103J Contemp Med Sci | Vol. 6, No. 3, May–June 2020: 103–108 Original ISSN 2413-0516 Introduction Stroke is the most prevalent vascular accident of central ner- vous system among middle aged individuals. The sensory- motor dysfunction, cognitive impairments, and declined quality of life are the side effects of stroke in suffered patients.1 Unfortunately, no effective pharmaceutical treatment has been introduced, only endovascular approaches or rehabilitation may reduce the severity of symptoms.2 In the field of regen- erative medicine, there are numerous preclinical and clinical studies that demonstrated the therapeutic effect of stem cell transplantation in various neurodegenerative diseases such as ischemic stroke.3-5 Embryonic stem cells (ESC), the plu- ripotent stem cells, can differentiate to different lineages. So that, under defined protocol, embryonic stem cells has been shown to differentiate to neural progenitor cells (NPC).6 After transplantation of embryonic derived neural progenitor cells (ES-NPCs) in model of middle cerebral artery occlusion (MCAO) in the rats, the cells are capable to migrate toward ischemic site, proliferate and differentiate to the neurons and glial cells, and replace the dead cells.7 On the other hand, they induce angiogenesis and neurogenesis, decrease the neuroin- flammation, and preserve the integrity of blood–brain barrier through bystander effects.8 Following the ES-NPCs injection, the size of ischemic area reduced and the sensory-motor func- tion improved. The histological investigation also revealed positive findings in favor of neural tissue repair.6, 9, 10 Moreover, different types of biomarkers such as micro- RNAs (miRNAs) appear in blood and brain tissue fol- lowing ischemia. The miRNAs are small non-coding and single-stranded RNAs that regulate many internal processes such as cell proliferation, differentiation, development, cell cycle, apoptosis, etc. In addition to tissues, they are present in serum or plasma in the form of complexes and macroves- icles.11 The previous studies exposed that a wide spectrum of miRNAs identified, in the blood and brain tissue after MCAO in rats by microarray with both up- and downregulated man- ner. Clinical studies also confirmed these findings in patients with ischemic stroke. Therefore, the miRNAs are considered as a promising biomarker for prognosis of stroke patients.12-16 miRNA-210 has been shown to prevent neuronal apopto- sis and with neuroprotective role by suppressing the caspase pathway, induce a balance between bcl-2 and bax expres- sion.17 In ischemic condition, mir-210 plays role as a proan- giogenic factor and involves in cell-cycle regulation, DNA damage reconstruction, and neural tissue restoration.17-19 Human embryonic derived neural progenitor cells improves neurological scores following brain ischemia/ reperfusion: Modulation of blood and brain tissue MicroRNA-210 Leila Arab1, Aslan Fanni2, Shiva Nemati#2, Ehsan Arefian3, Jafar Ai#4, Tahmineh Mokhtari 5,6, Maryam Farahmandfar7, Nasser Aghdami2, Gholamreza Hassanzadeh1,8,9 1Department of Neuroscience and Addiction Studies, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran. 2Department of Stem cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran. 3Department of Microbiology, School of Biology College of Science, University of Tehran, Tehran, Iran. 4Department of Tissue Engineering and Applied Cell Sciences, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran. 5CAS Key Laboratory of Mental Health, Institute of Psychology, Beijing, China; 6Department of Psychology, University of Chinese Academy of Sciences, Beijing, China 7Department of Neuroscience and Addiction Studies, School of Advanced Technologies in Medicine, Electrophysiology Research Center, Neuroscience Institute, Tehran University of Medical Sciences, Tehran, Iran. 8Legal Medicine Research Center, Legal Medicine Organization, Tehran, Iran. 9Department of Anatomy, School of Medicine, Tehran University of Medical Science, Tehran, Iran. #2 co-second author #3 co-third author Correspondence to: Gholamreza Hassanzadeh (hassanzadeh@tums.ac.ir ) (Submitted: 13 March 2020 – Revised version received: 28 March 2020 – Accepted: 11 May 2020 – Published online: 26 June 