Neuroprotection by Mesenchymal Stem Cell (MSC) Administration is Enhanced by Local Cooling Infusion (LCI) in Ischemia

Journal Pre-proofs Review Neuroprotection by Mesenchymal Stem Cell (MSC) Administration is Enhanced by Local Cooling Infusion (LCI) in Ischemia Wenjing Wei, Di Wu, Yunxia Duan, Kenneth B. Elkin, Ankush Chandra, Longfei Guan, Changya Peng, Xiaoduo He, Chuanjie Wu, Xunming Ji, Yuchuan Ding PII: DOI: Reference:

S0006-8993(19)30460-3 https://doi.org/10.1016/j.brainres.2019.146406 BRES 146406

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Brain Research

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31 May 2019 20 August 2019 23 August 2019

Please cite this article as: W. Wei, D. Wu, Y. Duan, K.B. Elkin, A. Chandra, L. Guan, C. Peng, X. He, C. Wu, X. Ji, Y. Ding, Neuroprotection by Mesenchymal Stem Cell (MSC) Administration is Enhanced by Local Cooling Infusion (LCI) in Ischemia, Brain Research (2019), doi: https://doi.org/10.1016/j.brainres.2019.146406

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Neuroprotection by Mesenchymal Stem Cell (MSC) Administration is Enhanced by Local Cooling Infusion (LCI) in Ischemia Wenjing Weia,b,c,d, Di Wua, Yunxia Duana, Kenneth B. Elkinc, Ankush Chandrac, Longfei Guanc,d, Changya Pengc,d, Xiaoduo Hea, Chuanjie Wub, Xunming Jia,b*,Yuchuan Dingc,d

a. China-America Institute of Neuroscience, Xuanwu Hospital, Capital Medical University, Beijing 100053, China b. Department of Neurology, Xuanwu Hospital, Capital Medical University, Beijing 100053, China c. Department of Neurosurgery, Wayne State University School of Medicine, Detroit, MI, USA d. Department of Research & Development Center, John D. Dingell VA Medical Center, Detroit, Michigan, USA

* Corresponding author. Corresponding author at: China-America Institute of Neuroscience, Xuanwu Hospital, Capital Medical University, Beijing 100053, China. E-mail address: [email protected] (X. Ji)

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Abstract Objective: The present study aimed to determine if hypothermia augments the neuroprotection conferred by MSC administration by providing a conducive micro-environment. Methods: Sprague-Dawley rats were subjected to 1.5 h middle cerebral artery occlusion (MCAO) followed by 6 or 24 h of reperfusion for molecular analyses, as well as 1, 14 and 28 days for brain infarction or functional outcomes. Rats were treated with either MSC (1×105), LCI (cold saline, 0.6 ml/min, 5min) or both. Brain damage was determined by Infarct volume and neurological deficits. Long-term functional outcomes were evaluated using foot-fault and Rotarod testing. Human neural SHSY5Y cells were investigated in vitro using 2 h oxygen-glucose deprivation (OGD) followed by MSC with or without hypothermia (HT) (34°C, 4 h). Mitochondrial transfer was assessed by confocal microscope, and cell damage was determined by cell viability, ATP, and ROS level. Protein levels of IL-1β, BAX, Bcl-2, VEGF and Miro1 were measured by Western blot following 6 h and 24 h of reperfusion and reoxygenation. Results: MSC, LCI, and LCI+MSC significantly reduced infarct volume and deficit scores. Combination therapy of LCI+MSC precipitated better long-term functional outcomes than monotherapy. Upregulation of Miro1 in the combination group increased mitochondrial transfer and lead to a greater increase in neuronal cell viability and ATP, as well as a decrease in ROS. Further, combination therapy significantly decreased expression of IL-1β and BAX while increasing Bcl-2 and VEGF expression. Conclusion: Therapeutic hypothermia upregulated Miro1 and enhanced MSC mitochondrial transfer-mediated neuroprotection in ischemic stroke. Combination of LCI with MSC therapy may facilitate clinical translation of this approach.

Keywords: Ischemia/reperfusion injury, Local cooling infusion (LCI), Middle cerebral artery, MSC therapy, Mitochondrial transfer 2

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1. Introduction Though adjunct stroke therapies are well-studied, tissue plasminogen activator (tPA) remains the only effective, FDA-approved drug for stroke and is limited by a narrow therapeutic window (Detante et al., 2018, Li et al., 2017). Recently, stem cell therapy has emerged as a promising treatment strategy for stroke (Lindvall et al., 2011, Shinozuka et al., 2013). Among stem cell therapies, bone marrow-derived mesenchymal stem cells (BM-MSC) are the most well-characterized, likely as a result of various sources of, and ease of, production (Shinozuka et al., 2013). Although MSC administration has potential to be therapeutic in ischemic stroke, the optimal quantity of MSC remains unknown, and inefficient delivery to affected tissue has limited its applicability. Very recently, the role of MSC mitochondrial transfer in stroke therapy has been identified, though the precise mechanism remains unclear (Nzigou Mombo et al., 2017, Hsu et al., 2016, Boukelmoune et al., 2018). Previous studies have demonstrated that Miro1, a key participant in mitochondrial traffic, promotes mitochondrial transfer from MSC and suggests that genetic modification of stem cells may improve cerebral injury therapies (Babenko et al., 2018, Russo E. et al., 2018a, b). Several studies have reported adverse events resulting from MSC therapy including embolization (Borlongan et al., 2009, Chopp et al., 2009), infection, and immune-inflammatory response (Borlongan et al., 2011a, b, Tauskela et al., 2017) which all limitt the surviving amount, and functionality, of circulating MSCs. Such effects can result in low homing rates and inefficient delivery of mitochondria and secretory factors. Consequently, there has been slow clinical progress in improving the safety and the therapeutic effects of MSC therapy (Vu, Q. et al., 2014, Diamandis et al., 2015, Zhang et al., 2009). To mitigate these problems, the present study aimed to improve the efficacy of MSC by using it in conjunction with local cooling infusion (LCI). There is a substantial amount of preclinical and clinical studies that report strong neuroprotective effects conferred by hypothermia following stroke (Han et al., 2015). 4

