Ultrasound-Microbubble Transplantation of Bone Marrow Stromal Cells Improves Neurological Function after Forebrain Ischemia in Adult Mice
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- Gong, Z., Ran, H., Wu, S. et al. Cell Biochem Biophys (2014) 70: 499. doi:10.1007/s12013-014-9947-y
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In this study, bone marrow stromal cells (MSCs) were transplanted into the brain of adult rats after forebrain ischemia induced by 4VO. SD rats (n = 60) were randomly divided into three groups: (I) rats (n = 20) were subjected to 4VO and transplanted with MSCs into the ischemic region using ultrasound-microbubble method, (2) rats (n = 20) were subjected to 4VO and transplanted with MSCs into the ischemic region (n = 20), and (3) 4VO alone (n = 20). Rats were sacrificed 28 days after treatment. Neurological functions of rats were evaluated by Morris Water Maze. The current findings suggest that the ultrasound microbubble transplanted MSCs survived in the ischemic brain and significantly improved functional recovery of adult rats compared to regular transplantation.
KeywordsBone marrow stromal cellsUltrasound microbubbleForebrain ischemia
Previous study has showed that intracerebrally transplanted embryonic mesencephalic precursors could provide therapeutic benefit in rat model of Parkinsonian disease . Previous studies showed that host-derived signals play a key role in directing differentiation of embryonic cells by demonstrating embryonic cells can differentiate into various phenotypes when placed in different regions of the brain [2, 3]. When grafted into homologous regions of adult animal model of cerebral ischemic infarction, grafted fetal tissue establishes extensive interconnections with the host [4, 5]. However, the application of transplantation in the treatment of neurodegeneration [6, 7], brain injury, and brain ischemia [8, 9] is restricted because of ethical and logistical problems. Therefore, studies have been focused on the alternatives to human fetal tissue. A few studies have highlighted the potential of bone marrow cells as a source for cell transplantation therapy [10, 11].
Adult BM contains stem and progenitor cells, which have multiple differentiation potentials and give rise to blood elements [12, 13]. In addition to stem and progenitor cells, bone marrow also contains a subset of heterogeneous non-hematopoietic cells, marrow stromal cells (MSCs) . These MSCs are regarded as hematopoietic support cells and a source of continual renewal of cells in a number of non- hematopoietic tissues . Furthermore, MSCs have attracted interest because of their ability to differentiate into various cell types and potential for gene therapy [15–19]. In vitro studies demonstrated that adult rat and human BM stromal cells can differentiate into neurons . MSCs can be induced to differentiate into astrocytes, microglia and macroglia in the brain of adult mice [10, 21, 22]. After injection into neonatal mouse brain, MSCs migrate throughout forebrain and cerebellum, and differentiate into astrocytes and neurons . Collectively, these results have led to the hypothesis that MSCs have the potential to attenuate neurological injury.
A few previous studies have reported that MSCs delivered to ischemic brains of animals were therapeutically beneficial [22, 24–26]. However, the effect of MSCs graft on neurological function recovery in ischemic brain remains to be investigated. In the current study, the authors tested the hypothesis that intracranial transplantation of adult MSCs into adult rat model of forebrain ischemia could restore the neurological function of ischemic damaged tissue.
Materials and Methods
Cell isolation and Cell Culture
Rats were treated with 5-fluorouracil (5-FU, 15 mg/kg) 48 h before primary cultures of BM were obtained as previously described . Briefly, fresh BM was harvested aseptically from tibias and femurs, and was then mechanically dissociated to form a milky homogenous single-cell suspension. Red blood cells were removed with NH4Cl and nucleated cells were cultured in Iscove’s Modified Dulbecco’s medium (IMDM) supplemented with 10 % fetal bovine serum (FBS) and stem cell factor (SCF, 100 ng/ml). After 72 h, non-adherent cells and adherent cells MSCs were collected, and MSCs were easily isolated in the medium by their tendency to adhere to plastic. The culture was supplemented with NGF (200 ng/ml) and cells were kept for 14 days for serial passages and cells of 2nd and 3rd passage were used for transplantation. Cells were isolated by treating with 0.25 % trypsin and 0.5 mM EDTA at 37 °C for 10 min at room temperature and washed three times with PBS before transplantation.
