Delayed injury of hippocampal interneurons after neonatal hypoxia‐ischemia and therapeutic hypothermia in a murine model

Abstract

Delayed hippocampal injury and memory impairments follow neonatal hypoxia-ischemia (HI) despite the use of therapeutic hypothermia (TH). Death of hippocampal pyramidal cells occurs acutely after HI, but characterization of delayed cell death and injury of interneurons (INs) is unknown. We hypothesize that injury of INs after HI is: (i) asynchronous to that of pyramidal cells, (ii) independent of injury severity, and (iii) unresponsive to TH. HI was induced in C57BL6 mice at p10 with unilateral right carotid ligation and 45 min of hypoxia (FiO = 0.08). Mice were randomized to normothermia (36 °C, NT) or TH (31 °C) for 4 hr after HI and anesthesia-exposed shams were use as controls. Brains were studied at 24 hr (p11) or 8 days (p18) after HI. Vglut1, GAD65/67, PSD95, parvalbumin (PV) and calbindin-1 (Calb1) were measured. Cell death was assessed using cresyl violet staining and TUNEL assay. Hippocampal atrophy and astroglyosis at p18 were used to assess injury severity and to correlate with number of PV + INs. VGlut1 level decreased by 30% at 24 hr after HI, while GAD65/67 level decreased by ∼50% in forebrain 8 days after HI, a decrease localized in CA1 and CA3. PSD95 levels decreased in forebrain by 65% at 24 hr after HI and remained low 8 days after HI. PV + INs increased in numbers (per mm ) and branching between p11 and p18 in sham mice but not in NT and TH mice, resulting in 21-52% fewer PV + INs in injured mice at p18. Calb1 protein and mRNA were also reduced in HI injured mice at p18. At p18, somatodendritic attrition of INs was evident in all injured mice without evidence of cell death. Neither hippocampal atrophy nor astroglyosis correlated with the number of PV + INs at p18. Thus, HI exposure has long lasting effects in the hippocampus impairing the development of the GABAergic system with only partial protection by TH independent of the degree of hippocampal injury. © 2018 Wiley Periodicals, Inc.

Connexin Hemichannel Mimetic Peptide Attenuates Cortical Interneuron Loss and Perineuronal Net Disruption Following Cerebral Ischemia in Near-Term Fetal Sheep

“…In the present study, cerebral ischemia in near-term fetal sheep was associated with a pattern of parasagittal cortical damage after 7 days of recovery, which included a marked reduction in the survival of cortical GAD + , parvalbumin + , calretinin + , and calbindin + interneuron populations. These findings are broadly consistent with previous studies showing loss and disrupted development of interneurons in the cerebral cortex and subcortical grey matter following HI in preterm and term fetal sheep [17,[32][33][34][35] and neonatal rodents [36][37][38][39][40][41][42][43] and following ventilatory support in preterm baboons [44]. Although there are no comparable term human data, limited pathology and imaging studies in preterm infants have shown reductions in the numbers and complexity of cortical interneurons, refs [25,45] cortical interneuron migration, [7] interneuron neurogenesis, [46] and cortical GABAergic signaling [4].…”

“…Of note, we previously reported that connexin43 hemichannel blockade could also reduce loss of striatal interneurons following HI in term fetal sheep [22]. Further, a reduction in interneuron loss in the cortex with erythropoietin treatment, [142] hippocampus with therapeutic hypothermia, [42] and striatum with bone marrow-derived mesenchymal stem cells [36] was reported following HI in neonatal rats and in the striatum with nitric oxide inhibition [33] or therapeutic hypothermia [143] following HI in preterm fetal sheep. Future studies are required to determine the exact timing of interneuron loss, altered PNN development, and expression of ECM remodeling enzymes after HI to help optimize treatment strategies.…”

Transient increase in CSF GAP-43 concentration after ischemic stroke

“…Also, GAP-43 expressing neurons may be more resistant to the direct ischemic event. It has been suggested that different types of neurons are differently susceptible to early and delayed cell death after ischemic injuries [ 31 ].…”

Evidence for Sexual Dimorphism in the Response to TLR3 Activation in the Developing Neonatal Mouse Brain: A Pilot Study

“…Sections obtained from animals killed 24 h or 7 days after normal saline or poly I:C exposure were used to assess astroglia (GFAP) and microglia (Iba1) activation and overall microscopic evidence of injury (Nissl counterstaining for GFAP IHC). Floating IHC was performed as previously described (Chavez-Valdez et al, 2018) with whole rabbit antisera anti-GFAP (DAKO/Agilent Technologies, Santa Clara, CA, United States; 1:1000), or anti-Iba1 (Wako Chemicals USA, Inc., Richmond, VA, United States; 1:500) followed by goat anti-rabbit antibody (1:200) used as the secondary antibody using DAB as the chromogen for GFAP and using Alexa Fluor 568 for immunofluorescence for Iba1. Cresyl-violet (CV) counterstaining was performed in those sections previously immunostained for GFAP to assess histological structure.…”