Convergence of sensory pathways in the development of somatic and visceral hypersensitivity

Sensory neurons innervating different tissues converge onto second-order neurons in the spinal cord. We examined whether inflammation or transient overexpression of nerve growth factor (NGF) in one tissue triggers hypersensitivity in referral sites. Thresholds to mechanical and thermal stimulation of the hindpaw, visceromotor responses to colorectal distension, and cystometrograms were performed in appropriate controls and mice with experimentally induced cystitis, inflammation of the hindpaw or front paw, or injection of viral vectors encoding NGF or green fluorescent protein (GFP). Cystitis and NGF but not GFP overexpression in the bladder triggered bladder hyperactivity associated with mechanical and thermal hypersensitivity in cutaneous referral sites and enhanced responses to colorectal distension. Hindpaw inflammation and injection of the NGF- but not GFP-encoding viral vector or front paw inflammation induced mechanical and thermal hyperalgesia in the affected hindpaw and increased responses to colorectal distension without altering the micturition reflex. In conclusion, sensitization of sensory pathways by inflammation or NGF contributes to the development of hypersensitivity in neighboring organs and cutaneous referral sites and provides a potential mechanism underlying the coexistence of pain syndromes in patients with functional diseases.     

Modulation of visceral hypersensitivity by glial cell line-derived neurotrophic factor family receptor α-3 in colorectal afferents

Irritable bowel syndrome is characterized by colorectal hypersensitivity and contributed to by sensitized mechanosensitive primary afferents and recruitment of mechanoinsensitive (silent) afferents. Neurotrophic factors are well known to orchestrate dynamic changes in the properties of sensory neurons. Although pain modulation by proteins in the glial cell line-derived neurotrophic factor (GDNF) family has been documented in various pathophysiological states, their role in colorectal hypersensitivity remains unexplored. Therefore, we investigated the involvement of the GDNF family receptor α-3 (GFRα3) signaling in visceral hypersensitivity by quantifying visceromotor responses (VMR) to colorectal distension before and after intracolonic treatment with 2,4,6-trinitrobenzene sulfonic acid (TNBS). Baseline responses to colorectal distension did not differ between C57BL/6 and GFRα3 knockout (KO) mice. Relative to intracolonic saline treatment, TNBS significantly enhanced the VMR to colorectal distension in C57BL/6 mice 2, 7, 10, and 14 days posttreatment, whereas TNBS-induced visceral hypersensitivity was significantly suppressed in GFRα3 KO mice. The proportion of GFRα3 immunopositive thoracolumbar and lumbosacral colorectal dorsal root ganglion neurons was significantly elevated 2 days after TNBS treatment. In single fiber recordings, responses to circumferential stretch of colorectal afferent endings in C57BL/6 mice were significantly increased (sensitized) after exposure to an inflammatory soup, whereas responses to stretch did not sensitize in GFRα3 KO mice. These findings suggest that enhanced GFRα3 signaling in visceral afferents may contribute to development of colorectal hypersensitivity.     

改良CCI大鼠痛行为学检测与损伤区自发放电活动再观察

目的：应用改良CCI模型研究外周神经损伤后痛觉过敏和自发放电各自特征及相互关系。方法：雄性SD大鼠,随机分为CCI组和Sham组,分别于术前1天和术后1、4、7、9、11、14天测定机械刺激缩足反射阈值和热缩腿反射潜伏期,同时选取术侧机械刺激缩腿反射阈值低于4g或者术侧和健侧热缩腿反射潜伏期差异大于2s的CCI模型大鼠观察术后4-14天损伤区自发放电活动。结果：神经纤维损伤后,机械痛敏和热痛敏随时间表现为逐渐增强,同时在损伤区观察到三类放电模式：整数倍放电﹑阵发放电和周期放电。结论：术后大鼠机械痛阈和热痛阈逐渐降低,机械痛敏的产生和损伤区自发放电活动关系密切,不同的放电模式可能代表不同的传入信息。     

