The differential effects of pelvic and vagal inputs on the supraspinal cystitis viscero(noci)ceptive-related axis.

Recently the development of the cyclophosphamide (CP, 100 mg/kg/i.p.) model has added an important element to the study of neural activities accompanying cystitis genesis. CP cystitis genesis results in the dual activation of the pelvic and vagal sensory afferent systems, which in turn activate a supraspinal network comprising the ventrocaudal bulbar reticular formation (vcBRF), the sensory subdivisions of the dorsal vagal complex (DVC) and its subcortical telencephalic targets, the dorsolateral subdivision of the bed nucleus of the stria terminalis (BSTLd) and the nucleus centralis of the amygdala (CeL). Altogether these structures form the sensory neural axis of the CP cystitis. However, both clinical and experimental observations have given evidence that only the pelvic afferents are at the origin of the painful sensation and related behaviour. Because of this, and for a better understanding of the nervous network that subserves cystitis painful information, we sought to determine whether the structures that constitute the cystitis sensory neural pathway have the same reactivity depending on the origin of the sensory afferent inputs, either pelvic or vagal. Using c-fos expression, which permits quantitative analysis of neural activity, we have demonstrated that the supraspinal CP cystitis responding structures do not form an homogeneous population in terms of sources of inputs. Although all structures are predominantly driven by vagal inputs, only the vcBRF, the DVC and the BSTLd respond to pelvic inputs. Consequently, and by referring to clinical observations, we have concluded that, it is these three areas, excluding the CeL, which constitute the main framework of the supraspinal pain sensory neural pathway of CP-induced cystitis. The activation of the vagus nerve would more probably relate to the other side effects that accompany CP injections such as nausea and headache attacks.

The uroprotection of mesna on cyclophosphamide cystitis in rats. Its consequences on behaviour and brain activities.

We studied the uroprotective effect of mesna, at doses of 40-300 mg/kg/i.p., in single or fractioned injections, on the development of cyclophosphamide (CP, 100 mg/kg/i.p.) cystitis in rats. The study concerns the histological, behavioural and nervous aspects of the disease. The specific effects of mesna, when injected alone, have also been considered. The mesna itself does not have specific deleterious effects, except at a dose of 300 mg/kg which provokes a moderate vesical inflammation although without consequence on the animal's behaviour. Mesna offers good protection against CP cystitis for only certain posologies. The uroprotective effects of mesna reach maxima at doses of 40-100 mg/kg and for fractioned injections given over the entire time frame of the urinary toxic release. The uroprotective effects of other posologies are only partial. The nervous activities were studied through the expression of Fos protein. The repetitive intraperitoneal injection of mesna induced a spinal activity and a preferential contralateral activity of the trigemino/reticular areas of the brainstem spinal cord junction--an effect which was reduced in the presence of CP. The prevention of cystitis by mesna was accompanied only by a reduction in spinal Fos activity, the supraspinal activities remaining high and in strict relationship with the vagal afferent activity. In conclusion, the uroprotective effect of mesna, which requires appropriate posologies, has led to the confirmation of the spinal actions of the CP cystitis, probably via the pelvic nerve, but did not allow a clear distinction between the consequences of the systemic (vagal) and local (spinal, pelvic) actions of CP at supraspinal level.

Heritability of nociception I: responses of 11 inbred mouse strains on 12 measures of nociception.

It is generally acknowledged that humans display highly variable sensitivity to pain, including variable responses to identical injuries or pathologies. The possible contribution of genetic factors has, however, been largely overlooked. An emerging rodent literature documents the importance of genotype in mediating basal nociceptive sensitivity, in establishing a predisposition to neuropathic pain following neural injury, and in determining sensitivity to pharmacological agents and endogenous antinociception. One clear finding from these studies is that the effect of genotype is at least partially specific to the nociceptive assay being considered. In this report we begin to systematically describe and characterize genetic variability of nociception in a mammalian species, Mus musculus. We tested 11 readily-available inbred mouse strains (129/J, A/J, AKR/J, BALB/cJ, C3H/HeJ, C57BL/6J, C58/J, CBA/J, DBA/2J, RIIIS/J and SM/J) using 12 common measures of nociception. These included assays for thermal nociception (hot plate, Hargreaves' test, tail withdrawal), mechanical nociception (von Frey filaments), chemical nociception (abdominal constriction, carrageenan, formalin), and neuropathic pain (autotomy, Chung model peripheral nerve injury). We demonstrate the existence of clear strain differences in each assay, with 1.2 to 54-fold ranges of sensitivity. All nociceptive assays display moderate-to-high heritability (h2 = 0.30-0.76) and mediation by a limited number of apparent genetic loci. Data comparing inbred strains have considerable utility as a tool for understanding the genetics of nociception, and a particular relevance to transgenic studies.

