Chronic peripheral administration of corticotropin-releasing factor causes colonic barrier dysfunction similar to psychological stress

Chronic psychological stress causes intestinal barrier dysfunction and impairs host defense mechanisms mediated by corticotrophin-releasing factor (CRF) and mast cells; however, the exact pathways involved are unclear. Here we investigated the effect of chronic CRF administration on colonic permeability and ion transport functions in rats and the role of mast cells in maintaining the abnormalities. CRF was delivered over 12 days via osmotic minipumps implanted subcutaneously in wild-type (+/+) and mast cell-deficient (Ws/Ws) rats. Colonic segments were excised for ex vivo functional studies in Ussing chambers [short-circuit current (Isc), conductance (G), and macromolecular permeability (horseradish peroxidase flux)], and analysis of morphological changes (mast cell numbers and bacterial host-interactions) was determined by light and electron microscopy. Chronic CRF treatment resulted in colonic mucosal dysfunction with increased Isc, G, and horseradish peroxidase flux in+/+but not in Ws/Ws rats. Furthermore, CRF administration caused mast cell hyperplasia and abnormal bacterial attachment and/or penetration into the mucosa only in+/+rats. Finally, selective CRF agonist/antagonist studies revealed that stimulation of CRF-R1 and CRF-R2 receptors induced the elevated secretory state and permeability dysfunction, respectively. Chronic CRF causes colonic barrier dysfunction in rats, which is mediated, at least in part, via mast cells. This information may be useful in designing novel treatment strategies for stress-related gastrointestinal disorders.     

Stress signaling pathways activated by weaning mediate intestinal dysfunction in the pig

Weaning in the piglet is a stressful event associated with gastrointestinal disorders and increased disease susceptibility. Although stress is thought to play a role in postweaning intestinal disease, the mechanisms by which stress influences intestinal pathophysiology in the weaned pig are not understood. The objectives of these experiments were to investigate the impact of weaning on gastrointestinal health in the pig and to assess the role of stress signaling pathways in this response. Nineteen-day-old pigs were weaned, and mucosal barrier function and ion transport were assessed in jejunal and colonic tissues mounted on Ussing chambers. Weaning caused marked disturbances in intestinal barrier function, as demonstrated by significant (P < 0.01) reductions in transepithelial electrical resistance and increases in intestinal permeability to [3H]mannitol in both the jejunum and colon compared with intestinal tissues from age-matched, unweaned control pigs. Weaned intestinal tissues exhibited increased intestinal secretory activity, as demonstrated by elevated short-circuit current that was sensitive to treatment with tetrodotoxin and indomethacin, suggesting activation of enteric neural and prostaglandin synthesis pathways in weaned intestinal tissues. Western blot analyses of mucosal homogenates showed increased expression of corticotrophin-releasing factor (CRF) receptor 1 in the jejunum and colon of weaned intestinal tissues. Pretreatment of pigs with the CRF receptor antagonist alpha-helical CRF(9-41), which was injected intraperitoneally 30 min prior to weaning, abolished the stress-induced mucosal changes. Our results indicate that weaning stress induces mucosal dysfunction mediated by intestinal CRF receptors and activated by enteric nerves and prostanoid pathways.     

