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Migration of neural stem cells to ischemic brain regions in ischemic stroke in rats
Neuroscience Letters 552 (2013) 124–128
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Migration of neural stem cells to ischemic brain regions in ischemic stroke in rats Jiong Dai, Shan-Quan Li ∗ , Yong-Ming Qiu, Wen-Hao Xiong, Yu-Hua Yin, Feng Jia, Ji-Yao Jiang Department of Neurosurgery, Renji Hospital, Shanghai Jiaotong University School of Medicine, 1630 Dongfang Road, Shanghai 200127, China
h i g h l i g h t s • • • •
Use of a model of ischemic stroke to evaluate the therapeutic effects of NSCs. NSCs migrated to the frontal and parietal lobes, caudate, and putamen. Rat NSCs can differentiate into astrocytes and neurons. Transplantation of NSCs can potentially improve neurologic function after cerebral ischemia.
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Article history: Received 10 May 2013 Received in revised form 4 July 2013 Accepted 27 July 2013 Keywords: Ischemia Stroke Neural stem cells Transplantation
a b s t r a c t An established rat model of ischemic stroke, produced by temporary middle cerebral artery occlusion and reperfusion (MCAO/R), was used in the evaluation of organ migration of intra-arterial (IA) transplantation of neural stem cells (NSCs). Immediately after transplantation, ischemic rats (n = 8) transplanted with either NSCs (MCAO/R + NSC group) or NSC growth medium (MCAO/R + medium group) exhibited neurological dysfunction but rats in a sham + NSCs group (n = 5) did not. During the postoperative period, neurological function improved to a similar extent in both MCAO/R groups. At 10 and 14 days post-transplantation, neurological function in the MCAO/R + NSC group was superior to that in the MCAO/R + medium group (p < 0.001). Hematoxylin–eosin staining showed neuronal degeneration and necrosis in ischemic rats. Immunofluorescence staining revealed that NSCs had migrated to the frontal and parietal lobes, caudate, and putamen. Some cells had begun differentiating into neurons and astrocytes. Rat NSCs can migrate into the ischemic region, survive, and differentiate into astrocytes and neurons, and thereby potentially improve neurologic function after cerebral ischemia. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Stroke remains a significant clinical unmet condition. Only 3% of the ischemic patient population benefits from current treatment modalities. The only approved therapy for stroke is strongly limited by the short therapeutic window and hemorrhagic complications, therefore excluding most patients from its benefits [21]. Stem cells have the potential for neurogenesis, angiogenesis, and synaptic plasticity that can potentially restore function after stroke [13]. Transplantation of stem cells or their derivatives in animal models of cerebral ischemia can improve function by replacing lost neurons and glial cells and by mediating remyelination and modulation of inflammation [7,16]. In animal models of stroke, transplantation of adipose tissuederived stem cells [27], human umbilical mesenchymal stem cells
[12], human induced pluripotent stem cells [8], bone marrow stromal cells [6], human bone marrow-derived mesenchymal stem cells [3], and neuronal stem cells (NSCs) [22] have proved effective in stroke treatment. NSCs possess characteristics that suggest good potential in the treatment of stroke [1,17]: NSCs (1) proliferate and divide exponentially; (2) have multiple potentials of differentiation and can differentiate into mature neuron, astrocytes and oligodendrocytes; (3) migrate and differentiate in response to signals that cause damage. We postulated that an ischemic rat model of stroke could be used to evaluate the organ migration pattern of NSCs administered by intra-arterial (IA) injection. 2. Materials and methods 2.1. Animal care
∗ Corresponding author. Tel.: +86 021 68383658. E-mail addresses: [email protected], [email protected] (S.-Q. Li). 0304-3940/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2013.07.044
Healthy adult male Sprague–Dawley (SD) rats (250 g and 280 g) were purchased from the Experimental Animal Center of Shanghai
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University of Traditional Chinese Medicine. The rats were randomly assigned into a middle cerebral artery occlusion and reperfusion (MCAO/R) + NSC group, a MCAO/R + growth medium (medium) group, and sham + NSC group. Before the experiment, rats were food-deprived for 12 h. The surgical procedures and postoperative animal care were conducted according to the Guide for the Care and Use of Laboratory Animals (National Research Council 1996) and the Guidelines and Policies for Rodent Survival Surgery provided by the Animal Care and Use Committees for Shanghai Jiaotong University. The rat model of ischemic stroke was produced using a modified right MCAO/R procedure as described previously [14]. Animals were anesthetized with intraperitoneal 10% chloral hydrate (350 mg/kg). A thicker, 3-0 surgical nylon suture than that used previously [11] and with a smooth, non-enlarged end was created by heating near a flame. The modified suture moved more smoothly from the external carotid artery (ECA) into the lumen of the internal carotid artery (ICA) where it blocked the origin of the MCA. Once the suture entered the pterygopalatine artery (PPA) (about 10 mm in length), evident resistance was felt. Then, the suture was retracted and the PPA was temporarily clamped. Then, the direction of the head of nylon suture was slightly adjusted, and this suture was further inserted. When the head of the suture was 17–20 cm away from the stump (from the bifurcation of CCA to the head), the resistance was absent. Then, the clamp on CCA was released and the skin was closed. The suture was left in place for 2 h. The common carotid artery (CCA) and pterygopalatine artery (PPA) were thus occluded temporarily.
clamped followed by injection of the NSC suspension. The dose and the procedure for injection were identical to those described above. Then, the clamp was removed and wound was closed. Rats were placed back in their cage and given ad libitum access to water and food.
