Capping Protein Regulator and Myosin 1 Linker 3 (CARMIL3) as a Molecular Signature of Ischemic Neurons in the DWI-T2 Mismatch Areas After Stroke

Abstract

Ischemic stroke with a mismatch between diffusion-weighted imaging (DWI) and fluid-attenuated inversion recovery (FLAIR) or T2-weighted images indicates onset within 4.5 h, but the pathological substrates in the DWI-T2 mismatch and T2(+) areas remain elusive. In this study, proteomics was used to explore (1) the protein expression profiles in the T2(+), mismatch, and contralateral areas, and (2) the protein with the highest expression in the T2(+) area in the brains of male Sprague-Dawley rats within 4.5 h after middle cerebral artery occlusion (MCAO). The expression of the candidate protein was further validated in (1) rat brain subjected to MCAO, (2) rat primary cortical neuronal culture with oxygen-glucose deprivation (OGD), and (3) infarcted human brain tissues. This study showed that apoptosis was observed in the T2(+) and mismatch regions and necroptosis in the T2(+) region of rat brains after MCAO. We identified capping protein regulator and myosin 1 linker 3 (CARMIL3) as the candidate molecule in the T2(+) and mismatch areas, exclusively in neurons, predominantly in the cytoplasm, and most abundant in the mismatch area. The CARMIL3(+) neurons and neurites in the mismatch and T2(+) areas were larger than those in the control area, and associated with (1) increased expression of sulfonylurea receptor 1 (SUR1), indicating edema, (2) accumulation of p62, indicating impaired autophagy, and (3) increase in 8-hydroxy-2′-deoxyguanosine (8-OHdG), indicating oxidative stress. The increased expression of CARMIL3 was validated in a cell model of cortical neurons after OGD and in infarcted human brain tissues. In conclusion, this study shows that the mismatch and T2(+) areas within 4.5 h after ischemia are characterized by upregulated expression of CARMIL3 in neurons, particularly the mismatch area, which is associated with neuronal edema, impaired autophagy, and oxidative stress, indicating that CARMIL3 serves as a molecular signature of brain ischemia.

Nerve pathology of microangiopathy and thromboinflammation in hereditary transthyretin amyloidosis

“…The procedures of immunohistochemistry and immunofluorescence staining and quantification of image signals followed our established protocol 26,27 . Briefly, sural nerve specimens were cryosectioned to 10 μm‐thickness sections and immunostained with specific antibodies (Supplemental Table S1).…”

“…The procedures of immunohistochemistry and immunofluorescence staining and quantification of image signals followed our established protocol. 26 , 27 Briefly, sural nerve specimens were cryosectioned to 10 μm‐thickness sections and immunostained with specific antibodies (Supplemental Table S1 ). Immunofluorescence images were acquired using a confocal microscope (Leica TCS SP8 × STED 3X, Wetzlar, Germany) and quantified using ImageJ software (National Institute of Mental Health, Bethesda, MD).…”

Differences between cultured cortical neurons by trypsin and papain digestion

“…With advancements in scientific research, in order to better elucidate the mechanism, the research needs to be done not only in living animal (in vivo), but also using cell culture in−vitro experiments for verification. The culture of neurons has enabled great convenience in the study of neuron growth, development, and pathophysiology mechanisms in vitro, which is helpful for medical staff and researchers to gain further understanding of a variety of neurological diseases, such as Alzheimer's disease, Parkinson's disease, neonatal hypoxic–ischemic encephalopathy, stroke, cerebral ischemia/reperfusion injury, and so on 1–5 . Primary culture of neurons can fulfill such demands.…”

“…The culture of neurons has enabled great convenience in the study of neuron growth, development, and pathophysiology mechanisms in vitro, which is helpful for medical staff and researchers to gain further understanding of a variety of neurological diseases, such as Alzheimer's disease, Parkinson's disease, neonatal hypoxic–ischemic encephalopathy, stroke, cerebral ischemia/reperfusion injury, and so on. 1 , 2 , 3 , 4 , 5 Primary culture of neurons can fulfill such demands. Primary cultured neurons are very similar to in vivo neurons, the experimental conditions are stable and controllable, and the experimental environment is relatively simple.…”