Ontogeny of pituitary responsiveness to corticotropin-releasing hormone in rat

The ontogeny of the pituitary's responsiveness to synthetic rat corticotropin-releasing hormone (CRH) in the late prenatal and early postnatal periods of rats was studied by a superfusion system using whole pituitaries. A significant increase of immunoreactive beta-endorphin (IR-beta-Ep) secretion in response to 10(-10) M CRH but not to 10(-11) M CRH was observed in pituitaries from the 15th day of gestation, the earliest day that we tested, whereas 10(-11) M CRH stimulated IR-beta-Ep release from the pituitaries of 17.5-day-old fetuses. Dose-related IR-beta-Ep secretions induced by 10(-12) M to 10(-10) M CRH were observed in pituitaries of 19.5- and 21.5-day-old fetuses, and 1-, 3- and 9-day-old newborn pups. CRH stimulated not only IR-beta-Ep and IR-adrenocorticotropic hormone (ACTH) but also IR-alpha-melanocyte-stimulating hormone (IR-alpha-MSH) secretions from fetal pituitaries. The content of IR-CRH in the hypothalamic extract from 15-day-old fetus was 6.6 +/- 3.6 pg/hypothalamus (mean +/- S.E.M.) and it gradually increased to reach 212.7 +/- 20.3 pg/hypothalamus on the 21.5th day of gestation. However, the content of IR-CRH in the hypothalamus dramatically decreased just after birth and then rapidly increased again from the 5th day after birth. These data indicate that the responsiveness of corticotrophs to CRH is already present on the 15th day of gestation, when the content of IR-CRH in the hypothalamus is extremely low and that the amount of hypothalamic IR-CRH dramatically dropped for several days just after birth in rats.     

Dehydration-induced drinking decreases Fos expression in hypothalamic paraventricular neurons expressing vasopressin but not corticotropin-releasing hormone

Water-restricted (WR) rats exhibit a rapid suppression of plasma corticosterone following drinking. The present study monitored Fos-like immunoreactivity (Fos) to assess the effect of WR-induced drinking on the activity of vasopressin (VP)-positive magnocellular and parvocellular neurons and corticotropin-releasing hormone (CRH)-positive parvocellular neurons in the paraventricular nucleus of the hypothalamus. Adult male rats received water for 30 min (WR) in the post meridiem (PM) each day for 6 days and were killed without receiving water or at 1 h after receiving water for 15 min. In WR rats, Fos increased in VP magnocellular and parvocellular neurons but not CRH neurons. After drinking, Fos was reduced in VP magnocellular and parvocellular neurons but did not change in CRH neurons. To assess the severity of osmotic stress, rats were sampled throughout the final day of WR. Plasma osmolality, hematocrit and plasma VP were increased throughout the day before PM rehydration, and plasma ACTH and corticosterone were elevated at 1230 and 1430, respectively, showing that WR activates hypothalamic-pituitary-adrenal activity during the early PM before the time of rehydration. To determine the effects of WR-induced drinking on CRH neurons activated by acute stress, WR rats underwent restraint. Restraint increased plasma ACTH and corticosterone and Fos in CRH neurons; although rehydration reduced plasma ACTH and Fos expression in VP neurons, Fos in CRH neurons was not affected. These results suggest that inhibition of VP magnocellular and parvocellular neurons, but not CRH parvocellular neurons, contributes to the suppression of corticosterone after WR-induced drinking.     

Maternal deprivation affects the neuromuscular protein profile of the rat colon in response to an acute stressor later in life

Early life stress as neonatal maternal deprivation (MD) predisposes rats to alter gut functions in response to acute psychological stressors in adulthood, mimicking features of irritable bowel syndrome (IBS). We applied proteomics to investigate whether MD permanently changes the protein profile of the external colonic neuromuscular layer that may condition the molecular response to an acute stressor later in life.

Male rat pups were separated 3 h/day from their mothers during the perinatal period and further submitted to water avoidance (WA) stress during adulthood. Proteins were extracted from the myenteric plexus-longitudinal muscle of control (C), WA and MD + WA rat colon, separated on 2D gels, and identified by mass spectrometry. MD amplified the WA-induced protein changes involved in muscle contractile function, suggesting that stress accumulation along life imbalances the muscle tone towards hypercontractility. Our results also propose a stress dependent regulation of gluconeogenesis. Secretogranin II – the secretoneurin precursor – was induced by MD. The presence of secretoneurin in myenteric ganglia may partially explain the stress-mediated modulation of gastrointestinal motility and/or mucosal inflammation previously described in MD rats.

In conclusion, our findings suggest that neonatal stress alters the responses to acute stress in adulthood in intestinal smooth muscle and enteric neurons.     

