Afferent Reflex Research Articles

Standards in Pupillography.

The number of research groups studying the pupil is increasing, as is the number of publications. Consequently, new standards in pupillography are needed to formalize the methodology including recording conditions, stimulus characteristics, as well as suitable parameters of evaluation. Since the description of intrinsically photosensitive retinal ganglion cells (ipRGCs) there has been an increased interest and broader application of pupillography in ophthalmology as well as other fields including psychology and chronobiology. Color pupillography plays an important role not only in research but also in clinical observational and therapy studies like gene therapy of hereditary retinal degenerations and psychopathology. Stimuli can vary in size, brightness, duration, and wavelength. Stimulus paradigms determine whether rhodopsin-driven rod responses, opsin-driven cone responses, or melanopsin-driven ipRGC responses are primarily elicited. Background illumination, adaptation state, and instruction for the participants will furthermore influence the results. This standard recommends a minimum set of variables to be used for pupillography and specified in the publication methodologies. Initiated at the 32nd International Pupil Colloquium 2017 in Morges, Switzerland, the aim of this manuscript is to outline standards in pupillography based on current knowledge and experience of pupil experts in order to achieve greater comparability of pupillographic studies. Such standards will particularly facilitate the proper application of pupillography by researchers new to the field. First we describe general standards, followed by specific suggestions concerning the demands of different targets of pupil research: the afferent and efferent reflex arc, pharmacology, psychology, sleepiness-related research and animal studies.

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.

Non-invasive transcutaneous auricular vagus nerve stimulation prevents postoperative ileus and endotoxemia in mice.

The cholinergic anti-inflammatory pathway comprises the perception of peripheral inflammation by afferent sensory neurons and reflex activation of efferent vagus nerve activity to regulate inflammation. Activation of this pathway was shown to reduce the inflammatory response and improve outcome of postoperative ileus (POI) and sepsis in rodents. Herein, we tested if a non-invasive auricular electrical transcutaneous vagus nerve stimulation (tVNS) affects inflammation in models of POI or endotoxemia. Mice underwent tVNS or sham stimulation before and after induction of either POI by intestinal manipulation (IM) or endotoxemia by lipopolysaccharide administration. Some animals underwent a preoperative right cervical vagotomy. Neuronal activation of the solitary tract nucleus (NTS) and the dorsal motor nucleus of the vagus nerve (DMV) were analyzed by immunohistological detection of c-fos+ cells. Gene and protein expression of IL-6, MCP-1, IL-1β as well as leukocyte infiltration and gastrointestinal transit were analyzed at different time points after IM. IL-6, TNFα, and IL-1β serum levels were analyzed 3hours after lipopolysaccharide administration. tVNS activated the NTS and DMV and reduced intestinal cytokine expression, reduced leukocyte recruitment to the manipulated intestine segment, and improved gastrointestinal transit after IM. Endotoxemia-induced IL-6 and TNF-α release was also reduced by tVNS. The protective effects of tVNS on POI and endotoxemia were abrogated by vagotomy. tVNS prevents intestinal and systemic inflammation. Activation of the DMV indicates an afferent to efferent central circuitry of the tVNS stimulation and the beneficial effects of tVNS depend on an intact vagus nerve. tVNS may become a non-invasive approach for treatment of POI.

Focal selective chemo-ablation of spinal cardiac afferent nerve by resiniferatoxin protects the heart from pressure overload-induced hypertrophy

Resiniferatoxin (RTX), a selective transient receptor potential vanilloid 1 (TRPV1) receptor agonist, can eliminate TRPV1+ primary sensory afferents and blunt cardiac sympathetic afferent reflex for a relatively long period. The present study determined the effects of intrathecal RTX administration on transverse aortic constriction (TAC)-induced cardiac dysfunction and cardiac remodeling in rats. Five days before TAC, RTX (2 μg/10 μl) was injected intrathecally into the T2/T3 interspace of rats. Cardiac sympathetic nerve activities (CSNAs) and cardiac structure and function were determined eight weeks after TAC. Intrathecal RTX administration abolished TRPV1 expression in the dorsal horn and reduced over-activated CSNA in the TAC rat model. Hemodynamic analysis revealed that RTX reduced left ventricular end-diastolic pressure, indicating the improvement of cardiac compliance. Histologic analysis, real-time reverse transcription-polymerase chain reaction, and Western blots showed that RTX prevented TAC-induced cardiac hypertrophy, cardiac fibrosis, and cardiac apoptosis and reduced the expression of apoptotic proteins and myocardial mRNAs. In conclusion, these results demonstrate that focal chemo-ablation of TRPV1+ afferents in the spinal cord protects the heart from pressure overload-induced cardiac remodeling and cardiac dysfunction, which suggest a novel promising therapeutic method for cardiac hypertrophy and diastolic dysfunction.