Behavioral and Electrophysiological Evidence for the Differential Functions of TRPV1 at Early and Late Stages of Chronic Inflammatory Nociception in Rats

We previously reported that vanilloid receptor type 1 (VR1, or TRPV1) was up-regulated in dorsal root ganglion (DRG) and the spinal dorsal horn after chronic inflammatory pain produced by complete Freund’s adjuvant (CFA) injection into the plantar of rat hind paw. In the present study, we found that subcutaneous or intrathecal application of capsazepine (CPZ), a TRPV1 competitive antagonist, could inhibit thermal hyperalgesia on day 1 and on day 14 but not on day 28 after CFA injection. With extracellular electrophysiological recording, the effect of CPZ on noxious electrical or heat stimulation evoked responses of wide dynamic range (WDR) neurons in the deep layers of the spinal dorsal horn was evaluated. Under noxious electrical stimulation to sciatic nerve, CPZ applied to the spinal cord produced an inhibition on Aδ- and C-fiber evoked responses of WDR neurons on day 1 and 14, but not on day 28. Under radiant heat stimulation to the receptive field skin, subcutaneous application of CPZ significantly inhibited the background activity and extended the response latency of WDR neurons on day 14. These results provide new evidence for the functional significance of TRPV1 at the early stage, but not the late stage, in the rat model of CFA-induced inflammatory pain. Special issue article in honor of Dr. Ji-Sheng Han. Hao Luo, Isabella Shi Xu, Yi Chen are Co-first authors.     

GLT-1 overexpression attenuates bladder nociception and local/cross-organ sensitization of bladder nociception

Glutamatergic pathways mediate transmission of pain. Strategies to reduce glutamatergic neurotransmission may have beneficial effects to mitigate nociception. Recent work revealed that overexpression of the astrocytic glutamate transporter (GLT-1) by transgenic or pharmacologic approaches produced a diminished visceral nociceptive response to colonic distension. The purpose of this study was to determine the effect of GLT-1 overexpression on the visceromotor response to bladder distension. Increased glutamate uptake activity produced by 1-wk ceftriaxone (CTX) treatment attenuated 60-64% the visceromotor response to graded bladder distension compared with vehicle-treated mice. One-hour pretreatment with selective GLT-1 antagonist dihydrokainate reversed the blunted visceromotor response to bladder distension produced by 1-wk CTX, suggesting that GLT-1 overexpression mediated the analgesic effect of CTX. Moreover, sensitization of the visceromotor response to bladder distension produced by local bladder irritation (acrolein) was also attenuated by 1-wk CTX treatment. A model of cross-organ sensitization of bladder visceromotor response to distension was next studied to determine whether increased expression of GLT-1 can mitigate colon to bladder sensitization. Intracolonic trinitrobenzene sulfonic acid (TNBS) administered 1 h before eliciting the visceromotor response to graded bladder distension produced a 75-138% increase in visceromotor response compared with animals receiving intracolonic vehicle. In marked contrast, animals treated with 1-wk CTX + intracolonic TNBS showed no enhanced visceromotor response compared with the 1-wk vehicle + intracolonic vehicle group. The study suggests that GLT-1 overexpression attenuates the visceromotor response to bladder distension and both local irritant-induced and cross-organ-sensitized visceromotor response to bladder distension.     