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hypothermia, there is reduced oxygen demand, increased preservation of energy stores, and reduced lactate production which is likely precipitated by decreased metabolic rate and stimulation of brain glucose utilization (Han et al., 2015, Kim et al., 2011) in addition to modulation of inflammatory and apoptotic pathways (Wu et al., 2017, Zhao et al., 2018). Presently, we determined the neuroprotective effect of MSC therapy following ischemic stroke when used in conjunction with hypothermia in vivo and in vitro. A local cooling infusion (LCI) method was utilized to minimize the negative effects of hypothermia (Ding et al., 2002, 2004) and optimize additional therapy by providing a micro-environment with low level of metabolism, apoptosis, and inflammation.

2. Results 2.1 Infarct volume and neurological deficits Ischemia produced a large infarct volume at 24 h (42.7 ± 2.3%), which was significantly reduced by LCI (28.8 ± 4.0%) (P<0.05), MSC (34.7 ± 3.2%) (P<0.05), and the combination therapy of LCI and MSC (28.5 ± 4.0%) (P<0.05, Fig. 1a, b). The differential reduction between the combination group and either monotherapy of LCI or MSC was not significant (P=0.95; P=0.26). A high neurological deficits score (10.6 ± 0.4) was observed in stroke group at 24 h. The deficits were significantly reduced by LCI (7.1 ± 0.9) and MSC (7.7 ± 0.6) (P<0.05). The combination group exhibited a significant reduction in scores (7.1 ± 0.6) (P<0.05, Fig. 1c), while there was no significant difference between the combination group and the monotherapies of LCI or MSC (P=0.98; P=0.48) In addition, there was no significant difference between the early combination group (LCI Day 0 + MSC Day 0) (30.4 ± 4.4%) and the late combination group (LCI Day 0 + MSC Day 1) (28.5 ± 4.0%) at 24 h in infarct volume (Fig. 1d) or neurological deficits between the early (7.3 ± 0.9) and the late (7.1 ± 0.6) combination group (Fig. 1e). 2.2 Long-term Neuroprotection determined by functional outcome up-to 28 days 5

Although there was no difference among groups for short-term neuroprotection, long-term functional outcomes were enhanced by combination therapy. In the Rota-rod test (Fig. 2a), ischemia led to a decrease in latency time to fall at Day 1 (27.5 ± 9.0). Compared with the control group (60.0 ± 18.6, 25.3 ± 14.0), LCI (83.3 ± 20.5, 99 ± 42.6) and MSC (106.5 ± 24.1, 83.7 ± 14.1) significantly decreased in running time at Day 14 and 28, respectively (P<0.05). The combination treatment significantly increased the running time (120.9 ± 20.2, 28.3 ± 24.1) compared with either monotherapy at Day 28 (P<0.05). Similarly, ischemia in the foot-fault test (Fig. 2b) led to a high fault steps at Day 1 (15.3 ± 1.1). Compared to the control group (6.8 ± 1.3, 5.0 ± 1.1), LCI (5.2 ± 1.5, 3.3 ± 0.6) and MSC (4.1 ± 1.2, 3.6 ± 0.7) groups showed a significant decrease in fault steps at Day 14 and 28, respectively (P<0.05). The combination treatment significantly lowered the foot-faults (3.1 ± 1.0, 1.7 ± 0.3) as compared with either monotherapy at Day 14 and 28 (P<0.05). Moreover, the late combination group (LCI Day 0 + MSC Day 1) showed even further neuroprotection when compared with the early combination group (LCI Day 0 + MSC Day 0) with an increase in running time by the Rota-rod test (P<0.05, Fig. 2c) at Day 14 and 28, and a decrease in fault steps by the foot fault test (P<0.05, Fig. 2d) at Day 28. 2.3 IL-1β, BAX, Bcl-2 and VEGF expression in rats There was a significant increase in IL-1β and BAX protein expression after ischemic stroke as compared to sham group at both 6 h and 24 h of reperfusion (P<0.05, Fig. 3a-d). The ischemia-induced increase in IL-1β and BAX expression was significantly reversed by LCI, MSC, and combination therapy at both 6 h (P<0.001, Fig. 3a, c) and 24 h (P<0.05, Fig. 3b, d) of reperfusion. Furthermore, combination therapy induced an even greater reduction in IL-1β (P<0.01, Fig. 3b) and BAX (P<0.05, Fig. 3d) level than either monotherapy at 24 h. Conversely, there was a significant decrease in Bcl-2 and VEGF levels after ischemic stroke as compared to sham group at 6 and 24 h of reperfusion (P<0.01, Fig. 3e-h). The ischemia-induced decrease in Bcl-2 and VEGF expression was significantly reversed by LCI, 6