Chronic heart failure: a disease of the brain.

The underlying mechanism for clinical and biochemical manifestations of chronic heart failure (HF) may be due in part to neurohumoral adaptations, such as activation of the renin-angiotensin-aldosterone and sympathetic nervous systems in the periphery and the brain. Internet search and discussion with colleagues are the methods for this study. Since chronic HF is associated with autonomic imbalance with increased sympathetic nerve activity and a withdrawal of parasympathetic activity, it may be considered a brain disease. This phenomenon may be the result of an increased systemic and cerebral angiotensin II signaling because plasma angiotensin II is increased in humans and animals with chronic HF. The increase in angiotensin II signaling enhances sympathetic nerve activity through actions on both central and peripheral sites during chronic HF. Activation of angiotensin II signaling in different brain sites such as the paraventricular nucleus (PVN), rostral ventrolateral medulla (RVLM), and area postrema (AP) may increase the release of norepinephrine, oxidative stress, and inflammation leading to increased cardiac contractility. It is possible that blocking angiotensin II type 1 receptors decreases sympathetic nerve activity and cardiac sympathetic afferent reflex when therapy is administered to the PVN. The administration of an angiotensin receptor blocker by injection into the AP activates the sympatho-inhibitory baroreflex indicating that receptor blockers act by increasing parasympathetic activity. In chronic HF, in peripheral regions, angiotensin II elevates both norepinephrine release and synthesis and inhibits norepinephrine uptake at nerve endings, which may contribute to the increase in sympathetic nerve activity. Increased circulating angiotensin II during chronic HF may enhance the sympatho-excitatory chemoreflex and inhibit the sympatho-inhibitory baroreflex resulting in worsening of HF. Increased circulating angiotensin II signaling can directly act on the central nervous system via the subfornical organ and the AP to increase sympathetic outflow resulting in to neurohumoral dysfunction, resulting in to heart failure.

Abstract P159: Neural Inflammation Results in Cardiac Sympathetic Afferent Sensitization in Post-Myocardial Infarction Rats

Sympatho-excitation is a hallmark of the chronic heart failure (CHF) state. The enhanced cardiac sympathetic afferent reflex (CSAR) contributes to the elevated sympathetic tone in CHF. However, the mechanisms underlying the CSAR sensitization have not been fully discovered. We hypothesized that myocardial infarction (MI) will trigger a neuro-inflammatory cascade that includes macrophage activation in thoracic dorsal root ganglia (DRG), which results in cardiac spinal afferent sensitization. Our immunofluorescence data ( Figure 1 ) demonstrated that, the number of Iba-1-positive (a macrophage marker) cells in T1-T4 DRGs of MI rats was significantly increased at 1 week and lasted at least 8 weeks post MI. RNA-seq analysis data in T1-T4 DRGs of MI rats also showed upregulated mRNA expressions of several macrophage-activation related genes such as IRF7, RGD1309362, slfn4 and lfit1. In the in vitro experiments, after the 50B11 DRG cells were co-cultured with LPS-pretreated BV2 cells (activated microglia/macrophage), the protein expressions of voltage-gated potassium channel isoforms (Kv1.4, Kv4.2, Kv4.3 and Kv3.4) were largely reduced, which explains how macrophage activation cause DRG neuronal sensitization. Lastly, our data showed that 4-week treatment with minocycline (a macrophage inhibitor, 40 mg/kg/day) via drinking water markedly reduced the enhanced CSAR in the post-MI rats (MAP, 30.2±2.6 vs. 18.0±1.9 mmHg, n=6, p<0.01; HR, 43.5±5.5 vs. 12.5±2.0 bpm, n=6, p<0.01; RSNA, 169.2±19.5 vs. 77.8±4.6% baseline, n=6, p<0.01). These data suggested that macrophage infiltration in sensory ganglia contributes to the sensitization of the CSAR in the post-MI state.

