Key Role of Cytochrome C for Apoptosis Detection Using Raman Microimaging in an Animal Model of Brain Ischemia with Insulin Treatment

Abstract

Brain ischemia represents a leading cause of death and disability in industrialized countries. To date, therapeutic intervention is largely unsatisfactory and novel strategies are required for getting better protection of neurons injured by cerebral blood flow restriction. Recent evidence suggests that brain insulin leads to protection of neuronal population undergoing apoptotic cell death via modulation of oxidative stress and mitochondrial cytochrome c (CytC), an effect to be better clarified. In this work, we investigate on the effect of insulin given intracerebroventricular (ICV) before inducing a transient global ischemia by bilateral occlusion of the common carotid arteries (BCCO) in Mongolian gerbils (MG). The transient (3 min) global ischemia in MG is observed to produce neurodegenerative effect mainly into CA3 hippocampal region, 72 h after cerebral blood restriction. Intracerebroventricular microinfusion of insulin significantly prevents the apoptosis of CA3 hippocampal neurons. Histological observation, after hematoxylin and eosin staining, puts in evidence the neuroprotective role of insulin, but Raman microimaging provides a clearer insight in the CytC mechanism underlying the apoptotic process. Above all, CytC has been revealed to be an outstanding, innate Raman marker for monitoring the cells status, thanks to its resonant scattering at 530 nm of incident wavelength and to its crucial role in the early stages of cells apoptosis. These data support the hypothesis of an insulin-dependent neuroprotection and antiapoptotic mechanism occurring in the brain of MG undergoing transient brain ischemia. The observed effects occurred without any peripheral change on serum glucose levels, suggesting an alternative mechanism of insulin-induced neuroprotection.

Small Sample Stress: Probing Oxygen-Deprived Ammonia-Oxidizing Bacteria with Raman Spectroscopy In Vivo

“…In contrast, Russo et al observed a 5 cm −1 upshift in the line at 1587 rel. cm −1 and used it to differentiate between healthy and apoptotic and pre-apoptotic brain cells of Mongolian gerbils, associating the line shift with cytochrome c deformation due to the protein binding to membrane elements [35]. A similar observation was reported earlier by Murgida and Hildebrandt [46,47], who saw a 10 cm −1 upshift in the line of the ν 19 vibration in ferrous compared to ferric cytochrome c adsorbed to a surface-enhanced Raman active substrate.…”

“…cm −1 , which at 532 nm excitation is one of the strongest Raman lines in ferrous cytochrome c, but is very weak if the protein is in ferric state. Recently, Russo et al used Raman microscopy with cytochrome-c-resonant excitation at 532 nm, using the redox state of cytochrome c as a crucial indicator for cell apoptosis [35], while Kakita et al used 532 nm excitation to quantify the redox state of cytochromes b and c in yeast [36]. Shin et al monitored photothermally induced mitochondrially mediated apoptosis in situ based on the Raman spectrum of cytochrome c, using surface-enhanced resonant Raman microscopy with gold nanoparticles and 785 nm excitation [37].…”

Data-Driven Identification of Biomarkers for In Situ Monitoring of Drug Treatment in Bladder Cancer Organoids

“…The most prominent peaks in correlation with vtx treatment are depicted in purple boxes in the loading plots. The peaks around 1308-1315 cm −1 , 1125 cm −1 and 747 cm −1 can be assigned to cytochrome c [28,38]. The bands in the region between 1447-1450 cm −1 are indicative of changes in the CH 2 conformation of proteins [30,31,39].…”

Hot Spots for the Use of Intranasal Insulin: Cerebral Ischemia, Brain Injury, Diabetes Mellitus, Endocrine Disorders and Postoperative Delirium

“…The neuroprotective properties of insulin in ischemic injury have been studied for more than 30 years [ 164 , 165 , 166 , 167 ]. It has been shown that insulin with various delivery methods (subcutaneous, intraperitoneal, intravenous, and intracerebroventricular) positively affects the survival and cognitive functions of animals that have undergone cerebral ischemia [ 165 , 168 , 169 , 170 , 171 , 172 , 173 , 174 , 175 , 176 ]. The antiapoptotic signaling pathway, including IR/IRS/PI3K/AKT, plays a leading role in the implementation of the neuroprotective action of insulin.…”