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Adipose-derived mesenchymal stem cells markedly attenuate brain infarct size and improve neurological function in rats.

Leu S, Lin YC, Yuen CM, Yen CH, Kao YH, Sun CK, Yip HK - J Transl Med (2010)

Bottom Line: The results showed that BIA was larger in control group than in treatment group (p < 0.001).Immunohistochemical staining showed that cellular proliferation and number of small vessels were lower but glial fibrillary acid protein was higher in control group than in treatment group (all p < 0.01).ADMSC therapy significantly limited BIA and improved sensorimotor dysfunction after acute IS.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Cardiology, Department of Internal Medicine, Chang Gung Memorial Hospital-Kaohsiung Medical Center, Chang Gung University College of Medicine, Kaohsiung, Taiwan.

ABSTRACT

Background: The therapeutic effect of adipose-derived mesenchymal stem cells (ADMSCs) on brain infarction area (BIA) and neurological status in a rat model of acute ischemic stroke (IS) was investigated.

Methods: Adult male Sprague-Dawley (SD) rats (n = 30) were divided into IS plus intra-venous 1 mL saline (at 0, 12 and 24 h after IS induction) (control group) and IS plus intra-venous ADMSCs (2.0 x 106) (treated interval as controls) (treatment group) after occlusion of distal left internal carotid artery. The rats were sacrificed and brain tissues were harvested on day 21 after the procedure.

Results: The results showed that BIA was larger in control group than in treatment group (p < 0.001). The sensorimotor functional test (Corner test) identified a higher frequency of turning movement to left in control group than in treatment group (p < 0.05). mRNA expressions of Bax, caspase 3, interleukin (IL)-18, toll-like receptor-4 and plasminogen activator inhibitor-1 were higher, whereas Bcl-2 and IL-8/Gro were lower in control group than in treatment group (all p < 0.05). Western blot demonstrated a lower CXCR4 and stromal-cell derived factor-1 (SDF-1) in control group than in treatment group (all p < 0.01). Immunohistofluorescent staining showed lower expressions of CXCR4, SDF-1, von Willebran factor and doublecortin, whereas the number of apoptotic nuclei on TUNEL assay was higher in control group than in treatment group (all p < 0.001). Immunohistochemical staining showed that cellular proliferation and number of small vessels were lower but glial fibrillary acid protein was higher in control group than in treatment group (all p < 0.01).

Conclusions: ADMSC therapy significantly limited BIA and improved sensorimotor dysfunction after acute IS.

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Related in: MedlinePlus

Flow cytometric analysis of rat adipose-derived mesenchymal stem cells (ADMSCs). Flow cytometry results of ADMSCs (the percentage shown in figure was mean value of n = 3) on day 14 after cell culturing showed the CD29 + and CD90+ cells were the highest population of stem cells. Spindle-shaped morphological feature of the stem cells were shown in the right lower corner (200×).
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Figure 1: Flow cytometric analysis of rat adipose-derived mesenchymal stem cells (ADMSCs). Flow cytometry results of ADMSCs (the percentage shown in figure was mean value of n = 3) on day 14 after cell culturing showed the CD29 + and CD90+ cells were the highest population of stem cells. Spindle-shaped morphological feature of the stem cells were shown in the right lower corner (200×).

Mentions: The rats were anesthetized with inhalational isoflurane. Adipose tissue surrounding the epididymis was carefully dissected and excised. Then 200-300 μL of sterile saline was added to every 0.5 g of tissue to prevent dehydration. The tissue was cut into < 1 mm3 size pieces using a sharp, sterile surgical scissors. Sterile saline (37°C) was added to the homogenized adipose tissue in a ratio of 3:1 (saline: adipose tissue), followed by the addition of stock collagenase solution to a final concentration of 0.5 Units/mL. The tubes with the contents were placed and secured on a Thermaline shaker and incubated with constant agitation for 60 ± 15 min at 37°C. After 40 minutes of incubation, the content was triturated with a 25 mL pipette for 2-3 min. The cells obtained were placed back to the rocker for incubation. The contents of the flask were transferred to 50 mL tubes after digestion, followed by centrifugation at 600 g, for 5 minutes at room temperature. The fat layer and saline supernatant from the tube were poured out gently in one smooth motion or removed using vacuum suction. The cell pellet thus obtained was resuspended in 40 mL saline and then centrifuged again at 600 g for 5 minutes at room temperature. After being resuspended again in 5 mL saline, the cell suspension was filtered through a 100 μm filter into a 50 mL conical tube to which 2 mL of saline was added to rinse the remaining cells through the filter. The flow-through was pipetted to a 40 μm filter into a new 50 mL conical tube. The tubes were centrifuged for a third time at 600 g for 5 minutes at room temperature. The cells were resuspended in saline. An aliquot of cell suspension was then removed for cell culture in DMEM-low glucose medium contain 10% FBS for two weeks. Flow cytometric analysis was performed for identification of cellular characteristics after cell-labeling with appropriate antibodies 30 minutes before transplantation (Figure 1).


