Neonatal hypoxic ischemic encephalopathy : inflammation and therapies

Neonatal hypoxic ischemia (HI) is a severe condition characterized by a complex pathophysiology. The lack of oxygen (hypoxia) and blood flow (ischemia) leads to neuronal cell death via necrosis and apoptosis, and a consequent post-ischemic inflammation. HI brain injury may lead to seizures, cognitive and motor impairments, and death. Worse neurodevelopmental outcomes have been observed in male than in female survivors, thus underlining sex-dependent differences. To date, hypothermia is the only available evidence-based treatment for neonatal HI that shows neuroprotection if applied within six hours after the insult. Although hypothermia reduces production of cytokines and metabolic stress, it was shown to not be effective in severe cases of neonatal HI. Additional therapies meant to alleviate the HI outcomes are therefore needed.

In the present thesis we investigated the role of two key players involved in post-ischemic inflammation, namely resident microglia and infiltrating macrophages, and studied the effects of drug- and cell-based treatments aimed at reducing injury. HI was induced by occluding the common carotid artery in mouse pups that were then subjected to hypoxia. By investigating the dynamics of inflammatory cells in the hippocampus of injured mice, sex-specific differences were observed in microglia and infiltrating peripheral-macrophages. Sequencing data revealed that macrophages are the drivers of the post-ischemic inflammation through significant upregulation of cytokine, chemokine and sensome markers, as well as activation genes. In addition, microglial cells, which were shown to downregulate unique signature genes upon inflammation, restored their homeostatic role within three days after injury in males, suggesting a different mechanistic effect in response to the neuroinflammatory cascade. The role of resident microglia was further investigated in a Tamoxifen-based depletion model in which HI was then performed. While no difference in the speed of microglial repopulation was observed between males and females, the injury progression and cytokine production changed in a sex-dependent fashion. Specifically, depletion aggravated neuronal damage and apoptosis in male mice following HI.

In order to reduce inflammation and to induce neuroprotection, therapies involving caffeine or bone marrow-derived macrophage administration were assessed in this thesis. Caffeine, which is an adenosine-receptor competitor, is currently used in the clinic as treatment for neonatal apnea. As long-term follow-up studies of apneic babies treated with caffeine showed a reduced incident of cerebral palsy, this drug was administered at different time points after HI. Our results revealed a reduced lesion and improved behavioral outcomes after a single dose of 5 mg/kg caffeine immediately post-HI, with a reduction of the lesion and glial scar extent, and modulation of microglia activation and pro-inflammatory genes. Bone marrow-derived macrophages were adoptively transferred 5 days after HI to investigate their immunomodulatory and wound healing properties. Our results showed a clear difference when anti-inflammatory macrophages (M2) or unpolarized control cells (M0) were administered. While M2 cell therapy led to functional recovery, we observed that M0 macrophages worsened behavioral outcomes and increased the injury size. In addition, in vitro studies in organotypic hippocampal slices co-cultured with these macrophages showed that, while M2 maintained memory of their phenotype, the M0 cells became polarized towards a pro-inflammatory state, thus suggesting how unpolarized cells could lead to exacerbation of the inflammation and the consequent worsening of injury extent and behavioral performance observed in vivo.

In summary, in this thesis I highlight the importance of microglia and infiltrating macrophages in the post-ischemic inflammatory cascade, and how caffeine and bone-marrow derived macrophages may be of potential therapeutic interest in future studies.

List of scientific papers

I. Elena Di Martino, Takashi Umekawa, Anoop Ambikan, Sarantis Giatrellis, Daniel Ramsköld, Davide Vacondio, Ahmed Osman, Qiaolin Deng, Jonas Frisen, Rickard Sandberg, Ujjwal Neogi, Ulrika Ådén, Volker M. Lauschke, Klas Blomgren and Julianna Kele. Sex-dependent and -independent inflammatory patterns in resident microglia and infiltrated macrophages after neonatal asphyxia. [Manuscript]

II. Shunichiro Tsuji, Elena Di Martino, Takeo Mukai, Shoko Tsuji, Takashi Murakami, Robert A. Harris, Klas Blomgren, Ulrika Åden. Aggravated brain injury after neonatal hypoxic ischemia in microglia-depleted mice. Journal of Neuroinflammation. 17(1):111 (April 2020).
https://doi.org/10.1186/s12974-020-01792-7

III. Elena Di Martino, Erica Bocchetta, Shunichiro Tsuji, Takeo Mukai, Robert A. Harris, Klas Blomgren, and Ulrika Ådén. Defining a time window for neuroprotection and glia modulation by caffeine after neonatal hypoxia-ischaemia. Molecular Neurobiology. 57(5):2194-2205 (May 2020).
https://doi.org/10.1007/s12035-020-01867-9

IV. Elena Di Martino, Davide Vacondio, Luigi Balasco, Luis Enrique Arroyo-Garcia, Takeo Mukai, Melanie Pieber, Anne-Kristin Kukla, Shunichiro Tsuji, André Fisahn, Xingmei Wang, Ronny Wickström, Klas Blomgren, Robert A. Harris and Ulrika Ådén. Adoptive transfer of bone-marrow-derived macrophages modulates the post-ischemic inflammation in a model of neonatal hypoxia-ischemia. [Manuscript]