6 research outputs found
Pharmacologic Neuromodulation Targeting Neuroinflammation as a Novel Therapeutic Strategy for Experimental Pulmonary Hypertension
Pharmacologic Neuromodulation Targeting Neuroinflammation as a Novel Therapeutic Strategy for Experimental Pulmonary Hypertension
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Allyl Isothiocyanate-Induced Rapid Sensitization of Heart Rate in Larval Zebrafish (Danio rerio)
Among the most protuberant behaviors during early developmental stages in the zebrafish is changes in heart rate.�In particular, past studies demonstrated behavioral sensitization by producing a range of behavioral modifications and often activating the autonomic nervous system. Previous work has shown that�allyl isothiocyanate�(AITC or mustard oil, MO) most likely elicits its sensitization effects through activation of TRP channels expressed on the trigeminal and Rohon Beard sensory neurons in larval zebrafish. To determine if AITC exposure activates the sympathetic nervous system in larval zebrafish, we used heart rate as a proxy for the activation of the autonomic nervous system. To confirm that our behavioral sensitization was induced by activation of TRP channels, we used Ruthenium Red (RR), which was previously shown to antagonize these receptors in zebrafish. In the present study, we found that 10��M total bath concentration of AITC significantly increased the heart rate activity in 5-day post fertilization (dpf), agarose-restrained larval zebrafish compared to control treated animals. On the other hand, 10��M total bath concentration of RR exposure prior to 10��M AITC exposure significantly decreased the heart rate activity, whereas, the equal concentration RR exposure after the AITC exposure had no suppressive effect on AITC induced increased heart rate in 5 DPF, agarose-restrained larval zebrafish. Further, we used behavioral pharmacology to dissect the molecular underpinnings of the sensitization memory. To determine whether the different neural circuits contributing to enhanced heart rate depend upon one or many different neuromodulators and ascertain how these neuromodulators influence distinct neural circuits we have conducted various extracellular receptor blockade experiments. We used D-APV, a competitive NMDAR antagonist, MK801, a non-competitive NMDAR antagonist, Methiothepin, a selective serotonin receptor antagonist, Haloperidol, a dopamine D2 receptor antagonist, Mecamylamine, a non-competitive nicotinic acetylcholine receptor antagonist, Propranolol, a non-selective beta-adrenergic receptor blocker and Atropine, a non-selective muscarinic acetylcholinergic antagonist. We discovered that AITC induced sensitization is accompanied by increase in heart rate, which implicates serotonin, dopamine receptors as well as β-adrenergic, nicotinic and muscarinic acetylcholine receptors from cardiac autonomic nervous system, but NMDA receptors don’t seem to play any role in this short term memory. Together, these results indicate the enormous potential that zebrafish hold as an animal model for the study of learning and memory, especially non-associative memory
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Pharmacologic Neuromodulation Targeting Neuroinflammation as a Novel Therapeutic Strategy for Experimental Pulmonary Hypertension
Pulmonary hypertension (PH) is a rare, progressive and debilitating disease characterized by elevated pressure in the pulmonary circulation coupled with right ventricular hypertrophy and failure. Microglial and astrocytic activation mediated neuroinflammation in the brain and spinal cord has been implicated in the sympathetic nervous system (SNS) hyperactivity in experimental PH, however, the role and precise mechanism of the intricate interplay between neuroinflammation and SNS activation in PH is still unknown. In Chapter 2, we discovered that cardiopulmonary afferent signaling to the thoracic DRG and dorsal horn is mediated via TRPV1 and substance P in PH. Our first-ever transcriptomic analysis on the TSC of 2 clinically relevant rat models (MCT and SuHx) of severe PH and RVF delineated common dysregulated genes and pathways highlighting neuroinflammation and apoptosis. We observed microgliosis, astrogliosis and increased fractalkine expression in the TSC of both rat models and in human PAH. Elevated plasma norepinephrine in both rat models confirmed increased sympathoexcitation. Finally, intrathecal minocycline decreased TSC microglial count, activation, and expression of proinflammatory cytokines, and reduced sympathoexcitation resulting in rescue of PH-RVF. In Chapter 3, we demonstrated that intrathecal RTX administration abolished TRPV1 expression in the TSC and attenuated PH-RVF by decreasing cardiac sympathetic tone in MCT and SuHx rats. We also revealed that mechanistically, NMDAR-NOS1 mediated neuroinflammation and neuronal apoptosis in the TSC in PH is dependent on TRPV1 activation. Lung and RV RNASeq from RTX-treated MCT rats demonstrated reversal of fibrosis, inflammation and apoptosis. In Chapter 4, we expanded on our findings by investigating downstream of TRPV1 gene and found increased signature of glutamatergic and NOS1 signaling in the TSC of MCT and SuHx rats and patients with PAH. We demonstrated that NMDAR inhibition decreased TSC NOS1 expression, neuroinflammation, neuronal apoptosis and associated sympathoexcitation and rescued PH-RVF in MCT rats. Finally, pharmacological inhibition of NOS1 attenuated PH-RVF by reducing TSC neuroinflammation, neuronal apoptosis and sympathoexcitation. Moreover, lung and RV RNA-Seq from SMTC-treated MCT rats demonstrated reversal fibrosis, inflammation and apoptosis. Invitro analysis indicated that norepinephrine and SP may play major role in lung and RV reversal of EndMT and fibrosis in SMTC-treated MCT Rats.
