Medullary raphe nuclei activate the lumbosacral defecation center through the descending serotonergic pathway to regulate colorectal motility in rats.

Colorectal motility is regulated by two defecation centers located in the brain and spinal cord. In previous studies, we have shown that administration of serotonin (5-HT) in the lumbosacral spinal cord causes enhancement of colorectal motility. Because spinal 5-HT is derived from neurons of the medullary raphe nuclei, including the raphe magnus, raphe obscurus, and raphe pallidus, we examined whether stimulation of the medullary raphe nuclei enhances colorectal motility via the lumbosacral defecation center. Colorectal pressure was recorded with a balloon in vivo in anesthetized rats. Electrical stimulation of the medullary raphe nuclei failed to enhance colorectal motility. Because GABAergic neurons can be simultaneously activated by the raphe stimulation and released GABA masks accelerating actions of the raphe nuclei on the lumbosacral defecation center, a GABAA receptor antagonist was preinjected intrathecally to manifest excitatory responses. When spinal GABAA receptors were blocked by the antagonist, electrical stimulation of the medullary raphe nuclei increased colorectal contractions. This effect of the raphe nuclei was inhibited by intrathecal injection of 5-hydroxytryptamine type 2 (5-HT2) and type 3 (5-HT3) receptor antagonists. In addition, injection of a selective 5-HT reuptake inhibitor in the lumbosacral spinal cord augmented the raphe stimulation-induced enhancement of colorectal motility. Transection of the pelvic nerves, but not transection of the colonic nerves, prevented the effect of the raphe nuclei on colorectal motility. These results demonstrate that activation of the medullary raphe nuclei causes augmented contractions of the colorectum via 5-HT2 and 5-HT3 receptors in the lumbosacral defecation center. NEW & NOTEWORTHY We have shown that electrical stimulation of the medullary raphe nuclei causes augmented contractions of the colorectum via pelvic nerves in rats. The effect of the medullary raphe nuclei on colorectal motility is exerted through activation of 5-hydroxytryptamine type 2 and type 3 receptors in the lumbosacral defecation center. The descending serotoninergic raphespinal tract represents new potential therapeutic targets against colorectal dysmotility such as irritable bowel syndrome.

A radiologic and anatomic assessment of injectate spread following transmuscular quadratus lumborum block in cadavers.

We performed bilateral transmuscular quadratus lumborum blocks in six cadavers using iodinated contrast and methylene blue. Computed tomography imaging was performed in four cadavers and anatomical dissection was completed in five. This demonstrated spread to the lumbar paravertebral space in 63% of specimens, laterally to the transversus abdominis muscle in 50% and caudally to the anterior superior iliac spine in 63% of specimens. There was no radiographic evidence of spread to the thoracic paravertebral space. Anatomical dissection revealed dye staining of the upper branches of the lumbar plexus and the psoas major muscle in 70% of specimens. Further clinical studies are required to confirm if the quadratus lumborum block might be a suitable alternative to lumbar plexus block.

An evaluation of contrast medium spread on caudal epidurography with the needle positioned toward the affected side in patients with unilateral lumbosacral radiculopathy.

OBJECTIVE: We used caudal epidurography to compare the spread of contrast medium when the needle was inserted toward the affected side in patients with unilateral radiculopathy undergoing caudal steroid injection. MATERIALS AND METHODS: We enrolled 24 patients with unilateral radiculopathy. A block needle was positioned toward the affected side in the sacral epidural space. After 5 mL of iodinated contrast medium was injected, a standardized anteroposterior view was imaged. Using Adobe Photoshop software, contrast medium spread was assessed by counting pixels within the areas spread on the affected side and on the opposite side, and the pixel counts of the two sides were compared. Spinal nerve root filling was also assessed. RESULTS: The pixel count within the area of contrast medium spread on the side with the needle was significantly greater than that of the opposite side (mean [SD] 41,368.6 [13,143.1] vs 15,165.3 (10,698.1), P < 0.001]. However, 13.6% of the study patients had greater spread on the opposite side. The rates of L5 and S1 nerve root filling in the affected side were 18.2% and 36.4% respectively. CONCLUSIONS: When a needle was intentionally inserted toward the side with radiculopathy, the spread of contrast medium and number of delineated roots tended to be greater on the side with the needle, compared with those on the opposite side. However, the pattern of contrast medium spread in the sacral epidural space varied and some patients even had greater spread on the opposite side.

