Early administration of umbilical cord blood cells following brief high tidal volume ventilation in preterm sheep: a cautionary tale.

BACKGROUND: Umbilical cord blood (UCB) cells are a promising treatment for preterm brain injury. Access to allogeneic sources of UCB cells offer the potential for early administration to optimise their therapeutic capacities. As preterm infants often require ventilatory support, which can contribute to preterm brain injury, we investigated the efficacy of early UCB cell administration following ventilation to reduce white matter inflammation and injury. METHODS: Preterm fetal sheep (0.85 gestation) were randomly allocated to no ventilation (SHAM; n = 5) or 15 min ex utero high tidal volume ventilation. One hour following ventilation, fetuses were randomly allocated to i.v. administration of saline (VENT; n = 7) or allogeneic term-derived UCB cells (24.5 ± 5.0 million cells/kg; VENT + UCB; n = 7). Twenty-four hours after ventilation, lambs were delivered for magnetic resonance imaging and post-mortem brain tissue collected. Arterial plasma was collected throughout the experiment for cytokine analyses. To further investigate the results from the in vivo study, mononuclear cells (MNCs) isolated from human UCB were subjected to in vitro cytokine-spiked culture medium (TNFα and/or IFNγ; 10 ng/mL; n = 3/group) for 16 h then supernatant and cells collected for protein and mRNA assessments respectively. RESULTS: In VENT + UCB lambs, systemic IFNγ levels increased and by 24 h, there was white matter neuroglial activation, vascular damage, reduced oligodendrocytes, and increased average, radial and mean diffusivity compared to VENT and SHAM. No evidence of white matter inflammation or injury was present in VENT lambs, except for mRNA downregulation of OCLN and CLDN1 compared to SHAM. In vitro, MNCs subjected to TNFα and/or IFNγ displayed both pro- and anti-inflammatory characteristics indicated by changes in cytokine (IL-18 & IL-10) and growth factor (BDNF & VEGF) gene and protein expression compared to controls. CONCLUSIONS: UCB cells administered early after brief high tidal volume ventilation in preterm fetal sheep causes white matter injury, and the mechanisms underlying these changes are likely dysregulated responses of the UCB cells to the degree of injury/inflammation already present. If immunomodulatory therapies such as UCB cells are to become a therapeutic strategy for preterm brain injury, especially after ventilation, our study suggests that the inflammatory state of the preterm infant should be considered when timing UCB cells administration.

Umbilical cord blood therapy modulates neonatal hypoxic ischemic brain injury in both females and males.

Preclinical and clinical studies have shown that sex is a significant risk factor for perinatal morbidity and mortality, with males being more susceptible to neonatal hypoxic ischemic (HI) brain injury. No study has investigated sexual dimorphism in the efficacy of umbilical cord blood (UCB) cell therapy. HI injury was induced in postnatal day 10 (PND10) rat pups using the Rice-Vannucci method of carotid artery ligation. Pups received 3 doses of UCB cells (PND11, 13, 20) and underwent behavioural testing. On PND50, brains were collected for immunohistochemical analysis. Behavioural and neuropathological outcomes were assessed for sex differences. HI brain injury resulted in a significant decrease in brain weight and increase in tissue loss in females and males. Females and males also exhibited significant cell death, region-specific neuron loss and long-term behavioural deficits. Females had significantly smaller brains overall compared to males and males had significantly reduced neuron numbers in the cortex compared to females. UCB administration improved multiple aspects of neuropathology and functional outcomes in males and females. Females and males both exhibited injury following HI. This is the first preclinical evidence that UCB is an appropriate treatment for neonatal brain injury in both female and male neonates.

Multiple doses of umbilical cord blood cells improve long-term brain injury in the neonatal rat.