Heritability of nociception II. 'Types' of nociception revealed by genetic correlation analysis.

Clinical pain syndromes, and experimental assays of nociception, are differentially affected by manipulations such as drug administration and exposure to environmental stress. This suggests that there are different 'types' of pain. We exploited genetic differences among inbred strains of mice in an attempt to define these primary 'types'; that is, to identify the fundamental parameters of pain processing. Eleven randomly-chosen inbred mouse strains were tested for their basal sensitivity on 12 common measures of nociception. These measures provided for a range of different nociceptive dimensions including noxious stimulus modality, location, duration and etiology, among others. Since individual members of inbred strains are identical at all genetic loci, the observation of correlated strain means in any given pair of nociceptive assays is an index of genetic correlation between these assays, and hence an indication of common physiological mediation. Obtained correlation matrices were subjected to multivariate analyses to identify constellations of nociceptive assays with common genetic mediation. This analysis revealed three major clusters of nociception: (1) baseline thermal nociception, (2) spontaneously-emitted responses to chemical stimuli, and (3) baseline mechanical sensitivity and cutaneous hypersensitivity. Many other nociceptive parameters that might a priori have been considered closely related proved to be genetically divergent.

Cyclophosphamide cystitis as a model of visceral pain in rats: a c-fos and Krox-24 study at telencephalic levels, with a note on pituitary adenylate cyclase activating polypeptide (PACAP).

This article is the fifth of a series aimed at mapping brain activities as they result from the development of cyclophosphamide (CP) cystitis in behaving rats using c-fos and Krox-24 expression. The inactive hepatic metabolites of CP are metabolized in the kidney to produce acrolein, which generates cystitis. Data come from animals which were injected once i.p. with either 1 ml saline (sham) or 100 mg/kg CP in 1 ml saline under transient volatile anesthesia and which behaved freely for 1-4 h postinjection, 4 h being the minimum time for cystitis to completely develop. Survival times longer than 4 h were not studied owing to ethical considerations. The first 2 h postinjection cover a period of time over which inputs of multifactorial origin (stress and pain due to the intraperitoneal injection process, possible effects due to the presence of hepatic CP metabolites in blood, cystitis onset) interact in an indistinguishable way; the last 2 h are more cystitis specific as the other effects have vanished. Complete screening of telencephalic levels has been performed. These data complete previously published data at both spinal and subtelencephalic levels. Of all the telencephalic structures, only the bed nucleus of the stria terminalis in the dorsal part of its lateral division (BSTLd) and, to a lesser degree, the nucleus centralis of the amygdala, mostly in its caudal portion (cCeA), appeared to be significantly driven over the most specific cystitis period. Both of these structures had related, but not identical patterns of expression. They both reacted shortly after CP injection, but, while cCeA maintained its activity throughout cystitis development, BSTLd showed a rebound, reaching a peak value when cystitis was fully developed. Both of these areas are the only telencephalic areas to contain high PACAP38 immunoreactivity. This is evidence that, (1) both the BSTLd and cCeA could be the most rostral areas that visceronociceptive inflow would reach when cystitis genesis is under way, and (2) PACAP38 could be one of the neurochemical agents involved in telencephalic visceronociceptive processing. From our complete mapping of brain activities under a fully developed cystitis situation (4 h postinjection), it appears that the activities in BSTLd and cCeA are concomitant with those of both the dorsal vagal complex (DVC), paratrigeminal nucleus (PaT), and the ventrocaudal bulbar reticular formation (vcBRF) at brainstem levels, suggesting they all form the main part of the neural network that subserves the central processing of cystitis-related inputs, comprising pain and associated pseudoaffective responses. Both the DVC and BSTLd, which are the most powerfully driven areas, would be particularly important in such a way. The origin of these activities should be found in both vagal (as sensed through PaT activity) and spinal (pelvic) influences. This network profoundly differs from those reported for painful situations, either somatic or visceral, which controversally accompany positive cardiac inotropism.

