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Mild hypothermia protects rat neuronal injury after intracerebral hemorrhage via attenuating endoplasmic reticulum response induced neuron apoptosis
Neuroscience Letters 635 (2016) 17–23
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Research article
Mild hypothermia protects rat neuronal injury after intracerebral hemorrhage via attenuating endoplasmic reticulum response induced neuron apoptosis Cen Guo, Yang Geng, Feifei Song, Yajing Huo, Xuqing Wu, Jianing Lv, Anyan Ge, Wei Fan ∗ Department of Neurology, Zhongshan Hospital, Fudan University, 200032, Shanghai, China
h i g h l i g h t s • ICH injury induces endoplasmic reticulum (ER) stress response. • Mild hypothermia could attenuate intracerebral hemorrhage (ICH) caused neuron injury by decreasing neuron apoptosis. • Mild hypothermia decreased ER response after ICH.
a r t i c l e
i n f o
Article history: Received 30 August 2016 Received in revised form 6 October 2016 Accepted 18 October 2016 Available online 19 October 2016 Keywords: Intracerebral hemorrhage Hypothermia ER stress Apoptosis
a b s t r a c t Background and purpose: Mild hypothermia has been proved to reduce global and focal cerebral ischemic injury in rodents by preventing cellular apoptosis through several pathways. However, whether hypothermia will be beneficial for intracerebral hemorrhage (ICH) and its underlying mechanisms haven’t reached a consensus. It has been implicated that endoplasmic reticulum (ER) stress plays a role in the secondary injury after ICH in rats. In this study, we aimed to investigate whether mild hypothermia would attenuate ICH induced neuronal injury via regulating ER stress. Methods: The model of ICH was induced by injecting autologous blood (120 l) into the rat striatum. Rats were divided into sham, normothermic (NT) and hypothermic (HT) groups. HT group were subjected to mild hypothermia (33 ◦ C–35 ◦ C) for 2 days starting from 6 h after ICH. Neurological deficits were evaluated. The ER stress related proteins (GRP78, CHOP and p-eIF2␣) and the apoptosis associated indicators (cleaved caspase3, Bcl-2 and Bim) around hematoma were assessed by western blot, qRT-PCR (quantificational real-time polymerase chain reaction), immunofluorescence and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling) assay. Results: Neurological deficits following ICH were reduced in HT group compared to NT group. Protein levels of GRP78, CHOP and p-eIF2␣ significantly increased after ICH in both NT and HT group compared to sham group, which was consistent with the trend of cleaved-caspase3 at protein level, and Bim, Bcl-2 at gene level. In comparison to NT group, GRP78, CHOP, p-eIF2␣, cleaved caspase-3 and Bim all decreased, while Bcl-2 increased in HT group. Additionally, apoptotic cells detected by TUNEL staining significantly decreased in the HT group. Conclusion: Mild hypothermia could attenuate ICH caused neuron injury by decreasing ER responseinduced neuron apoptosis. © 2016 Published by Elsevier Ireland Ltd.
1. Introduction
Abbreviations: ICH, intracerebral hemorrhage; ER, endoplasmic reticulum; NT, normothermic; HT, hypothermic; qRT- PCR, quantificational real-time polymerase chain reaction; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. ∗ Corresponding author. E-mail address: [email protected] (W. Fan). http://dx.doi.org/10.1016/j.neulet.2016.10.031 0304-3940/© 2016 Published by Elsevier Ireland Ltd.
Stroke is a leading cause of human death and disability across the world. Intracerebral hemorrhage (ICH) comprises 10–30% of all stroke cases. Patients suffering from ICH often have abysmal outcomes which are worse than ischemic stroke, with 30-day mortality estimated as high as 44%, and survivors typically suffer life limiting disability [1,2]. To date, there is no effective therapy for this subtype of stroke.
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Several clinical works [3,4] and experimental studies [5,6] have shown the neuroprotective effects of mild hypothermia in ischemic stroke. However, studies in ICH are fewer than those on ischemic stroke and the underlying mechanisms remain unknown both in ischemic stroke and ICH. The endoplasmic reticulum (ER), cell’s manufacturing machinery, is involved in protein synthesis, folding and many other important processes. Perturbation in ER functions leads to ER stress and it triggers a host response known as the unfolded protein response (UPR) to restore cellular homeostasis by transiently reducing protein translation and inducing ER repairing mechanisms within a short period; however, prolonged ER stress can activate irreversible signaling pathways leading to cell death [7]. ER stress-induced apoptosis has been implicated in several neurological diseases [8]. A recent study showed that ER stress was associated tightly with cell apoptosis after ICH [9]. In the present study, we tested the hypothesis that hypothermia had a protective effect on ICH by attenuating ER stress induced neuron apoptosis around hematoma. 2. Material and methods 2.1. Animals All studies were carried out in accordance with the guidelines for animal care and use established by our University Animal Care and Use Committee. The protocols were approved by the Committee on the Ethics of Animal Experiments of our university. A total of 68 male, Sprague Dawley rats (300 ± 50 g) were obtained from the Shanghai Fudan University. All animals were housed individually after surgery with freely access to food and water. Housing rooms were temperature, humidity, and light (12-h light-12-h dark cycle) controlled. 2.2. Experimental groups Animals were divided into SHAM (n = 16), normothermic (NT) (n = 26), hypothermic (HT) (n = 26) groups. Animals in the SHAM group were euthanized at day 3 or day 7 and the other two groups at day 2, 3, 5 or 7 after ICH. For the rats euthanized at day 7, behavioral tests were performed at day 2, 3, 5 and 7 (n = 8 per group). The harvested brains were used for histology (n = 3 per group, at day 3) and western blotting assays (n = 5 of NT and HT group at day 2, 3, 5, 7; n = 5 of SHAM group at day 3). 2.3. ICH model [10] Rats were anesthetized by injecting 10% chloral hydrate (0.4 ml/100 g) intraperitoneally. Rectal temperature was maintained within 37 ± 1 ◦ C during the surgery. Rats were positioned in a stereotactic frame and the scalp was incised along the midline. Using a sterile technique, a 1 mm burr hole was opened in the skull on the right 3 mm lateral, 1 mm posterior to the bregma. A blunt 26-gauge needle was inserted into the right basal ganglia under stereotactic guidance. Then, a total of 120 l of autologous whole blood withdrawn fast from the tail was infused at a rate of 5 l/min using a microinjector. A pause for 8 min was given after every 40 l injection. After completion of the infusion, the needle was paused for another 10 min, then withdrawn slowly and bone wax was placed in the burr hole. The skin incision was closed with sutures. Sham animals underwent the same surgical procedures, but only received a pause of microinjector for 10 min without infusion of blood. After the surgery, rats were divided into NT and HT groups randomly. HT treatment started from 6 h after the induction of ICH. NT and sham rats were housed in a room maintained at
