Korean Journal of Cerebrovascular Surgery 2007;9(4):277-285.
Published online December 1, 2007.
Increase of Angiogenesis and Neurogenesis, and Neurobehavioral Recovery by Transplanted Mesenchymal Stem Cells in the Ischemic Rat Brain.
Lee, Woo Sung , Huh, Pil Woo
Department of Biomedical Science, 1Department of Neurosurgery, Graduate School, The Catholic University of Korea, Korea.
Abstract
OBJECT: In this study, murine mesechymal stem cells (MSC) was grafted to cerebral ischemia rats, the differentiation of the transplanted MSC was assessed, and at that time, the association of the level of the recovery of neurological function. METHODS: As experimental animals, the transient middle cerebral artery occlusion (MCAo) rat model was used, and MSC collected from the bone marrow of C57/Bl6 mice were cultured, 5microliter(105 microliter) cells marked with the fluorescent substance 3.3'-dilinoleyloxacarbocyanine(DIO) were transplanted into the ischemic cortex. Angiogenesis and neurogenesis were examined and the neurobehavioral evaluation were done. To assess the differentiation level of MSC, and it was evaluated as the percentage of the mean area of stained cells within each region of interest (ROI). RESULTS: In the MCA transplanted group, Neurobehavioral score was improved to twelve points one hour, one day, and three days after transplant, and seven days and fourteen days after transplant, to eleven points and nine points, respectively. The result of immunohistochemical staining showed that the angiogenesis on the day seven was 3.15% and on the day fourteen was 4.26%, the neurogenesis on the day seven was 2.29% and on the day fourteen was 3.82%. CONCLUSION: On the day seven and fourteen after MCS transplantation, the phenomenon of the actively ongoing angiogenesis and neurogenesis was detected, and it was found that at that time, the recovery of neurological function occurred. However, the level of differentiation from MSC was small, and thus it was not statistically significant.
Key Words: Mesenchymal stem cells, Cb.infraction, Angiogenesis, Neurogenesis

Introduction 


  
With the expectation that cell transplant would be of help to brain damage caused by cerebral infract, numerous investigators are conducing research. Reviewing the result of experimental animals, Hodges et al. showed that in a global ischemia model, fetal hippocampal tissues were transplanted to the C1 area of the damaged hippocampus, graft-host connectivity was formed and the recovery of function in a certain level was confirmed.1)2) In addition, in several experiments using fetal brain cells, a certain level of success was observed, nonetheless, because of the basic problem of the difficulty of obtaining human brain tissues that could be transplanted, other cells were explored.3) 
   Explosive development of stem cell biology in the last few years allows the culture and differentiation of stem cells in vitro and elevates the interest on rehabilitation medicine to rehabilitate damaged tissues and organs using such cells. The mammalian central nervous system lacks the ability to regenerate, and thus it has been considered that spontaneous restoration of lost nervous functions caused by diverse neurological diseases is difficult to expect. Therefore, more interests were focused on the development of the therapeutic methods for neurological diseases using stem cells than diseases in other organs. From the late 19th century, some investigators have speculated that in bone marrow, in addition to hematopoetic blasts, so called mesenchymal stem cells that form the bone, the cartilage, and the muscle may be present, and their presence was confirmed by the experiment performed by Friedenstein in 1976.4) It has been reported that bone marrow stromal cells could generate the bone, the cartilage, the muscle, hepatocytes, neurons and glia cells.5)6)7) 
   It has been reported that upon direct administration of bone marrow stromal cells stria vascularis, to the stria vascularis, the carotid artery and the vein, they migrate to the ischemic region in rats directly, differentiate to neurons and glial cells, and improved neurological function.8)9)10) 
   In our study, in an ischemic rat brain model, it was examined whether murine MSC transplanted to the ischemic cortex could survive in the brain, differentiate to which direction between angiogenesis and neurogenesis, and such phenomena influence the recovery of neurological function with time. 

Materials & Methods 

1. MSC harvest 
  
C57/Bl6 mice femur and tibia were harvested, and bone marrow was collected by flushing with PBS. The bone marrow was mixed with Histopaque-1077 at 1:1 ratio, centrifuged at RT, 1,800rpm for 30min, the MN cell layer was separated, and cultured in DMEM (high glucose) medium (10% FBS + 1% antibiotics). One week after the harvest, their serial culture was initiated. After 3
~5 times serial culture, they were used in experiments. 

2. Surgical preparation for MCA occlusion 
  
Fifty adult male Sprague-Dawley rats (220
~240g, Orient, Korea) for MCAo were used in these studies. All protocol were approved by the Catholic University of Korea Medical College's Animal care and Use Committee. Rats were fasted overnight but allowed free access to water. Following intraperitoneal atropine sulfate (0.5mg/kg), anesthesia was induced with isoflurane (Fluovac, Harvard apparatus, England) in a mixture of 70% nitrous oxide and a abalance of oxygen. Rats were intubated, immobilized with pancuronium bromide (0.6mg/kg. i.v.), and mechanically ventilated with a artificial ventilator (Ventilator animal, Ugo Basile, USA). Temperature probes were inserted into the rectum and left temporalis muscle (Homeothermic Blanket Control Unit, Harvard Apparatus, England), and separate head heating lamps were used to maintain rectal and cranial temperatures at 37~37.3 . Polyethylene catheters were introduced into the right femoral artery and vein for blood pressure monitoring, drug infusion, and blood sampling. Mean arterial blood pressure (MABP) was measured via an indwelling femoral arterial catheter connected to a precalibrated pressure transducer (Advanced blood pressure monitor, Harvard apparatus, England) and was recorded continuously. Serial measurements were made of arterial blood gases and pH (V.M.C machine, MDS Matrix medical, USA), plasma glucose. 

