Cerebral parenchymal arterioles (PAs) play a critical role in assuring appropriate blood flow and perfusion pressure within the brain. PAs are unique in contrast to upstream pial arteries, as defined by their critical roles in neurovascular coupling, distinct sensitivities to vasoconstrictors, and enhanced myogenic responsiveness. Dysfunction of these blood vessels is implicated in numerous cardiovascular diseases. However, treatments are limited due to incomplete understanding of the fundamental control mechanisms at this level of the circulation. One of the key elements within most vascular networks, including the cerebral circulation, is the presence of myogenic tone, an intrinsic process whereby resistance arteries constrict and reduce their diameter in response to elevated arterial pressure. This process is centrally involved in the ability of the brain to maintain nearly constant blood flow over a broad range of systemic blood pressures. The overall goal of this dissertation was to investigate the unique mechanisms of myogenic tone regulation in the cerebral microcirculation. To reveal the contributions of various signaling factors in this process, measurements of diameter, intracellular Ca2+ concentration ([Ca2+]i), membrane potential and ion channel activity were performed. Initial work determined that two purinergic G protein-coupled receptors, P2Y4 and P2Y6 receptors, play a unique role in mediating pressure-induced vasoconstriction of PAs in a ligand-independent manner. Moreover, a particular transient receptor potential (TRP) channel in the melastatin subfamily, i.e. TRPM4, was also identified as a mediator of PA myogenic responses. Notably, the observations that inhibiting TRPM4 channels substantially reduces P2Y receptor-mediated depolarization and vasoconstriction, and that P2Y receptor ligands markedly activate TRPM4 currents provide definitive evidence that this ion channel functions as an important link between mechano-sensitive P2Y receptor activation and the myogenic response in PAs. Next, the signaling cascades that mediate stretch-induced TRPM4 activation in PA myocytes were explored. Interestingly, these experiments determined that the RhoA/Rho kinase signaling pathway is involved in this mechanism by facilitating pressure-induced, P2Y receptor-mediated stimulation of TRPM4 channels, leading to subsequent smooth muscle depolarization, [Ca2+]i increase and contraction. Since Rho kinase is generally accepted as a 'Ca2+-sensitization' mediator, the present, contrasting observations point to an underappreciated role of RhoA/Rho kinase signaling in the excitation-contraction mechanisms within the cerebral microcirculation. Overall, this dissertation provides evidence that myogenic regulation of cerebral PAs is mediated by mechano-sensitive P2Y receptors, which initiate the RhoA/Rho kinase signaling pathway, subsequent TRPM4 channel opening, and concomitant depolarization and contraction of arteriolar smooth muscle cells. Revealing the unique mechanochemical coupling mechanisms in the cerebral microcirculation may lead to development of innovative therapeutic strategies for prevention and treatment of microvascular pathologies in the brain.

As lesões dos nervos periféricos, que inervam os músculos esqueléticos, evoluem para perdas da propriocepção e alterações na morfologia e função das fibras musculares, causando um impacto negativo na qualidade de vidas dos indivíduos. Tais lesões implicam em alteração na expressão de genes específicos do músculo, como por exemplo, na MyoD e Miogenina, atuantes na ativação de células satélites e reguladores da massa muscular A técnica cirúrgica de tubulização é um recurso empregado na prática clínica para tratamento de músculos que sofreram desnervação. O objetivo do presente estudo foi analisar se a técnica de tubulização com o preenchimento de gordura altera a expressão de Myod e Miogenina, a morfometria do músculo sóleo de ratos e localização da Myod e Miogenina. Para isso, 57 ratos Wistar foram separados em grupos: controle inicial (GCI); final 45 (GCF45), final 150 (GCF150), desnervado 45 dias (GD45), desnervado 150 dias (GCD150) e grupos experimentais com veia vazia 45 dias (GESP45) e 150 dias (GESP150) e com veia preenchida de gordura 45 dias (GEG45) e 150 dias (GEG150). Para os procedimentos cirúrgicos de desnervação e reinervação e coleta do músculo os animais foram profundamente anestesiados. Após os devidos tempos experimentais, os animais foram sacrificados, o músculo sóleo foi dissecado, envolvido em meio de criopreservação e estocado a -80°C. A quantificação de mRNA do MyoD e Miogenina foi realizada por amplificação por reação em cadeia de polimerase (PCR) em tempo real (RealTimePCR) e a localização da produção de Myod e Miogenina foi realizada por microscopia confocal a laser e imunofluorescência. A morfometria foi realizada em lâminas coradas com HE, observadas em microscópio ótico e calculadas pelo software Image Pro-Plus 6.2. Os resultados do presente estudo mostraram que houve aumento da expressão do Myod e Miogenina nos grupos experimentais 45 dias quando comparados ao grupo controle inicial e um decréscimo da expressão de Myod e Miogenina para os grupos experimentais com 150 dias. A área da secção transversa nos grupos experimentais com 45 dias (GESP45 e GEG45) não apresentaram diferença estatística, quando comparado com grupo desnervado 45 dias (GCD45), enquanto que o grupo experimental com preenchimento de gordura 150 dias (GEG150) obteve