Sacral spinal cord neurons responsive to bladder pelvic and perineal inputs in cats.

In chloralose-anesthetized or decerebrate male cats, 70% of 73 sacral spinal cord neurons activated from the bladder branch of the pelvic nerve also received excitatory inputs from urethra and/or perineal cutaneous nerves (sensory pudendal in 55% and superficial perineal in 84% of neurons). Only 29% of these neurons were excited by the hindlimb skin and muscle nerves tested. The pelvic nerve-responsive neurons received monosynaptic urethral/perineal input in 25% of cases and required temporal summation of this input in 47% of cases. Of 211 neurons responding to superficial perineal nerve stimulation, 101 were not excited by the other nerves tested. Neurons activated by superficial perineal nerve stimulation were found predominantly in S2. It is likely that the superficial perineal nerve represents an important pathway whereby perineal stimulation influences bladder function.

An immunohistochemical study of nerve structures in the anulus fibrosus of human normal lumbar intervertebral discs.

STUDY DESIGN: The innervation of the anulus fibrosus of human macroscopically normal intervertebral discs from five patients was investigated immunohistochemically. OBJECTIVES: Immunoreactivity to general nerve markers (synaptophysin and protein gene product 9.5) and to neuropeptides (substance P and C-flanking peptide of neuropeptide Y) was studied. SUMMARY OF BACKGROUND DATA: In the lumbar disc of a newborn, free nerve endings have been demonstrated in the outer layers of anulus fibrosus. In degenerated and herniated discs, nerve structures have been shown to penetrate deeper into the anulus fibrosus. There are only a few studies on the innervation of normal adult intervertebral disc tissue. METHODS: Thin frozen sections of human normal lumbar intervertebral disc tissue were immunostained for general nerve markers and neuropeptides. RESULTS: Synaptophysin and protein gene product 9.5 immunoreactive nerve structures were observed penetrating 3.5 mm and 1.1 mm into the anulus, respectively. Immunoreactivity to C-flanking peptide of neuropeptide Y and substance P were observed at a maximum depth of 0.9 and 0.5 mm in the anulus, respectively. Antibodies to the former have been used to study sympathetic nerves, whereas substance P is a transmitter present in sensory nerves. CONCLUSIONS: In anulus fibrosus samples from macroscopically normal discs, a general marker for nerve endings can be found at a depth of a few millimeters, whereas neuropeptide markers show nerves only in the outermost layers of the anulus fibrosus. This absence of demonstrable nerves in deeper anulus fibrosus in normal discs is probably not a methodologic artifact, because blood vessels have also been demonstrated only at the disc surface. It is, however, possible that neuropeptide nerves also penetrate to a depth of a few millimeters, but that methodologic limitations permit the visualization of only the neuropeptide nerves closest to the disc surface. The results of the present study lend support to previous suggestions that, except at the surface, a normal intervertebral disc is almost without innervation.

Latent turkey herpesvirus infection in lymphoid, nervous, and feather tissues of chickens.

In earlier studies, we found that a late gene product, glycoprotein B (gB) was highly expressed in lymphoid tissues of chickens inoculated with turkey herpesvirus (HVT). The objectives of the present study were twofold. First, we wanted to expand on our previous research and determine if gB expression declines or disappears during later time periods of HVT infection. Second, we wanted to correlate gB expression with presence of HVT, i.e. if gB expression is absent, can HVT still be detected? Fifteen 1-day-old chicks were inoculated by intraperitoneal inoculation with 2000 plaque forming units of strain FC126 HVT. Thymus, spleen, bursa, brachial plexus, sciatic plexus, and feather tips were harvested at 21, 28, 35, 70, and 105 days postinoculation (PI). Brachial plexus and sciatic plexus were examined at 21, 28, and 35 days PI, and feather tips were examined at 21 and 28 days PI. An indirect immunofluorescence assay was used to detect HVT gB expression, and an in situ hybridization assay was used to detect HVT. At 21 days PI, gB expression was present in the thymus, spleen, and bursa. At 28 and 35 days PI, gB expression was detected in the thymus and spleen. At 70 days PI, gB expression was detected only in the spleen, and at 105 days PI, gB expression was not detected in any of the lymphoid tissue (thymus, spleen, or bursa). gB expression was not detected in the brachial plexus, sciatic plexus, or feather tips at any of the five time points. The bursa contained HVT only at 21 and 28 days PI. However, HVT was demonstrated in all other tissues from 21 to 105 days PI. Progression from a productive HVT infection to a latent HVT infection results in the loss of gB expression. Throughout this progression, a region of the HVT genome can be detected by appropriate methods.

