Chiara Giuli

Protective and worsening peripheral nociceptin/orphanin FQ receptor-mediated effect in a rat model of experimental colitis.

Nociceptin/orphanin FQ (N/OFQ) and nociceptin orphanin peptide (NOP) receptors represent an endogenous system modulating gastrointestinal functions and inflammation. We investigated the peripheral effect of N/OFQ and of UFP-101, the NOP antagonist, in a model of colitis induced by TNBS (2,4,6 trinitrobenzenesulphonic acid; 60mg/kg). Male rats received two intraperitoneal injections per day of N/OFQ, UFP-101 or saline for 3 days after colitis induction. Four days after TNBS, animals were sacrificed and colonic histological damage, myeloperoxidase (MPO) activity and cytokine (IL-1β and IL-10) levels were evaluated. N/OFQ plasmatic levels were assessed by radioimmunoassay. TNBS increased all the inflammatory variables considered. In colitic rats, N/OFQ (0.02 and 0.2nmol/kg) improved microscopic damage, MPO activity and decreased IL-1β levels in comparison with TNBS group, whereas at the highest dose (20nmol/kg) the peptide worsened colitis. UFP-101 at the dose of 1nmol/kg, without pharmacological activity, antagonised the protective effect of N/OFQ (0.2nmol/kg) on colitis, but at a dose level of 3 and 10nmol/kg worsened inflammation, revealing the endogenous N/OFQergic system protective role. N/OFQ plasmatic levels were not modified in TNBS-treated rats compared with controls, whereas they were reduced in rats treated with the doses of UFP-101 aggravating colitis. In conclusion, peripheral low doses of N/OFQ have a beneficial effect on colonic inflammation in rats. In contrast, N/OFQ at a dose 100-1000-fold higher than those that protect worsens colitis, probably through different mechanisms. The peripheral N/OFQergic system can represent a new field of investigation in some intestinal inflammatory conditions.

Cannabinoid agonist WIN55,212 in vitro inhibits interleukin‐6 (IL‐6) and monocyte chemo‐attractant protein‐1 (MCP‐1) release by rat pancreatic acini and in vivo induces dual effects on the course of acute pancreatitis

Background  Cannabinoids (CBs) evoke their effects by activating the cannabinoid receptor subtypes CB1‐r and CB2‐r and exert anti‐inflammatory effects altering chemokine and cytokine expression. Various cytokines and chemokines are produced and released by rodent pancreatic acini in acute pancreatitis. Although CB1‐r and CB2‐r expressed in rat exocrine pancreatic acinar cells do not modulate digestive enzyme release, whether they modulate inflammatory mediators remains unclear. We investigated the CB‐r system role on exocrine pancreas in unstimulated conditions and during acute pancreatitis. Methods We evaluated in vitro and in vivo changes induced by WIN55,212 on the inflammatory variables amylasemia, pancreatic edema and morphology, and on acinar release and content of the cytokine interleukin‐6 (IL‐6) and chemokine monocyte chemo‐attractant protein‐1 (MCP‐1) in untreated rats and rats with caerulein (CK)‐induced pancreatitis. Key Results In the in vitro experiments, WIN55,212 (10−6 mol L−1) inhibited IL‐6 and MCP‐1 release from acinar cells of unstimulated rats and after CK‐induced pancreatitis. In vivo, when rats were pretreated with WIN55,212 (2 mg kg−1, intraperitoneally) before experimentally‐induced pancreatitis, serum amylase, pancreatic edema and IL‐6 and MCP‐1 acinar content diminished and pancreatic morphology improved. Conversely, when rats with experimentally‐induced pancreatitis were post‐treated with WIN55,212, pancreatitis worsened. Conclusions & Inferences These findings provide new evidence showing that the pancreatic CB1‐r/CB2‐r system modulates pro‐inflammatory factor levels in rat exocrine pancreatic acinar cells. The dual, time‐dependent WIN55,212‐induced changes in the development and course of acute pancreatitis support the idea that the role of the endogenous CB receptor system differs according to the local inflammatory status.

Maternal Exposure to Low Levels of Corticosterone during Lactation Protects against Experimental Inflammatory Colitis-Induced Damage in Adult Rat Offspring