2.2. Transplantation
At 14 days after transplantation, rats were anesthetized and transcardially perfused with 4% paraformaldehyde followed by decapitation and dissection of the brain. The brain was fixed in 4% paraformaldehyde in sterile water for more than 24 h and then in 30% sucrose in sterile water for 24–48 h for dehydration. When the brain sank, the 1.5-mm brain tissues in front of and back of bregma were collected for frozen sectioning with a freezing microtome (CM1900, Leica, Germany) at −20 ◦ C. Tissues were cut into 5-m sections from the caudal to cranial side and sections were preserved at −20 ◦ C for hematoxylin–eosin (HE) staining and immunofluorescence staining. Sections undergoing HE staining were observed under an BX 40 microscope (Olympus, Japan) and those receiving immunofluorescence staining under an IX 70 fluorescence microscope (Olympus, Japan) and a LSM 510 META Laser scanning confocal microscope (Carl Zeiss, Germany). The primary antibodies used for immunofluorescence staining were a mouse anti--tubulin-III antibody for the detection of neurons (1:800, Sigma, USA) and a mouse anti-glial fibrillary acidic protein (GFAP) (1:200, Sigma, USA) antibody for detection of astrocytes. The secondary antibody was a fluorescein-conjugated goat anti-mouse secondary antibody (Rhodamine, 1:100; Sigma). All procedures for immunohistochemical staining were as described by Xu et al. [26].
NSCs and growth medium were provided by the Department of Neurobiology, School of Medicine of Shanghai Jiaotong University. NSCs were collected from the E16 embryos of SD transgenic rats carrying the gene for green fluorescent protein [15,26]. Forebrains were dissected using fine forceps, transferred separately into two 10-ml tubes that contained growth medium (mitogen-free), and near-single cell suspensions were obtained by dissociation with gentle mechanical trituration. The dissociated cells were filtered through a cell strainer (BD Falcon, Two Oak Park, Bedford, MA, USA) and seeded into a T25 culture flask (Corning Inc., Corning, NY, USA) at a density of 0.1 × 106 cells/mL. Cells were grown in 1× Dulbecco’s Modified Eagle’s Medium/F12 (DMEM/F12; Gibco), 1× N2 supplement (Gibco), 0.06% glucose, and 2 mM glutamine (Gibco) supplemented with epidermal growth factor (20 ng/mL), basic fibroblast growth factor (20 ng/mL), heparin (2 g/mL; Sigma, St. Louis, USA), and 2% B27 supplement (Gibco). All cells were incubated in a humidified atmosphere of 5% CO2 at 37 ◦ C. NSCs grown in spheres (50–200 cells/sphere) were passaged by mechanical dissociation once every 3–4 days. For passaging, cells were centrifuged (1000 rpm, 10 min) at 4 ◦ C. The cells were resuspended in 200 L medium and mechanically dissociated into single cells by triturating 100 times with a 200-L Pipetteman, which produced near-single cell suspensions with an average viability over 90%, determined by Trypan Blue staining. Cells were then reseeded at 0.1 × 106 cells/mL. At 24 h after MCAO/R, rats were anesthetized and the right CCA, ICA, and PPA were exposed, and clamping transiently blocked the CCA and PPA. Then, 200 L of NSC suspension (2 × 106 cells) and 200 L of NCS growth medium were injected at the beginning of ICA in the MCAO/R + NSC group and MCAO/R + medium group, respectively. The injection was completed within 5 min. Muscular pieces and medical glue were used to close the wound in the ICA followed by removal of the clamp and wound closure. In the sham + NSC group, animals were anesthetized and the right CCA, ECA, ICA, and PPA were exposed, and the CCA, ECA, and PPA were transiently
2.3. Assessment of neurological function Immediately before and following infusion of NSCs and at 1, 2, 3, 7, 10, and 14 days after transplantation, the behavior and neurological function of the rats in all treatment groups were evaluated. Neurological function was assessed using a 14-point neurological function severity scale (NFSS) in which 1 point was awarded for failure to perform a task or for the absence of a tested reflex [11]. Neurological function assessments included the following: forelimb bending, hind limb bending, and raising the head up at an angle of >10◦ in 30 s (all after pulling the rats up by the tail); ability to walk straight, circularly move toward the hemiplegic side, or fall toward the hemiplegic side (all after placing the rat on the floor); presence of abnormal movement such as immobility and staring, tremor, myodystony, irritability, or seizures; sense of touch or sight; proprioception; Pinna reflex; corneal reflex; startle reflex. Severe neurological dysfunction was defined as a score of 10–14, moderate neurological dysfunction as a score of 5-9 and mild neurological dysfunction as a score of 1–4. 2.4. Pathological examination
2.5. Statistical analysis Mean ± standard deviation (SD) NFSS scores for given follow-up days were graphed as a line plot. A two-sample t-test was performed to compare the mean change in NFSS score from pre-OP to post-OP conditions between two groups. Differences between groups over time were compared using repeated measurement analysis of variance (ANOVA). Statistical assessments were twotailed and considered significant at p < 0.05. Statistical analyses were performed using SPSS 15.0 statistics software (SPSS, Inc., Chicago, IL, USA).
