Canadian Multicentric Pan-TRK (CANTRK) Immunohistochemistry Harmonization Study.

Tumor-agnostic testing for NTRK1-3 gene rearrangements is required to identify patients who may benefit from TRK inhibitor therapies. The overarching objective of this study was to establish a high-quality pan-TRK immunohistochemistry (IHC) screening assay among 18 large regional pathology laboratories across Canada using pan-TRK monoclonal antibody clone EPR17341 in a ring study design. TRK-fusion positive and negative tumor samples were collected from participating sites, with fusion status confirmed by panel next-generation sequencing assays. Each laboratory received: (1) unstained sections from 30 cases of TRK-fusion-positive or -negative tumors, (2) 2 types of reference standards: TRK calibrator slides and IHC critical assay performance controls (iCAPCs), (3) EPR17341 antibody, and (4) suggestions for developing IHC protocols. Participants were asked to optimize the IHC protocol for their instruments and detection systems by using iCAPCs, to stain the 30 study cases, and to report the percentage scores for membranous, cytoplasmic, and nuclear staining. TRK calibrators were used to assess the analytical sensitivity of IHC protocols developed by using the 2 reference standards. Fifteen of 18 laboratories achieved diagnostic sensitivity of 100% against next-generation sequencing. The diagnostic specificity ranged from 40% to 90%. The results did not differ significantly between positive scores based on the presence of any type of staining vs the presence of overall staining in ≥1% of cells. The median limit of detection measured by TRK calibrators was 76,000 molecules/cell (range 38,000 to >200,000 molecules/cell). Three different patterns of staining were observed in 19 TRK-positive cases, cytoplasmic-only in 7 samples, nuclear and cytoplasmic in 9 samples, and cytoplasmic and membranous in 3 samples. The Canadian multicentric pan-TRK study illustrates a successful strategy to accelerate the multicenter harmonization and implementation of pan-TRK immunohistochemical screening that achieves high diagnostic sensitivity by using laboratory-developed tests where laboratories used centrally developed reference materials. The measurement of analytical sensitivity by using TRK calibrators provided additional insights into IHC protocol performance.

Voltage-gated potassium channel dysfunction in dorsal root ganglia contributes to the exaggerated exercise pressor reflex in rats with chronic heart failure.

An exaggerated exercise pressor reflex (EPR) causes excessive sympathoexcitation and exercise intolerance during physical activity in the chronic heart failure (CHF) state. Muscle afferent sensitization contributes to the genesis of the exaggerated EPR in CHF. However, the cellular mechanisms underlying muscle afferent sensitization in CHF remain unclear. Considering that voltage-gated potassium (Kv) channels critically regulate afferent neuronal excitability, we examined the potential role of Kv channels in mediating the sensitized EPR in male rats with CHF. Real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting experiments demonstrate that both mRNA and protein expressions of multiple Kv channel isoforms (Kv1.4, Kv3.4, Kv4.2, and Kv4.3) were downregulated in lumbar dorsal root ganglions (DRGs) of CHF rats compared with sham rats. Immunofluorescence data demonstrate significant decreased Kv channel staining in both NF200-positive and IB4-positive lumbar DRG neurons in CHF rats compared with sham rats. Data from patch-clamp experiments demonstrate that the total Kv current, especially IA, was dramatically decreased in medium-sized IB4-negative muscle afferent neurons (a subpopulation containing mostly Aδ neurons) from CHF rats compared with sham rats, indicating a potential functional loss of Kv channels in muscle afferent Aδ neurons. In in vivo experiments, adenoviral overexpression of Kv4.3 in lumbar DRGs for 1 wk attenuated the exaggerated EPR induced by muscle static contraction and the mechanoreflex by passive stretch without affecting the blunted cardiovascular response to hindlimb arterial injection of capsaicin in CHF rats. These data suggest that Kv channel dysfunction in DRGs plays a critical role in mediating the exaggerated EPR and muscle afferent sensitization in CHF.NEW & NOTEWORTHY The primary finding of this manuscript is that voltage-gated potassium (Kv) channel dysfunction in DRGs plays a critical role in mediating the exaggerated EPR and muscle afferent sensitization in chronic heart failure (CHF). We propose that manipulation of Kv channels in DRG neurons could be considered as a potential new approach to reduce the exaggerated sympathoexcitation and to improve exercise intolerance in CHF, which can ultimately facilitate an improved quality of life and reduce mortality.

Skeletal Muscle Nrf2 Contributes to Exercise-Evoked Systemic Antioxidant Defense Via Extracellular Vesicular Communication.