MSC, and combined therapy at 6 h (P<0.01, Fig. 3e, g) and 24 h (P<0.01, Fig. 3f, h), excluding VEGF expression treated by LCI therapy at 6 h. The combination therapy enhanced expression of Bcl-2 and VEGF proteins compared with either monotherapy at 6 and 24 h, excluding comparison with MSC therapy in VEGF expression at 6 h (P<0.05, Fig. 3e-h). 2.4 Cell function determined by cell viability The level of cell viability was greatly decreased in the OGD group compared to the shamoperated group (referenced as 1) at 6 h (0.26 ± 0.13) and 24 h (0.34 ± 0.02) after reperfusion (P<0.001, Fig. 4a, b). Compared with OGD group, the HT (0.65 ± 0.01), MSC (0.60 ± 0.06), and combination groups (0.60 ± 0.03) significantly increased cell viability at 6 h (P<0.05) and 24 h (P<0.01). Moreover, the combination therapy (0.84 ± 0.02) further increased cell viability as compared to the MSC group (0.65 ± 0.05) and the HT group (0.66 ± 0.03) at 24 h (P<0.05, Fig. 4b). 2.5 Mitochondrial function determined by ATP level When compared with the sham-operated group, ATP level was significantly decreased at 6 h (0.44 ± 0.08) and 24 h (0.55 ± 0.03) (P<0.01, Fig. 4c, d). At 6 h, HT therapy (0.70 ± 0.03) significantly increased ATP levels (P<0.05) while the MSC and combination therapy had no effect. At 24 h, the HT (0.81 ± 0.02), MSC (1.26 ± 0.12), and combination groups (1.6 ± 0.05) exhibited a significant increase in ATP levels compared with the OGD group (P<0.05). Moreover, the combination group produced a greater increase than the either monotherapy group at 24 h (P<0.05, Fig. 4d). 2.6 Cellular injury determined by ROS level Oxidative stress following stroke was significantly increased at 6 h (7.6 ± 0.6) and remained elevated at 24 h (10.5 ± 0.3) following reperfusion as compared to sham group (P<0.001, Fig. 4e, f). At 6 h, both the HT group (5.6 ± 0.3) and combination group (5.4 ± 0.3) reduced ROS levels (P<0.05) while the MSC group remained unchanged (6.3 ± 0.4, P=0.17). The combination group did not show enhanced reduction in ROS than the HT group at 6 h. 7

At 24 h and

compared with the OGD group, the HT (6.4 ± 0.2), MSC (8.0 ± 0.3), and combination groups (5.2 ± 0.3) exhibited a significant reduction in ROS levels (P<0.001). Once again, the combination group exhibited a more significant decrease in ROS level than either monotherapy group (P<0.05, Fig. 4f). 2.7 IL-1β, BAX, Bcl-2 and VEGF expression in SHSY5Y cells There was a significant increase in IL-1β and BAX protein levels after OGD as compared to sham group at both 6 h and 24 h of reperfusion (P<0.05, Fig. 5a-d). At 6 h, the OGD-induced upregulation of IL-1β was significantly reversed in the HT and the combination groups (P<0.01, Fig. 5a) while BAX expression was significantly reversed by both HT and MSC therapy (P<0.05, Fig. 5c). At 24 h, the OGD-induced upregulation of IL-1β and BAX expression was significantly reversed by HT, MSC and combination therapy (P<0.001, Fig. 5b, d). Furthermore, the combination group induced a greater reduction in IL-1β and BAX level than either monotherapy (P<0.01, Fig. 5b, d). Conversely, there was a significant decrease in Bcl-2 and VEGF levels after OGD at both 6 h and 24 h of reperfusion (P<0.01, Fig. 5e-h). At 6 h, the OGD-induced decrease in Bcl-2 and VEGF levels was significantly reversed in the MSC (P<0.05) and combination groups (P<0.01). At 24 h, the OGD-induced decrease in Bcl-2 and VEGF expression was significantly reversed by HT, MSC, and combined therapy (P<0.01, Fig. 5f, h). Again, the combination group had even greater expression of Bcl-2 and VEGF levels than either monotherapy (P<0.01, Fig. 5e-h). 2.8 Transportation number of mitochondria determined by confocal microscopy image In co-culture experiments, confocal microscopy (Zeiss LSM 780, Germany) revealed that MSC-derived mitochondria (red, MitoTracker Red CMXRos) were present in SHSY5Y cells in a co-cultured group (Fig. 6a). The transferred mitochondria survived primarily in the soma and axon of SHSY5Y cells after OGD (Fig. 6b). In these conditions, MSC mitochondrial transfer (7.9 ± 0.2) was significantly enhanced by HT (22.3 ± 0.4) at 24 h (P<0.001, Fig. 6c). 2.9 Mitochondria transfer associated protein expression 8

Miro1, a mitochondrial transfer-associated protein located on microtubules, was significantly upregulated in MSC by HT (34°C, 4 h) when compared to no treatment at 24 h of reoxygenation (P<0.01, Fig. 6d).

