Topically applied adipose-derived mesenchymal stem cell treatment in experimental focal cerebral ischemia

Journal of Clinical Neuroscience xxx (xxxx) xxx

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Experimental study

Topically applied adipose-derived mesenchymal stem cell treatment in experimental focal cerebral ischemia Ping Kuen Lam a,b, Kevin Ka Wang Wang c, Don Wai Ching Chin a,b, Cindy See Wai Tong a,b, Yixiang Wang d, Kin Ki Yan Lo a,b, Paul Bo San Lai a,b, Hui Ma a,b, Vera Zhi Yuan Zheng a,b, Wai Sang Poon a,b, George Kwok Chu Wong a,b,⇑ a

Department of Surgery, The Chinese University of Hong Kong, Hong Kong Special Administrative Region Chow Tai Fook-Cheng Yu Tung Surgical Stem Cell Research Center, The Chinese University of Hong Kong, Hong Kong Special Administrative Region c Program for Neurotrauma, Neuroproteomics & Biomarkers Research, Departments of Psychiatry, McKnight Brain Institute, University of Florida, Gainesville, FL, USA d Department of Imaging and Interventional Radiology, The Chinese University of Hong Kong, Hong Kong Special Administrative Region b

a r t i c l e

i n f o

Article history: Received 11 June 2019 Accepted 6 August 2019 Available online xxxx Keywords: Cerebral ischemia Experimental model Mesenchymal stem cell Neurosurgery Stroke

a b s t r a c t In this study, the neuro-modulation effect of topical mesenchymal stem cells (MSCs) was tested in a rodent middle carotid artery occlusion (MCAO) model. Twenty-four hours after MCAO, craniotomy was made and 0.8  106 GFP-MSCs were topically applied to the exposed parietal cortex. The MSCs were fixed in position by a thin layer of fibrin glue (N = 30). In the control group, saline were topically applied to the ipsilateral parietal cortex (N = 30). Three days after topical application, few GFP-positive cells were found in the ischemic penumbra. They expressed GFAP and NeuN. Topical MSCs triggered microglial activation, astrocytosis and cellular proliferation at day 3. The recovery of neurological functions were significantly enhanced as determined in Rotarod test and Morris Water Maze test with smaller infarct volume. PCR array showed that expressions of ten genes of neurogenesis were altered in the penumbra region (fold change > 1.25, p < 0.05) in MSCs group: Apoe, Ascl1, Efnb1, Mef2c, Nog, A100a6 and B2m were upregulated; Pax2, Pax3 and Th were down-regulated. In conclusion, topical application provided a direct and effective transplant method for the delivery of MSCs to the surface of ipsilateral cerebral cortex and the topical MSCs could improve the neurological function from cerebral ischemia resulting from a major cerebral artery occlusion in a rodent experimental model. Ó 2019 Elsevier Ltd. All rights reserved.

1. Introduction Mortality after stroke is high, with stroke ranked as the third most common cause of death in developed countries after ischemic heart disease and malignancy, and stroke is the fourth most common cause of death in Hong Kong. Disability after severe stroke from a major cerebral artery occlusion is common and is associated with prolonged hospital stay and rehabilitation, and these are definite burden for resources to cater for the patient’s long-term care [1]. Despite the recent adoption of prophylactic decompressive craniectomy within 48 h to counter the deleterious effect of cerebral edema after hemispheric infarction (typically middle cerebral artery territory infarction and cerebellar infarction), the rates of death and disability remained high [1–3]. Pathologically, cerebral ⇑ Corresponding author at: 4/F Lui Che Wo Clinical Sciences Building, Department of Surgery, Prince of Wales Hospital, 30-32 Ngan Shing Street, Shatin, Hong Kong Special Administrative Region. E-mail address: [email protected] (G.K.C. Wong).

ischemia progresses to neuronal and glial cell death, resulting in infarction. To date, there is no effective treatment to reverse or replenish these damaged cells. The ability of mesenchymal stem cells (MSCs) to undergo prolonged self-renewal and differentiate into different cell lineages provides the possibility of their use in regenerative medicine [4,5]. Besides, MSCs also execute their reparative effects through paracrine signaling by secreting various trophic and immunomodulatory factors that suppress inflammation and apoptosis, enhance angiogenesis, stimulate proliferation and cellular differentiation [6,7]. Large amount of MSCs could be obtained from adipose tissue easily with limited invasiveness, and the cells grow fast for transplantation [8,9]. Experimental studies showed that intracerebral injection and intravenous infusion of MSCs improved neurological recovery in focal cerebral ischemia [10–12]. Homing of MSCs to its target organ is a prerequisite for cell therapy to differentiate into desirable cell types and to execute their biological functions. Although a large amount of MSCs can be infused intravenously, most of them are trapped in the

https://doi.org/10.1016/j.jocn.2019.08.051 0967-5868/Ó 2019 Elsevier Ltd. All rights reserved.

