Mesenchymal Stem Cells Induce T-Cell Tolerance and Protect the Preterm Brain after Global Hypoxia-Ischemia

Hypoxic-ischemic encephalopathy (HIE) in preterm infants is a severe disease for which no curative treatment is available. Cerebral inflammation and invasion of activated peripheral immune cells have been shown to play a pivotal role in the etiology of white matter injury, which is the clinical hallmark of HIE in preterm infants. The objective of this study was to assess the neuroprotective and anti-inflammatory effects of intravenously delivered mesenchymal stem cells (MSC) in an ovine model of HIE. In this translational animal model, global hypoxia-ischemia (HI) was induced in instrumented preterm sheep by transient umbilical cord occlusion, which closely mimics the clinical insult. Intravenous administration of 2 x 106MSC/kg reduced microglial proliferation, diminished loss of oligodendrocytes and reduced demyelination, as determined by histology and Diffusion Tensor Imaging (DTI), in the preterm brain after global HI. These anti-inflammatory and neuroprotective effects of MSC were paralleled by reduced electrographic seizure activity in the ischemic preterm brain. Furthermore, we showed that MSC induced persistent peripheral T-cell tolerance in vivo and reduced invasion of T-cells into the preterm brain following global HI. These findings show in a preclinical animal model that intravenously administered MSC reduced cerebral inflammation, protected against white matter injury and established functional improvement in the preterm brain following global HI. Moreover, we provide evidence that induction of T-cell tolerance by MSC might play an important role in the neuroprotective effects of MSC in HIE. This is the first study to describe a marked neuroprotective effect of MSC in a translational animal model of HIE

Mesenchymal stem cells induce T-cell tolerance and protect the preterm brain after global hypoxia-ischemia

\u3cp\u3eHypoxic-ischemic encephalopathy (HIE) in preterm infants is a severe disease for which no curative treatment is available. Cerebral inflammation and invasion of activated peripheral immune cells have been shown to play a pivotal role in the etiology of white matter injury, which is the clinical hallmark of HIE in preterm infants. The objective of this study was to assess the neuroprotective and anti-inflammatory effects of intravenously delivered mesenchymal stem cells (MSC) in an ovine model of HIE. In this translational animal model, global hypoxia-ischemia (HI) was induced in instrumented preterm sheep by transient umbilical cord occlusion, which closely mimics the clinical insult. Intravenous administration of 2 x 10(6) MSC/kg reduced microglial proliferation, diminished loss of oligodendrocytes and reduced demyelination, as determined by histology and Diffusion Tensor Imaging (DTI), in the preterm brain after global HI. These anti-inflammatory and neuroprotective effects of MSC were paralleled by reduced electrographic seizure activity in the ischemic preterm brain. Furthermore, we showed that MSC induced persistent peripheral T-cell tolerance in vivo and reduced invasion of T-cells into the preterm brain following global HI. These findings show in a preclinical animal model that intravenously administered MSC reduced cerebral inflammation, protected against white matter injury and established functional improvement in the preterm brain following global HI. Moreover, we provide evidence that induction of T-cell tolerance by MSC might play an important role in the neuroprotective effects of MSC in HIE. This is the first study to describe a marked neuroprotective effect of MSC in a translational animal model of HIE. \u3c/p\u3

Reproducibility of 25 min umbilical cord occlusion (UCO).

<p>(<b>A</b>) Fetal mean arterial blood pressure (MABP) and (<b>B</b>) fetal heart rate (FHR) measurements indicated that all animals exposed to global HI experienced the same degree of hypotension and bradycardia, respectively, at the end of UCO; means (thick line) ± SD (shaded areas) of n=8 animals per experimental group are shown. MABP and FHR normalized within one hour after the end of UCO and were stable throughout the rest of the study period. All sham operated animals had similar MABP and FHR parameters during the entire duration of the study. For clarity reasons the sham-SAL and sham-MSC groups are depicted as one sham group. (<b>C</b>–<b>E</b>) Blood gas analysis indicated that twenty five minutes of umbilical cord occlusion induced comparable acidosis, hypoxemia and hypercarboxemia in all animals exposed to global HI, as demonstrated by (<b>C</b>) arterial pH, (<b>D</b>) arterial partial oxygen pressure (pO₂) and (<b>E</b>) arterial partial carbon dioxide pressure (pCO₂), respectively; means (lines) ± SD (error bars) of n=8 animals are depicted. Blood gas indices in animals exposed to global HI normalized within one hour after the end of UCO and stayed within the normal range throughout the rest of the study period.</p

Intravenous MSC treatment prevented white matter injury and reduced electrographic seizure activity after global HI.

<p>(<b>A</b>) Regions of interest; SCWM and hippocampus in FA map. FA values in the hippocampus were not altered by global HI (data not shown). (<b>B</b>) Global HI caused a significant decrease in FA values in the SCWM, which was prevented by intravenous MSC administration; means ± 95% CI and levels of significance are depicted, which were calculated with ANOVA (sham-SAL n=8, sham-MSC n=4, HI-SAL n=8, HI-MSC n=8). Dots show each measurement (i.e. non-repeated measures) per animal. * <i>P</i>≤0.05, ‡ <i>P</i>≤0.01, # <i>P</i>≤0.001. FA = fractional anisotropy, SAL = saline, MSC = mesenchymal stem cells, HI = hypoxia-ischemia. (<b>C</b>) Representative image of an aEEG trace from a HI-SAL animal. The two upper traces represent aEEG traces of the C₃-C₄ and P₃-P₄ channel, respectively. The aEEG signal is time-compressed, showing a 120-min period. The amplitude is displayed semi-logarithmic, indicating a LMA of approximately 4-3 µV and UMA of approximately 10 µV. The arrows indicate seizure activity. The two lower traces depict the corresponding EEG signal (30 s) of one electrographic seizure activity indicated by the diamond. (<b>D</b>) Intravenous MSC administration significantly reduced electrographic seizure burden following global HI; medians ± IQR are depicted (sham-SAL n=6, sham-MSC n=6, HI-SAL n=6, HI-MSC n=6). * <i>P</i>≤0.05, ‡ <i>P</i>≤0.01, # <i>P</i>≤0.001. SAL = saline, MSC = mesenchymal stem cells, HI = hypoxia-ischemia.</p

Intravenous MSC reduced proliferation of microglia after global HI.

