Low Endogenous G-Protein-Coupled Receptor Kinase 2 Sensitizes the Immature Brain to Hypoxia–Ischemia-Induced Gray and White Matter Damage

Abstract

Hypoxic-ischemic brain injury is regulated in part by neurotransmitter and chemokine signaling via G-protein-coupled receptors (GPCRs). GPCR-kinase 2 (GRK2) protects these receptors against overstimulation by inducing desensitization. Neonatal hypoxic-ischemic brain damage is preceded by a reduction in cerebral GRK2 expression. We determined the functional importance of GRK2 in hypoxic-ischemic brain damage.Nine-day-old wild-type and GRK2 ϩ/Ϫ mice with a ϳ50% reduction in GRK2 protein were exposed to unilateral carotid artery occlusion and hypoxia. In GRK2 ϩ/Ϫ animals, gray and white matter damage was aggravated at 3 weeks after hypoxia-ischemia. In addition, cerebral neutrophil infiltration was increased in GRK2 ϩ/Ϫ animals. Neutrophil depletion reduced brain damage, but neuronal loss was still more pronounced in GRK2 ϩ/Ϫ animals. Onset of neuronal loss was advanced in GRK2 ϩ/Ϫ animals regardless of neutrophil depletion. White matter injury was advanced in GRK2 ϩ/Ϫ animals and was not affected by neutrophil depletion. Activation/infiltration of microglia/macrophages was stronger in GRK2 ϩ/Ϫ brains but only occurred 24 h after hypoxia-ischemia and is therefore not the primary cause of increased damage. During hypoxia, cerebral blood flow was reduced to the same extent in both genotypes. In vitro, GRK2 ϩ/Ϫ hippocampal slices and cerebellar granular neurons were more sensitive to glutamate-induced death.We propose the novel concept that the kinase GRK2 regulates onset and magnitude of hypoxic-ischemic brain damage. Increased gray and white matter damage in GRK2 ϩ/Ϫ animals was not dependent on infiltrating neutrophils and occurred before microglia/macrophage activation was detected. Collectively, our data suggest that cerebral GRK2 has an important endogenous neuroprotective role in ischemic cerebral damage.

Phosphorylation-independent Regulation of Metabotropic Glutamate Receptor 5 Desensitization and Internalization by G Protein-coupled Receptor Kinase 2 in Neurons

“…The observed differences in GRK2 protein expression and mGluR5 endocytosis may be important physiologically in determining the sensitivity of neurons to excitotoxic cell death. For example, heterozygous GRK2 knock-out mice exhibit increased neuronal cell loss following unilateral carotid artery occlusion and hypoxia (39). Moreover, hippocampal slices and cerebellar granular neurons derived from heterozygous GRK2 knock-out mice are more sensitive to glutamate-induced death.…”

Repeated Mesenchymal Stem Cell Treatment after Neonatal Hypoxia-Ischemia Has Distinct Effects on Formation and Maturation of New Neurons and Oligodendrocytes Leading to Restoration of Damage, Corticospinal Motor Tract Activity, and Sensorimotor Function

“…Experiments were performed according to international guidelines and approved by the University Medical Center Utrecht experimental animal committee. At postnatal day 9 (P9), C57BL/6J mice underwent HI by right common carotid artery occlusion under isoflurane anesthesia [3% induction, 1% maintenance in O 2 :N 2 O (1:1)] followed by 45 min exposure to 10% oxygen in nitrogen (Nijboer et al, 2008). This procedure induced a lesion involving hippocampus, neocortex, and striatum (van der Kooij et al, 2010).…”

“…Brain damage was analyzed at a location equivalent to Ϫ1.58 mm from bregma in adult mice by outlining both hemispheres on full section images using ImageJ software (W. S. Rasband, ImageJ, National Institutes of Health, Bethesda, MD; http://rsb.info.nih.gov/ij/; 1997-2009). Ipsilateral MAP2 and MBP area loss were calculated as described previously (Nijboer et al, 2008).…”

Nasal administration of stem cells: a promising novel route to treat neonatal ischemic brain damage

“…The animal committee of Academic Biomedical Center Utrecht approved all experiments. At postnatal d 9 (p9), C57Bl/6 mice pups of both sexes underwent HI by permanent right common carotid artery occlusion under isoflurane anesthesia [3% induction and 1% maintenance in O 2 :N 2 O (1:1)] followed by exposure of 10% oxygen in nitrogen for 45 min (16). This procedure induced a lesion involving hippocampus, neocortex, and striatum.…”

“…Ipsilateral MAP2 area loss was calculated as follows: [1 Ϫ (area ipsilateral MAP2 staining/area contralateral MAP2 staining)] times 100%. Similarly, MBP area loss was determined (16).…”