Human Microglia Transplanted in Rat Focal Ischemia Brain Induce Neuroprotection and Behavioral Improvement

BACKGROUND AND PURPOSE: Microglia are resident immunocompetent and phagocytic cells of central nervous system (CNS), which produce various cytokines and growth factors in response to injury and thereby regulate disease pathology. The purpose of this study is to investigate the effects of microglial transplantation on focal cerebral ischemia model in rat. METHODS: Transient middle cerebral artery occlusion (MCAO) in rats was induced by the intraluminal filament technique. HMO6 cells, human microglial cell line, were transplanted intravenously at 48 hours after MCAO. Functional tests were performed and the infarct volume was measured at 7 and 14 days after MCAO. Migration and cell survival of transplanted microglial cells and host glial reaction in the brain were studied by immunohistochemistry. Gene expression of neurotrophic factors, cytokines and chemokines in transplanted cells and host rat glial cells was determined by laser capture microdissection (LCM) and quantitative real time-PCR. RESULTS: HMO6 human microglial cells transplantation group demonstrated significant functional recovery compared with control group. At 7 and 14 days after MCAO, infarct volume was significantly reduced in the HMO group. In the HMO6 group, number of apoptotic cells was time-dependently reduced in the infarct core and penumbra. In addition, number of host rat microglia/macrophages and reactive astrocytes was significantly decreased at 7 and 14 days after MCAO in the penumbra. Gene expression of various neurotrophic factors (GDNF, BDNF, VEGF and BMP7) and anti-inflammatory cytokines (IL4 and IL5) was up-regulated in transplanted HMO6 cells of brain tissue compared with those in culture. The expression of GDNF and VEGF in astrocytes in penumbra was significantly up-regulated in the HMO6 group. CONCLUSIONS: Our results indicate that transplantation of HMO6 human microglial cells reduces ischemic deficits and apoptotic events in stroke animals. The results were mediated by modulation of gliosis and neuroinflammation, and neuroprotection provided by neurotrophic factors of endogenous and transplanted cells-origin

Distribution of human nuclei positive cells after HMO6 cell transplantation.

<p>Human nuclei (HuN, FITC) positive cells were detected by fluorescence microscope at 5 (A and B) and 14 (C and D) days after MCAO in the infarct core (A and C) and the penumbra (B and D) of the brain. Histogram (E) shows the number of HuN-positive cells in infarct core (empty bars) and penumbra (solid bars), counted at X400 magnification. HuN-positive cells in internal organs were shown at 5 (F and G) and 7 (H and I) days in the spleen (F and H) and lung (G and I). Histogram (J) shows the number of HuN-positive cells in spleen, liver, kidney and lung, counted at X400 magnification in the time course after MCAO. Number of HuN-positive cells is expressed as the mean ± SEM. Bar  = 20 µm, * P<0.05 vs. infarct core at 3 and 14 days, ** P<0.01. vs. penumbra at 3, 7 and 14 days. ^# P<0.05 vs. spleen at 7 and 14 days, ^## P<0.05 vs. lung at 7 and 14 days.</p

Primers for real time PCR.

<p>Primers for real time PCR.</p

Gene expression in HMO6 cell <i>in vitro</i> and <i>in vivo</i>.

<p>Gene expression of neurotrophic factors, cytokines and chemokines in the HMO6 cell of culture (open bars) and brain tissue (solid bars) was analyzed by quantitative real-time PCR. Glyceraldehyde-3- phosphate dehydrogenase (GAPDH) was used as a reaction standard. Gene expression was determined in culture HMO6 cells under normal condition. HMO6 cells of brain tissue were isolated from infarct core and penumbra at 5 days after MCAO by laser capture microdissection (LCM). Values represent mean ± SEM from three experiments.</p

Gene expression in rat glial cells.

<p>Gene expression of neurotrophic factors, cytokines and chemokines in rat ED1-positive microglia/macrophages (A) and GFAP-positive astrocytes (B) was analyzed by quantitative real-time PCR. ED1- and GFAP-positive cells were isolated from penumbra of the HMO6 transplantation and control groups at 5 days after MCAO by laser capture microdissection. Glyceraldehyde-3- phosphate dehydrogenase (GAPDH) was used as a reaction standard. Expressional levels of each factor were calculated in the transplantation and control groups. Values represent mean ± SEM from three experiments. **P<0.01 compared to the control group.</p

Effect of HMO6 transplantation on neurological function, infarct volume and tissue damage.

<p>(A) Neurological function of the experimental groups after MCAO was determined by the modified Neurological Severity Scores (mNSS). The mNSS summarizes the results of motor, sensory, reflex, and balance tests. Transplantation of HMO6 human microglial cells enhanced neurological functional recovery at 7, 10 and 14 days after MCAO. (B) Infarct volume was calculated at 7 and 14 days after MCAO in the HMO6 transplantation and control groups based on TTC staining and NIH image software. Data of mNSS and infarct volume is expressed as the mean ± SEM. *P<0.05, ** P<0.01, as compared with the control group. H.E. staining of the penumbra lesion in a control rat (C) and a transplanted rat (D) at 7 days after MCAO. Bar  = 100 µm.</p

ED1 and GFAP positive cells in the penumbra.

<p>Immunohistochemical reactions to ED1 (A-D: FITC) and GFAP (E-H: Texas red) were shown at 7 (A, C, E and G) and 14 (B, D, F and H) days after MCAO. Double staining with anti-ED1 and anti-GFAP antibodies was detected in the penumbra of the HMO6 transplantation group (J) and control group (I) at 7 days after MCAO. Histograms show the density of ED1-positive microglia/macrophages (K) and GFAP-positive astrocytes (L) in the HMO6 transplantation and control groups of 7 and 14 days after MCAO. Number of positive cells is expressed as the mean ± SEM. Bar  = 20 µm, ** P<0.01 compared to the control group.</p

Modified NSS.

<p>One point is awarded for inability to perform the tasks or for the lack of a tested reflex.</p

Effect of HMO6 transplantation on apoptosis.

<p>TUNEL positive cells were shown in the HMO6 transplantation (C, D, H and I) and control group (A, B, F and G) at 3 (A-D) and 14 (F-I) days after MCAO. Number of apoptotic cells was determined in the infarct core (E) and the penumbra (J). Number of apoptotic cells is expressed as the mean ± SEM. Bar  = 20 µm, *P<0.05, ** P<0.01, as compared with the control group.</p