Repeated exposure to water avoidance stress in rats: a new model for sustained visceral hyperalgesia

Chronic stress plays an important role in the development and exacerbation of symptoms in functional gastrointestinal disorders. To better understand the mechanisms underlying this relationship, we aimed to characterize changes in visceral and somatic nociception, colonic motility, anxiety-related behavior, and mucosal immune activation in rats exposed to 10 days of chronic psychological stress. Male Wistar rats were submitted daily to either 1-h water avoidance (WA) stress or sham WA for 10 consecutive days. The visceromotor response to colorectal distension, thermal somatic nociception, and behavioral responses to an open field test were measured at baseline and after chronic WA. Fecal pellets were counted after each WA stress or sham WA session as a measure of stress-induced colonic motility. Colonic samples were collected from both groups and evaluated for structural changes and neutrophil infiltration, mast cell number by immunohistochemistry, and cytokine expression by quantitative RT-PCR. Rats exposed to chronic WA (but not sham stress) developed persistent visceral hyperalgesia, whereas only transient changes in somatic nociception were observed. Chronically stressed rats also exhibited anxiety-like behaviors, enhanced fecal pellet excretion, and small but significant increases in the mast cell numbers and the expression of IL-1beta and IFN-gamma. Visceral hyperalgesia following chronic stress persisted for at least a month. Chronic psychological stress in rats results in a robust and long-lasting alteration of visceral, but not somatic nociception. Visceral hyperalgesia is associated with other behavioral manifestations of stress sensitization but was only associated with minor colonic immune activation arguing against a primary role of mucosal immune activation in the maintenance of this phenomenon.     

Corticotropin releasing hormone (CRH) increases beta-endorphin (beta-end like) concentration in cerebrospinal fluid of rats with vasospasm following subarachnoid hemorrhage.

The chronic stage of vasospasm occurring several days after subarachnoid hemorrhage (SAH) is characterized by the development of histopathologic changes in cerebral arteries causing cerebral ischemia. Numerous experimental data indicate the involvement of immune mechanisms in the angiopathy caused by SAH. Endogenous opioids play also an important role in the ischemic lesions of the brain. Corticotropin releasing hormone (CRH) induces the release of beta-endorphin (beta-END) from hypothalamic neurons and also from mononuclear white blood cells. The function of CRH and beta-END in vasospasm following SAH and the interrelationship between neuroendocrine and immune changes requires further elucidation. In the present study we investigated the influence of CRH injected into cerebral cisterna magna (CM) of rats on beta-END-like level in cerebrospinal fluid (CSF) in acute and chronic phase of cerebral vasospasm following artificial SAH. Acutely CRH induced a significant rise of beta-END-like in CSF both in SAH and sham SAH rats. However, in rats subjected to SAH, a single injection of CRH caused a prolonged rise of 5-END in CSF, which was also seen 2 days after SAH, during the chronic phase of vasospasm. The obtained results indicate that CRH increases neuroendocrine changes induced by SAH, probably by an activation of immune cells involved in the patomechanism of chronic vasospasm.     

Psychomotor responsiveness to corticoliberin in rats with active and passive adaptive strategy in water-immersion paradigm of depression

A single intranasal injection of CRH alters adaptive behavior only in active rats. In particular this procedure results in increase of their behavioral passivity and reduction of psychomotor responsiveness. This neurohormone causes the same effect in active rats being injected by means of the same routine 20 days after animals have been exposed to a single water-immersion stress. But this effect of CHR is not observed after injection of the neurohormone to depressed animals in the stress-restress paradigm. Passive rats, intact or depressed, do not show any behavioral changes, while active ones develop signs of depression after repeated CRH intranasal injection.     

