Hypothermia followed by rapid rewarming exacerbates ischemia-induced brain injury and augments inflammatory response in rats

Hypothermia followed by rapid rewarming exacerbates ischemia-induced brain injury and augments inflammatory response in rats

Biochemical and Biophysical Research Communications xxx (2016) 1e7 Contents lists available at ScienceDirect Biochemical and Biophysical Research Co...

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Biochemical and Biophysical Research Communications xxx (2016) 1e7

Contents lists available at ScienceDirect

Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc

Hypothermia followed by rapid rewarming exacerbates ischemiainduced brain injury and augments inflammatory response in rats Shu-Zhen Zhu a, b, Yong Gu a, Zhou Wu a, Ya-Fang Hu a, Su-Yue Pan a, * a b

Department of Neurology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, PR China Department of Neurology, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 13 April 2016 Accepted 17 April 2016 Available online xxx

Hypothermia followed by slow rewarming is neuroprotective for ischemic stroke. However, slow rewarming causes patients' longer stay in intensive care unit and increases the risk of hypothermic complications. Hypothermia followed by rapid rewarming (HTRR) is more convenient; but it exacerbates intracranial hypertension for patients with massive hemispheric infarcts. The present study aims to investigate in detail how HTRR exacerbates ischemic brain injury and what are underlying mechanisms. Rats subjected to transient focal ischemia by middle cerebral artery occlusion were treated with normothermia or hypothermia followed by rapid rewarming. Neurological outcome, neuronal injury, bloodebrain barrier integrity and expressions of inflammatory cytokines were observed. Results showed that HTRR at a rate of 3  C/20 min increased both neurological deficit score and Longa score, enhanced the loss of neurons and the plasma level of neuron-specific enolase. Rapid rewarmed rats also displayed increased Evans blue dye extravasation, matrix metalloproteinase 9 level and tight junction impairment. Meanwhile, interleukin-1b, -6, tumor necrosis factor a and cyclooxygenase-2 were markedly elevated in rapid rewarmed rats. Anti-inflammatory agent minocycline suppressed HTRR-induced elevation of inflammatory cytokines and improved neurological outcome. These results indicated that HTRR significantly impaired neurovascular unit and augmented proinflammatory response in stroke. © 2016 Published by Elsevier Inc.

Keywords: Hypothermia Rapid rewarming Ischemic stroke Bloodebrain barrier Inflammatory response

1. Introduction Ischemic stroke is one of the major causes of death and disability worldwide [1]. Hypothermia (HT) followed by slow rewarming has been proved to be neuroprotective for experimental ischemic stroke [2,3]. The neuroprotective effect of hypothermia (2 h duration) confers significant smaller ischemic volumes and less infiltration of neutrophils in experimental stroke models [4]. In patients with ischemic stroke and successful recanalization, HT for 48 h followed by slow rewarming at 0.05  C/h reduces the risk of cerebral edema and hemorrhagic transformation and decreases 3-

Abbreviation: BBB, blood brain barrier; COX-2, cyclooxygenase-2; HT, hypothermia; HTRR, hypothermia followed by rapid rewarming; IL, interleukin; M, minocycline; MMP9, matrix metalloproteinase 9; mNSS, modified neurological severity score; NSE, neuron-specific enolase; NT, normothermia; PI, Propidium iodide; TNF-a, tumor necrosis factor a; V, vehicle. * Corresponding author. Department of Neurology, Nanfang Hospital, Southern Medical University, 1838 Guangzhou Avenue North, Guangzhou, Guangdong 510515, PR China. E-mail address: [email protected] (S.-Y. Pan).