Taken together, this dissertation is a compilation of the first work investigating the mechanistic evidence of TSC neuroinflammation and its role in SNS hyperactivity in PH
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Transcriptomic Analysis of Right Ventricular Remodeling in Two Rat Models of Pulmonary Hypertension: Identification and Validation of Epithelial-to-Mesenchymal Transition in Human Right Ventricular Failure.
BackgroundRight ventricular (RV) dysfunction is a significant prognostic determinant of morbidity and mortality in pulmonary arterial hypertension (PAH). Despite the importance of RV function in PAH, the underlying molecular mechanisms of RV dysfunction secondary to PAH remain unclear. We aim to identify and compare molecular determinants of RV failure using RNA sequencing of RV tissue from 2 clinically relevant animal models of PAH.MethodsWe performed RNA sequencing on RV from rats treated with monocrotaline or Sugen with hypoxia/normoxia. PAH and RV failure were confirmed by catheterization and echocardiography. We validated the RV transcriptome results using quantitative real-time polymerase chain reaction, immunofluorescence, and Western blot. Immunohistochemistry and immunofluorescence were performed on human RV tissue from control (n=3) and PAH-induced RV failure patients (n=5).ResultsWe identified similar transcriptomic profiles of RV from monocrotaline- and Sugen with hypoxia-induced RV failure. Pathway analysis showed genes enriched in epithelial-to-mesenchymal transition, inflammation, and metabolism. Histological staining of human RV tissue from patients with RV failure secondary to PAH revealed significant RV fibrosis and endothelial-to-mesenchymal transition, as well as elevated cellular communication network factor 2 (top gene implicated in epithelial-to-mesenchymal transition/endothelial-to-mesenchymal transition) expression in perivascular areas compared with normal RV.ConclusionsTranscriptomic signature of RV failure in monocrotaline and Sugen with hypoxia models showed similar gene expressions and biological pathways. We provide translational relevance of this transcriptomic signature using RV from patients with PAH to demonstrate evidence of epithelial-to-mesenchymal transition/endothelial-to-mesenchymal transition and protein expression of cellular communication network factor 2 (CTGF [connective tissue growth factor]). Targeting specific molecular mechanisms responsible for RV failure in monocrotaline and Sugen with hypoxia models may identify novel therapeutic strategies for PAH-associated RV failure
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Induction of Short-Term Sensitization by an Aversive Chemical Stimulus in Zebrafish Larvae
Larval zebrafish possess a number of molecular and genetic advantages for rigorous biological analyses of learning and memory. These advantages have motivated the search for novel forms of memory in these animals that can be exploited for understanding the cellular and molecular bases of vertebrate memory formation and consolidation. Here, we report a new form of behavioral sensitization in zebrafish larvae that is elicited by an aversive chemical stimulus [allyl isothiocyanate (AITC)] and that persists for ≥30 min. This form of sensitization is expressed as enhanced locomotion and thigmotaxis, as well as elevated heart rate. To characterize the neural basis of this nonassociative memory, we used transgenic zebrafish expressing the fluorescent calcium indicator GCaMP6 (Chen et al., 2013); because of the transparency of larval zebrafish, we could optically monitor neural activity in the brain of intact transgenic zebrafish before and after the induction of sensitization. We found a distinct brain area, previously linked to locomotion, that exhibited persistently enhanced neural activity following washout of AITC; this enhanced neural activity correlated with the behavioral sensitization. These results establish a novel form of memory in larval zebrafish and begin to unravel the neural basis of this memory