3. Discussion The present study revealed that combined hypothermia and MSC administration enhanced neuroprotection to a greater degree than either monotherapy, evidenced by assessment of longterm neurological outcomes, cellular functions, and apoptotic proteins and regulatory molecules in the peri-infarct area. Further, the increased transfer of mitochondria from MSC to neurons via Miro1 upregulation by hypothermia may partially comprise the responsible molecular mechanism. In our study, intra-arterial (IA) MSC delivery achieved a significant decrease in infarct volume with a lower dose, and a higher migrated number, into the ipsilateral parenchyma compared to intravenous (IV) administration (Ishizaka et al., 2013). Compared with IA delivery, IV administration of MSC was more likely to target the spleen, lymphatic tissue, and other immune organs in lieu of the brain in adult rats with chronic stroke (Acosta et al., 2015, Byun et al., 2013, Liu et al., 2006). Moreover, IV administration has been reported to produce very little cerebral migration in long term observation (Borlongan et al., 2004). In this study, though, a highly selective endovascular delivery method was employed via an intra-carotid microcatheter, which is a more effective method for transporting low-dose MSCs to the ischemic territory. The quantity and speed of delivery was strictly controlled to avoid secondary blockage (Shi et al., 2017). The use of BM-MSC has been shown to prompt functional recovery of neurological deficits following cerebral ischemia in stroke models (Song et al., 2004, Chopp et al., 2002, Rempe et al., 2002). Rather than targeting the differentiation of MSC to replace damaged neurons (Chen et al., 2003), current research emphasizes the capacity of MSCs to secrete several growth 9

factors, cytokines, and chemokines which reduce inflammation and increase neurogenesis (Zhu et al., 2015, Wakabayashi et al., 2010, Jeong et al., 2014, Huang et al., 2013, Hao et al., 2014, Acosta et al., 2015). While MSC administration in animal stroke models is generally safe and exhibits favorable effects on behavioral outcome (Lindvall et al., 2011, Ishizaka et al., 2013), many clinical trials have failed in their translation to the clinic (Diamandis et al., 2015, Dulamea et al., 2015). Two recently concluded clinical trials indicated stem cells are safe, but ineffective in stroke patients (Borlongan, 2019). Still, bone marrow-derived MSC remains an optimal source for cell therapy (Abraham et al., 2019). Recognizing the multiple cell death processes associated with stroke, we consider the need for novel strategies and, potentially, the need for combination therapy to realize safe and effective translation of stem cell therapy for stroke. The number of MSC homing into the penumbra and the function of MSCs are essential for clinical outcomes. Therefore, a pro-survival micro-environment, which hypothermia therapy may provide, is necessary for MSCs to stay and support cell survival. In various studies, local cooling infusion and washing have afforded substantial neuroprotective effects following stroke by decreasing metabolic rate, preventing lactate production (Han et al., 2015, Kim et al., 2011), and removing excessive toxic products between tissues (Ding et al., 2004, Liu et al., 2018), all of which establish a pro-survival microenvironment (Zhang et al., 2018). The findings of the LCI group in the present study are consistent with these effects. The benefits of LCI+MSC combination therapy in ischemic rats was supported by a previous study (Kaneko et al., 2012). In this study, the authors first employed moderate hypothermia and MSC transplantation to protect against hypoxic-ischemic-like injury in vitro. Their initial results showed that this combination treatment of hypothermia and MSC had greater neuroprotective effects compared to stand-alone treatments. Based on their combination strategy and hypoxic-ischemic-like injury model, we explored the novel mechanism of combination therapy and further expanded in animal models. 10

In the present study, the delivery of MSC on Day 1 conferred greater neuroprotection than on Day 0, which was supported by Toyoshima’s study. Toyoshima et al. injected MSC intraarterially at 1, 6, 24, or 48 hours after MCAO and observed that MSC transplantation at 24 hours was optimal timing for their ischemic stroke model (Toyoshima et al., 2015). This observation was accompanied by MSC survival and migration observations, with the highest number of integrated MSCs detected in the 24h group. Moreover, bFGF and SDF-1α levels of the infarcted cortex were highly elevated in the 24h group. This may suggest that delivery time is a significant factor in the neurological recovery of the combined therapy. Thus, we prefer the late combination therapy when designing clinical trials of cell transplantation for stroke patients. However, the present study needs to be developed further in future experiments. In vivo, a delayed MSC delivery group (on Day 1) should to be added to determine whether hypothermia is beneficial for delayed MSC treatment as it is in early MSC treatment. In parallel, cellular experiments should add the delayed MSC delivery group (Day 1) and delayed combination group to verify the mechanism of how hypothermia affects MSC. Stem cell therapy, once colonized in peri-infarct and core area in brain, sustains its therapeutic effect through the entire post-stroke period. Unsurprisingly, the key of MSC therapy is the number and function of survival stem cells. Considering this, hypothermia was utilized prior to MSC administration to increase MSC plantation number and the function. Though this is speculation in part, our study demonstrated that our LCI+MSC combination protocol enhanced long-term functional outcome, but not short-time recovery. The combination group worked better than monotherapy group in the long-term outcomes as a result. In short-time observation, the changes in molecular levels were not reflected in infarct volume and 12 scores, though the mitochondrial transfer and some protein expression exhibited enhanced variation in combination group in vivo and vitro at 24 h. One possible explanation is that histologic and behavioral recovery requires more time to improve function. However, further study is needed to determine why hypothermia had greater effect on the molecular level than on infarct volume. 11