Does calcitonin gene-related peptide act as a chemoattractant for rat gubernacular cells?

Calcitonin gene-related peptide (CGRP) receptors are located in the cremaster muscle of the gubernaculum of rats, and causes gubernacular contraction in vitro, suggesting that CGRP plays an important role in testicular descent. It has been postulated that CGRP released from the genitofemoral nerve acts as a "chemoattractant" for gubernacular migration to the scrotum. The aim of this study is to investigate whether the gubernacular cells of rats exhibit a chemotactic response to CGRP in vitro. Chemotaxis of gubernacular cells from Sprague-Dawley rats (1 to 6 days old) was measured using blind-well chambers. The migration of cells, which passed from the upper to the lower compartment through a polycarbonate filter, were counted using microscopy. The lower compartment included 10(-11), 10(-10), 10(-9), and 10(-8) M CGRP or phosphate buffered saline (PBS) as control. The chemotactic index (CI) was defined as the ratio of migration toward CGRP versus PBS as control. There was no significant migration even at varying CGRP concentrations. Isolated rat gubernacular cells therefore did not exhibit a chemotactic response to CGRP and the role CGRP plays in testicular descent still remains unclear. This result does not exclude the possibility that the gubernaculum responds to CGRP as a whole organ rather than as individual cells.

Population pharmacokinetics of bupivacaine in combined lumbar and sciatic nerve block.

OBJECTIVES: The primary aim of this study was to establish the population pharmacokinetic (PPK) model of bupivacaine after combined lumbar plexus and sciatic nerve blocks and secondary aim is to assess the effect of patient's characteristics including age, body weight and sex on pharmacokinetic parameters. MATERIALS AND METHODS: A total of 31 patients scheduled for elective lower extremity surgery with combined lumbar and sciatic nerve block using plain bupivacaine 0.5% were included. The total bupivacaine plasma concentrations were measured before injection and after two blocks placement and at selected time points. Monitoring of bupivacaine was made by high performance liquid chromatography (HPLC) with ultraviolet detection. Non-linear mixed effects modeling was used to analyze the PPK of bupivacaine. RESULTS: One compartment model with first order absorption, two input compartments and a central elimination was selected. The Shapiro-Wilks test of normality for normalized prediction distribution errors for this model (P = 0.156) showed this as a valid model. The selected model predicts a population clearance of 930 ml/min (residual standard error [RSE] = 15.48%, IC 95% = 930 ± 282.24) with inter individual variability of 75.29%. The central volume of distribution was 134 l (RSE = 12.76%, IC = 134 ± 33.51 L) with inter individual variability of 63.40%. The absorption of bupivacaine in two sites Ka1 and Ka2 were 0.00462/min for the lumbar site and 0.292/min for the sciatic site. Age, body weight and sex have no effect on the bupivacaine pharmacokinetics in this studied population. CONCLUSION: The developed model helps us to assess the systemic absorption of bupivacaine at two injections sites.

The effects of lidocaine used in sciatic nerve on the pharmacodynamics and pharmacokinetics of ropivacaine in sciatic nerve combined with lumbar plexus blockade: a double-blind, randomized study.

In this controlled, randomized, double-blind study, we compared the pharmacodynamics and pharmacokinetics of ropivacaine and staged injection of lidocaine and ropivacaine in a combined lumbar plexus-sciatic nerve block. The experiment was performed in two parts: pharmacodynamics study (Group r, n = 20; Group lr, n = 20) and pharmacokinetics study (Group R, n = 10; Group LR, n = 10). The sciatic nerve blockade was performed using either (1) 10 mL of 2% lidocaine and then 10 mL of 0.75% ropivacaine (Group lr and Group LR) or (2) 10 mL of normal saline (N.S.) and then 10 mL of 0.75% ropivacaine (Group r and Group R). Two kinds of solutions were 'staged' injection. The sensory onset time and sensory recovery time were assessed in the pharmacodynamics study. Arterial blood samples were collected for the pharmacokinetics study. Sciatic sensory block onset times were reduced, and the sensory recovery times were decreased in Group lr. C(max) of ropivacaine in Group LR was significantly higher than that in Group R. A significant increase in AUC((0-t)) and AUC((0-∞)) was observed in Group LR compared with Group R. When 2% lidocaine and 0.75% ropivacaine are used for a combined sciatic nerve-lumbar plexus block by 'staged' injection, lidocaine induced faster onset times, decreased the block duration and increased the AUC and C(max) of ropivacaine.