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Fig. 1. Mean ± SD change in NFSS scores for MCAO/R + NSC (closed circles) and MCAO/R + medium (open circles) transplantation groups (n = 8) at various times post-OP, including Day 0 (assessed immediately post-OP), and Days 1, 2, 3, 7, 10, and 14. The p-value represents the difference of NFSS scores over time between groups, analyzed using repeated measurement ANOVA; ***denotes significant differences in change of NFSS scores between pre-OP and post-OP groups (p < 0.001).
3. Results Rats were assigned to 3 treatment groups. Eight rats were in the MCAO/R + NSC group, 8 in the MCAO/R + medium group, and 5 in the sham + NSC group. The mean body weight of the rats in each group was 265.5 ± 8.5 g, 269.4 ± 9.5 g, and 264.4 ± 10.6 g, respectively. The effect of NSCs on brain function was assessed by comparing NFSS scores for MCAO/R + NSC, MCAO/R + medium, and sham + NSC groups over a 14-day post-OP period. The mean from pre-OP values in NFSS score for the MACO/R + NSC and MACO/R + medium groups on Day 0 (immediately post-OP), and on Days 1, 2, 3, 7, 10, and 14 are presented in Fig. 1. NFSS scores immediately post-OP on Day 0 increased to a similar extent for both the MCAO/R + NSC and MCAO/R + medium groups. At Days 10 and 14, the mean change in NFSS score was significantly lower in the MACO/R + NSC group than in the MACO/R + medium group (p < 0.001). In a repeated measure ANOVA, the difference between the mean change in NFSS score for the MCAO/R + NSC group and that for the MCAO/R + medium group was not significant (p = 0.105). The mean NFSS score remained unchanged (from pre-OP) values for the control sham + NSC group at all post-OP time points assessed (data not shown).
The ability of NSCs to restore neurological function was also assessed by examining the number of rats that showed a shift in severity of dysfunction over a 14-day period. Seven of 8 rats and 1/8 rats in the MCAO/R + NCS and MCAO/R + medium groups, respectively, had a moderate or severe level of neurological dysfunction, respectively, on Day 0 immediately following transplantation. A clear trend toward improvement in neurological function was evident in the MCAO/R + NCS and MCAO/R + medium groups over the subsequent14-day post-OP period. More rats in the MCAO/R + NCS group showed a shift toward mild dysfunction (a measure of neurological improvement) than did rats in the MCAO/R + medium group. On Days 7, 10, and 14 post-OP, 3/8, 8/8, and 8/8 rats in the MCAO/R + NCS group, respectively, had mild dysfunction whereas only 1/8, 3/8, and 4/8 rats in the MCAO/R + medium group, respectively, were in this category. As expected, 5/5 rats in the sham + NSC group had an NFSS score of 0 when assessed immediately postOP on Day 0. None of the rats in this group showed a changed in neurological function at any time post-OP from Day 0 through Day 14. 3.1. Pathological examination As shown by hematoxylin–eosin staining (Fig. 2), rats in the MCAO/R + NSC and MCAO/R + medium groups had a shrinkage of neurons in the frontal and parietal lobes and at the location in the caudate and putamen that was fed by the right MCA. These neurons were irregular and eosinophilic in the cytoplasm. Their nuclei were darkly stained or absent and cellular interstitium was loose. Hemorrhage was absent. By contrast, in the sham + NSC group, the brain was intact, the neurons were full in the cell body, and the nuclei were located at the center. As shown by immunofluorescence staining (Fig. 3), rats in the MCAO/R + NSC group had NSCs with green fluorescence in the frontal and parietal lobes and caudate and putamen; some NSCs had started to differentiate into neurons (positive for -tubulin-III, red fluorescence) and astrocytes (positive for GFAP, red fluorescence). In the left hemisphere, NSCs with green fluorescence were absent but normal neurons and astrocytes were observed. In the MCAO/R + medium group, NSCs with green fluorescence were not found in bilateral hemispheres. Normal neurons and astrocytes were found in the left hemisphere, and the neurons and astrocytes in the frontal and parietal lobes, caudate, and putamen were loosely distributed. In the sham + NSC group, NSCs with green fluorescence
Fig. 2. Hematoxylin–eosin stained 5 m sections of pre-frontal lobe, caudate, and putamen of rats from the MCAO/R + NSC, MCAO/R + medium, and sham + NSC groups.