Opposing emotional events (negative/trauma or positive/maternal care) during the postnatal period may differentially influence vulnerability to the effects of stress later in life. The development and course of intestinal disorders such as inflammatory bowel disease are negatively affected by persistent stress, but to date the role of positive life events on these pathologies has been entirely unknown. In the present study, the effect of early life beneficial experiences in the development of intestinal dysfunctions, where inflammation and stress stimuli play a primary role, was investigated. As a “positive” experimental model we used adult male rat progeny nursed by mothers whose drinking water was supplemented with moderate doses of corticosterone (CORT) (0.2 mg/ml) during the lactation period. Such animals have been generally shown to cope better with different environmental situations during life. The susceptibility to inflammatory experimental colitis induced by intracolonic infusion of TNBS (2,4,6-trinitrobenzenesulphonic acid) was investigated in CORT-nursed rats in comparison with control rats. This mild increase in maternal corticosterone during lactation induced, in CORT-nursed rats, a long lasting protective effect on TNBS-colitis, characterized by improvements in some indices of the disease (increased colonic myeloperoxidase activity, loss of body weight and food intake) and by the involvement of endogenous peripheral pathways known to participate in intestinal disorder development (lower plasma corticosterone levels and colonic mast cell degranulation, alterations in the colonic expression of both corticotrophin releasing factor/CRF and its receptor/CRH-1R). All these findings contribute to suggesting that the reduced vulnerability to TNBS-colitis in CORT-nursed rats is due to recovery from the colonic mucosal barrier dysfunction. Such long lasting changes induced by mild hormonal manipulation during lactation, making the adult also better adapted to colonic inflammatory stress, constitute a useful experimental model to investigate the etiopathogenetic mechanisms and therapeutic treatments of some gastrointestinal diseases.

Transplacental Exposure to AZT Induces Adverse Neurochemical and Behavioral Effects in a Mouse Model: Protection by L-Acetylcarnitine

Maternal-fetal HIV-1 transmission can be prevented by administration of AZT, alone or in combination with other antiretroviral drugs to pregnant HIV-1-infected women and their newborns. In spite of the benefits deriving from this life-saving prophylactic therapy, there is still considerable uncertainty on the potential long-term adverse effects of antiretroviral drugs on exposed children. Clinical and experimental studies have consistently shown the occurrence of mitochondrial dysfunction and increased oxidative stress following prenatal treatment with antiretroviral drugs, and clinical evidence suggests that the developing brain is one of the targets of the toxic action of these compounds possibly resulting in behavioral problems. We intended to verify the effects on brain and behavior of mice exposed during gestation to AZT, the backbone of antiretroviral therapy during human pregnancy. We hypothesized that glutamate, a neurotransmitter involved in excitotoxicity and behavioral plasticity, could be one of the major actors in AZT-induced neurochemical and behavioral alterations. We also assessed the antioxidant and neuroprotective effect of L-acetylcarnitine, a compound that improves mitochondrial function and is successfully used to treat antiretroviral-induced polyneuropathy in HIV-1 patients. We found that transplacental exposure to AZT given per os to pregnant mice from day 10 of pregnancy to delivery impaired in the adult offspring spatial learning and memory, enhanced corticosterone release in response to acute stress, increased brain oxidative stress also at birth and markedly reduced expression of mGluR1 and mGluR5 subtypes and GluR1 subunit of AMPA receptors in the hippocampus. Notably, administration during the entire pregnancy of L-acetylcarnitine was effective in preventing/ameliorating the neurochemical, neuroendocrine and behavioral adverse effects induced by AZT in the offspring. The present preclinical findings provide a mechanistic hypothesis for the neurobehavioral effects of AZT and strongly suggest that preventive administration of L-acetylcarnitine might be effective in reducing the neurological side-effects of antiretroviral therapy in fetus/newborn.

Maternal exposure to environmental enrichment before and during gestation influences behaviour of rat offspring in a sex-specific manner

The beneficial effects of Environmental Enrichment (EE) applied immediately after weaning or even in adulthood have been widely demonstrated. Less is known about the possible changes in behaviour and brain development of the progeny following the exposure of dams to EE. In order to further investigate this matter, female rats were reared in EE for 12weeks, from weaning until delivery. After having confirmed the presence of relevant behavioural effects of EE, both control and EE females underwent mating. Maternal behaviour was observed and male and female offspring were then administered a battery of behavioural test at different ages. EE mothers showed a decreased frequency of total nursing and, during the first 2days of lactation, an increase in licking/grooming behaviour. Maternal exposure to EE affected offspring behaviour in a sex-specific manner: social play behaviour and anxiety-like behaviour were increased in males but not in females and learning ability was improved only in females. As a general trend, maternal EE had a marked influence on motility in male and female offspring in both locomotor activity and swimming speed. Overall, this study highlights the importance of environmental stimulation, not only in the animals directly experiencing EE, but for their progeny too, opening the way to new hypothesis on the heritability mechanisms of behavioural traits.

