Hemoglobin-induced neuronal degeneration in the hippocampus after neonatal intraventricular hemorrhage

Abstract

Neuronal degeneration following neonatal intraventricular hemorrhage (IVH) is incompletely understood. Understanding the mechanisms of degeneration and cell loss may point toward specific treatments to limit injury. We evaluated the role of hemoglobin (Hb) in cell death after intraventricular injection in neonatal rats. Hb was injected into the right lateral ventricle of post-natal day 7 rats. Rats exposed to anesthesia were used for controls. The CA-1 region of the hippocampus was analyzed via immunohistochemistry, hematoxylin and eosin (H&E) staining, Fluoro-Jade C staining, Western blots, and double-labeling stains. Compared to controls, intraventricular injection of Hb decreased hippocampal volume (27% decrease; p<0.05), induced neuronal loss (31% loss; p<0.01), and increased neuronal degeneration (2.7 fold increase; p<0.01), which were all significantly reduced with the iron chelator, deferoxamine. Hb upregulated p-JNK (1.8 fold increase; p<0.05) and increased expression of the Hb/haptoglobin endocytotic receptor CD163 in neurons in vivo and in vitro (cultured cortical neurons). Hb induced expression of the CD163 receptor, which co-localized with p-JNK in hippocampal neurons, suggesting a potential pathway by which Hb enters the neuron to result in cell death. There were no differences in neuronal loss or degenerating neurons in Hb-injected animals that developed hydrocephalus versus those that did not. Intraventricular injection of Hb causes hippocampal neuronal degeneration and cell loss and increases brain p-JNK levels. p-JNK co-localized with the Hb/haptoglobin receptor CD163, suggesting a novel pathway by which Hb enters the neuron after IVH to result in cell death.

Pathophysiology of extracellular haemoglobin: use of animal models to translate molecular mechanisms into clinical significance

“…3). Deferoxamine, a potent free iron chelator, has been shown to attenuate hippocampal cell loss following IVH [39]. Of further interest, it was recently shown that deletion of Hpx, a natural hemescavenger, leads to aggravated brain injury following intracranial bleeding and increased iron deposition [40].…”

CD163, a Hemoglobin/Haptoglobin Scavenger Receptor, After Intracerebral Hemorrhage: Functions in Microglia/Macrophages Versus Neurons

“…However, several recent studies have reported evidence of CD163 + neurons following hemorrhage. Our lab documented the first finding of CD163 expression in neurons [2,4]. A neonatal rat model was used to simulate intraventricular hemorrhage, after which CD163 was found in hippocampal pyramidal neurons.…”

“…When not bound to O 2 , heme-bound iron exists in a ferrous (Fe 2+ ) state capable of performing radical chemistry to create dangerous reactive oxygen species. Several studies have highlighted the hemoglobin-induced damage caused to neuronal populations during hemorrhagic stroke, suggesting the existence of pathways by which hemoglobin-associated iron can enter neurons and inflict oxidative injury [1][2][3]. While there are several known mechanisms by which free iron and heme can enter cells, the number of known hemoglobin uptake mechanisms is limited.…”

“…The best characterized is CD163, long thought to be a marker of monocytes/macrophages. Recent evidence shows that CD163 can also be expressed in neuronal populations following ICH, providing a mechanism for hemoglobin uptake into neurons [2,4]. This commentary discusses the potential beneficial roles of CD163 in microglia/ macrophages after ICH and compares them to potentially detrimental effects the receptor may have in neurons.…”

In vivoquantification of intraventricular hemorrhage in a neonatal piglet model using an EEG-layout based electrical impedance tomography array

“…The bleed may become severe enough to occupy >50% of the ventricle producing dilation of the ventricles (Grade III). Large IVHs are associated with unfavorable neurological outcomes (Alderliesten et al 2013, Noori et al 2014, Garton et al 2016). Adverse neurodevelopmental sequelae include cerebral palsy, seizures, and hydrocephalus requiring a shunt, blindness, deafness, and cognitive impairment (Goldenberg and Jobe 2001, Hintz and O’Shea 2008).…”