This review explores the hypothesis that the repetitive contraction-relaxation that occurs during chronic exercise activates skeletal myocyte nuclear factor erythroid-derived 2-like 2 (Nrf2) to upregulate antioxidant enzymes. These proteins are secreted into the circulation within extracellular vesicles and taken up by remote cells, thus providing remote organs with cytoprotection against subsequent oxidative stress.

Functional, proteomic and bioinformatic analyses of Nrf2- and Keap1- null skeletal muscle.

KEY POINTS: Nrf2 is a master regulator of endogenous cellular defences, governing the expression of more than 200 cytoprotective proteins, including a panel of antioxidant enzymes. Nrf2 plays an important role in redox haemostasis of skeletal muscle in response to the increased generation of reactive oxygen species during contraction. Employing skeletal muscle-specific transgenic mouse models with unbiased-omic approaches, we uncovered new target proteins, downstream pathways and molecular networks of Nrf2 in skeletal muscle following Nrf2 or Keap1 deletion. Based on the findings, we proposed a two-way model to understand Nrf2 function: a tonic effect through a Keap1-independent mechanism under basal conditions and an induced effect through a Keap1-dependent mechanism in response to oxidative and other stresses. ABSTRACT: Although Nrf2 has been recognized as a master regulator of cytoprotection, its functional significance remains to be completely defined. We hypothesized that proteomic/bioinformatic analyses from Nrf2-deficient or overexpressed skeletal muscle tissues will provide a broader spectrum of Nrf2 targets and downstream pathways than are currently known. To this end, we created two transgenic mouse models; the iMS-Nrf2flox/flox and iMS-Keap1flox/flox , employing which we demonstrated that selective deletion of skeletal muscle Nrf2 or Keap1 separately impaired or improved skeletal muscle function. Mass spectrometry revealed that Nrf2-KO changed expression of 114 proteins while Keap1-KO changed expression of 117 proteins with 10 proteins in common between the groups. Gene ontology analysis suggested that Nrf2 KO-changed proteins are involved in metabolism of oxidoreduction coenzymes, purine ribonucleoside triphosphate, ATP and propanoate, which are considered as the basal function of Nrf2, while Keap1 KO-changed proteins are involved in cellular detoxification, NADP metabolism, glutathione metabolism and the electron transport chain, which belong to the induced effect of Nrf2. Canonical pathway analysis suggested that Keap1-KO activated four pathways, whereas Nrf2-KO did not. Ingenuity pathway analysis further revealed that Nrf2-KO and Keap1-KO impacted different signal proteins and functions. Finally, we validated the proteomic and bioinformatics data by analysing glutathione metabolism and mitochondrial function. In conclusion, we found that Nrf2-targeted proteins are assigned to two groups: one mediates the tonic effects evoked by a low level of Nrf2 at basal condition; the other is responsible for the inducible effects evoked by a surge of Nrf2 that is dependent on a Keap1 mechanism.

Sympathoexcitation in response to cardiac and pulmonary afferent stimulation of TRPA1 channels is attenuated in rats with chronic heart failure.

Excessive sympathoexcitation characterizes the chronic heart failure (CHF) state. An exaggerated cardiac sympathetic afferent reflex (CSAR) contributes to this sympathoexcitation. Prior studies have demonstrated that the CSAR to capsaicin [transient receptor potential (TRP) vanilloid 1 agonist] is exaggerated in CHF animal models. We recently discovered that capsaicin application to the lung visceral pleura in anesthetized, vagotomized, open-chested rats increases mean arterial pressure (MAP), heart rate (HR), and renal sympathetic nerve activity (RSNA). We named this response the pulmonary spinal afferent reflex (PSAR). Due to the similarities between TRP vanilloid 1 and TRP ankyrin 1 (TRPA1) channels as well as the excessive sympathoexcitation of CHF, we hypothesized that stimulation of the CSAR and PSAR with a specific TRPA1 agonist would result in an augmented response in CHF rats (coronary ligation model) compared with sham control rats. In response to a TRPA1 agonist, both CSAR and PSAR in sham rats resulted in biphasic changes in MAP and increases in HR and RSNA 10-12 wk postmyocardial infarction (post-MI). These effects were blunted in CHF rats. Assessment of TRPA1 expression levels in cardiopulmonary spinal afferents by immunofluorescence, quantitative RT-PCR, and Western blot analysis 10-12 wk post-MI all indicates reduced expression in CHF rats but no reduction at earlier time points. TRPA1 protein was reduced in a dorsal root ganglia cell culture model of inflammation and simulated tissue ischemia, raising the possibility that the in vivo reduction of TRPA1 expression was, in part, caused by CHF-related tissue ischemia and inflammation. These data provide evidence that reflex responses to cardiopulmonary spinal afferent TRPA1 stimulation may be attenuated in CHF rather than enhanced. NEW & NOTEWORTHY Excessive sympathoexcitation characterizes chronic heart failure (CHF). The contribution of transient receptor potential ankyrin 1 (TRPA1) channel-mediated reflexes to this sympathoexcitation is unknown. We found that application of TRPA1 agonist to the heart and lung surface resulted in increased heart rate and sympathetic output and a biphasic change in mean arterial pressure in control rats. These effects were attenuated in CHF rats, decreasing the likelihood that TRPA1 channels contribute to cardiopulmonary afferent sensitization in CHF.

