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 multi sensory 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 I 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 postrema) and the caudal part of the bulbar reticular formation. In contrast, more rostral subtelencephalic levels contain a variety of areas, in which maximal reaction precedes the complete development of cystitis and appears to be more involved in vegetative functions.