Please cite this article as: P. K. Lam, K. K. W. Wang, D. W. C. Chin et al., Topically applied adipose-derived mesenchymal stem cell treatment in experimental focal cerebral ischemia, Journal of Clinical Neuroscience, https://doi.org/10.1016/j.jocn.2019.08.051

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P.K. Lam et al. / Journal of Clinical Neuroscience xxx (xxxx) xxx

pulmonary capillaries, producing pneumonitis and reducing the overall efficiency of the cell transplantation procedure [13]. Intracerebral injection of MSCs through Hamilton syringe allows MSCs delivered to the injured site but the method itself induces additional brain damage. The intra-arterial application increased the cerebral lesions and failed to improve functional recovery in a rodent model [14]. Most importantly, without repeated injections, the injectable stem cell volume is limited. Because of the aforementioned disadvantages in these methods of delivery, we have developed topical application of adipose-derived MSCs and tested in ischemia-reperfusion injury experimental models of liver and kidney [15], as well as traumatic brain injury experimental model [16]. Topical MSC treatment is appealing with the current recommendation of early decompressive craniectomy after middle cerebral artery infarction and cerebellar infarction, which provides an ideal window of opportunity to apply topical cell transplantation therapy at the same surgical procedure. In this rodent study of focal cerebral ischemia, we aimed to investigate the neuromodulation effects of adipose-derived MSCs delivered in a single procedure by topically application.

2.2. Animal experiments 2.2.1. Focal cerebral ischemic model Female SD rats (250–300 g) were anesthetized with intraperitoneal administration of ketamine (50 mg/kg) and xylazine (10 mg/kg). The right carotid bifurcation was exposed. A 40-mm 3–0 nylon monofilament with a heat-blunted spherical tip was advanced from external carotid artery (ECA) to the lumen of internal carotid artery (ICA) until it blocks the origin of the middle carotid artery (MCA) [17]. After transient MCA occlusion (MCAO) for 75 min, the monofilament was gently withdrawn, the ligature around the ECA is tightened, perfusion was restored, and the wound was closed. Rectal temperature of the animals was

2. Methods The animal experiment was performed in accordance with the guidelines of the Animals (Control of Experiments) Ordinance Chapter 340, Department of Health, Hong Kong and was approved by the Animal Experimentation Ethnics Committee of the Chinese University of Hong Kong.

2.1. Adipose derived mesenchymal stem cells (MSCs) 2.1.1. Cultivation of adipose tissue-derived mesenchymal stem cells (MSCs) The MSCs used in this study were derived from the subcutaneous adipose tissue of male transgenic Sprague-Dawley (SD) rats with green fluorescence protein (GFP) expression. Briefly, the adipose tissue was washed with phosphate buffered saline (PBS) and treated with 0.1% collagenase (type I; Sigma-Aldrich) for 30 min at 37 °C. After the enzymatic digestion, the cell suspension was filtrated through 100-lm mesh filter to remove debris. Then the filtrate was washed and resuspended in Dulbecco’s modified Eagle’s medium (DMEM) which was supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 lg/ml streptomycin, and 2 mM L-glutamine. The MSCs were cultivated in a humidified 5% CO2 incubator.

2.1.2. Characterization of adipose tissue-derived MSCs 2.1.2.1. Cell phenotyping. The cell phenotype of MSCs was characterized with flow cytometer (BD Bioscience, San Jose, CA). MSCs were stained with phycoerythrin-conjugated antibodies against CD45 and CD90 (Abcam Inc., Cambridge, UK) and CD29 (Biolegend, San Diego, CA). Isotype-matched negative controls were used to balance the background fluorescence.

2.1.2.2. Adipogenic, chondrogenic and osteogenic differentiation potential. MSCs were cultured with adipogenic chondrogenic and osteogenic differentiation culture media (Invitrogen, Life TechnologiesTM). The differentiated adipocytes were identified with Oil Red O. Alcian Blue and Alizarin Red S were used to stain the extracellular glycosaminoglycans in chondrocytes and calcium deposits in osteocytes

Fig. 1. 7 days after primary culture of rat adipose derived MSCs culture showed a spindle-shaped morphology (A), phase-contrast microscopy, 100). Flow cytometric analysis of surface markers expression on adipose derived MSCs (CD29, CD90 and CD45) of the MSCs population. The MSCs were cultured in Dulbecco’s modified Eagle’s medium with 10% FBS. CD29 and CD90 surface markers showed 64.3% and 68.8% respectively. CD45 surface marker showed 0.05%. (B).

Please cite this article as: P. K. Lam, K. K. W. Wang, D. W. C. Chin et al., Topically applied adipose-derived mesenchymal stem cell treatment in experimental focal cerebral ischemia, Journal of Clinical Neuroscience, https://doi.org/10.1016/j.jocn.2019.08.051

P.K. Lam et al. / Journal of Clinical Neuroscience xxx (xxxx) xxx

maintained at 37 °C with a warming pad throughout the experiment. Ointment was applied to protect their eyes.

