Background
Ischemic stroke is one of the most frequent causes of injury to the central nervous system (CNS). Complex pathological mechanisms are involved in brain injury after cerebral ischemia. Inflammatory processes have a fundamental role in the pathophysiology of ischemic injury stroke. Microglial cells, which are rapidly and time-dependently activated after ischemia, are considered as the major cellular contributors to post-injury inflammation [
1,
2]. Stroke-induced microglial activation causes release of a variety of inflammatory mediators many of which are cytotoxic and/or cytoprotective. Emerging evidence demonstrates that alternatively activated microglia promotes tissue repair by secreting anti-inflammatory mediators and growth factors [
3,
4]. In contrast, overactivated microglia following ischemia stroke can secrete a wide range of inflammatory factors and neurotoxic compounds, including nitric oxide, tumor necrosis factor (TNF)-α, interleukin (IL)-6, and reactive oxygen species (ROS), which are deleterious to bystander neurons and impact their processes [
5]. As a result of findings such as these, it is increasingly well-accepted that microglia activation has been known to exert dual effects on ischemic injury. An improved understanding of the mechanisms underlying microglial activation and their functional modulation by the local microenvironment is likely to advance our knowledge of many CNS pathological states.
Homocysteine (Hcy) is an intermediate sulfhydryl-containing amino acid derived from methionine. It has been proved that patients suffering with severe hyperhomocysteinemia manifest typical clinical cardiovascular symptoms as well as neurological disorders, such as cerebral atrophy, dementia, and seizures [
6]. The mechanism underlying Hcy mediated-pathogenesis of nervous system disorders is not yet fully understood. The toxicity of Hcy to CNS neurons is widely recognized affecting both the neuronal survival rate and the ability of neurons to transmit signal and thus to form functional neural networks [
7]. Moreover, Hcy is deemed to affect microglia proliferation in vitro [
8]. However, whether microglia activation and microglia-mediated inflammatory responses involve neurotoxicity of Hcy remains largely unknown.
The signal transducer and activator of transcription 3 (STAT3), a member of the STAT protein family of transcription factors, has been extensively described as a central signaling molecule that controls cellular adaption in response to environmental stimuli or stress. Several groups have shown that the STAT3 is activated in vitro and in vivo experimental models of stroke and subsequently activated STAT3 promotes numerous genes responsible for many cellular functions that may play a critical role in both neural injury and repair [
9,
10]. There are conflicting data whether this pathway activation leads to improved neurological recovery. Some findings suggest that treatments that activate the STAT3 signaling after experimental stroke may lead to improved functional performance and/or decreased cell death [
11]. On the contrary, accumulating evidence suggests that activation of STAT3 leads to decreased cerebral recovery and that blocking this pathway leads to better neurological outcomes [
12]. Thus, the precise contribution of STAT3 activation after stroke remains incompletely understood. On the other hand, STAT3, an important regulator of inflammatory gene expression, has been shown to play an important regulatory role in microglial reactivity to various stimuli and mediate pro-inflammatory responses in microglia in response to various CNS insults. Activated STAT3 can promote the transcription and expression of many genes that encode proinflammatory mediators, including cytokines, chemokines, adhesion molecules, and inflammatory enzymes [
2]. In addition, it has been demonstrated that neuroinflammatory processes after cerebral ischemia involve aberrantly activated STAT3. The strong induction of pSTAT3 is predominantly localized in the macrophages/microglia in the post-ischemic brain [
12]. Given the importance of STAT3 in activating microglia and inducing cytokine/chemokine expression, we investigated whether Hcy activates STAT3 in microgla and whether STAT3 is involved in the Hcy-induced inflammatory responses.
Accordingly, the present study was designed to elucidate the molecular mechanism of neurotoxicity by Hcy in relation to microglial-mediated neuroinflammation and microglial STAT3 overactivation using a rat model of transient focal cerebral ischemia. This study demonstrates for the first time that STAT3 overactivation located in microglia cells plays an important role in Hcy-induced microglia activation and the inflammatory responses both in the brain cortex and the dentate gyrus (DG) region of the hippocampus following ischemic injury.
