LC/MS-guided isolation and GNPS procedures to identify flavonoid of HCoV-OC43 inhibitors.

The screening of based target compounds supported by LC/MS, MS/MS and Global Natural Products Social (GNPS) used to identify the compounds 1-10 of Butea monsperma. They were evaluated in human malignant embryonic rhabdomyoma cells (RD cells) infected with Human coronavirus OC43 (HCoV-OC43) and showed significant inhibitory activity. Target inhibition tests showed that compounds 6 and 8 inhibited the proteolytic enzyme 3CLpro, which is widely present in coronavirus and plays an important role in the replication process, with an effective IC50 value. The study confirmed that dioxymethylene of compound 8 may be a key active fragment in inhibiting coronavirus (EC50 7.2 µM, SI > 139.1). The results have led to identifying natural bioactive compounds for possible inhibiting HCoV-OC43 and developing drug for Traditional Chinese Medicine (TCM).

FDA Perspectives on the Regulation of Neuromodulation Devices.

The United States Food and Drug Administration (FDA) ensures that patients in the United States have access to safe and effective medical devices. The division of neurological and physical medicine devices reviews medical technologies that interface with the nervous system, including many neuromodulation devices. This article focuses on neuromodulation devices and addresses how to navigate the FDA's regulatory landscape to successfully bring devices to patients.

Absence of Neuropathology With Prolonged Isoflurane Sedation in Healthy Adult Rats.

BACKGROUND: The use of isoflurane sedation for prolonged periods in the critical care environment is increasing. However, isoflurane-mediated neurotoxicity has been widely reported. The goal of the present study was to determine whether long-term exposure to low-dose isoflurane in mechanically ventilated rodents is associated with evidence of neurodegeneration or neuroinflammation. METHODS: Adult female Sprague-Dawley rats were used in this study. Experimental animals (n=11) were induced with 1.5% isoflurane, intubated, and given a neuromuscular blockade with α-cobratoxin. EEG electrodes were surgically implanted, subcutaneous precordial EKG Ag wire electrodes, and bladder, femoral artery, and femoral vein cannulas permanently placed. After these procedures, the isoflurane concentration was reduced to 0.5% and, in conjunction with the neuromuscular blockade, continued for 7 days. Arterial blood gases and chemistry were measured at 3 time points and core body temperature servoregulated and maintenance IV fluids were given during the 7 days. Experimental animals and untreated controls (n=9) were euthanized on day 7. RESULTS: Immunohistochemical and cytochemical assays did not detect evidence of microgliosis, astrocytosis, neuronal apoptosis or necrosis, amyloidosis, or phosphorylated-tau accumulation. Blood glucose levels were significantly reduced on days 3/4 and 6/7 and partial pressure of oxygen was significantly reduced, but still within the normal range, on day 6/7. All other blood measurements were unchanged. CONCLUSIONS: No neuropathologic changes consistent with neurotoxicity were detected in the brain after 1 week of continuous exposure to 0.5% isoflurane in healthy rats. These data suggest that even long exposures to low concentrations of isoflurane have no overt consequences on neuropathology.

FDA Regulation of Neurological and Physical Medicine Devices: Access to Safe and Effective Neurotechnologies for All Americans.

The United States Food and Drug Administration (FDA) ensures that patients in the U.S. have access to safe and effective medical devices. The Division of Neurological and Physical Medicine Devices reviews medical technologies that interface with the nervous system. This article addresses how to navigate the FDA's regulatory landscape to successfully bring medical devices to patients.

Baroreflex and chemoreflex controls of sympathetic activity following intermittent hypoxia.

There is a large amount of evidence linking obstructive sleep apnea (OSA), and the associated intermittent hypoxia that accompanies it, with the development of hypertension. For example, cross-sectional studies demonstrate that the prevalence of hypertension increases with the severity of OSA (Bixler et al., 2000; Grote et al., 2001) and an initial determination of OSA is associated with a three-fold increase for future hypertension (Peppard et al., 2000). Interestingly, bouts of intermittent hypoxia have also been shown to affect sympathetic output associated with the baroreflex and chemoreflex, important mechanisms in the regulation of arterial blood pressure. As such, the possibility exists that changes in the baroreflex and chemoreflex may contribute to the development of chronic hypertension observed in OSA patients. The aim of the current article is to briefly review the response of the baroreflex and chemoreflex to intermittent hypoxic exposure and to evaluate evidence for the hypothesis that modification of these autonomic reflexes may, at least in part, support the comorbidity observed between chronic hypertension and OSA.