2.2.2. Radiological assessments after MCAO Six hours after MCAO, the rats were anesthetized with a mixture of ketamine (50 mg/kg) and xylazine (10 mg/kg). Each rat was placed in an animal holder/MRI probe apparatus and the rat’s head was hold in placed inside the imaging coil. T2 weighted images (T2WI) were measured by a MR system (1.5 T Siemens Sonata, Erlangen, Germany) using echo time (TE) = 100 ms, repetition time (TR) = 2.0 s, FOV = 40 mm. The ischemic lesion area was marked if the signal intensity was 1.25 times higher than that of contralateral side of the brain [18]. The infarction volume was calculated by a summation of image slice area times thickness. Rats with lesion volume greater than 100 mm3 were selected for the experiments. The infarct volumes were measured at days 3, 7, and 14.

2.2.3. MSCs transplantation Twenty-four hours after the occlusion, sixty eligible rats were randomized into two groups. The skull of all rats was exposed after a cranial incisions. A circular (4 mm) craniotomy and durotomy on the right side (same) were conducted. 0.8  106 MSCs were topically applied to ipsilateral parietal cortex (N = 30). The MSCs were fixed in position by a thin layer of fibrin glue (Tisseel, Baxter Healthcare SA, Wallisellen, Switzerland). In the control group, saline was added to the ipsilateral parietal cortex (N = 30).

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2.3. Neurological assessments 2.3.1. Rotarod training assessment Before MCAO, all rats were trained for 3 days to stay on an linearly accelerating, computer-driven rotating, horizontal cylinder (Stoelting Company, USA) until they remained on the rotarod for 100 s. There were ten animals in each group. The degree of motor impairment was studied by measuring the time the rats stayed on the rotarod with speed increasing from 10 to 30 rpm in 100 s. The time (in seconds) of animal remained on the accelerating rotating rod in each of the trials was recorded at days 3, 7, and 10 post transplantation. 2.3.2. Morris water maze The visuospatial learning and memory were examined by Morris water maze (ANY-mazeTM, Stoelting Company, USA) which consisted of a circular water pool, 1.8 m in diameter, and a submerged 9-cm-diameter platform in one quadrant. Ten rats in each group were trained to find the platform daily after treatment from day 1 to day 10. The distance travelled was recorded as parameter. At day 11, the platform was taken away and the visuospatial memory was assessed by the examination of the swimming path (probe test). 2.4. Microscopic examination At days 3, 7, and 14, animals were sacrificed for microscopic examinations, a series of coronal paraffin sections of 4-um in

Fig. 2. Few GFP-positive cells (marked with arrow) were seen at the periphery of the infarct 3 days after topical application (A, ICH 100; B H&E 100). Under fluorescence microscope, MSCs pre-labelled with CM-DIL (red) expressed markers of Neuronal nuclei (NeuN, red) and Glial fibrillary acidic protein (GFAP, red). Nuclei were stained with DAPI (blue) (C, D, IFS 400). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Please cite this article as: P. K. Lam, K. K. W. Wang, D. W. C. Chin et al., Topically applied adipose-derived mesenchymal stem cell treatment in experimental focal cerebral ischemia, Journal of Clinical Neuroscience, https://doi.org/10.1016/j.jocn.2019.08.051

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thickness were collected from the center of the lesion (bregma 1 to +1 mm). Homing and migration of MSCs were detected by antiGFP (Abcam, 1:5000) antibody staining. For the determination of cellular differentiation, MSCs from wild-type SD rats were labeled with red fluorescence CM-DIL (Life Technologies). The co-expressions of CM-DIL with FITC conjugated anti-GFAP and anti-NeuN (Millipore) were examined under conventional fluorescence microscope. Immunohistochemistry staining of anti-GFAP, anti-Iba1 (Abcam), PCNA (Wako) antibodies were performed. Cresyl Violet staining was performed to assess the neuronal death. 2.5. PCR array related to neurogenesis At day 3, eighty-four neurogenesis genes were analyzed using RT2 ProfilerTM PCR Array (Qiagen). RNA was extracted from the penumbral cortex (with estimated 2 mm zone around the border of infarct), empirically amplified and undergone RT-PCR according to the manufacturer’s protocol.

256 ± 62 mm3 at day 3; 99 ± 14 mm3 vs 130 ± 12mm3at day 7; 51 ± 3 mm3 vs 73 ± 12 mm3 at day 14), when compared with the control. 3.5. Microscopic examinations Microscopic examination was focused in five different sites of the penumbra which was the brain parenchyma, 2 mm from the peripheral of the infarct. Five different sections of each brain sample were examined microscopically. In the immunochemistry staining, the number of Iba1 + ve cells and GFAP + ve cells were counted in five different high-power field per section. Immunohistochemistry staining showed more Iba1 + ve cells (marker of microglia) in the penumbra of topical MSC treatment group at day 3 than the control group (p < 0.01) (Fig. 3A). There was no significant difference in Iba1 expression at day 7 and day 14. The number of GFAP + ve cells (a marker of astrocyte) was significantly higher at day 3 (p < 0.05), day 7 (p < 0.05) and day 14 (p < 0.001)