25 ◦ C. They were all kept freely moving and free access to food and water after waking up from anesthesia of operation. 2.4. Hypothermia application and temperature monitor All the animals were maintained over 36 ◦ C after ICH. Rats of NT and Sham group were kept above 36 ◦ C during the next 48 h. HT rats were housed in a cold room maintained at 4 ◦ C starting from 6 h after ICH, with 75% ethyl alcohol spraying, back hair shaving and a heating pad to maintain their rectal temperature at 33–35 ◦ C.They were brought to room temperature 48 h after ICH and then slowly warmed. Since brain temperature was about 1 ◦ C higher than that measured rectally[11], we measured rectal temperature with a special thermometer inserted 5 cm deep into the animals’ rectum instead, and it was recorded every 15 min. Temperature monitoring was stopped once the rats regained normal temperature. 2.5. Behavioral test 2.5.1. Z −longa score [12] All animals underwent longa test 6 h after surgery and were scored by experimenters who were blinded to both neurological and treatment conditions. The neurologic findings were scored on a five-point scale as previously described: 0: no neurologic deficit; 1: failure to extend left forepaw fully; 2: circling to the left; 3: falling to the left; 4: rats couldn’t walk spontaneously and had a depressed level of consciousness. Rats were kicked out when the score was below 1 or over 3. 2.5.2. Garcia score [13] The neurobehavioral study consisted of the following six tests evaluating both motor function and sensory performance: spontaneous activity, symmetry in the movement of four limbs, forepaw outstretching, wire cage climbing, body proprioception and response to vibrissae touch. The total score ranged from 3 to 18 and a higher score indicated better performance. The test was performed at day 2, 3, 5 and 7. 2.6. Tissue harvesting Animals were deeply anesthetized with 10% chloral hydrate (0.4 ml/100 g) intraperitoneally and intracardially perfused with ice-cold saline. For biochemical analyses, tissues around hematoma with a diameter of 4 mm were rapidly dissected, snap frozen by liquid nitrogen and then stored in the −80 ◦ C freezer prior to RNA isolation, or protein analyses. For histology analyses, 4% paraformaldehyde (PFA) was given followed saline perfusion for fixation. Subsequently, the whole brains were isolated. Then they were incubated in 4%PFA for another 6 h, and immersed in a solution of 10%, 20%, 30% sucrose in turn for gradient dehydration. Serial coronal sections were cut using a cryotome (Leica; Germany) with a thickness of 10um.Sections were stored in the −80 ◦ C freezer prior to Nissl, immunofluorescent and TUNEL staining. 2.7. Western-blots Equal amount of protein samples was loaded in each well and subjected to 10–15% sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Separated proteins were then transferred from the gel to polyvinylidinene fluoride (PVDF) (Millipore, USA) membranes and blocked in 5% non-fat dry milk for 1.5 h. The membranes were incubated with the primary antibodies overnight at 4 ◦ C. Following primary antibodies were used: anti ribbit-GRP78 (1:500, Santa Cruz Biotechnology, USA),anti rabbit-CHOP (1:500, Bioworld, USA),anti rabbit-p-eIF2␣ (1:500, Sangong, China), anti rabbit-cleaved caspase 3(1:500,CST,USA) and anti rabbit-GAPDH
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antibody (1:500, Bioworld, USA).After washing primary antibodies with 1*TBST, the membranes were incubated with appropriate secondary antibodies for 1 h at room temperature. The blots were developed using ECL (NCM Biotechnology, China). 2.8. RNA isolation and qRT-PCR Total RNA was isolated. Reverse transcription was performed using a PrimeScript RT reagent kit (TaKaRa Bio Inc.,Dalian China), and the reaction mixture was subjected to quantitative real-time PCR Bim, Bcl-2, using SYBR Premix Ex Taq (Tli RnaseH Plus) (Takara, Dalian, China). Primers were as follows: Bim: (FP 5 -ACAAACCCCAAGTCCTCCTT-3 ; RP 5 -GCCCTCCTCGTGTAAGTCTC-3 ) Bcl-2: (FP5 - ACAGCCAGGAGAAATCAAACA-3 ; RP5 - ACAGCCAGGAGAAATCAAACA-3 ) Product specificity was confirmed by melting curve analysis. Data were normalized to GAPDH housekeeping gene. Foldinduction was calculated using the 2−CT method. 2.9. Nissl’s staining Nissl’s staining was performed to identify the basic neuronal structure of necrotic neurons in the brain. Sections were rinsed in tap and distilled water, and subsequently stained in toluidine blue solution for 2 min. After rinsing in distilled water, the sections were differentiated in 95% ethyl alcohol, cleared with xylene, prior to mounting with neutral balsam. 2.10. Immunofluorescence staining Sections were incubated in 0.3% Triton X-100 for 10 min and blocked in 10% normal sheep serum for 30 min at room temperature. They were then incubated overnight at 4 ◦ C with the following antibodies: rabbit anti-GRP78(1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-CHOP (1:200, Bioworld, USA), rabbit anti-p-eIF2␣(1:200, Sangong, China).The sections were incubated for 1 h at RT in dark with FITC-conjugated secondary antibodies (1:200, Yeasen, China), and then incubated in DAPI (1:1000,SIGMA,USA) for 10 min.They were coverslipped with 50% glycerinum. Images were captured using fluorescence microscope (DP71, Olympus, Japan). 2.11. TUNEL staining TUNEL staining was performed using an in situ cell death detection kit in accordance with the manufacturer’s instructions (Roche Applied Science, USA). Frozen sections were incubated with reaction buffer containing enzyme at 37 ◦ C for 1 h in the dark and then incubated in DAPI. Images were captured using fluorescence microscope (DP71, Olympus, Japan). The positive cells and total cells were counted on three fields at ×200 magnifications. The result was presented as a ratio of positive cell number to the total cell number. 2.12. Statistics All data were expressed as mean ± standard error of the mean (SEM) and analyzed by SPSS 19.0(IBM Inc., USA). A p-value less than 0.05 was considered significant. A one-way ANOVA with LSD post hoc test for multiple comparisons was used to compare the results between different groups. Levene’s test for equality of variances was used to confirm the multiple comparison procedure used. Nonparametric statistics was used for any such data that violated the assumption of homogeneity of variance.