The para-secretion of factors by MSCs likely play an essential role in neuroprotection. The inflammatory factor IL-1β, the pro-apoptotic protein BAX, the anti-apoptotic protein Bcl-2, and the neurotrophic factor VEGF have been implicated in pathways which: directly protect against cell death, induce endogenous cerebral reparative processes (Cosky and Ding, 2018, Yasuhara et al., 2009, Hanson et al., 2008), and activate neurogenesis and angiogenesis (Goldman et al., 2011). Similarly, hypothermia has been shown to upregulate growth factors in animals and patients (Fan et al., 2010, Schmitt et al., 2010) and regulate other inflammatory and apoptotic factors (Wu et al., 2017). Thus, combination therapy of hypothermia and MSC likely results in an elevated level of these therapeutic substances. In addition, MSCs are capable of transferring mitochondria to cardiomyocytes, cancer cells and others (Cho et al., 2012, Acquistapace et al., 2011, Pasquier et al., 2013). The protective effect of mitochondrial transfer from MSCs to injured areas in animal models have been widelystudied in various conditions, including acute lung injury and heart ischemia (Islam et al., 2012, Sun et al., 2015, Davis et al., 2014, Mishra et al., 2016, Tan et al., 2015). MSC have been observed to provide functional mitochondria to OGD neurons and increase survival (Babenko et al., 2018), though the mechanism of the mitochondrial transfer is unclear. A very recent study (Nguyen et al., 2019) used a combination of in vitro cell culture and in vivo rat models to examine the role of mitochondria dysfunction in stroke-related retinal ischemia, and of stem cells in the repair of mitochondria and the rescue of ischemic retinal cells. The authors demonstrated that MSC transplantation afforded functional benefits against cerebral and retinal ischemia by abrogating mitochondrial dysfunction. The mechanism, in part, was stem cellmediated mitochondrial transfer, which supports our conclusion of mitochondrial protection through upregulation of Miro1. There is a growing body of evidence that Miro1 precipitates mitochondrial transfer (Las et al., 2018，Ahmad, 2014). Previous studies have shown that Miro1 physically connects the

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mitochondrion to the KIF5 motor protein through accessory proteins like Miro2, TRAK1, TRAK2, and Myo19, thus organizing intracellular mitochondrial movement along microtubules (Fransson et al., 2006; Saotome et al., 2008; Macaskill et al., 2009). A recent study demonstrated that Miro1 increased the efficiency of mitochondrial transfer from MSCs to astrocytes, and that the introduction of MSCs with overexpressed Miro1 led to significant recovery of neurological functions in animals that had undergone experimental stroke (Babenko et al., 2018). In our study, Miro1 expression was significantly upregulated by hypothermia which, we hypothesize, resulted in an increase in the number of transferred mitochondria. It is likely that the increased mitochondrial transfer played a key role in elevating cell viability and ATP levels while reducing ROS levels. In summary, our study demonstrated that hypothermia compounds the neuroprotective effect of MSC therapy, especially in long-term functional recovery after stroke. The increased mitochondrial transfer from MSC to neurons via Miro1 upregulation with hypothermia is implicated as the underlying mechanism. Furthermore, the suppression of IL-1β and BAX as well as elevation of Bcl-2 and VEGF may further explain the greater neuroprotection that was observed. Our conclusion supports the use of MSC as cell therapy and further justifies a need for novel strategies, such as combination therapy with hypothermia, to enhance success in clinical translation. Ultimately, the novel combination therapy of hypothermia and MSC administration has revealed potential insights in stroke pathology and may advance the clinical therapy of ischemic diseases.

4. Experimental procedures 4.1 Subjects Adult male Sprague-Dawley rats (300 ± 20 g weight, Vital River Laboratory Animal Co.) were used in this study. All experimental procedures were approved by the Institutional Animal Investigation Committee of Capital Medical University (Beijing, China) in accordance with the 13

National Institutes of Health (USA) guidelines for care and use of laboratory animals. Throughout the study, all animals were housed in the same animal care facility with 12-h light/dark cycles. 198 rats were randomly divided into 5 groups by animal tag number: (1) Sham, in which the rats underwent the operative processes except the middle cerebral artery occlusion (MCAO); (2) Stroke, in which the rats received 1.5 h MCAO followed by the reperfusion without treatment; (3) LCI, in which ischemic rats received IA cold saline infusion into the ischemic territory at the onset of reperfusion by slightly withdrawing the catheter; (4) MSC group, in which ischemic rats received MSC delivery at the onset of reperfusion through the catheter; (5) LCI + MSC (combination) group, in which ischemic rats received both LCI and MSC delivery. Rats that died before expected time points, due to brain hemorrhage, and those with no signs of infarct based on 2,3,5-triphenyltetrazolium chloride (TTC) staining and neurological deficits were excluded for further analyses. In parallel, human SHSY5Y cells were divided into 5 groups: (1) Sham, in which the cells underwent the operative process except OGD; (2) OGD, in which the cells received 2 h OGD followed by the reoxygenation without treatment; (3) HT, in which OGD cells were placed in a 34°C incubator for 4 h before the regular incubator; (4) MSC group, in which OGD cells were co-cultured with the inserts of MSC; (5) LCI + MSC (combination) group, in which OGD cells received both LCI and MSC treatments. The procedures and data analysis were performed in blinded and randomized manner. 4.2 Preparation of MSC MSC was obtained from femur and tibia marrow of 4-week male SD rats (total 20) (An et al., 2017a). Briefly, after 0.5 h of UV sterilization of the room, the rats' femur and tibia were excised, rinsed 3 times with PBS containing 5% penicillin and streptomycin, and then transferred to the cell hood. The ends of the bone were cut with sterilized bone scissors and the marrow was slowly washed with a syringe containing DMEM/F12 (Gibco,12634028) 2-3 times into a cell culture dish placed on an ice pad. The dish was then placed into the incubator and whole DMEM/F12 was replaced at 24 h, following which half of the media was replaced every three 14