Increased Brain-Derived Neurotrophic Factor in Lumbar Dorsal Root Ganglia Contributes to the Enhanced Exercise Pressor Reflex in Heart Failure.

An exaggerated exercise pressor reflex (EPR) is associated with excessive sympatho-excitation and exercise intolerance in the chronic heart failure (CHF) state. We hypothesized that brain-derived neurotrophic factor (BDNF) causes the exaggerated EPR via sensitizing muscle mechanosensitive afferents in CHF. Increased BDNF expression was observed in lumbar dorsal root ganglia (DRGs) from CHF rats compared to sham rats. Immunofluorescence data showed a greater increase in the number of BDNF-positive neurons in medium and large-sized DRG subpopulations from CHF rats. Patch clamp data showed that incubation with BDNF for 4⁻6 h, significantly decreased the current threshold-inducing action potential (AP), threshold potential and the number of APs during current injection in Dil-labeled isolectin B4 (IB4)-negative medium-sized DRG neurons (mainly mechano-sensitive) from sham rats. Compared to sham rats, CHF rats exhibited an increased number of APs during current injection in the same DRG subpopulation, which was significantly attenuated by 4-h incubation with anti-BDNF. Finally, chronic epidural delivery of anti-BDNF attenuated the exaggerated pressor response to either static contraction or passive stretch in CHF rats whereas this intervention had no effect on the pressor response to hindlimb arterial injection of capsaicin. These data suggest that increased BDNF in lumbar DRGs contributes to the exaggerated EPR in CHF.

Cardiac sympathetic afferent reflex control of cardiac function in normal and chronic heart failure states.

KEY POINTS: Cardiac sympathetic afferents are considered to be essential pathways for transmission of cardiac nociception to the central nervous system during myocardial ischaemia. However, a potential contribution of the CSAR control of cardiac dysfunction in both normal and chronic heart failure (CHF) states remains unknown. We found that activation of the CSAR evokes little increase in cardiac contractility with an exaggerated peripheral vasoconstriction in the CHF state. CSAR inhibition by epicardial lidocaine decreased cardiac contractility to a greater extent in CHF rats than sham rats. Furthermore, we also found that epicardial lidocaine paradoxically decreased left ventricular end-diastolic pressure (LVEDP) and left ventricular end-diastolic volume (preload) in CHF rats, which was not observed in sham rats. Chronic ablation of the CSAR by epicardial application of the afferent neurotoxin, RTX, selectively lowered diastolic blood pressure CHF rats. The observation suggests that CSAR has a differential effect on cardiac function in normal and CHF states. CSAR activation in normal state causes significant increase in cardiac contractility and cardiac output. ABSTRACT: The enhanced 'cardiac sympathetic afferent reflex' (CSAR) critically contributes to the exaggerated global sympathetic tone in chronic heart failure (CHF). However, a potential contribution of the cardio-cardiac reflex control of cardiac function in both normal and CHF states remains unknown. In this study, we evaluated the effects of direct activation or inhibition of the CSAR on cardiac function by pressure-volume (P-V) loop analysis in ∼12-week sham-operated and myocardial infarcted (MI) rats. In sham rats, acute CSAR activation by epicardial application of bradykinin (BK) increased heart rate (HR), left ventricular systolic pressure (LVSP), the maximum first derivative of left ventricular pressure (dp/dtmax ), and the slope of the end-systolic P-V relationship (ESPVR), suggesting that acute CSAR activation in the normal state enhances myocardial contractility. CSAR activation also decreased left ventricular (LV) systolic and diastolic volumes with little effect on LV end-diastolic pressure (LVEDP) or the end-diastolic P-V relationship (EDPVR) in sham rats. Compared to sham, CHF rats exhibit a reduced increase in the slope of the ESPVR and dp/dtmax in response to BK, indicating a poor contractile response to CSAR activation. Interestingly, BK application in CHF rats increased cardiac systolic and diastolic volumes and further increased the elevated LVEDP, neither of which was seen in sham rats. Following CSAR inhibition by epicardial lidocaine, blood pressure, HR, LVSP, dp/dt, LVEDP and ESPVR decreased in CHF rats whereas lidocaine had little effect in sham rats, indicating that the CSAR is tonically active in CHF and contributes to cardiac dysfunction. Furthermore, we found that epicardial lidocaine paradoxically decreased LV end-diastolic volume (preload) in CHF rats, which was not observed in sham rats. The decreased preload by lidocaine in CHF rats may be due to a reduction in peripheral vascular resistance since epicardial lidocaine significantly lowered peripheral (renal) sympathetic nerve activity in CHF rats but not in sham rats. Furthermore, chronic ablation of CSAR by epicardial application of a selective afferent neurotoxin, resiniferatoxin, selectively lowered diastolic blood pressure both at daytime and night-time with less effect on systolic blood pressure in CHF rats. Our data suggest that there is an imbalance between cardiac and peripheral responses to CSAR in CHF animals compared to sham-operated controls.