Mechanisms by which the serotonergic system inhibits micturition in rats.

AIMS: Serotonergic neurons and amino acid neurons are involved in the central nervous control of lower urinary tract function. We investigated the role of the serotonergic system in the central regulation of micturition, as well as the relationship between serotonergic neurons and amino acid neurons in the lumbosacral cord of rats. MAIN METHODS: Under urethane anesthesia, bladder and urethral activity were recorded before and after intrathecal injection of serotonin (5-hydroxytryptamine: 5-HT), a 5-HT(2A) receptor antagonist (ketanserin: KET), or KET+5-HT by isovolumetric cystometry and measurement of the urethral pressure in intact rats and rats with hypogastric nerve transection (HGNT). Amino acid levels in the lumbosacral cord were also measured after intrathecal injection of 5-HT in intact rats. KEY FINDINGS: In intact rats, intrathecal injection of 5-HT transiently abolished rhythmic bladder contractions, decreased the maximal bladder contraction pressure, and increased the intravesical baseline pressure and the urethral baseline pressure. Intrathecal injection of KET+5-HT also transiently abolished rhythmic bladder contractions. In HGNT rats, intrathecal injection of 5-HT transiently abolished rhythmic bladder contractions and increased the urethral baseline pressure. Intrathecal injection of 5-HT decreased the level of glycine in the lumbosacral cord. SIGNIFICANCE: The serotonergic system may be involved in blocking the afferent pathway of the micturition reflex, increasing sympathetic activity, and secondary promotion of urethral contraction through inhibition of glycinergic neurons in the lumbosacral cord. 5-HT(2A) receptors may be involved in these effects on the bladder and urethra. Therefore, the serotonergic system may play a role of the maintenance of urine storage.

Inflammatory mediators in diabetic and non-diabetic lumbosacral radiculoplexus neuropathy.

Nerve microvasculitis and ischemic injury appear to be the primary and important pathogenic alterations in lumbosacral radiculoplexus neuropathy of patients with (DLRPN) and without (LRPN) diabetes mellitus (DM). Here, we examine the involvement of inflammatory mediators in DLRPN and LRPN. Paraffin sections of sural nerves from 19 patients with DLRPN, 13 patients with LRPN, and 20 disease control patients were immunostained for intercellular adhesion molecule-1 (ICAM-1), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and nuclear factor kappaB (NF-kappaB). The findings were correlated with histopathology. The pathologic and immunohistochemical alterations of DLRPN and LRPN nerves were indistinguishable. The nerves of both types of LRPN had a significantly greater number of ICAM-1 positive vessels than did the controls (P < 0.01). TNF-alpha expression was seen in Schwann cells and some macrophages of DLRPN and LRPN nerves, whereas IL-6 expression was minimal. There was greater NF-kappaB immunoreactivity in vessels and endoneurial cells of DLRPN and LRPN nerves than of the controls (P < 0.001). NF-kappaB expression correlated with the number of empty nerve strands (P < 0.01) and the frequency of axonal degeneration (P < 0.05), whereas TNF-alpha expression correlated inversely with the number of empty nerve strands of teased fibers (P < 0.05). Our findings suggest that up-regulation of inflammatory mediators target different cells at different disease stages and that these mediators may be sequentially involved in an immune-mediated inflammatory process that is shared by both DLRPN and LRPN. Up-regulated inflammatory mediators may be immunotherapeutic targets in these two conditions.

Interstitial norepinephrine concentrations in skeletal muscle of ischemic heart failure.