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Fig. 3. Immunofluorescence of brain sections from rats in the MCAO/R + NSC (left column), MCAO/R + medium (middle column), and sham + NSC (right column) groups. Green fluorescence showed NSCs expressing GFP. Neurons (positive for -tubulin-III) and astrocytes (positive for GFAP) showed red fluorescence. Images were captured under an Olympus IX 70 fluorescence microscope. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
were not found but normal neurons and astrocytes were noted in the bilateral hemispheres. 4. Discussion Ischemic rats transplanted with NSCs displayed significant improvement in neurological function at 10 and 14 days following transplantation. Rats transplanted with NSCs shifted from severe/moderate neurological dysfunction to mild dysfunction between 7 and 14 days post-OP. These results demonstrate that IA transplantation of NSCs promotes functional neurological recovery after cerebral ischemia. Statistical analysis (i.e., paired data analyzed by a two-tailed Student’s t-test) followed the approach taken in other studies of a similar nature [15,26]. A statistical analysis of the comparison between groups at each time point was performed and presented as the change from pre-OP to post-OP. Because all values of pre-OP were zero, the changes are equal to values for post-OP. A twosample t-test was therefore a valid statistical approach. To account for multiple comparisons, a repeated measurement ANOVA was performed for the trend of NHSS. This analysis showed no significant difference between the 2 groups (p = 0.105). A post hoc analysis was not carried out because the ANOVA did not show significance. Although ANOVA may not be ideal for the analysis of data based on scoring, a previous study of a similar nature to that described here used ANOVA to compare Basso, Beattie and Bresnahan (BBB) open-field locomotion Locomotor Rating Scale scores [26]. In ischemic rats that had been transplanted with NSCs, hematoxylin–eosin staining showed neuronal degeneration and necrosis and immunofluorescence staining revealed that NSCs with green fluorescence had migrated to the frontal and parietal lobes,
caudate, and putamen. Some of these cells had begun to differentiate into neurons and several NSCs started to differentiate into astrocytes. Following NSC transplantation for the treatment of ischemic stroke, the transplanted NSCs migrate into the ischemic region, survive, and differentiate into astrocytes and neurons. The specific mechanisms whereby transplanted NSCs enter, migrate, and distribute in the ischemic region following transcarotid artery transplantation remain poorly understood. At approximately 5–6 h after ischemic stroke, the blood brain barrier opens, an occurrence that may partially account for the entry of NSCs into the brain. Cellular adhesion molecules are secreted in the local microenvironment, facilitating the migration of inflammatory cells and possibly also transplanted NSCs into the ischemic region [24]. The relatively small number of NSCs that migrate into regions of ischemic injury appears to be insufficient to account for the reversal of neurological dysfunction following ischemic stroke [4]. In the present study, the NSCs migrating into the brain were not quantified. In a previous study [11], approximately 21% of transplanted NSCs migrated into the ischemic region, and of these about 10% differentiated into astrocytes and about 1% into neurons. The expression of biomarkers (e.g., glial activation, oxidative stress, or neuronal injury) has been measured in previous studies as a way of tracking the migration of transplanted cells in ischemic injury models [10]. Future studies in the model of ischemic injury described here could include such biomarkers as measures for quantifying the migration of NSCs. Stem cell therapy may provide trophic support to the injured tissue, fostering neurogenesis and angiogenesis [9]. Thus, paracrine effects of secreted or released factors (e.g., nerve growth factor, salvianolic acid B), rather than direct cell replacement, may be
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responsible for most of the benefits observed after cell transplantation [2,12,22,23,28]. Alternatively, studies using the well established MCAO/R model could be designed to explore the role of specific signaling pathways (i.e., the Wnt/-catenin pathway) that have been shown to control the expansion of the stem cell pool in the adult brain [20]. This study did not examine infarct volume, a critical factor that has been shown to correlate strongly with behavioral improvement and neuronal sparing in the MCAO rat stroke model [25]. It will be important to determine whether lesion volumes are similar between treatments. More extensive histological assessments could be done to quantify neuron cell death (i.e., using TUNEL staining) after NSC transplantation [2]. Future studies could be aimed at quantifying the proportion of neurons versus glia that are generated by NSC transplantation and if cells that had migrated from the blood show a similar differentiation pattern. Currently, the optimal time of NSC transplantation following ischemic cerebral injury is a matter of controversy. This study did not assess the effect of the time interval between the MCAO/R procedure and transplantation on the ability of NSCs to restore neurological function. NSC transplantation was carried out at 24 h after MCAO/R. This may be the optimum time interval for transplantation on the basis that it coincides with the time of opening of the blood–brain barrier following ischemic stroke. Several reports emphasized that NSC transplantation should be performed as soon as possible during or after ischemic brain injury (i.e., ≤24 h) whereas others revealed that the survival rate of transplants is greatest when transplantation is performed at about 7 days after injury when [5,9–11,18,19] the levels of neurotoxic substances are reduced, neurotrophic factors are released, and angiogenesis occurs. Thus, some physicians have recommended stem cell transplantation at ≥7 days after injury [10,18]. The results of the present study suggest that the migration of NSCs to ischemic brain regions is potentially associated with an improvement in neurological function. The effect of dosage, treatment time point, and observation period on the migration and differentiation of NSCs and on the extent of neurological improvement in cerebral ischemia are important areas for further research. Conflict of interest statement The authors declare that they have no conflict of interest. Acknowledgements We would like to express our gratitude to Sai-Li Fu and Zheng-Wen Ma in Laboratory of Neurobiology, Shanghai Jiaotong University, Shanghai, China for their generous help. References [1] J.M. Abrahams, S. Gokhan, E.S. Flamm, M.F. Mehler, De novo neurogenesis and acute stroke: are exogenous stem cells really necessary? Neurosurgery 54 (2004) 150–156. [2] G. Andsberg, Z. Kokaia, A. Björklund, O. Lindvall, A. Martínez-Serrano, Amelioration of ischaemia-induced neuronal death in the rat striatum by NGF-secreting neural stem cells, European Journal of Neuroscience 10 (1998) 2026–2036. [3] X. Bao, J. Wei, M. Feng, S. Lu, G. Li, W. Dou, W. Ma, S. Ma, Y. An, C. Qin, R.C. Zhao, R. Wang, Transplantation of human bone marrow-derived mesenchymal stem cells promotes behavioral recovery and endogenous neurogenesis after cerebral ischemia in rats, Brain Research 1367 (2011) 103–113. [4] J. Chen, P.R. Sanberg, Y. Li, L. Wang, M. Lu, A.E. Willing, J. Sanchez-Ramos, M. Chopp, Intravenous administration of human umbilical cord blood reduces behavioral deficits after stroke in rats, Stroke 32 (2001) 2682–2688.