Influence of brain-derived neurotrophic factor-tyrosine receptor kinase B signalling in the nucleus tractus solitarius on baroreflex sensitivity in rats with chronic heart failure.

KEY POINTS: Impairment of baroreflex function is associated with the progression of chronic heart failure (CHF) and a poor prognosis. The baroreflex desensitization in CHF is at least partly the result of central neuronal network dysfunction. The dorsal medial nucleus tractus solitarius (dmNTS) has long been appreciated as a primary site of baroreceptor afferent termination in the central nervous system. However, the influence of neurotransmitters and neuromodulators in the dmNTS on baroreflex function both in normal and CHF states is not fully understood. The present study provides the first evidence showing a tonic sympatho-inhibitory role for brain-derived neurotrophic factor (BDNF) neurotransmission in the dmNTS. Most importantly, BDNF- tyrosine receptor kinase B (TrkB) signalling in the dmNTS is integral for normal baroreflex function as indicated by the blunting of baroreflex sensitivity (BRS) following the antagonization of TrkB, which inhibited baroreflex gain and range. Furthermore, we found that the tonic sympatho-inhibition of BDNF was withdrawn in the CHF state, thus contributing to the increased sympathetic tone associated with CHF. Consistent with this finding, BDNF/TrkB antagonism had little effect on reducing BRS in CHF animals, which is corroborated by the observation of decreased TrkB expression in the dmNTS during CHF. Taken together, these results implicate a reduction in BDNF-TrkB signalling in the dmNTS during CHF that contributes to sympatho-excitation and baroreflex desensitization. The observation that the BDNF/TrkB pathway is impaired in the dmNTS during CHF provides a novel mechanism for understanding the central alterations that contribute to baroreflex desensitization during CHF. ABSTRACT: Chronic heart failure (CHF) results in blunting of arterial baroreflex sensitivity (BRS), which arises from alterations to both peripheral baroreceptors and central autonomic nuclei such as the nucleus tractus solitarius (NTS). Although glutamate is known to be an important neurotransmitter released from baroreceptor afferent synapses in the NTS, the influence of other neurotransmitters and neuromodulators remains unclear. Alterations to NTS signalling in CHF remain particularly undefined. The present study aimed to evaluate the role of brain-derived neurotrophic factor (BDNF) and tyrosine receptor kinase B (TrkB) receptor signalling in the NTS on baroreflex control both in healthy and CHF rats. To this end, we microinjected BDNF or the highly selective TrkB receptor antagonist [N2-2-2-oxoazepan-3-yl amino] carbonyl phenyl benzo (b)thiophene-2-carboxamide (ANA-12) into the dorsal medial NTS (dmNTS) of male Sprague-Dawley rats with coronary artery ligation-induced CHF and sham operated controls and recorded blood pressure and renal sympathetic nerve activity responses. We subsequently measured BRS before and after bilateral dmNTS microinjections of ANA-12. In sham rats, BDNF evoked a dose-dependent depressor and sympatho-inhibitory effect and ANA-12 produced the opposite response. Both of these responses were significantly blunted in CHF rats. Furthermore, bilateral microinjection of ANA-12 into the dmNTS greatly diminished baroreflex sensitivity in sham rats, whereas it had less of an effect in CHF rats. We observed decreased levels of TrkB protein and mRNA in the dmNTS of CHF rats. These data indicate that endogenous BDNF signalling in the NTS is integral for the maintenance of BRS and that BDNF/TrkB signalling is impaired in the NTS in the CHF state.

Glutamatergic receptor dysfunction in spinal cord contributes to the exaggerated exercise pressor reflex in heart failure.