Faecal corticosterone metabolite assessment in socially housed male and female Wistar rats

Knowledge of animals’ hormonal status is important for conservation studies in wild or semi-free-ranging conditions as well as for behavioural and clinical experiments conducted in laboratory research, mostly performed on rats and mice. Faecal sampling is a useful non-invasive method to obtain steroid hormone assessments. Nevertheless, in laboratory studies, unlike other contexts, faecal sampling is less utilised. One of the issues raised is the necessity to collect samples belonging to different animals, separately. Usually, researchers using faecal sampling solve this problem through the isolation of animals or taking the cage rather than single animal as unit of study. These solutions though, could lead to unreliable measurements, and cannot be applied in many studies. Our aim was to show the biological reliability of individual faecal corticosterone metabolite (FCM) assessments in socially housed male and female Wistar rats. We analytically validated the enzyme immunoassay kit used for FCM assessments. Then, we exposed the animals to two different stress stimuli that are known to activate the hypothalamus–pituitary–adrenal axis and the following release of corticosterone to biologically validate the EIA kit: environmental enrichment and predator odour. Individual faecal sampling from social animals was collected through short-time handling. The results demonstrated that both the stimuli increased FCM levels in male and female rats showing the reliability of EIA kit assessment and the applicability of our sampling method. We also found a diurnal rhythm in FCM levels. These results could help to increase the use of faecal hormone metabolite determinations in studies conducted on rats.

Relocation stress induces short-term fecal cortisol increase in Tonkean macaques (Macaca tonkeana).

The level of glucocorticoids, especially if obtained from noninvasive sampling, can be used as an index of animal well-being, allowing evaluation of the animal’s response to environmental modifications. Despite evidence that these hormones play a relevant role in energy metabolism regulation in perceived or real stress events, little is known regarding the factors that could modify the capability of animals to cope with relocation events. The aim of this research was to assess fecal cortisol metabolite concentrations before, during and after acute stress (transfer and relocation event) in two well-established social groups of Tonkean macaques (Macaca tonkeana). The results showed that the fecal levels of cortisol increased in individuals of both groups in response to the stress event, with a similar trend in males and females. Hormone levels were back to baseline values in both groups a few days after transfer and relocation. The presence of known social partners could be one of the factors that possibly facilitated the adaptation process.

Maternal exposure to low levels of corticosterone during lactation increases social play behavior in rat adolescent offspring

Abstract Although costly in energy and time, social play is present and evolutionarily conserved in nearly all young mammals. Ontogenetic factors responsible for this particular form of supposed rewarding behavior are incompletely understood. Here, we have focused our attention on maternal glucocorticoid hormone. We used a model in which neonate rats are fed by mothers in which drinking water has been supplemented with 0.2 mg/ml corticosterone. The control groups were lactated by water-drinking mothers. Both male and female adolescent offspring of corticosterone (CORT) supplemented dams (CORT-nursed) showed an increase in social play behavior (i.e., pinning, pouncing, wrestling/boxing and social exploration) when compared to controls. No differences were observed between CORT-nursed progeny of both sexes and controls in the exploration of the arena during the social encounter. Finally, no differences were found in CORT plasma levels in basal conditions and following a social play session in both male and female CORT-nursed rats. These results indicate that variations in the maternal glucocorticoid status are able, directly or indirectly, to influence social play behavior in the offspring, although there is no direct relationship between the level of social play behavior and the intensity of adrenocortical activation.

Maternal exposure to low levels of corticosterone during lactation protects adult rat progeny against TNBS-induced colitis: A study on GR-mediated anti-inflammatory effect and prokineticin system

The early phase of life represents a critical period for the development of an organism. Interestingly, early life experiences are able to influence the development of the gastrointestinal tract and the reactivity to colonic inflammatory stress. We recently demonstrated that adult male rats exposed to low doses of corticosterone during lactation (CORT-nursed rats) are protected against experimental colitis induced by the intracolonic infusion of 2,4,6-trinitrobenzenesulfonic acid (TNBS). Based on these interesting results, we wanted to better investigate which cellular actors could be involved in the protection of CORT-nursed rats from TNBS-induced experimental colitis. Therefore, in the present work, we focused our attention on different factors implicated in GR-mediated anti-inflammatory effect. To address this issue, colonic tissues, collected from control and CORT-nursed healthy animals and from control and CORT-nursed colitic rats, were processed and the following inflammatory factors were evaluated: the expression of (i) glucocorticoid receptors (GR), (ii) glucocorticoid-induced leucine zipper (GILZ), (iii) phospho-p65NF-κB, (iv) the pro-inflammatory cytokines IL-1β and TNF-α, (v) the prokineticins PK2 and PK2L and (vi) their receptors PKR1 and PKR2. We found that adult CORT-nursed rats, in comparison to controls, showed increased expression of colonic GR and reduced expression of pro-inflammatory molecules (IL-1β, TNF-α, PK2 and PK2L) in response to inflammatory colitis. The observed changes were associated with an increase in GILZ colonic expression and with a reduction in phospo-p65NF-κB colonic expression.