Methods
Experimental animals and administration
One hundred adult male Sprague-Dawley rats (180–220 g) (Grade SPF, Certificate Number SCXK (jing) 2012–0001) were purchased from Vital River Laboratory Co. Ltd. (Beijing, China). The experimental protocols were approved by the Tianjin Medical University Animal Ethics Committee and performed in compliance with institutional guidelines under approved protocols. The rats were randomly allocated into six groups: SHAM operation control group (SHAM), middle cerebral artery occlusion reperfusion group (MCAO), Hcy-treated SHAM group (SHAM + Hcy), Hcy-treated MCAO group (MCAO + Hcy), Hcy combined with AG490(α-cyano-(3,4-dihydroxy)-N-benzylcin-namide)-treated SHAM group (SHAM+ Hcy + AG490), and Hcy combined with AG490-treated MCAO group (MCAO + Hcy + AG490). Hcy (1.6 mg/kg/day, Sigma-Aldrich, St. Lous, Mo, USA) was administered by tail vein injection (Additional file
1: Video S1) for 21 days prior to SHAM or MCAO operation. The AG490 (3 mg/kg, Abcam, Cambridge, MA, USA) was administered by intraperitoneal injection before 1 h of SHAM or MCAO operation.
Induction of focal cerebral ischemia-reperfusion
The MCAO rats were induced by using the intraluminal filament technique as we described previously [
13]. A nylon thread was advanced by the left common carotid artery, through the left internal carotid artery and into the origin of the middle cerebral artery. Two hours after cerebral ischemia, the thread was carefully withdrawn to establish reperfusion. Animals assigned to SHAM operation were treated similarly, except that the thread was not advanced to the origin of the middle cerebral artery.
Measurement of infarct volume
At 24 h after reperfusion, three rats in each group were euthanized and the brains were rapidly removed and frozen at −20 °C for 15 min. Serial coronal brain slices with a 2 mm thickness each were stained with 2% 2,3,5-triphenyltetrazolium chloride (TTC) at 37 °C for 15 min followed by fixation with 4% paraformaldehyde for 24 h. Infarct volume measurements were carried out by an investigator blinded to the treatment groups as previously described [
14].
Hematoxylin and eosin (HE) staining
Rats were euthanized at 72 h after the MCAO operation for HE staining. Left brains were quickly removed and further fixed using 4% paraformaldehyde overnight. Then brain tissues were equilibrated in a phosphate-buffered 30% sucrose solution, embedded in paraffin, and cut into 6-μm coronal sections.
Three rats of each group were assigned to HE staining. Sections of brain tissues embedded in paraffin were stained with HE for routine histological examinations and the morphological changes were observed under a light microscope (IX81; Olympus, Tokyo, Japan).
Fluoro-jade B staining
Fluoro-Jade B is a high affinity fluorescent marker for the localization of neuronal degeneration. Fluoro-Jade B staining was performed using a modification of a previously published procedure [
15]. Briefly, the sections were immersed in a solution containing 1% sodium hydroxide in 80% alcohol for 5 min, followed by 2 min in 70% alcohol and 2 min in distilled water. The slides were then transferred to a solution containing 0.06% potassium permanganate for 10 min, then incubated in the staining solution of Fluoro-Jade B (Millipore, Chemicon, USA) for 20 min. After washing 3 times in distilled water, the sections were allowed to dry, immersed in xylene, coverslipped, and examined using a fluorescence microscope with 450–490 nm excitation light.
Immunofluorescence analysis
Immunofluorescence analysis of the paraffin embedded tissues was performed using standard protocols provided by the antibody manufacturers. Briefly, sections were fixed in 4% paraformaldehyde, blocked with 10% normal goat serum, and then incubated overnight at 4 °C with the primary antibodies: rabbit anti-TNF-α (Abcam, Cambridge, MA; 1:200), mouse anti-Iba1 (Abcam; 1:100), mouse anti-IL-6 (Abcam; 1:200,), mouse anti-OX-42 (Abcam; 1:100), rabbit anti-pSTAT3 (Cell Signaling Technology, Danvers, MA, USA; 1:100). Thereafter, sections were briefly washed with PBS and incubated for 1 h at room temperature with appropriate secondary antibodies (tetramethylrhodamine -conjugated anti-mouse IgG; fluorescein isothiocyanate -labeled anti-rabbit IgG; Abcam; 1:100). 4′, 6-diamidino-2- phenylinedole (DAPI; Solarbio, Beijing, China; 1 μg/ml) was used to dye the nuclei before 10 min of mounting. The sections were then analyzed with an Olympus IX81 microscope (Olympus, Tokyo, Japan) and Image-Pro Plus 6.0 software.