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Migration of neural stem cells to ischemic brain regions in ischemic stroke in rats Jiong Dai, Shan-Quan Li ∗ , Yong-Ming Qiu, Wen-Hao Xiong, Yu-Hua Yin, Feng Jia, Ji-Yao Jiang Department of Neurosurgery, Renji Hospital, Shanghai Jiaotong University School of Medicine, 1630 Dongfang Road, Shanghai 200127, China
h i g h l i g h t s • • • •
Use of a model of ischemic stroke to evaluate the therapeutic effects of NSCs. NSCs migrated to the frontal and parietal lobes, caudate, and putamen. Rat NSCs can differentiate into astrocytes and neurons. Transplantation of NSCs can potentially improve neurologic function after cerebral ischemia.
a r t i c l e
i n f o
Article history: Received 10 May 2013 Received in revised form 4 July 2013 Accepted 27 July 2013 Keywords: Ischemia Stroke Neural stem cells Transplantation
a b s t r a c t An established rat model of ischemic stroke, produced by temporary middle cerebral artery occlusion and reperfusion (MCAO/R), was used in the evaluation of organ migration of intra-arterial (IA) transplantation of neural stem cells (NSCs). Immediately after transplantation, ischemic rats (n = 8) transplanted with either NSCs (MCAO/R + NSC group) or NSC growth medium (MCAO/R + medium group) exhibited neurological dysfunction but rats in a sham + NSCs group (n = 5) did not. During the postoperative period, neurological function improved to a similar extent in both MCAO/R groups. At 10 and 14 days post-transplantation, neurological function in the MCAO/R + NSC group was superior to that in the MCAO/R + medium group (p < 0.001). Hematoxylin–eosin staining showed neuronal degeneration and necrosis in ischemic rats. Immunofluorescence staining revealed that NSCs had migrated to the frontal and parietal lobes, caudate, and putamen. Some cells had begun differentiating into neurons and astrocytes. Rat NSCs can migrate into the ischemic region, survive, and differentiate into astrocytes and neurons, and thereby potentially improve neurologic function after cerebral ischemia. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Stroke remains a significant clinical unmet condition. Only 3% of the ischemic patient population benefits from current treatment modalities. The only approved therapy for stroke is strongly limited by the short therapeutic window and hemorrhagic complications, therefore excluding most patients from its benefits [21]. Stem cells have the potential for neurogenesis, angiogenesis, and synaptic plasticity that can potentially restore function after stroke [13]. Transplantation of stem cells or their derivatives in animal models of cerebral ischemia can improve function by replacing lost neurons and glial cells and by mediating remyelination and modulation of inflammation [7,16]. In animal models of stroke, transplantation of adipose tissuederived stem cells [27], human umbilical mesenchymal stem cells
[12], human induced pluripotent stem cells [8], bone marrow stromal cells [6], human bone marrow-derived mesenchymal stem cells [3], and neuronal stem cells (NSCs) [22] have proved effective in stroke treatment. NSCs possess characteristics that suggest good potential in the treatment of stroke [1,17]: NSCs (1) proliferate and divide exponentially; (2) have multiple potentials of differentiation and can differentiate into mature neuron, astrocytes and oligodendrocytes; (3) migrate and differentiate in response to signals that cause damage. We postulated that an ischemic rat model of stroke could be used to evaluate the organ migration pattern of NSCs administered by intra-arterial (IA) injection. 2. Materials and methods 2.1. Animal care
∗ Corresponding author. Tel.: +86 021 68383658. E-mail addresses: [email protected], [email protected] (S.-Q. Li). 0304-3940/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2013.07.044
Healthy adult male Sprague–Dawley (SD) rats (250 g and 280 g) were purchased from the Experimental Animal Center of Shanghai
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University of Traditional Chinese Medicine. The rats were randomly assigned into a middle cerebral artery occlusion and reperfusion (MCAO/R) + NSC group, a MCAO/R + growth medium (medium) group, and sham + NSC group. Before the experiment, rats were food-deprived for 12 h. The surgical procedures and postoperative animal care were conducted according to the Guide for the Care and Use of Laboratory Animals (National Research Council 1996) and the Guidelines and Policies for Rodent Survival Surgery provided by the Animal Care and Use Committees for Shanghai Jiaotong University. The rat model of ischemic stroke was produced using a modified right MCAO/R procedure as described previously [14]. Animals were anesthetized with intraperitoneal 10% chloral hydrate (350 mg/kg). A thicker, 3-0 surgical nylon suture than that used previously [11] and with a smooth, non-enlarged end was created by heating near a flame. The modified suture moved more smoothly from the external carotid artery (ECA) into the lumen of the internal carotid artery (ICA) where it blocked the origin of the MCA. Once the suture entered the pterygopalatine artery (PPA) (about 10 mm in length), evident resistance was felt. Then, the suture was retracted and the PPA was temporarily clamped. Then, the direction of the head of nylon suture was slightly adjusted, and this suture was further inserted. When the head of the suture was 17–20 cm away from the stump (from the bifurcation of CCA to the head), the resistance was absent. Then, the clamp on CCA was released and the skin was closed. The suture was left in place for 2 h. The common carotid artery (CCA) and pterygopalatine artery (PPA) were thus occluded temporarily.
clamped followed by injection of the NSC suspension. The dose and the procedure for injection were identical to those described above. Then, the clamp was removed and wound was closed. Rats were placed back in their cage and given ad libitum access to water and food.