Acute colonic inflammation triggers detrusor instability via activation of TRPV1 receptors in a rat model of pelvic organ cross-sensitization

Chronic pelvic pain of unknown etiology is a common clinical condition and may develop as a result of cross-sensitization in the pelvis when pathological changes in one of the pelvic organs result in functional alterations in an adjacent structure. The aim of the current study was to compare transient receptor potential vanilloid 1 (TRPV1) activated pathways on detrusor contractility in vivo and in vitro using a rat model of pelvic organ cross-sensitization. Four groups of male Sprague-Dawley rats (N = 56) were included in the study. Animals received intracolonic saline (control), resiniferatoxin (RTX, TRPV1 agonist, 10(-7) M), 2,4,6-trinitrobenzene sulfonic acid (TNBS, colonic irritant), or double treatment (RTX followed by TNBS). Detrusor muscle contractility was assessed under in vitro and in vivo conditions. Intracolonic RTX increased the contractility of the isolated detrusor in response to electric field stimulation (EFS) by twofold (P ≤ 0.001) and enhanced the contractile response of the bladder smooth muscle to carbachol (CCh). Acute colonic inflammation reduced detrusor contractility upon application of CCh in vitro, decreased bladder capacity by 28.1% (P ≤ 0.001), and reduced micturition volume by 60% (P ≤ 0.001). These changes were accompanied by an increased number of nonmicturition contractions from 3.7 ± 0.7 to 15 ± 2.7 (N = 6 in both groups, P ≤ 0.001 vs. control). Desensitization of intracolonic TRPV1 receptors before the induction of acute colitis restored the response of isolated detrusor strips to CCh but not to EFS stimulation. Cystometric parameters were significantly improved in animals with double treatment and approximated the control values. Our data suggest that acute colonic inflammation triggers the occurrence of detrusor instability via activation of TRPV1-related pathways. Comparison of the results obtained under in vitro vs. in vivo conditions provides evidence that intact neural pathways are critical for the development of an overactive bladder resulting from pelvic organ cross talk.     

Distension of urinary bladder induces exaggerated coronary constriction in smokers with early atherosclerosis

Distension of the urinary bladder causes an increase in efferent sympathetic activity, which can precipitate myocardial ischemia. Smoking has been shown to modulate activities of afferent nerves from the distended urinary bladder and to impair endothelial function in response to sympathetic activation. To assess the effect of bladder distension on coronary dynamics in smokers, we measured epicardial and microvascular responses in 24 patients with early atherosclerosis (< 50% diameter stenosis). Patients were classified into habitual smokers (group 1, n = 14) and nonsmokers (group 2, n = 10). Habitual smokers were randomized into two subgroups on the basis of the use of doxazosin, as follows: subgroup 1A (n = 7), without administration of doxazosin before catheterization; subgroup 1B (n = 7), with dosing doxazosin. In response to bladder distension (mean intravesical pressure 21.5 mmHg), bladder distension significantly decreased coronary diameter at the stenotic segments, coronary blood flow, and increased coronary resistance compared with baseline values, in subgroup 1A patients. In subgroup 1B patients during bladder distension, coronary diameter, coronary blood flow, and coronary resistance did not show significant changes compared with baseline values. There were significant differences of coronary diameter at the stenotic segments, coronary blood flow, and of changes of coronary vascular resistance between subgroup 1A and group 2 during bladder distension, despite similar changes in rate-pressure product. The present study showed that urinary bladder distension caused an abnormal vasomotor response of epicardial vasoconstriction and a concomitant increased coronary resistance, which leads to reduction in coronary blood flow in patients with early atherosclerosis. Smoking may further impair the response, implying that smoking has exaggerated response to sympathetic stimulation of conduit and resistance vessels. The abnormal response was abolished by pretreated administration of doxazosin, suggesting that the involved mechanisms are related to alpha(1)-adrenoceptors.     

