Daniela Grandi

Morphological and functional alterations of the myenteric plexus in rats with TNBS-induced colitis

The 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced model of experimental colitis was used to investigate the time-course of alterations in enteric neurotransmission and/or smooth muscle function that occur in chronic inflammation. Myenteric plexus morphology (immunocytochemical markers), functional integrity of cholinergic neurons (3H-choline uptake, acetylcholine release and contractile response to electrical field stimulation) and smooth muscle integrity (contractile response to exogenous acetylcholine) were determined 2, 7, 15, and 30 days after TNBS treatment. In TNBS-treated rats extensive ulcerations of the mucosa and/or the submucosa and increase in colonic weights were accompanied by significant reduction in 3H-choline uptake, acetylcholine release and contractile response to stimulation of enteric nerves. These changes were maximal 7 and 15 days after TNBS treatment. Immunocytochemical marker (PGP 9.5, SNAP 25, synaptophysin and S100 protein) expression was absent in necrotic areas of colons removed 7 days post-injury and partially reduced in colons removed 15 days after TNBS treatment. By contrast, the contractile response to exogenous acetylcholine was significantly increased after 7 days in both inflamed and uninflamed regions and returned to control values by day 30. Likewise, an almost complete recovery of neural cholinergic function and of myenteric plexus morphology was observed 30 days after TNBS treatment. These data suggest that TNBS-induced colitis is associated with progressive and selective alterations in myenteric plexus structure and function, with consequent reduction of cholinergic neurotransmission and abnormality in colonic contractility. The reversibility of myenteric plexus disruption is a clear indication of neuronal plasticity within enteric nervous system as an adaptative mechanism against inflammatory challenges.

(R)-Alpha-Methylhistamine Inhibits Ethanol-lnduced Gastric Lesions in the Rat: Involvement of Histamine H3 Receptors?

This study examined the gastroprotective effect of (R)-alpha-methylhistamine (MHA), a selective agonist of histamine H3 receptors. Gastric lesions were induced in the rat by administering absolute ethanol in a volume of 1 ml. Stomachs were removed 1 h later and lesions evaluated both macroscopically and histologically. MHA dose-dependently inhibited ethanol-induced lesions in the range 1-100 mg/kg both by the intragastric and intraperitoneal route. Histological findings indicated that the number of mucous granules in surface and neck cells was increased and the process of reepithelialization was rapidly promoted by MHA. Thioperamide, at 10 mg/kg, inhibited the protective effect of 10 but not of 100 mg/kg of MHA. Larger doses of thioperamide could not be tested because of an interaction with ethanol causing central nervous system effects. Famotidine and indomethacin pretreatment only partially counteracted the MHA effect. The results indicate that MHA is highly effective in preventing ethanol-induced lesions but the exact mechanism is still uncertain.

Strain-dependent effects of the histamine H(4) receptor antagonist JNJ7777120 in a murine model of acute skin inflammation

Abstract:  The effects of the histamine H4 receptor antagonist JNJ7777120 were evaluated in a model of acute skin inflammation induced by local application of croton oil. The influence of strain on the effect of JNJ7777120 was investigated in four different mouse strains (CD‐1, NMRI, BALB/c and C57BL/6J). In CD‐1 mice, JNJ777720 (30–100 mg/kg subcutaneously, s.c.) exerted a dose‐dependent inhibition of croton oil‐induced ear inflammation and polymorphonuclear leucocyte infiltration, as confirmed by histological evaluation of ear tissues. JNJ7777120 (30–100 mg/kg) did not reduce ear oedema in NMRI, BALB/c or C57BL/6J mice. The positive control, dexamethasone (2 mg/kg s.c.) induced significant anti‐inflammatory effects only in CD‐1 and NMRI mice. In these strains, also the histamine H1‐receptor blocker pyrilamine (30 mg/kg s.c.) significantly reduced ear oedema at 2 h after croton oil challenge, being as effective as JNJ7777120 in CD‐1 mice. Taken together, these data demonstrate that the H4 receptor antagonist JNJ7777120 may reduce acute croton oil‐induced skin inflammation as effectively as H1 receptor blockade. However, present experiments evidenced for the first time marked strain‐related differences in the JNJ7777120 pharmacological activity, which have to be carefully considered when using this ligand to characterize histamine H4 receptor functions in murine models and translating preclinical data to clinical human settings.