os melhores resultados na medida da área da secção transversal do músculo sóleo. As lâminas observadas no microscópio confocal mostram a MyoD e Miogen localizadas no mionúcleo. Concluiu-se que o uso da gordura na técnica de tubulização do nervo ciático de ratos, interfere na regeneração do músculo sóleo. / Peripheral nerve injuries can result in the loss of propioception, morphological and functional alterations of muscle fibers which causes a negative impact on the quality of life. These injuries elicit an alteration on the expression of muscle specific genes, like MyoD and Myogenin, involved in the satellite cell activation and muscle mass regulation. The vein graft tubulization is a well known technique for treatment of denervated muscle. The aim of this work was to investigate if vein graf tubulization filled with fat tissue changes the expression and localization of MyoD and Myogenin and to study if it can modify the morphometry of soleus muscle. Fifty seven Wistar rats were divided in initial control group (ICG), final control group 45 days and 150 days (FCG45; FCG150), denervated 45 days and 150 days (D45; D150) and experimental groups with vein graft 45 days and 150 days (VG45; VG150). and vein graft filled with fat tissue 45 days and 150 days (VF45; VF150). For denervation and reinervation procedures and muscle biopsy the animals were submitted to anaesthesia and after the experimental time they were euthanized. Soleus muscle was dissected, involved in criopreservation medium and stored at -80oC. It was performed RealTime polymerase chain reaction (RealTimePCR) for MyoD and Myogenin mRNA quantification. The localization of its production was analysed by laser confocal microscopy and immunofluorescence staining. The morphometric analysis were done by Hematoxilin-Eosin staining and examined at optical microscopy using the Image ProPlus 6.2 software. There was an upregulation on the expression of MyoD and Myogenin for the experimental groups at 45 days when compared to the initial control group. On the other hand, we found a downregulation on the MyoD and Myogenin expression in the same groups with 150 days. The area of transversal section in the 45 days experimental groups (VF45, VG45) did not show statistical difference compared with denervated group with 45 days (D45). Moreover, the group filled with fat tissue at 150 days (VF150) presented the best results in the transversal section area of soleus muscle. In addition, the slides analysed under confocal microscopy showed the localization of MyoD and Myogenin in the mionuclei. In conclusion, the application of vein graft filled fat tissue improves the soleus muscle regeneration.

This session was developed by Special Interest Division #6: Hearing & Hearing Disorders. Cervical vestibular evoked myogenic potentials (cVEMP) supplement the current vestibular test battery by providing diagnostic information about saccular and/ or inferior vestibular nerve function. The session will provide background, recording method, and clinical application of the cVEMP

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Integrins are transmembrane heterodimeric proteins that link extracellular matrix (ECM) to cytoskeleton and have been shown to function as mechanotransducers in non-muscle cells. Synthetic integrin-binding peptide triggers Ca2+ mobilization and contraction in vascular smooth muscle cells (VSMCs) from rat afferent arteriole, indicating that interactions between ECM and integrins modulate vascular tone. RGD, an integrin binding peptide, triggered contraction in cultured VSMCs as observed by Electric Cell-Substrate Impedance Sensing technique. To examine whether integrins transduce extracellular mechanical stress into intracellular Ca2+ signaling events in VSMCs, unidirectional mechanical force was applied to freshly isolated renal VSMCs through paramagnetic beads coated with fibronectin (FN, natural ligand of α5β1 integrin in VSMCs). Pulling of fibronectin-coated beads with electromagnet triggered Ca2+ sparks, followed by global Ca2+ mobilization. Paramagnetic beads coated with low-density lipoprotein (LDL), whose receptors are not linked to cytoskeleton, were minimally effective in triggering Ca2+ sparks and global Ca2+ mobilization. Pre-incubation with ryanodine, cytochalasin-D, or colchicine substantially reduced the occurrence of Ca2+ sparks triggered by fibronectin-coated beads. Binding of VSMCs with antibodies specific to the extracellular domains of alpha5 and beta1 integrins triggered Ca2+ sparks simulating the effects of fibronectin-coated beads. Anti-β2- integrin antibody served as the negative control. Traction force microscopy studies showed that only the force transduced via integrins could potentially trigger cytoskeletal remodeling in cultured VSMCs. Atomic force microscopy revealed a significant increase in surface roughness in VSMCs when treated with RGD peptide though there was no difference in the maximum deflection of the force curves. Pre-incubation of microperfused afferent arterioles with ryanodine or integrin specific binding peptide inhibited pressure-induced myogenic constriction. In conclusion, integrins transduce mechanical force into intracellular Ca2+ signaling events in renal VSMCs. Integrin-mediated mechanotransduction is probably involved in myogenic response of afferent arterioles. Thus, integrins can potentially act as sensors for myogenic response phenomenon and affect the autoregulatory mechanism in the vasculature.