Adipose-derived mesenchymal stem cells markedly attenuate brain infarct size and improve neurological function in rats.

Leu S, Lin YC, Yuen CM, Yen CH, Kao YH, Sun CK, Yip HK - J Transl Med (2010)

Flow cytometric analysis of rat adipose-derived mesenchymal stem cells (ADMSCs). Flow cytometry results of ADMSCs (the percentage shown in figure was mean value of n = 3) on day 14 after cell culturing showed the CD29 + and CD90+ cells were the highest population of stem cells. Spindle-shaped morphological feature of the stem cells were shown in the right lower corner (200×).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2913939&req=5

Figure 1: Flow cytometric analysis of rat adipose-derived mesenchymal stem cells (ADMSCs). Flow cytometry results of ADMSCs (the percentage shown in figure was mean value of n = 3) on day 14 after cell culturing showed the CD29 + and CD90+ cells were the highest population of stem cells. Spindle-shaped morphological feature of the stem cells were shown in the right lower corner (200×).
Mentions: The rats were anesthetized with inhalational isoflurane. Adipose tissue surrounding the epididymis was carefully dissected and excised. Then 200-300 μL of sterile saline was added to every 0.5 g of tissue to prevent dehydration. The tissue was cut into < 1 mm3 size pieces using a sharp, sterile surgical scissors. Sterile saline (37°C) was added to the homogenized adipose tissue in a ratio of 3:1 (saline: adipose tissue), followed by the addition of stock collagenase solution to a final concentration of 0.5 Units/mL. The tubes with the contents were placed and secured on a Thermaline shaker and incubated with constant agitation for 60 ± 15 min at 37°C. After 40 minutes of incubation, the content was triturated with a 25 mL pipette for 2-3 min. The cells obtained were placed back to the rocker for incubation. The contents of the flask were transferred to 50 mL tubes after digestion, followed by centrifugation at 600 g, for 5 minutes at room temperature. The fat layer and saline supernatant from the tube were poured out gently in one smooth motion or removed using vacuum suction. The cell pellet thus obtained was resuspended in 40 mL saline and then centrifuged again at 600 g for 5 minutes at room temperature. After being resuspended again in 5 mL saline, the cell suspension was filtered through a 100 μm filter into a 50 mL conical tube to which 2 mL of saline was added to rinse the remaining cells through the filter. The flow-through was pipetted to a 40 μm filter into a new 50 mL conical tube. The tubes were centrifuged for a third time at 600 g for 5 minutes at room temperature. The cells were resuspended in saline. An aliquot of cell suspension was then removed for cell culture in DMEM-low glucose medium contain 10% FBS for two weeks. Flow cytometric analysis was performed for identification of cellular characteristics after cell-labeling with appropriate antibodies 30 minutes before transplantation (Figure 1).

Bottom Line: The results showed that BIA was larger in control group than in treatment group (p < 0.001).Immunohistochemical staining showed that cellular proliferation and number of small vessels were lower but glial fibrillary acid protein was higher in control group than in treatment group (all p < 0.01).ADMSC therapy significantly limited BIA and improved sensorimotor dysfunction after acute IS.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Cardiology, Department of Internal Medicine, Chang Gung Memorial Hospital-Kaohsiung Medical Center, Chang Gung University College of Medicine, Kaohsiung, Taiwan.

ABSTRACT

Background: The therapeutic effect of adipose-derived mesenchymal stem cells (ADMSCs) on brain infarction area (BIA) and neurological status in a rat model of acute ischemic stroke (IS) was investigated.

Methods: Adult male Sprague-Dawley (SD) rats (n = 30) were divided into IS plus intra-venous 1 mL saline (at 0, 12 and 24 h after IS induction) (control group) and IS plus intra-venous ADMSCs (2.0 x 106) (treated interval as controls) (treatment group) after occlusion of distal left internal carotid artery. The rats were sacrificed and brain tissues were harvested on day 21 after the procedure.

Results: The results showed that BIA was larger in control group than in treatment group (p < 0.001). The sensorimotor functional test (Corner test) identified a higher frequency of turning movement to left in control group than in treatment group (p < 0.05). mRNA expressions of Bax, caspase 3, interleukin (IL)-18, toll-like receptor-4 and plasminogen activator inhibitor-1 were higher, whereas Bcl-2 and IL-8/Gro were lower in control group than in treatment group (all p < 0.05). Western blot demonstrated a lower CXCR4 and stromal-cell derived factor-1 (SDF-1) in control group than in treatment group (all p < 0.01). Immunohistofluorescent staining showed lower expressions of CXCR4, SDF-1, von Willebran factor and doublecortin, whereas the number of apoptotic nuclei on TUNEL assay was higher in control group than in treatment group (all p < 0.001). Immunohistochemical staining showed that cellular proliferation and number of small vessels were lower but glial fibrillary acid protein was higher in control group than in treatment group (all p < 0.01).

Conclusions: ADMSC therapy significantly limited BIA and improved sensorimotor dysfunction after acute IS.

Show MeSH
Related in: MedlinePlus