ABSTRACT
Following middle cerebral artery (MCA) stroke, enhanced contralesional evoked responses have been consistently reported both in man and rodents as part of plastic processes thought to influence motor recovery. How early this marker of large-scale network reorganization develops has however been little addressed, yet has clinical relevance for rehabilitation strategies targeting plasticity. Previous work in mice has reported enhanced contralesional responses to unaffected-side forepaw stimulation as early as 45 min after MCA small branch occlusion. Using functional ultrasound imaging (fUSi) in anesthetized rats subjected to distal temporary MCA occlusion (MCAo), we assessed here (i) whether enhanced contralesional responses also occurred with unaffected-side whisker pad stimulation, and if so, how early after MCAo; and (ii) the time course of this abnormal response during occlusion and after reperfusion. We replicate in a more proximal MCA occlusion model the earlier findings of ultra-early enhanced contralesional evoked responses. In addition, we document this phenomenon within minutes after MCAo, and its persistence throughout the entire 90-min occlusion as well as 90-min reperfusion periods studied. These findings suggest that plastic processes may start within minutes following MCAo in rodents. If replicated in man, they might have implications regarding how early plasticity-enhancing therapies can be initiated after stroke.
Subject(s)
Electric Stimulation , Functional Laterality/physiology , Infarction, Middle Cerebral Artery/physiopathology , Neuronal Plasticity/physiology , Animals , Male , Rats , Rats, Sprague-Dawley , Ultrasonography , Vibrissae/physiologyABSTRACT
The ability of the gut hormone ghrelin to promote positive energy balance is mediated by the growth hormone secretagogue receptor (GHSR). GHSR is a G protein-coupled receptor (GPCR) that is found centrally and peripherally and that can signal in a ligand-independent manner basally or when heterodimerized with other GPCRs. However, current Ghsr knockout models cannot dissect ghrelin-dependent and ghrelin-independent signaling, precluding assessment of the physiological importance of these signaling pathways. An animal model carrying a Ghsr mutation that preserves GHSR cell surface abundance, but selectively alters GHSR signaling, would be a useful tool to decipher GHSR signaling in vivo. We used rats with the Ghsr(Q343X) mutation (Ghsr(M/M)), which is predicted to delete the distal part of the GHSR carboxyl-terminal tail, a domain critical for the signal termination processes of receptor internalization and ß-arrestin recruitment. In cells, the GHSR-Q343X mutant showed enhanced ligand-induced G protein-dependent signaling and blunted activity of processes involved in GPCR signal termination. Ghsr(M/M)rats displayed enhanced responses to submaximal doses of ghrelin or GHSR agonist. Moreover, Ghsr(M/M)rats had a more stable body weight under caloric restriction, a condition that increases endogenous ghrelin tone, whereas under standard housing conditions,Ghsr(M/M)rats showed increased body weight and adiposity and reduced glucose tolerance. Overall, our data stress the physiological role of the distal domain of GHSR carboxyl terminus as a suppressor of ghrelin sensitivity, and we propose using the Ghsr(M/M)rat as a physiological model of gain of function in Ghsr to identify treatments for obesity-related conditions.
Subject(s)
Adiposity/drug effects , Appetite/drug effects , Ghrelin/pharmacology , Mutation , Receptors, Ghrelin/genetics , Adiposity/genetics , Administration, Intravenous , Animals , Appetite/genetics , Blood Glucose/metabolism , Body Weight/drug effects , Body Weight/genetics , Caloric Restriction , Eating/drug effects , Eating/genetics , Female , Ghrelin/administration & dosage , Ghrelin/metabolism , Glucose Tolerance Test , Growth Hormone/metabolism , HEK293 Cells , Humans , Male , Microscopy, Confocal , Oligopeptides/pharmacology , Rats , Receptors, Ghrelin/agonists , Receptors, Ghrelin/metabolism , Signal Transduction/genetics , beta-Arrestin 1/genetics , beta-Arrestin 1/metabolismABSTRACT
A sensitive and accurate liquid chromatography method with mass spectrometry detection was developed and validated for the quantification of dabigatran (Pradaxa(®)) and rivaroxaban (Xarelto(®)). (13)C6-dabigatran and (13)C6-rivaroxaban were used as the internal standard. A single-step protein precipitation was used for plasma sample preparation. This method was validated with respect to linearity, selectivity, inter- and intra-day precision and accuracy, limit of quantification and stability. The lower limit of quantification was 2.5ng/mL for both drugs in plasma.