2.2. Transplantation
At 14 days after transplantation, rats were anesthetized and transcardially perfused with 4% paraformaldehyde followed by decapitation and dissection of the brain. The brain was fixed in 4% paraformaldehyde in sterile water for more than 24 h and then in 30% sucrose in sterile water for 24–48 h for dehydration. When the brain sank, the 1.5-mm brain tissues in front of and back of bregma were collected for frozen sectioning with a freezing microtome (CM1900, Leica, Germany) at −20 ◦ C. Tissues were cut into 5-m sections from the caudal to cranial side and sections were preserved at −20 ◦ C for hematoxylin–eosin (HE) staining and immunofluorescence staining. Sections undergoing HE staining were observed under an BX 40 microscope (Olympus, Japan) and those receiving immunofluorescence staining under an IX 70 fluorescence microscope (Olympus, Japan) and a LSM 510 META Laser scanning confocal microscope (Carl Zeiss, Germany). The primary antibodies used for immunofluorescence staining were a mouse anti--tubulin-III antibody for the detection of neurons (1:800, Sigma, USA) and a mouse anti-glial fibrillary acidic protein (GFAP) (1:200, Sigma, USA) antibody for detection of astrocytes. The secondary antibody was a fluorescein-conjugated goat anti-mouse secondary antibody (Rhodamine, 1:100; Sigma). All procedures for immunohistochemical staining were as described by Xu et al. [26].
NSCs and growth medium were provided by the Department of Neurobiology, School of Medicine of Shanghai Jiaotong University. NSCs were collected from the E16 embryos of SD transgenic rats carrying the gene for green fluorescent protein [15,26]. Forebrains were dissected using fine forceps, transferred separately into two 10-ml tubes that contained growth medium (mitogen-free), and near-single cell suspensions were obtained by dissociation with gentle mechanical trituration. The dissociated cells were filtered through a cell strainer (BD Falcon, Two Oak Park, Bedford, MA, USA) and seeded into a T25 culture flask (Corning Inc., Corning, NY, USA) at a density of 0.1 × 106 cells/mL. Cells were grown in 1× Dulbecco’s Modified Eagle’s Medium/F12 (DMEM/F12; Gibco), 1× N2 supplement (Gibco), 0.06% glucose, and 2 mM glutamine (Gibco) supplemented with epidermal growth factor (20 ng/mL), basic fibroblast growth factor (20 ng/mL), heparin (2 g/mL; Sigma, St. Louis, USA), and 2% B27 supplement (Gibco). All cells were incubated in a humidified atmosphere of 5% CO2 at 37 ◦ C. NSCs grown in spheres (50–200 cells/sphere) were passaged by mechanical dissociation once every 3–4 days. For passaging, cells were centrifuged (1000 rpm, 10 min) at 4 ◦ C. The cells were resuspended in 200 L medium and mechanically dissociated into single cells by triturating 100 times with a 200-L Pipetteman, which produced near-single cell suspensions with an average viability over 90%, determined by Trypan Blue staining. Cells were then reseeded at 0.1 × 106 cells/mL. At 24 h after MCAO/R, rats were anesthetized and the right CCA, ICA, and PPA were exposed, and clamping transiently blocked the CCA and PPA. Then, 200 L of NSC suspension (2 × 106 cells) and 200 L of NCS growth medium were injected at the beginning of ICA in the MCAO/R + NSC group and MCAO/R + medium group, respectively. The injection was completed within 5 min. Muscular pieces and medical glue were used to close the wound in the ICA followed by removal of the clamp and wound closure. In the sham + NSC group, animals were anesthetized and the right CCA, ECA, ICA, and PPA were exposed, and the CCA, ECA, and PPA were transiently
2.3. Assessment of neurological function Immediately before and following infusion of NSCs and at 1, 2, 3, 7, 10, and 14 days after transplantation, the behavior and neurological function of the rats in all treatment groups were evaluated. Neurological function was assessed using a 14-point neurological function severity scale (NFSS) in which 1 point was awarded for failure to perform a task or for the absence of a tested reflex [11]. Neurological function assessments included the following: forelimb bending, hind limb bending, and raising the head up at an angle of >10◦ in 30 s (all after pulling the rats up by the tail); ability to walk straight, circularly move toward the hemiplegic side, or fall toward the hemiplegic side (all after placing the rat on the floor); presence of abnormal movement such as immobility and staring, tremor, myodystony, irritability, or seizures; sense of touch or sight; proprioception; Pinna reflex; corneal reflex; startle reflex. Severe neurological dysfunction was defined as a score of 10–14, moderate neurological dysfunction as a score of 5-9 and mild neurological dysfunction as a score of 1–4. 2.4. Pathological examination
2.5. Statistical analysis Mean ± standard deviation (SD) NFSS scores for given follow-up days were graphed as a line plot. A two-sample t-test was performed to compare the mean change in NFSS score from pre-OP to post-OP conditions between two groups. Differences between groups over time were compared using repeated measurement analysis of variance (ANOVA). Statistical assessments were twotailed and considered significant at p < 0.05. Statistical analyses were performed using SPSS 15.0 statistics software (SPSS, Inc., Chicago, IL, USA).