BACKGROUND: Hypoxic ischemic (HI) insults during pregnancy and birth can result in neurodevelopmental disorders, such as cerebral palsy. We have previously shown that a single dose of umbilical cord blood (UCB) cells is effective at reducing short-term neuroinflammation and improves short and long-term behavioural outcomes in rat pups. A single dose of UCB was not able to modulate long-term neuroinflammation or brain tissue loss. In this study we examined whether multiple doses of UCB can modulate neuroinflammation, decrease cerebral tissue damage and improve behavioural outcomes when followed up long-term. METHODS: HI injury was induced in postnatal day 10 (PND10) rat pups using the Rice-Vannucci method of carotid artery ligation. Pups received either 1 dose (PND11), or 3 doses (PND11, 13, 20) of UCB cells. Rats were followed with behavioural testing, to assess both motor and cognitive outcomes. On PND50, brains were collected for analysis. RESULTS: HI brain injury in rat pups caused significant behavioural deficits. These deficits were significantly improved by multiple doses of UCB. HI injury resulted in a significant decrease in brain weight and left hemisphere tissue, which was improved by multiple doses of UCB. HI resulted in increased cerebral apoptosis, loss of neurons and upregulation of activated microglia. Multiple doses of UCB modulated these neuropathologies. A single dose of UCB at PND11 did not improve behavioural or neuropathological outcomes. CONCLUSIONS: Treatment with repeated doses of UCB is more effective than a single dose for reducing tissue damage, improving brain pathology and restoring behavioural deficits following perinatal brain injury.

Intranasal Delivery of Mesenchymal Stromal Cells Protects against Neonatal Hypoxic⁻Ischemic Brain Injury.

Cerebral palsy (CP) is a permanent motor disorder that results from brain injury and neuroinflammation during the perinatal period. Mesenchymal stromal cells (MSCs) have been explored as a therapy in multiple adult neuroinflammatory conditions. Our study examined the therapeutic benefits of intranasal delivery of human umbilical cord tissue (UC) derived-MSCs in a rat model of neonatal hypoxic-ischemic (HI) brain injury. To do this, HI was performed on postnatal day 10 Sprague-Dawley rat pups via permanent ligation of the left carotid artery, followed by a hypoxic challenge of 8% oxygen for 90 min. A total of 200,000 UC-MSCs (10 million/kg) were administered intranasally 24 h post-HI. Motor control was assessed after seven days, followed by post-mortem. Analysis included brain immunohistochemistry, gene analysis and serum cytokine measurement. Neonatal HI resulted in brain injury with significant loss of neurons, particularly in the hippocampus. Intranasal administration of UC-MSCs significantly reduced the loss of brain tissue and increased the number of hippocampal neurons. HI significantly upregulated brain inflammation and expression of pro-inflammatory cytokines, while intranasal UC-MSCs significantly reduced markers of neuroinflammation. This study demonstrated that a clinically relevant dose (10 million/kg) of UC-MSCs was neuroprotective following HI by restoring neuronal cell numbers and reducing brain inflammation. Therefore, intranasal delivery of UC-MSCs may be an effective therapy for neonatal brain injury.

Effects of umbilical cord blood cells, and subtypes, to reduce neuroinflammation following perinatal hypoxic-ischemic brain injury.

BACKGROUND: It is well understood that hypoxic-ischemic (HI) brain injury during the highly vulnerable perinatal period can lead to cerebral palsy, the most prevalent cause of chronic disability in children. Recently, human clinical trials have reported safety and some efficacy following treatment of cerebral palsy using umbilical cord blood (UCB) cells. UCB is made up of many different cell types, including endothelial progenitor cells (EPCs), T regulatory cells (Tregs), and monocyte-derived suppressor cells (MDSCs). How each cell type contributes individually towards reducing neuroinflammation and/or repairing brain injury is not known. In this study, we examined whether human (h) UCB, or specific UCB cell types, could reduce peripheral and cerebral inflammation, and promote brain repair, when given early after perinatal HI brain injury. METHODS: HI brain injury was induced in postnatal day (PND) 7 rat pups and cells were administered intraperitoneally on PND 8. Behavioral testing was performed 7 days post injury, and then, brains and spleens were collected for analysis. RESULTS: We found in vitro that all UCB cell types, except for EPCs, were immunomodulatory. Perinatal HI brain injury induced significant infiltration of CD4+ T cells into the injured cerebral hemisphere, and this was significantly reduced by all hUCB cell types tested. Compared to HI, UCB, Tregs, and EPCs were able to reduce motor deficits, reduce CD4+ T cell infiltration into the brain, and reduce microglial activation. In addition to the beneficial effects of UCB, EPCs also significantly reduced cortical cell death, returned CD4+ T cell infiltration to sham levels, and reduced the peripheral Th1-mediated pro-inflammatory shift. CONCLUSION: This study highlights that cells found in UCB is able to mediate neuroinflammation and is an effective neuroprotective therapy. Our study also shows that particular cells found in UCB, namely EPCs, may have an added advantage over using UCB alone. This work has the potential to progress towards tailored UCB therapies for the treatment of perinatal brain injury.