Abstract P202: Targeting the Cardiac Sympathetic Afferent Reflex in Hypertrophic Cardiomyopathy

Sarcomere gene mutations cause hypertrophic cardiomyopathy (HCM). Hallmark features include cardiac hypertrophy, fibrosis, excessive cardiac sympathetic tone, and increased risk of arrhythmias and sudden death. The mechanism(s) of increased sympathetic tone is poorly understood. We hypothesized that cardiac spinal (sympathetic) afferents are sensitized by metabolites (e.g., H+) produced during myocyte energy depletion in HCM, resulting in an enhanced cardiac sympathetic afferent reflex (CSAR). We studied mice with cardiac-specific expression of mutated alpha-tropomyosin (Glu180Gly), an established model of HCM. The CSAR was assessed by measuring mean arterial pressure (MAP) and heart rate (HR) responses to epicardial application of the TRPV1 agonist capsaicin (2.5mM, 2μL) to the left ventricle in anesthetized, ventilated HCM (n=5) and wild-type (WT) littermate control (n=5) mice. Pressor and tachycardic responses to capsaicin were markedly enhanced in HCM mice (Table). Blocking the tonic influence of cardiac afferent activity by epicardial application of the local anesthetic lidocaine (2%) caused small, significant decreases in MAP in both groups of mice; but decreased HR markedly in HCM mice only (n=5) (Table). mRNA expression (qPCR) of acid-sensing ion channel 3 was increased by 2-fold in dorsal root ganglia (T1-T9) of HCM vs. WT mice (n=6 each, P<0.05), suggesting a mechanism for sensitization of cardiac sympathetic afferents in HCM. We conclude that the CSAR is enhanced in HCM mice and speculate that increased activity of cardiac ‘sympathetic’ afferent nerves contributes to increased sympathetic tone, arrhythmias and disease progression in HCM.

Sciatic nerve stimulation and its effects on upper airway resistance in the anesthetized rabbit model relevant to sleep apnea.

Obstructive sleep apnea (OSA) is a disorder characterized by collapse of the velopharynx and/or oropharynx during sleep when drive to the upper airway is reduced. Here, we explore an indirect approach for activation of upper airway muscles that might affect airway dynamics, namely, unilateral electrical stimulation of the afferent fibers of the sciatic nerve, in an anesthetized rabbit model. A nerve cuff electrode was placed around the sciatic and hypoglossal nerves to deliver stimulus while airflow, air pressure, and alae nasi electromyogram (EMG) were monitored both before and after sciatic transection. Sciatic nerve stimulation increased respiratory effort, rate, and alae nasi EMG, which persisted for seconds after stimulation; however, upper airway resistance was unchanged. Hypoglossal stimulation reduced resistance without altering drive. Although sciatic nerve stimulation is not ideal for treating OSA, it remains a target for altering respiratory drive. NEW & NOTEWORTHY Previously, sciatic nerve stimulation has been shown to activate upper airway and chest wall muscles. The supposition that resistance through the upper airway would be reduced with this afferent reflex was disproven. Findings were in contrast with the effect of hypoglossal nerve stimulation, which was shown to decrease resistance without changing muscle activation or ventilatory drive.

Sympatho-excitatory response to pulmonary chemosensitive spinal afferent activation in anesthetized, vagotomized rats.