Regulation of 6-phosphofructo-1-kinase from the epithelial cells of rat small intestine during pregnancy and lactation

The regulation of 6-phosphofructo-1-kinase (PFK) in the epithelial cells of rat small intestine was studied during pregnancy and lactation. The total activities and activity ratios (activity at 0.5 mM fructose 6-phosphate at pH 7.0/activity at pH 8.0 (nu 0.5/V] of the partially purified mucosal PFK were found to increase initially in early pregnant rats (11-12 days of gestation) and to fall back to normal in late pregnant rats (19-20 days of gestation). These changes in enzyme activity during pregnancy were associated with similar changes in the circulating levels of progesterone. The maximal activity and activity ratio (nu 0.5/V) were increased in male and female rats injected with progesterone. An increase in the total activity and activity ratio of mucosal PFK was also obtained in lactating rats. However, the enzyme was not strongly activated by inorganic phosphate, fructose 2,6-bisphosphate or glucose 1,6-bisphosphate either in early pregnant or lactating rats. These results indicate that mucosal PFK is already present as an active form during early pregnancy and lactation. Therefore, it is suggested that female sex hormones increase the circulating levels of insulin during early pregnancy which, in turn, positively affect the activity of mucosal PFK which could be also stimulated by the increased levels of fructose 2,6-bisphosphate. The increased activity of PFK in the peak lactating rats could be possible because of an increased demand for lactate production from glucose together with the stimulation of PFK by the increased concentrations of fructose 2,6-bisphosphate which therefore increases the rate of glycolysis.     

Effect of cyclooxygenase inhibitors on the CRH-induced pituitary-adrenocortical activity during crowding stress.

The aim of the present study was to determine the effect of social stress and significance of prostaglandins (PG) generated by constitutive and inducible cyclooxygenase (COX-1 and COX-2) in the stimulation of hypothalamic-pituitary-adrenal (HPA) axis by corticotropin releasing hormone (CRH) under basal and social crowding stress conditions. The stressed rats were crowded in groups of 24 to a cage for 3 or 7 days, whereas the control animals were haused in groups of 7 to a cage of the same size. The activity of HPA axis was determined by measuring plasma ACTH and serum corticosterone levels 1 h after i.p. CRH administration. Inhibitors of COX-1, piroxicam (0.2, 2.0, and 5.0 mg/kg), and COX-2, compound NS-398 (0.2 and 2.0 mg/kg), were administered i.p. 15 min prior to CRH (0.1 microg/kg i.p.) to control or crowded rats. The obtained results indicate that social stress for 3 and 7 days markedly intensifies the stimulatory action of CRH on ACTH secretion. Neither piroxicam nor NS-398 induce any significant effect on the CRH-elicited ACTH and corticosterone secretion in non-stressed or crowded rats. Therefore, PG generated by COX-1 or COX-2 do not participate to a significant extent in the stimulation of HPA axis by CRH under either basal conditions or during crowding stress. These results also indicate that the stimulatory action of CRH on ACTH secretion is not only completely resistant to desensitization but is sensitized during social crowding stress. The results contrast with a significant involvement of PG in the vasopressin-induced stimulation of HPA response during crowding stress.     

Prenatal exposure to dexamethasone alters hippocampal drive on hypothalamic-pituitary-adrenal axis activity in adult male rats