Western Blot assay
The protein levels of pSTAT3, two microglia markers Iba1and OX-42, and proinflammatory cytokines TNF-α and IL-6 in the ipsilateral ischemic hemisphere (including brain cortex and hippocampus) were measured by Western blot. The protein concentration in the supernatant was measured with the bicinchoninic acid (BCA) protein assay Kit (Pierce, Rockford, IL, USA). Equivalent amounts of protein were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose blotting membranes (Millipore, Bedford, MA, USA) was performed according to the wet electrical transfer method. The membranes were blocked with 5% non- fat dry milk in tris-buffered saline and tween 20 (TBST) and subsequently incubated with mouse anti-STAT3 antibody (Cell Signaling Technology; 1:1000), rabbit anti-phosphorylated STAT3 at Tyr705 (1:1000, Cell Signaling Technology), Mouse anti- Iba-1 (Abcam; 1:1000), mouse anti- OX-42 (Abcam; 1:1000), and mouse anti-β-actin (Cell Signaling Technology; 1:2000). After washing in TBST, immunoblots were incubated with horseradish peroxidase-conjugated secondary antibodies (HRP-linked anti-rabbit IgG; HRP-linked anti-mouse IgG; Cell Signaling Technology; 1:2000) for 1 h at room temperature. The immunoblots were developed with chemiluminescence reagents (Millipore, Bedford, MA), and then observed using the ChemiDoc™ XRS+ Imaging System (Bio-Rad, Hercules, California, USA). The densities of the immunobands were quantitated by Image J 1.4.3.67.
Statistical analysis
Data were expressed as mean ± S.D. Variance was analyzed by a one-way ANOVA followed by Student-Newman-Keuls test. Statistical analysis was performed with SPSS v.16.0. Values of p < 0.05 were considered statistically significant.
Discussion
The ischemic stroke is one of the common causes of morbidity and mortality worldwide. There are many causes of ischemic stroke, and hyperhomocysteinemia is one of the documented causes. Hyperhomocysteinemia originates from a deviation in the methionine-homocysteine metabolism including disturbances of enzymes, vitamin deficiencies, and different other factors [
21,
22]. Although it has been shown that Hcy has a neurotoxic effect on ischemic brains, its roles in modulating microglial activation and inducing inflammation have not been documented. The present study reveals that Hcy treatment enhanced brain injury, induced activation of microglia, and triggered the expression of pro-inflammatory cytokines in the brain cortex and the DG region of the hippocampus using a rat MCAO model. Moreover, to our best knowledge, this is the first study to provide evidence that changes of STAT3 activities located in microglia involve Hcy-induced microglia activation and inflammation responses following stroke.
The previous studies demonstrated that the main cell types of the central nervous system involve Hcy neurotoxicity. High level of Hcy can cause the toxic effect, leading to neuron and neural cell death [
23,
24] while low dose can promote glial cell proliferation [
25]. In addition, some studies suggest that the neurotoxicity of Hcy may involve negative regulation of neural stem cell (NSC) proliferation [
26]. Several reports have also found that Hcy promotes proliferation and activation of microglia through induction of NAD(P)H oxidase in vitro [
25,
27]. Low-dose Hcy not only induced Bv2 cell proliferation but also activated these cells in a dose- and time-dependent manner. In our study, Hcy induced microglia activation in ischemic brain. This result is in line with in vitro study. Meanwhile, since Hcy affected the activities of several main brain cells, complex pathogenetic mechanisms may be involved in brain injury caused by Hcy after cerebral ischemia.