2.6. Statistical analyses Numerical results were expressed as mean ± standard deviation. The histology examination and RT-PCR results were analyzed statically with IBM SPSS Statistics. The improvement of the behavior recovery was evaluated using group-based ANOVA and pairwise Turkey test. PCR array data were exported to an Excel file to create a table of CT values. They were then analyzed with web portal (http:www.quiagen.com/genelobe). p < 0.05 was considered statistically significant. 3. Results 3.1. MCAO rodent model We performed a total of 70 rodent MCAO experiments. Among these rats, 8 died within 24 h and 2 had lesion volume less than 100 mm3. These 10 rats were thus excluded for further analyses. Sixty rats had eligible large territory cerebral infarction for recruitment and randomized into topical MSC treatment group (n = 30) and control group (n = 30). 3.2. MSC characterization The MSCs demonstrated spindle-shape morphology (Fig. 1A) and were adherent to the plastic culture flasks (Fig. 1A). Under specific environments, the MSCs could differentiate into adipocytes, chondroblasts and osteoblasts (data not shown). In flow cytometric analysis, the MSCs expressed CD29 and CD90, and were negative for CD45 (Fig. 1B). 3.3. Homing of topical MSCs Animals were sacrificed at days 3, 7 and 14. Three days after topical application, few GFP positive cells were found in the penumbra of the infarct (Fig. 2A, B). They co-expressed NeuN and GFAP in immunofluorescence staining (Fig. 2C, D). 7 days post topical application of MSCs, no viable GFP-positive cells were observed in the brain parenchyma. No GFP-positive cells were detected in other somatic organs including the lungs (data not shown) throughout the study. 3.4. Reduction of infarct volume Upon topical application of MSCs, there was a significant difference (p < 0.05) of the infarct volumes (163 ± 20 mm3 vs

Fig. 3. Representative pictures of IHC staining with Iba-1, GFAP and PCNA antibodies (A, B, C). (A, B) On day 3 after treatment, there were more Iba-1 positive and GFAP positive cells in penumbra than control group under 400 microscope. (C) More cell proliferation was observed in penumbra after MSCs treatment on day 3 (400). (D) Cresyl violet staining was performed to detect cell death. It showed less cell death in penumbra after MSCs treatment on day 3. The arrow indicated normally living cells after C.V. staining under 400 microscope.

Please cite this article as: P. K. Lam, K. K. W. Wang, D. W. C. Chin et al., Topically applied adipose-derived mesenchymal stem cell treatment in experimental focal cerebral ischemia, Journal of Clinical Neuroscience, https://doi.org/10.1016/j.jocn.2019.08.051

P.K. Lam et al. / Journal of Clinical Neuroscience xxx (xxxx) xxx

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(Fig. 3B). Total number of PCNA + ve cells was measured. More proliferative cells were observed at day 3 (Fig. 3C). Moreover, more viable neuronal cells were found at the infarct of MSC-treated group at day 14 (Fig. 3D). Compares with the control group, there were less apoptotic cells in the penumbra of MSC-treated group at days 3, 7, and 14, but the difference was not statistically significant (p > 0.05).

regulated (Fig. 5B). These genes were related to neuronal migration (Ascl1); neuronal differentiation (Asc1, Mef2C, Nog); neural cell fate determination (Ascl1); regulation of synaptic plasticity (Apoe); synaptic transmission (Apoe, Th) axonogensis (S100a6); growth factors (S100a6); cell adhesion (Efnb1) and apoptosis (Pax2, Pax3). Using the Gene Network CentralTM provided by Qiagen, a molecular pathway network was predicted. (Fig. 5C)

3.6. Neurological assessments

4. Discussion

In the Rotarod test, MSCs-treated rats demonstrated better recovery of balance and motor function after MCAO with a longer latency to fall from the accelerating motor rod at days 3, 7, and 10 as compared to the control group (Fig. 4A). In the Morris Water Maze test, the MSCs-treated rats required a shorter learning period (day 3 vs day 10 in the control group) as they took a shorter swimming path to reach the platform. (Fig. 4B). During the probe test (the platform was removed) at day 11, the rats treated with topical MSCs kept staying in the quadrant at least for 7 s, where the platform was originally placed (Fig. 4C). In contrast, the control group did not show preference on any particular quadrant of the water maze (Fig. 4D).