month modified Rankin Scale, indicating the promising strategy of HT for the treatment of ischemic stroke patients [5]. Although slow rewarming following hypothermia is less rewarming injurious [6], it is inevitably associated with longer hypothermic duration and longer stay in intensive care units, which increase the risk of pneumonia [7]. In addition, slow rewarming is not suitable for patients with epileptic seizures which occurred in about 6.93% of people with stroke [8] or with severe hypothermic complications such as fatal arrhythmia [2,9] during hypothermic therapy. Thus, increasing the rewarming velocity is necessary for hypothermia therapy in clinical practice. Unfortunately, hypothermia followed by rapid rewarming (HTRR) is reported to exacerbate intracranial hypertension. For example, intracranial pressure was reduced from 36 to 14 mmHg during hypothermia maintenance stage in patients with massive hemispheric infarcts; but it was rebounded to 52 mmHg, even higher than before hypothermia treatment, after rapid rewarming, associating with half of deaths died of cerebral herniation during rewarming period [10]. How rapid rewarming exacerbates brain injury in ischemic stroke is unclear. Exploring the characteristics and mechanisms of rapid-

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rewarming induced-deterioration of ischemic brain injury will provide the basis for the intervention strategy to prevent the detrimental effect of rapid rewarming. Excessive inflammatory response might be one of the critical events that exacerbates brain injury caused by rapid rewarming [11,12]. Hypothermia followed by slow rewarming can inhibit the expression of pro-inflammatory cytokines, such as interleukin-1b (IL-1b), interleukin-6 (IL-6) and tumor necrosis factor a (TNF-a), and thereby protecting the brain [13e15]. However, the role of inflammation during rapid rewarming period is unknown. To address this question, we detected the brain injury and inflammation reactions in response to rapid rewarming at a rate of 3  C/ 20 min after hypothermia therapy. Our data revealed that HTRR aggravated neurological outcome and neuronal loss and exacerbated bloodebrain barrier (BBB) breakdown, and the deterioration of ischemic brain damage was associated with augmented inflammatory response. 2. Materials and methods 2.1. Animals A total of 136 male SpragueeDawley rats weighing 280e320 g were used. Animals were provided by the Experimental Animal Centre of Southern Medical University, Guangzhou, China. They were allowed free access to food and water before and after treatment. All surgical procedures were approved by the Ethics Committee for Animal Experimentation of Nanfang Hospital. Animals were divided into seven groups: (1) sham-operated rats (n ¼ 33); (2) normothermia (NT) control group (n ¼ 33): ischemiareperfused rats were maintained at 37  C; (3) HTRR group (n ¼ 34): ischemia-reperfused rats received therapeutic hypothermia at 34  C for 2 h followed by rapid rewarming from 34  C to 37  C within 20 min; (4) NT rats received vehicle control (n ¼ 6); (5) NT rats received minocycline (n ¼ 6); (6) HTRR rats received vehicle control (n ¼ 6); (7) HTRR rats received three different doses of minocycline treatment (n ¼ 6 in each dose, 18 in total). 2.2. Transient middle cerebral artery occlusion (MCAO) in rats Rat MCAO was performed following our previously described method [16,17]. Briefly, after the rats were anesthetized with isoflurane, a silicone-coated suture was gently inserted through the external carotid artery to the internal carotid artery to occlude the middle cerebral artery for 2 h. The right femoral vein was cannulated for blood sampling. A burr hole (1 mm in diameter) was drilled bilaterally 6 mm lateral and 1 mm posterior from the bregma and a laser Doppler Flowmeter (Moor Instruments Ltd., Devon, UK) probe was positioned above the surface of hemisphere to monitor the cerebral blood flow. The rats without a reduction in blood flow below 30% of the base-line value after filament insertion were eliminated from the study. The success of reperfusion was determined by an increase in blood flow above 80% of the baseline value after removal of the filament.