The sensory innervation of the lung is well known to be innervated by nerve fibers of both vagal and sympathetic origin. Although the vagal afferent innervation of the lung has been well characterized, less is known about physiological effects mediated by spinal sympathetic afferent fibers. We hypothesized that activation of sympathetic spinal afferent nerve fibers of the lung would result in an excitatory pressor reflex, similar to that previously characterized in the heart. In this study, we evaluated changes in renal sympathetic nerve activity (RSNA) and hemodynamics in response to activation of TRPV1‐sensitive pulmonary spinal sensory fibers by agonist application to the visceral pleura of the lung and by administration into the primary bronchus in anesthetized, bilaterally vagotomized, adult Sprague‐Dawley rats. Application of bradykinin (BK) to the visceral pleura of the lung produced an increase in mean arterial pressure (MAP), heart rate (HR), and RSNA. This response was significantly greater when BK was applied to the ventral surface of the left lung compared to the dorsal surface. Conversely, topical application of capsaicin (Cap) onto the visceral pleura of the lung, produced a biphasic reflex change in MAP, coupled with increases in HR and RSNA which was very similar to the hemodynamic response to epicardial application of Cap. This reflex was also evoked in animals with intact pulmonary vagal innervation and when BK was applied to the distal airways of the lung via the left primary bronchus. In order to further confirm the origin of this reflex, epidural application of a selective afferent neurotoxin (resiniferatoxin, RTX) was used to chronically ablate thoracic TRPV1‐expressing afferent soma at the level of T1–T4 dorsal root ganglia pleura. This treatment abolished all sympatho‐excitatory responses to both cardiac and pulmonary application of BK and Cap in vagotomized rats 9–10 weeks post‐RTX. These data suggest the presence of an excitatory pulmonary chemosensitive sympathetic afferent reflex. This finding may have important clinical implications in pulmonary conditions inducing sensory nerve activation such as pulmonary inflammation and inhalation of chemical stimuli.

Airway Rapidly Adapting Receptors

By the turn of the millennium, only three major types of receptors were recognized in the airways [C‐fiber receptors (CFRs), rapidly adapting receptors (RARs), and slowly adapting receptors (SARs)]. RARs and SARs are mechanical receptors with myelinated afferents. RARs were thought to be sensitive to a wide variety of mechanical (inflation and deflation) and chemical stimuli, and display a spectrum of responses. It is perplex because such characteristics make the RAR reflex system non‐specific. Careful review of literature shows RARs are not strictly defined. Many investigators ascribe observed sensory properties and reflex effects to RARs when SARs and CFRs were ruled out. Thus, RARs are credited more.In this report, we did a series experiments to characterize ‘RARs' in rabbits. We found that some ‘RARs' share many characteristics of CFRs, including low resting discharge frequency, chemosensitivity, and insensitivity to mechanical stimulation. These fibers have conduction velocities of 4–15 m/s, belong to the Aδ afferent fibers, and are termed high threshold Ad receptors (HTARs). Using histochemical staining [antibodies against Na+/K+‐ATPase and myelin basic protein (MBP)], we found that many mechanical receptor structures share a single axon, indicating that what we assessed is not a sensory receptor but a sensory unit that contains many receptors. By recording sensory unit activities, we found that many units had multiple receptive fields. Blocking a field with lidocaine, we found that each field contained at least a receptor. Moreover, both SARs and RARs can respond to lung inflation and deflation. Unit responses to lung inflation and deflation can be selectively blocked by injection of lidocaine into a field. This indicates that lung deflation response is elicited from deflation activated receptors (DARs). Furthermore, a single sensory unit may contain both RARs and SARs, each can be selectively blocked by injection of lidocaine into a field. Based on these characteristics of airway mechanical sensory units, we conclude that a sensory unit consists of many different receptors for sensing different mechanical forces. Three major types of mechanical sensors operate in the lung: SARs, RARs, and DARs. Many SAR and RAR units also respond to lung deflation because they contain DARs. Pure DARs, which respond to lung deflation only, are rare in large animals but are easily identified in small animals. Actually, some chemosensitive responses ascribed to RARs may instead belong to HTARs, while some mechanical sensitive responses belong to DARs.In summary, with identification of DARs and HTARs, afferent properties and reflex functions of RARs should be redefined. RARs, by definition, are mechanoreceptors. They are stimulated by lung inflation but not deflation. Their afferent properties may not include chemosensitivities, which are belong to HTARs. Therefore, lung deflation and chemical stimulation induced reflex effects also should be ascribed to those sensors.Support or Funding InformationSupported by a VA Merit Review Award PULM‐029‐10SThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Chemosensitive Cardiac Afferent Reflexes in Mice: Are they Altered in Hypertrophic Cardiomyopathy?

