Multipotent adult progenitor cells for hypoxic-ischemic injury in the preterm brain

Abstract

Background: Preterm infants are at risk for hypoxic-ischemic encephalopathy. No therapy exists to treat this brain injury and subsequent long-term sequelae. We have previously shown in a well-established pre-clinical model of global hypoxia-ischemia (HI) that mesenchymal stem cells are a promising candidate for the treatment of hypoxic-ischemic brain injury. In the current study, we investigated the neuroprotective capacity of multipotent adult progenitor cells (MAPC®), which are adherent bone marrow-derived cells of an earlier developmental stage than mesenchymal stem cells and exhibiting more potent anti-inflammatory and regenerative properties.

Methods: Instrumented preterm sheep fetuses were subjected to global hypoxia-ischemia by 25 min of umbilical cord occlusion at a gestational age of 106 (term ~147) days. During a 7-day reperfusion period, vital parameters (e.g., blood pressure and heart rate; baroreceptor reflex) and (amplitude-integrated) electroencephalogram were recorded. At the end of the experiment, the preterm brain was studied by histology.

Results: Systemic administration of MAPC therapy reduced the number and duration of seizures and prevented decrease in baroreflex sensitivity after global HI. In addition, MAPC cells prevented HI-induced microglial proliferation in the preterm brain. These anti-inflammatory effects were associated with MAPC-induced prevention of hypomyelination after global HI. Besides attenuation of the cerebral inflammatory response, our findings showed that MAPC cells modulated the peripheral splenic inflammatory response, which has been implicated in the etiology of hypoxic-ischemic injury in the preterm brain.

Conclusions: In a pre-clinical animal model MAPC cell therapy improved the functional and structural outcome of the preterm brain after global HI. Future studies should establish the mechanism and long-term therapeutic effects of neuroprotection established by MAPC cells in the developing preterm brain exposed to HI. Our study may form the basis for future clinical trials, which will evaluate whether MAPC therapy is capable of reducing neurological sequelae in preterm infants with hypoxic-ischemic encephalopathy.

Fig. 1

Study design. Fetuses were instrumented at a gestational age (GA) of 102 days. After a recovery period of 4 days, fetuses were subjected to 25 min of umbilical cord occlusion (UCO) or sham. One hour and 4 days after UCO or sham, fetuses received either intravenous MAPC (10 million cells, closed arrow) or saline 0.9 % (open arrow). After a 7-day reperfusion period, brain tissue was collected. Abbreviations: in instrumentation, HI hypoxia-ischemia, SAL saline, MAPC multipotent adult progenitor cells

Fig. 2

Reproducibility of 25 min umbilical cord occlusion (UCO) as evidenced by comparable vital parameters and blood gasses in animals exposed to global HI. Fetal heart rate (FHR) (a) and fetal mean arterial blood pressure (MABP) (b) measurements indicated that all animals exposed to global HI experienced the same degree of bradycardia and hypotension, respectively; means (thick line) ± SD (shaded areas) of n = 8 animals per experimental group are shown. HI hypoxia-ischemia, SAL saline, MAPC multipotent adult progenitor cells, min minutes

Fig. 3

MAPC reduced cerebral inflammation in the subcortical white matter (SCWM), but not in the hippocampus. a–b Immunohistochemical IBA-1 staining in the SCWM at ×20 (a) and ×200 (b) magnification and hippocampus of the four experimental groups (sham-SAL n = 8, sham-MAPC n = 5, HI-SAL n = 8, HI-MAPC n = 5). Global HI induced a profound increase of IBA-1 immunoreactivity and amoeboid morphology in both regions, which was significantly reduced by MAPC in the SCWM, but not in the hippocampus. c–d Graphical presentation of area fraction of IBA-1 immunoreactivity in SCWM and hippocampus. Geometric 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. e–f Immunohistochemical IBA-1 staining in the hippocampus at ×20 (e) and ×200 (f) magnification of the four experimental groups. IBA-1 IR was markedly increased and accompanied by amoeboid morphology following global HI, which remained unaffected after MAPC treatment (sham-SAL n = 8, sham-MAPC n = 5, HI-SAL n = 8, HI-MAPC n = 5). *P ≤ 0.05, §P ≤ 0.01, #P ≤ 0.001. IBA-1 ionized calcium-binding adaptor molecule 1, HI hypoxia-ischemia, SAL saline, MAPC multipotent adult progenitor cells, IR immunoreactivity. a–b Scale bars: a–e represent 1 mm, b–f 100 μm