Pancreatic insulin and exocrine secretion are under the modulatory control of distinct subpopulations of vagal motoneurones in the rat.

Brainstem vago-vagal neurocircuits modulate upper gastrointestinal functions. Derangement of these sensory-motor circuits is implicated in several pathophysiological states, such as gastroesophageal reflux disease (GERD), functional dyspepsia and, possibly, pancreatitis. While vagal circuits controlling the stomach have received more attention, the organization of brainstem pancreatic neurocircuits is still largely unknown. We aimed to investigate the in vitro and in vivo modulation of brainstem vagal circuits controlling pancreatic secretion. Using patch clamp techniques on identified vagal pancreas-projecting neurones, we studied the effects of metabotropic glutamate receptor (mGluR) agents in relation to the effects of exendin-4, a glucagon-like peptide 1 analogue, cholecystokinin (CCK) and pancreatic polypeptide (PP). An in vivo anaesthetized rat preparation was used to measure pancreatic exocrine secretion (PES) and plasma insulin following microinjection of metabotropic glutamate receptor (mGluR) agonists and exendin-4 in the brainstem. Group II and III mGluR agonists (2R,4R-4-aminopyrrolidine-2,4-dicarboxylate (APDC) and L(+)-2-amino-4-phosphonobutyric acid (L-AP4), respectively) decreased the frequency of miniature inhibitory and excitatory postsynaptic currents (mIPSCs and mEPSCs, respectively) in the majority of the neurones tested. All neurones responsive to L-AP4 were also responsive to APDC, but not vice versa. Further, in neurones where L-AP4 decreased mIPSC frequency, exendin-4 increased, while PP had no effect upon, mIPSC frequency. Brainstem microinjection of APDC or L-AP4 decreased plasma insulin secretion, whereas only APDC microinjections increased PES. Exendin-4 microinjections increased plasma insulin. Our results indicate a discrete organization of vagal circuits, which opens up promising avenues of research aimed at investigating the physiology of homeostatic autonomic neurocircuits.

Neural control of arterial pressure variability in the neuromuscularly blocked rat.

The baroreflexes stabilize moment-to-moment arterial pressure. Sinoaortic denervation (SAD) of the baroreflexes results in a large increase in arterial pressure variability (APV) across various species. Due to an incomplete understanding of the nonlinear interactions between central and peripheral systems, the major source of APV remains controversial. While some studies suggested that the variability is endogenous to the central nervous system (CNS), others argued that peripheral influences may be the main source. For decades, abnormal cardiovascular variability has been associated with a number of cardiovascular diseases including hypertension, heart failure, and stroke. Delineating mechanisms of the APV is critical for the improvement of current strategies that use APV as a clinical tool for the diagnosis and prognosis of cardiovascular diseases. In this study, with a unique chronic neuromuscularly blocked (NMB) rat preparation that largely constrains peripheral influences, we determined the CNS contribution to the post-SAD APV. First, we confirmed that SAD significantly increased APV in the NMB rat, then demonstrated that post-SAD ganglionic blockade substantially reduced APV, and subsequent intravenous infusions of phenylephrine and epinephrine (in presence of ganglionic blockade) only slightly increased APV. These data suggest that the CNS is an important source, and skeletal activity, thermal challenges or other forms of peripherally generated cardiovascular stress are not required for the post-SAD APV. In addition, we showed that bilateral aortic denervation produced a larger increase in APV than bilateral carotid sinus denervation, suggesting that the aortic baroreflex plays a more dominant role in the control of APV than the carotid sinus.

Preferential skeletal muscle myosin loss in response to mechanical silencing in a novel rat intensive care unit model: underlying mechanisms.