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3. Results 3.1. Physiologic parameters The weight, blood pressure (BP), heart rates (HR), rectal temperature before surgery of the rats enrolled in were all in the expected range and did not differ significantly among groups(Table 1). Data of the rectal temperature continually supervised after ICH of each group were shown (Fig. 1a). All the animals’ body temperature in the SHAM and NT group after ICH were beyond 36 ◦ C; those in the HT group had a temperature fluctuation between 33 and 35 ◦ C starting from 6 h after ICH and they reached this target temperature within 1 h after the beginning of the intervention. The Longa scores at 6 h after ICH of the rats enrolled in were within 1–3.There was no significant difference between the NT and HT group (Fig. 1b). The mortality rate between the HT group (6/32) and NT group (5/31) was similar. 3.2. Mild hypothermia reduced neurological deficits after ICH Neurological deficits according to Garcia score of the SHAM group were nearly normal. The score of the HT group was higher at day 2 and day 3 compared with the NT group after ICH (p < 0.05)(Fig. 1c). 3.3. Mild hypothermia induced less neuronal loss and shrinkage around hematoma SHAM group had a nearly normal neuronal morphology. Compared with SHAM group, abnormal neurons around hematoma increased both in NT and HT group. Compared with NT group, HT rats had less extensive neuron loss and neuronal shrinkage was less severe (Fig. 1d–f). 3.4. Mild hypotherima induced less ER stress after ICH To explore the role of ER stress response in the neuroprotection of hypothermia, the expression of GRP78, CHOP and p-eIF2␣ was investigated in different groups at different time points with Western blot and at day3 with immunofluorescence staining. Western blot showed that the three proteins all increased at day 2 after ICH and peaked at day 3 in the NT group, and then they gradually decreased thereafter. However, in HT group, levels of the three proteins around hematoma in the basal ganglia were lower than those in NT group till 7 days after ICH. (Fig. 2a). Quantitative analysis of western blot showed that hypothermia significantly decreased GRP78 and CHOP expression on day 2, 3 and 5, and decreased peIF2␣ on day 3 and day 5(p < 0.05). Consistent with the Western blot results, GRP78, CHOP and p-eIF2␣ expression decreased at day 3 compared with the NT group upon immunofluorescence staining (Fig. 2c–e). 3.5. Mild hypothermia reduced apoptosis after ICH TUNEL staining performed 3d after ICH showed that apoptotic cells were barely detected in the sham group, while they increased significantly in both of the NT and HT group. However, mild hypothermia significantly ameliorated cell apoptosis around hematoma compared with the NT group (P < 0.005, Fig. 2f, g).Western blot showed the expression level of the apoptosis determined protein cleaved-caspase 3 increased apparently after ICH, while it decreased in HT group at different time points compared with NT group after ICH (Fig. 3a). Quantitative analysis of western blot showed that hypothermia significantly decreased cleavedcaspase3 expression 3 days after ICH (p < 0.05)(Fig. 3b). Moreover, the qRT-PCR data analysis showed that the expression level of
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Table 1 Physiological measurements (mean ± SEM) taken before the ICH surgery. Body weight(g)
SHAM NT HT
285.00 ± 21.27 294.25 ± 34.97 278.35 ± 28.93
Body temperature(◦ C)
37.4 ± 0.21 37.6 ± 0.08 37.6 ± 0.07
BP(mmHg)
HR(bpm)
SBP
DBP
117.8 ± 7.57 133.45 ± 3.18 133.3 ± 4.66
89.6 ± 3.17 102.1 ± 3.71 94.15 ± 2.89
382.4 ± 8.52 384.28 ± 7.92 363.95 ± 6.53
All values are in the appropriate physiological ranges, and not significantly different among groups. (n = 20 per group).
Fig. 1. (a) Rectal temperature was monitored. Rats of NT and Sham group were kept above 36 ◦ C during the next 48 h after ICH. HT rats maintained their rectal temperature between 33 and 35 ◦ C 6 h after ICH. (b)Longa score tested 6 h after ICH before any interference. The difference between sham group and the other two was significant. There was no significant difference between the NT and HT groups. (***P < 0.005, n = 20 per group) (c) Garcia score evaluated at day 2 to day 7. Hypothermia improved the neurological score on day 2 and day 3 after ICH. (*P < 0.05, n = 8 per group)(d–f) Nissl staining on day 3. The short arrows showed injuried neurons with character of cytoplasmic shrinkage, nuclear pyknosis, and hyperchromasia. Long arrow denotes normal neurons. SHAM group had a nearly normal neuronal morphology. Abnormal neurons in the perihematomal area increased both in NT and HT group. Compared with NT group, HT rats had less extensive neuronal loss and exhibited less neuronal shrinkage. Scale bar = 50 m, magnification *400.
anti-apoptosis gene Bim was down regulated from day 2 to day 5 (p < 0.05) and the pro-apoptosis gene Bcl-2 was up-regulated on day 3 and day 5 (P < 0.05) after ICH by hypothermia when they were compared to the normal temperature treatment (Fig. 3c, d).