Histamine H3 and H4 receptors are expressed on distinct endocrine cell types in the rat fundic mucosa.

The histamine H3 receptor (H3R) is expressed in the central nervous system of different mammalian species [1] and is also localized to the endocrine cells of rat gastric mucosa [2]. In this study, the expression and the distribution of histamine H4 receptor (H4R) within the rat gastric fundus and its possible coexistence with H3R have been explored by immunohistochemistry using our unique immunological probes [3, 4].

Ligands for histamine H3 receptors modulate cell proliferation and migration in rat oxyntic mucosa

(R)‐α‐methylhistamine, a selective agonist of histamine H3 receptors, promotes mucus secretion and increases the number and volume of mucus‐secreting cells. The hypothesis that the increased number of mucous cells could reside in an alteration of homeostasis in the gastric epithelium was investigated. (R)‐α‐methylhistamine was administered to rats 1 h (10–100 mg kg−1 by intragastric and by intraperitoneal route) and 24 h (100 mg kg−1 by intragastric route) prior to killing. The (S)‐isomer of α‐methylhistamine (55.4 mg kg−1), 100 times less potent than the (R)‐isomer at H3 receptors, and the H3‐receptor agonist FUB 407 (9.14–91.35 mg kg−1) were intragrastically administered 1 h prior to killing. The H1‐receptor antagonist mepyramine (30 mg kg−1), the H2‐receptor antagonist famotidine (3 mg kg−1), and the H3‐receptor antagonists ciproxifan (3 mg kg−1) and clobenpropit (30 mg kg−1) were intragastrically administered 30 min before (R)‐α‐methylhistamine. Gastric tissue was processed for histology and immunohistochemistry. Within 1 h, (R)‐α‐methylhistamine and FUB 407 dose‐dependently increased the number of BrdU‐positive cells and of apoptotic cells. (S)‐α‐methylhistamine failed to modify proliferation and apoptosis. The increase in proliferation by (R)‐α‐methylhistamine was reversed by ciproxifan and clobenpropit, but not by mepyramine and famotidine. (R)‐α‐methylhistamine accelerated the differentiation towards pit cells and their outward migration 24 h after its administration. These effects were counteracted by ciproxifan. The apoptosis rate was unaffected at 24 h. These findings reveal a primary role of histamine H3‐receptor ligands in modulating cell proliferation and migration in rat fundic mucosa.

Gastrosparing Effect of New Antiinflammatory Drug Amtolmetin Guacyl in the Rat (Involvement of Nitric Oxide)

The effect of the nonsteroidal antiinflammatorydrug (NSAID) amtolmetin guacyl (AMG) on the gastricmucosa was studied in the rat by means of histologicaland functional techniques. AMG administered at 50-300 mg/kg intragastrically was virtuallydevoid of gastrolesive properties after either acute orrepeated treatment. By contrast, its metabolite,tolmetin (TOL, 15-60 mg/kg, intragastrically) caused dose-dependent gastric damage after bothtreatments. Light and electron microscopy revealed thatAMG induced minimal changes in the surface epitheliumlayer, without signs of vasocongestion or leukocytes adherence. AMG (50 mg/kg intragastrically) didnot change basal gastric potential difference (PD),whereas acetylsalicylic acid and ibuprofen induced fallsin PD of 22 and 27 mV, respectively. AMG (50 mg/kg intragastrically) reduced by 60% the fall in PDinduced by 50% ethanol; this inhibition was dependent onthe incubation time, and was maximal when AMG was given4 hr before ethanol. AMG (100 mg/kg intragastrically) induced an increase in NO synthase type 2(NOS2) activity, which was significantly different fromcontrol values, when AMG was administered 4 hr beforethe test. The metabolites of AMG, tolmetin, MED 5, and guaiacol were ineffective. Pharmacokineticanalysis of the residence time of AMG in the differentareas of the gastrointestinal tract, revealed that AMGremains in the gastrointestinal tract at least for 4 hr, the time necessary for a maximalinduction of NOS2 and for maximal protection againstethanol-induced damage. In conclusion, these dataindicate that the nonsteroidal antiinflammatory drugamtolmetin guacyl is devoid of gastrolesive properties;this gastrosparing effect seems to involve theproduction of nitric oxide, which can counteract thedamaging effects due to prostaglandin inhibition. Thepresence in the stomach of the native molecule ofamtolmetin guacyl seems to be necessary for theprotective effect observed.