Ischemic stroke occurs when the cerebral blood flow is occluded by thrombus or embolism. Cerebral ischemia not only destroys neurons and glial cells, but also leads to the neuroinflammation, which involves the interaction of activated resident cells including microglia, astrocytes and endothelial cells [18]. The early prophylactic decompressive craniectomy does not alleviate the detrimental effects of cerebral edema after hemispheric infarction. In fact, the rates of death and disability of ischemic stroke remain high [2,19]. In addition to the self-renewal and differentiation abilities, MSCs potentially work through multiple mechanisms of immunological, inflammatory, vascular and regenerative pathways in contrast to the pharmacological agents that usually target only a single event or pathway in the pathophysiology of a given disease. MSC application has emerged as a promising neurorestorative strategy to reduce brain damage and facilitate neurological function recovery. Recent experimental studies have also suggested that intracerebral injections or intravenous infusions of MSCs improved neurological recovery in focal cerebral ischemia [10–12,20–22]. Clinical studies have shown that intravenous infusion of MSCs to patients with ischemic stroke was safe but long-term clinical outcomes need further investigation [23,24]. One of the hurdles to stem cell therapy for the neurologic disorders is the lack of an effective and minimally invasive

3.7. Gene expression profile The expression of 84 genes commonly associated with neurogenesis was studied using PCR array on day 3 samples (Fig. 5A). The expressions of 10 genes in the penumbra were significantly altered in the topical MSCs group (fold change > 1.25, p < 0.05). Apolipoprotein E(Apoe), Achoete-scute complex homolog 1(Ascl1), Ephrin B1(Efnb1), Myocyte enhancer factor 2C (Mef2c), Noggin (Nog), S100 calcium binding protein A6(S100a6) and Beta-2 macroglobulin (B2m) were up-regulated. Paired box 2(Pax2), Paired box 3 (Pax3) and Tyrosine hydroxylase (Th) were down-

Fig. 4. Animal behavioral tests after MCAO. Parameters were shown in mean ± standard deviation. MSCs-treated rats stayed longer in the accelerating motor rod (A) and took a shorter swimming distance before arriving at the hidden platform (B); kept staying in the quadrant where the platform was originally placed (C). In contrast, the control group could not find the location and randomly swan in the water tank (D).

Please cite this article as: P. K. Lam, K. K. W. Wang, D. W. C. Chin et al., Topically applied adipose-derived mesenchymal stem cell treatment in experimental focal cerebral ischemia, Journal of Clinical Neuroscience, https://doi.org/10.1016/j.jocn.2019.08.051

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Fig. 5. Results of PCR-array for genes related to neurogenesis in the penumbra cortex with topical MSCs vs the control (A). Seven genes were up-regulated, and three genes were down-regulated (fold change >1.25; p < 0.05) (B). The network formed by the ten genes which was predicted for molecular mechanisms under topical MSCs treatment for MCAO. Square genes associated to the network but not examined by the PCR-array (C).

transplantation method. Although systemic delivery of ex vivo expanded MSCs has been considered relatively safe, less than 0.001% of infused MSCs homed to the brain because most of the them were captured by pulmonary capillaries [25]. We did not intravenously transplant MSCs because our previous studies (unpublished) showed that approximately 25% rats died immediately after systemic infusion of equivalent amounts of MSCs via tail vein. Severe pneumonic reactions to the trapped MSCs were observed in the lung. The additional invasiveness and risk of intra-cerebral injection and intra-arterial infusion were considered a major obstacle to repeated injections and hence limited the total stem cell volume injectable and thus these methods were not pursued. Nevertheless, the possible benefit of bypassing the pulmonary circulation was suggested by pilot studies using intraarterial injections of bone marrow mononuclear cells with a dose-outcome correlation [26,27]. Along a similar line to bypass the pulmonary circulation and to take advantage of the current standard treatment of decompressive craniectomy for patients with middle cerebral artery territory infarction and cerebellar infarction, we developed the techniques of topical transplantation of MSCs to brain parenchyma that was based on our previously transplantation technique to deliver cultured epidermal skin graft to burn wound/ chronic wounds [28].