All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and approved by the Institutional Animal Care and Use Committee of Chongqing Xinqiao Hospital of Third Military Medical University, China. A total of 60 male SD star rats (Experimental Animal Center, Third Military Medical University) weighing 250–260 g were used for this study. 20 rats were randomly allocated into each experimental group: ultrasound microbubble transplantation group, transplantation group, and forebrain ischemia group. Severe forebrain ischemia was produced by a modified 4-VO surgery . Briefly, under sodium pentobarbital (50 mg/kg body weight i. p.) anesthesia, a ventral midline cervical incision was performed to expose both common carotid arteries. A silk thread was loosely placed around each artery without interrupting carotid blood flow, and the incision was closed with a single suture. A second incision was made in the dorsal midline of the neck, and both vertebral arteries were electrocauterized through the alar foramen of the first cervical vertebra. After 24 h, both common carotid arteries were reexposed and occluded with aneurismal clips to induce forebrain ischemia for 20 min under light ether anesthesia. Then, the clips were released and blood flow was restored. Rats that became unresponsive and lost the righting reflex during bilateral carotid artery occlusion and that showed no seizure during and after ischemia were considered to have met the criteria for adequate ischemia and were used for the experiment .
Focused Ultrasound-Induced Blood–Brain Barrier Opening
Blood brain barrier was opened as previously described and a FUS transducer (Imasonics, Besancon, France) was used in this study . SonoVue® SF6-coated ultrasound microbubbles (Bracco Diagnostics Inc., Milan, Italy) were injected through vein in tail before treatment (0.1 ml/kg bolus of microbubbles mixed with 0.2 ml of saline). The rat brain site was then exposed to burst-tone mode ultrasound to locally open the BBB for 5 min .
Transplantation of MSCs
All transplantation procedures were performed under aseptic conditions. Intracerebral transplantation of MSCs was carried out according to the methods described by Goto et al. . Only ultrasound microbubble transplantation group and transplantation group rats received transplantation.
Morris Water Maze Test
Morris water maze test was performed to evaluate the spatial learning performance as previously described [32, 33]. The Morris water maze consisted of a circular pool (130 cm diameter, 50 cm height, and 30 cm depth) filled with water at 25–27 °C. The water surface was covered with floating white milk, and the head of each rat was dyed black. Before training, a 180 s free swim trial was run without the platform. For training, a submerged platform (12 cm diameter, 29 cm height) was fixed 1.5 cm below the surface of the water in the center of a quadrant. A video camera positioned 3 meters above the center of the pool was used to record the swimming distances (cm) and escape latency (second). A video camera mounted at the height of 180 cm above the center of the maze and data were stored in a computer system. Rats were allowed to swim freely for 2 min to adopt themselves to the environment. Each animal participated in 4 trials each morning and 4 trials each afternoon for 4 days. Each animal was given 60 s to reach to the platform, upon which it remained for 10 s. If the platform was not located within 60 s the animal was placed on it by the experimenter. The next trial started immediately after removal from the platform. After completion of the all trials, the animal was placed in its home cage. Escape latency and path length to find the hidden platform were recorded.
Spatial memory was evaluated 5 days after the completion of the learning performance tests using the probe test. The platform was removed, and the animals were placed in a novel starting position. The time spent in quadrant 4 (platform removed) was calculated as the index for spatial memory.
All data were presented as mean ± standard deviation and analyzed using one-way ANOVA methods. SPSS 10.0 software was used for statistical analysis. The level of significance was set as P < 0.05.
FUS-Induced BBB Opening
Morris Water Maze Results
Results of probe test (Platform located in quadrant III)
Times of passing target
Percentage of time in each quadrant (%)
7.34 ± 3.1
12.73 ± 3.0
22.97 ± 8.24
45.82 ± 4.27
17.62 ± 4.59
7.53 ± 3.56
12.16 ± 2.13
20.34 ± 3.58
45.21 ± 6.08
21.66 ± 2.97
6.82 ± 4.6
13.87 ± 2.48
22.32 ± 4.37
46.22 ± 4.45
17.02 ± 2.52
Currently, cerebral stroke is the leading cause of disability and mortality and cerebral ischemia accounts for the majority of cerebral stroke. However, treatment of cerebral ischemia is unsatisfactory. Cell transplantation has attracted more and more attention as a strategy of neurological function recovery following ischemia. MSCs are a subgroup of bone marrow cells in addition to hematopoietic stem cells. MScs have potential to differentiate into a various cells, which make it a promising cell for transplantation. Previous studies have showed that MSCs are: (1) easy to isolate from the small aspirates of bone marrow that can be obtained under local anesthesia, (2) capable of rapid proliferation in culture, (3) immunologically inert, (4) capable of transfection and stably expressing exogenous gene, and (5) amenable to survival and integration in the host brain.
