Intracerebroventricular insulin injection acutely normalizes the augmented exercise pressor reflex in male rats with type 2 diabetes mellitus.

The exercise pressor reflex (EPR) is exaggerated in type 2 diabetes mellitus (T2DM), but the underlying central nervous system aberrations have not been fully delineated. Stimulation of muscle afferents within working skeletal muscle activates the EPR, by sending information to neurons in the brainstem, where it is integrated and results in reflexively increased mean arterial pressure (MAP) and sympathetic nerve activity. Brain insulin is known to regulate neural activity within the brainstem. We hypothesize that brain insulin injection in T2DM rats attenuates the augmented EPR, and that T2DM is associated with decreased brain insulin. Using male Sprague-Dawley rats, T2DM and control rats were generated via an induction protocol with two low doses of streptozotocin (35 and 25 mg/kg, i.p.) in combination with a 14-23-week high-fat diet or saline injections and a low-fat diet, respectively. After decerebration, MAP and renal sympathetic nerve activity (RSNA) were evaluated during EPR stimulation, evoked by electrically induced muscle contraction via ventral root stimulation, before and after (1 and 2 h post) intracerebroventricular (i.c.v.) insulin microinjections (500 mU, 50 nl). i.c.v. insulin decreased peak MAP (ΔMAP Pre (36 ± 14 mmHg) vs. 1 h (21 ± 14 mmHg) vs. 2 h (11 ± 6 mmHg), P < 0.05) and RSNA (ΔRSNA Pre (107.5 ± 40%), vs. 1 h (75.4 ± 46%) vs. 2 h (51 ± 35%), P < 0.05) responses in T2DM, but not controls. In T2DM rats, cerebrospinal fluid insulin was decreased (0.41 ± 0.19 vs. 0.11 ± 0.05 ng/ml, control (n = 14) vs. T2DM (n = 4), P < 0.01). The results demonstrated that insulin injections into the brain normalized the augmented EPR in brain hypoinsulinaemic T2DM rats, indicating that the EPR can be regulated by brain insulin. KEY POINTS: The reflexive increase in blood pressure and sympathetic nerve activity mediated by the autonomic nervous system during muscle contractions is also known as the exercise pressor reflex. The exercise pressor reflex is dangerously augmented in type 2 diabetes, in both rats and humans. In type 2 diabetic rats both cerebrospinal fluid insulin and phosphoinositide 3-kinase signalling within cardiovascular brainstem neurons decrease in parallel. Brain insulin injections decrease the magnitude of the reflexive pressor and sympathetic responses to hindlimb muscle contraction in type 2 diabetic rats. Partial correction of low insulin within the central nervous system in type 2 diabetes may treat aberrant exercise pressor reflex function.

Blockade of endogenous insulin receptor signaling in the nucleus tractus solitarius potentiates exercise pressor reflex function in healthy male rats.

Insulin not only regulates glucose and/or lipid metabolism but also modulates brain neural activity. The nucleus tractus solitarius (NTS) is a key central integration site for sensory input from working skeletal muscle and arterial baroreceptors during exercise. Stimulation of the skeletal muscle exercise pressor reflex (EPR), the responses of which are buffered by the arterial baroreflex, leads to compensatory increases in arterial pressure to supply blood to working muscle. Evidence suggests that insulin signaling decreases neuronal excitability in the brain, thus antagonizing insulin receptors (IRs) may increase neuronal excitability. However, the impact of brain insulin signaling on the EPR remains fully undetermined. We hypothesized that antagonism of NTS IRs increases EPR function in normal healthy rodents. In decerebrate rats, stimulation of the EPR via electrically induced muscle contractions increased peak mean arterial pressure (MAP) responses 30 min following NTS microinjections of an IR antagonist (GSK1838705, 100 µM; Pre: Δ16 ± 10 mmHg vs. 30 min: Δ23 ± 13 mmHg, n = 11, p = .004), a finding absent in sino-aortic baroreceptor denervated rats. Intrathecal injections of GSK1838705 did not influence peak MAP responses to mechano- or chemoreflex stimulation of the hindlimb muscle. Immunofluorescence triple overlap analysis following repetitive EPR stimulation increased c-Fos overlap with EPR-sensitive nuclei and IR-positive cells relative to sham operation (p < .001). The results suggest that IR blockade in the NTS potentiates the MAP response to EPR stimulation. In addition, insulin signaling in the NTS may buffer EPR stimulated increases in blood pressure via baroreflex-mediated mechanisms during exercise.