Fibrin glue has been long used as a matrix hemostatic sealant in various areas of surgery. Fibrin enhances the anchorage of MSCs to the recipient surface. It acts as a template for the MSC proliferation and allows a slow release of MSCs. Topical MSC treatment is feasible during decompressive craniectomy and no additional invasive procedure is required. The exact underlying mechanism for the topical MSCs to home the penumbra remains to be determined. Only a few MSCs were found in the peri-infarct area at day 3 but not at days 7 and 14 after topical MSC treatment, suggesting that the homed MSCs did not survive in the hypoxic, nutrient-poor, inflammatory microenvironment after homing to the infarct penumbra. The neurological assessment improvement was not accountable by MSC engraftment and differentiation. This is also in line with previous studies that the positive therapeutic effect of short-lived stem cells might be related to an increase in trophic factors and the subsequent trophic factor expression [29,30]. Topical MSCs treatment improved motor coordination, balance, visuospatial learning and memory, and reduced infarct volume as compared to the control group. Microglia are the major resident macrophages of central nervous system. Upon ischemic stroke, microglia in the penumbra are activated. However, microglia have a dual role after acute neural disorders [31]. They produce proinflammatory cytokines, toxic metabolites (free oxygen radicles) and matrix metalloproteinases, which disrupt the blood brain barrier [32]. On the other hand, they also release anti-inflammatory cytokines and neuroprotective factors such as insulin-like growth factor, TNF-alpha. The phagocytic function of activated microglia is crucial for the remodeling of the necrotic tissue after ischemia injury [33]. In this study, more microglia were found in the MSCs-treatment group at day 3 but not at days 7 and 14, as compared to control group. Previous studies have showed that activated microglia may lead to the subsequent activation of astrocytes following the injury in central nervous system and might explain the mechanism of astrocytosis after topical MSC treatment in our study [34,35]. Although in early stage after stroke, reactive astrocytosis in response to neuro-inflammation may aggravate the ischemia, it contributes to neuro-protection through limitation of ischemia extension and secretion of neurotrophies [36]. Similarly, during the late phase, the glial scar formation blocks axonal regeneration, but the astrocytes also contribute to angiogenesis, neurogenesis, synaptogenesis, and axonal remodelling [37]. The infarction volume was smaller in the topical MSC treatment group as compared to the control group although the number of apoptotic cells in the penumbra showed no significant difference in MSC-treatment group and control group. The reduction in infarct volume and less neuronal death as confirmed by Cresyl Violet staining suggested that at least part of the action of topical MSC treatment was through neuroprotection. There were ten genes whose expression significantly showed more than 1.25-fold increase or decrease in the penumbra of MSCs treatment group. The seven up-regulated genes (Apoe, Ascl1, Efnb1, Mef2c, Nog, S100a6, B2m) involve neuronal migration, neuronal differentiation, neuronal cell fate determination, regulation of synaptic plasticity, axonogenesis, growth factors, and cell adhesion. Pax2, Pax3 and Th were downregulated. Pax2 and Pax3 are related to apoptosis. Both Apoe and Thl involve synaptic transmission. Apoe, the major lipid transport protein in the brain, plays a key role in the assembly and stabilization of synaptic connection [38]. Thl is the rate-limiting enzyme in the biosynthesis of catecholamine neurotransmitters [39]. The balance of the roles of Apoe and Thl in the synaptic transmission in this study remains to be determined. This study has limitations. Firstly, our study did not directly compare topical application to other routes of applications such as intravenously, intra-arterially, or intra-cerebrally. Secondly, we

Please cite this article as: P. K. Lam, K. K. W. Wang, D. W. C. Chin et al., Topically applied adipose-derived mesenchymal stem cell treatment in experimental focal cerebral ischemia, Journal of Clinical Neuroscience, https://doi.org/10.1016/j.jocn.2019.08.051

P.K. Lam et al. / Journal of Clinical Neuroscience xxx (xxxx) xxx

did not explore the dose relationship or whether repeated treatments could further enhance the neurological recovery. We empirically selected the maximal dose volume that was considered feasible to apply topically. Thirdly, we did not study the impact of the time of application. We selected 24 h after the MCAO to topically apply MSCs as we considered this would be the equivalent time window for decompressive craniectomy in hemispheric infarction patients. Fourthly, we used a rat model and transient MCAO, and results could differ between species or between transient and permanent vessel occlusion [10]. We did not include a fibrin control group in this study because fibrin applied alone to the cerebral cortex did not enhance the neuro-recovery in our previous study. In our current study, for the topically applied MSC group, fibrin glue was applied together with MSCs on the cortex as a slow release carrier and an adhesive agent [40], and the fibrin glue was not injected intracerebrally or used as scaffold for neural regeneration. The impression of the neutral effect of topically applied fibrin glue should be formally assessed in future study. Fifthly, our focal cerebral ischemia model with transient MCA occlusion (MCAO) for 75 min was only one of the commonly employed models. There was another reported malignant cerebral infarction model with a 35% mortality rate [41]. The model should be ideal for assessment of the effects of topically applied MSCs with or without decompressive craniectomy. Sixthly, a more extensive battery of functional tests might be needed to differentiate between function recovery and compensation [42]. Seventhly, we did not carry out quantitative analyses on topically applied MSCs homed to the penumbra. The impression was that less than 0.01% of MSCs would be in the penumbra.

5. Conclusion In conclusion, topically applied MSCs reduced cerebral infarction volume and improved the neurological function from cerebral ischemia in a rodent MCAO model mimicking severe stroke due to a major cerebral artery occlusion. Topical MSC treatment should be further investigated and optimized for safety and efficacy in severe stroke due to major cerebral artery occlusion.

Disclosure The abstract of this paper was presented at the International Stroke Conference 2017 as a poster presentation. The poster’s abstract was published in ‘poster abstracts’ in Journal of Stroke: https://www.ahajournals.org/doi/abs/10.1161/str.48.suppl_1.tp93.

Author contribution statements Wong GK, Lam PK, and Wang K conceived the study. Lam PK, Chin DW, Tong CS, Wang YX, Kwong ST, Lo KK, Ma Hui, Zheng VZ performed the experiments. Wang K, Poon WS, Lai PB, and Wong GK supervised the study. Lam PK and Wong GK wrote the manuscript. Lam PK and Don DW prepared the figures. All authors reviewed and commented on the manuscript. All authors approved the final version of the manuscript.

Funding source The study was supported by the Health and Medical Research Fund (HMRF01120666) of the Food and Health Bureau of Hong Kong and Chow Tai Fook-Cheng Yu Tung Surgical Stem Cell Research Centre of the Chinese University of Hong Kong.