Irritable bowel syndrome: methods, mechanisms, and pathophysiology. Neural and neuro-immune mechanisms of visceral hypersensitivity in irritable bowel syndrome

Irritable bowel syndrome (IBS) is characterized as functional because a pathobiological cause is not readily apparent. Considerable evidence, however, documents that sensitizing proinflammatory and lipotoxic lipids, mast cells and their products, tryptases, enteroendocrine cells, and mononuclear phagocytes and their receptors are increased in tissues of IBS patients with colorectal hypersensitivity. It is also clear from recordings in animals of the colorectal afferent innervation that afferents exhibit long-term changes in models of persistent colorectal hypersensitivity. Such changes in afferent excitability and responses to mechanical stimuli are consistent with relief of discomfort and pain in IBS patients, including relief of referred abdominal hypersensitivity, upon intra-rectal instillation of local anesthetic. In the aggregate, these experimental outcomes establish the importance of afferent drive in IBS, consistent with a larger literature with respect to other chronic conditions in which pain is a principal complaint (e.g., neuropathic pain, painful bladder syndrome, fibromyalgia). Accordingly, colorectal afferents and the environment in which these receptive endings reside constitute the focus of this review. That environment includes understudied and incompletely understood contributions from immune-competent cells resident in and recruited into the colorectum. We close this review by highlighting deficiencies in existing knowledge and identifying several areas for further investigation, resolution of which we anticipate would significantly advance our understanding of neural and neuro-immune contributions to IBS pain and hypersensitivity.     

Subdiaphragmatic vagal afferent nerves modulate visceral pain

Activation of the vagal afferents by noxious gastrointestinal stimuli suggests that vagal afferents may play a complex role in visceral pain processes. The contribution of the vagus nerve to visceral pain remains unresolved. Previous studies reported that patients following chronic vagotomy have lower pain thresholds. The patient with irritable bowel syndrome has been shown alteration of vagal function. We hypothesize that vagal afferent nerves modulate visceral pain. Visceromotor responses (VMR) to graded colorectal distension (CRD) were recorded from the abdominal muscles in conscious rats. Chronic subdiaphragmatic vagus nerve sections induced 470, 106, 51, and 54% increases in VMR to CRD at 20, 40, 60 and 80 mmHg, respectively. Similarly, at light level of anesthesia, topical application of lidocaine to the subdiaphragmatic vagus nerve in rats increased VMR to CRD. Vagal afferent neuronal responses to low or high-intensity electrical vagal stimulation (EVS) of vagal afferent Adelta or C fibers were distinguished by calculating their conduction velocity. Low-intensity EVS of Adelta fibers (40 microA, 20 Hz, 0.5 ms for 30 s) reduced VMR to CRD at 40, 60, and 80 mmHg by 41, 52, and 58%, respectively. In contrast, high-intensity EVS of C fibers (400 microA, 1 Hz, 0.5 ms for 30 s) had no effect on VMR to CRD. In conclusion, we demonstrated that vagal afferent nerves modulate visceral pain. Low-intensity EVS that activates vagal afferent Adelta fibers reduced visceral pain. Thus EVS may potentially have a role in the treatment of chronic visceral pain.     

Endogenous enkephalin does not contribute to the cerebral anti-hyperalgesic action of gabapentin

The aim of this study was to investigate the role of endogenous enkephalin in the cerebral antihyperalgesic action of gabapentin. Neuropathic pain models and antihyperalgesic effect of gabapentin were confirmed by the presentation and changes of mechanical allodynia and thermal hyperalgesia of operated mouse hind paws. The results suggested that endogenous enkephalin may not be involved in the antihyperalgesic effect of gabapentin.     

改良CCI 大鼠痛行为学检测与损伤区自发放电活动再观察

目的:应用改良CCI模型研究外周神经损伤后痛觉过敏和自发放电各自特征及相互关系.方法:雄性SD大鼠,随机分为CCI组和Sham组,分别于术前1天和术后1、4、7、9、11、14天测定机械刺激缩足反射阈值和热缩腿反射潜伏期,同时选取术侧机械刺激缩腿反射阈值低于4g或者术侧和健侧热缩腿反射潜伏期差异大于2s的CCI模型大鼠观察术后4-14天损伤区自发放电活动.结果:神经纤维损伤后,机械痛敏和热痛敏随时间表现为逐渐增强,同时在损伤区观察到三类放电模式:整数倍放电阵发放电和周期放电.结论:术后大鼠机械痛阈和热痛阈逐渐降低,机械痛敏的产生和损伤区自发放电活动关系密切,不同的放电模式可能代表不同的传入信息.     

