Aggravated brain injury after neonatal hypoxic ischemia in microglia-depleted mice

Shunichiro Tsuji,Takeo Mukai,Takashi Murakami,Elena Di Martino,Klas Blomgren,Robert A Harris,Ulrika Åden,Shoko Tsuji

doi:10.1186/s12974-020-01792-7

Shunichiro Tsuji, Takeo Mukai + Show 6 more

Open Access

https://doi.org/10.1186/s12974-020-01792-7

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Abstract

BackgroundNeuroinflammation plays an important role in neonatal hypoxic-ischemic encephalopathy (HIE). Although microglia are largely responsible for injury-induced inflammatory response, they play beneficial roles in both normal and disease states. However, the effects of microglial depletion on neonatal HIE remain unclear.MethodsTamoxifen was administered to Cx3cr1CreER/+Rosa26DTA/+ (microglia-depleted model) and Cx3cr1CreER/+Rosa26DTA/− (control) mice at P8 and P9 to assess the effect of microglial depletion. The density of microglia was quantified using Iba-1 staining. Moreover, the proportion of resident microglia after the HI insult was analyzed using flow cytometric analysis. At P10, the HI insult was conducted using the Rice-Vannucci procedure at P10. The infarct size and apoptotic cells were analyzed at P13. Cytokine analyses were performed using quantitative polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA) at P13.ResultsAt P10, tamoxifen administration induced > 99% microglial depletion in DTA+ mice. Following HI insult, there was persisted microglial depletion over 97% at P13. Compared to male DTA− mice, male DTA+ mice exhibited significantly larger infarct volumes; however, there were no significant differences among females. Moreover, compared to male DTA− mice, male DTA+ mice had a significantly higher density of TUNEL+ cells in the caudoputamen, cerebral cortex, and thalamus. Moreover, compared to female DTA− mice, female DTA+ mice showed a significantly greater number of TUNEL+ cells in the hippocampus and thalamus. Compared to DTA− mice, ELISA revealed significantly lower IL-10 and TGF-β levels in both male and female DTA+ mice under both normal conditions and after HI (more pronounced).ConclusionWe established a microglial depletion model that aggravated neuronal damage and apoptosis after the HI insult, which was predominantly observed in males.

Highlights

Neuroinflammation plays an important role in neonatal hypoxic-ischemic encephalopathy (HIE)
These cells engage in intimate cross-talk with other intrinsic brain cells and infiltrating leukocytes from the periphery that enter the brain through the compromised blood-brain barrier (BBB) [6,7,8]
Microglial depletion and repopulation using tamoxifen in Cx3cr1CreERRosa26DTA neonatal mice After tamoxifen administration at P8 and P9, Iba-1+ cells were counted at P10, P13, P17, and P24 to evaluate microglia depletion and repopulation (Fig. 1b)

Summary

Introduction

Neuroinflammation plays an important role in neonatal hypoxic-ischemic encephalopathy (HIE). Microglia are largely responsible for injury-induced inflammatory response, they play beneficial roles in both normal and disease states. HI insults are known to induce inflammatory reactions within hours in rodent models with microglia playing an important role [1,2,3,4,5]. Microglia are resident cells in the brain that are the first responders to ischemia. In response to brain ischemia, microglia initiate reactive oxygen species generation, antigen presentation, phagocytosis, and the production of inflammatory mediators, including interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6, and matrix metalloproteinases [8,9,10]. The response to HIE largely differs between the adult and neonatal brain [7, 18,19,20,21]

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