Impaired Neurofilament Integrity and Neuronal Morphology in Different Models of Focal Cerebral Ischemia and Human Stroke Tissue

Abstract

As part of the neuronal cytoskeleton, neurofilaments are involved in maintaining cellular integrity. In the setting of ischemic stroke, the affection of the neurofilament network is considered to mediate the transition towards long-lasting tissue damage. Although peripheral levels of distinct neurofilament subunits are shown to correlate with the clinically observed severity of cerebral ischemia, neurofilaments have so far not been considered for neuroprotective approaches. Therefore, the present study systematically addresses ischemia-induced alterations of the neurofilament light (NF-L), medium (NF-M), and heavy (NF-H) subunits as well as of α-internexin (INA). For this purpose, we applied a multi-parametric approach including immunofluorescence labeling, western blotting, qRT-PCR and electron microscopy. Analyses comprised ischemia-affected tissue from three stroke models of middle cerebral artery occlusion (MCAO), including approaches of filament-based MCAO in mice, thromboembolic MCAO in rats, and electrosurgical MCAO in sheep, as well as human autoptic stroke tissue. As indicated by altered immunosignals, impairment of neurofilament subunits was consistently observed throughout the applied stroke models and in human tissue. Thereby, altered NF-L immunoreactivity was also found to reach penumbral areas, while protein analysis revealed consistent reductions for NF-L and INA in the ischemia-affected neocortex in mice. At the mRNA level, the ischemic neocortex and striatum exhibited reduced expressions of NF-L- and NF-H-associated genes, whereas an upregulation for Ina appeared in the striatum. Further, multiple fluorescence labeling of neurofilament proteins revealed spheroid and bead-like structural alterations in human and rodent tissue, correlating with a cellular edema and lost cytoskeletal order at the ultrastructural level. Thus, the consistent ischemia-induced affection of neurofilament subunits in animals and human tissue, as well as the involvement of potentially salvageable tissue qualify neurofilaments as promising targets for neuroprotective strategies. During ischemia formation, such approaches may focus on the maintenance of neurofilament integrity, and appear applicable as co-treatment to modern recanalizing strategies.

Acute Neurofilament Light Chain Plasma Levels Correlate With Stroke Severity and Clinical Outcome in Ischemic Stroke Patients

“…NF immunoreactivity has previously been shown to increase in the ischemic brain following experimental ischemia in rats and mice and the postmortem human ischemic brain (24,25). In the present study, we also observed intense neurofilament staining in ischemic neurons and in the infarcted brain parenchyma in two postmortem ischemic stroke cases with >7-day-old parietal and 3-7-day-old temporal lobe infarcts (Figure 1).…”

“…In the present study, we observed increased NF immunoreactivity in the ischemia-affected area compared to normal-appearing tissue in a 3-7-day-old temporal lobe infarct and a >7-day-old right parietal lobe infarct. The increased NF immunoreactivity is possibly related to an increase in detectable NF degradation products in the ischemic core due to structural cytoskeletal alterations in neurons following ischemia (25). Previous studies on postmortem tissues from victims of stroke and other focal lesions using immunostaining with antibodies specific for phosphorylated NF-H have demonstrated that NF phosphorylation (normally confined to axons) occurs aberrantly in perikaria and dendrites of neurons with axons projecting to damaged brain areas (26).…”

“…Statistical analyses were carried out using R 3.5.1. Data are presented as medians and interquartile ranges [25,75], and p ≤ 0.05 was considered statistically significant. Graphs were generated using the Prism 5 software for Mac (GraphPad).…”

Increased Immunosignals of Collagen IV and Fibronectin Indicate Ischemic Consequences for the Neurovascular Matrix Adhesion Zone in Various Animal Models and Human Stroke Tissue

“…Quantitative analyses of the immunofluorescence signals from collagen IV and fibronectin were based on five brain sections from each mouse, while these sections were selected concerning the following criteria: first, an unequivocal ischemic affection as indicated by a regional change of the fluorescence signal typically located in the right-sided neocortex and subcortical areas as known from own earlier studies (e.g., Mages et al, 2018), and second, the absence of tissue damage that prevents immunofluorescence-based analyses in large parts of the ischemia-affected hemisphere. This selection process allowed the analysis of six mice with an ischemia duration of 4 h and five mice with an ischemia duration of 24 h. As prerequisite for the following quantification, in each of the selected brain sections, seven regions of interests (ROIs) were arranged on the ischemia-affected hemisphere including six neocortical ROIs covering the ischemic border zone with most evident changes of the collagen IV immunosignal between ROIs 3 and 4, and a further ROI (number 7) in the subcortex, i.e., the striatum.…”

Characterization of Hippocampal Histomorphology Following R. vomitoria Root Extract Treatment

“…NF is a neuronal cytoskeletal protein, an intermediate filament that provides structural support for axons, as well as regulating their diameter (Iwanaga et al, 1989). Decreased NF expression indicates adverse protein rearrangement arising prior to alterations in axonal cytoskeleton (Mages et al, 2018), and RV is reported with adverse neuronal activities (Ekong et al, 2020;Nduohosewo and Ekong, 2020), which may have resulted in decreased expression and population in the present study. The present result is in line with Ekong et al (2016b), who reported that RV cause decreased NF expression in the olfactory bulb.…”