3. MCA occlusion and transplantation of MSC 
  
This MCAo model follows that developed by Koizumi and colleagues11) and Nishino and colleagues12) with minor modifications.13)14) In Brief, the right common carotid artery (CCA) was exposed through midline neck incision and dissected free of surrounding nerves; the occipital branches of the external carotid artery (ECA) were coagulated; and the pteryropalatine artery was ligated. A 4cm length of 3-0 monofilament nylon suture was then inserted via the proximal ECA into the internal carotid artery (ICA) and MCA, a distance of 19
~20mm from the CCA bifurcation according the animal's weight, thereby occluding the MCA. Prior to use, the tip of the suture was heat-blunted, and a 20- mm distal segment of the suture coated with poly-L-lysin solution (0.1%[w/v]) and dried at 60 for one h. After suture placement, the neck incision was closed, and animals were allowed to awaken from anesthesia. After two hour of MCAo, rats were reanesthetized, temperature probes were reinserted, and the intraluminal suture was carefully removed. The transplantation of MSC into the ischemic cortex using a stereotactic guide system was done after two hour of MCAo and reperfusion. The transplanted site of ischemia area was navigated on the stereotatic flame 4mm from bregma posteriorly, 3mm from midline right laterally, and 3mm deep from cortical surface. Using 26-gauge Hamilton syringe, 5X105/5㎕ MSC were infused in 5 minutes (Fig. 1). 

4. Neurobehavioral evaluation 
  
To examine whether ischemic stroke animals transplanted adult stem cells indeed improved neurologically, they were examined by comparing with the control group. After the establishment of the MCAo model, neurolobehavioral evaluation was performed after one hour, one day, three days, seven days, and fourteen days. According to the protocol of our laboratory for evaluating the postural reflex, the placing sense, proprioceptive placing test (Table 1), in each category, normal was scored as 0 point, and other cases were scored as 1 point or 2 points (Table 1). Neurological Evaluation Protocol of Rats in MCAo Model. 

5. H-E stain 
  
To assess transplanted MSC, H-E staining was performed. Tissue sections were deparaffined, hydrated, treated with harris hematoxylin for 7 minutes, and immersed in acid alcohol (1% HCL) several times. After changed blue, immersed in Eosin Y for 20
~30 seconds. After sealing, examined (Fig. 1). 

6. Immunohistochemical staining method 
  
As immunohistochemical staining method, brain tissue sections were treated with 3% hydroxidase peroxide for 30 minutes to block endogenous peroxidase, a blocking step was performed for two hours, and the blocking solution consisted of 10% normal horse serum (NHS), 1% bovine serum albumin (BSA), and 0.3% tritonX-100. Reacted with the first monoclonal antibody anti-rabbit-vWF (1:5000,Chemicon,USA) and anti-rabbit-GFAP (1:500, DAKO Cytomation, Denmark) at 4 for 16
~18 hours and washed. Treated with the second antibody, anti rabbit-Alexa Fluor 555 (1:5000, Molecular Probes, USA), at room temperature for two hours, sealed and examined. For the analysis of results, under microscope, 5 ROIs in the cortex where MSC was transplant were selected, the mean area was calculated, and the mean area of stained cells within each ROI was evaluated by the percentage of the mean area of ROIs (mean area of stained cell of ROI/mean area of ROI%) (Adobe Image ready7.0). 

Results 

1. Neurological behavior 
  
After one hour, both PBS group and MSC group were 12 points, and identical scores were obtained. However, after seven days, the experiment group was 9 points and 7 points, and differences began to be shown noticeably (Fig. 2). 

2. Increase of angiogenesis 
  
The result of immunohistochemical staining of normal rats and the MCAo model at each time point was compared (Fig. 3). In the PBS injected group, at one hour was 0.38%, one day was 0.74%, three days were 1.19%, seven days were 1.67%, and fourteen days were 2.04%, and in the MSC transplanted group, at one hour was 0.32%, one day was 1.02%, three days were 1.81%, seven days were 3.15%, and fourteen days showed the result of 4.26% (Fig. 5). 

3. Increase of neurogenesis 
  
The result of immunohistochemical staining of normal rats and the MCAo model was evaluated (Fig. 4), the PBS injected group at 1 hour was 0.14 %, one day was 0.57%, three days were 0.91%, seven days were 1.23 %, and fourteen days were 1.85%, and in the MSC transplanted group, at one hour was 0.28%, one day was 0.76%, three days were 1.38%, seven days were 2.29%, and fourteen days were 3.82% (Fig. 5). 

