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 (uninjected), 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. Differential basal and evoked labeling between c-Fos and Krox-24 allowed a functional distinction between these spinal subregions: Krox-24 was an indicator of the first signs of inflammation (simple chorionic edema); c-Fos was an indicator of more pronounced impairments. DGC, which displayed a high level of basal Krox-24 expression and in which both Krox-24 and c-Fos were evoked in relation to disease evolution, probably codes inputs from both physiological (progressive bladder filling) and all types of pathological (inflammatory processes, overdistension) conditions. SPN, which, except for basal Krox-24 expression, responded like DGC and contains preganglionic motor cells, probably codes inputs involved in eliciting motor activity that results in bladder emptying as needed from natural filling and/or pathological situations. Laminae I and II, which responded like DGC and SPN with respect to Krox-24 expression, may code inputs from physiopathological changes of the bladder, though lamina I would be primarily involved in pain processing as it was the only region in which c-Fos expression increased during abrasion and complete inflammation. The cyclophosphamide-cystitis model has the following unique features compared with other visceral models: first, the stimulus is a ''pure'' visceral one, confined to one viscus (bladder; no somatic stimulation is induced by either introducing a stimulating device or from surgical wounds, and no anesthesia is required); second, it is the exact replica of a human disease, and its evolution can be efficently monitored through behavioral and histological observations; third, it can be used in behaving animals without departing from ethical rules.