Antagonism of TRPV4 channels partially reduces mechanotransduction in rat skeletal muscle afferents.

Mechanical distortion of working skeletal muscle induces sympathoexcitation via thin fibre afferents, a reflex response known as the skeletal muscle mechanoreflex. However, to date, the receptor ion channels responsible for mechanotransduction in skeletal muscle remain largely undetermined. Transient receptor potential vanilloid 4 (TRPV4) is known to sense mechanical stimuli such as shear stress or osmotic pressure in various organs. It is hypothesized that TRPV4 in thin-fibre primary afferents innervating skeletal muscle is involved in mechanotransduction. Fluorescence immunostaining revealed that 20.1 ± 10.1% of TRPV4 positive neurons were small dorsal root ganglion (DRG) neurons that were DiI-labelled, and among them 9.5 ± 6.1% of TRPV4 co-localized with the C-fibre marker peripherin. In vitro whole-cell patch clamp recordings from cultured rat DRG neurons demonstrated that mechanically activated current amplitude was significantly attenuated after the application of the TRPV4 antagonist HC067047 compared to control (P = 0.004). Such reductions were also observed in single-fibre recordings from a muscle-nerve ex vivo preparation where HC067047 significantly decreased afferent discharge to mechanical stimulation (P = 0.007). Likewise, in an in vivo decerebrate rat preparation, the renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) responses to passive stretch of hindlimb muscle were significantly reduced by intra-arterial injection of HC067047 (ΔRSNA: P = 0.019, ΔMAP: P = 0.002). The findings suggest that TRPV4 plays an important role in mechanotransduction contributing to the cardiovascular responses evoked by the skeletal muscle mechanoreflex during exercise. KEY POINTS: Although a mechanical stimulus to skeletal muscle reflexively activates the sympathetic nervous system, the receptors responsible for mechanotransduction in skeletal muscle thin fibre afferents have not been fully identified. Evidence suggests that TRPV4 is a mechanosensitive channel that plays an important role in mechanotransduction within various organs. Immunocytochemical staining demonstrates that TRPV4 is expressed in group IV skeletal muscle afferents. In addition, we show that the TRPV4 antagonist HC067047 decreases the responsiveness of thin fibre afferents to mechanical stimulation at the muscle tissue level as well as at the level of dorsal root ganglion neurons. Moreover, we demonstrate that intra-arterial HC067047 injection attenuates the sympathetic and pressor responses to passive muscle stretch in decerebrate rats. These data suggest that antagonism of TRPV4 attenuates mechanotransduction in skeletal muscle afferents. The present study demonstrates a probable physiological role for TRPV4 in the regulation of mechanical sensation in somatosensory thin fibre muscle afferents.

Sex-dependent attenuating effects of capsaicin administration on the mechanoreflex in healthy rats.