previously described [16]. The brain and rectal temperature were shown in Supplemental Fig. 1. 2.4. Minocycline administration Minocycline (SigmaeAldrich, St. Louis, MO) was used to alleviate inflammation [18]. It was dissolved in 0.4% DMSO saline solution and injected via femoral vein immediately after MCAO onset with dose of 1.5, 3 or 6 mg/kg. The same amount of 0.4% DMSO saline solution was used in the vehicle control group. 2.5. Neurological outcome assessment Neurological function was assessed in rats from different groups at 24 h after the onset of MCAO by an investigator who was blinded to grouping. Neurological deficits were determined using Longa five-grade scale [19] and modified neurological severity score (mNSS) [20] which were widely used in ischemic stroke studies and considered to effectively evaluate the functional deficits of MCAO rats. 2.6. Histological analysis Twenty 4 h after MCAO onset or sham surgery, rats (n ¼ 3 per group) were deeply anesthetized, transcardially perfused and the brains were fixed with paraffin-embedded 4% paraformaldehyde. Coronal brain sections located at 3.5 mm posterior to bregma were obtained (Leica CM1800, Heidelberg, Germany). Neuronal damage was assessed with H&E and Nissl staining. Histological analyses were performed by a researcher who was blinded to the animal grouping. Propidium iodide (PI; 1 mg/mL; Sigma) was used to label necrotic cells in rats (n ¼ 3 per group). At 1 h prior to killing, PI was intraperitoneally injected (1 mg/kg) to rats. Rat Brains were removed and fixed in 100% ethanol, and observed with excitation/ emission filters 568/585. Five fields in ischemic region of cortex, from eight brain sections were selected for analysis. The mean counts of PI-positive cells per field were automatically calculated using Image-Pro Plus version 6.0 (Media Cybernetics, Warren dale, PA). 2.7. Evans blue extravasation BBB Permeability was evaluated by measuring the extravasation of Evans blue dye in rats (n ¼ 6). Right after reperfusion onset, 2% Evans blue (SigmaeAldrich) was injected through the tail vein. At 24 h after MCAO onset, the hemisphere was removed and immersed in 1 mL of 60% trichloroacetic acid. Evans blue concentration was quantified by measuring supernatant fluorescence (590/645) using a multiplate reader (SpectraMax M5, Molecular Devices, Sunnyvale, CA). The fold increase of Evans blue dye extravasation was calculated using the formula: dye contents in the (ipsilateral hemispheric tissue e contralateral hemispheric tissue)/ contralateral hemisphere [21].

2.3. Hypothermia (HT) and rewarming 2.8. ELISA Hypothermia was conducted after MCAO onset. The target rectum temperature (34  C) in HTRR rats were reached within a median of 20 min (range 15e25 min) and kept at 34  C automatically by a temperature controller (RWD Life Science, Shenzhen, China). After 2 h, the rapid rewarming was started by the temperature controller from 34  C to 37  C within about 20 min (at a speed of 1  C per 6e7 min). Brain temperature was monitored with temperature probe (Physitemp Instruments, Clifton, NJ) as we

Blood samples were collected via femoral vein at 8 and 24 h after the onset of MCAO (n ¼ 6). Plasma was obtained by centrifugation at 1000 g for 15 min. Rat neuron-specific enolase (NSE), matrix metalloproteinase-9 (MMP-9), TNF-a, IL-1b, IL-6 and Cox-2 ELISA kits were purchased from CUSABIO Biotechnology (Wuhan, China). The optical density was detected using a microplate reader (SpectraMax M5; Molecular Devices) set to 450 nm.