2020) Objective In this study, we evaluated the effects of human embryonic derived neural progenitor cells on neurological score, histopathological changes, and miRNA-210 as biomarkers of regeneration. Methods The animals were randomly divided into the four groups: Sh (sham), MCAO (middle cerebral artery occlusion), MCAO+PBS, and MCAO+Cell. One day after MCAO induction, embryonic derived neural progenitor cells (hESC-NPCsGFP) or PBS were injected intracerebroventriculary in MCAO+Cell or MCAO+PBS groups. On day 1, 2, 3, and 7 after ischemia induction, the neurological score was tested in each rat. At 48 h, the expression of miRNA-210 was evaluated and 7 days after, the pathological assessments were performed by H&E staining. Results Neurological score showed the promotion of functional recovery in MCAO+Cell group. Based on H&E staining, the percentage of neural death in ischemic region reduced in MCAO+Cell group. The miRNA-210 significantly upregulated in both brain and blood samples. Conclusion According to the findings, hESC-NPCsGFP injection could upregulate the miRNA-210 of tissue and blood to support the neuroprotective and regenerative effect of hESC-NPCsGFP in the ischemic lesion and improved the neurological score and reduce the neural death in ischemic region. Keywords Embryonic stem cell; Neural death; Micro-RNA-210; Brain ischemia; Rat 104 Original hESC-NPCsGFP after brain ischemia/ reperfusion Leila Arab et al. J Contemp Med Sci | Vol. 6, No. 3, May–June 2020: 103–108 Also, miRNA-210 improves the stem cell survival via regula- tion of apoptosis-related protein (caspase 8 associated protein 2).20, 21 Also, overexpression of Mir-210 induces angiogenesis and neurogenesis in ischemic tissues to compensate decrease hypoperfusion.22 We designed this study to evaluate the effect of intrace- rebroventricul injection (ICVI) of ES-NPCs on fold changes of miRNA-210 in ischemic brain tissue and compared with MCAO and MCAO+Placebo groups, 24 h, and 48 h after ischemia. Method and Material Animals The male Wistar albino rats (260–300 g, 12-week-old) were enrolled into the study, purchased from laboratory animal department of Royan Institute. They were kept in animal room with temperature of 18-24°C, 40-–70% humidity, 12h light–12h dark cycle and free access to food and water. They were treated according to the guidelines of Iranian council for use and care of animals and approved by Ethical Committee of Tehran University of Medical Sciences. The rats were ran- domly divided into four groups: 1. Sham group (Sh): Operated rats without any vascular occlusion which underwent stereotaxic intervention and with experience of ICVI injection (n=8 rats). 2. MCAO: Rats only with MCAO for 60 min (n= 8 rats). 3. MCAO + PBS (phosphate buffered saline) group: Rats with MCAO for 60 min which followed by ICVI injection of PBS (5 µl) (n= 8 rats). 4. MCAO + Cell group: Rats with MCAO for 60 min which was followed by ICVI injection of cell suspension. (105 cells in 5 µl PBS) (n=8 rats). Model Induction Before MCAO induction, each rat was held in the induction chamber with vaporization of 5% isoflurane. Then, it was put immediately in supine position on heating pad and heating light with nosecone mask to inhalation of 1–2% isoflurane during surgery. With a middle neck incision and dissection of the neck soft tissues and muscles, we accessed to the common carotid artery (CCA). In next step, the proximal of CCA and external carotid artery (ECA) were ligated, and an intralumi- nal 4-0 nylon monofilament (Doccol Co., USA) was inserted into the MCA to occlude its origin under monitoring of blood flow by Laser Doppler flowmeter (Moor Instruments). After 60 min, the filament was removed and the neck incision was sutured.23 The body temperature was monitored with rectal temperature probe to remain at 37°C.24 Cell Preparation and Generating of hESC-NPCsGFP The hESC (RH6) was received from Royan cell bank, then, their differentiation procedure toward hNPCs has been done according to a standard procedure.25 The characteristics of hESC-NPCsGFP were evaluated using immunofluorescence staining. Immunofluorescence Staining To perform immunofluorescence staining, hNPC-GFP were fixed using 4% paraformaldehyde (Mallinckrodt, Phillipsburg, NJ), and permeabilized with 0.1% Triton X-100 (Sigma) for 15 min at ambient temperature. The cells were incubated with primary antibody for 1 h at room temperature (RT), washed, and incubated with fluorescein isothiocyanate-conjugated secondary antibodies, antimouse immunoglobulin M (IgM) (1:100), antimouse IgG (1:200), and antirat IgM (1:200), as appropriate, for 1 h at RT. Primary antibodies were Nestin (1:100), SOX1(1:100), GFAP (1:400) to confirm the undif- ferentiated stage. The cells were analyzed with a fluorescent microscope (Olympus). ICV Injection To perform the ICV injection, after 24 h the rats were anes- thetized with Isoflurane (5% for induction and 2% for mainte- nance), then fixed in stereotaxic frame. The ES-NPCs (1×105 cells in 5 µl PBS) or PBS was injected with using Hamilton syringe into the right cerebral ventricle at: bregma: AP=-0.12 mm, ML=1.6 mm, and DV=4.3 mm. Modified Neurological Severity Score (mNSS) To assess the sensory, motor, reflexes, and balance of rats after MCAO, we use mNSS test,26 while the worst score is 0 and the best one is 18. The test was performed for each rat on day 1, 2, 3, and 7 after ischemia induction. miRNA Real-Time Quantitative PCR The miRNA expression was measured in the ischemic area and blood samples 48 h after MCAO. The rats were anesthe- tized with isoflurane 5%. The cardiac blood samples were taken and the ischemic area of the right hemisphere was iso- lated and stored in -80°C freezer. Total RNA (plus miRNA) was extracted from brain samples. Single-strand cDNA was synthesized using universal cDNA synthesis kit (Exiqon, Vedbaek, Denmark). Quantification of miRNA-210 was per- formed with stem-loop real-time PCR. qPCR was performed in triplicate in three separate experiments on an Applied Biosystems Step One Plus real-time PCR machine. The rela- tive expression of miRNA was normalized to the endogenous control U6 expression using the comparative cycle threshold (CT) method. Hematoxylin and Eosin Staining For light microscopy study, the rats were anesthetized with ketamine/xylazine (RaziCo, Iran), and perfused by 0.9% saline and 4% paraformaldehyde (PFA, sigma), respectively. The brains were dissected and cut into the sections with 3–5 mm thickness. Then, the sections were post-fixed in 10% formalin 72 h at 4°C. In order to light microscopy analysis, the samples were embedded in paraffin and 5 μm coronal sections were prepared by using a rotary microtome (Leica Biosystems, Milan, Italy). One section from each five section was selected and the tissue sections stained with Hematoxylin and eosin (H&E). Afterward, graded alcohols (70, 80, 90, and 100% [2 times]) was used to dehydrate the sections. Finally, they were mounted in Canada balsam and prepared for analysis. Study and survey of sections was performed by using a light field microscope (Olympus, CX31, Tokyo, Japan). In cortex field, the intact and ischemic cells considered as dark neurons, were counted in the ×400 images by using a connected camera to the microscope.27 Statistical Analysis Data analysis was performed with standard statistical software GraphPad Prism, version 6 (GraphPad, La Jolla, CA). One-way 105 Original hESC-NPCsGFP after brain ischemia/ reperfusionLeila Arab et al. J Contemp Med Sci | Vol. 6, No. 3, May–June 2020: 103–108 ANOVA followed by Bonferroni’s post-hoc comparisons tests were performed in all statistical analyses. To test the feasibility, we built statistical model by regression analysis. Correlations were estimated by Pearson correlation test. Differences were considered significant at p < 0.05. Result Characterization of hESC-NPCsGFP Generated hNPC (Fig. 1) was evaluated for expression of neu- ral progenitor markers by immunofluorescence staining. The phase contrast microscopy photograph of normal hNPC was shown in Fig. 1a. The hNPC population had highly expressed NESTIN (Fig. 1b), SOX1 (Fig. 1d) with the lower expression of GFAP (Fig. 1c) at their progenitor stage. Effects of ICV Injection of hESC-NPCsGFP on the Modified Neurological Severity Score (mNSS) Following I/R Injury The results of behavioral functional test (mNSS) showed that the neurological function outcomes significantly improved in MCAO+Cell during a week after