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Supplemental material, sj-pdf-10-jcb-10.1177_0271678X221098811 for Temporal brain transcriptome analysis reveals key pathological events after germinal matrix hemorrhage in neonatal rats by Juan Song, Gisela Nilsson, Yiran Xu, Aura Zelco, Eridan Rocha-Ferreira, Yafeng Wang, Xiaoli Zhang, Shan Zhang, Joakim Ek, Henrik Hagberg, Changlian Zhu and Xiaoyang Wang in Journal of Cerebral Blood Flow & Metabolism

Preterm newborns are particularly susceptible to complications such as hypoxia-ischemia (HI), which can result in brain injury and subsequent cognitive and/or motor function disabilities, including cerebral palsy. Immune cells have been shown to be involved in the development of perinatal brain damage, commonly with detrimental effects. There is recent evidence that the membranes around the brain parenchyma, the meninges, might also have important roles in the immune response after injury in the adult brain, for example, by being a site of peripheral immune cell infiltration into the brain parenchyma. However, the role of the meninges in preterm brain injury is not known. Thus, the aim of this doctoral thesis was to identify the roles of immune cells in the meninges and brain parenchyma after preterm brain injury using a mouse model of HI-induced preterm brain injury. In Paper I we found that T and B cells accumulate in post-mortem brains and meninges in preterm infants with brain injury. Similarly, in mouse experiments we found that T and B cells respond to the HI injury and infiltrate into the parenchyma. Additionally, genetic deletion of T and B cells resulted in reduced white matter tissue loss 7 days after HI. Paper II shows that innate lymphoid cells subtype 2 (ILC2s) also accumulate in the meninges 7 days after HI, but ILC2-impaired mice show no differences in inflammatory response, tissue loss, or glial immunoreactivity compared to wild type mice after HI, demonstrating a non-essential role for this immune cell subtype after preterm brain injury. Using single cell RNA sequencing, Paper III presents the cellular composition and the unique transcriptional identities of meningeal immune cells in neonatal mice such as border-associated macrophages, monocytes, and microglia. We also identify the possible involvement of neutrophils in the injury process 6 hours after HI. To conclude, the findings of this thesis reveal the participation of immune cells in the brain parenchyma and in the meninges to the development of HI injury. We provide insights into the unique single cell profile in the meninges in the immature mouse brain and thus contribute to the understanding of immune cell involvement in the injury process and the inflammatory reactions after preterm brain injury.

Hypoxic-ischaemic encephalopathy (HIE) causes 25% of neonatal deaths worldwide. Rocha-Ferreira et al. demonstrate that a diabetes drug protects the neonatal brain in a model of acute HIE, and confirm that the required receptor is found in human perinatal brain tissue. Synergistic combination with clinical hypothermia enhances therapy, supporting potential translation., Hypoxic-ischaemic encephalopathy remains a global health burden. Despite medical advances and treatment with therapeutic hypothermia, over 50% of cooled infants are not protected and still develop lifelong neurodisabilities, including cerebral palsy. Furthermore, hypothermia is not used in preterm cases or low resource settings. Alternatives or adjunct therapies are urgently needed. Exendin-4 is a drug used to treat type 2 diabetes mellitus that has also demonstrated neuroprotective properties, and is currently being tested in clinical trials for Alzheimer’s and Parkinson’s diseases. Therefore, we hypothesized a neuroprotective effect for exendin-4 in neonatal neurodisorders, particularly in the treatment of neonatal hypoxic-ischaemic encephalopathy. Initially, we confirmed that the glucagon like peptide 1 receptor (GLP1R) was expressed in the human neonatal brain and in murine neurons at postnatal Day 7 (human equivalent late preterm) and postnatal Day 10 (term). Using a well characterized mouse model of neonatal hypoxic-ischaemic brain injury, we investigated the potential neuroprotective effect of exendin-4 in both postnatal Day 7 and 10 mice. An optimal exendin-4 treatment dosing regimen was identified, where four high doses (0.5 µg/g) starting at 0 h, then at 12 h, 24 h and 36 h after postnatal Day 7 hypoxic-ischaemic insult resulted in significant brain neuroprotection. Furthermore, neuroprotection was sustained even when treatment using exendin-4 was delayed by 2 h post hypoxic-ischaemic brain injury. This protective effect was observed in various histopathological markers: tissue infarction, cell death, astrogliosis, microglial and endothelial activation. Blood glucose levels were not altered by high dose exendin-4 administration when compared to controls. Exendin-4 administration did not result in adverse organ histopathology (haematoxylin and eosin) or inflammation (CD68). Despite initial reduced weight gain, animals restored weight gain following end of treatment. Overall high dose exendin-4 administration was well tolerated. To mimic the clinical scenario, postnatal Day 10 mice underwent exendin-4 and therapeutic hypothermia treatment, either alone or in combination, and brain tissue loss was assessed after 1 week. Exendin-4 treatment resulted in significant neuroprotection alone, and enhanced the cerebroprotective effect of therapeutic hypothermia. In summary, the safety and tolerance of high dose exendin-4 administrations, combined with its neuroprotective effect alone or in conjunction with clinically relevant hypothermia make the repurposing of exendin-4 for the treatment of neonatal hypoxic-ischaemic encephalopathy particularly promising.

Background Periventricular leukomalacia (PVL) is the most common form of preterm brain injury affecting the cerebral white matter. This type of injury involves a multiphase process and is induced by many factors, including hypoxia–ischemia (HI) and infection. Previous studies have suggested that lymphocytes play a significant role in the pathogenesis of brain injury, and the aim of this study was to determine the contribution of lymphocyte subsets to preterm brain injury. Methods Immunohistochemistry on brain sections from neonatal mice was performed to evaluate the extent of brain injury in wild-type and T cell and B cell-deficient neonatal mice (Rag1−/− mice) using a mouse model of HI-induced preterm brain injury. Flow cytometry was performed to determine the presence of different types of immune cells in mouse brains following HI. In addition, immunostaining for CD3 T cells and CD20 B cells was performed on postmortem preterm human infant brains with PVL. Results Mature lymphocyte-deficient Rag1−/− mice showed protection from white matter loss compared to wild type mice as indicated by myelin basic protein immunostaining of mouse brains. CD3+ T cells and CD20+ B cells were observed in the postmortem preterm infant brains with PVL. Flow cytometry analysis of mouse brains after HI-induced injury showed increased frequency of CD3+ T, αβT and B cells at 7 days after HI in the ipsilateral (injured) hemisphere compared to the contralateral (control, uninjured) hemisphere. Conclusion Lymphocytes were found in the injured brain after injury in both mice and humans, and lack of mature lymphocytes protected neonatal mice from HI-induced brain white matter injury. This finding provides insight into the pathology of perinatal brain injury and suggests new avenues for the development of therapeutic strategies.