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Fig. 1. Mean ± SD change in NFSS scores for MCAO/R + NSC (closed circles) and MCAO/R + medium (open circles) transplantation groups (n = 8) at various times post-OP, including Day 0 (assessed immediately post-OP), and Days 1, 2, 3, 7, 10, and 14. The p-value represents the difference of NFSS scores over time between groups, analyzed using repeated measurement ANOVA; ***denotes significant differences in change of NFSS scores between pre-OP and post-OP groups (p < 0.001).
3. Results Rats were assigned to 3 treatment groups. Eight rats were in the MCAO/R + NSC group, 8 in the MCAO/R + medium group, and 5 in the sham + NSC group. The mean body weight of the rats in each group was 265.5 ± 8.5 g, 269.4 ± 9.5 g, and 264.4 ± 10.6 g, respectively. The effect of NSCs on brain function was assessed by comparing NFSS scores for MCAO/R + NSC, MCAO/R + medium, and sham + NSC groups over a 14-day post-OP period. The mean from pre-OP values in NFSS score for the MACO/R + NSC and MACO/R + medium groups on Day 0 (immediately post-OP), and on Days 1, 2, 3, 7, 10, and 14 are presented in Fig. 1. NFSS scores immediately post-OP on Day 0 increased to a similar extent for both the MCAO/R + NSC and MCAO/R + medium groups. At Days 10 and 14, the mean change in NFSS score was significantly lower in the MACO/R + NSC group than in the MACO/R + medium group (p < 0.001). In a repeated measure ANOVA, the difference between the mean change in NFSS score for the MCAO/R + NSC group and that for the MCAO/R + medium group was not significant (p = 0.105). The mean NFSS score remained unchanged (from pre-OP) values for the control sham + NSC group at all post-OP time points assessed (data not shown).
The ability of NSCs to restore neurological function was also assessed by examining the number of rats that showed a shift in severity of dysfunction over a 14-day period. Seven of 8 rats and 1/8 rats in the MCAO/R + NCS and MCAO/R + medium groups, respectively, had a moderate or severe level of neurological dysfunction, respectively, on Day 0 immediately following transplantation. A clear trend toward improvement in neurological function was evident in the MCAO/R + NCS and MCAO/R + medium groups over the subsequent14-day post-OP period. More rats in the MCAO/R + NCS group showed a shift toward mild dysfunction (a measure of neurological improvement) than did rats in the MCAO/R + medium group. On Days 7, 10, and 14 post-OP, 3/8, 8/8, and 8/8 rats in the MCAO/R + NCS group, respectively, had mild dysfunction whereas only 1/8, 3/8, and 4/8 rats in the MCAO/R + medium group, respectively, were in this category. As expected, 5/5 rats in the sham + NSC group had an NFSS score of 0 when assessed immediately postOP on Day 0. None of the rats in this group showed a changed in neurological function at any time post-OP from Day 0 through Day 14. 3.1. Pathological examination As shown by hematoxylin–eosin staining (Fig. 2), rats in the MCAO/R + NSC and MCAO/R + medium groups had a shrinkage of neurons in the frontal and parietal lobes and at the location in the caudate and putamen that was fed by the right MCA. These neurons were irregular and eosinophilic in the cytoplasm. Their nuclei were darkly stained or absent and cellular interstitium was loose. Hemorrhage was absent. By contrast, in the sham + NSC group, the brain was intact, the neurons were full in the cell body, and the nuclei were located at the center. As shown by immunofluorescence staining (Fig. 3), rats in the MCAO/R + NSC group had NSCs with green fluorescence in the frontal and parietal lobes and caudate and putamen; some NSCs had started to differentiate into neurons (positive for -tubulin-III, red fluorescence) and astrocytes (positive for GFAP, red fluorescence). In the left hemisphere, NSCs with green fluorescence were absent but normal neurons and astrocytes were observed. In the MCAO/R + medium group, NSCs with green fluorescence were not found in bilateral hemispheres. Normal neurons and astrocytes were found in the left hemisphere, and the neurons and astrocytes in the frontal and parietal lobes, caudate, and putamen were loosely distributed. In the sham + NSC group, NSCs with green fluorescence
Fig. 2. Hematoxylin–eosin stained 5 m sections of pre-frontal lobe, caudate, and putamen of rats from the MCAO/R + NSC, MCAO/R + medium, and sham + NSC groups.