Excitatory amino acids (e.g., glutamate) released by contraction-activated skeletal muscle afferents into the dorsal horn of the spinal cord initiate the central component of the exercise pressor reflex (EPR) in physiological conditions. However, the role of glutamate and glutamate receptors in mediating the exaggerated EPR in the chronic heart failure (CHF) state remains to be determined. In the present study, we performed microinjection of glutamate receptor antagonists into ipisilateral L4/L5 dorsal horns to investigate their effects on the pressor response to static contraction induced by stimulation of the peripheral end of L4/L5 ventral roots in decerebrate sham-operated (sham) and CHF rats. Microinjection of glutamate (10 mM, 100 nl) into the L4 or L5 dorsal horn caused a greater pressor response in CHF rats compared with sham rats. Furthermore, microinjection of either the broad-spectrum glutamate receptor antagonist kynurenate (10 mM, 100 nl) or the N-methyl-d-aspartate (NMDA) receptor antagonist dl-2-amino-5-phosphonovalerate (50 mM, 100 nl) or the non-NMDA-sensitive receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (5 mM, 100 nl) into L4/5 dorsal horns decreased the pressor response to static contraction in CHF rats to a greater extent than in sham rats. Molecular evidence showed that the protein expression of glutamate receptors (both non-NMDA and NMDA) was elevated in the dorsal horn of the lumbar spinal cord in CHF rats. In addition, data from microdialysis experiments demonstrated that although basal glutamate release at the dorsal horn at rest was similar between sham and CHF rats (225 ± 50 vs. 260 ± 63 nM in sham vs. CHF rats, n = 4, P > 0.05), CHF rats exhibit greater glutamate release into the dorsal horn during muscle contraction compared with sham rats (549 ± 60 vs. 980 ± 65 nM in sham vs. CHF rats, n = 4, P < 0.01). These data indicate that the spinal glutamate system contributes to the exaggerated EPR in the CHF state.

Spinal cord GABA receptors modulate the exercise pressor reflex in decerebrate rats.

Neurotransmitters and neuromodulators released by contraction-activated skeletal muscle afferents into the dorsal horn of the spinal cord initiate the central component of the exercise pressor reflex (EPR). Whether γ-aminobutyric acid (GABA), a major inhibitory neurotransmitter within the mammalian central nervous system, is involved in the modulation of the EPR at the level of dorsal horn remains to be determined. We performed local microinjection of either the GABA(A) antagonist bicuculline or the GABA(B) antagonist CGP 52432 into the ipisilateral L4/L5 dorsal horns to investigate the effect of GABA receptor blockade on the pressor response to either static contraction induced by stimulation of the peripheral end of L4/L5 ventral roots, passive stretch, or hindlimb arterial injection of capsaicin (0.1 µg/0.2 ml) in decerebrate rats. Microinjection of either bicuculline (1 mM, 100 nl) or CGP 52432 (10 mM, 100 nl) into the L4/5 dorsal horns significantly increased the pressor and cardioaccelerator responses to all stimuli. Microinjection of either bicuculline or CGP 52432 into the L5 dorsal horn significantly increased the pressor and cardioaccelerator responses to direct microinjection of l-glutatmate (10 mM, 100 nl) into this spinal segment. The disinhibitory effect of both GABA receptor antagonists on the EPR was abolished by microinjection of the broad-spectrum glutamate receptor antagonist kynurenate (10 mM/100 nl). These data suggest that 1) GABA exerts a tonic inhibition of the EPR at the level of dorsal horn; and 2) that an interaction between glutamatergic and GABAergic inputs exist at the level of dorsal horn, contributing to spinal control of the EPR.

Potential Neuromodulation of the Cardio-Renal Syndrome.

The cardio-renal syndrome (CRS) type 2 is defined as a progressive loss of renal function following a primary insult to the myocardium that may be either acute or chronic but is accompanied by a decline in myocardial pump performance. The treatment of patients with CRS is difficult, and the disease often progresses to end-stage renal disease that is refractory to conventional therapy. While a good deal of information is known concerning renal injury in the CRS, less is understood about how reflex control of renal sympathetic nerve activity affects this syndrome. In this review, we provide insight into the role of the renal nerves, both from the afferent or sensory side and from the efferent side, in mediating renal dysfunction in CRS. We discuss how interventions such as renal denervation and abrogation of systemic reflexes may be used to alleviate renal dysfunction in the setting of chronic heart failure. We specifically focus on a novel cardiac sensory reflex that is sensitized in heart failure and activates the sympathetic nervous system, especially outflow to the kidney. This so-called Cardiac Sympathetic Afferent Reflex (CSAR) can be ablated using the potent neurotoxin resinferitoxin due to the high expression of Transient Receptor Potential Vanilloid 1 (TRPV1) receptors. Following ablation of the CSAR, several markers of renal dysfunction are reversed in the post-myocardial infarction heart failure state. This review puts forth the novel idea of neuromodulation at the cardiac level in the treatment of CRS Type 2.