Top-Down Effect of Direct Current Stimulation on the Nociceptive Response of Rats

Transcranial direct current stimulation (tDCS) is an emerging, noninvasive technique of neurostimulation for treating pain. However, the mechanisms and pathways involved in its analgesic effects are poorly understood. Therefore, we investigated the effects of direct current stimulation (DCS) on thermal and mechanical nociceptive thresholds and on the activation of the midbrain periaqueductal gray (PAG) and the dorsal horn of the spinal cord (DHSC) in rats; these central nervous system areas are associated with pain processing. Male Wistar rats underwent cathodal DCS of the motor cortex and, while still under stimulation, were evaluated using tail-flick and paw pressure nociceptive tests. Sham stimulation and naive rats were used as controls. We used a randomized design; the assays were not blinded to the experimenter. Immunoreactivity of the early growth response gene 1 (Egr-1), which is a marker of neuronal activation, was evaluated in the PAG and DHSC, and enkephalin immunoreactivity was evaluated in the DHSC. DCS did not change the thermal nociceptive threshold; however, it increased the mechanical nociceptive threshold of both hind paws compared with that of controls, characterizing a topographical effect. DCS decreased the Egr-1 labeling in the PAG and DHSC as well as the immunoreactivity of spinal enkephalin. Altogether, the data suggest that DCS disinhibits the midbrain descending analgesic pathway, consequently inhibiting spinal nociceptive neurons and causing an increase in the nociceptive threshold. This study reinforces the idea that the motor cortex participates in the neurocircuitry that is involved in analgesia and further clarifies the mechanisms of action of tDCS in pain treatment.     

P2X3 Receptors Mediate Visceral Hypersensitivity during Acute Chemically-Induced Colitis and in the Post-Inflammatory Phase via Different Mechanisms of Sensitization

Objectives

Experiments using P2X₃ knock-out mice or more general P2X receptor antagonists suggest that P2X₃ receptors contribute to visceral hypersensitivity. We aimed to investigate the effect of the selective P2X₃ antagonist A-317491 on visceral sensitivity under physiological conditions, during acute colitis and in the post-inflammatory phase of colitis.

Methods

Trinitrobenzene sulphonic-acid colitis was monitored by colonoscopy: on day 3 to confirm the presence of colitis and then every 4 days, starting from day 10, to monitor convalescence and determine the exact timepoint of endoscopic healing in each rat. Visceral sensitivity was assessed by quantifying visceromotor responses to colorectal distension in controls, rats with acute colitis and post-colitis rats. A-317491 was administered 30 min prior to visceral sensitivity testing. Expression of P2X₃ receptors (RT-PCR and immunohistochemistry) and the intracellular signalling molecules cdk5, csk and CASK (RT-PCR) were quantified in colonic tissue and dorsal root ganglia. ATP release in response to colorectal distension was measured by luminiscence.

Results

Rats with acute TNBS-colitis displayed significant visceral hypersensitivity that was dose-dependently, but not fully, reversed by A-317491. Hypersenstivity was accompanied by an increased colonic release of ATP. Post-colitis rats also displayed visceral hypersensitivity that was dose-dependently reduced and fully normalized by A-317491 without increased release of ATP. A-317491 did not modify visceral sensitivity in controls. P2X₃ mRNA and protein expression in the colon and dorsal root ganglia were similar in control, acute colitis and post-colitis groups, while colonic mRNA expression of cdk5, csk and CASK was increased in the post-colitis group only.

Conclusions

These findings indicate that P2X₃ receptors are not involved in sensory signaling under physiological conditions whereas they modulate visceral hypersensitivity during acute TNBS-colitis and even more so in the post-inflammatory phase, albeit via different mechanisms of sensitization, validating P2X₃ receptors as potential new targets in the treatment of abdominal pain syndromes.     

