Di ff usion-weighted magnetic resonance imaging re fl ects activation of signal transducer and activator of transcription 3 during focal cerebral ischemia/ reperfusion

Wen-juan Wu, Chun-juan Jiang Zhui-yang Zhang Kai Xu, Wei Li

1 Department of Radiology, Nanjing Medical Unversity A ffi liated Wuxi Second People’s Hospital, Wuxi, Jiangsu Province, China

2 Department of Radiology, A ffi liated Hospital of Xuzhou Medical University, Xuzhou, Jiangsu Province, China

Di ff usion-weighted magnetic resonance imaging re fl ects activation of signal transducer and activator of transcription 3 during focal cerebral ischemia/ reperfusion

Wen-juan Wu1,2, Chun-juan Jiang1, Zhui-yang Zhang1, Kai Xu2, Wei Li1,＊

1 Department of Radiology, Nanjing Medical Unversity A ffi liated Wuxi Second People’s Hospital, Wuxi, Jiangsu Province, China

2 Department of Radiology, A ffi liated Hospital of Xuzhou Medical University, Xuzhou, Jiangsu Province, China

How to cite this article:Wu WJ, Jiang CJ, Zhang ZY, Xu K, Li W (2017) Di ff usion-weighted3 magnetic resonance imaging re fl ects activation of signal transducer and activator of transcription 3 during focal cerebral ischemia/reperfusion. Neural Regen Res 12(7):1124-1130.

Graphical Abstract

orcid: 0000-0002-3808-4399 (Wei Li)

Signal transducer and activator of transcription (STAT) is a unique protein family that binds to DNA, coupled with tyrosine phosphorylation signaling pathways, acting as a transcriptional regulator to mediate a variety of biological e ff ects. Cerebral ischemia and reperfusion can activate STATs signaling pathway, but no studies have con fi rmed whether STAT activation can be veri fi ed by di ff usion-weighted magnetic resonance imaging (DWI) in rats aer cerebral ischemia/reperfusion. Here, we established a rat model of focal cerebral ischemia injury using the modi fi ed Longa method. DWI revealed hyperintensity in parts of the lehemisphere before reperfusion and a low apparent di ff usion coe ffi cient. STAT3 protein expression showed no signi fi cant change aer reperfusion, but phosphorylated STAT3 expression began to increase after 30 minutes of reperfusion and peaked at 24 hours. Pearson correlation analysis showed that STAT3 activation was correlated positively with the relative apparent di ff usion coe ffi cient and negatively with the DWI abnormal signal area.ese results indicate that DWI is a reliable representation of the infarct area and re fl ects STAT phosphorylation in rat brain following focal cerebral ischemia/reperfusion.

nerve regeneration; cerebral ischemia/reperfusion; magnetic resonance imaging; di ff usion weighted imaging; signal transducer and activator of transcription 3; phosphorylated signal transducer and activator of transcription 3; apparent di ff usion coe ffi cient; relative apparent di ff usion coe ffi cient; immunohistochemistry; western blot assay; neural regeneration

Introduction

Cerebral ischemia/reperfusion injury is an important pathophysiological process that underlies cerebrovascular disease. Magnetic resonance imaging (MRI) can reveal ischemic brain tissue. In hyperacute cerebral infarction (＜ 6 hours), the infarcted area can be seen in diffusion-weighted MRI (DWI), and magnetic resonance perfusion imaging can show the location and extent of the ischemic zone at around 10 minutes (Beck et al., 2014).

Cerebral ischemia/reperfusion can activate signal transducers and activators of transcription (STATs) (Li et al., 2015b).e Janus kinase (JAK)-STAT pathway is activated after cerebral ischemia. Membrane receptor signaling by various ligands induces activation of JAK kinases, which then leads to tyrosine phosphorylation of various STAT transcription factors (Kim et al., 2017). STAT1 and STAT3 are members of the STAT family, and phosphorylated (p-) STAT3 is the activated form of STAT3 (Jia et al., 2017). These proteins play an important role in neuronal survival and antiapoptosis. p-STAT3 is a mediator of growth factors, hormones and cytokines, and exerts its protective and regenerative effects in cerebral ischemia/reperfusion partly through transcriptional upregulation of neuroprotective and neurotrophic genes (Jiang et al., 2012). In the present study, we analyzed the changes in DWI, STAT3 and p-STAT3 in the ischemic injury zone in a rat model of focal cerebral ischemia/reperfusion injury.

Materials and Methods

Animals

A total of 110 healthy male Sprague-Dawley rats, 45–60 days old and weighing 290–330 g, were provided by the Animal Center of Xuzhou Medical University, Jiangsu Province, China. The rats were randomized into three groups: sham (n= 10), 2-hour ischemia (n= 50), and 6-hour ischemia (n= 50). Rats in the ischemia groups underwent 2- or 6-hour ischemia followed by reperfusion for 0, 0.5, 2, 6, or 24 hours (n= 10 rats per time point). All rats were housed under diurnal lighting and had free access to food and water before the experiments. The protocols were approval by the Committee on Animal Experimental Guidelines of the A ffi liated Hospital of Xuzhou Medical University (XZMU-A201204-057R).