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Fig. 3. Immunofluorescence of brain sections from rats in the MCAO/R + NSC (left column), MCAO/R + medium (middle column), and sham + NSC (right column) groups. Green fluorescence showed NSCs expressing GFP. Neurons (positive for -tubulin-III) and astrocytes (positive for GFAP) showed red fluorescence. Images were captured under an Olympus IX 70 fluorescence microscope. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
were not found but normal neurons and astrocytes were noted in the bilateral hemispheres. 4. Discussion Ischemic rats transplanted with NSCs displayed significant improvement in neurological function at 10 and 14 days following transplantation. Rats transplanted with NSCs shifted from severe/moderate neurological dysfunction to mild dysfunction between 7 and 14 days post-OP. These results demonstrate that IA transplantation of NSCs promotes functional neurological recovery after cerebral ischemia. Statistical analysis (i.e., paired data analyzed by a two-tailed Student’s t-test) followed the approach taken in other studies of a similar nature [15,26]. A statistical analysis of the comparison between groups at each time point was performed and presented as the change from pre-OP to post-OP. Because all values of pre-OP were zero, the changes are equal to values for post-OP. A twosample t-test was therefore a valid statistical approach. To account for multiple comparisons, a repeated measurement ANOVA was performed for the trend of NHSS. This analysis showed no significant difference between the 2 groups (p = 0.105). A post hoc analysis was not carried out because the ANOVA did not show significance. Although ANOVA may not be ideal for the analysis of data based on scoring, a previous study of a similar nature to that described here used ANOVA to compare Basso, Beattie and Bresnahan (BBB) open-field locomotion Locomotor Rating Scale scores [26]. In ischemic rats that had been transplanted with NSCs, hematoxylin–eosin staining showed neuronal degeneration and necrosis and immunofluorescence staining revealed that NSCs with green fluorescence had migrated to the frontal and parietal lobes,
caudate, and putamen. Some of these cells had begun to differentiate into neurons and several NSCs started to differentiate into astrocytes. Following NSC transplantation for the treatment of ischemic stroke, the transplanted NSCs migrate into the ischemic region, survive, and differentiate into astrocytes and neurons. The specific mechanisms whereby transplanted NSCs enter, migrate, and distribute in the ischemic region following transcarotid artery transplantation remain poorly understood. At approximately 5–6 h after ischemic stroke, the blood brain barrier opens, an occurrence that may partially account for the entry of NSCs into the brain. Cellular adhesion molecules are secreted in the local microenvironment, facilitating the migration of inflammatory cells and possibly also transplanted NSCs into the ischemic region [24]. The relatively small number of NSCs that migrate into regions of ischemic injury appears to be insufficient to account for the reversal of neurological dysfunction following ischemic stroke [4]. In the present study, the NSCs migrating into the brain were not quantified. In a previous study [11], approximately 21% of transplanted NSCs migrated into the ischemic region, and of these about 10% differentiated into astrocytes and about 1% into neurons. The expression of biomarkers (e.g., glial activation, oxidative stress, or neuronal injury) has been measured in previous studies as a way of tracking the migration of transplanted cells in ischemic injury models [10]. Future studies in the model of ischemic injury described here could include such biomarkers as measures for quantifying the migration of NSCs. Stem cell therapy may provide trophic support to the injured tissue, fostering neurogenesis and angiogenesis [9]. Thus, paracrine effects of secreted or released factors (e.g., nerve growth factor, salvianolic acid B), rather than direct cell replacement, may be
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responsible for most of the benefits observed after cell transplantation [2,12,22,23,28]. Alternatively, studies using the well established MCAO/R model could be designed to explore the role of specific signaling pathways (i.e., the Wnt/-catenin pathway) that have been shown to control the expansion of the stem cell pool in the adult brain [20]. This study did not examine infarct volume, a critical factor that has been shown to correlate strongly with behavioral improvement and neuronal sparing in the MCAO rat stroke model [25]. It will be important to determine whether lesion volumes are similar between treatments. More extensive histological assessments could be done to quantify neuron cell death (i.e., using TUNEL staining) after NSC transplantation [2]. Future studies could be aimed at quantifying the proportion of neurons versus glia that are generated by NSC transplantation and if cells that had migrated from the blood show a similar differentiation pattern. Currently, the optimal time of NSC transplantation following ischemic cerebral injury is a matter of controversy. This study did not assess the effect of the time interval between the MCAO/R procedure and transplantation on the ability of NSCs to restore neurological function. NSC transplantation was carried out at 24 h after MCAO/R. This may be the optimum time interval for transplantation on the basis that it coincides with the time of opening of the blood–brain barrier following ischemic stroke. Several reports emphasized that NSC transplantation should be performed as soon as possible during or after ischemic brain injury (i.e., ≤24 h) whereas others revealed that the survival rate of transplants is greatest when transplantation is performed at about 7 days after injury when [5,9–11,18,19] the levels of neurotoxic substances are reduced, neurotrophic factors are released, and angiogenesis occurs. Thus, some physicians have recommended stem cell transplantation at ≥7 days after injury [10,18]. The results of the present study suggest that the migration of NSCs to ischemic brain regions is potentially associated with an improvement in neurological function. The effect of dosage, treatment time point, and observation period on the migration and differentiation of NSCs and on the extent of neurological improvement in cerebral ischemia are important areas for further research. Conflict of interest statement The authors declare that they have no conflict of interest. Acknowledgements We would like to express our gratitude to Sai-Li Fu and Zheng-Wen Ma in Laboratory of Neurobiology, Shanghai Jiaotong University, Shanghai, China for their generous help. References [1] J.M. Abrahams, S. Gokhan, E.S. Flamm, M.F. Mehler, De novo neurogenesis and acute stroke: are exogenous stem cells really necessary? Neurosurgery 54 (2004) 150–156. [2] G. Andsberg, Z. Kokaia, A. Björklund, O. Lindvall, A. Martínez-Serrano, Amelioration of ischaemia-induced neuronal death in the rat striatum by NGF-secreting neural stem cells, European Journal of Neuroscience 10 (1998) 2026–2036. [3] X. Bao, J. Wei, M. Feng, S. Lu, G. Li, W. Dou, W. Ma, S. Ma, Y. An, C. Qin, R.C. Zhao, R. Wang, Transplantation of human bone marrow-derived mesenchymal stem cells promotes behavioral recovery and endogenous neurogenesis after cerebral ischemia in rats, Brain Research 1367 (2011) 103–113. [4] J. Chen, P.R. Sanberg, Y. Li, L. Wang, M. Lu, A.E. Willing, J. Sanchez-Ramos, M. Chopp, Intravenous administration of human umbilical cord blood reduces behavioral deficits after stroke in rats, Stroke 32 (2001) 2682–2688.
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