Modeling the spinal pudendo-vesical reflex for bladder control by pudendal afferent stimulation

Electrical stimulation of the pudendal nerve (PN) is a promising approach to restore continence and micturition following bladder dysfunction resulting from neurological disease or injury. Although the pudendo-vesical reflex and its physiological properties are well established, there is limited understanding of the specific neural mechanisms that mediate this reflex. We sought to develop a computational model of the spinal neural network that governs the reflex bladder response to PN stimulation. We implemented and validated a neural network architecture based on previous neuroanatomical and electrophysiological studies. Using synaptically-connected integrate and fire model neurons, we created a network model with realistic spiking behavior. The model produced expected sacral parasympathetic nucleus (SPN) neuron firing rates from prescribed neural inputs and predicted bladder activation and inhibition with different frequencies of pudendal afferent stimulation. In addition, the model matched experimental results from previous studies of temporal patterns of pudendal afferent stimulation and selective pharmacological blockade of inhibitory neurons. The frequency- and pattern-dependent effects of pudendal afferent stimulation were determined by changes in firing rate of spinal interneurons, suggesting that neural network interactions at the lumbosacral level can mediate the bladder response to different frequencies or temporal patterns of pudendal afferent stimulation. Further, the anatomical structure of excitatory and inhibitory interneurons in the network model was necessary and sufficient to reproduce the critical features of the pudendo-vesical reflex, and this model may prove useful to guide development of novel, more effective electrical stimulation techniques for bladder control.     

Spatio-temporal changes of SDF1 and its CXCR4 receptor in the dorsal root ganglia following unilateral sciatic nerve injury as a model of neuropathic pain

There is a growing evidence that chemokines and their receptors play a role in inducing and maintaining neuropathic pain. In the present study, unilateral chronic constriction injury (CCI) of rat sciatic nerve under aseptic conditions was used to investigate changes for stromal derived factor-1 (SDF1) and its CXCR4 receptor in lumbal (L4–L5) and cervical (C7–C8) dorsal root ganglia (DRG) from both sides of naïve, CCI-operated and sham-operated rats. All CCI-operated rats displayed mechanical allodynia and thermal hyperalgesia in hind paws ipsilateral to CCI, but forepaws exhibited only temporal changes of sensitivity not correlated with alterations in SDF1 and CXCR4 proteins. Naïve DRG displayed immunofluorescence for SDF1 (SDF1-IF) in the satellite glial cells (SGC) and CXCR4-IF in the neuronal bodies with highest intensity in small- and medium-sized neurons. Immunofluorescence staining and Western blot analysis confirmed that unilateral CCI induced bilateral alterations of SDF1 and CXCR4 proteins in both L4–L5 and C7–C8 DRG. Only lumbal DRG were invaded by ED-1+ macrophages exhibiting SDF1-IF while elevation of CXCR4-IF was found in DRG neurons and SGC but not in ED-1+ macrophages. No attenuation of mechanical allodynia, but reversed thermal hyperalgesia, in ipsi- and contralateral hind paws was found in CCI-operated rats after i.p. administration of CXCR4 antagonist (AMD3100). These results indicate that SDF1/CXCR4 changes are not limited to DRG associated with injured nerve but that they also spread to DRG non-associated with such nerve. Functional involvement of these alterations in DRG non-associated with injured nerve in neuropathic pain remains to be elucidated.     