<p>(<b>A</b>) Immunohistochemical IBA-1 staining in the SCWM of the four experimental groups with squares in the first panel indicating the regions where immunoreactivity was assessed. Global HI induced a profound increase of IBA-1 immunoreactivity, which was significantly reduced by intravenous MSC treatment. (<b>B</b>) Immunohistochemical IBA-1 staining in the hippocampus of the four experimental groups. Profound proliferation of microglia was observed in the hippocampus following global HI. MSC partially reduced the inflammatory response of microglia in the hippocampus after global HI. (<b>C</b>–<b>D</b>) Graphical presentation of area fraction of IBA-1 immunoreactivity in SCWM and hippocampus; (<b>C</b>) geometric means ± 95% CI and (<b>D</b>) means ± 95% CI and levels of significance are depicted, which were calculated by the random intercept model with all repeated measures (i.e. brain sections) per animal (sham-SAL n=6, sham-MSC n=3, HI-SAL n=6, HI-MSC n=6). Dots show the averaged results of the repeated measures (i.e. brain sections) per animal. * <i>P</i>≤0.05, ‡ <i>P</i>≤0.01, # <i>P</i>≤0.001. IBA-1 = ionized calcium binding adaptor molecule 1, HI = hypoxia-ischemia, SAL = saline, MSC = mesenchymal stem cells, IR = immunoreactivity. (<b>A</b>–<b>B</b>) Scale bars represent 1 mm.</p

MSC were detected in the fetal ovine brain.

<p>A FISH probe specific for the human Y-chromosome detected the presence of systemically delivered MSC in the ovine brain. (<b>A</b>–<b>D</b>) Representative fluorescent images of the SCWM in the different experimental groups; (<b>A</b>) sham-SAL, (<b>B</b>) sham-MSC, (<b>C</b>) HI-SAL and (<b>D</b>) HI-MSC. (<b>A</b>–<b>B</b>) In saline treated animals the FISH probe did not react with any nucleus. (<b>C</b>–<b>D</b>) The FISH probe was detected in MSC treated animals indicating that MSC were present in the preterm brain 7 d after intravenous administration. Scale bars: (<b>A</b>–<b>D</b>) 50 µm, scale bars inserts: (<b>A</b>–<b>D</b>) 5 µm.</p

Intravenous MSC modulated the peripheral T-cell response after global HI.

<p>MSC persistently suppressed the proliferative capacity of CD4-positive splenocytes and reduced the number of invading T-cells into the preterm brain follow global HI and. (<b>A</b>) Representative flow cytometry histograms showing proliferation (increased CFSE dilution) of CD4-positive splenocytes (green) and CD8-positive splenocytes (blue) derived from the different experimental groups. Global HI did not alter the proliferative capacity of CD4-positive splenocytes (HI-SAL <i>versus</i> sham-SAL). MSC significantly suppressed the proliferation of CD4-positive splenocytes in sham operated animals (sham-MSC <i>versus</i> sham-SAL) and in animals exposed to global HI (HI-MSC <i>versus</i> HI-SAL). (<b>B</b>) Immunohistochemical CD3 (T-cell) staining in the subcortical white matter (SCWM) of all four experimental groups. In sham conditions, T-cells were primarily detected in the intravascular space (open arrows). Following global HI, a significant increase in the number of perivascular and interstitial (black arrows) CD3-positive cells was observed. T-cell invasion after global HI was significantly reduced by MSC. (<b>C</b>–<b>D</b>) Graphical presentation of (<b>C</b>) the percentage of non-proliferative CD4-positive splenocytes and (<b>D</b>) number of CD3-positive cells in SCWM; (<b>C</b>) means ± 95% CI and (<b>D</b>) geometric means ± 95% CI and levels of significance are depicted, which were calculated by ANOVA for CD4 proliferation (<b>C</b>) and the random intercept model with all repeated measures (i.e. brain sections) for CD3 counts (<b>D</b>). Dots show each measurement (i.e. non-repeated measures) per animal for CD4 proliferation (<b>C</b>) and the averaged results of the repeated measures (i.e. brain sections) per animal for CD3 counts (<b>D</b>). * <i>P</i>≤0.05, ‡ <i>P</i>≤0.01, # <i>P</i>≤0.001. SAL = saline, MSC = mesenchymal stem cells, HI = hypoxia-ischemia, FOV = field of view.</p

Study design.

<p>Fetuses were instrumented at a gestational age (GA) of 102 d. After a recovery period of 4 d fetuses were subjected to 25 min of umbilical cord occlusion (UCO) or sham. One hour after UCO or sham, fetuses received either intravenous mesenchymal stem cells (2.0 million/kg BW) (closed arrow) or saline 0,9% (open arrow). After a 7 d reperfusion period brain tissue was collected. Abbreviations: in = instrumentation, HI = hypoxia-ischemia, SAL = saline, MSC = mesenchymal stem cells.</p

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