4. Discussion The neuroprotective effects of hypothermia have been demonstrated over the past decades. In the present study, we further investigated the protective effect of hypothermia on ICH and the role of ER stress as a possible underlying mechanism. We found that the neurological deficit was most apparent at day 2 and mild hypothermia improved the behavior score at day 2 and day 3. We also found that ICH injury induced ER stress and the response was most apparent at day 3. Furthermore, we confirmed that hypothermia induced a remarkable decrease in the expression of the ER stress associated protein, GRP78, CHOP and p-eIF2␣ compared with the NT treatment, indicating that the protective effect of hypothermia might be exerted by attenuating ER stress. Then we found hypothermia could reduce apoptosis after ICH and decrease the number of degenerative neurons. It suggested that hypothermia might reduce neuron apoptosis through the ER stress dependent apoptosis pathway.
Accumulating evidence implicates ER stress-induced cellular dysfunction and cell apoptosis as major contributors to many diseases [8], including brain ischemia injury [14], traumatic brain injury (TBI) [15] and some neurodegenerative diseases [16]. ER stress activates the UPR, a signaling pathway that aims to clear unfolded proteins and restore ER homeostasis, involves both proadaptive and pro-death cellular responses, depending on the strength, cell type and duration of the inflicted stress. It is initially protective, while prolonged ER stress can be deleterious. The UPR is initiated by three signal proteins located at the ER lumen: PKR-like sensors ER kinase (PERK), activating transcription factor 6(ATF6) and Inositol-requiring enzyme-1␣ (IRE1␣). It eventually acts on CHOP, caspase-12 and JNK to show its proapoptotic function. Under normal conditions, PERK is bound to the ER resident chaperone protein glucose-regulated protein (GRP78), which keeps them inactive. When unfolded proteins accumulate in the ER, GRP78 is released from these complexes to bind to the unfolded or misfolded proteins to help correct or clear them. GRP78 is widely used as a sentinel marker for ER stress under pathologic conditions. Accumulating evidence suggests that the induction of GRP78 prevents neuronal death induced by ER stress [17–19]. However, severe stress leads to excessive GRP78 release and apoptotic response [20]. In our study, we found that hypothermia treatment to ICH significantly down regulated the expression of GRP78.
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Fig. 2. Hypothermia induced less ER stress response and apoptosis. (a) The expression level of protein GRP78, CHOP and p-eIF2␣ around hematoma was assessed at different time points after ICH by Western blot. GAPDH was used as a loading control. (b) Densitometric evaluation of Western blot. (n = 5, *P < 0.05, **P < 0.01, ***P < 0.005) (c-e) Images of brain sections harvested on day 3 stained with double immunofluorescence. GRP78(c), CHOP (d). p-eIF2␣ (e). The targeted proteins were stained green, while nuclei blue (DAPI). Scale bar = 50 m, magnification *400. (f) Apoptotic cells detected by TUNEL staining around hematoma on day3. (g) Quantification of the percentage of TUNEL positive cells. Scale bar = 100 m, magnification *200. (n = 3, ***P < 0.005). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Therapeutic hypothermia protects the brain via multiple mechanisms from the injury of ischemia stroke, TBI, cardiac arrest and hypoxic-ischemic encephalopathy (HIE). It can reduce cell death, attenuate intracranial pressure, alleviate brain edema, suppress inflammatory reaction, and reduce the overall cerebral metabolism [25]. Clinically, hypothermia has been considered an approved therapy for patients after cardiac arrest [26] and in children with HIE [27]. Given the overlap in mechanisms contributing to injury after ischemia and ICH, we supposed HT could protect neurons from ICH induced injury and our findings support our hypothesis. However, the current study has some limitations. The collagenase and whole blood intracerebral ICH models are most widely used to identify mechanisms of injury and to evaluate treatments. However, some studies showed that 6 h delayed mild hypothermia in the collagenase model did not reduce behavior deficits and might increase hematoma volume [28]. Thus, whether our finding is modeling method dependent needs further investigated in the collagenase ICH model. Additionally, in our study, we found a slight temperature increase approximately from 6 h after ICH in the NT group while we didn’t give it any intervention. More rigorous temperature control should be considered in further studies. In conclusion, hypothermia attenuates ICH injury by inhibiting ER-stress mediated neuron apoptosis. Further researches are deserved to explore whether hypothermia could be applied as an effective treatment for ICH. Acknowledgments The authors have no commercial interests or conflicts regarding this work. Conceived and designed the experiments: WF. Performed the experiments: CG, YG, FFS, YJH, XQW, JNL, AYG. Wrote the paper: CG. This work was supported by the Shanghai Municipal Science and Technology Commission (Grant nos. 12140903300). References
Fig. 3. Hypothermia reduced apoptosis after ICH. (a)The expression level of protein cleaved-caspase 3 around hematoma was assessed at different time points after ICH by Western blot. GAPDH was used as a loading control. (b) Densitometric evaluation of Western blot. (c-d) mRNA levels of pro-apoptosis protein Bcl-2 (c) and anti-apoptosis protein Bim (d) relative to GAPDH, and expressed as fold change vs. SHAM. (n = 5 per group, *P < 0.05, ***P < 0.005).
Once PERK is activated, it phosphorylates the initiation factor eIF2␣, which activates the transcription factor ATF4. ATF4 controls CHOP expression which is a transcription factor that controls expression of a set of stress-induced target genes involved in programmed cell death. In our study, p-eIF2␣ and CHOP were both significantly reduced by HT. It is not clear how UPR response elements globally coordinated that leads to a delicate balance between adaptive and pro-apoptotic responses. But recent studies have suggested that prolonged activation of PERK signaling would promote apoptosis [21,22]. These all supports our findings that HT may exert its protective role by suppressing the ER stress. H. Puthalakath et al. points out that CHOP may induce apoptosis via inhibition of Bcl-2 transcription [23] and induct Bim expression [24]. ER stress induced apoptosis is similar with mitochondrialmediated apoptosis. Thus in our study, we tested the expression level of mRNA of anti-apoptotic gene Bim and pro-apoptotic gene Bcl-2 by qRT-PCR and investigated the expression of protein cleaved-caspase 3. Bim was significantly up-regulated while Bcl-2 was down regulated, and the expression of cleaved-caspase 3 was suppressed.