Microglia represent 5–20% of the total glial population and are key modulators of the immune response in the brain [
28,
29]. Similar to the role of peripheral macrophages, Microglia are now known as the first line of immune defense against central nervous system (CNS) injuries and disorders. These highly plastic cells play dualistic roles in neuronal injury and recovery. Some studies have found that under physiologic conditions, resident microglia are quiescent and scattered throughout the CNS [
30]. Occasionally microglia are moderately activated to play a classic role as “scavenger” for the maintenance and restoration of the CNS [
31]. The moderate activation of microglia contributes to nervous system repair by engulfing pathogens within brain tissue and releasing neurotrophic factors. However, excessive and prolonged activation of microglial cells can lead to a variety of pathological damages in the central nervous system [
3,
32]. Vehmas et al. [
33] have confirmed that the amount of activated microglial cells is positively correlated with the progress of Alzheimer’s disease. In our models, Hcy treatment activated microglial cells, markedly enlarged the infarct volume and induced cell injury, so the hyperactivation of microglial cells caused by Hcy appeared to be harmful for the ischemic brain. Thus, whether the activation of microglial cells is beneficial or detrimental appears to depend on the extent of cell activation.
In most cases, activated microglia initiate neuroinflammation by producing cytotoxic and inflammatory factors such as the cytokines IL-1β, TNF-α and IL-6, thereby aggravating brain damage [
34]. High concentration of TNF-α has direct toxic effect on neurons and neural cells. TNF-α transgenic mice have severe inflammation and exhibit brain and neurodegenerative diseases [
35]. Regarding IL-6, its high expression can accelerate the pathological processes of central nervous system disorders [
36]. Meanwhile, there is increasing evidence that demonstrating inflammation not only immediately affects the infarcted tissue but also causes long-term damage in the ischemic penumbra after cerebral ischemia. The present study confirms that Hcy treatment induced activation of microglia and a significant up-regulation of the inflammatory factors TNF-α and IL-6 in the cortex and hippocampus of ischemic brains. Moreover, based on results of TTC staining and morphological assays, Hcy may exert a neurotoxic effect by microglia-mediated neuroinflammation injury.
To explore the potential mechanisms of the microglia-inflammatory enhancing effect of Hcy, we focused on investigating JAK2/STAT3 pathway. JAK2/STAT3, as critical immunological signaling molecules, are normally expressed in the brain and play a vital role in regulating microglial activation and inflammatory response [
37,
38]. STAT3, an important downstream regulatory molecule of the JAK/STAT pathway, is a well-established regulator of inflammatory gene expression and a marker of CNS damage [
39]. Expression levels of STAT3 have been shown to be enhanced in reactive microglia cells when the brain suffers focal ischemic injury [
40] Aberrantly activated STAT3 has also been reported to involved in the neuroinflammatory injury after ischemic stroke. To investigate the potential function of the JAK2-STAT3 pathway in Hcy-induced microglia activation and inflammatory response following MCAO, we examine whether the JAK2 inhibitor AG490 could affect Hcy-induced pSTAT3, the microglia specific markers Iba-1and OX-42, and the proinflammatory mediators TNF-α and IL-6 expression. Our results revealed that the activation of STAT3 in microglia and secretion of TNF-α and IL-6 were significantly upregulated in Hcy-treated ischemic brains and this effect was reversed by AG490. These data provide further evidence that that the STAT3 expression raised by Hcy treatment might be involved in microglial activation and the neuroinflammatory injury in MCAO rats.
Focal ischemia-induced STAT3 phosphorylation was previously reported to be localized in various cell types including microglia/macrophages, astrocytes and neurons [
39,
41,
42]. Activated microglia/macrophages (ED1 and OX42/Cd11b positive, microglia/macrophage markers) were observed to be the predominant cell type that showed pSTAT3 immunostaining at 24 and 72 h after brain ischemia/reperfusion injury [
12]. Moreover, it has been reported that pSTAT3 immunoreactivity was also present in different brain regions following stroke. For instance, Planas et al. [
39] showed that a transient episode of MCAO induced a strong microglial response. This was accompanied by increased expression of STAT3 in the ipsilateral cortex and striatum. However, little is known about changes in activation of microglial STAT3 in rat hippocampus after brain ischemia, despite the finding that hippocampus is one of the most vulnerable brain regions to ischemic damages. The present study demonstrated that a strong induction of microglial STAT3 occurred in rat hippocampus after cerebral ischemia by immunohistochemistry. In future study, two points will still need to be clarified: whether the activation of STAT3 in neurons, reactive astrocytes and microglia would play different roles in ischemia-induced injury and whether STAT3 activation in different brain regions may serve different functions.