Hypertrophic cardiomyopathy (HCM) is a life‐threatening disease caused by sarcomere gene mutations. Hallmark features include myocyte disarray, left ventricular hypertrophy, fibrosis, and increased risk of arrhythmias and sudden death. Cardiac norepinephrine spillover is augmented in humans with HCM. Similarly, our group has previously reported that cardiac sympathetic tone is increased and vasomotor sympathetic tone is decreased in transgenic mice with cardiac‐specific expression of mutated alpha‐tropomyosin (Glu180Gly), a model of human HCM. Specific aims of this study were to develop protocols to evaluate cardiac reflexes evoked by activation of vagal and ‘sympathetic’ afferents in anesthetized mice, and test the hypothesis that alpha‐tropomyosin mutant HCM mice exhibit enhanced heart rate responses to activation of cardiac ‘sympathetic’ afferents. Cardiac reflexes were evoked via injection of the TRPV1 receptor agonist capsaicin (25 & 50 ng/g, IV) in mice anesthetized with ketamine (91μg/g) and xylazine (9.1 μg/g). Injection of capsaicin (50 ng/g, IV) in C57BL6 mice evoked rapid decreases in heart rate (HR) (−131±17bpm) and mean arterial pressure (MAP) (−30.1±2.7 mmHg) followed by delayed increases in HR (+12.5±7.6bpm) and MAP (+12.7±3.7 mmHg) (n=6, p<0.05). Responses were dose‐dependent and repeatable. Bilateral cervical vagotomy abolished the bradycardic/depressor responses to capsaicin and enhanced the delayed increases in HR (+22.6±4.1bpm) and MAP (+27.6±1.7 mmHg) (n=5). All responses to capsaicin were essentially abolished after ganglionic blockade (p<0.05, n=3) confirming the responses were neurally mediated. The tachycardic response to capsaicin (25ng/g, IV) after vagotomy was significantly (p<0.05) augmented in HCM mice (+32±11bpm, n=5) compared with their wild‐type (WT) controls (+11±3bpm, n=6). In contrast, the MAP response to capsaicin was similar between HCM (+8.5±2.5 mmHg, n=5) and WT (+6.3±1.6 mmHg, n=6). We conclude: (1) Measurements of reflex responses to capsaicin before and after vagotomy enable assessment of both inhibitory and excitatory reflexes in mice, and (2) HCM mice demonstrate an exaggerated tachycardic response to injection of capsaicin, presumably reflecting an increased sensitivity of cardiac ‘sympathetic’ afferent nerves.Support or Funding InformationNIH T32HL007121, AAS‐Lundbeck Fellowship, NIH HL14388, VA 1BX001414This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

TRPA1‐Induced Pulmonary Spinal Sympathetic Afferent Activation is Attenuated in Rats with Chronic Heart Failure

In a recent preliminary study (Association of the University Anesthesiologists 64th annual meeting, Abstract #1109, 2017), we reported that activation of pulmonary spinal afferents by topical application of bradykinin (BK) and the TRPV1 agonist capsaisin on to lung surfaces (especially on the ventral surface) evokes a previously undocumented potent sympatho‐excitatory reflex that includes increased bood pressure (BP), heart rate (HR) and renal sympathetic nerve activity (RSNA) in urethane‐α‐chloralose anesthetized, vagotomized rats. These data suggested a new pulmonary sympathetic afferent reflex (PSAR) in rats. However, it remains unclear whether pulmonary sympathetic afferents can also be activated by the transient receptor potential ankyrin 1 (TRPA1) agonist, allyl isothiocyanate (AITC). In the current study, we examined the TRPA1‐induced PSAR in both normal and chronic heart failure (CHF) states. After bilateral vagotomy, we applied filter paper (3 × 3 mm) saturated with 30 mM AITC to the ventral lung surfaces to stimulate the PSAR in urethane‐ α‐chloralose anesthetized sham‐operated and CHF (8 weeks post myocardial infarction) rats. We found that application of AITC to the lungs resulted in a biphasic BP response whereas HR and RSNA remained elevated during the AITC application in sham rats (Table 1). Compared to sham rats, AITC caused a predominantly depressor response in CHF rats. The increases in HR and RSNA in response to AITC were also attenuated in CHF rats. These data suggest that activation of the TRPA1 receptor in pulmonary spinal afferents can cause sympatho‐excitation in normal rats whereas this response was blunted in CHF rats. Ongoing studies are validating TRPA1 receptor protein expression in pulmonary sensory neurons in sham and CHF rats.Support or Funding InformationThis work was, in part, supported by grants from the National Heart, Lung, and Blood Institute (1R01HL121012‐01 and 1R01HL126796‐01).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Exercise intolerance in Type 2 diabetes: is there a cardiovascular contribution?