Tachykinins as modulators of the micturition reflex in the central and peripheral nervous system

In the normal urinary bladder, tachykinins (TKs) are expressed in a population of bladder nociceptors that is sensitive to the excitatory and desensitizing effects of capsaicin (i.e., capsaicin-sensitive primary afferent neurons (CSPANs)). Several endobiotics or xenobiotics excite CSPANs and release TKs and other mediators at both the peripheral and spinal cord level. The peripheral release of TKs determines a set of responses (known as neurogenic inflammation) that includes vasodilatation, plasma protein extravasation, smooth muscle contraction and stimulation of afferent nerves. Following chronic inflammation, both immune cells and capsaicin-resistant sensory neurons can de novo express TKs: whether these pools of TKs are releasable and contribute to inflammatory processes is presently unsettled. At the spinal cord level, the release of TKs contributes in determining an altered pattern of vesicourethral reflexes in response to nociceptive stimulation of the bladder by conveying: (a) the afferent transmission to supraspinal sites, and (b) descending or sensory inputs to the sacral parasympathetic nucleus (SPN). Recent evidence also attribute a synergetic role of TKs in the supraspinal modulation of the sensory arm of the micturition reflex.The overall available information suggests that TK receptor antagonists may affect bladder motility/reflexes which occur during different pathological states, while having little influence on the normal motor bladder function.     

Long-term sensitization of mechanosensitive and -insensitive afferents in mice with persistent colorectal hypersensitivity

Afferent input contributes significantly to the pain and colorectal hypersensitivity that characterize irritable bowel syndrome. In the present study, we investigated the contributions of mechanically sensitive and mechanically insensitive afferents (MIAs; or silent afferents) to colorectal hypersensitivity. The visceromotor response to colorectal distension (CRD; 15-60 mmHg) was recorded in mice before and for weeks after intracolonic treatment with zymosan or saline. After CRD tests, the distal colorectum with the pelvic nerve attached was removed for single-fiber electrophysiological recordings. Colorectal afferent endings were located by electrical stimulation and characterized as mechanosensitive or not by blunt probing, mucosal stroking, and circumferential stretch. Intracolonic zymosan produced persistent colorectal hypersensitivity (>24 days) associated with brief colorectal inflammation. Pelvic nerve muscular-mucosal but not muscular mechanosensitive afferents recorded from mice with colorectal hypersensitivity exhibited persistent sensitization. In addition, the proportion of MIAs (relative to control) was significantly reduced from 27% to 13%, whereas the proportion of serosal afferents was significantly increased from 34% to 53%, suggesting that MIAs acquired mechanosensitivity. PGP9.5 immunostaining revealed no significant loss of colorectal nerve fiber density, suggesting that the reduction in MIAs is not due to peripheral fiber loss after intracolonic zymosan. These results indicate that colorectal MIAs and sensitized muscular-mucosal afferents that respond to stretch contribute significantly to the afferent input that sustains hypersensitivity to CRD, suggesting that targeted management of colorectal afferent input could significantly reduce patients' complaints of pain and hypersensitivity.     

Morphological and Functional Alterations of the Myenteric Plexus in Rats with TNBS-Induced Colitis

The 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced model of experimental colitis was used to investigate the time-course of alterations in enteric neurotransmission and/or smooth muscle function that occur in chronic inflammation. Myenteric plexus morphology (immunocytochemical markers), functional integrity of cholinergic neurons (³H-choline uptake, acetylcholine release and contractile response to electrical field stimulation) and smooth muscle integrity (contractile response to exogenous acetylcholine) were determined 2, 7, 15, and 30 days after TNBS treatment. In TNBS-treated rats extensive ulcerations of the mucosa and/or the submucosa and increase in colonic weights were accompanied by significant reduction in ³H-choline uptake, acetylcholine release and contractile response to stimulation of enteric nerves. These changes were maximal 7 and 15 days after TNBS treatment. Immunocytochemical marker (PGP 9.5, SNAP 25, synaptophysin and S100 protein) expression was absent in necrotic areas of colons removed 7 days post-injury and partially reduced in colons removed 15 days after TNBS treatment. By contrast, the contractile response to exogenous acetylcholine was significantly increased after 7 days in both inflamed and uninflamed regions and returned to control values by day 30. Likewise, an almost complete recovery of neural cholinergic function and of myenteric plexus morphology was observed 30 days after TNBS treatment. These data suggest that TNBS-induced colitis is associated with progressive and selective alterations in myenteric plexus structure and function, with consequent reduction of cholinergic neurotransmission and abnormality in colonic contractility. The reversibility of myenteric plexus disruption is a clear indication of neuronal plasticity within enteric nervous system as an adaptative mechanism against inflammatory challenges.     