Involvement of the dorsal paratrigeminal nucleus in visceral pain-related phenomena.

Cyclophosphamide is an antitumor agent that generates evolving cystitis through the release of toxic urinary by-products, mostly acrolein, that attack the bladder walls. Using c-fos expression, which permits quantitative analysis of neural activity, we demonstrated that the paratrigeminal nucleus is involved in processing the inputs that this disease generates. c-Fos staining in the paratrigeminal nucleus increases regularly reaching a plateau over the 4 h postinjection period during which the disease develops. The degree of staining is directly correlated with that of the subnucleus medialis of the nucleus of the solitary tract, which is one of the main structures that processes cystitis-related inputs at the supraspinal level.

Involvement of the dorsal paratrigeminal nucleus in visceral pain-related phenomena.

Cyclophosphamide is an antitumor agent that generates evolving cystitis through the release of toxic urinary by-products, mostly acrolein, that attack the bladder walls. Using c-fos expression, which permits quantitative analysis of neural activity, we demonstrated that the paratrigeminal nucleus is involved in processing the inputs that this disease generates. c-Fos staining in the paratrigeminal nucleus increases regularly reaching a plateau over the 4 h postinjection period during which the disease develops. The degree of staining is directly correlated with that of the subnucleus medialis of the nucleus of the solitary tract, which is one of the main structures that processes cystitis-related inputs at the supraspinal level.

Cyclophosphamide cystitis as a model of visceral pain in rats: minor effects at mesodiencephalic levels as revealed by the expression of c-fos, with a note on Krox-24.

The evoked expression of the immediate-early gene-encoded proteins c-Fos and Krox-24 was used to study activation of mesodiencephalic structures as a function of the development of cyclophosphamide (CP) cystitis in behaving rats. This article is the third of a series and completes previously published data obtained at both spinal and hindbrain levels. CP-injected animals received a single dose of 100 mg/kg i.p. under transient volatile anesthesia and survived for 1-4 h in order to cover the entire postinjection period during which the disease develops. Survival times longer than 4 h were not used owing to ethical considerations. Results from CP-injected groups are compared with those from either noninjected controls or saline-injected animals having survived for the same times as CP-injected ones. Quantitative results come from c-fos expression. At mesodiencephalic levels a high and widespread basal c-fos expression was observed in control animals; maximum staining was observed at the midthalamic level. Four groups of nuclei were identified with regard to the density of staining. The first group included nuclei showing clustered, intensely labeled cells; these areas were restricted in extent and related to the maintenance of circadian rythms (intergeniculate leaf, suprachiasmatic nucleus, dorsal parts of either paraventricular thalamic nuclei or central gray), sleep-arousal cycle (supramamillary nucleus), or changes in arterial pressure (laterodorsal tegmental nucleus). The second group included nuclei showing scattered, moderately labeled cells; these areas were widespread at all rostrocaudal levels and related to either autonomic/neuroendocrine regulations (central gray, lateral habenula, hypothalamus) or motor behavior, orienting reflex and oculomotor coordination (unspecific subdivisions of both colliculi and their adjoining mesencephalic regions, zona incerta dorsal). The third group included nuclei with evenly distributed, faintly labeled cells; these areas, which, with few exceptions, covered almost the entire diencephalon, mainly concerned nuclei of multisensory convergence having functions in either discriminative tasks (laterodorsal and lateroposterior thalamic nuclei) or emotional responses (intralaminar and midline thalamic nuclei). The fourth group included nuclei free of labeling; these were areas that received the bulk of unimodal sensory/motor inputs (central inferior colliculus, pretectal optic nuclei, ventral medial geniculate nucleus, ventral anterior pretectal nucleus, dorsal lateral geniculate nucleus, ventrobasal complex; zona incerta ventral, parafascicular thalamic nucleus) and are thus the most discriminative regarding specific modalities. Variations in staining were of the same magnitude in both saline- and CP-injected animals. A sequential study spanning every postinjection hour revealed maximum staining at 1 h postinjection, which was followed by a progressive, time-related decrease. Increases in the number of labeled cells 1 h postinjection were significant in only a restricted number of nuclei showing low basal expression (Edinger-Westphal nucleus and paraventricular, supraoptic, and lateral hypothalamic nuclei); time-related reductions in staining that were correlated to sleep or quiescence behaviors finally resulted in staining equal to or below that seen in control animals. No structures showed significantly increased staining in relation to the full development of cystitis, i.e., with the increase of visceronociceptive inputs. Comparing the present results with those previously obtained at more caudal levels, it appears that subtelencephalic levels primarily driven by visceronociceptive inputs, i.e., those that increase and/or maintain their activity in parallel with the degree of nociception, are confined to brainstem-spinal cord junction levels and only comprise certain subdivisions of the nucleus of the solitary tract (nucleus medialis, nucleus commissuralis, and ventralmost part of area po