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Research article
Mild hypothermia protects rat neuronal injury after intracerebral hemorrhage via attenuating endoplasmic reticulum response induced neuron apoptosis Cen Guo, Yang Geng, Feifei Song, Yajing Huo, Xuqing Wu, Jianing Lv, Anyan Ge, Wei Fan ∗ Department of Neurology, Zhongshan Hospital, Fudan University, 200032, Shanghai, China
h i g h l i g h t s • ICH injury induces endoplasmic reticulum (ER) stress response. • Mild hypothermia could attenuate intracerebral hemorrhage (ICH) caused neuron injury by decreasing neuron apoptosis. • Mild hypothermia decreased ER response after ICH.
a r t i c l e
i n f o
Article history: Received 30 August 2016 Received in revised form 6 October 2016 Accepted 18 October 2016 Available online 19 October 2016 Keywords: Intracerebral hemorrhage Hypothermia ER stress Apoptosis
a b s t r a c t Background and purpose: Mild hypothermia has been proved to reduce global and focal cerebral ischemic injury in rodents by preventing cellular apoptosis through several pathways. However, whether hypothermia will be beneficial for intracerebral hemorrhage (ICH) and its underlying mechanisms haven’t reached a consensus. It has been implicated that endoplasmic reticulum (ER) stress plays a role in the secondary injury after ICH in rats. In this study, we aimed to investigate whether mild hypothermia would attenuate ICH induced neuronal injury via regulating ER stress. Methods: The model of ICH was induced by injecting autologous blood (120 l) into the rat striatum. Rats were divided into sham, normothermic (NT) and hypothermic (HT) groups. HT group were subjected to mild hypothermia (33 ◦ C–35 ◦ C) for 2 days starting from 6 h after ICH. Neurological deficits were evaluated. The ER stress related proteins (GRP78, CHOP and p-eIF2␣) and the apoptosis associated indicators (cleaved caspase3, Bcl-2 and Bim) around hematoma were assessed by western blot, qRT-PCR (quantificational real-time polymerase chain reaction), immunofluorescence and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling) assay. Results: Neurological deficits following ICH were reduced in HT group compared to NT group. Protein levels of GRP78, CHOP and p-eIF2␣ significantly increased after ICH in both NT and HT group compared to sham group, which was consistent with the trend of cleaved-caspase3 at protein level, and Bim, Bcl-2 at gene level. In comparison to NT group, GRP78, CHOP, p-eIF2␣, cleaved caspase-3 and Bim all decreased, while Bcl-2 increased in HT group. Additionally, apoptotic cells detected by TUNEL staining significantly decreased in the HT group. Conclusion: Mild hypothermia could attenuate ICH caused neuron injury by decreasing ER responseinduced neuron apoptosis. © 2016 Published by Elsevier Ireland Ltd.
1. Introduction
Abbreviations: ICH, intracerebral hemorrhage; ER, endoplasmic reticulum; NT, normothermic; HT, hypothermic; qRT- PCR, quantificational real-time polymerase chain reaction; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. ∗ Corresponding author. E-mail address: [email protected] (W. Fan). http://dx.doi.org/10.1016/j.neulet.2016.10.031 0304-3940/© 2016 Published by Elsevier Ireland Ltd.
Stroke is a leading cause of human death and disability across the world. Intracerebral hemorrhage (ICH) comprises 10–30% of all stroke cases. Patients suffering from ICH often have abysmal outcomes which are worse than ischemic stroke, with 30-day mortality estimated as high as 44%, and survivors typically suffer life limiting disability [1,2]. To date, there is no effective therapy for this subtype of stroke.
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Several clinical works [3,4] and experimental studies [5,6] have shown the neuroprotective effects of mild hypothermia in ischemic stroke. However, studies in ICH are fewer than those on ischemic stroke and the underlying mechanisms remain unknown both in ischemic stroke and ICH. The endoplasmic reticulum (ER), cell’s manufacturing machinery, is involved in protein synthesis, folding and many other important processes. Perturbation in ER functions leads to ER stress and it triggers a host response known as the unfolded protein response (UPR) to restore cellular homeostasis by transiently reducing protein translation and inducing ER repairing mechanisms within a short period; however, prolonged ER stress can activate irreversible signaling pathways leading to cell death [7]. ER stress-induced apoptosis has been implicated in several neurological diseases [8]. A recent study showed that ER stress was associated tightly with cell apoptosis after ICH [9]. In the present study, we tested the hypothesis that hypothermia had a protective effect on ICH by attenuating ER stress induced neuron apoptosis around hematoma. 2. Material and methods 2.1. Animals All studies were carried out in accordance with the guidelines for animal care and use established by our University Animal Care and Use Committee. The protocols were approved by the Committee on the Ethics of Animal Experiments of our university. A total of 68 male, Sprague Dawley rats (300 ± 50 g) were obtained from the Shanghai Fudan University. All animals were housed individually after surgery with freely access to food and water. Housing rooms were temperature, humidity, and light (12-h light-12-h dark cycle) controlled. 2.2. Experimental groups Animals were divided into SHAM (n = 16), normothermic (NT) (n = 26), hypothermic (HT) (n = 26) groups. Animals in the SHAM group were euthanized at day 3 or day 7 and the other two groups at day 2, 3, 5 or 7 after ICH. For the rats euthanized at day 7, behavioral tests were performed at day 2, 3, 5 and 7 (n = 8 per group). The harvested brains were used for histology (n = 3 per group, at day 3) and western blotting assays (n = 5 of NT and HT group at day 2, 3, 5, 7; n = 5 of SHAM group at day 3). 2.3. ICH model [10] Rats were anesthetized by injecting 10% chloral hydrate (0.4 ml/100 g) intraperitoneally. Rectal temperature was maintained within 37 ± 1 ◦ C during the surgery. Rats were positioned in a stereotactic frame and the scalp was incised along the midline. Using a sterile technique, a 1 mm burr hole was opened in the skull on the right 3 mm lateral, 1 mm posterior to the bregma. A blunt 26-gauge needle was inserted into the right basal ganglia under stereotactic guidance. Then, a total of 120 l of autologous whole blood withdrawn fast from the tail was infused at a rate of 5 l/min using a microinjector. A pause for 8 min was given after every 40 l injection. After completion of the infusion, the needle was paused for another 10 min, then withdrawn slowly and bone wax was placed in the burr hole. The skin incision was closed with sutures. Sham animals underwent the same surgical procedures, but only received a pause of microinjector for 10 min without infusion of blood. After the surgery, rats were divided into NT and HT groups randomly. HT treatment started from 6 h after the induction of ICH. NT and sham rats were housed in a room maintained at