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2.9. Western blot analysis At 8 h after MCAO onset, rats (n ¼ 4) were deeply anesthetized and transcardially perfused. Protein samples were loaded for electrophoresis and trans-blotted and immunoprobed with primary antibody IL-1b (1:100; Santa Cruz Biotech, Santa Cruz, CA), IL6 (1:100; Santa Cruz), TNF-a (1:100; Santa Cruz), Cox-2 (1:200; Invitrogen, Carlsbad, CA), occludin (1:200; Invitrogen) and claudin 5 (1:200; Invitrogen). The blots were then incubated with horseradish peroxidase-conjugated secondary antibody (1:5000; Santa Cruz) for 1 h. Imaging was recorded using Automated Kodak InVivo Imaging system (Carestream Health, NH). 2.10. Statistical analysis Analyses were performed using the program SPSS version 13. Parametric data were analyzed using one way analysis of variance with post hoc test of Bonferroni comparisons. Graphs were made using GraphPad Prism 6 software (GraphPad Software, San Diego, CA). P-value <0.05 was considered significant. 3. Results 3.1. HTRR worsened neurological function of MCAO rats First of all, neurological deficit of each rat was observed at 24 h after the onset of MCAO. As shown in Fig. 1A, neurological impairment indicated by Longa five-grade scoring showed that rats in HTRR group displayed higher mean score than those in NT group (3.75 vs 2.45). Similarly, mNSS was also elevated by HTRR, with the mean score of 8.71 in the HTRR group compared with 6.67 in NT control group (Fig. 1B). These observations indicate rapid rewarming aggravates neurological outcome of MCAO rats. 3.2. HTRR deteriorated neuronal impairment in ischemiareperfused rats We next studied whether HTRR induced neuronal insult. The H&E staining pictures in Fig. 2A revealed more injured neurons that were loosely located in infarct regions in rats from HTRR group than those in NT group. The Nissl staining showed decreased number of neurons in HTRR rats compared with NT rats (Fig. 2B). There were obvious differences between NT and HTRR group in the cortex, subcortex and hippocampus (Fig. 2C). The PI staining showed more necrotic cells in ischemic cortex of HTRR rats compared with NT

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rats (Fig. 2DeE). NSE is expressed in neurons whose elevation in plasma represents the neuronal injury. Fig. 2F revealed that brain ischemiareperfusion significantly increased the release of NSE from the central nervous system to peripheral blood at 8 and 24 h after the onset of MCAO. The NSE concentration was further augmented by HTRR at 8 h, but not at 24 h after MCAO onset. These results indicated that HTRR could augment neuronal impairments at early phase of ischemic stroke. 3.3. HTRR exacerbated the BBB breakdown in ischemia-reperfused rats Then we explored whether HTRR exacerbated the impairment of BBB. As shown in Fig. 3AeB, Evans blue dye extravasation, indicative of BBB breakdown, was almost undetectable in shamoperated rats. However, it was clearly observed in ischemic cortex of NT rats at 24 h after MCAO. HTRR further increased the dye extravasation compared with the NT control rats subjected to ischemia-reperfusion. To understand how HTRR deteriorated the BBB, the levels of tight junction proteins, occludin and claudin-5, were detected (Fig. 3CeD). Results showed that both of them were obviously reduced in the HTRR group compared with the NT control group at 8 h after MCAO. The level of MMP-9, an enzyme that digests extracellular matrix and disrupts BBB, was also examined. As shown in Fig. 3E, plasma MMP-9 was significantly elevated after ischemic insult and was further increased after HTRR at 8 h after MCAO. These results indicated that HTRR could deteriorate the disruption of BBB by augmenting the loss of tight junction and the release of MMP-9. 3.4. HTRR augmented inflammatory response in the ischemiareperfused brain Finally, considering proinflammatory factors contributes to acute ischemic brain injury, we hypothesized HTRR augmented inflammatory cytokines expression during acute stroke insult. The levels of proinflammatory cytokines between the NT control and HTRR group were compared. As shown in Fig. 4AeB, TNF-a, IL-1b, IL-6 and Cox-2 levels in brain tissue were higher in the rats from HTRR group than those in the NT control group at 8 h after MCAO. To address whether inflammatory response contributed to HTRRaggravated neuronal injury, the anti-inflammatory agent minocycline was injected to MCAO rats to attenuate inflammation and see

Fig. 1. Hypothermia followed by rapid rewarming (HTRR) worsened neurological outcome of brain ischemia-reperfused rats. (A) Longa neurological score at 24 h in the rats from Sham operative, normothermia (NT) control and HTRR groups. (B) Neurological deficit score at 24 h in different groups. n ¼ 11 in Sham, NT control and n ¼ 12 in HTRR group. * P < 0.05 versus sham group, #P < 0.05 versus NT control.

