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.
Neonatal hypoxic-ischemic (HI) encephalopathy (HIE) remains a major cause of mortality and persistent neurological disabilities in affected individuals. At present, hypothermia is considered to be the only applicable treatment option, although growing evidence suggests that cell-based therapy might achieve better outcomes. Dedifferentiated fat (DFAT) cells are derived from mature adipocytes via a dedifferentiation strategy called ceiling culture. Their abundance and ready availability might make them an ideal therapeutic tool for the treatment of HIE. In the present study, we aimed to determine whether the outcome of HIE can be improved by DFAT cell treatment. HI injury was achieved by ligating the left common carotid artery in 7-day-old rat pups, followed by 1-h exposure to 8% O2. Subsequently, the severity of damage was assessed by diffusion-weighted magnetic resonance imaging to assign animals to equivalent groups. 24 h after hypoxia, DFAT cells were injected at 105 cells/pup into the right external jugular vein. To evaluate brain damage in the acute phase, a group of animals was sacrificed 48 h after the insult, and paraffin sections of the brain were stained to assess several acute injury markers. In the chronic phase, the behavioral outcome was measured by performing a series of behavioral tests. From the 24th day of age, the sensorimotor function was examined by evaluating the initial forepaw placement on a cylinder wall and the latency to falling from a rotarod treadmill. The cognitive function was tested with the novel object recognition (NOR) test. In vitro conditioned medium (CM) prepared from cultured DFAT cells was added at various concentrations to neuronal cell cultures, which were then exposed to oxygen-glucose deprivation (OGD). The number of cells that stained positive for the apoptosis marker active caspase-3 decreased by 73 and 52% in the hippocampus and temporal cortex areas of the brain, respectively, in the DFAT-treated pups. Similarly, the numbers of ED-1-positive cells (activated microglia) decreased by 66 and 44%, respectively, in the same areas in the DFAT-treated group. The number of cells positive for the oxidative stress marker 4-hydroxyl-2-nonenal decreased by 68 and 50% in the hippocampus and the parietal cortex areas, respectively, in the DFAT-treated group. The HI insult led to a motor deficit according to the rotarod treadmill and cylinder test, where it significantly affected the vehicle group, whereas no difference was confirmed between the DFAT and sham groups. However, the NOR test indicated no significant differences between any of the groups. DFAT treatment did not reduce the infarct volume, which was confirmed immunohistochemically. According to in vitro experiments, the cell death rates in the DFAT-CM-treated cells were significantly lower than those in the controls when DFAT-CM was added 48 h prior to OGD. The treatment effect of adding DFAT-CM 24 h prior to OGD was also significant. Our results indicate that intravenous injection with DFAT cells i...
Background/Objective: Perinatal hypoxic-ischemia (HI) causes neonatal death and permanent neurological deficits. Cell therapy using various cell sources has been recently identified as a novel therapy for perinatal HI. Among the available types of cell sources, bone marrow-derived mononuclear cells (BMMNCs) have unique features for clinical application. For example, stem cells can be collected after admission, thus enabling us to perform autologous transplantation. This study aimed to investigate whether the administration of BMMNCs ameliorated HI brain injury in a neonatal rat model.Methods: Seven-day-old rats underwent left carotid artery ligation and were exposed to 8% oxygen for 60 min. BMMNCs were collected from the femurs and tibias of juvenile rats using the Ficoll–Hypaque technique and injected intravenously 24 h after the insult (1 × 105 cells). Active caspase-3, as an apoptosis marker, and ED1, as an activated microglia/macrophage marker, were evaluated immunohistochemically 48 h after the insult (vehicle, n = 9; BMMNC, n = 10). Behavioral assessments using the rotarod treadmill, gait analysis, and active avoidance tests were initiated 3 weeks after the insult (sham, n = 9, vehicle, n = 8; BMMNC, n = 8). After these behavioral tests (6 weeks after the insult), we evaluated the volumes of their hippocampi, cortices, thalami, striata, and globus pallidus.Results: The mean cell densities of the sum of four parts that were positive for active caspase-3 significantly decreased in the BMMNC group (p < 0.05), whereas in the hippocampi, cortices, thalami, and striata cell densities decreased by 42, 60, 56, and 47%, respectively, although statistical significance was not attained. The number of ED1 positive cells for the sum of the four parts also significantly decreased in the BMMNC group compared to the vehicle group (p < 0.05), whereas in each of the four parts the decrease was 35, 39, 47, and 36%, respectively, although statistical significance was not attained. In gait analysis, the BMMNC normalized the contact area of the affected hind paw widened by HI. The volumes of the affected striata and globus pallidus were significantly larger in the BMMNC group than in the control group.Conclusion: These results indicated that the injection of BMMNCs ameliorated HI brain injury in a neonatal rat model.
Background: Hyperpolarized 13 C spectroscopic magnetic resonance spectroscopy (MRS) is an advanced imaging tool that may provide important real-time information about brain metabolism. Methods: Mice underwent unilateral hypoxia-ischemia (HI) on postnatal day (P)10. Injured and sham mice were scanned at P10, P17, and P31. We used hyperpolarized 13 C MRS to investigate the metabolic exchange of pyruvate to lactate in real time during brain development following HI. 13 C-1-labeled pyruvate was hyperpolarized and injected into the tail vein through a tail-vein catheter. Chemical-shift imaging was performed to acquire spectral-spatial information of the metabolites in the brain. A voxel placed on each of the injured and contralateral hemispheres was chosen for comparison. The difference in pyruvate delivery and lactate to pyruvate ratio was calculated for each of the voxels at each time point. The normalized lactate level of the injured hemisphere was also calculated for each mouse at each of the scanning time points. Results: There was a significant reduction in pyruvate delivery and a higher lactate to pyruvate ratio in the ipsilateral (HI) hemisphere at P10. The differences decreased at P17 and disappeared at P31. The normalized lactate level in the injured hemisphere increased from P10 to P31 in both sham and HI mice without brain injury. Conclusion: We describe a method for detecting and monitoring the evolution of HI injury during brain maturation which could prove to be an excellent biomarker of injury.
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