Hypothermia modulates myeloid cell polarization in neonatal hypoxic–ischemic brain injury

Background Neonatal encephalopathy due to hypoxia–ischemia (HI) is a leading cause of death and disability in term newborns. Therapeutic hypothermia (HT) is the only recommended therapy. However, 30% still suffer from neurological deficits. Inflammation is a major hallmark of HI pathophysiology with myeloid cells being key players, participating either in progression or in resolution of injury-induced inflammation. In the present study, we investigated the impact of HT on the temporal and spatial dynamics of microglia/macrophage polarization after neonatal HI in newborn mice. Methods Nine-day-old C57BL/6 mice were exposed to HI through occlusion of the right common carotid artery followed by 1 h hypoxia. Immediately after HI, animals were cooled for 4 h or kept at physiological body core temperature. Analyses were performed at 1, 3 and 7 days post HI. Brain injury, neuronal cell loss, apoptosis and microglia activation were assessed by immunohistochemistry. A broad set of typical genes associated with classical (M1) and alternative (M2) myeloid cell activation was analyzed by real time PCR in ex vivo isolated CD11b+ microglia/macrophages. Purity and composition of isolated cells was determined by flow cytometry. Results Immediate HT significantly reduced HI-induced brain injury and neuronal loss 7 days post HI, whereas only mild non-significant protection from HI-induced apoptosis and neuronal loss were observed 1 and 3 days after HI. Microglia activation, i.e., Iba-1 immunoreactivity peaked 3 days after HI and was not modulated by HT. However, ex vivo isolated CD11b+ cells revealed a strong upregulation of the majority of M1 but also M2 marker genes at day 1, which was significantly reduced by HT and rapidly declined at day 3. HI induced a significant increase in the frequency of peripheral macrophages in sorted CD11b+ cells at day 1, which deteriorated until day 7 and was significantly decreased by HT. Conclusion Our data demonstrate that HT-induced neuroprotection is preceded by acute suppression of HI-induced upregulation of inflammatory genes in myeloid cells and decreased infiltration of peripheral macrophages, both representing potential important effector mechanisms of HT. Supplementary Information The online version contains supplementary material available at 10.1186/s12974-021-02314-9.


Fig. S1: Iba-1 immunoreactivity does not change over time in sham-operated animals.
Nine-day-old C57Bl/6 mice were exposed to sham operation, i.e. unilateral ligation of the common carotid artery followed by analyses of microglia activation via immunohistochemistry 1, 3 and 7 days after HI in the depicted brain regions. n=5/time point. Quantiles of pooled data from all time points are presented as dashed lines (median) and grey shadows (25 th and 75 th percentiles) in Fig. 3 of the main manuscript.

Fig. S2
: HI-induced neuronal injury correlates with Iba-1 immunoreactivity 3 and 7 days after HI. Nine-day-old C57BL/6 mice were exposed to HI followed by 4 h hypothermia or normothermia. Animals were sacrificed and analysed via immunohistochemistry 1, 3 and 7 days after HI. Apoptotic cell death, neuronal loss and microglia activation were assessed via immunohistochemistry by TUNEL-labelling, staining of NeuN and Iba-1, respectively.
Correlation analyses was performed on pooled data from both treatment groups and all analysed brain regions, demonstrating a significant and positive correlation 3 and 7 days after injury (a). Separate correlation analyses for each treatment group including all analysed brain regions (b) revealed a more pronounced correlation in HT-treated animals 3 days after HI. n=7/time point and treatment and brain region. r=correlation coefficient, p=p-value Fig. S3. Impact of HI and hypothermia on expression of typical marker proteins associated with M1 and M2 polarization. Nine-day-old C57BL/6 mice were exposed to HI followed by 4 h hypothermia or normothermia. Animals were sacrificed and analysed 1, 3 or 7 days after HI. Myeloid cell polarization was assessed via immunohistochemistry for the prototypical M1 marker CD86 (green) and the M2 marker CD206 (red) in the cortex (a) and thalamus (b). CD86 single (left), CD206 single (middle) and CD86/CD206 double (right) positive cells were quantified. Dashed lines and grey shadows in the graphs indicate quantiles (i.e. median and 25 th , 75 th percentiles) of sham-operated animals across all time points, statistical comparisons to sham groups were performed for animals of the same time point. Detailed time course analyses of sham-operated animals are provided in this Additional file 1: Fig. S4b,c. *p<0.05; *p<0.05, **p<0.01, ***p<0.001, # p<0.05, ## p<0.01 vs. sham. n=5 (sham)/time point, n=7 (HI)/treatment and time point

Fig. S4: Microglia polarization is comparable between different time points of analyses in sham-operated mice.
Nine-day-old C57Bl/6 mice were exposed to sham operation, i.e. unilateral ligation of the common carotid artery followed by analyses of microglia polarization via immunohistochemistry for the M1 marker CD86 and the M2 marker CD206 1, 3 and 7 days after HI in the hippocampus (a), thalamus (b) and cortex (c). Except of a slightly increased number of CD86/CD206 double positive cells in the thalamus day 1 after surgery (b), no significant time-dependent differences were observed for CD86 and CD206 expression in the hippocampus (a) and cortex (c) of sham animals.  One day after HI, animals were deeply anesthetized by i.p. injections of an overdose chloralhydrate followed by transcardial perfusion with ice-cold PBS and removal of brains. For comparison of the impact of different tissue homogenization and cell isolation procedures on the composition of isolated cells, brain hemispheres were dissected and homogenized through a 70 µm cell strainer (BD Biosciences) by continuous rinsing with 15 mL of cold HEPES-buffered RPMI1640. Brain samples were centrifuged at 400xg for 10 min at 18°C. The supernatants were discarded and the pellets were resuspended in 15 mL of 37% Percoll in 0.01 N HCl/PBS and centrifuged at 2800xg for 20 min. Myelin was removed and the remaining cell pellet was washed twice in PBS followed by flow cytometry (a). For preparation of cell suspensions to be used for magnetic activated cell sorting (MACS) of CD11b + cells, brain tissues were mechanically and enzymatically dissociated using the Neural tissue dissociation kit followed by myelin removal according the manufacturer's instructions (Miltenyi Biotech, Germany) (b). The cell suspension was incubated with anti-CD11b coupled magnetic microbeads followed by magnetic separation of CD11b + cells. A small aliquot of sorted cells (1-2 x 10 5 ) was used for flow cytometry to determine the cellular composition via flow cytometry (c). Contour plots are representative for at least 3 animals per condition. Dead cells were excluded by staining with propidiumiodide (PI) prior to measurement.

Fig. S6: Sex-stratified analyses of HI-induced hippocampal brain injury and inflammation.
Nine-day-old C57BL/6 mice were exposed to HI followed by 4 h hypothermia or normothermia. Brain injury was assessed in cresyl violet stained tissue sections for neurophatological changes and tissue atrophy (a). Apoptotic cell death (b) and neuronal loss (c) were determined via immunohistochemistry by TUNEL labelling (b) and staining for NeuN (c), respectively. Microglia activation was analysed via immunohistochemistry for Iba-1 (d). Myeloid cell polarization was evaluated by quantification of CD86 single (e), CD206 single (f) and CD86/CD206 double positive (g) cells via immunohistochemistry. *p<0.05, **p<0.01, n=3-4/group and time point.    Fold change values were calculated relative to sham-operated animals, reg=immunoregulatory markers