Physical activity is critically important for Type 2 diabetes management, yet adherence levels are poor. This might be partly due to disproportionate exercise intolerance. Submaximal exercise tolerance is highly sensitive to muscle oxygenation; impairments in exercising muscle oxygen delivery may contribute to exercise intolerance in Type 2 diabetes since there is considerable evidence for the existence of both cardiac and peripheral vascular dysfunction. While uncompromised cardiac output during submaximal exercise is consistently observed in Type 2 diabetes, it remains to be determined whether an elevated cardiac sympathetic afferent reflex could sympathetically restrain exercising muscle blood flow. Furthermore, while deficits in endothelial function are common in Type 2 diabetes and are often cited as impairing exercising muscle oxygen delivery, no direct evidence in exercise exists, and there are several other vasoregulatory mechanisms whose dysfunction could contribute. Finally, while there are findings of impaired oxygen delivery, conflicting evidence also exists. A definitive conclusion that Type 2 diabetes compromises exercising muscle oxygen delivery remains premature. We review these potentially dysfunctional mechanisms in terms of how they could impair oxygen delivery in exercise, evaluate the current literature on whether an oxygen delivery deficit is actually manifest, and correspondingly identify key directions for future research.

Application of non-invasive electric stimulation of the spinal cord in motor rehabilitation of children with consequences of vertebral and cerebrospinal injury (preliminary report)

Introduction. Vertebral and cerebrospinal injury and its consequences constitute an important problem in modern medicine. In recent years, studies have shown that percutaneous electric stimulation in patients with these injuries can influence the neuronal networks of different parts of the spinal cord to activate afferent and efferent reflex connections with complete or partial disorders of supraspinal influences of various geneses.
 Aim. To investigate the effect of percutaneous electric stimulation of the spinal cord on the dynamics of recovery of neurological functions in children with vertebral and cerebrospinal injury.
 Materials and methods. Seven patients aged 4 to 18 years with lesions of the spinal cord from C5-C6 to Th12-L1 and who mainly had a marked neurological deficit were examined from 1 month to 9 years after surgical treatment. All patients underwent neurophysiological studies, including electroneuromyography, electromyography, and somatosensory-evoked potentials. The patients and their parents kept a diary of urination.
 Results. This clinical study showed that percutaneous electric stimulation of the spinal cord contributed to the rapid and complete restoration of the neurological functions in patients with vertebral and medullar conflict and depended directly on the early terms of surgical intervention.
 Conclusion. The positive results obtained in the complex rehabilitation of children with vertebral and cerebrospinal injuries by using non-invasive percutaneous electric stimulation of the spinal cord support the use of this method in the early stages after surgical intervention.

Superoxide Anions and NO in the Paraventricular Nucleus Modulate the Cardiac Sympathetic Afferent Reflex in Obese Rats.

This study was conducted to explore the hypothesis that the endogenous superoxide anions (O2−) and nitric oxide (NO) system of the paraventricular nucleus (PVN) regulates the cardiac sympathetic afferent reflex (CSAR) contributing to sympathoexcitation in obese rats induced by a high-fat diet (42% kcal as fat) for 12 weeks. CSAR was evaluated by monitoring the changes of renal sympathetic nerve activity (RSNA) and the mean arterial pressure (MAP) responses to the epicardial application of capsaicin (CAP) in anaesthetized rats. In obese rats with hypertension (OH group) or without hypertension (OB group), the levels of PVN O2−, angiotensinII (Ang II), Ang II type 1 receptor (AT1R), and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase were elevated, whereas neural NO synthase (nNOS) and NO were significantly reduced. Moreover, CSAR was markedly enhanced, which promoted the elevation of plasma norepinephrine levels. The enhanced CSAR was attenuated by PVN application of the superoxide scavenger polyethylene glycol-superoxide dismutase (PEG-SOD) and the NO donor sodium nitroprusside (SNP), and was strengthened by the superoxide dismutase inhibitor diethyldithiocarbamic acid (DETC) and the nNOS inhibitor N(ω)-propyl-l-arginine hydrochloride (PLA); conversely, there was a smaller CSAR response to PLA or SNP in rats that received a low-fat (12% kcal) diet. Furthermore, PVN pretreatment with the AT1R antagonist losartan or with PEG-SOD, but not SNP, abolished Ang II-induced CSAR enhancement. These findings suggest that obesity alters the PVN O2− and NO system that modulates CSAR and promotes sympathoexcitation.