The muscle wasting and impaired muscle function in critically ill intensive care unit (ICU) patients delay recovery from the primary disease, and have debilitating consequences that can persist for years after hospital discharge. It is likely that, in addition to pernicious effects of the primary disease, the basic life support procedures of long-term ICU treatment contribute directly to the progressive impairment of muscle function. This study aims at improving our understanding of the mechanisms underlying muscle wasting in ICU patients by using a unique experimental rat ICU model where animals are mechanically ventilated, sedated and pharmacologically paralysed for duration varying between 6 h and 14 days. Results show that the ICU intervention induces a phenotype resembling the severe muscle wasting and paralysis associated with the acute quadriplegic myopathy (AQM) observed in ICU patients, i.e. a preferential loss of myosin, transcriptional down-regulation of myosin synthesis, muscle atrophy and a dramatic decrease in muscle fibre force generation capacity. Detailed analyses of protein degradation pathways show that the ubiquitin proteasome pathway is highly involved in this process. A sequential change in localisation of muscle-specific RING finger proteins 1/2 (MuRF1/2) observed during the experimental period is suggested to play an instrumental role in both transcriptional regulation and protein degradation. We propose that, for those critically ill patients who develop AQM, complete mechanical silencing, due to pharmacological paralysis or sedation, is a critical factor underlying the preferential loss of the molecular motor protein myosin that leads to impaired muscle function or persisting paralysis.

Baroreflexes of the rat. VI. Sleep and responses to aortic nerve stimulation in the dmNTS.

The sensitivity of the baroreflex determines its stability and effectiveness in controlling blood pressure (BP). Sleep and arousal are reported to affect baroreflex sensitivity, but the findings are not consistent across studies. After statistically correcting the effect of sleep on the baselines in chronically neuromuscular-blocked (NMB) rats, we found that sleep affects BP and heart period (HP) baroreflex gain similarly. This finding is consistent with baroreflex modulation of HP and BP before divergence of the sympathetic and parasympathetic pathways. Therefore, we hypothesized that the gain modulation occurs in the dorsal medial nucleus of the solitary tract (dmNTS). The present study used long-term dmNTS recordings in NMB rats and single-pulse aortic depressor nerve stimulation. Under these conditions, the magnitude of A-fiber evoked responses (ERs), recorded from second- or higher-order dmNTS baroreflex neurons, was reliably augmented during high-amplitude low-frequency EEG activity (slow-wave sleep) and reduced during low-amplitude high-frequency EEG activity (arousal; DeltaER = 11%, t = 9.49, P < 0.001, degrees of freedom = 1,016). This result has methodological implications for techniques that use changes in HP to estimate baroreflex BP gain and general implications for understanding the relationship between sleep and cardiovascular control.

The dmNTS is not the source of increased blood pressure variability in baroreflex denervated rats.

A consistent and prominent feature, observed across many species, including our neuromuscular blocked (NMB) rat preparation, is that obliterating the baroafferent inputs to the brainstem, e.g., by sinoaortic denervation (SAD), significantly increases blood pressure variability (BPV). The sources of the BPV, however, are not completely understood, but involve both the central and the peripheral mechanisms. The key central noise source is likely in the brainstem. Previously, in NMB rats, we showed that the maximum gain of the baroreflex system is in the very low frequency (VLF) range of 0.01-0.2 Hz. In this study, using the same NMB preparation, we demonstrated that, after SAD, there was a significant increase in the VLF power of the expiratory systolic blood pressure (EsBP) spectrum, but a decrease in the VLF power of the expiratory heart inter-beat-interval (EIBI) spectrum. Because dmNTS is the only major common anatomic node for the vascular sympathetic and the cardiac parasympathetic pathways, the opposite changes in the post-SAD VLF powers of the EsBP and EIBI spectra suggest that dmNTS is unlikely the major noise source for the post-SAD BPV. Supporting this finding, we found that the dmNTS evoked response to single pulse baroreflex afferent aortic depressor nerve (ADN) stimuli was substantially more reliable than the evoked systolic blood pressure responses to the same stimuli.

Baroreflexes of the rat. V. Tetanus-induced potentiation of ADN A-fiber responses at the NTS.