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Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.jocn.2019.08.051. References [1] Balami JS, Chen RL, Grunwald, Buchan AM. Neurological complications of acute ischemic stroke. Lancet Neurol 2011;10:357–71. [2] Cruz-Flores S, Berge E, Whittle IR. Surgical decompression for cerebral oedema in acute ischemic stroke. Cochrane Database Syst Rev 2012. 1:CD003435. [3] Wong GK, Mak CW, Po YC, Poon WS. Decompressive surgery for large territory anterior circulation cerebral infarction: the earlier, the better? ANZ J Surg 2012;80:115. [4] Barry FP, Murphy JM. Mesenchymal stem cells: clinical applications and biological characterization. J Biochem Cell Bio 2004;36:568–84. [5] Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999;284:143–7. [6] Meirelles LD, Fontes AM, Covas DT, Caplan AI. Mechanisms involved in the therapeutic properties of mesenchymal stem cells. Cytokine Growth Factor Rev 2009;20:419–27. [7] Gnecchi M, Zhang Z, Ni A, Dzau V. Paracrine mechanisms in adult stem cell signalling and therapy. Circ Res 2008;103:1204–19. [8] Schaffler A, Buchler C. Concise review: Adipose tissue-derived stromal cellsbasic and clinical implications for novel cell-based therapies. Stem Cells 2007;25:818–27. [9] Kokai LE, Rubin JP, Marra K. The potential of adipose-derived adult stem cells as a source of neural progenitor cells. Plast Reconstr Surg 2005;116:1453–60. [10] Nam HS, Kwon I, Lee BH, Kim H, An S, Lee PH, et al. Effects of mesenchymal stem cell treatment on the expression of matrix metalloproteinanses and angiogenesis during ischemic stroke recovery. PLoS One 2015;10:e0144218. [11] Park HW, Chang JW, Yang YS, Oh W, Hwang JH, Kim DG, et al. The effects of donor-dependent administration of human umbilical cord blood-derived mesenchymal stem cells following focal cerebral ischemia in rats. Exp Neurobiol 2015;24:358–65. [12] Chi K, Fu RH, Huang YC, Chen SY, Lin SZ, Huang PC, et al. Therapeutic effect of ligustilide-stimulated adipose-derived stem cells in a mouse thromboembolic stroke model. [published inline ahead of print January 18, 2016]. Cell Transplantation. 2016. DOI: http://dx.doi.org/10.3727/096368916X690539 (accessed April 12), 2016. [13] Gao J, Dennis JE, Muzic R, Lundberg M, Caplan AI. The dynamic in vivo distribution of bone marrow-derived mesenchymal stem cells after infusion. Cells Tissues Organs 2001;169:12–20. [14] Argibay B, Trekker J, Himmelreich U, Beiras A, Topete A, Taboada P, et al. Intraarterial route increases the risk of cerebral lesions after mesenchymal cell administration in animal model of ischemia. Sci Rep 2017;7:40758. [15] Lam PK, Ng CF, To KF, Ng SS, Mak TW, Chan ES, et al. Topical application of mesenchymal stem cells to somatic organs-a preliminary report. Transplantation 2011;19:e9–e11. [16] Lam PK, Lo AW, Wang KK, Lau HC, Leung KK, Li KT, et al. Transplantation of mesenchymal stem cells to the brain by topical application in an experimental traumatic injury. J Clin Neurosci 2013;20(2):306–9. [17] Longa EZ, Weinstein PR, Carlson S, Cummins R. Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke 1989;20:84–91. [18] Honma T, Honmou O, Iihoshi S, Harada K, Houkin K, Hamada H, et al. Intravenous infusion of immortalized human mesenchymal stem cells protects against in a cerebral ischemia model in adult rat. Exp Neurol 2006;199:56–66. [19] Agarwalla PK, Stapleton CJ, Ogilvy CS. Craniectomy in acute ischemic stroke. Neurosurgery 2014;74:S151–62. [20] Pavlichenko N, Sokolova I, Vijde S, Shvedova E, Alexandrov G, Krouglyakov P, et al. Mesenchymal stem cells transplantation could be beneficial for treatment of experimental ischemic stroke in rats. Brain Res 2008;1233:203–13. [21] Leu S, Lin YC, Yuen CM, Yen CH, Kao YH, et al. Adipose-derived mesenchymal stem cells markedly attenuate brain infarct size and improve neurological function in rats. J Trans Med 2010;8:63–79. [22] Omori Y, Honmou O, Harada K, Suzuki J, Houkina K, Kocsis. Optimization of therapeutic protocol for intravenous injection of human mesenchymal stem cells after cerebral ischemia in adult rats. Brain Res 2008;1236:30–8. [23] Kim SJ, Moon GJ, Chang WH, Bang OY. Intravenous transplantation of mesenchymal stem cells preconditioned with early phase stroke serum: current evidence and study protocol for a randomized trial. Trials 2013;14:317–29. [24] Bang OY, Lee JS, Lee PH, Lee G. Autologous mesenchymal stem cell transplantation in stroke patients. Ann Neurol 2005;57:874–82.

