Michael A. Pezzone

Induction of c-Fos immunoreactivity in the rat forebrain by conditioned and unconditioned aversive stimuli.

The protein product of the c-fos proto-oncogene was immunocytochemically localized in forebrain regions of adult male Lewis rats subjected to a physically aversive footshock stimulus or a Pavlovian-conditioned, non-aversive, auditory stimulus. Animals receiving the conditioned stimulus were first conditioned by repeatedly pairing electric footshock, the unconditioned stimulus (US), with an auditory cue, the conditioned stimulus (CS). These animals were later tested with the CS in the absence of the US, a procedure which, like footshock itself, suppresses immune function. In animals exposed to the conditioned or unconditioned stressor, c-Fos was strongly expressed in cells of the paraventricular nuclei (PVN) of the hypothalamus, some of which contain corticotropin-releasing hormone (CRH), and other forebrain areas directly associated with autonomic function, the ventral lateral septal nuclei (LSV), the medial amygdaloid nuclei (AME), the sensorimotor cortex, the basal ganglia and thalamic nuclei. Control animals exhibited very little or no c-Fos in the above areas. The identified forebrain nuclei can now be targeted for further study aimed at elucidating their role in stress-induced immune alteration.

Activation of brainstem catecholaminergic neurons by conditioned and unconditioned aversive stimuli as revealed by c-Fos immunoreactivity

In an attempt to define areas of the brain that respond to stressors and influence immune function, we have previously identified stress-induced, c-Fos-immunoreactive areas of the diencephalon. We found that c-Fos was strongly expressed in cells of the paraventricular nuclei (some of which contain corticotropin-releasing hormone (CRH)) and other hypothalamic areas directly associated with autonomic function. To further characterize the presumptive pathways mediating stress-induced immune alterations, including the assessment of brainstem catecholaminergic neuron involvement, the induction of c-Fos immunoreactivity was examined in the brainstem of rats exposed to conditioned and unconditioned, immunomodulating stimuli. In response to electric footshock (the unconditioned stimulus (US)), c-Fos immunoreactivity was strongly induced in the noradrenergic neurons of the locus ceruleus (A6), the nucleus of the solitary tract (A2/C2), the ventral lateral medulla (A1/C1), A5, and A7, as well as in unidentified neurons of the dorsal and ventral subdivisions of the periaqueductal gray (PAG), and in the serotonergic neurons of the dorsal raphe nuclei. Conditioned animals re-exposed to the conditioned stimulus showed c-Fos induction in these same areas but to a lesser degree. Control animals exposed only to the conditioning stimulus (CS) (electronic tone) in the absence of the US, expressed very little, if any, c-Fos activity in the above loci except for a small degree of baseline expression in the PAG. These results further confirm the role of autonomic and endocrine pathways as mediators of the stress response and will help to more fully characterize the pathways of stress-induced immune alteration.

Convergence of Bladder and Colon Sensory Innervation Occurs at the Primary Afferent Level

Abstract Dichotomizing afferents are individual dorsal root ganglion (DRG) neurons that innervate two distinct structures thereby providing a form of afferent convergence that may be involved in pelvic organ cross‐sensitization. To determine the distribution of dichotomizing afferents supplying the distal colon and bladder of the Sprague–Dawley rat and the C57Bl/6 mouse, we performed concurrent retrograde labeling of urinary bladder and distal colon afferents using cholera toxin subunit B (CTB) fluorescent conjugates. Animals were perfused 4–5 days after sub‐serosal organ injections, and the T10‐S2 DRG were removed, sectioned, and analyzed using confocal microscopy. In the rat, CTB‐positive afferents retrogradely labeled from the bladder were nearly three times more numerous than those labeled from the distal colon, while in the mouse, each organ was equally represented. In both species, the majority of colon and bladder afferents projected from lumbosacral (LS) ganglia and secondarily from thoracolumbar (TL) ganglia. In the rat, 17% of the total CTB‐positive neurons were retrogradely labeled from both organs with 11% localized in TL, 6% in LS, and 0.8% in thoracic (TH) ganglia. In the mouse, 21% of the total CTB‐positive neurons were dually‐labeled with 12% localized in LS, 4% in TH, and 4% in TL ganglia. These findings support the existence of dichotomizing pelvic afferents, which provide a pre‐existing neuronal substrate for possible immediate and maintained pelvic organ cross‐sensitization and ultimately may play a role in the overlap of pelvic pain disorders.