days. After approximately 10 days, the primary cells reached 80% confluence and were passaged to P1. The 3-5th generation of the cells were used for the present study. 4.3 Surgical preparation Anesthesia was induced and maintained using 1.5–3.5% enflurane in 70% nitrous oxide and 30% oxygen (Bickford veterinary anesthesia equipment model 61010; AM Bickford Inc., Wales Center, NY, USA). Rectal temperature was monitored throughout the procedure and was maintained at 36.5–37.5°C using a feedback-controlled heating blanket as needed. 4.4 Focal ischemic and therapeutic methods 1.5 h MCAO was induced using a similar approach to previously reported studies (Wu et al., 2019; Luan et al., 2004). Briefly, a modified PE-50 catheter was inserted into the right external carotid artery via an arteriotomy and passed up the lumen of the internal carotid artery into the intracranial circulation. The filament was lodged in the narrow proximal anterior cerebral artery (ACA) and blocked the MCA at its origin. 1.5 h after MCA occlusion, animals were reanesthetized and reperfusion was established by filament withdrawal. In LCI group, the catheter was withdrawn 1mm from the origin of the MCA. During and after the withdrawal, 3 ml of cold saline (4°C) was injected at the junction of the MCA and ACA at an infusion rate of 0.6 ml/min for 5 min. In MSC group, 5×105 MSC diluted in 0.2 ml phosphate buffered saline (PBS) was delivered with an infusion rate of 0.1 ml/min for 2 min. After infusion, the catheter was completely withdrawn and perfusion was re-established. 4.5 Infarct volume Infarct volume was evaluated at 24 h following reperfusion as previously described (Wu et al., 2017, Cai et al., 2016). Six coronal brain slices with a 2-mm thickness were cut for the treatment with TTC (Sigma, St. Louis, MO, USA) at 37°C for 20 min and then fixed in a 10% formalin solution. The percentage of infarct volume relative to non-infarcted area was calculated to minimize errors due to edema. 4.6 Neurological deficits 15

The 12-point scoring scale was used to valuate sensorimotor integration of forelimbs before surgery at 2 h after MCAO as well as 24 h after reperfusion (An et al., 2017b). Functional outcomes were evaluated by the accelerating Rota-rod test (motor coordination) and foot-fault test (sensorimotor function). Animals were pre-trained for 3 days, and performed at pre-MCAO and 1, 14, and 28 days post-MCAO by an independent investigator blinded to the experimental groups. The Rota-rod (R03-1, Xin Ruan Instruments, Inc., Shanghai, China) was used as a training platform in the present study (Shen et al., 2016, Shi et al., 2017). The Rota-rod is a horizontallyoriented, mechanically-driven cylinder upon which the animals were forced to run. The rod is 7 cm in diameter, 11 cm in length, and is covered with smooth rubber. The apparatus delivered an electric shock (0.1 mA, 3 s) to animals that fell from the rotating cylinder (5–30 rpm incrementally), forcing the animals to exercise continuously for the duration of the 0.5 h exercise period. For pre-conditioning, rats performed Rota-rod training at constant speeds of 15 rpm for 20 min/day for 3 days prior to MCA occlusion. Non-exercise controls and exercised animals were housed in groups of three in standard cages for equal amounts of time. The grid-walking apparatus was manufactured as previously described, using 4 cm2 square wire mesh with a grid area 120 cm x 40 cm x 50 cm (Clarkson et al., 2010). A mirror was placed beneath the apparatus to allow video footage to assess the animals’ stepping errors. Each rat was placed individually atop of the elevated wire grid and allowed to freely walk through the whole length of grid. Video footage was analyzed offline by raters blind to the treatment groups. The total number of foot faults for each limb in addition to the total number of non-foot fault steps were counted. 4.7 The SHSY5Y cell culture The SHSY5Y cell line was purchased from ATCC (CRL-2266, ATCC) and incubated in a 75‐cm2 culture flask (T75) containing 12–15 mL of DMEM/F12 (Olivieri et al., 2002). Briefly, the cells were centrifuged (1100rpm, 4°C, 4 min) after quick melting by 37°C water bath. 16

Resuspended cells with DMEM/F12 in a T75 and placed into a regular incubator. After 24 h, the cells were adhered to the bottom. The media was replaced every three days. After approximately 7 days, the cells reached 80% confluence and were passaged to P1. Generations 3-5 of the cells were used for experiments. 4.8 OGD and therapeutic methods OGD experiments were performed using an anaerobic chamber that was flushed with 5% CO2 and 95% N2 (v/v) kept at 37°C. To initiate OGD, culture medium was replaced with deoxygenated, glucose-free DMEM (Gibco, 11966025). After 2 h, SHSY5Y cells were removed from the anaerobic chamber and the OGD solution was replaced with maintenance medium and followed by different therapy. In HT group, the cells were placed into a 34°C incubator for 4 h before the regular incubator. In MSC group, transparent inserts were used (Corning, 353091, 353096) to achieve a co-culture system between MSC in the upper chamber and SHSY5Y cells in the lower chamber (Hayakawa et al., 2016). In the combination group, MSC inserts were added and then the whole plate was placed into a 34°C incubator for 4 h and then into the regular incubator. 4.9 Cell Viability Assay Cell viability was measured by cell proliferation reagent WST-1 (Roche, 05015944001). Transparent, 96-well plates with culture media (100 μl) were prepared and cell proliferation reagent WST-1 (10 μl) was added. The plates were incubated for 1 h in a regular incubator and shook for 1 min on a shaker. Absorbance signal was determined using a microplate reader at 450 nm. 4.10 ATP Measurement Intracellular ATP was determined by CellTiter-Glo luminescence kit (Promega, G7570), which can be performed by cell lysis and generate a luminescent signal proportional to the amount of ATP present. Opaque-walled 96-well plates with culture media (50 μl) or cell lysate (50 μl) were prepared. CellTiter-Glo luminescence test solution (50 μl) was added, and then the 17