Focal cerebral ischemia injury modeling

A rat model of unilateral middle cerebral artery occlusion was established using the modi fi ed Longa method (Longa et al., 1989). Rats were anesthetized intraperitoneally with 10% chloral hydrate (3 mL/kg).e lecommon, external, and internal carotid arteries were exposedviaan incision in the neck and separated under a surgical microscope. The external carotid artery was ligated 0.8–1.0 cm from the common carotid artery, and the internal carotid artery was occluded. A 3-0 surgical mono fi lament nylon suture, blunted at the end, was gently inserted into the internal carotid artery through a small incision at the bifurcation. When the thread was extended 17–19 mm from the bifurcation and a slight resistance was felt, this indicated that the thread had been inserted into the origin of the middle cerebral artery at the circle of Willis, blocking blood fl ow in the middle cerebral artery trunk.e thread was ligated with a slipknot in the internal carotid artery, and the incision was sutured.e rats were returned to their cages with food and water and allowed to recover. Body temperature was maintained near 37°C using a heat pad. Aer 2 or 6 hours, the thread was withdrawn by approximately 10 mm to begin reperfusion. For the sham group, the procedure was identical except the thread was only inserted to a depth of 5 mm.

MRI scan

3.0 T MRI examination (Signa HD 3.0, GE Healthcare, Chicago, IL, USA) was performed at various time points aer ischemia/perfusion. A rat coil (Chenguang Medical Technology Co., Ltd., Shanghai, China) was used as the receiver coil.e rats were anesthetized by an intraperitoneal injection of 10% chloral hydrate.eir heads were then placed in the center of the coil in the prone position. Echo planar imaging was used with the following parameters: repetition time, 6,800 ms; echo time, 93 ms; field of view, 8 cm × 6 cm; matrix, 64 × 64; number of excitations, 2; thickness, 2.4 mm; slice gap, 0.2 mm. Aer the scan, DWI data were transmitted to the workstation for postprocessing to obtain apparent diffusion coefficient (ADC) profiles. The relative ADC (rADC) for the abnormal signal area on a slice with marked ischemia in the region of interest (ROI) was calculated as follows: rADC = ADCROI/ ADCcontralateral× 100%.e ratio of the DWI abnormal signal area (rS-DWI) on the selected slice (with marked ischemia) to that in the wholebrain slice area was calculated. Brains were removed aer scanning.

Immunohistochemistry

Figure 1 Brain di ff usion-weighted magnetic resonance imaging (DWI) and apparent di ff usion coeffi cient (ADC) in rat models of focal cerebral ischemia/reperfusion injury.

Figure 2 Immunohistochemical staining of STAT3 and p-STAT3 in the brain 24 h aer reperfusion (× 400).

Western blot assay

Statistical analysis

Figure 3 Changes in STAT3 expression and activation in the brain aer acute cerebral ischemia/reperfusion.

Figure 4 Correlation of phosphorylated signal transducer and activator of transcription 3 (p-STAT3) with relative apparent di ff usion coeffi cient (rADC) and relative abnormal signal area in di ff usion-weighted magnetic resonance imaging at di ff erent reperfusion time points.

SPSS 16.0 for Windows (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. Experimental data are expressed as the mean ± SD. One-way analysis of variance was used to compare DWI and ADC before and aer cerebral ischemia/ reperfusion, and STAT3 and p-STAT3 expression.e relationship between p-STAT3 expression and rADC or rS-DWI was analyzed by Pearson correlation analysis.P＜ 0.05 was considered statistically signi fi cant.

Results

DWI in rat brain before and aer acute cerebral ischemia/ reperfusion

In the sham group, DWI fi ndings were as expected, and ADC values were similar across both cerebral hemispheres. In both ischemia groups, before reperfusion, DWI showed patchy hyperintensity in the right corpus striatum and frontoparietal cortex, and ADC pseudocolor images showed an abnormal blue signal with blurred edges.e 6-hour ischemia group had larger rS-DWI and smaller ADC values than the 2-hour ischemia group (Figure 1). In the 2-hour ischemia group, aer 24 hours of reperfusion, the rS-DWI was signi fi cantly smaller than before reperfusion, and the ADC was partially restored. Aer 24 hours of reperfusion in the 6-hour group, the rS-DWI was slightly lower than before reperfusion, and the ADC value was slightly greater (Figure 1).

Changes in STAT3 expression and activation aer acute cerebral ischemia/reperfusion

Immunohistochemistry showed that p-STAT3-positive cells were rarely expressed in brain tissue from sham-operated rats, but in rats with ischemia, expression in the ischemic area increased with reperfusion time.e positive cells were circular or oval, and mainly astrocytes, followed in number by neurons. Aer 24 hours of reperfusion, there was neural nuclear condensation, cell body shrinkage and deformation, and larger astrocytes with abundant cytoplasm (Figure 2).

In the western blot assay, a low level of p-STAT3 was detected in brain tissue from sham-operated rats. In rats with ischemia, expression increased significantly after 0.5 hours of reperfusion, decreased slightly aer 2 hours, and peaked at 24 hours (Figure 3).

