Interaction between hypothermia and delayed mesenchymal stem cell therapy in neonatal hypoxic-ischemic brain injury

Abstract

Acute hypothermia treatment (HT) is the only clinically established intervention following neonatal hypoxic-ischemic brain injury. However, almost half of all cooled infants still die or suffer from long-lasting neurological impairments. Regenerative therapies, such as mesenchymal stem cells (MSC) appear promising as adjuvant therapy. In the present study, we hypothesized that HT combined with delayed MSC therapy results in augmented protection, improving long-term neurological outcome. Postnatal day 9 (P9) C57BL/6 mice were exposed to hypoxia-ischemia followed by 4 h HT. Murine bone marrow-derived MSC (1 × 10 cells/animal) were administered intranasally at P12. Cytokine and growth factor levels were assessed by ELISA and Luminex® multiplex assay 24 h following MSC delivery. One week after HI, tissue injury and neuroinflammatory responses were determined by immunohistochemistry and western blot. Long-term motor-cognitive outcome was assessed 5 weeks post injury. MSC responses to the brains' environment were evaluated by gene expression analysis in MSC, co-cultured with brain homogenates isolated at P12. Both, MSC and HT improved motor deficits, while cognitive function could only be restored by MSC. Compared to each single therapy, combined treatment led to increased long-lasting motor-cognitive deficits and exacerbated brain injury, accompanied by enhanced endothelial activation and peripheral immune cell infiltration. MSC co-cultured with brain extracts of HT-treated animals revealed increased pro-inflammatory cytokine and decreased growth factor expression. In vivo protein analysis showed higher pro-inflammatory cytokine levels after combined treatment compared to single therapy. Furthermore, HI-induced increase in growth factors was normalized to control levels by HT and MSC single therapy, while the combination induced a further decline below control levels. Our results suggest that alteration of the brains' microenvironment by acute HT modulates MSC function resulting in a pro-inflammatory environment combined with alteration of the homeostatic growth factor milieu in the neonatal hypoxic-ischemic brain. This study delineates potential unexpected side effects of cell-based therapies as add-on therapy for acute hypothermia treatment.

RETRACTED ARTICLE:Bone mesenchymal stem cell-derived exosomal microRNA-29b-3p prevents hypoxic-ischemic injury in rat brain by activating the PTEN-mediated Akt signaling pathway

“…MSCs have gained much interest in the therapy for various disorders and have also shown augmented protection in the treatment of neonatal hypoxic-ischemic brain injury [35]. Recently, exosomes derived from MSCs have been found to carry various kinds of mediators, miRNAs and proteins, which can mediate the function of MSCs [10,36,37].…”

Non‐additive effects of adjunct erythropoietin therapy with therapeutic hypothermia after global cerebral ischaemia in near‐term fetal sheep

“…There is concerning evidence that in P9 mice combined therapy with hypothermia and mesenchymal stem cells after hypoxia‐ischaemia was less protective than either alone (Herz et al . ), illustrating the high importance of formally testing combined effects. Overall, these findings strongly suggest overlapping protective mechanisms of rEpo and therapeutic hypothermia.…”

Hypoxia and myelination deficits in the developing brain

“…It has been reported that bone marrow derived mesenchymal stem cells treatment after hypoxic injury in neonatal murine model promoted neuronal and oligodendrocyte regeneration (van Velthoven et al, 2010). Mesenchymal stem cell therapy in combination with hypothermia resulted in reduced hypoxia induced hypomyelination and memory deficits in neonatal C57BL/6 mice (Herz et al, 2018). Intraperitoneal injection of human cord blood derived mesenchymal stem cells in neonatal rats increased the count of mature oligodendrocytes and reduced the number of reactive astrocytes as well as reactive microglia, ameliorating the hypoxia induced brain damage (Zhu et al, 2014).…”