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Fig. 2. HTRR deteriorated neuronal damage in MCAO rats. (A) Representative H&E staining photomicrographs showing the injured neurons that were loosely arranged in infarct cortex, sub-cortex and hippocampus at 24 h after MCAO onset in rats, n ¼ 3. (BeC) Representative Nissl staining photomicrographs and quantification of viable neurons indicating the survived neurons in the cortical, sub-cortical and hippocampal regions at 24 h after ischemia. n ¼ 3, the same batch of rats with H&E staining. (DeE) Representative PI staining pictures and the quantification of PI-positive cell numbers in different groups. n ¼ 3. (F) Plasma NSE level at 8 h and 24 h after the onset of MCAO. n ¼ 6. *P < 0.05 versus sham group. #P < 0.05 versus NT control. Magnification:  200 in a, c and e;  400 in b, d and f. Bars indicate 40 mm in (AeB) and 500 mm in (E).

the effects. As expected, the levels of proinflammatory cytokines in HTRR rats were significantly decreased after minocycline treatment compared to vehicle treatment at 8 h after the onset of MCAO (Fig. 4CeF, n ¼ 6). More importantly, the neurological function was

improved after minocycline treatment (Fig. 4GeH, n ¼ 6). These results suggested that HTRR-induced elevation of inflammatory cytokines contributed to the deterioration of the neurological function caused by HTRR.

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Fig. 3. HTRR aggravated BBB disruption in MCAO rats. (AeB) Representative brains showing Evans blue extravasation and the quantification data of dye contents in the ischemic area at 24 h after the onset of MCAO in sham, NT control and HTRR groups. n ¼ 6. (CeD) Western blot analysis of occludin and claudin-5 and the quantification in different groups at 8 h after the onset of MCAO. n ¼ 4. (E) ELISA analysis of plasma MMP9 level in these groups at 8 h and 24 h after the onset of MCAO. n ¼ 6. *P < 0.05 versus sham group. #P < 0.05 versus NT control.

4. Discussion The present study demonstrates that rapid rewarming following hypothermia therapy markedly worsened neurological functions, aggravated neuronal injury and BBB disruption, as well as augmented inflammatory responses in a rat model of ischemiareperfusion. These data indicate that HTRR after ischemic stroke is detrimental to neurovascular units and hence deteriorates neurological outcome. Inflammatory response triggered by HTRR may be the mechanism underlying the neurovascular unit damage at the acute phase. Hypothermia has become one of the most potent neuroprotective strategies because it simultaneously targets multiple mechanisms [22,23]. HT followed by slow rewarming at a rate of 0.05  C/h can preserve neurologic function and reduce cerebral edema in ischemic stroke patients [5]. Although slowing the rewarming rate can ameliorate rewarming injury, it is associated with longer stay in intensive care unit and higher incidences of hypothermic complications. This indicates accelerating velocity of rewarming is necessary, but rapid rewarming at about 0.17  C/h (from 34  C to 37  C within 18 h) is associated with the rebounded intracranial pressure [24]. Therefore, intervention strategy and injurious mechanism study of rapid rewarming are necessary and urgent in stroke therapy. In our study, HTRR deteriorates