Fig. 4

MAPC reduced white matter injury after global HI. a–b Immunohistochemical MBP staining in the SCWM of the four experimental groups at (a) ×100 and (b) ×200 magnification. c MBP immunoreactivity in SCWM. Global HI induced marked hypomyelination in the SCWM. MAPC significantly prevented the decrease in MBP reactivity after global HI. The area fraction of MBP was similar in sham conditions. Geometric 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 = 8, sham-MAPC n = 5, HI-SAL n = 8, HI-MAPC n = 5). d Numbers of Caspase-3-positive cells in the four experimental groups; geometric means ± 95 % CI and levels of significance are depicted, which were calculated with ANOVA (sham-SAL n = 8, sham-MAPC n = 8, HI-SAL n = 8, HI-MAPC n = 8). *P ≤ 0.05, §P ≤ 0.01, #P ≤ 0.001. MBP myelin basic protein 1, HI hypoxia-ischemia, SAL saline, MAPC multipotent adult progenitor cells, IR immunoreactivity. Scale bars: (a) 200 μm, (b) 100 μm

Fig. 5

MAPC prevented splenic involution and modulated the peripheral inflammatory response. a Splenic weight corrected for body weight (BW). Global HI induced significant splenic involution, which was significantly prevented by MAPC; means ± 95 % CI and levels of significance are depicted, which were calculated with ANOVA (sham-SAL n = 8, sham-MAPC n = 8, HI-SAL n = 8, HI-MAPC n = 8). b IL-10 mRNA levels were significantly increased in the spleen 7 days after global HI. MAPC significantly attenuated the HI-induced IL-10 response in the spleen. c Splenic IFNγ mRNA levels were significantly increased 7 days following global HI. MAPC treatment tended to reduce IFNγ mRNA levels. However, statistical significance was not reached. d TNFα mRNA levels showed a trend to increase following global HI, which was significantly prevented by MAPC; geometric means ± 95 % CI and levels of significance are depicted, which were calculated with ANOVA (sham-SAL n = 8, sham-MAPC n = 6, HI-SAL n = 8, HI-MAPC n = 8). *P ≤ 0.05, §P ≤ 0.01, #P ≤ 0.001. HI hypoxia-ischemia, SAL saline, MAPC multipotent adult progenitor cells, IFNγ interferon gamma, TNFα tumor necrosis factor alpha

Fig. 6

MAPC induced functional neuroprotection after global HI. Global HI caused a significant seizure burden indicated by an increased total number (a) and duration of seizures (b) compared to controls. Administration of MAPC significantly reduced electrographic seizure number and duration. Medians ± interquartile ranges (IQR) and levels of significance of the treatment effect (HI-SAL vs. HI-MAPC) are depicted, which were calculated by Mann-Whitney test (HI-SAL n = 8, HI-MAPC n = 8). No electrographic seizure activity was detected under sham conditions. For clarity purposes these sham groups (sham-SAL n = 8 and sham-MAPC n = 6) are not shown. *P ≤ 0.05, §P ≤ 0.01, #P ≤ 0.001. HI hypoxia-ischemia, SAL saline, MAPC multipotent adult progenitor cells

Fig. 7

MAPC prevented loss of baroreflex sensitivity. Global HI caused a significant gradual decline of baroreflex sensitivity over time, which was prevented by MAPC treatment; means ± 95 % CI and levels of significance of the treatment effect (HI-SAL vs. HI-MAPC) are depicted, which were calculated by the Bayesian multi-level model. There were no differences in baroreflex sensitivity between the sham-SAL and sham-MAPC groups. For clarity purposes, all sham-treated animals (sham-SAL n = 8 and sham-MAPC n = 6) were grouped and depicted as one sham group. HI-SAL n = 8, HI-MAPC n = 8. *P ≤ 0.05, §P ≤ 0.01, #P ≤ 0.001. HI hypoxia-ischemia, SAL saline, MAPC multipotent adult progenitor cells