In a long-term neuromuscular blocked (NMB) rat preparation, tetanic stimulation of the aortic depressor nerve (ADN) enhanced the A-fiber evoked responses (ERs) in the cardiovascular region, the nucleus of the solitary tract (dmNTS). The potentiation persisted for at least several hours and may be a mechanism for adaptive adjustment of the gain of the baroreflex, with functional implications for blood pressure regulation. Using a capacitance electrode, we selectively stimulated A-fibers and acquired a stable 10-h "A-fiber only" ER baseline at the dmNTS. Following baseline, an A+C-fiber activating tetanus was applied to the ADN. The tetanus consisted of 1,000 "high current" pulses (10 trains; 300 mus, 100 Hz, 1 s), with intertrain interval of 9 s. A 10-h A-fiber only posttetanic test phase repeated the stimulus pattern of the baseline. Fourteen tetanus experiments were done in 12 rats. Compared with the baseline before tetanus, the A-fiber ER magnitudes of posttetanus hours were larger [F(13, 247) = 3.407, P < .001]; additionally, the 10-h posttetanus magnitude slopes were more positive than during 10 h before tetanus (df = 13; t = -3.47; P < 0.005); thus, an ADN A+C fiber-activating tetanus produced increases in the magnitude of the A-fiber ERs in the dmNTS that persisted for several hours. In an additional rat, application of an NMDA receptor antagonist, prior to the tetanus, blocked the potentiation effect. The stimulus protocols, magnitude and duration of the effect, and pharmacology resemble associative long-term potentiation (LTP).

Baroreflexes of the rat. IV. ADN-evoked responses at the NTS.

In a long-term (7-21 days) neuromuscular blocked (NMB) rat preparation, using precise single-pulse aortic depressor nerve (ADN) stimulation and stable chronic evoked response (ER) recordings from the dorsal-medial solitary nucleus (dmNTS), two different response patterns were observed: continuous and discrete. For the continuous pattern, activity began approximately 3 ms after the stimulus and persisted for 45 ms; for the discrete pattern, two complexes were separated by a gap from approximately 17 to 25 ms. The early complex was probably transmitted via A-fibers: it had a low stimulus current threshold and an average conduction velocity (CV) of 0.58-5.5 m/s; the high threshold late (HTL) complex had a CV = 0.26-0.58 m/s. The average stimulus amplitude-ER magnitude transduction curves for the A and HTL complexes were sigmoidal. For individual rats, in the linear range, mean r2 = 0.96 +/- 0.03 for both complexes. The average stimulus amplitude vs. the systolic blood pressure change (delta sBP) transduction curve was also approximately linear; however, for individual rats, the relationship was not consistently reliable: mean r2 = 0.48 +/- 0.19. Approximately 90% of recording sites had respiratory, and 50% had cardiac synchronism. The NMB preparation is useful for studying central baroreflex mechanisms that operate on time scales of days or weeks, such as adaptation and other kinds of neural plasticity.

Transcription factors in muscle atrophy caused by blocked neuromuscular transmission and muscle unloading in rats.

The muscle wasting associated with long-term intensive care unit (ICU) treatment has a negative effect on muscle function resulting in prolonged periods of rehabilitation and a decreased quality of life. To identify mechanisms behind this form of muscle wasting, we have used a rat model designed to mimic the conditions in an ICU. Rats were pharmacologically paralyzed with a postsynaptic blocker of neuromuscular transmission, and mechanically ventilated for one to two weeks, thereby unloading the limb muscles. Transcription factors were analyzed for cellular localization and nuclear concentration in the fast-twitch muscle extensor digitorum longus (EDL) and in the slow-twitch soleus. Significant muscle wasting and upregulation of mRNA for the ubiquitin ligases MAFbx and MuRF1 followed the treatment. The IkappaB family-member Bcl-3 displayed a concomitant decrease in concentration, suggesting altered kappaB controlled gene expression, although NFkappaB p65 was not significantly affected. The nuclear levels of the glucocorticoid receptor (GR) and the thyroid receptor alpha1 (TRalpha1) were altered and also suggested as potential mediators of the MAFbx- and MuRF1-induction in the absence of induced Foxo1. We believe that this model, and the strategy of quantifying nuclear proteins, will provide a valuable tool for further, more detailed, analyses of the muscle wasting occurring in patients kept on a mechanical ventilator.