25 ◦ C. They were all kept freely moving and free access to food and water after waking up from anesthesia of operation. 2.4. Hypothermia application and temperature monitor All the animals were maintained over 36 ◦ C after ICH. Rats of NT and Sham group were kept above 36 ◦ C during the next 48 h. HT rats were housed in a cold room maintained at 4 ◦ C starting from 6 h after ICH, with 75% ethyl alcohol spraying, back hair shaving and a heating pad to maintain their rectal temperature at 33–35 ◦ C.They were brought to room temperature 48 h after ICH and then slowly warmed. Since brain temperature was about 1 ◦ C higher than that measured rectally[11], we measured rectal temperature with a special thermometer inserted 5 cm deep into the animals’ rectum instead, and it was recorded every 15 min. Temperature monitoring was stopped once the rats regained normal temperature. 2.5. Behavioral test 2.5.1. Z −longa score [12] All animals underwent longa test 6 h after surgery and were scored by experimenters who were blinded to both neurological and treatment conditions. The neurologic findings were scored on a five-point scale as previously described: 0: no neurologic deficit; 1: failure to extend left forepaw fully; 2: circling to the left; 3: falling to the left; 4: rats couldn’t walk spontaneously and had a depressed level of consciousness. Rats were kicked out when the score was below 1 or over 3. 2.5.2. Garcia score [13] The neurobehavioral study consisted of the following six tests evaluating both motor function and sensory performance: spontaneous activity, symmetry in the movement of four limbs, forepaw outstretching, wire cage climbing, body proprioception and response to vibrissae touch. The total score ranged from 3 to 18 and a higher score indicated better performance. The test was performed at day 2, 3, 5 and 7. 2.6. Tissue harvesting Animals were deeply anesthetized with 10% chloral hydrate (0.4 ml/100 g) intraperitoneally and intracardially perfused with ice-cold saline. For biochemical analyses, tissues around hematoma with a diameter of 4 mm were rapidly dissected, snap frozen by liquid nitrogen and then stored in the −80 ◦ C freezer prior to RNA isolation, or protein analyses. For histology analyses, 4% paraformaldehyde (PFA) was given followed saline perfusion for fixation. Subsequently, the whole brains were isolated. Then they were incubated in 4%PFA for another 6 h, and immersed in a solution of 10%, 20%, 30% sucrose in turn for gradient dehydration. Serial coronal sections were cut using a cryotome (Leica; Germany) with a thickness of 10um.Sections were stored in the −80 ◦ C freezer prior to Nissl, immunofluorescent and TUNEL staining. 2.7. Western-blots Equal amount of protein samples was loaded in each well and subjected to 10–15% sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Separated proteins were then transferred from the gel to polyvinylidinene fluoride (PVDF) (Millipore, USA) membranes and blocked in 5% non-fat dry milk for 1.5 h. The membranes were incubated with the primary antibodies overnight at 4 ◦ C. Following primary antibodies were used: anti ribbit-GRP78 (1:500, Santa Cruz Biotechnology, USA),anti rabbit-CHOP (1:500, Bioworld, USA),anti rabbit-p-eIF2␣ (1:500, Sangong, China), anti rabbit-cleaved caspase 3(1:500,CST,USA) and anti rabbit-GAPDH
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antibody (1:500, Bioworld, USA).After washing primary antibodies with 1*TBST, the membranes were incubated with appropriate secondary antibodies for 1 h at room temperature. The blots were developed using ECL (NCM Biotechnology, China). 2.8. RNA isolation and qRT-PCR Total RNA was isolated. Reverse transcription was performed using a PrimeScript RT reagent kit (TaKaRa Bio Inc.,Dalian China), and the reaction mixture was subjected to quantitative real-time PCR Bim, Bcl-2, using SYBR Premix Ex Taq (Tli RnaseH Plus) (Takara, Dalian, China). Primers were as follows: Bim: (FP 5 -ACAAACCCCAAGTCCTCCTT-3 ; RP 5 -GCCCTCCTCGTGTAAGTCTC-3 ) Bcl-2: (FP5 - ACAGCCAGGAGAAATCAAACA-3 ; RP5 - ACAGCCAGGAGAAATCAAACA-3 ) Product specificity was confirmed by melting curve analysis. Data were normalized to GAPDH housekeeping gene. Foldinduction was calculated using the 2−CT method. 2.9. Nissl’s staining Nissl’s staining was performed to identify the basic neuronal structure of necrotic neurons in the brain. Sections were rinsed in tap and distilled water, and subsequently stained in toluidine blue solution for 2 min. After rinsing in distilled water, the sections were differentiated in 95% ethyl alcohol, cleared with xylene, prior to mounting with neutral balsam. 2.10. Immunofluorescence staining Sections were incubated in 0.3% Triton X-100 for 10 min and blocked in 10% normal sheep serum for 30 min at room temperature. They were then incubated overnight at 4 ◦ C with the following antibodies: rabbit anti-GRP78(1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-CHOP (1:200, Bioworld, USA), rabbit anti-p-eIF2␣(1:200, Sangong, China).The sections were incubated for 1 h at RT in dark with FITC-conjugated secondary antibodies (1:200, Yeasen, China), and then incubated in DAPI (1:1000,SIGMA,USA) for 10 min.They were coverslipped with 50% glycerinum. Images were captured using fluorescence microscope (DP71, Olympus, Japan). 2.11. TUNEL staining TUNEL staining was performed using an in situ cell death detection kit in accordance with the manufacturer’s instructions (Roche Applied Science, USA). Frozen sections were incubated with reaction buffer containing enzyme at 37 ◦ C for 1 h in the dark and then incubated in DAPI. Images were captured using fluorescence microscope (DP71, Olympus, Japan). The positive cells and total cells were counted on three fields at ×200 magnifications. The result was presented as a ratio of positive cell number to the total cell number. 2.12. Statistics All data were expressed as mean ± standard error of the mean (SEM) and analyzed by SPSS 19.0(IBM Inc., USA). A p-value less than 0.05 was considered significant. A one-way ANOVA with LSD post hoc test for multiple comparisons was used to compare the results between different groups. Levene’s test for equality of variances was used to confirm the multiple comparison procedure used. Nonparametric statistics was used for any such data that violated the assumption of homogeneity of variance.
