Tricellulin, α-Catenin and Microfibrillar-Associated Protein 5 Exhibit Concomitantly Altered Immunosignals along with Vascular, Extracellular and Cytoskeletal Elements after Experimental Focal Cerebral Ischemia.

Along with initiatives to understand the pathophysiology of stroke in detail and to identify neuroprotective targets, cell-stabilizing elements have gained increasing attention. Although cell culture experiments have indicated that tricellulin, α-catenin and microfibrillar-associated protein 5 (MFAP5) contribute to cellular integrity, these elements have not yet been investigated in the ischemic brain. Applying immunofluorescence labeling, this study explored tricellulin, MFAP5 and α-catenin in non-ischemic and ischemic brain areas of mice (24, 4 h of ischemia) and rats (4 h of ischemia), along with collagen IV and fibronectin as vascular and extracellular matrix constituents and microtubule-associated protein 2 (MAP2) and neurofilament light chain (NF-L) as cytoskeletal elements. Immunosignals of tricellulin and notably MFAP5 partially appeared in a fiber-like pattern, and α-catenin appeared more in a dotted pattern. Regional associations with vascular and extracellular constituents were found for tricellulin and α-catenin, particularly in ischemic areas. Due to ischemia, signals of tricellulin, MFAP5 and α-catenin decreased concomitantly with MAP2 and NF-L, whereby MFAP5 provided the most sensitive reaction. For the first time, this study demonstrated ischemia-related alterations in tricellulin, MFAP5 and α-catenin along with the vasculature, extracellular matrix and cytoskeleton. Confirmatory studies are needed, also exploring their role in cellular integrity and the potential for neuroprotective approaches in stroke.

Combining atomic force microscopy and fluorescence-based techniques to explore mechanical properties of naive and ischemia-affected brain regions in mice.

Knowledge of the brain's structure and function is essential for understanding processes in health and disease. Histochemical and fluorescence-based techniques have proven beneficial in characterizing brain regions and cellular compositions in pre-clinical research. Atomic force microscopy (AFM) has been introduced for mechanical tissue characterization, which may also help investigate pathophysiological aspects in disease-related models such as stroke. While combining AFM and fluorescence-based techniques, this study explored the mechanical properties of naive and ischemic brain regions in mice. Ischemia-affected regions were identified by the green signal of fluorescein isothiocyanate-conjugated albumin. A semi-automated protocol based on a brain atlas allowed regional allocations to the neocortex, striatum, thalamus, hypothalamus, hippocampus, and fiber tracts. Although AFM led to varying measurements, intra-individual analyses indicated a gradually increased tissue stiffness in the neocortex compared to subcortical areas, i.e., the striatum and fiber tracts. Regions affected by ischemia predominantly exhibited an increased tissue stiffness compared to those of the contra-lateral hemisphere, which might be related to cellular swelling. This study indicated intra-individual differences in mechanical properties among naive and ischemia-affected brain regions. The combination of AFM, semi-automated regional allocations, and fluorescence-based techniques thus qualifies for mechanical characterizations of the healthy and disease-affected brain in pre-clinical research.

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.

In the setting of stroke, ischemia not only impairs neuronal function, but also detrimentally affects the different components of the neurovascular unit, which are shown to be involved in the transition from reversible to long-lasting tissue damage. In this context, the glial proteins myelin basic protein (MBP) and the 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNP) as well as the vasculature-associated basement membrane proteins laminin and collagen IV have been identified as ischemia-sensitive elements. However, available data from immunofluorescence and Western blot analyses are often found to be contradictory, which renders interpretation of the respective data rather difficult. Therefore, the present study investigates the impact of tissue pre-treatment and antibody clonality on immunofluorescence measurements of the mentioned proteins in a highly reproducible model of permanent middle cerebral artery occlusion. Here, immunofluorescence labeling using polyclonal antibodies revealed an increased immunofluorescence intensity of MBP, CNP, laminin and collagen IV in ischemic areas, although Western blot analyses did not reveal increased protein levels. Importantly, contrary to polyclonal antibodies, monoclonal ones did not provide increased fluorescence intensities in ischemic areas. Further, we were able to demonstrate that different ways of tissue pre-treatment including paraformaldehyde fixation and antigen retrieval may not only impact on fluorescence intensity measurements in general, but rather one-sidedly affect either ischemic or unaffected tissue. Therefore, immunofluorescence intensity measurements do not necessarily correlate with the actual protein levels, especially in ischemia-affected tissue and should always be complemented by different techniques to enhance reproducibility and to hopefully overcome the translational roadblock from bench to bedside.