Spinal interneurons infected by renal injection of pseudorabies virus in the rat.

The potency of spinal sympathetic reflexes is increased after spinal injury, and these reflexes may result in life-threatening hypertensive crises in humans. Few, if any, primary afferents project directly to sympathetic preganglionic neurons (SPN). Therefore, spinal sympathetic interneurons (IN) must play a major role in generating dysfunctional sympathetic activity after spinal cord injury. Furthermore, these IN are potentially aberrant targets, either for ascending and descending axons that may sprout after spinal cord injury or for axons that regenerate after spinal cord injury. We identified IN via the transsynaptic retrograde transport of pseudorabies virus (PRV) injected into the kidneys of rats. The proportion of infected IN ranged from approximately 1/3 to approximately 2/3 of the number of infected SPN. IN were heavily concentrated among the SPN in spinal lamina VII. However, IN were located in all lamina of the dorsal horn. The longitudinal distribution of infected IN was closely correlated with the longitudinal distribution of infected SPN. Few infected IN were found rostral or caudal to the longitudinal range of infected SPN. Infected IN were heterogeneous in both their sizes and the extent of their dendritic trees. The strong correlation between longitudinal distributions of infected IN and SPN supports physiological data demonstrating a segmental organization of spinal sympathetic reflexes. The paucity of infected IN in segments distant from SPN suggests that multisegmental sympathetic reflexes are mediated by projections onto IN rather than onto SPN themselves. The morphological heterogeneity of IN probably manifests the variety of systems that affect spinal sympathetic regulation.

Sympathetic activity and the underlying action potentials in sympathetic nerves: a simulation.

Understanding the relationship between activity recorded in sympathetic nerves and the action potentials of the axons that contribute to that activity is important for understanding the processing of sympathetic activity by the central nervous system. Because this relationship cannot be determined experimentally and is difficult to predict analytically, we simulated the summed action potentials of 300 axons. This simulation closely resembled actual sympathetic activity and permitted us to know how many action potentials contributed to each burst of simulated sympathetic activity and the durations and amplitudes of each burst. We used these simulated data to examine a statistical method (cluster analysis) that has been used to identify and quantify bursts of sympathetic activity. Simulation indicated that the integrals of bursts, whether determined directly from the simulation or by integrating bursts detected by cluster analysis, were linearly correlated to the number of action potentials contributing to bursts. The variances of samples of the simulated signal were also linearly correlated to the number of action potentials. The amplitudes of bursts of sympathetic activity were less well correlated to the number of underlying action potentials. A linear relationship existed between the average number of action potentials contributing to simulated bursts and the integral of the amplitude spectra obtained by Fourier transform of the simulated activity. Finally, simulated experiments indicated that relatively brief recordings might be sufficient to detect statistically significant changes in sympathetic activity.

Locations and morphologies of sympathetically correlated neurons in the T(10) spinal segment of the rat.

We precisely localized and morphologically characterized sympathetically correlated neurons in the acutely transected spinal cord of the rat. We have shown that these neurons are likely members of the spinal networks that generate sympathetic activity after spinal cord transection. In humans with injured spinal cords, these networks are responsible for hypertensive crises that occur in response to ordinarily innocuous stimuli. We recorded from neurons in the dorsal horn of the T(10) spinal segment of anesthetized rats after acute spinal cord transection at C(2). Neurons with activities closely correlated to renal sympathetic nerve activity (RSNA) were considered to be putative components of spinal sympathetic systems. These neurons had receptive fields on the left flank and abdomen. After characterizing their ongoing activities, receptive fields, and degrees of correlation with RSNA, we juxtacellularly labeled neurons with biotinamide and subsequently reconstructed their somas and dendrites histologically. Confirming our earlier studies, sympathetically correlated neurons were found in dorsal horn laminae III, IV, and V. For the first time, we also identified sympathetically correlated neurons in laminae I and II. The dendrites of all sympathetically correlated neurons projected to multiple lamina. By virtue of the positions of their somas and the broad projections of their dendrites, we concluded that sympathetically correlated neurons may receive direct input both from supraspinal systems and from nociceptive and non-nociceptive primary afferents.