Colonic Postoperative Inflammatory Ileus in the Rat

ObjectiveTo investigate local inflammatory events within the colonic muscularis as a causative factor of postoperative ileus. Summary Background DataSurgically induced intestinal muscularis inflammation has been hypothesized as a mechanism for postoperative ileus. The colon is a crucial component for recovery of gastrointestinal motor function after surgery but remains unaddressed. The authors hypothesize that colonic manipulation initiates inflammatory events that directly mediate postoperative smooth muscle dysfunction. MethodsRats underwent colonic manipulation. In vivo transit and colonic motility was estimated using geometric center analysis and intraluminal pressure monitoring. Leukocyte extravasation was investigated in muscularis whole mounts. Mediator mRNA expression was determined by real-time reverse transcriptase–polymerase chain reaction. In vitro circular muscle contractility was assessed in a standard organ bath. The relevance of iNOS and COX-2 inhibition was determined using DFU or L-NIL perfusion. ResultsColonic manipulation resulted in a massive leukocyte recruitment and an increase in inflammatory mRNA expression. This inflammatory response was associated with an impairment of in vivo motor function and an inhibition of in vitro smooth muscle contractility (56%). L-NIL but not DFU significantly ameliorated smooth muscle dysfunction. ConclusionsThe results provide evidence for a surgically initiated local inflammatory cascade within the colonic muscularis that mediates smooth muscle dysfunction, which contributes to postoperative ileus.

Cross-Talk and Sensitization of Bladder Afferent Nerves

The coordination of pelvic physiologic function requires complex integrative sensory pathways that may converge both peripherally and/or centrally. Following a focal, acute irritative or infectious pelvic insult, these same afferent pathways may produce generalized pelvic sensitization or cross‐sensitization as we show bi‐directionally for the bladder and bowel in an animal model. Single unit bladder afferent recordings following intracolonic irritation reveal direct sensitization to both chemical and mechanical stimuli thats dependent upon both intact bladder sensory (C‐fiber) innervation and neuropeptide content. Concurrent mastocytosis (preponderantly neurogenic) likely plays a role in long‐term pelvic organ sensitization via the release of nociceptive and afferent‐modulating molecules. Prolonged pelvic sensitization as mediated by these convergent and antidromic reflexive pathway may likewise lead to chronic pelvic pain and thus the overlap of chronic pelvic pain disorders. Neurourol. Urodynam. 29: 77–81, 2010.

Lack of serotonin1B receptor expression leads to age-related motor dysfunction, early onset of brain molecular aging and reduced longevity

Normal aging of the brain differs from pathological conditions and is associated with increased risk for psychiatric and neurological disorders. In addition to its role in the etiology and treatment of mood disorders, altered serotonin (5-HT) signaling is considered a contributing factor to aging; however, no causative role has been identified in aging. We hypothesized that a deregulation of the 5-HT system would reveal its contribution to age-related processes and investigated behavioral and molecular changes throughout adult life in mice lacking the regulatory presynaptic 5-HT1B receptor (5-HT1BR), a candidate gene for 5-HT-mediated age-related functions. We show that the lack of 5-HT1BR (Htr1bKO mice) induced an early age-related motor decline and resulted in decreased longevity. Analysis of life-long transcriptome changes revealed an early and global shift of the gene expression signature of aging in the brain of Htr1bKO mice. Moreover, molecular changes reached an apparent maximum effect at 18-months in Htr1bKO mice, corresponding to the onset of early death in that group. A comparative analysis with our previous characterization of aging in the human brain revealed a phylogenetic conservation of age-effect from mice to humans, and confirmed the early onset of molecular aging in Htr1bKO mice. Potential mechanisms appear independent of known central mechanisms (Bdnf, inflammation), but may include interactions with previously identified age-related systems (IGF-1, sirtuins). In summary, our findings suggest that the onset of age-related events can be influenced by altered 5-HT function, thus identifying 5-HT as a modulator of brain aging, and suggesting age-related consequences to chronic manipulation of 5-HT.

Gastrointestinal pain: unraveling a novel endogenous pathway through uroguanylin/guanylate cyclase-C/cGMP activation.