Na+/Ca2+ exchanger (NCX) is a membrane protein which can mediate either Ca2+ entry (reverse mode) or exit (forward mode) in cells. As one of the major Ca2+ transport systems, NCX is postulated to play a critical role in the vascular smooth muscle cell. The aims of the present study are to firstly demonstrate the functional existence of NCX in vascular smooth muscle (including aorta and arteriole); to clarify the modulation of NCX; to explore the selectivity of NCX inhibitor KB-R7943; and lastly to investigate the role of NCX in the myogenic response. KB-R7943 has been widely used as a NCX inhibitor. The study investigated its pharmacological actions in rat aorta on a variety of Ca2+ dependent systems. Rat aortic rings were used. The constriction to low extracellular [Na+] is a functional response mediated by NCX operating in reverse mode. The data demonstrate that 10 µM KB-R7943 inhibited L-type Ca2+ channel, the capacitative Ca2+ entry and  adrenergic receptor pathway. Nevertheless, KB-R7943 can be used as a selective inhibitor of NCX at the lower concentration of 1 µM in rat aortic rings. The study investigated whether the endothelium could modulate NCX in rat aortic rings. Lowering extracellular [Na+] to 1.18 mM induced constriction in endothelium denuded rat aortic rings, but only a small constriction in endothelium intact rat aortic rings. In endothelium intact rat aortic rings, the guanylate cyclise inhibitor ODQ (1 µM) and the nitric oxide synthase inhibitor L-NAME (50 µM) greatly amplified the vasoconstriction to lowering extracellular [Na+], but had no effect when the endothelium was removed. The adenylate cyclise inhibitor SQ 22536 (100 µM) and the cyclooxygenase inhibitor indomethacin (10 M) showed no significant effect on the low-Na+ induced vasoconstriction in either endothelium denuded or intact aortic rings. The results suggest that endothelium modulated the NCX operation via the nitric oxide/guanylate cyclase, not the adenylate cyclase system; further prostanoids including prostacyclin was not involved. The interaction between nitric oxide and NCX was furt her explored using the nitric oxide donor sodium nitroprusside. Endothelium denuded rat aortic rings were preconstricted to the same extent with either low Na+ (1.18 mM), or the thromboxane A2 agonist U46619 (0.1 µM) or high K+ (80 mM). The vasorelaxation of SNP (30 nM) in low Na+ constriction was significantly larger compared to other agents. This indicates that NO has a special antagonism of low Na+ constriction and a hypothesis is proposed involving Na+/K+ ATPase. The investigation of NCX is mainly conducted in large vessels; much less evidence is available for small resistance vessels. The study investigated the role of NCX on myogenic response in pressurized cremaster muscle arterioles. Reducing extracellular [Na+] resulted in graded vasoconstriction which was inhibited by NCX inhibitor SEA0400 (1 µM). Myogenic vasoconstriction and the concomitant rise in internal [Ca2+] were induced by a transmural pressure increase from 70 to 120 mmHg which was prevented by NCX inhibitor: SEA0400 (1 µM). In conclusion, the present study suggests that NCX contributes to the myogenic response in cremaster arteriole.