The superior cervical ganglion is involved in chronic chemoreflex sensitization during recovery from acute lung injury.

Introduction: Acute lung injury (ALI) initiates an inflammatory cascade that impairs gas exchange, induces hypoxemia, and causes an increase in respiratory rate (fR). This stimulates the carotid body (CB) chemoreflex, a fundamental protective reflex that maintains oxygen homeostasis. Our previous study indicated that the chemoreflex is sensitized during the recovery from ALI. The superior cervical ganglion (SCG) is known to innervate the CB, and its electrical stimulation has been shown to significantly sensitize the chemoreflex in hypertensive and normotensive rats. We hypothesized that the SCG is involved in the chemoreflex sensitization post-ALI. Methods: We performed a bilateral SCG ganglionectomy (SCGx) or sham-SCGx (Sx) in male Sprague Dawley rats 2 weeks before inducing ALI (Week -2 i.e., W-2). ALI was induced using a single intra-tracheal instillation of bleomycin (bleo) (day 1). Resting-fR, Vt (Tidal Volume), and V̇ E (Minute Ventilation) were measured. The chemoreflex response to hypoxia (10% O2, 0% CO2) and normoxic-hypercapnia (21% O2, 5% CO2) were measured before surgery on W (-3), before bleo administration on W0 and on W4 post-bleo using whole-body plethysmography (WBP). Results: SCGx did not affect resting fR, Vt and V̇E as well as the chemoreflex responses to hypoxia and normoxic hypercapnia in either group prior to bleo. There was no significant difference in ALI-induced increase in resting fR between Sx and SCGx rats at W1 post-bleo. At W4 post-bleo, there were no significant differences in resting fR, Vt, and V̇E between Sx and SCGx rats. Consistent with our previous study, we observed a sensitized chemoreflex (delta fR) in response to hypoxia and normoxic hypercapnia in Sx rats at W4 post-bleo. However, at the same time, compared to Sx rats, the chemoreflex sensitivity was significantly less in SCGx rats in response to either hypoxia or normoxic hypercapnia. Discussion: These data suggest that SCG is involved in the chemoreflex sensitization during ALI recovery. Further understanding of the underlying mechanism will provide important information for the long-term goal of developing novel targeted therapeutic approaches to pulmonary diseases to improve clinical outcomes.

Systemic mapping of organ plasma extravasation at multiple stages of chronic heart failure.

Introduction: Chronic Heart failure (CHF) is a highly prevalent disease that leads to significant morbidity and mortality. Diffuse vasculopathy is a commonmorbidity associated with CHF. Increased vascular permeability leading to plasma extravasation (PEx) occurs in surrounding tissues following endothelial dysfunction. Such micro- and macrovascular complications develop over time and lead to edema, inflammation, and multi-organ dysfunction in CHF. However, a systemic examination of PEx in vital organs among different time windows of CHF has never been performed. In the present study, we investigated time-dependent PEx in several major visceral organs including heart, lung, liver, spleen, kidney, duodenum, ileum, cecum, and pancreas between sham-operated and CHF rats induced by myocardial infarction (MI). Methods: Plasma extravasation was determined by colorimetric evaluation of Evans Blue (EB) concentrations at 3 days, ∼10 weeks and 4 months following MI. Results: Data show that cardiac PEx was initially high at day 3 post MI and then gradually decreased but remained at a moderately high level at ∼10 weeks and 4 months post MI. Lung PEx began at day 3 and remained significantly elevated at both ∼10 weeks and 4 months post MI. Spleen PExwas significantly increased at ∼10 weeks and 4 months but not on day 3 post MI. Liver PEx occurred early at day 3 and remain significantly increased at ∼10 weeks and 4 months post MI. For the gastrointestinal (GI) organs including duodenum, ileum and cecum, there was a general trend that PEx level gradually increased following MI and reached statistical significance at either 10 weeks or 4 months post MI. Similar to GI PEx, renal PEx was significantly elevated at 4 months post MI. Discussion: In summary, we found that MI generally incites a timedependent PEx of multiple visceral organs. However, the PEx time window for individual organs in response to the MI challenge was different, suggesting that different mechanisms are involved in the pathogenesis of PEx in these vital organs during the development of CHF.