Nociceptin/orphanin FQ prevents gastric damage induced by cold-restraint stress in the rat by acting in the periphery

The influence of peripheral nociceptin/orphanin FQ (N/OFQ) on cold restraint-induced gastric mucosal damage in the rat was investigated. Exposure to cold-restraint for 3 and 4h caused the formation of hemorrhagic lesions in the glandular portion of the stomach. N/OFQ dose-dependently decreased lesion formation, in the range 0.03-1 microg/kg/h i.p. Its effect was reversed by the selective NOP receptor antagonist [Nphe(1)Arg(14)Lys(15)]N/OFQ-NH(2) (UFP-101), 30 microg/kg/h ip. The selective NOP receptor agonist [(pF)Phe(4)Aib(7)Arg(14)Lys(15)]N/OFQ-NH(2) (UFP-112), 0.01-0.3 microg/kg/h i.p., similarly reduced lesion formation. Light and scanning electron microscopy confirmed the protective activity of N/OFQ. Cold-restraint stress causes a reduction in mucus content and in adhering mucus layer, partly counteracted by N/OFQ. These results suggest that N/OFQ counteracts acute stress-induced gastric mucosal damage by interacting with NOP receptor and by influencing mucous cell activity.

Histological effect of (R)-α-methylhistamine on ethanol damage in rat gastric mucosa influence on mucus production

Abstract(R)-α-Methylhistamine, a selective agonistof histamine H3 receptors, preventsmacroscopically visible gastric lesions by absoluteethanol in the rat. A further insight into its activitywas the aim of our study. Rats were given saline or(R)-α-methylhistamine (100 mg/kg)intragastrically. After 30 min, absolute ethanol wasgiven and gastric mucosa was sampled 60 min later.Histologic damage and intracellular and adherent mucus werequantified. Luminal surface and mucous cells wereexamined by scanning and transmission electronmicroscopy. (R)-α-Methylhistamine reduced theextent of lesions by ethanol from 96 to 18%. Surface mucous cellsand mucous neck cells were increased in volume andnumber, packaging of intracellular mucus was modified,and the secretory processes were promoted by(R)-αmethylhistamine itself, although these modifications weremostly evident in stomachs subsequently exposed toethanol. Adherent mucus layer thickness was increased by(R)-αmethylhistamine only after ethanol exposure. It is concluded that(R)-α-methylhistamine predisposes mucous cells toreact to ethanol.

Gastric mucosal injury by nonsteroidal anti-inflammatory drugs is reduced by (R)- -methylhistamine and its pro-drugs in the rat

reduced by (R)-a-methylhistamine and its pro-drugs in the rat G. Morini , D. Grandi, M. Krause and W. Schunack Institute of Pharmacology, University of Parma, Via Gramsci 14, Ospedale Maggiore, I-43100 Parma, Italy Institute of Anatomy, University of Parma, Via Gramsci 14, Ospedale Maggiore, I-43100 Parma, Italy Institut für Pharmazie, Freie Universita ̈t Berlin, Königin-Luise-Strasse 2-4, D-14195 Berlin, Germany

Effect of the Macrolide Antibacterial Drug, Tylosin, on TNBS-Induced Colitis in the Rat

Bacterial antigens, such as intestinal microflora, are known to play a role in the pathogenesis of human inflammatory bowel disease (IBD). Tylosin, a macrolide antimicrobial agent, has proven to be effective in cat and dog chronic colitis, but the reasons underlying this efficacy are still unclear. In the present study we evaluated the effects of tylosin on 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis in the rat, in comparison with the antibacterial drug metronidazole and the corticosteroid budesonide. Colitis was induced by a single intrarectal administration of 10 mg TNBS under light ether anesthesia. Tylosin (20 mg/kg twice a day), metronidazole (160 mg/kg twice a day) and budesonide (500 µg/kg once a day) were given orally for up to 6 days to separate groups of rats. The animals were sacrificed after 6 days and colonic lesions evaluated (colon weight, macroscopic and histologic damage, myeloperoxidase activity). Tylosin and metronidazole significantly lowered macroscopic lesion score, reduced colon weight, the severity of histologic lesions and myeloperoxidase activity; budesonide did not significantly change the parameters of colonic inflammation. These data indicate a protective effect of tylosin against intestinal inflammation, suggesting a major role for bacteria, anaerobes in particular, in the development of TNBS-induced mucosal damage.