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.

Keywords: MCAO; axonal spheroids; cerebral ischemia; neurofilaments; stroke; α-internexin.

FIGURE 1

Affection of neurofilament subunits in mice 24 h after experimental stroke induction. Representative overview showing double immunofluorescence labeling of NF-L and NF-H (A) as well as of INA and NF-M (B) 24 h after fMCAO. Ischemia-affected areas are demarcated by an increase of NF-L immunoreactivity, while the immunosignals for INA, NF-M, and NF-H are found to be decreased. Dashed lines outline the border of increased NF-L immunoreactivity within ischemia-affected areas (i). Scale bars: 1 mm.

FIGURE 2

Opposite alterations of neurofilament subunits at the infarct border after fMCAO in mice. Triple immunofluorescence labeling of NF-L, INA, and NF-M (A) as well as of NF-L, INA, and NF-H (B) is shown at the ischemic border zone and in respective contralateral control areas in the neocortex of mice 24 h after ischemia induction. Notably, NF-L appears to be upregulated in the ischemia-affected area, whereas the same area is characterized by decreased immunoreactivities of INA, NF-M, and slightly of NF-H. Dashed lines outline the border of increased NF-L immunoreactivity within ischemia-affected areas (i). Scale bars: (A) 100 μm; (B) 200 μm.

FIGURE 3

Opposite alterations of neurofilament subunits at the infarct border after eMCAO in rats. Triple immunofluorescence labeling of NF-L, INA, and NF-M (A) as well as of NF-L, INA, and NF-H (B) is shown at the ischemic border zone and in respective contralateral control areas in the model of eMCAO in rats 24 h after ischemia induction. Notably, in the ischemia-affected area NF-L appears to be upregulated, whereas the same area is characterized by decreased immunoreactivities of INA and NF-M. While the reduction of NF-H related immunolabeling appears to be faint, structural deformations of fibers and cellular processes became visible (B). Dashed lines outline the border of increased NF-L immunoreactivity within ischemia-affected areas (i). Scale bars: (A) 100 μm; (B) 200 μm.

FIGURE 4

Co-labeling of NF-L with ischemia-sensitive neuronal markers Double immunofluorescence labeling illustrates the alternate affection of NF-L and MAP2-related immunoreactivity. The border of the ischemic area (i) is characterized by a clear-cut loss of MAP2-related immunolabeling (dashed line), whereas the immunoreactivity of NF-L is increased (A). The ischemic penumbra is demarked by upregulation of HSP70 in NeuN-positive neurons (white arrows), which co-localizes with upregulated NF-L immunoreactivity, demonstrating ischemia-derived affection of NF-L in potentially salvageable tissue (B). The corresponding contralateral area serves as control. Scale bars: (A) 100 μm; (B) 75 μm.

FIGURE 5

Semi-quantification of NF-L, INA, NF-M, and NF-H immunofluorescence intensity in the model of fMCAO. Coronal mouse brain sections labeled with NF-L (red) and NF-H (green) illustrate the predefined ROIs in the cortex (A), as well as the peripheral (P), transitional (T), and central (C) areas of the striatum (B). Semi-quantification reveals a significant increase of NF-L immunoreactivity, as well as a significant decrease of INA immunoreactivity in the ischemic tissue of the neocortex (A) and striatum (B). NF-M immunoreactivity is significantly decreased in the ischemic neocortex, while NF-H immunoreactivity is not significantly altered (A). NF-M and NF-H show a trend towards decreased immunoreactivities in the ischemic striatum (B). Data are given as mean values; error bars indicate SEM. ^∗p < 0.05; n = 5.

FIGURE 6

Western blot and qRT-PCR analyses of fMCAO tissue. Western blot analyses reveal a significantly lower protein level of NF-L in the ischemic neocortex and a slightly lower level in the ischemic striatum (A,B). The ischemia-affected striatum and neocortex exhibit increased bands of lower molecular weight (∼40–65 kDa) compared to control areas (B,C). Western blot analysis reveals a trend towards decreased INA expression in the ischemic neocortex compared to control areas (D). qRT-PCR shows significantly decreased mRNA levels of Nefl and Nefh in the cortex (E), while the mRNA level of Ina is significantly increased in the striatum (F). Actb served as endogenous control gene (E,F). Bars and squares indicate mean values; error bars indicate SEM. ^∗p < 0.05; n = 5.

FIGURE 7

Opposite affections of neurofilament subunits and morphological alterations in human post-mortem stroke tissue. Overview of human autoptic brain stem tissue with histopathologically verified infarct (i) delineated by a dashed line (A). Higher magnification reveals alterations comparable to the findings in animal MCAO models with most profound ischemia-derived affection of NF-L and INA. NF-L shows an increased immunoreactivity within the ischemic area (B), while NF-M (B) and INA (C) appear to be decreased. A representative image illustrates characteristic morphological bead-like neuronal alterations, which can be visualized using each of the applied neuronal markers at high magnification (D). Dashed lines indicate ischemia-affected areas. Rectangle in (A) illustrates the area captured in (B) at higher magnification. Abbreviations: ICP, inferior cerebellar peduncle; NXII, hypoglossal nerve; ML, medial lemniscus; IO, inferior olive; PT, pyramidal tract. Scale bars: (A) 2 mm; (B) 200 μm; (C) 750 μm; (D) 100 μm.

FIGURE 8

Morphological alterations of neuronal processes due to ischemia demonstrated by immunofluorescence and electron microscopy. Morphological alterations as demarcated by NF-L, NF-M, and NF-H labeling observed in human stroke tissue (A) are detectable throughout the applied animal models of MCAO (B–F). NF-L immunolabeling reveals swollen and morphologically altered axons (C) and bulb-like vacuolization of NF-L-positive structures (D). Of note, bead-like deformations (arrow heads) are visible in continuity with neuronal processes (E) as well as apparently discontinuous to respective axons or dendrites (arrow heads) (F). Electron microscopy images obtained from the neocortex in the model of fMCAO illustrate severe alterations of the neuronal cytoskeleton resulting in vacuolization of axons (arrows) (G) comparable to the bead-like structures captured by immunofluorescence microscopy (D–F). Further, non-myelinated dendrites often appear less electron dense and swollen (transparently highlighted in yellow) indicative of a cellular edema, resulting in an impaired cytoskeletal order (insets) with lost alignment of microtubules and neurofilaments (H). Swollen and structurally altered cellular processes with an impaired cytoskeleton are also observed in the ischemia-affected striatum (I). Generally, a cellular edema is also detectable in surrounding ischemia affected glial cells (asterisks). Scale bars: (A–F) 25 μm; (G–I) 2 μm.