Cervical vestibular evoked myogenic potential (cVEMP) testing is widely used in the assessment of vestibular disorders in clinical practice (Welgampola & Colebatch, 2003). Ocular vestibular evoked myogenic potentials (oVEMPs) are similar to the cervical VEMPs in that the vestibular system is also stimulated by a loud sound. The difference is that the response is measured on the inferior oblique muscle of the eye as opposed to the sternocleidomastoid muscle (SCM) of the neck (Chihara, Iwasaki, Ushio, & Murofushi, 2007). The current study compares the standard cervical VEMP to the ocular VEMP in both control subjects and participants with “clinically certain” Ménière’s disease. By investigating cervical VEMPs in comparison to ocular VEMPs we aimed to improve the ability to stage and diagnose Ménière’s disease using the ocular VEMP. 22 control participants and 19 participants with confirmed unilateral Ménière’s disease took part in the study. The peak latency and amplitudes of the ocular and cervical VEMP tests were recorded and analysed. In addition, the background electromyographic (EMG) activity of both the inferior oblique and sternocleidomastoid muscles was recorded throughout testing. A questionnaire was also distributed to all participants to compare the relative difficulty of the VEMP tests. Statistical analysis using the paired t-test, standard t-test and the one-way ANOVA on ranks test was applied to determine a difference between the control and patient groups for both the ocular and cervical VEMP tests. Overall, the threshold and IAD ratio measures did not produce any significant results when sound was presented to the affected ear for the cervical and ocular VEMP tests. A significant reduction in amplitude of the VEMPs from the Ménière’s groups was found compared to the control groups for the ocular the cervical VEMPs. Overall, an increase in P2 and N3 latency of the ocular VEMP response in Ménière’s patients was determined. Results from the questionnaire suggest that the ocular VEMP test was more tolerable to the cervical VEMP test in this current study. Furthermore, statistical analyses revealed no significant differences in EMG level between the control and Ménière’s group for both the ocular and cervical VEMP data. Overall, results suggest that both the cervical and ocular VEMP tests provide information regarding the integrity of the saccule, owing to the abnormal VEMP findings in the participants with Ménière’s disease. In addition, this study provides evidence that the ocular VEMP is as useful a tool in diagnosing Ménière’s disease as the cervical VEMP.

Cervical vestibular evoked myogenic potential (cVEMP) testing is widely used in the assessment of vestibular disorders in clinical practice (Welgampola & Colebatch, 2003). Ocular vestibular evoked myogenic potentials (oVEMPs) are similar to the cervical VEMPs in that the vestibular system is also stimulated by a loud sound. The difference is that the response is measured on the inferior oblique muscle of the eye as opposed to the sternocleidomastoid muscle (SCM) of the neck (Chihara, Iwasaki, Ushio, & Murofushi, 2007). The current study compares the standard cervical VEMP to the ocular VEMP in both control subjects and participants with “clinically certain” Ménière’s disease. By investigating cervical VEMPs in comparison to ocular VEMPs we aimed to improve the ability to stage and diagnose Ménière’s disease using the ocular VEMP. 22 control participants and 19 participants with confirmed unilateral Ménière’s disease took part in the study. The peak latency and amplitudes of the ocular and cervical VEMP tests were recorded and analysed. In addition, the background electromyographic (EMG) activity of both the inferior oblique and sternocleidomastoid muscles was recorded throughout testing. A questionnaire was also distributed to all participants to compare the relative difficulty of the VEMP tests. Statistical analysis using the paired t-test, standard t-test and the one-way ANOVA on ranks test was applied to determine a difference between the control and patient groups for both the ocular and cervical VEMP tests. Overall, the threshold and IAD ratio measures did not produce any significant results when sound was presented to the affected ear for the cervical and ocular VEMP tests. A significant reduction in amplitude of the VEMPs from the Ménière’s groups was found compared to the control groups for the ocular the cervical VEMPs. Overall, an increase in P2 and N3 latency of the ocular VEMP response in Ménière’s patients was determined. Results from the questionnaire suggest that the ocular VEMP test was more tolerable to the cervical VEMP test in this current study. Furthermore, statistical analyses revealed no significant differences in EMG level between the control and Ménière’s group for both the ocular and cervical VEMP data. Overall, results suggest that both the cervical and ocular VEMP tests provide information regarding the integrity of the saccule, owing to the abnormal VEMP findings in the participants with Ménière’s disease. In addition, this study provides evidence that the ocular VEMP is as useful a tool in diagnosing Ménière’s disease as the cervical VEMP.