Intracranial transplantation of MSCs has been tested on animal models, showing promising results. Li et al.  performed intrastriatal transplantation of MSCs on stroke adult mice established by occlusion of middle cerebral artery (MCAO). 28 days, later, MSCs were found to migrate 2.2 mm toward ischemic region with 1 % cells expressing NeuN and 8 % cells expressing GFAP, indicating that MSCs differentiated into neurons and neuroglial cell. In addition, experimental groups showed better neurological recovery compared with control group. Then, MSCs were transplantation through vein in the mice tail, which did not induce the production of cytotoxic T lymphocyte from the spleen. 14 days after treatment, the mNSS (including motor, sensory and reflex) were significantly improved compared with control group (mice did not receive hMSC or receive hepatic connective tissue cells instead of hMSC) (P < 0.05). hMSC were labeled with mAb1281 with 1 % of the cell population expressing NEuN, 1 % expressing MAP-2, 5 % expressing GFAP, and 2 % expressing vWF. Results from TUNEL and HE staining showed that apoptotic cells decreased surrounding ischemic region while VZ/SVZ Brdu (+) cells were significantly increased. Moreover, secretions of BDNF and NGF from the brain were significantly elevated at 7 days after treatment (P < 0.05) .
In present study, we found that FUS could significantly promote the migration of MSCs into brain, leading to improvement of the neurological functions of rats. Compared with transplantation group and control group (forebrain ischemia model), ultrasound microbubble transplantation group showed significant improved spatial learning capability, while difference was found in spatial memory, indicating that MSCs were therapeutic beneficial for neurological function recovery following forebrain ischemia.
The mechanism how ultrasound microbubble induces blood brain barrier (BBB) opening is unclear now. However, a few theories have been proposed: (1) ultrasound-induced contraction and dilation will produce mechanical polvent drag, leading to the opening of contact region; (2) ultrasound-induced change in internal pressure of capillary will cause associated biochemical reaction, resulting in opening of BBB; (3) ultrasound-induced local movement of microbubbles will elicit the microjet and pulse from local sensor, which then induce the reversible opening of BBB; and (4) ultrasound with microbubbles causes the contraction of blood vessels and consequently temporarily local ischemia around sensor, leading to temporary opening of BBB.
Based on previous studies, scientists believed that the neuroprotective effect of MSCs on ischemia-refusion injury was caused by the following mechanisms: (1) MSCs could replace the dead neurons. MSCs are stem cells, which can express NeuN, GFAP, MAP-2 although at low level when transplantation into brain. However, whether the small amount of differentiated cells could replace the neurological function need to be investigated. (2)The interactions between MSCs and its surrounding neural tissues induces the production of cellular factors, such as NTF, cytokines (including IL-6, 7,8,11,12,14 and 15), CSF, Fit-3 ligand, and Stem Cell Growth Factors, which is essential to the survival, growth and/or differentiation of hippocampal neurons. In addition, MSCs could secrete catecholamines and release special neurotransmitters [35, 36]. All these cellular factors can promote the neurological function. Following cerebral ischemia, all kinds of neural factors such as BDNF, bFGF, GDNF, NGF, and TGF-β1 was induced, which facilitated the survival, proliferation, and migration of MSCs, decreased the cell apoptosis in ischemic penumbra, increased the proliferation and differentiation of the endogenous neural stem cells and progenitor cells [10, 34, 37]. Previous studies have found that NTF could promote the development of neural, synapse formation and neural signal transmission and neurotransmitters release . (3) promoting angiogenesis or directly involved in angiogenesis. Li et al.  have treated local cerebral ischemia by MSCs transplantation and used histochemical VIII afctor staining and FVIII staining to detect the density of local microvessels. Their results indicated that the density of microvessels around ischemia region was significantly increased compared with control group. Chen et al.  proposed that transplantation of BMSCs increased the expression of VEGF in ischemia region, leading to the proliferation of endothelial cells and angiogenesis. However, Hess et al.  proposed that part of BMSCs differentiated into endothelial cells and directly participated in the process of angiogenesis.
In conclusion, our results demonstrated that ultrasound microbubble significantly promoted the migration of MSCs to intracranial space, leading to the improvement of neurological function of rats with forebrain ischemic injury. Intracranial transplantation of MSCs showed therapeutic beneficial on spatial learning, while no effect on spatial memory. MSCs are easy to obtain with rich sources and rapidly proliferation. Therefore, MSCs are ideal candidate for clinical applications. However, the mechanisms how ultrasound mircrobubbles open BBB and how MSCs promote the recovery of neurological function need further study.
This study was supported by Chongqing Natural science Foundation CSTC201JJA10011, National Natural Science Foundation Nos. 81271331 and 81000507.