Sex, time-of-day and estrous variations in behavioral and bladder histological consequences of cyclophosphamide-induced cystitis in rats.

This study examined how cyclophosphamide (CP)-induced cystitis related manifestations (bladder inflammation and behavioral impairment) differed in female and male Sprague-Dawley rats. Under transient halothane-O2-N2O gas anesthesia, a single dose of CP was injected (100 mg/kg i.p. in 1 ml saline) and the animal's behaviors analyzed for a period of 4 h using a protocol that permits quantitative analysis of behavioral impairment. The rats were then sacrificed and their bladders removed for histological quantification of inflammation. All CP-injected, but not control rats, exhibited a range of impairment behaviors that increased rapidly over a period of 2 h, gradually reaching plateau levels over the next 2 h. Female rats initially developed behavioral responses faster than male rats, but reached the same mean peak values overall as males. No sex differences were observed in CP-induced bladder inflammation. Influences of time-of-day and estrous stage were further examined in females. Time-of-day had no effect on the degree of bladder inflammation. Although there were also no significant time-of-day differences in behavioral impairments, impairment scores from 90 min after the injection consistently tended to be lower for rats injected 5 h versus 9 h after lights on. Overall, the effects of estrous stage were also insignificant. However, a subset of rats who were in the estrous stage of their cycle early in the morning of the experimental day developed the most severe degree of bladder inflammation, but failed to develop the severe behavioral impairments shown by all the other rats. These results show that there are seemingly only minor sex differences in the overall behavioral and inflammatory consequences of CP injections, as evidenced by similar final degrees of behavioral impairment and inflammation. These results also suggest, however, that there are sex differences in the etiology of the disease process. These differences are evidenced by the more rapid development of behavioral symptoms in females and the susceptibility of some of those having shown morning estrous smears to develop very severe bladder inflammation in absence of corresponding behavioral impairment. The multiple influences of sex and estrous condition on CP-induced cystitis related manifestations observed here underline the complexity of the etiological factors associated with the cystitis disease process.

Cyclophosphamide cystitis as a model of visceral pain in rats. A survey of hindbrain structures involved in visceroception and nociception using the expression of c-Fos and Krox-24 proteins.