neurological outcome in the rat model of ischemic stroke, which is consistent with previous studies conducted in the models of traumatic brain injury and hypoxic-ischemic encephalopathy [6,25]. For the first time, we found rapid rewarming enhanced the Longa and neurological deficit score, and exacerbated neuronal impairments, BBB breakdown and augmented inflammatory response in ischemic stroke rats. NSE is released to extracellular space by injured neurons and has high predictive value for determining severity of neurobehavioral outcome after acute stroke. Although prior studies have reported that hypothermic therapy following slow rewarming decreased the NSE levels [26], in the present study, we found that HTRR not only failed to decrease but further increase the concentration of plasma NSE at 8 h after the onset of ischemia. A similar conclusion was drawn in a prior study that rapid rewarming deteriorated axons in traumatic brain injury animals [27]. Deterioration of BBB may induce vasogenic edema and high intracranial pressure. Whether deterioration of BBB was involved in rapid rewarming is unclear. In line with the cerebro-protective role, slow rewarming after hypothermia protects BBB integrity, inhibits MMP-9 expression and tight junction loss [23]. Rapid rewarming, however, plays an opposite role which is proved in our study by the evidence that hypothermia followed by rapid rewarming increased BBB permeability, augmented the release of MMP-9 and the loss of

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Fig. 4. The expressions of proinflammatory cytokines contributed to the detrimental effects caused by HTRR in ischemia-reperfused rats. (AeB) Protein levels of TNF-a, IL-6, IL-1b and Cox-2 were detected by Western blot at 8 h after MCAO in different groups. n ¼ 4. *P < 0.05 versus sham group. #P < 0.05 versus NT control. (CeF) Circulating TNF-a, IL-6, IL-1b and Cox-2 levels in rats from NT and HTRR treated with vehicle or minocycline were determined by ELISA at 8 h and 24 h after MCAO. (GeH) Longa and Neurological deficit score at 24 h in the NT and HTRR rats treated with vehicle or minocycline. n ¼ 6. *P < 0.05 versus vehicle control of HTRR groups. #P < 0.05 versus vehicle control of NT group.

TJ proteins at acute stage of ischemic stroke. These findings imply that exacerbation of BBB leakage induced by HTRR might be the explanation of vasogenic edema and rebounded intracranial pressure. Understanding the molecular mechanism of rapid rewarming-

mediated brain damage would be helpful for drug intervention in clinical settings. Excessive inflammatory response is crucial for BBB breakdown and neuronal death [11,28]. In focal ischemia rats, TNFa levels in ischemic brain tissue increased at 24 h after reperfusion and reached its peak at day 3 [29], IL-6 levels increased on day 3

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and remained elevated up to day 14 [30]. Higher expressions of cytokines in brain tissues lead to neuronal and vascular injury, and correlate with worse neurological outcome [31e33]. In the present study, TNF-a levels in brain tissues were up-regulated by HTRR at 8 h after ischemia onset. Similarly, HTRR robustly enhanced the expression levels of IL-6, -1b and Cox-2 in the rat brain. To further elucidate the inflammatory response induced the brain injury caused by rapid rewarming, minocycline was employed to inhibit neuroinflammation [18]. As expected, administration of minocycline significantly reduced cytokines expression in HTRR rats. More importantly, neurological function of HTRR rats was improved by minocycline, suggesting minocycline may attenuate the side effects brought by rapid rewarming. The augmented cytokines expressions may provide a partial explanation for the BBB breakdown and high morbidity associated with rapid rewarming in clinical settings. These inflammatory factors may be potential predictors for rewarming injury and serve as biomarkers for guiding an optimal rewarming protocol in future. Furthermore, combination of minocycline and rapid rewarming might be a good strategy for hypothermic therapy. Disclosures None.

[8] [9] [10]

[11] [12]

[13]

[14]

[15]

[16]

[17]

[18]

Acknowledgments This study was supported by the National Natural Science Foundation of China Projects 81271521 and 81471339 and the Science and Technology Commission of Guangdong Province 2012A030400011.

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Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2016.04.095.

[22] [23]

Transparency document [24]

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Please cite this article in press as: S.-Z. Zhu, et al., Hypothermia followed by rapid rewarming exacerbates ischemia-induced brain injury and augments inflammatory response in rats, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/ j.bbrc.2016.04.095

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