Stimulation of mechanically sensitive channels on the sensory endings of group III and IV thin fiber muscle afferents activates the mechanoreflex, which contributes to reflex increases in sympathetic nerve activity (SNA) and blood pressure during exercise. Accumulating evidence suggests that activation of the nonselective cation channel transient receptor potential vanilloid-1 (TRPV1) on the sensory endings of thin fiber afferents with capsaicin may attenuate mechanosensation. However, no study has investigated the effect of capsaicin on the mechanoreflex. We tested the hypothesis that in male and female decerebrate, unanesthetized rats, the injection of capsaicin (0.05 µg) into the arterial supply of the hindlimb reduces the pressor and renal SNA (RSNA) response to 30 s of 1 Hz rhythmic hindlimb muscle stretch (a model of isolated mechanoreflex activation). In male rats (n = 8), capsaicin injection significantly reduced the integrated blood pressure (blood pressure index or BPI: pre, 363 ± 78; post, 211 ± 88 mmHg·s; P = 0.023) and RSNA [∫ΔRSNA; pre, 687 ± 206; post, 216 ± 80 arbitrary units (au), P = 0.049] response to hindlimb muscle stretch. In female rats (n = 8), capsaicin injection had no significant effect on the pressor (BPI; pre: 277 ± 67; post: 207 ± 77 mmHg·s; P = 0.343) or RSNA (∫ΔRSNA: pre, 697 ± 123; post, 440 ± 183 au; P = 0.307) response to hindlimb muscle stretch. The data suggest that the injection of capsaicin into the hindlimb arterial supply to stimulate TRPV1 on the sensory endings of thin fiber muscle afferents attenuates the mechanoreflex in healthy male, but not female, rats. The findings may carry important implications for chronic conditions in which an exaggerated mechanoreflex contributes to aberrant sympathoexcitation during exercise.NEW & NOTEWORTHY Recent evidence in isolated sensory neurons indicates that capsaicin-induced stimulation of TRPV1 attenuates mechanosensitivity. Here we demonstrate for the first time that capsaicin exposure/administration reduces the reflex pressor and renal sympathetic nerve response to mechanoreflex activation in male rats, but not female rats, in vivo. Our data may carry important clinical implications for chronic diseases which have been linked to an exaggerated mechanoreflex, at least in males.

Neural discharge of muscle afferents and pressor response to mechanical stimulation are associated with muscle deformation velocity in rats.

Skeletal muscle reflexes play a crucial role in determining the magnitude of the cardiovascular response to exercise. However, evidence supporting an association between the magnitude of the pressor response and the velocity of muscle deformation has remained to be elucidated. Thus, we investigated the impact of different muscle deformation rates on the neural discharge of muscle afferents and pressor and sympathetic responses in Sprague-Dawley rats. In an ex vivo muscle-nerve preparation, action potentials elicited by sinusoidal mechanical stimuli (137 mN) at different frequencies (0.01, 0.05, 0.1, 0.2, and 0.25 Hz) were recorded in mechanosensitive group III and IV fibers. The afferent response magnitude to sine-wave stimulation significantly varied at different frequencies (ANOVA, P = 0.01). Specifically, as compared with 0.01 Hz (0.83 ± 0.96 spikes/s), the response magnitudes were significantly greater at 0.20 Hz (4.07 ± 5.04 spikes/s, P = 0.031) and 0.25 Hz (4.91 ± 5.30 spikes/s, P = 0.014). In an in vivo decerebrated rat preparation, renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) responses to passive stretch (1 kg) of hindlimb skeletal muscle at different velocities of loading (slow, medium, and fast) were measured. Pressor responses to passive stretch were significantly associated with the velocity of muscle deformation (ANOVA, P < 0.001). The MAP response to fast stretch (Δ 56 ± 12 mmHg) was greater than slow (Δ 33 ± 11 mmHg, P = 0.006) or medium (Δ 30 ± 11 mmHg, P < 0.001) stretch. Likewise, the RSNA response was related to deformation velocity (ANOVA, P = 0.024). These findings suggest that the muscle neural afferent discharge and the cardiovascular response to mechanical stimulation are associated with muscle deformation velocity.

Exaggerated renal sympathetic nerve and pressor responses during spontaneously occurring motor activity in hypertensive rats.

