Chronic cerebral hypoperfusion (CCH) evokes mild cognitive impairment (MCI) and contributes to the progression of vascular dementia and Alzheimer's disease (AD). How CCH 2 induces these neurodegenerative processes that may spread along the synaptic network and whether they are detectable at the synaptic proteome level of the cerebral cortex remains to be established.In the present study, we report the synaptic protein changes in the cerebral cortex after stepwise bilateral common carotid artery occlusion (BCCAO) induced CCH in the rat. The occlusions were confirmed with Magnetic Resonance Angiography 5 weeks after the surgery.Synaptosome fractions were prepared using sucrose gradient centrifugation from cerebral cortex dissected 7 weeks after the occlusion. The synaptic protein differences between the sham operated and CCH groups were analysed with label free nanoUHPLC-MS/MS.
During chronic cerebral hypoperfusion (CCH), the cerebral blood flow gradually decreases, leading to cognitive impairments and neurodegenerative disorders, such as vascular dementia. The reduced oxygenation, energy supply induced metabolic changes, and insufficient neuroplasticity could be reflected in the synaptic proteome. We performed stepwise bilateral common carotid occlusions on rats and studied the synaptic proteome changes of the hippocampus, occipital and frontal cortices. Samples were prepared and separated by 2-D DIGE and significantly altered protein spots were identified by HPLC–MS/MS. We revealed an outstanding amount of protein changes in the occipital cortex compared to the frontal cortex and the hippocampus with 94, 33, and 17 proteins, respectively. The high alterations in the occipital cortex are probably due to the hypoxia-induced retrograde degeneration of the primary visual cortex, which was demonstrated by electrophysiological experiments. Altered proteins have functions related to cytoskeletal organization and energy metabolism. As CCH could also be an important risk factor for Alzheimer’s disease (AD), we investigated whether our altered proteins overlap with AD protein databases. We revealed a significant amount of altered proteins associated with AD in the two neocortical areas, suggesting a prominent overlap with the AD pathomechanism.
Systemic administration of cholinergic antagonist scopolamine (SCO) is a widely used model to induce experimental cognitive impairment during preclinical drug testing of putative procognitive compounds. This model, however, has limited predictive validity partly due to its peripheral side-effects, therefore objective neuroimaging measures would greatly enhance its translational value. To this end, we administered SCO and peripherally acting butylscopolamine (BSCO) in preclinical functional MRI (fMRI, 9.4T) studies and measured their effects on whisker stimulation induced fMRI activation in Wistar rats. Beside the commonly used gradient echo echo-planar imaging (GE EPI) an arterial spin labeling method was also used to elucidate the vasculature-related properties of the BOLD-response. To account for anesthesia-effects, in two separate experiments we used isoflurane and combined (isoflurane + i.p. dexmedetomidine) anesthesia. In the second experiment with GE EPI and spin echo (SE) EPI sequences the effect of donepezil (DON) was also examined. In isoflurane anesthesia with GE EPI, SCO decreased the evoked BOLD response in the barrel cortex (BC), while BSCO increased it in the anterior cingulate cortex. In the combined anesthesia SCO decreased the activation in the BC as well as in the inferior colliculus (IC) while BSCO reduced the response merely in the IC. Our results revealed that SCO attenuated the evoked BOLD activation in the BC as a probable central effect in both experiments while its other detected actions depended on the type of anesthesia or dose and the given fMRI sequences.
Declining cerebral blood flow leads to chronic cerebral hypoperfusion which can induce neurodegenerative disorders, such as vascular dementia. The reduced energy supply of the brain impairs mitochondrial functions that could trigger further damaging cellular processes. We carried out stepwise bilateral common carotid occlusions on rats and investigated long-term mitochondrial, mitochondria-associated membrane (MAM), and cerebrospinal fluid (CSF) proteome changes. Samples were studied by gel-based and mass spectrometry-based proteomic analyses. We found 19, 35, and 12 significantly altered proteins in the mitochondria, MAM, and CSF, respectively. Most of the changed proteins were involved in protein turnover and import in all three sample types. We confirmed decreased levels of proteins involved in protein folding and amino acid catabolism, such as P4hb and Hibadh in the mitochondria by western blot. We detected reduced levels of several components of protein synthesis and degradation in the CSF as well as in the subcellular fractions, implying that hypoperfusion-induced altered protein turnover of brain tissue can be detected in the CSF by proteomic analysis.
During preclinical drug testing, the systemic administration of scopolamine (SCO), a cholinergic antagonist, is widely used. However, it suffers important limitations, like non-specific behavioural effects partly due to its peripheral side-effects. Therefore, neuroimaging measures would enhance its translational value. To this end, in Wistar rats, we measured whisker-stimulation induced functional MRI activation after SCO, peripherally acting butylscopolamine (BSCO), or saline administration in a cross-over design. Besides the commonly used gradient-echo echo-planar imaging (GE EPI), we also used an arterial spin labeling method in isoflurane anesthesia. With the GE EPI measurement, SCO decreased the evoked BOLD response in the barrel cortex (BC), while BSCO increased it in the anterior cingulate cortex. In a second experiment, we used GE EPI and spin-echo (SE) EPI sequences in a combined (isoflurane + i.p. dexmedetomidine) anesthesia to account for anesthesia-effects. Here, we also examined the effect of donepezil. In the combined anesthesia, with the GE EPI, SCO decreased the activation in the BC and the inferior colliculus (IC). BSCO reduced the response merely in the IC. Our results revealed that SCO attenuated the evoked BOLD activation in the BC as a probable central effect in both experiments. The likely peripheral vascular actions of SCO with the given fMRI sequences depended on the type of anesthesia or its dose.
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