Glucocorticoids are essential for normal hypothalamic-pituitary-adrenal (HPA) axis activity; however, recent studies warn that exposure to excess endogenous or synthetic glucocorticoid during a specific period of prenatal development adversely affects HPA axis stability. We administered dexamethasone (DEX) to pregnant rats during the last week of gestation and investigated subsequent HPA axis regulation in adult male offspring in unrestrained and restraint-stressed conditions. With the use of real-time PCR and RIA, we examined the expression of regulatory genes in the hippocampus, hypothalamus, and pituitary, including corticotropin-releasing hormone (CRH), arginine vasopressin (AVP), glucocorticoid receptors (GR), mineralcorticoid receptors (MR), and 11-beta-hydroxysteroid dehydrogenase-1 (11beta-HSD-1), as well as the main HPA axis hormones, adrenal corticotropic hormone (ACTH) and corticosterone (CORT). Our results demonstrate that the DEX-exposed group exhibited an overall change in the pattern of gene expression and hormone levels in the unrestrained animals. These changes included an upregulation of CRH in the hypothalamus, a downregulation of MR with a concomitant upregulation of 11beta-HSD-1 in the hippocampus, and an increase in circulating levels of both ACTH and CORT relative to unrestrained control animals. Interestingly, both DEX-exposed and control rats exhibited an increase in pituitary GR mRNA levels following a 1-h recovery from restraint stress; however, the increased expression in DEX-exposed rats was significantly less and was associated with a slower return to baseline CORT compared with controls. In addition, circulating levels of ACTH and CORT as well as hypothalamic CRH and hippocampal 11beta-HSD-1 expression levels were significantly higher in the DEX-exposed group compared with controls following restraint stress. Taken together, these data demonstrate that late-gestation DEX exposure in rats is associated with persistent changes in both the modulation of HPA axis activity and the HPA axis-mediated response to stress.     

Effects of the corticoliberin on synaptic transmission in the rat olfactory cortex slices in water-immersion model depression

Corticoliberin (corticotrophin-releasing hormone, CRH) regulated of endocrine, autonomic and immune response to stress and is a mediator of anxiety in behavioral response. We studied the effect of corticoliberin on neuronal activity after microstimulation of olfactory cortex slices. Wistar rats strain were selected in T-maze labyrinth according to active and passive strategy of the adaptive behavior. The rats were exposed to water-immersions stress and after 10 days from their brain the olfactory cortex slices were prepared. The evoked focal potential were registered after perfusion with 0.1 mcM of CRH. It was revealed that in 60% of the slices of the active rats CRH induced the small decrease of excitatory amplitude but the increase amplitude inhibitory postsynaptic potential. In 40%, CRH induced the depression of synaptic transmission. Addition of CRH in incubation medium of the passive rat slices related, blockade the synaptic transmission.     

Chronic melatonin treatment and the hypothalamo-pituitary-adrenal axis in the rat: Attenuation of the secretory response to stress and effects on hypothalamic neuropeptide content and release

The pituitary-adrenal secretory response to acute and chronic stress, suppressibility of adrenocortical secretions by exogenous glucocorticoids, and hypothalamic content and in vitro release of the two major peptidergic activators of the hypothalamo-pituitary-adrenal (HPA) axis, corticotropinreleasing hormone (CRH) and arginine-vasopressin (AVP), were examined in rats receiving daily melatonin (MEL) injections coincident with the circadian increment of endogenous pineal and adrenocortical secretory activity. After 7 days of MEL administration, the rats displayed a significant attenuation of the adrenocortical secretory response to acute and chronic stress. Chronic MEL treatment also prevented the decline in adrenocorticotropic hormone (ACTH) release resulting from chronic stress exposure. Hypothalamic CRH content was significantly lower in rats receiving MEL treatment, while AVP remained largely unaltered; however, MEL administration counteracted the chronic stress-induced decrease in hypothalamic AVP content and in vitro release. When exposed to dexamethasone in vitro, hypothalamic explants from MEL-treated rats responded with a stronger suppression of CRH and AVP release than those originating from vehicle-injected animals. These observations indicate that MEL attenuates the adrenocortical response to stress and influences the biosynthesis, release and glucocorticoid responsiveness of hypothalamic ACTH secretagogues.     

Corticotropin-releasing hormone inhibits maternal behavior and induces pup-killing

Behavioral responses to stressors and the effects of stressors on maternal behavior change with mothering experience. Corticotropin-releasing hormone (CRH) is released by stressors and produces stress-like behavioral effects. We tested the effects of ICV infusion of ovine CRH (0.5-4 ug) on pup-directed behaviors in ovariectomized, ovarian steroid-treated virgin rats that were either naive to pups or that had three days of mothering experience. CRH inhibited maternal behavior in naive and experienced rats in a dose-related manner. The magnitude and duration of inhibition, especially at the 1 ug dose, were less in rats with mothering experience. Higher doses of CRH (1 - 4 ug) significantly increased pup-killing in rats that were naive to pups. In contrast, CRH produced no pup-killing in rats with mothering experience.     