Stimulation of the mesencephalic locomotor region elicits exaggerated sympathetic nerve and pressor responses in spontaneously hypertensive rats (SHR) as compared with normotensive Wistar-Kyoto rats (WKY). This suggests that central command or its influence on vasomotor centers is augmented in hypertension. The decerebrate animal model possesses an ability to evoke intermittent bouts of spontaneously occurring motor activity (SpMA) and generates cardiovascular responses associated with the SpMA. It remains unknown whether the changes in sympathetic nerve activity and hemodynamics during SpMA are altered by hypertension. To test the hypothesis that the responses in renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) during SpMA are exaggerated with hypertension, this study aimed to compare the responses in decerebrate, paralyzed SHR, WKY, and normotensive Sprague-Dawley (SD) rats. In all strains, an abrupt increase in RSNA occurred in synchronization with tibial motor discharge (an index of motor activity) and was followed by rises in MAP and heart rate. The centrally evoked increase in RSNA and MAP during SpMA was much greater (306 ± 110%) in SHR than WKY (187 ± 146%) and SD (165 ± 44%). Although resting baroreflex-mediated changes in RSNA were not different across strains, mechanically or pharmacologically induced elevations in MAP attenuated or abolished the RSNA increase during SpMA in WKY and SD but had no effect in SHR. It is likely that the exaggerated sympathetic nerve and pressor responses during SpMA in SHR are induced along a central command pathway independent of the arterial baroreflex and/or result from central command-induced inhibition of the baroreflex.

An alternative method to evaluate lumbar interbody fusion status focusing on position change of screw-rod constructs.

OBJECTIVES: The evaluation of lumbar interbody fusion status is generally subjective and may differ among raters. The authors examined whether the assessment of position change of screw-rod constructs could be an alternative method for the evaluation of fusion status. METHODS: Sixty-three patients undergoing lumbar interbody single-level fusion were retrospectively reviewed. Three-dimensional images of screw-rod constructs were created from baseline CT examination on the day after surgery and follow-up CT examinations (3-5 months, 6-11 months, and ≥ 12 months) and superposed, with position change of screw-rod constructs being evaluated by the distance between the 3-dimensional images at baseline and follow-up. The evaluation was repeated twice to confirm the reproducibility. Fusion status on follow-up CT examinations was assessed by three raters, where inter-rater reliability was evaluated with Fleiss' kappa. The results of the fusion status were classified into fusion and incomplete fusion groups in each timing of follow-up CT examinations, where the amount of position change was compared between the two groups. RESULTS: The evaluation of position change was completely reproducible. The Fleiss' kappa (agreements) was 0.481 (69.4%). The medians of the amount of position change in fusion and incomplete fusion groups were 0.134 mm and 0.158 mm at 3-5 months (p = 0.21), 0.160 mm and 0.190 mm at 6-11 months (p = 0.02), and 0.156 mm and 0.314 mm at ≥ 12 months (p = 0.004). CONCLUSIONS: The assessment of position change of screw-rod constructs at 6 months or more after surgery can be an alternative method for evaluating lumbar interbody fusion status. KEY POINTS: • Lumbar interbody fusion status (satisfactory, incomplete, or failed) is associated with the quantification of position change of screw-rod in this study. • Reference values for the evaluation of position change in identifying interbody fusion status are provided. • Position change of screw-rod could be a supportive method for evaluating interbody fusion status.

Insulin potentiates the response to capsaicin in dorsal root ganglion neurons in vitro and muscle afferents ex vivo in normal healthy rodents.