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3. Results 3.1. Physiologic parameters The weight, blood pressure (BP), heart rates (HR), rectal temperature before surgery of the rats enrolled in were all in the expected range and did not differ significantly among groups(Table 1). Data of the rectal temperature continually supervised after ICH of each group were shown (Fig. 1a). All the animals’ body temperature in the SHAM and NT group after ICH were beyond 36 ◦ C; those in the HT group had a temperature fluctuation between 33 and 35 ◦ C starting from 6 h after ICH and they reached this target temperature within 1 h after the beginning of the intervention. The Longa scores at 6 h after ICH of the rats enrolled in were within 1–3.There was no significant difference between the NT and HT group (Fig. 1b). The mortality rate between the HT group (6/32) and NT group (5/31) was similar. 3.2. Mild hypothermia reduced neurological deficits after ICH Neurological deficits according to Garcia score of the SHAM group were nearly normal. The score of the HT group was higher at day 2 and day 3 compared with the NT group after ICH (p < 0.05)(Fig. 1c). 3.3. Mild hypothermia induced less neuronal loss and shrinkage around hematoma SHAM group had a nearly normal neuronal morphology. Compared with SHAM group, abnormal neurons around hematoma increased both in NT and HT group. Compared with NT group, HT rats had less extensive neuron loss and neuronal shrinkage was less severe (Fig. 1d–f). 3.4. Mild hypotherima induced less ER stress after ICH To explore the role of ER stress response in the neuroprotection of hypothermia, the expression of GRP78, CHOP and p-eIF2␣ was investigated in different groups at different time points with Western blot and at day3 with immunofluorescence staining. Western blot showed that the three proteins all increased at day 2 after ICH and peaked at day 3 in the NT group, and then they gradually decreased thereafter. However, in HT group, levels of the three proteins around hematoma in the basal ganglia were lower than those in NT group till 7 days after ICH. (Fig. 2a). Quantitative analysis of western blot showed that hypothermia significantly decreased GRP78 and CHOP expression on day 2, 3 and 5, and decreased peIF2␣ on day 3 and day 5(p < 0.05). Consistent with the Western blot results, GRP78, CHOP and p-eIF2␣ expression decreased at day 3 compared with the NT group upon immunofluorescence staining (Fig. 2c–e). 3.5. Mild hypothermia reduced apoptosis after ICH TUNEL staining performed 3d after ICH showed that apoptotic cells were barely detected in the sham group, while they increased significantly in both of the NT and HT group. However, mild hypothermia significantly ameliorated cell apoptosis around hematoma compared with the NT group (P < 0.005, Fig. 2f, g).Western blot showed the expression level of the apoptosis determined protein cleaved-caspase 3 increased apparently after ICH, while it decreased in HT group at different time points compared with NT group after ICH (Fig. 3a). Quantitative analysis of western blot showed that hypothermia significantly decreased cleavedcaspase3 expression 3 days after ICH (p < 0.05)(Fig. 3b). Moreover, the qRT-PCR data analysis showed that the expression level of
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Table 1 Physiological measurements (mean ± SEM) taken before the ICH surgery. Body weight(g)
SHAM NT HT
285.00 ± 21.27 294.25 ± 34.97 278.35 ± 28.93
Body temperature(◦ C)
37.4 ± 0.21 37.6 ± 0.08 37.6 ± 0.07
BP(mmHg)
HR(bpm)
SBP
DBP
117.8 ± 7.57 133.45 ± 3.18 133.3 ± 4.66
89.6 ± 3.17 102.1 ± 3.71 94.15 ± 2.89
382.4 ± 8.52 384.28 ± 7.92 363.95 ± 6.53
All values are in the appropriate physiological ranges, and not significantly different among groups. (n = 20 per group).
Fig. 1. (a) Rectal temperature was monitored. Rats of NT and Sham group were kept above 36 ◦ C during the next 48 h after ICH. HT rats maintained their rectal temperature between 33 and 35 ◦ C 6 h after ICH. (b)Longa score tested 6 h after ICH before any interference. The difference between sham group and the other two was significant. There was no significant difference between the NT and HT groups. (***P < 0.005, n = 20 per group) (c) Garcia score evaluated at day 2 to day 7. Hypothermia improved the neurological score on day 2 and day 3 after ICH. (*P < 0.05, n = 8 per group)(d–f) Nissl staining on day 3. The short arrows showed injuried neurons with character of cytoplasmic shrinkage, nuclear pyknosis, and hyperchromasia. Long arrow denotes normal neurons. SHAM group had a nearly normal neuronal morphology. Abnormal neurons in the perihematomal area increased both in NT and HT group. Compared with NT group, HT rats had less extensive neuronal loss and exhibited less neuronal shrinkage. Scale bar = 50 m, magnification *400.
anti-apoptosis gene Bim was down regulated from day 2 to day 5 (p < 0.05) and the pro-apoptosis gene Bcl-2 was up-regulated on day 3 and day 5 (P < 0.05) after ICH by hypothermia when they were compared to the normal temperature treatment (Fig. 3c, d).