Corticotropin-releasing hormone acts on CRH-R1 to inhibit the spontaneous contractility of non-labouring human myometrium at term

AIMS: Corticotropin-releasing hormone (CRH) has been implicated in the mechanisms controlling human parturition. The aims of the present study were to explore effects of CRH on contractility of human term myometrium and compare these effects in labouring and non-labouring myometrial strips. MAIN METHODS: The cumulative effects of CRH (10(-10) to 10(-7) mol/l) on the spontaneous contractility of labouring and non-labouring myometrial samples were evaluated using isometric tension recordings. KEY FINDINGS: CRH exhibited a concentration-dependent relaxant effect on spontaneous contractions in non-labouring term myometrium. This effect was mediated principally via a reduction in the amplitude rather than any changes in the frequency of contractions. The CRH-induced inhibitory effect on contractility could be blocked by pre-treatment with a CRH-R1 antagonist antalarmin, but not by pre-treatment with the CRH-R2 antagonist astressin 2B. CRH had no effect on spontaneous contractions in the labouring myometrium, as no change in either the amplitude or the frequency was observed. SIGNIFICANCE: Our findings indicate that CRH acts on CRH-R1 to inhibit spontaneous contractions in term myometrium from women who were not undergoing labour, but not those who were undergoing labour, supporting the hypothesis that CRH exerts dual effect on myometrium during pregnancy.     

Subacute toxicity of anilofos, a new organophosphorus herbicide in male rats: effect on lipid peroxidation and ATPase activity

Effects of anilofos on lipid peroxidation--an index of oxidative stress, ATPase activity--an integral part of active transport mechanisms for cations, GSH level and GST activity were evaluated in blood (erythrocyte/plasma), brain and liver of male rats after daily oral exposure to 50, 100 or 200 mg/kg for 28 days. None of the doses increased lipid peroxidation. The lowest dose, rather, produced marginally significant decrease in peroxidation in liver. Different doses of anilofos decreased GSH content and activities of GST and ATPases. Inhibition of total ATPase (34-44%) and Na+-K+-ATPase (45-52%) activities was maximum in liver, while that of Mg2+-ATPase (46-56%) was more in erythrocyte. Results indicate that anilofos may not cause oxidative damage to cell membrane in repeatedly exposed animals and may cause neuronal/cellular dysfunction by affecting ionic transport across cell membrane.     

The role of corticotropin-releasing hormone in alteration of adaptive behavior of the active and passive rats after inescapable stress

Effects of corticotropin-releasing hormone (CRH) on the formation of post-stress psychopathology were studied using of two genetic strains KHA (Koltushi high Avoidance) and KLA (Koltushi low Avoidance) selected on high or low acquisition of active avoidance, respectively. These strains are characterized by higher (KHA) and lower (KLA) behavioral activity in open field and adopted, respectively, active and passive strategies in stressful conditions. A widely used experimental paradigm of learned helplessness where behavioral depression was produced by inescapable uncontrollable footshock has been applied in our study. KHA rats demonstrated psychopathology already 1st day following exsposure to the stress faktor, and the depression progressed by the 5th and 10th post-stress days. Intranasal application of CRH facilitated the development of depression in active rats. In KLA rats, which originally displayed low exploratory activity associated with high anxiety, the inescapable stress at first enhanced the exploratory behavior but 10 days later these rats displayed a progressive decline of exploration and locomotion. Initially, the application of CRH also enhanced the exploratory behavior in these rats, but to 10th post-stress day promoted development of depressive state. The results suggest that CRH in different ways affects the formation of depressive state in rats with different strategies of adaptive behavior.