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. 2018 Sep 11;9:757.
doi: 10.3389/fneur.2018.00757. eCollection 2018.

Intravenous Administration of Bone Marrow-Derived Mesenchymal Stem Cell, but Not Adipose Tissue-Derived Stem Cell, Ameliorated the Neonatal Hypoxic-Ischemic Brain Injury by Changing Cerebral Inflammatory State in Rat

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Free PMC article

Intravenous Administration of Bone Marrow-Derived Mesenchymal Stem Cell, but Not Adipose Tissue-Derived Stem Cell, Ameliorated the Neonatal Hypoxic-Ischemic Brain Injury by Changing Cerebral Inflammatory State in Rat

Yuichiro Sugiyama et al. Front Neurol. .
Free PMC article

Abstract

Perinatal hypoxic-ischemic (HI) brain injury occurs in 1 in 1,000 live births and remains the main cause of neurological disability and death in term infants. Cytotherapy has recently emerged as a novel treatment for tissue injury. In particular, mesenchymal stem cells (MSCs) are thought to have therapeutic potential, but little is known about the differences according to their origin. In the current study, we investigated the therapeutic effects and safety of intravenous injection of allogeneic bone marrow-derived MSCs (BM-MSCs) and adipose-derived stem cells (ADSCs) in a rat model of HI brain injury. HI models were generated by ligating the left carotid artery of postnatal day 7 Wistar/ST rats and exposing them to 8% hypoxia for 60 min. Bone marrow and adipose tissue were harvested from adult green fluorescent protein transgenic Wistar rats, and cells were isolated and cultured to develop BM-MSCs and ADSCs. At passaging stages 2-3, 1 × 105 cells were intravenously injected into the external right jugular vein of the HI rats at 4 or 24 h after hypoxia. Brain damage was evaluated by counting the number of cells positive for active caspase-3 in the entire dentate gyrus. Microglial isotypes and serum cytokines/chemokines were also evaluated. Distribution of each cell type after intravenous injection was investigated pathologically and bio-optically by ex vivo imaging (IVIS®) with a fluorescent lipophilic tracer DiR. The mortality rate was higher in the ADSC group compared to the BM-MSC group, in pups injected with cells 4 h after hypoxia. The number of active caspase-3-positive cells significantly decreased in the BM-MSC group, and the percentage of M1 microglia (a proinflammatory isotype) was also lower in the BM-MSC vs control group in the penumbra of the cortex. Moreover, BM-MSC administration increased anti-inflammatory cytokine and growth factor levels, while ADSCs did not. Each injected cell type was mainly distributed in the lungs and liver, but ADSCs remained in the lungs longer. Pathologically, pulmonary embolisms and diffuse alveolar hemorrhages were seen in the ADSC group. These results indicated that injection of allogeneic BM-MSCs ameliorated neonatal HI brain injury, whereas ADSCs induced severe lung hemorrhage and higher mortality.

Keywords: M1 microglia; cell distribution; cytotherapy; multiplex; neonatal encephalopathy; regenerative medicine; serum chemokine.

Figures

Figure 1
Figure 1
Photomicrographs of anti-active caspase-3 staining of the hippocampus. Representative photomicrographs of the hippocampus 48 h after HI insult. Sections are from rats injected with vehicle (PBS), ADSCs, or BM-MSCs; bar = 500 μm. Insets show higher magnification; bar = 50 μm.
Figure 2
Figure 2
Number of active caspase-3-positive cells in the CA3 and dentate gyrus of the affected side. Impact of vehicle (PBS) (n = 7), ADSCs (n = 7), and BM-MSCs (n = 8) on apoptosis 24 h after injection. The number of active caspase-3-positive cells was significantly decreased in BM-MSC rats but not ADSC rats compared with vehicle. *P < 0.05.
Figure 3
Figure 3
Impact of BM-MSCs and ADSCs on microglial M1 polarization in the cortex, hippocampus, and basal ganglia. (A) Representative photomicrographs of the affected side cortex stained with anti-Iba-1 (pan-microglia marker) and anti-iNOS (M1 phenotype marker) 24 h after injection. Bar = 50 μm. (B) Number of Iba-1/iNOS positive cells, and the ratio of iNOS to Iba-1 in the penumbra of the cortex, hippocampus, and basal ganglia. In the penumbra of the cortex, iNOS-positive cell and the iNOS/Iba-1 ratio were significantly decreased in BM-MSC rats (n = 8), but not in ADSC rats (n = 7), compared to those in the vehicle group (n = 7). These parameters in hippocampus basal ganglia, and the number of Iba-1 in penumbra of the cortex and hippocampus showed the same trend. *P < 0.05.
Figure 4
Figure 4
Impact of BM-MSCs and ADSCs on serum cytokines/chemokines. Various serum cytokines/chemokines were measured by multiplex assay 24 h after injection. Serum levels of various cytokines/chemokines were significantly altered in the BM-MSC group but not the ADSC group. BM-MSCs, but not ADSCs, increased anti-inflammatory cytokine IL-2 (A) and granulocyte colony stimulating factor (B) levels. BM-MSCs also decreased chemotactic chemokines CCL2 (C), CCL3 (D), CX3CL1 (E), CXCL1 (F), CXCL2 (G), CXCL3 (H), and CXCL10 (I). On the other hand, levels of inflammatory cytokines IL-12p70 (J), IL-17a (K), and tumor necrosis factor (L) were increased by BM-MSC administration, but not ADSC. *P < 0.05.
Figure 5
Figure 5
Distribution of ADSCs and BM-MSCs after intravenous injection. The distribution of ADSCs (n = 3) and BM-MSC (n = 3) after injection was detected by ex vivo imaging (IVIS®). Cells were labeled with DiR and injected 24 h after HI. ADSCs (red line) and BM-MSCs (blue line) mainly distributed into the lungs and liver in the first 3 d (A,C,D). From 7 d after injection, the radiant efficiency of the lungs in ADSC-injected rats was significantly higher than those in the BM-MSC group (A,C). Neither ADSCs nor BM-MSCs were detected in the brain at any time point (B,E). There were no difference of fluorescence in the kidney (F). *P < 0.05.
Figure 6
Figure 6
Pathological findings. Macroscopic evaluations after injection of 1 × 105 ADSCs (A) and 1 × 104 ADSCs (B) showed diffuse lung hemorrhages were markedly more severe in the ADSC group compared with rats injected with 1 × 105 (C) and 1 × 104 (D) BM-MSCs. Microscopic evaluation of the lungs after ADSC (1 × 106 cells) injection [40 × magnification, HE] (E) revealed cell emboli (arrowhead) and fibrin deposition (arrow) in the large vessel. Fibrin deposition suggests embolism, inflammation, and coagulation. In the lungs of rats injected with 1 × 105 ADSCs [200 × magnification, HE] (F), diffuse alveolar hemorrhages were seen. Immunohistochemistry with anti-GFP (G) in the same blood vessel as (E) showed that cells with large nuclei filling pulmonary vessels were GFP-positive (arrowhead). The micrograph of lungs injected with 1 × 105 ADSCs (H) showed the presence of many GFP-positive cells in the alveolar vessel (arrowhead).

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