During exercise, sympathetic nerve responses are accentuated in heart failure (HF), and this enhances norepinephrine (NE) release and evokes vasoconstriction. Two key pathophysiological responses could contribute to the greater NE release: 1) increased sympathetic nerve discharge and 2) increased NE in the neurovascular junction for a given level of sympathetic discharge. In this report, we focus on the second of these two general issues and test the following hypotheses: 1) in HF for a given level of sympathetic nerve stimulation, NE concentration in the interstitium (an index of neurovascular NE) would be greater, and 2) the greater interstitial NE concentration would be linked to reduced NE uptake. Studies were performed in rats 8-10 wk after induction of myocardial infarction (MI). Interstitial NE samples were collected from microdialysis probes inserted into the hindlimb muscle. Dialysate concentration of NE was determined by the HPLC method. First, interstitial NE concentration increased during electrical stimulation of the lumbar sympathetic nerves in eight control rats. An increase in interstitial NE concentration was significantly greater in 10 rats with severe MI. Additionally, an NE uptake-1 inhibitor (desipramine, 1 microM) was injected into the arterial blood supply of the muscle in six control and eight MI rats. Desipramine increased interstitial NE concentration by 24% in control and by only 3% (P < 0.05 vs. control) in MI rats. In conclusion, given levels of electrical stimulation of the lumbar sympathetic nerve lead to higher interstitial NE concentration in HF. This effect is due, in part, to reduced NE uptake-1 in HF.

Mouse colon sensory neurons detect extracellular acidosis via TRPV1.

Extracellular acidification contributes to pain by activating or modulating nociceptor activity. To evaluate acidic signaling from the colon, we characterized acid-elicited currents in thoracolumbar (TL) and lumbosacral (LS) dorsal root ganglion (DRG) neurons identified by content of a fluorescent dye (DiI) previously injected into the colon wall. In 13% of unidentified LS DRG neurons (not labeled with DiI) and 69% of LS colon neurons labeled with DiI, protons activated a sustained current that was significantly and reversibly attenuated by the transient receptor potential vanilloid receptor 1 (TRPV1) antagonist capsazepine. In contrast, 63% of unidentified LS DRG neurons and 4% of LS colon neurons exhibited transient amiloride-sensitive acid-sensing ion channel (ASIC) currents. The peak current density of acid-elicited currents was significantly reduced in colon sensory neurons from TRPV1-null mice, supporting predominant expression of TRPV1 in LS colon sensory neurons, which was also confirmed immunohistochemically. Similar to LS colon DRG neurons, acid-elicited currents in TL colon DRG neurons were mediated predominantly by TRPV1. However, the pH producing half-activation of responses significantly differed between TL and LS colon DRG neurons. The properties of acid-elicited currents in colon DRG neurons suggest differential contributions of ASICs and TRPV1 to colon sensation and likely nociception.

Spatiotemporal alterations of the NO/NOS neuronal pools following transient abdominal aorta occlusion: morphological and biochemical studies in the rabbit.

1. Brief interruption of spinal cord blood flow resulting from transient abdominal aortic occlusion may lead to degeneration of specific spinal cord neurons and to irreversible loss of neurological function. The alteration of nitric oxide/nitric oxide synthase (NO/NOS) pool occurring after ischemic insult may play a protective or destructive role in neuronal survival of affected spinal cord segments. 2. In the present study, the spatiotemporal changes of NOS following transient ischemia were evaluated by investigating neuronal NOS immunoreactivity (nNOS-IR), reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry, and calcium-dependent NOS (cNOS) conversion of [(3)H] l-arginine to [(3)H] l-citrulline. 3. The greatest levels of these enzymes and activities were detected in the dorsal horn, which appeared to be most resistant to ischemia. In that area, the first significant increase in NADPHd staining and cNOS catalytic activity was found immediately after a 15-min ischemic insult. 4. Increases in the ventral horn were observed later (i.e., after a 24-h reperfusion period). While the most intense increase in nNOS-IR was detected in surviving motoneurons of animals with a shorter ischemic insult (13 min), the greatest increase of cNOS catalytic activity and NADPHd staining of the endothelial cells was found after stronger insult (15 min). 5. Given that the highest levels of nNOS, NADPHd, and cNOS were found in the ischemia-resistant dorsal horn, and nNOS-IR in surviving motoneurons, it is possible that NO production may play a neuroprotective role in ischemic/reperfusion injury.