4. Discussion The neuroprotective effects of hypothermia have been demonstrated over the past decades. In the present study, we further investigated the protective effect of hypothermia on ICH and the role of ER stress as a possible underlying mechanism. We found that the neurological deficit was most apparent at day 2 and mild hypothermia improved the behavior score at day 2 and day 3. We also found that ICH injury induced ER stress and the response was most apparent at day 3. Furthermore, we confirmed that hypothermia induced a remarkable decrease in the expression of the ER stress associated protein, GRP78, CHOP and p-eIF2␣ compared with the NT treatment, indicating that the protective effect of hypothermia might be exerted by attenuating ER stress. Then we found hypothermia could reduce apoptosis after ICH and decrease the number of degenerative neurons. It suggested that hypothermia might reduce neuron apoptosis through the ER stress dependent apoptosis pathway.
Accumulating evidence implicates ER stress-induced cellular dysfunction and cell apoptosis as major contributors to many diseases [8], including brain ischemia injury [14], traumatic brain injury (TBI) [15] and some neurodegenerative diseases [16]. ER stress activates the UPR, a signaling pathway that aims to clear unfolded proteins and restore ER homeostasis, involves both proadaptive and pro-death cellular responses, depending on the strength, cell type and duration of the inflicted stress. It is initially protective, while prolonged ER stress can be deleterious. The UPR is initiated by three signal proteins located at the ER lumen: PKR-like sensors ER kinase (PERK), activating transcription factor 6(ATF6) and Inositol-requiring enzyme-1␣ (IRE1␣). It eventually acts on CHOP, caspase-12 and JNK to show its proapoptotic function. Under normal conditions, PERK is bound to the ER resident chaperone protein glucose-regulated protein (GRP78), which keeps them inactive. When unfolded proteins accumulate in the ER, GRP78 is released from these complexes to bind to the unfolded or misfolded proteins to help correct or clear them. GRP78 is widely used as a sentinel marker for ER stress under pathologic conditions. Accumulating evidence suggests that the induction of GRP78 prevents neuronal death induced by ER stress [17–19]. However, severe stress leads to excessive GRP78 release and apoptotic response [20]. In our study, we found that hypothermia treatment to ICH significantly down regulated the expression of GRP78.
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Fig. 2. Hypothermia induced less ER stress response and apoptosis. (a) The expression level of protein GRP78, CHOP and p-eIF2␣ around hematoma was assessed at different time points after ICH by Western blot. GAPDH was used as a loading control. (b) Densitometric evaluation of Western blot. (n = 5, *P < 0.05, **P < 0.01, ***P < 0.005) (c-e) Images of brain sections harvested on day 3 stained with double immunofluorescence. GRP78(c), CHOP (d). p-eIF2␣ (e). The targeted proteins were stained green, while nuclei blue (DAPI). Scale bar = 50 m, magnification *400. (f) Apoptotic cells detected by TUNEL staining around hematoma on day3. (g) Quantification of the percentage of TUNEL positive cells. Scale bar = 100 m, magnification *200. (n = 3, ***P < 0.005). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Therapeutic hypothermia protects the brain via multiple mechanisms from the injury of ischemia stroke, TBI, cardiac arrest and hypoxic-ischemic encephalopathy (HIE). It can reduce cell death, attenuate intracranial pressure, alleviate brain edema, suppress inflammatory reaction, and reduce the overall cerebral metabolism [25]. Clinically, hypothermia has been considered an approved therapy for patients after cardiac arrest [26] and in children with HIE [27]. Given the overlap in mechanisms contributing to injury after ischemia and ICH, we supposed HT could protect neurons from ICH induced injury and our findings support our hypothesis. However, the current study has some limitations. The collagenase and whole blood intracerebral ICH models are most widely used to identify mechanisms of injury and to evaluate treatments. However, some studies showed that 6 h delayed mild hypothermia in the collagenase model did not reduce behavior deficits and might increase hematoma volume [28]. Thus, whether our finding is modeling method dependent needs further investigated in the collagenase ICH model. Additionally, in our study, we found a slight temperature increase approximately from 6 h after ICH in the NT group while we didn’t give it any intervention. More rigorous temperature control should be considered in further studies. In conclusion, hypothermia attenuates ICH injury by inhibiting ER-stress mediated neuron apoptosis. Further researches are deserved to explore whether hypothermia could be applied as an effective treatment for ICH. Acknowledgments The authors have no commercial interests or conflicts regarding this work. Conceived and designed the experiments: WF. Performed the experiments: CG, YG, FFS, YJH, XQW, JNL, AYG. Wrote the paper: CG. This work was supported by the Shanghai Municipal Science and Technology Commission (Grant nos. 12140903300). References
Fig. 3. Hypothermia reduced apoptosis after ICH. (a)The expression level of protein cleaved-caspase 3 around hematoma was assessed at different time points after ICH by Western blot. GAPDH was used as a loading control. (b) Densitometric evaluation of Western blot. (c-d) mRNA levels of pro-apoptosis protein Bcl-2 (c) and anti-apoptosis protein Bim (d) relative to GAPDH, and expressed as fold change vs. SHAM. (n = 5 per group, *P < 0.05, ***P < 0.005).
Once PERK is activated, it phosphorylates the initiation factor eIF2␣, which activates the transcription factor ATF4. ATF4 controls CHOP expression which is a transcription factor that controls expression of a set of stress-induced target genes involved in programmed cell death. In our study, p-eIF2␣ and CHOP were both significantly reduced by HT. It is not clear how UPR response elements globally coordinated that leads to a delicate balance between adaptive and pro-apoptotic responses. But recent studies have suggested that prolonged activation of PERK signaling would promote apoptosis [21,22]. These all supports our findings that HT may exert its protective role by suppressing the ER stress. H. Puthalakath et al. points out that CHOP may induce apoptosis via inhibition of Bcl-2 transcription [23] and induct Bim expression [24]. ER stress induced apoptosis is similar with mitochondrialmediated apoptosis. Thus in our study, we tested the expression level of mRNA of anti-apoptotic gene Bim and pro-apoptotic gene Bcl-2 by qRT-PCR and investigated the expression of protein cleaved-caspase 3. Bim was significantly up-regulated while Bcl-2 was down regulated, and the expression of cleaved-caspase 3 was suppressed.
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