Short hairpin RNA interference targeting interleukin 1 receptor type I in the paraventricular nucleus attenuates hypertension in rats.

Blood pressure is controlled by tonic sympathetic activities, excessive activation of which contributes to the pathogenesis and progression of hypertension. Interleukin (IL)-1β in the paraventricular nucleus (PVN) is involved in sympathetic overdrive and hypertension. Here, we investigated the therapeutic effects of IL-1 receptor type I (IL-1R1) gene silencing in the PVN on hypertension. Recombinant lentivirus vectors expressing a short hairpin RNA (shRNA) targeting IL-1R1 (Lv-shR-IL-1R1) or a control shRNA were microinjected into PVN of spontaneously hypertensive rats (SHRs) and normotensive WKY rats. The fluorescence of green fluorescent protein-labelled vectors appeared at 2weeks after injection and persisted for at least 8weeks. IL-1R1 protein expression in the PVN was reduced 4weeks after Lv-shR-IL-1R1 injection in SHRs. IL-1R1 interference also reduced basal sympathetic activity, cardiac sympathetic afferent reflex in SHRs. Depressor effects were observed from week 2 to 10 after Lv-shR-IL-1R1 treatment in SHRs, with the most prominent effects seen at the end of week 4. Furthermore, Lv-shR-IL-1R1 treatment decreased the ratio of left ventricular weight to body weight and cross-sectional areas of myocardial cells in SHRs. Additionally, Lv-shR-IL-1R1 treatment prevented an increase in superoxide anion and pro-inflammatory cytokines (PICs, TNF-α and IL-1β) in the PVN of SHR, and upregulated anti-inflammatory cytokine (AIC, IL-10) expression. These results indicate that shRNA interference targeting IL-1R1 in the PVN decreases arterial blood pressure, attenuates excessive sympathetic activity and cardiac sympathetic afferent reflex, and improves myocardial remodelling in SHRs by restoring the balance between PICs and AICs to attenuate oxidative stress.

Periodic limb movements in complete spinal cord injuries: Important new insights into the pathophysiology of this symptom

Objective Periodic limb movements (PLM) are automatic stereotyped movements of the lower limbs which occurs predominantly at night or when lying down. In a previous study, we identified PLM in numerous patients with spinal cord injuries (SCI), falsely labeled as spasticity and spasms. But unlike spasticity and flexor afferent reflex, the pathophysiology of this movement disorder remains unclear, whether it has a cerebral or spinal origin. Material/Patients and methods Patients with a history of SCI and consecutively addressed for uncontrolled spasticity and spasms despite treatment from March 2014 to July 2016, were systematically assessed by a nocturnal polysomnography (PSG). PLM were defined as a PLM index above 15 events per hour of sleep, according to AASM guidelines. We extracted the data regarding patients presenting with a clinically complete SCI, defined as an ASIA impairment scale scored as A (AIS-A). Results Among the 24 patients initially included, 4 were AIS-A. They all had a thoracic level of injury above Th10. They were all diagnosed with PLM, with a median PLM index of 53.5/h (range [18.2;155]), and PLM arousals of 2.2/h (range [0;15.9]). The initiation of a dopaminergic agonist restored normal sleep motor behaviors in all 4 patients. Discussion/Conclusion The fact that PLM were observed in patients with a clinically complete disruption of the spinal cord, strongly suggested that these movements originates within the spinal cord, possibly below Th10. This hypothesis has been previously raised by the observation of persistent PLM during REM sleep in incomplete SCI patients. We therefore hypothesize the existence of a spinal generator of PLM, at least partly made up by dopaminergic neurons expressing D3 dopa-receptors. This sets the bases for future anatomical and physiological studies for the origination of these movement disorders.

