Regionally Altered Immunosignals of Surfactant Protein-G, Vascular and Non-Vascular Elements of the Neurovascular Unit after Experimental Focal Cerebral Ischemia in Mice, Rats, and Sheep

Abstract

The surfactant protein-G (SP-G) has recently been discovered in the brain and linked to fluid balance regulations. Stroke is characterized by impaired vessel integrity, promoting water influx and edema formation. The neurovascular unit concept (NVU) has been generated to cover not only ischemic affections of neurons or vessels but also other regionally associated cells. This study provides the first spatio-temporal characterization of SP-G and NVU elements after experimental stroke. Immunofluorescence labeling was applied to explore SP-G, vascular and cellular markers in mice (4, 24, and 72 h of ischemia), rats (24 h of ischemia), and sheep (two weeks of ischemia). Extravasated albumin indicated vascular damage within ischemic areas. Quantifications revealed decreasing SP-G signals in the ischemia-affected neocortex and subcortex. Inverse immunosignals of SP-G and vascular elements existed throughout all models. Despite local associations between SP-G and the vasculature, a definite co-localization was not seen. Along with a decreased SP-G-immunoreactivity in ischemic areas, signals originating from neurons, glial elements, and the extracellular matrix exhibited morphological alterations or changed intensities. Collectively, this study revealed regional alterations of SP-G, vascular, and non-vascular NVU elements after ischemia, and may thus stimulate the discussion about the role of SP-G during stroke.

Increasing reproducibility in preclinical stroke research: the correlation of immunofluorescence intensity measurements and Western blot analyses strongly depends on antibody clonality and tissue pre-treatment in a mouse model of focal cerebral ischemia

“…Similar results are reported for MBP, either describing a reduction of MBP at the protein level or insignificant changes 24 h after ischemic onset ( Chu et al, 2012 ; Buscemi et al, 2019 ; Zalewska et al, 2021 ). In contrast, studies using immunofluorescence labeling describe a post-ischemic increase of CNP and MBP-related signals ( Michalski et al, 2018 , 2022 ; Mages et al, 2019 , 2021 ). The available literature regarding post-ischemic levels of laminin and collagen IV is also indeterminate.…”

“…Although, methodological differences of ischemia induction and observation time points should always be considered, these aspects alone are unlikely to explain the described contradictions. On the contrary, increased immunofluorescence intensities of CNP and collagen IV have been consistently observed using polyclonal antibodies in various models of ischemia and human autoptic brain tissue over different time points (Härtig et al, 2017;Mages et al, 2018Mages et al, , 2019Mages et al, , 2021Michalski et al, 2020Michalski et al, , 2022.…”

“…Although other host species for monoclonal antibodies might be an alternative, the availability is often limited as rodents still represent the most common host. This may explain, why analyses based on immunolabeling in stroke tissue are mostly performed using polyclonal antibodies (CNP: Jing et al, 2013 ; Michalski et al, 2018 ; Mages et al, 2019 , 2021 ; MBP: Gregersen et al, 2001 ; Zhao et al, 2014 ; Michalski et al, 2018 ; collagen IV: Hamann et al, 2002 ; Vosko et al, 2006 ; Anik et al, 2011 ; Ji and Tsirka, 2012 ; Härtig et al, 2017 ; Michalski et al, 2017 , 2020 , 2022 ; laminin: Wagner et al, 1997 ; Gu et al, 2005 ; Ji and Tsirka, 2012 ).…”

Cellular and Molecular Targets in Acute Ischemic Stroke

“…A special focus was laid on the role of local vascular responses in acute stroke. The following studies paradigmatically delineate a crucial role of immune cell subsets and associated molecules/proteins on arterial thrombus formation [ 1 ], intravascular inflammation in hyper-acute stroke [ 2 ], ischemic affection/impairment of the NVU [ 3 , 4 ], and thus the blood-brain barrier (BBB) [ 5 ], as well as the direct translational approach of this special issue. Essig and co-workers characterized cerebral thromboemboli retrieved via endovascular thrombectomy regarding high-mobility group box 1 protein (HMGB1), a damage-associated molecular pattern (DAMP) involved in neutrophil extracellular trap (NET) formation and thrombosis.…”

“…The number of leukocytes (especially neutrophils) within the occluded cerebral vasculature was significantly higher compared to that in the systemic circulation and was associated with infarct extent [ 2 ]. Michalski et al, provide the first spatio-temporal characterization of the surfactant protein-G (SP-G) at the NVU after experimental focal cerebral ischemia in mice, rats, and sheep [ 3 ]. This study may stimulate further discussion about the role of SP-G within the ischemia affected NVU, which might be linked to the integrity of the vasculature.…”