Long-Lasting Actions of Progesterone Protect the Neonatal Brain Following Hypoxia-Ischemia

Abstract

Neonatal hypoxia-ischemia (HI) is the leading cause of mortality and morbidity in newborns, occurring in approximately 2% of live births. Neuroprotective actions of progesterone (PROG) have already been described in animal models of brain lesions. However, PROG actions on neonates are still controversial. Here, we treated male Wistar rats exposed to HI with PROG. Five experimental groups were defined (n = 6/group) according to the scheme of PROG administration (10 mg/kg): SHAM (animals submitted to a fictitious surgery, without ischemia induction, and maintained under normoxia), HI (animals undergoing HI), BEFORE (animals undergoing HI and receiving PROG immediately before HI), AFTER (animals undergoing HI and receiving PROG at 6 and 24 h after HI) and BEFORE/AFTER (animals undergoing HI and receiving PROG immediately before and 6 and 24 h after HI). At P14 (7 days following HI), the volumes of lesion of the cerebral hemisphere and the hippocampus ipsilateral to the cerebral ischemia were evaluated, along with p-Akt, cleaved caspase-3 and GFAP expression in the hippocampus. PROG reduces the loss of brain tissue caused by HI. Moreover, when administered after HI, PROG was able to increase p-Akt expression and reduce both cleaved caspase-3 and GFAP expression in the hippocampus. In summary, it was possible to observe a neuroprotective action of PROG on the brain of neonatal animals exposed to experimental HI. This is the first study suggesting PROG-dependent Akt activation is able to regulate negatively cleaved caspase-3 and GFAP expression protecting neonatal hypoxic-ischemic brain tissue from apoptosis and reactive gliosis.

Therapeutic hypothermia for the treatment of neonatal hypoxia-ischemia: sex-dependent modulation of reactive astrogliosis

“…Neonatal HI procedure was based on the Rice-Vannuci model (Rice et al, 1981) and our previous studies (Sanches et al 2013;Fabres et al 2018Fabres et al , 2020Tassinari et al 2020). At P7, under iso urane anaesthesia (5% for induction and 3% for maintenance), neonates underwent surgical occlusion of the right common carotid artery.…”

“…The following antero-posterior coordinates were used for brain analysis: +2.52 mm to -6.84 mm relative to bregma for analysis of the cerebral hemisphere; +1.70 mm to -4.16 mm for the cerebral cortex and − 2.04 mm to -6.12 mm for the hippocampus. All coordinates were obtained according to the Paxinos and Watson rat brain atlas (Paxinos and Watson 2007) and to a previous study from our group (Fabres et al 2020). In order to reduce possible errors due to occurrence of brain oedema, the equation described by Sun et al (2015) was used to calculate the volume of lesion (Sun et al 2015;Fabres et al 2020).…”

“…All coordinates were obtained according to the Paxinos and Watson rat brain atlas (Paxinos and Watson 2007) and to a previous study from our group (Fabres et al 2020). In order to reduce possible errors due to occurrence of brain oedema, the equation described by Sun et al (2015) was used to calculate the volume of lesion (Sun et al 2015;Fabres et al 2020).…”

“…To estimate the number of cells in degeneration (cells showing shrinkage and deformity of the cell body or karyorrhectic and pyknotic nuclei) we used slides stained with haematoxylin and eosin as described in a previous paper (Fabres et al 2020). CA1 sub elds and hilus of the dentate gyrus of the hippocampus ipsilateral to the lesion were evaluated.…”

“…Sections were depara nized using xylene (10 minutes) and rehydrated using a decreasing alcoholic series. After these procedures, staining was performed as previously reported (Fabres et al 2020). For identi cation of astrocytes, a primary antiglial brillary acidic protein antibody (anti-GFAP, #G9269, rabbit IgG, 1:200, Sigma-Aldrich) was used and the secondary antibody of choice was Alexa 488 anti-rabbit (1:500, Molecular Probes, Invitrogen).…”