Supplementary Material for: Dedifferentiated Fat Cells as a Novel Source for Cell Therapy to Target Neonatal Hypoxic-Ischemic Encephalopathy

<p>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 is effective for ameliorating HI brain injury, possibly via paracrine effects.</p

Supplementary Material for: “Mild’’ Hypoxic-Ischaemic Encephalopathy and Therapeutic Hypothermia: A Survey of Clinical Practice and Opinion from 35 Countries

Introduction: We aimed to determine global professional opinion and practice for the use of therapeutic hypothermia (TH) for treating infants with mild hypoxic-ischaemic encephalopathy (HIE). Methods: A web-based survey (REDCap) was distributed via emails, social networking sites, and professional groups from October 2020 to February 2021 to neonatal clinicians in 35 countries. Results: A total of 484 responses were obtained from 35 countries and categorized into low/middle-income (43%, LMIC) or high-income (57%, HIC) countries. Of the 484 respondents, 53% would provide TH in mild HIE on case-to-case basis and only 25% would never cool. Clinicians from LMIC were more likely to routinely offer TH in mild HIE (25% v HIC 16%, p v HIC 26%, p v HIC 32%, p v HIC 40%, p v HIC 95%, p Conclusions: This is the first survey of global opinion for TH in mild HIE. The overwhelming majority of professionals would consider “cooling” an infant with mild HIE, but LMIC respondents were more likely to routinely cool infants with mild HIE and use adjunctive tools for diagnosis and follow-up. There is wide practice heterogeneity and a sufficiently large RCT designed to examine neurodevelopmental outcomes, is urgently needed and widely supported