Exercise training prevents skeletal muscle afferent sensitization in rats with chronic heart failure.

An exaggerated exercise pressor reflex (EPR) contributes to exercise intolerance and excessive sympathoexcitation in the chronic heart failure (CHF) state, which is prevented by exercise training (ExT) at an early stage in the development of CHF. We hypothesized that ExT has a beneficial effect on the exaggerated EPR by improving the dysfunction of muscle afferents in CHF. We recorded the discharge of mechanically sensitive (group III) and metabolically sensitive (group IV) afferents in response to static contraction, passive stretch, and hindlimb intra-arterial injection of capsaicin in sham+sedentary (Sed), sham+ExT, CHF+Sed, and CHF+ExT rats. Compared with sham+Sed rats, CHF+Sed rats exhibited greater responses of group III afferents to contraction and stretch, whereas the responses of group IV afferents to contraction and capsaicin were blunted. ExT prevented the sensitization of group III responses to contraction or stretch and partially prevented the blunted group IV responses to contraction or capsaicin in CHF rats. Furthermore, we investigated whether purinergic 2X (P2X) and transient receptor potential vanilloid 1 (TRPV1) receptors mediate the altered sensitivity of muscle afferents by ExT in CHF. We found that the upregulated P2X and downregulated TRPV1 receptors in L4/5 dorsal root ganglia of CHF rats were normalized by ExT. Hindlimb intra-arterial infusion of a P2X antagonist attenuated the group III response to contraction or stretch in CHF rats to a greater extent than in sham rats, which was normalized by ExT. These findings suggest that ExT improves the abnormal sensitization of muscle afferents in CHF at least, in part, via restoring the dysfunction of P2X and TRPV1 receptors.

Timeline of Multi-Organ Plasma Extravasation After Bleomycin-Induced Acute Lung Injury.

Acute lung injury (ALI) is characterized by the abrupt onset of clinically significant hypoxemia in the context of non-hydrostatic pulmonary edema. Acute lung injury is associated with cytokine release and plasma extravasation (PEx) that can cause pulmonary edema and subsequently acute respiratory distress syndrome (ARDS). Therefore, it is critical we understand the relationship between ALI and lung PEx. In addition, it is also important to assess PEx in the lungs and other organs post-ALI since ALI/ARDS often causes multi-organ failure. We hypothesized that ALI induces time-dependent lung PEx, which promotes extravasation in the heart, liver, kidney, spleen, pancreas, and gastrointestinal (GI) tract, in a time-dependent manner. To test our hypothesis, we administered bleomycin or saline via tracheal intubation in 8-week-old Sprague Dawley rats. At the terminal experiments, Evans Blue was injected (IV) through the femoral vein to allow for the visualization of PEx. Plasma extravasation of desired organs was evaluated at 3-, 7-, 14-, 21-, and 28-days after bleomycin or saline treatment by evaluating Evans Blue concentrations calorimetrically at fluorescence excitation wavelength of 620 nm (bandwidth 10 nm) and an emission wavelength of 680 nm (bandwidth 40 nm). Data show that ALI induces lung PEx beginning at day 3 and peaking between 7 and 21 days. Extravasation was also seen in all organs at varying degrees beginning at day 3 and peaking between days 7 and 14. Resolution appears to start after day 21 and continues past day 28. We conclude that ALI caused by bleomycin incites a time-dependent PEx of the lungs and multiple other organs.

Time-dependent alteration in the chemoreflex post-acute lung injury.

