ann N Y Acad Sci 1997; 835:203–217. Wang Y, Ahmad N, Kudo M, Ashraf M. Contribution of Akt and endothelial nitric oxide synthase to diazoxide-induced late preconditioning. Am J Physiol Heart Circ Physiol 2004; 287:H1125–H1131. Miura T, Hamawaki M, Hazama S, Hashizume K, Ariyoshi T, Sumi M, et al. Outcome of surgical management for active mitral native valve infective endocarditis: a collective review of 57 patients. Gen Thorac Cardiovasc Surg 2014 [Epub ahead of print]. Cao Y, Mao X, Sun C, Zheng P, Gao J, Wang X, et al. Baicalin attenuates global cerebral ischemia/reperfusion injury in gerbils via anti-oxidative and anti-apoptotic pathways. Brain Res Bull 2011; 85:396–402. Richards EM, Fiskum G, Rosenthal RE, Hopkins I, McKenna MC. Hyperoxic reperfusion after global ischemia decreases hippocampal energy metabolism. Stroke 2007; 38:1578–1584. Wang GL, Jiang BH, Rue EA, Semenza GL. Hypoxia-inducible factor 1 is a basic-helix–loop–helix–PAS heterodimer regulated by cellular O2 tension. Proc Natl Acad Sci USA 1995; 92:5510–5514. Zeis T, Graumann U, Reynolds R, Schaeren-Wiemers N. Normal-appearing white matter in multiple sclerosis is in a subtle balance between inflammation and neuroprotection. Brain 2008; 131 (Pt 1):288–303. Chiamulera C, Albertini P, Valerio E, Reggiani A. Activation of metabotropic receptors has a neuroprotective effect in a rodent model of focal ischaemia. Eur J Pharmacol 1992; 216:335–336. Zhu XH, Yan HC, Zhang J, Qu HD, Qiu XS, Chen L, et al. Intermittent hypoxia promotes hippocampal neurogenesis and produces antidepressant-like effects in adult rats. J Neurosci 2010; 30:12653–12663. Cao CX, Yang QW, Lv FL, Cui J, Fu HB, Wang JZ. Reduced cerebral ischemia-reperfusion injury in Toll-like receptor 4 deficient mice. Biochem Biophys Res Commun 2007; 353:509–514. Zhang G, Zhou SM, Yuan C, Tian HJ, Li P, Gao YQ. The effects of short-term and long-term exposure to a high altitude hypoxic environment on neurobehavioral function. High Alt Med Biol 2013; 14:338–341. Tang Z, Gan Y, Liu Q, Yin JX, Liu Q, Shi J, Shi FD. CX3CR1 deficiency suppresses activation and neurotoxicity of microglia/macrophage in experimental ischemic stroke. J Neuroinflammation 2014; 11:26. Kyotani Y, Ota H, Itaya-Hironaka A, Yamauchi A, Sakuramoto-Tsuchida S, Zhao J, et al. Intermittent hypoxia induces the proliferation of rat vascular smooth muscle cell with the increases in epidermal growth factor family and erbB2 receptor. Exp Cell Res 2013; 319:3042–3050. Lu DY, Liou HC, Tang CH, Fu WM. Hypoxia-induced iNOS expression in microglia is regulated by the PI3-kinase/Akt/mTOR signaling pathway and activation of hypoxia inducible factor-1alpha. Biochem Pharmacol 2006; 72:992–1000. Del Bigio MR, Becker LE. Microglial aggregation in the dentate gyrus: a marker of mild hypoxic-ischaemic brain insult in human infants. Neuropathol Appl Neurobiol 1994; 20:144–151. Chavez-Valdez R, Martin LJ, Flock DL, Northington FJ. Necrostatin-1 attenuates mitochondrial dysfunction in neurons and astrocytes following neonatal hypoxia-ischemia. Neuroscience 2012; 219:192–203. Schaible EV, Windschügl J, Bobkiewicz W, Kaburov Y, Dangel L, Krämer T, et al. 2-Methoxyestradiol confers neuroprotection and inhibits a maladaptive HIF-1alpha response after traumatic brain injury in mice. J Neurochem 2014; 129:940–954.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