Abstract P179: Pulmonary Spinal Sympathetic Afferent Activation in Post-myocardial Infarction Rats

Recently, we reported activation of pulmonary spinal afferents by topical application of bradykinin (BK) to lung surfaces evokes a potent sympatho-excitatory reflex, including increased blood pressure (BP), heart rate (HR) and renal sympathetic nerve activity (RSNA) in urethane-α-chloralose anesthetized vagotomized rats. This finding suggests a new previously undocumented pulmonary sympathetic afferent reflex (PSAR). Preliminary data was reported in abstract form at the Association of University Anesthesiologists 64 th Annual meeting. Expanding this work, we examined the activation of the PSAR at different time points post myocardial infarction (1 month vs. 5 months post MI) and at different degrees of infarct size (15%-35% (moderate) vs. above 35% (large) of left ventricle). Following bilateral vagotomy , we applied filter paper (3 х 3 mm) saturated with BK (10 μg/ml) to the ventral lung surface to stimulate the PSAR in both urethane- α-chloralose anesthetized sham-operated and MI rats. Application of BK to the lungs resulted in increased BP, HR and RSNA in all sham and MI rats. Compared to sham rats, the BK-induced sympatho-excitatory response was significantly increased in 1-month moderate and 1-month large MI rats as well as in 5-month moderate MI rats. The BK-induced sympatho-excitatory response was significantly blunted in 5-month large MI rats (Table 1). These data suggest that the activation of pulmonary spinal afferents in the post-MI state may be an independent risk factor for sympatho-excitation. Further studies are needed to understand the differential effects of pulmonary spinal afferent activation at the transition from the post MI state to heart failure.

Multiple Reflex Pathways Contribute to Bladder Activation by Intraurethral Stimulation in Persons With Spinal Cord Injury

To measure the urodynamic effects of electrical co-stimulation of 2 individual sites in the proximal and distal urethra in persons with spinal cord injury (SCI). This work was motivated by preclinical findings that selective co-stimulation of the cranial urethral sensory nerve and the dorsal genital nerve, which innervate the proximal and distal portions of the urethra, respectively, increased reflex bladder activation and voiding efficiency. Electrical co-stimulation of urethral afferents was conducted in persons with chronic SCI during urodynamics. The effects of different frequencies of intraurethral stimulation at multiple urethral locations on bladder pressure and pelvic floor electromyographic activity were measured. Electromyographic activity indicated that multiple reflex pathways were recruited through stimulation that contributed to bladder activation. The size of reflex bladder contractions evoked by stimulation was dependent on stimulation location or reflex activated and stimulation frequency. Pudendal nerve afferents are a promising target to restore lost bladder control, as stimulation with different frequencies may be used to treat urinary incontinence and increase continent volumes or to generate stimulation-evoked bladder contractions for on-demand voiding. This work identified that co-stimulation of multiple afferent reflex pathways can enhance activation of spinal circuits and may enable improved bladder emptying in SCI when stimulation of a single pathway is not sufficient.

A review of human neuroimaging investigations involved with central autonomic regulation of baroreflex-mediated cardiovascular control.

Effective regulation of central blood volume and arterial pressure is critical for optimal cardiovascular homeostasis. Inadequate regulation of mean arterial pressure has important pathophysiological implications including syncope, end organ damage, and stroke. Such regulation requires appropriate central integration of barosensory afferents and reflex autonomic control of the heart and blood vessels. The neural pathways involved with the baroreflex include brainstem nuclei that receive modulatory input from higher brain centres. Studies in anesthetized animals have highlighted the role of a central autonomic network involved with baroreflex control. The refinement of functional neuroimaging techniques has provided the opportunity to confirm and extend these findings in awake humans. Such methods have provided information about the temporal and spatial neural patterns associated with changes in barosensory afferent activity and reflex autonomic and cardiovascular responses. This review focuses on human neuroimaging investigations that utilized volitional (e.g., respiratory challenges) and/or non-volitional (e.g. lower body suction) methods to study baroreflex control. The cumulative evidence points to the importance of a baroreflex autonomic network that includes the insular cortex, anterior cingulate cortex, medial prefrontal cortex, amygdala and cerebellum. Future work is required to further delineate the brain regions involved specifically with sensing barosensory afferents versus reflex efferent responses. The use of functional electrophysiological imaging techniques (e.g. MEG) may provide an opportunity to: 1) expand the methods and physiologic measures used to study central baroreflex function in humans, and 2) enhance the temporal precision required to delineate the order of activation within higher brain regions involved with baroreflex control.