Correlation between p-STAT3 expression and DWI

Data from different time points after reperfusion showed that p-STAT3 expression was positively correlated with rADC (2-hour ischemia group:r= 0.803,P＜ 0.05; 6-hour ischemia group:r= 0.697,P＜ 0.05) and negatively correlated with the rS-DWI (2-hour ischemia group:r= −0.680,P＜0.05; 6-hour ischemia group:r= −0.678,P＜ 0.05) (Figure 4).

Discussion

Semi-quantitative western blot assay revealed that STAT3 protein expression did not change signi fi cantly with di ff erent reperfusion periods following ischemia for 2 or 6 hours. By contrast, p-STAT3 expression increased gradually, whereas it was barely detectable in the sham group.e increase in p-STAT3 levels aer 0.5 hours of reperfusion may be related to the involvement of STAT3, c-Fos, and c-Jun in the transcriptional regulation of immediate early genes in neurons (Amantea et al., 2011).e signi fi cant increase in p-STAT3 levels after 24 hours of reperfusion may be related to ATP depletion during ischemia and significantly increased ATP levels after reperfusion; conversely, a marked proliferation of reactive glial cells and microglia was induced by ischemic brain damage, and increased cytokines and growth factors were released as reperfusion continued.

The basic pathology of the rS-DWI is that Na+/K+–ATP enzyme pump function is reduced due to ischemia and hypoxia, which leads to sodium retention and consequent cytotoxic edema, resulting in slowed molecular diffusion; this is demonstrated by the low ADC and DWI hyperintensity (Kim et al., 2006; Cereda et al., 2015; Lago et al., 2015; Song et al., 2015; Aoki et al., 2016; Freitag et al., 2016; Grams et al., 2016; Jiang et al., 2016; Kaseka et al., 2016; Kohno et al., 2016; Kvistad et al., 2016; Onofrj et al., 2016; Tamura et al., 2016; Xin and Han, 2016; Zhang et al., 2016; Zhou et al., 2016; Abdelgawad et al., 2017; Bekiesinska-Figatowska et al., 2017; Heiss and Zaro Weber, 2017). The decrease in ADC was highly consistent with the ATP-labeled defect area and decreased tissue pH area; and the decrease in ADC in ischemic brain damage was consistent with the level of cytotoxic edema caused by cell energy metabolism disorders (Anticoli et al., 2015; Baron et al., 2015; Brown et al., 2015; Eom et al., 2015; Gory et al., 2015; Kate et al., 2015; Landais, 2015; Li et al., 2015a; Makin et al., 2015; Mawet et al., 2015; Michałowska et al., 2015; Odland et al., 2015; Ostwaldt et al., 2015; Sasai et al., 2015; Yaghi et al., 2015). As infarction time increases (＞ 24 hours), there are corresponding increases in vasogenic edema, extracellular space water, di ff usion speed, and ADC values (Maruyama et al., 2015).

As the JAK-STAT signaling pathway is activated during cerebral ischemia, JAK1 expression in cortical pyramidal neurons and striatal cells increases, and STAT3 nuclear translocation also increases, in rats with focal cerebral ischemia/reperfusion injury; this results in extensive proliferation of reactive microglia and macrophages (Li and Zhang, 2003; Zechariah et al., 2010; Jiang et al., 2013; Feng et al., 2015; Jung et al., 2015; Deng et al., 2016). Previous studies have demonstrated that the gp130-STAT signaling pathway could be activated by the nuclear translocation of STAT3, and that this activation in astrocytes correlates closely with gp130 expression (Jang et al., 2014; Song et al., 2014; Xu et al., 2015; Zhang et al., 2015; Guo et al., 2016). Selection of the samples used in the present experiment was based on MRI fi ndings. STAT3 activation levels before and aer ischemia/reperfusion correlated with the rS-DWI in the ischemic region and rADC. This finding indicated that with aggravation of cytotoxic and vasogenic edema, thestatus of some membrane channels changed, which a ff ected certain cell signaling pathways and STAT3 activation.is correlation helps to identify biochemical changes of JAKSTAT signaling in brain tissue aer ischemia/reperfusion. Further research is needed to determine whether blocking STAT3 phosphorylation could prevent neuronal necrosis or apoptosis due to ischemia to achieve neuroprotective e ff ects and minimize ischemia/reperfusion injury, and whether it could be re fl ected in MRI.is correlation also provides a theoretical and experimental basis for the clinical treatment of cerebral ischemia.

Author contributions:WJW provided and analyzed data and wrote the paper. CJJ and KX participated in study conception and design, data analysis, statistical analysis, and provided technical or material support. ZZY guided the revision. WL was in charge of paper authorization and served as a principle investigator. All authors performed the experiments and approved the fi nal version of the paper.

Con fl icts of interest:

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Plagiarism check:This paper has been checked twice with duplication-checking soware ienticate.

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Copyedited by Slone-Murphy J, de Souza M, Yu J, Li CH, Qiu Y, Song LP, Zhao M

10.4103/1673-5374.211192

Accepted: 2017-04-14

＊Correspondence to: Wei Li, 409631600@qq.com.