4. Assessment of the survival of MSC 
  
MSC was marked with the fluorescent marker fluorescent DIO and injected to animals. Tissues were assessed at each time point, and the marked MSC could be detected. In comparison with the time of the initial injection, migration of MSC could be observed (Fig. 6). 

Discussion 

   Assuming that MSC could compensate the functional loss of rats caused by ischemic damage, we transplanted MSC directly to the ischemic cortex where regional ischemia occurred. Subsequently, the survival and differentiation of transplanted stem cells were assessed, and at that time, the improvement of neurological function of experimental rats was evaluated by neurobehavioral test. Regarding the choice of the transient MCA occlusion model, and SD rats that the hemiparesis and hemiparesthesia could be induced constantly were determined to be appropriate for the evaluation of neurobehavior associated with survivor of the transplanted adult stem cells. As the ischemic period, upon the reperfusion of the MCA territory, permanent ischemia occurred in more than 60% and thus become a useful condition for cell transplant, and to rule out the possibility of the functional recovery by reperfusion and the recovery of blood flow by the collateral circulation, two hours MCA occlusion period was selected. 
   In other experiments, diverse types of cells have been used experimentally. The representative examples are human mesenchymal stem cell,15) human cord blood cell,16) the neuroepithelial cell MHP36 generated in an embryonic mouse,17)18) neurons originated from human monster blastoma,19)20) etc. However, we obtained MSC from murine femur and tibia and performed experiments. The reason of using mouse MSC was that it is easy to handle, easy to obtain, its culture ability is excellent and thus serial culture could be performed readily, and its immunity to rats is absented thus used. 
   MSC marked with the fluorescent DIO was injected, and during the experiment period, a constant MSC in each tissue could be assessed. After MSC transplantation, after one day, neurogenesis and angiogenesis was 0.28% and 0.32%, respectively, and the result of neurobehavioral evaluation was 11points, and a noticeable level of the increasement of neurogenesis, angiogenesis, and the recovery of neurological function could not be observed. However, from the day seven, neurogenesis and angiogenesis were 2.29% and 3.15%, respectively, and the result of neurobehavioral evaluation was 9 points, and from the day seven, neurogenesis, angiogenesis, and the recovery of neurological function were shown noticeably, and on the day fourteen, neurogenesis and angiogenesis were 3.82% and 4.26%, respectively, and the result of neurobehavioral evaluation was 7 points, and the marked recovery of neurological function was shown. 
   However, the experiment period was short, fourteen days, and thus by the result of this experiment only, it cold not be confirmed whether it was improved markedly by the injection of MSC. However, the number of cells was not as high as expected, which could be considered that the damage of tissues in the ischemia area influenced the survival of the injected cells. 
   In addition, in the group injected MSC, the level of angiogenesis and neurogenesis could be assessed, and comparing the level of angiogenesis and neurogenesis, the result showed that angiogenesis on the day seven was 3.15% and the day fourteen was 4.26%, and neurogenesis on the day seven was 2.29% and the day fourteen was 3.82%. In both angiogenesis and neuogenesis, on the day seven, marked differences from the control group were shown. In addition, in the comparison of the angiogenesis and neurogenesis of the MSC group, it was found that angiogenesis was progressed slightly more than neurogenesis. However, based on this result alone, the expression direction of transplanted MSC could not be concluded, and concerning the improvement shown in neurobehavioral evaluation, it may be considered that functional recovery was due to substances released by MSC rather than transplanted MSC was integrated to the neurological circuit and functioned. This could be anticipated by the report that grafted cells secreted nerve growth factors without converting to neurons directly,21) and they played a role of stimulating the regeneration mechanism of stem cells already present, reducing cell death, and stimulating nerve regeneration and revascularization.22)23) In the study reported by Zhao et al., one week after local ischemia, human MSC (hMSC) were injected, and after the transplant, neurological improvement could be observed, and after six weeks, the volume of cerebral infarction was not different from the control group. In addition, it has been also reported that it took approximately six weeks for transplanted cells functioning completely.24)25) Based on the result of this experiment, it was confirmed that in future experiments, the period should be provided. 

Conclusion 

   The result our experiments that compared the PBS injected group and the MSC transplanted group directly showed that in neurobehavioral evaluation, the improved pattern in the MSC transplanted group than the control group could be observed. In addition, after MSC transplantation, on the day seven and the day fourteen, the phenomenon of actively ongoing angiogenesis and neurogenesis was confirmed, and it was found that the recovery of neurological function also occurred at that time. However, the difference of the level of angiogenesis and neurogenesis originated from MSC was meager, and thus it was difficult to be considered to be statistically significant. Consequently, it was true that the recovery of neurological function was occurring, nonetheless, between these angiogenesis and neurogenesis, it was difficult to assess with which the recovery of neurological function is associated more. Therefore, in future, the relation of the recovery of neurological function and the differentiation of transplanted cells should be examined 2
~3 months after the transplant of adult stem cells to cerebral ischemic area, and the changed milieu in the vicinity of the ischemia area due to transplanted cells should be examined. 


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