Acute lung injury (ALI) induces inflammation that disrupts the normal alveolar-capillary endothelial barrier which impairs gas exchange to induce hypoxemia that reflexively increases respiration. The neural mechanisms underlying the respiratory dysfunction during ALI are not fully understood. The purpose of this study was to investigate the role of the chemoreflex in mediating abnormal ventilation during acute (early) and recovery (late) stages of ALI. We hypothesized that the increase in respiratory rate (fR) during post-ALI is mediated by a sensitized chemoreflex. ALI was induced in male Sprague-Dawley rats using a single intra-tracheal injection of bleomycin (Bleo: low-dose = 1.25 mg/Kg or high-dose = 2.5 mg/Kg) (day 1) and respiratory variables- fR, Vt (Tidal Volume), and VE (Minute Ventilation) in response to 10% hypoxia (10% O2, 0% CO2) and 5% hypercapnia/21% normoxia (21% O2, 5% CO2) were measured weekly from W0-W4 using whole-body plethysmography (WBP). Our data indicate sensitization (∆fR = 93 ± 31 bpm, p < 0.0001) of the chemoreflex at W1 post-ALI in response to hypoxic/hypercapnic gas challenge in the low-dose bleo (moderate ALI) group and a blunted chemoreflex (∆fR = -0.97 ± 42 bpm, p < 0.0001) at W1 post-ALI in the high-dose bleo (severe ALI) group. During recovery from ALI, at W3-W4, both low-dose and high-dose groups exhibited a sensitized chemoreflex in response to hypoxia and normoxic-hypercapnia. We then hypothesized that the blunted chemoreflex at W1 post-ALI in the high-dose bleo group could be due to near maximal tonic activation of chemoreceptors, called the "ceiling effect". To test this possibility, 90% hyperoxia (90% O2, 0% CO2) was given to bleo treated rats to inhibit the chemoreflex. Our results showed no changes in fR, suggesting absence of the tonic chemoreflex activation in response to hypoxia at W1 post-ALI. These data suggest that during the acute stage of moderate (low-dose bleo) and severe (high-dose bleo) ALI, chemoreflex activity trends to be slightly sensitized and blunted, respectively while it becomes significantly sensitized during the recovery stage. Future studies are required to examine the molecular/cellular mechanisms underlying the time-course changes in chemoreflex sensitivity post-ALI.

Cardiac Spinal Afferent Denervation Attenuates Renal Dysfunction in Rats With Cardiorenal Syndrome Type 2.

Cardiorenal syndrome type 2 (CRS2) is defined as a chronic cardiovascular disease, usually chronic heart failure (CHF), resulting in chronic kidney disease. We hypothesized that the cardiac spinal afferent reflex (CSAR) plays a critical role in the development of CRS2. Our data suggest that cardiac afferent ablation by resiniferatoxin not only improves cardiac function but also benefits the kidneys and increases long-term survival in the myocardial infarction model of CHF. We also found that renal denervation has a similar reno-protective effect in CHF rats. We believe this novel work contributes to the development of a unique neuromodulation therapy to treat CHF patients.

Safety and efficacy of renal denervation in patients with heart failure with reduced ejection fraction (HFrEF): A systematic review and meta-analysis.

INTRODUCTION: A systematic analysis of clinical trials was performed in order to assess the effectiveness and risks of bilateral renal denervation (RDN) in patients with chronic heart failure with reduced ejection fraction (HFrEF). METHODS: A systematic review was conducted of all clinical trials exploring the effectiveness of RDN in patients with HF who had reduced (<50%) EF. Primary outcomes were NYHA class, 6-min walk test, N-terminal pro-B-type natriuretic peptide (NT-proBNP) levels, left ventricular ejection fraction (LVEF) and other cardiac parameters including left ventricular end-systolic diameter (LVESD), left ventricular end-diastolic diameter (LVEDD), and left atrium diameter (LAD). Secondary outcomes were systolic blood pressure (SBP), diastolic blood pressure (DBP), heart rate (HR), glomerular filtration rate (GFR), and creatinine. RESULTS: Seven studies were included in this analysis. From baseline to 6 months after RDN, the pooled mean NYHA class was decreased (mean difference [MD], -0.9; 95% confidence interval [CI], -1.6 to -0.2; P = 0.018), the mean 6-min walk test was increased (MD, 79.5 m; 95% CI, 26.9 to 132.1; P = 0.003), and the average NT-proBNP level was decreased (MD, -520.6 pg/mL; 95% CI, -1128.4 to 87.2; P = 0.093). Bilateral RDN increased the LVEF (MD, 5.7%; 95% CI, 1.6 to 9.6; P = 0.004), decreased the LVESD (MD, -0.4 cm; 95% CI, -0.5 to -0.2; P < 0.001), decreased the LVEDD (MD, -0.5 cm; 95% CI, -0.6 to -0.3; P < 0.001), and decreased the LAD (MD, -0.4 cm; 95% CI, -0.8 to 0; P = 0.045). In addition, RDN significantly decreased systolic BP (MD, -9.4 mmHg; 95% CI, -16.3 to -2.4; P = 0.008) and diastolic BP (MD, -4.9 mmHg; 95% CI, -9.5 to -0.4; P = 0.033), and decreased HR (MD, -4.5 bpm; 95% CI, -8.2to -0.9; P = 0.015). RDN did not significantly change GFR (MD, 7.9; 95% CI, -5.0 to 20.8; P = 0.230), or serum creatinine levels (MD, -7.2; 95% CI, -23.7 to 9.4; P = 0.397). CONCLUSION: Bilateral RDN appears safe and well-tolerated in patients with HF. RDN improved the signs and symptoms of HF and slightly decreased systolic and diastolic BP without affecting renal function in the clinical trials performed to date.