injection (3.71±0.76/18, p-value<0.001) compared with MCAO (8.29±1.11, p-value<0.059) and MCAO+PBS (7.71±1.11, p-value: 0.230) groups on day 7 (Table 1). Effects of ICV Injection of hESC-NPCsGFP on the Neural Cells Death of Ischemic Area Following I/R Injury Evaluation of apoptosis by H&E staining showed that the count of neural cell death (shrunken cells) in MCAO+Cell group is much lesser than (~50%) MCAO+PBS group (80%) and MCAO group (~80%) (Fig. 2). Effects of ICV Injection of hESC-NPCsGFP on miRNA-210 Profile (RT-PCR) of Blood and Brain Samples of Ischemic Area Following I/R Injury Brain and blood samples from rats in different groups were screened for a total of 72 Rattus norvegicus. The miRNA-210 was found to be present in both the blood and brain 48 h after reperfusion. Then, the correlation between MCAO-Blood/ Tissue-48H was examined (Fig. 3). Fig. 1 Characterization of hESC-NPCsGFP. (a) Phase contrast microscopy of normal hNPC after generation of RH6. (b–d) Immunofluorescence staining for neural progenitor markers (NESTIN, SOX1, and GFAP). 106 Original hESC-NPCsGFP after brain ischemia/ reperfusion Leila Arab et al. J Contemp Med Sci | Vol. 6, No. 3, May–June 2020: 103–108 Discussion In this study, we evaluated the effects of h-ESC-NPC on brain impairments induced by ischemia reperfusion. For this pur- pose, the neurological score, histopathological changes, and the miRNAs-120 level of ischemic area and peripheral blood were investigated in each group. The MCAO was applied by a similar method in all rats and the formation of ischemic tissue was confirmed by H&E staining. The MCAO+Cell group sig- nificantly had better sensory-motor function. As the previous studies showed, transplantation of ESC–NPCs in brain isch- emic lesion could promotes functional recovery after ischemia reperfusion via migration proliferation, and differentiation in the ischemic region. Human ESC-NPCs proliferate and differ- entiate to neurons and glia cells in one step. Administration of ESC-NPCs was approved to reduce the volume of isch- emic region via induction of neurogenesis and angiogenesis.8 On day 7, after cell injection, the rats were perfused and their brains were extracted for more studies. The H&E staining results confirmed the reduced percentage of neural death in ischemic region. The migration, proliferation and differen- tiation of h-ESC-NPCs in the ischemic lesion has been con- firmed in several studies.28-31 On the other hand, this amount of transplanted cells can- not be differentiated to replace the damaged tissue in the isch- emic region. So, they might apply their alterations via other mechanisms. The transplanted cells secrete different trophic factors including cytokines, chemokines, and extracellular proteins to the surrounding environments which act as anti- apoptotic factor, immunomodulators, angiogenesis factors, and antioxidant molecules. These progenitor cells are capa- ble to endogenous neurotropic factors such as brain-derived neurotrophic factor (BDNF), stromal cell-derived factor 1 (SDF1), vascular endothelial growth factor (VEGF), nerve growth factor (NGF), etc. All these factors play important and beneficial role to repair the ischemic impairments following stem cell transplantation. The analysis of neural stem cells’ secretome showed that they secrete different growth factors, Fig. 2 Effects of hESC-NPCsGFP on percentage of neural death of ischemic region in rat. (A) H&E staining in different groups (100×). (B) Comparing the percentage of neural death in different groups ****p < 0.0001 compared to Sh group; Sh: sham operated group; MCAO: Ischemia induction group, MCAO+PBS: Ischemia induction group with ICV injection of PBS; MCAO+Cell: Ischemia induction group with ICV injection of hESC-NPCsGFP. Table 1. Effects of ICV injection of hESC-NPCsGFP on the Modified Neurological Severity Score (mNSS) following I/R injury. Time (Day) Group Day 1 Day 2 Day 3 Day 7 p-Value Pairwise comparisons MCAO (M) 10.14 (1.86) 9.43 (1.27) 9.29 (0.95) 8.29 (1.11) 0.059 - MCAO+ PBC (P) 9.43 (1.81) 8.57 (2.23) 7.29 (3.09) 7.71 (1.11) 0.230 - MCAO+ Cell (C) 10.14 (2.27) 7.57 (1.40) 6.29 (1.38) 3.71 (0.76) <0.001 1>3; 1>7; 2>7 P-Value 0.744 0.164 0.044 <0.001 Pairwise Comparisons - - C