Analysis of the afferent limb of the vesicovascular reflex using neurotoxins, resiniferatoxin and capsaicin

The afferent limb of the vesicovascular reflex (VV-R) evoked by distension or contraction of the urinary bladder (UB) was studied in urethane-anesthetized female rats by examining the changes in VV-R after administration of C-fiber afferent neurotoxins [capsaicin and resiniferatoxin (RTX)]. Systemic arterial blood pressure increased parallel (5.1 to 53.7 mmHg) with graded increases in UB pressure (20 to 80 cm H(2)O) or during UB contractions. The arterial pressor response to UB distension was significantly reduced (60-85%) by acute or chronic (4 days earlier) intravesical administration of RTX (100-1,000 nM) or by capsaicin (125 mg/kg sc) pretreatment (4 days earlier). Chronic neurotoxin treatments also increased the volume threshold (>100%) for eliciting micturition in anesthetized rats but did not change voiding pressure. Acute RTX treatment (10-50 nM) did not alter the arterial pressor response during reflex UB contractions, whereas higher concentrations of RTX (100-1,000 nM) blocked reflex bladder contractions. It is concluded that VV-R is triggered primarily by distension- and contraction-sensitive C-fiber afferents located, respectively, near the luminal surface and deeper in the muscle layers of the bladder.     

Increased spinal expression of c-Fos following stimulation of the lower urinary tract in chronic spinal cord-injured rats

c-Fos expression was studied in the lumbar and sacral spinal cord regions involved in processing afferent input from the lower urinary tract and a comparison was made between spinal cord-injured (SCI) animals and control animals with intact neuraxes. Afferent pathways from the lower urinary tract were activated either by insertion of a catheter through the urethra into the urinary bladder or by catheterisation plus induction of reflex micturition contractions by intravesical saline infusion. Placement of a catheter alone elicited Fos expression in a similar number of neurones in SCI and control rats mainly in the medial dorsal horn (MDH) and dorsal commissure (DCM) in the segments L1–2 and L5–S1 with a maximum in L5. Additional saline infusion induced low-frequency, high-amplitude, rhythmic bladder contractions of long duration in the rats with intact spinal cords, whereas in SCI rats, bladder distension elicited reflex contractions at a higher frequency, smaller amplitude and shorter duration. However, the basal and mean bladder pressure, as well as the total contraction time relative to the whole recording time, was not significantly different. Distension-induced bladder contractions markedly increased Fos expression primarily in the spinal segments L5–S1 in the control rats, where the majority of bladder and urethral afferent fibres terminates. Fos-positive cells were located in the MDH, lateral dorsal horn (LDH), DCM and the lateral aspect of laminae V–VII. Compared to controls, Fos expression after spinal cord injury (SCI) occurred in a significantly greater number of neurones throughout the segments L3–S1 following induction of bladder reflexes. The greatest proportional increase in the number of Fos-positive cells occurred in L3–5 which normally receive only little afferent input from the urinary bladder. Cell numbers predominantly increased in the LDH and lateral lamina V–VII. The data are consistent with the concept of a neuroplastic reorganisation of spinal pathways after SCI. Unmasking of silent synapses or formation of new connections by afferent axonal sprouting caudal to the lesion, as evident from the increased numbers of cells expressing Fos after bladder distension, could be factors underlying the emergence of reflexogenic micturition in chronic SCI rats. Accepted: 27 May 1999