plates were incubated for 30 min at room temperature. Luminescent signal was determined by luminescence microplate reader. 4.11 ROS Levels To measure reactive oxygen species (ROS) production, we used DCFH-DA (2,7dichlorodihydrofluorescein diacetate) (Sigma, D6883) as previously described (Azad et al., 2014). In brief, cultures were washed once with PBS and stained with 25 μM DCFDA in 100 μl PBS for 30 min at 37°C. Cultures were washed 3 times with PBS before signal reading at Ex/Em: 485/535 nm. 4.12 Protein expression Western blot analysis was used to detect expression of pro-apoptotic (BAX) and antiapoptotic (Bcl-2) proteins, as well as inflammatory (IL-1β) and neurotrophic factors (VEGF). Tissue samples from the ischemic cerebral hemispheres of all experimental and control groups were harvested at 6 and 24 h after reperfusion, as previously described (Geng et al., 2015). The protein samples were loaded onto gels for electrophoresis, and the proteins were transferred to a PVDF membrane. Membranes were incubated with primary antibodies including rabbit monoclonal anti-BAX antibody (1:500, Abcam), rabbit monoclonal anti-Bcl-2 antibody (1:500, Abcam), rabbit polyclonal anti- IL-1β antibody (1:400, Abcam), rabbit polyclonal anti-VEGF antibody (1:500, Santa Cruz), mouse polyclonal anti-β-actin antibody (1:1200, Santa Cruz), mouse monoclonal anti-Miro1 antibody (1:500, Abcam) at 4°C for 24 h. Subsequently, the membranes were washed three times with 1% TBST for 10 min each and re-incubated with the relevant secondary antibody (goat anti-rabbit IgG, Santa Cruz) for 1 h at room temperature. Equal protein loading was adjusted using β-actin. An ECL system was used to detect immunoreactive bands by luminescence. Quantification of relative target protein expression was obtained using ImageJ 1.48 (National Institutes of Health, USA). 4.13 Statistical analysis

18

All values are expressed as means ± Standard Error (SE). Statistical analyses were performed using SPSS, version 16.0 (SPSS, Inc.). The differences between groups were assessed using 1-way analysis of variance (ANOVA), a 2-way ANOVA, or a student’s t-test with a significance level set at P<0.05. Post-hoc comparison between groups was conducted using Tukey or Bonferroni post-tests.

Disclosures None.

Acknowledgments This work was supported by National Key R&D Program of China (2017YFC1308401); Chang Jiang Scholars Program (#T2014251) from the Chinese Ministry of Education; National Natural Science Foundation of China (81701287;81871022;81500997); Beijing Municipal Administration of Hospitals Clinical Medicine Development of Special Funding Support （ZYLX201706）; The “mission” talent project of Beijing Municipal Administration of Hospitals (SML20150802);

Beijing

Municipal

Administration

of

Hospitals’

Youth

Programme

(QML20170802); and Ten Thousand Talent Program.

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2017.

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the

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Figure legend Figure 1. Infarct volume and deficit scores were reduced in treatment groups at 24 h. The sham group in each test served as a reference and was set to 1.0. (a, b) Compared with the stroke group (control), TTC histology demonstrated significant infarct volume reductions in LCI, MSC, and combination therapy groups. (c) LCI, MSC, and combination therapy all produced significant reductions in neurological deficits as assessed using the 12-point deficit score system. Combination therapy provided no additional reduction over monotherapy in this test of early outcomes. (d, e) There was no significant difference in infarct volume and 12 scores between the early combination group (LCI Day 0 + MSC Day 0) and the late combination group (LCI Day 0 + MSC Day 1) at 24 h (Data were shown as mean ± SE. One-way analysis of variance (ANOVA) followed by Tukey’s method were used for comparisons among and between groups. N= 8-10 in each group. *P<0.05, **P<0.01 vs control). Figure 2. Effect of LCI, MSC, and combined therapy on neurological function. Neurobehavioral analyses by the Rota-rod test and foot-fault test were performed at pre- (Day 0) and post-MCAO (Day 1-28). Compared with the control group, LCI, MSC and combination groups showed an increase in latency period to fall assessed by the Rota-rod test (a*) and a decrease in fault steps detected by foot-fault test (b*) at Day 14 and 28. Compared with either monotherapy group, the combination group significantly improved long-term neurological deficit assessed by the Rota-rod tests (a#) at Day 28, and foot-fault test (b#) at Day 14 and 28. Compared with the early combination group (LCI Day 0 + MSC Day 0), the late combination group (LCI Day 0 + MSC Day 1) showed a significant increase in running time by the Rota-rod test (c*) at Day 14 and 28 and a decrease in fault steps by the foot-fault test (d*) at Day 28 (Data were shown as mean ± SE. Two-way ANOVA followed by Bonferroni post-tests for comparisons of multiple groups. N= 8 in each group; *P<0.05, **P<0.01 vs control, #P<0.05, ##P<0.01 vs the combination group). Figure 3. Inflammatory and apoptotic protein expression with LCI and/ or MSC therapy at 6 h and 24 h. The sham group in each test served as a reference and was set to 1.0. ANOVA analyses indicated a significant increase in IL-1β and BAX levels after ischemic stroke at both 6 h and 24 h of reperfusion (P<0.05, a-d). The ischemia-induced increase in IL-1β and BAX expression was significantly reversed by LCI, MSC and combination therapy at both 6 h and 24 h of reperfusion(a-d*). Further, the combination group had a greater reduction in IL-1β and BAX level than either monotherapy at 24 h (b, d#). Conversely, there was a significant decrease in Bcl-2 and VEGF levels after ischemic stroke at 6 and 24 h of reperfusion (P<0.05, e-h). The ischemia-induced decrease in Bcl-2 and VEGF expression was significantly reversed by LCI and/ or MSC therapy at both 6 h and 24 h of reperfusion, excluding VEGF expression treated by LCI therapy at 6 h (e-h*). In addition, the combination group had an enhanced expression in Bcl26