Systemic insulin administration evokes sympathoexcitatory actions, but the mechanisms underlying these observations are unknown. We reported that insulin sensitizes the response of thin-fibre primary afferents, as well as the dorsal root ganglion (DRG) that subserves them, to mechanical stimuli. However, little is known about the effects of insulin on primary neuronal responses to chemical stimuli. TRPV1, whose agonist is capsaicin (CAP), is widely expressed on chemically sensitive metaboreceptors and/or nociceptors. The aim of this investigation was to determine the effects of insulin on CAP-activated currents in small DRG neurons and CAP-induced action potentials in thin-fibre muscle afferents of normal healthy rodents. Additionally, we investigated whether insulin potentiates sympathetic nerve activity (SNA) responses to CAP. In whole-cell patch-clamp recordings from cultured mice DRG neurons in vitro, the fold change in CAP-activated current from pre- to post-application of insulin (n = 13) was significantly (P < 0.05) higher than with a vehicle control (n = 14). Similar results were observed in single-fibre recording experiments ex vivo as insulin potentiated CAP-induced action potentials compared to vehicle controls (n = 9 per group, P < 0.05). Furthermore, insulin receptor blockade with GSK1838705 significantly suppressed the insulin-induced augmentation in CAP-activated currents (n = 13) as well as the response magnitude of CAP-induced action potentials (n = 9). Likewise, the renal SNA response to CAP after intramuscular injection of insulin (n = 8) was significantly (P < 0.05) greater compared to vehicle (n = 9). The findings suggest that insulin potentiates TRPV1 responsiveness to CAP at the DRG and muscle tissue levels, possibly contributing to the augmentation in sympathoexcitation during activities such as physical exercise. KEY POINTS: Evidence suggests insulin centrally activates the sympathetic nervous system, and a chemical stimulus to tissues activates the sympathetic nervous system via thin fibre muscle afferents. Insulin is reported to modulate putative chemical-sensitive channels in the dorsal root ganglion neurons of these afferents. In the present study, it is demonstrated that insulin potentiates the responsiveness of thin fibre afferents to capsaicin at muscle tissue levels as well as at the level of dorsal root ganglion neurons. In addition, it is demonstrated that insulin augments the sympathetic nerve activity response to capsaicin in vivo. These data suggest that sympathoexcitation is peripherally mediated via insulin-induced chemical sensitization. The present study proposes a possible physiological role of insulin in the regulation of chemical sensitivity in somatosensory thin fibre muscle afferents.

The Impact of Insulin Resistance on Cardiovascular Control During Exercise in Diabetes.

Patients with diabetes display heightened blood pressure response to exercise, but the underlying mechanism remains to be elucidated. There is no direct evidence that insulin resistance (hyperinsulinemia or hyperglycemia) impacts neural cardiovascular control during exercise. We propose a novel paradigm in which hyperinsulinemia or hyperglycemia significantly influences neural regulatory pathways controlling the circulation during exercise in diabetes.

A screw position change at an early postoperative stage preceding the subsequent occurrence of screw loosening.

PURPOSE: The authors recently proposed the novel radiologic assessment method to measure chronological screw position changes precisely. The aim of this study was to predict the late occurrence of screw loosening, which was diagnosed by the radiographic lucent zone, by evaluating screw position changes at an early postoperative stage using the novel method. METHODS: Forty-three patients who underwent thoracolumbar screw fixation and follow-up computed tomography (CT) scans on the day, between 1 and 5 weeks, and at more than 6 months after surgery were retrospectively evaluated. Screw images were generated from CT data. Screw position changes were evaluated by superposing screw images on the day and between 1 and 5 weeks after surgery. Screw loosening was diagnosed by the radiographic lucent zone on CT images at 6 months or later post-surgery, and patients were classified into screw loosening and non-loosening groups. The early screw position changes were compared between the two groups. RESULTS: Significant differences in early screw position changes were found between the screw loosening and non-loosening groups in Mann-Whitney U test (p = 0.001). On the receiver operating characteristic (ROC) curve analysis, the area under the ROC curve was 0.791, and the best cutoff value of early screw position change for the prediction of screw loosening was 0.83 mm with a sensitivity of 64.0% and a specificity of 88.9%. CONCLUSION: We calculated a cutoff value of the screw position changes at an early postoperative stage for the prediction of subsequent development of screw loosening with the radiographic lucent zone.