The evoked expression of the immediate early gene-encoded proteins c-Fos and Krox-24 was used to study activation of hindbrain neurons as a function of the development of cyclophosphamide (CP) cystitis in behaving rats. CP-injected animals received a single dose of 100 mg/kg i.p. under transient volatile anesthesia and survived for 1 to 4 h in order to cover the whole postinjection period during which the disease develops. CP-injected groups included: (1) animals with minor simple chorionic edema, an early characteristic of inflammation (1 h postinjection); (2) animals with well-developed simple chorionic edema (2 h postinjection); (3) animals with mild inflammation (chorionic edema accompanied by epithelial cleavage; 3 h postinjection); and (4) animals with complete inflammation (4 h postinjection). In addition to onset of chorionic edema, the earliest postinjection period also included the general aspects of the nervous reaction consecutive to the injection process (handling, transient volatile anesthesia and postanesthesia awakening, abdominal pinprick, CP-blood circulating effects). Controls included both noninjected animals and saline-injected animals surviving for the same times as CP-injected ones. Quantitative results come from c-Fos expression. It has been shown that: (1) saline injection is a significant stimulus for only nucleus O and central gray pars alpha and nucleus medialis of the dorsal vagal complex; (2) all structures driven by CP injection (nucleus O and central gray pars alpha, locus coeruleus, Barrington's nucleus and parabrachial area mostly in its ventral and lateral subdivisions, dorsal vagal complex, ventrocaudal portion of lateral bulbar reticular formation) responded vigorously shortly after injection, but only two (dorsal vagal complex, ventrocaudal portion of lateral bulbar reticular formation) showed increased or renewed activity when cystitis completely developed, i.e., when noxious visceral inputs reached highest levels. Regarding the sequential activation of these structures in relation to postinjection time, evidence is given that: (1) a large variety of hindbrain structures are differentially involved in either the general reaction consecutive to the injection process or to various degrees of cystitis; (2) these structures extend from the brain-spinal cord to the pons-mesencephalon transitional junction levels; (3) the two structures most powerfully driven by visceronociceptive inputs are also the most caudal ones, being located at the brain-spinal cord junction level; and (4) the dorsal vagal complex could be the main hindbrain visceral pain center, with three particular subdivisions, the nucleus medialis, nucleus commissuralis, and ventralmost part of area postrema, being involved.

Cyclophosphamide cystitis as a model of visceral pain in rats: model elaboration and spinal structures involved as revealed by the expression of c-Fos and Krox-24 proteins.

The evoked expression of the immediate early gene (IEG)-encoded proteins c-Fos and Krox-24 was used to monitor spinal visceronociceptive processing that results from cyclophosphamide cystitis in behaving rats. Animals received a single dose of 100 mg/kg i.p. of cyclophosphamide and survived for 30 min to 5 h. Longer survival times were not considered because of ethical considerations. Cyclophosphamide-injected animals developed characteristic behavioral signs in parallel with development of bladder lesions and spinal evoked expression of IEG-encoded proteins. Histological examination of the urinary bladder was used to evaluate the degree of cystitis and as a criterion for selection of groups of animals to be quantitatively analyzed. Controls consisted of freely behaving animals including control (un-injected), sham (saline-injected) or diuretic (furosemide-injected) animals. Behavioral modifications consisted of lacrimation, piloerection, assumption of a peculiar "rounded-back" posture, which was accompanied by head immobility and various brief "crises" (tail hyperextension, abdominal retractions, licking of the lower abdomen, backward withdrawal movements). Abnormal behaviors, which first appeared (lacrimation, piloerection) at the end of postinjection hour 1, progressively increased in severity (rounded-back posture) over the following 90 min to reach a plateau at about postinjection hour 2; the rounded-back posture was maintained up to time of death. Histological modifications of bladder tissue were assessed using a 4-grade scale in a blind setting. The 1st grade consisted of control or sham animals with no bladder lesion; 2nd grade, animals with simple chorionic edema; 3rd grade, animals with chorionic edema associated with mucosal abrasion, fibrin deposit, and onset of polymorphonuclear leukocyte infiltration; 4th grade, animals with complete cystitis corresponding to an increase in severity and spread of all the signs of cystitis described above plus petechial hemorrhage. Simple chorionic edema was observed from 30 min to 3 h postinjection, but with a progressive increase in severity over time. Edema accompanied by epithelial abrasion was observed for animals that survived 3-4 h postinjection; complete inflammation was observed in animals that survived 4-5 h postinjection. The study of c-Fos- and Krox-24-encoded protein expression demonstrated that few lumbosacral spinal areas were specifically involved in the processing of visceral inputs in response to bladder stimulation. These areas were the parasympathetic column (SPN), the dorsal gray commissure (DGC as the caudal extent of lamina X), and superficial layers of the dorsal horn.(ABSTRACT TRUNCATED AT 400 WORDS)