Summary Uroguanylin activation of the guanylate cyclase‐C/cyclic guanosine monophosphate pathway elicits analgesic effects in animal models of colonic hypersensitivity, unraveling a novel pathway to treat abdominal pain. ABSTRACT The natural hormone uroguanylin regulates intestinal fluid homeostasis and bowel function through activation of guanylate cyclase‐C (GC‐C), resulting in increased intracellular cyclic guanosine‐3′,5′‐monophosphate (cGMP). We report the effects of uroguanylin‐mediated activation of the GC‐C/cGMP pathway in vitro on extracellular cGMP transport and in vivo in rat models of inflammation‐ and stress‐induced visceral hypersensitivity. In vitro exposure of intestinal Caco‐2 cells to uroguanylin stimulated bidirectional, active extracellular transport of cGMP into luminal and basolateral spaces. cGMP transport was significantly and concentration dependently decreased by probenecid, an inhibitor of cGMP efflux pumps. In ex vivo Ussing chamber assays, uroguanylin stimulated cGMP secretion from the basolateral side of rat colonic epithelium into the submucosal space. In a rat model of trinitrobenzene sulfonic acid (TNBS)‐induced visceral hypersensitivity, orally administered uroguanylin increased colonic thresholds required to elicit abdominal contractions in response to colorectal distension (CRD). Oral administration of cGMP mimicked the antihyperalgesic effects of uroguanylin, significantly decreasing TNBS‐ and restraint stress–induced visceromotor response to graded CRD in rats. The antihyperalgesic effects of cGMP were not associated with increased colonic spasmolytic activity, but were linked to significantly decreased firing rates of TNBS‐sensitized colonic afferents in rats in response to mechanical stimuli. In conclusion, these data suggest that the continuous activation of the GC‐C/cGMP pathway along the intestinal tract by the endogenous hormones guanylin and uroguanylin results in significant reduction of gastrointestinal pain. Extracellular cGMP produced on activation of GC‐C is the primary mediator in this process via modulation of sensory afferent activity.

Corticosterone-independent alteration of lymphocyte mitogenic function by amphetamine.

Amphetamine, a neural stimulatory agent with acute effects mimicking those of stress, is shown here to elevate plasma corticosterone levels and suppress spleen and peripheral blood lymphocyte (PBL) mitogenic responses to concanavalin A (Con A) and phytohemagglutinin (PHA) when administered to rats. Pretreatment of the rats with propranolol, a nonselective beta-adrenergic receptor antagonist, totally prevented the amphetamine-induced suppression of lymphocyte mitogenic reactivity to Con A and PHA in the spleen and to PHA in the peripheral blood; however, the PBL mitogenic response to Con A was only partially restored. Although the amphetamine-induced alterations in immune function were prevented by propranolol pretreatment, the elevated plasma corticosterone response was not. This suggests that corticosterone is not modulating the mitogenic activity of splenic lymphocytes or PHA-reactive PBLs. On the other hand, Con A-reactive PBLs may be affected by corticosterone and/or other mechanisms, which may include the catecholamines.

Quantitative multispectral imaging of Herovici's polychrome for the assessment of collagen content and tissue remodelling.

Bioprosthetic devices, constructed from a variety of materials, are routinely implanted in a variety of anatomical locations. Essential to their success is the formation of a non‐destructive interface with the host tissue and appropriate tissue remodelling. Traditionally, the main method of assessing the host–material interface has been qualitative histological evaluation, using pattern recognition and comparative assessment to identify changes in the normal tissue architecture that are characteristic of scar tissue. In the present study, the recently developed technique of multispectral imaging was used to revisit a little‐described histological stain, Herovicis polychrome, which is capable of distinguishing between types I and III collagen. Combined, these techniques allowed quantification of collagen content and distribution of collagen types within a tissue sample. Samples of rat tail and human scar tissue were used to optimize the staining, while comparison with immunolabelled samples was used to develop a reproducible quantification system, based on the specific colour profiles for types I and III collagen. Finally the remodelling of rat abdominal wall defects repaired with crosslinked or non‐crosslinked extracellular matrix scaffolds derived from porcine urinary bladder was assessed with this technique. Compared to standard histological assessment, the combination of multispectral imaging and Herovicis polychrome staining presents a quick, simple, reliable technique that can provide accurate quantification of tissue remodelling and specifically identify the expression and distribution of types I and III collagen. Copyright

Effects of footshock stress upon spleen and peripheral blood lymphocyte mitogenic responses in rats with lesions of the paraventricular nuclei

To assess the role of the hypothalamic paraventricular nucleus (PVN) in mediating stressor-induced immune alterations, male Lewis rats were subjected to a 1-h session of intermittent footshock stress or home cage conditions 6 days after receiving bilateral or sham PVN lesions. Splenic and peripheral blood lymphocyte proliferative responses to the non-specific mitogens, concanavalin A (ConA) and phytohemagglutinin (PHA), were subsequently measured as were plasma corticosterone levels. In sham-operated rats, footshock markedly elevated plasma corticosterone levels and concurrently suppressed the proliferative responses of peripheral blood and splenic lymphocytes. In PVN-lesioned rats, however, the shock-induced suppression of lymphocyte proliferation in the peripheral blood and the elevation of plasma corticosterone were significantly attenuated, while lymphocyte proliferation in the spleen was suppressed below the level of the sham-treated animals. Thus, by utilizing ablation studies, we have determined that the PVN may play a direct role in the alteration of lymphocyte function during stress, and an intact PVN buffers the effect of stress on the responsiveness of spleen lymphocytes to non-specific mitogens.