2 and VEGF proteins compared with either monotherapy at 6 and 24 h, excluding comparison with MSC therapy in VEGF expression at 6 h (e-h#) (Data were shown as mean ± SE. One-way analysis of variance (ANOVA) followed by Tukey’s method. N= 8 in each group. *P<0.05, **P<0.01, ***P<0.001 vs control, #P<0.05, ##P<0.01, ###P<0.001 vs the combination group). Figure 4. Cell viability, and ROS and ATP level in HT, MSC, and combined therapy after OGD. The sham group in each test served as a reference and was set to 1.0. ANOVA analyses indicated a significant decrease in cell viability and ATP level after OGD at both 6 h and 24 h of reperfusion (P<0.05, a-d). The OGD-induced decrease in cell viability and ATP were restored by HT, MSC, and HT+MSC therapy at 6 h (a, c*) and 24 h (b, d*) after reperfusion. At 24 h, the combination group showed an even greater increase in cell viability and ATP level compared with the monotherapy groups (b, d#). ROS level was increased after OGD (P<0.05, e-f) and decreased by treatment (e, f*). At 24 h, the combination group showed a more significant decrease in ROS level than the monotherapy groups (f #) (n=9 from n=3 biological replicates, n=3 independent experiments; data were represented as mean ± SE, one-way ANOVA followed by Tukey’s test; *P<0.05, **P<0.01, ***P<0.001 vs OGD, #P<0.05, ###P<0.001 vs the combination group). Figure 5. Inflammatory and apoptotic protein expression with HT and/ or MSC therapy at 6 h and 24 h after OGD. The sham group in each test served as a reference and was set to 1.0. ANOVA analyses indicated a significant increase in IL-1β and BAX levels after OGD at both 6 h and 24 h of reperfusion (P<0.05, a-d). At 6 h, the OGD-induced increase in IL-1β was significantly reversed in the HT and combination groups (a*), while the BAX expression was significantly reversed by HT as well as MSC therapy (c*). At 24 h, the OGD-induced increase in IL-1β and BAX expression were significantly reversed by HT, MSC and the combination therapy (b, d*). Furthermore, the combination group had a greater reduction in IL-1β and BAX level than either monotherapy (b, d#). Conversely, there was a significant decrease in Bcl-2 and VEGF levels after OGD at both 6 h and 24 h of reperfusion (P<0.05, e-h). At 6 h, the OGD-induced decrease in Bcl-2 and VEGF levels were significantly reversed by the MSC and the combination groups (e, g*). At 24 h, the OGD-induced decrease in Bcl-2 and VEGF expression was significantly reversed by HT, MSC, and combined therapy (f, h*). In addition, the combination group had an even greater expression in Bcl-2 and VEGF levels than either monotherapy (f, h#) (Data were shown as mean ± SE. One-way analysis of variance (ANOVA) followed by Tukey’s method. N = 8 in each group. *P<0.05, **P<0.01, ***P<0.001 vs control, #P<0.05, ##P<0.01, ###P<0.001 vs the combination group). Figure 6. MSC mitochondrial transfer into SHSY5Y cells and Miro1 level at 24 h after OGD. (a) Confocal microscopy revealed MSC mitochondria (red, MitoTracker Red CMXRos) transferred into soma and axon (b) of SHSY5Y cells (nuclear, blue, DAPI) in the MSC and 27

SHSY5Y cell co-cultured group. (c) Transferred mitochondrial density in neuronal cells was significantly increased by HT (P<0.001) (n= 45 or 50 somas from n=2 biological replicates, n=3 independent experiments were counted; scale bars: 10 μm). (d) Expression of Miro1 was enhanced by HT at 24 h (P<0.01) (n=6 from n=3 biological replicates, n=3 independent experiments; data were represented as mean± SE, unpaired t test was used).

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Highlights 

Hypothermia enhances the neuroprotective effect of MSC therapy, especially in longterm functional recovery after stroke.

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Hypothermia could increase mitochondrial transfer from MSC to neurons via Miro1 upregulation, elevate Bcl-2 and VEGF as well as decrease IL-1β and BAX expression.

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The novel combination therapy of hypothermia and MSC administration has revealed potential insights in stroke pathology and may advance the clinical therapy of ischemic diseases.

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