Please cite this article as: P. K. Lam, K. K. W. Wang, D. W. C. Chin et al., Topically applied adipose-derived mesenchymal stem cell treatment in experimental focal cerebral ischemia, Journal of Clinical Neuroscience, https://doi.org/10.1016/j.jocn.2019.08.051

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[25] Acosta SA, Tajiri N, Hoover J, Kaneko Y, Borlongan CV. Intravenous bone marrow stem cell grafts preferentially migrate to spleen and abrogate chronic inflammation in stroke. Stroke 2015;46:2616–27. [26] Moniche F, Gonzalez A, Gonzalez-Marcos JR, Carmona M, Pinero P, Espigado I, et al. Intra-arterial bone marrow mononuclear cells in ischemic stroke. A pilot clinical trial. Stroke 2012;43:2242–4. [27] Moniche F, Montaner J, Gonzalez-Marcos JR, Carmona M, Pinero P, Espigado I, et al. Intra-arterial bone marrow mononuclear cell transplantation correlates with GM-CSF, PDGF-BB, and MMP-2 serum levels in stroke patients: Results from a clinical trial. Cell Transplant 2014;23:S57–64. [28] Chan ESY, Lam Lam PK, Liew CT, Yen Yen RSC, Lau LauWY. The use of Composite Laserskin graft and artificial skin for burns reconstruction. Plast Reconst Surg 2000;105:807–8. [29] Jablonska A, Drela K, Wojcik-Stanaszek L, Janowski M, Zalewska T, Lukomska B. Short-lived human umbilical cord-derived neural stem cells influence the endogenous secretome and increase the number of endogenous neural progenitors in a rat model of lacunar infarct. Mol Neurobiol 2016;53:6413–25. [30] Porambo M, Phillips AW, Marx J, Ternes K, Arauz E, Pletnikov M, et al. Transplanted glial restricted precursor cells improve neurobehavioral and neuropathological outcomes in a mouse model of neonatal white matter injury despite limited cell survival. Glia 2015;63:452–65. [31] Walace GL. Microglial physiopathology: how to explain the dual role of microglia after acute neural disorders? Brain Behav 2012;2:345–56. [32] Patel AR, Ritzel R, McCullough LD, Liu F. Microglia and ischemic stroke: a double-edged sword. Int J Physiology, Pathophysiol Pharmacol 2013;5: 73–90. [33] Loane DJ, Byrnes KR. Role of microglia in neurotrauma. Neurotherapeutics 2010;7:366–77.

[34] Ma Y, Wang J, Wang Y, Yang GY. The biphasic function of microglia in ischemic stroke. [published online ahead of print February 2, 2016]. Prog Neurobiol. 2016. DOI:10.1016/j.pneurobio.2016.01.005. Accessed April 12, 2016. [35] Liu Z, Chopp M. Astrocyte, therapeutic targets for neuroprotection and neurorestoration in ischemic stroke. [published online ahead of print February 2, 2016]. Prog Neurobiol. 2016. DOI: 10.1016/j.pneurobio.2015.09.008 (accessed April 12), 2016. [36] Anderson CM, Swanson RA. Astrocyte glutamate transport: review of properties, regulation, and physiological functions. Glia 2000;32:1–14. [37] Sofroniew MV, Vinters HV. Astrocytes: biology and pathology. Acta Neurobiol 2010;119:7–35. [38] Theendakara V, peters-Libeu CA, Spilman P, Poksay KS, Bredesen DE, Rao RV. Direct transcriptional effects of Apolipoprotein E. J Neurosci 2016;36:685–700. [39] Filippi A, Mahler J, Schweitzer J, Driever W. Expression of the paralogous tyrosine hydroxylase encoding genes th1 and th2 reveals the full complement of dopaminergic and noradrenergic neurons in zebrafish larval and juvenile brain. J Comp Neurol 2010;518:423–38. [40] Tsai MJ, Tsai SK, Huang MC, Liou DY, Huang SL, Hseih WH, et al. Acidic FGF promoted neurite outgrowth of cortical neurons and improves neuroprotective effect in a cerebral ischemic rat model. Neuroscience 2015;305:238–47. [41] Doerfler A, Forsting M, Reith W, Heiland CS, Schabitz WR, von Kummer R, et al. Decompressive craniectomy in a rat model of ‘‘malignant” cerebral hemispheric stroke: experimental support for an aggressive therapeutic approach. J Neurosurg 1996;85:853–9. [42] Boltze J, Kulomska B, Jolkkonen J. for the MEMS=IRBI consortium. Mesenchymal stromal cells in stroke: improvement of motor recovery or functional compensation? J Cereb Blood Flow Metab 2014;34:1420–1.

Please cite this article as: P. K. Lam, K. K. W. Wang, D. W. C. Chin et al., Topically applied adipose-derived mesenchymal stem cell treatment in experimental focal cerebral ischemia, Journal of Clinical Neuroscience, https://doi.org/10.1016/j.jocn.2019.08.051