dithizone and Disease-Models--Animal

Necrotizing enterocolitis is associated with decreased intestinal perfusion and ischemia. Paneth cells, specialized epithelial cells, have been shown to regulate the intestinal vasculature and disruption of these cells has been associated with NEC. We hypothesized that Paneth cell disruption in immature mice intestine would decrease the perfusion of the intestinal microvasculature.. Paneth cells were disrupted in P14-16 mice using chemical (dithizone) and transgenic (diphtheria toxin) methodology. Six hours after Paneth cell disruption, Dylight 488 was injected directly into the left ventricle and allowed to perfuse for 5 minutes prior to intestinal harvesting. Tissue samples were evaluated with confocal fluorescence microscopy to quantify intestinal perfusion and samples were quantified by real time RT-PCR for gene expression.. Dithizone treatment significantly decreased intestinal perfusion compared to controls (p < 0.01). However, diphtheria toxin treatment demonstrated no significant difference in perfusion (p > 0.21). Intestines from all treatment groups had similar PECAM staining, but intestines treated with dithizone had significantly decreased nNOS and iNOS gene expression compared to controls (p < 0.007).. Paneth cell disruption significantly decreases the perfusion of the small intestinal microvasculature in a dithizone-specific manner. Dithizone has no effect on the amount of microvasculature, but does impact genes critical to nitric oxide signaling likely contributing to mesenteric vasoconstriction.

Topics: Animals; Diphtheria Toxin; Disease Models, Animal; Dithizone; Enterocolitis, Necrotizing; Intestine, Small; Ischemia; Mice; Microcirculation; Nitric Oxide; Paneth Cells; Signal Transduction

Necrotizing enterocolitis (NEC) remains the leading cause of gastrointestinal morbidity and mortality in premature infants. Human and animal studies suggest a role for Paneth cells in NEC pathogenesis. Paneth cells play critical roles in host-microbial interactions and epithelial homeostasis. The ramifications of eliminating Paneth cell function on the immature host-microbial axis remains incomplete. Paneth cell function was depleted in the immature murine intestine using chemical and genetic models, which resulted in intestinal injury consistent with NEC. Paneth cell depletion was confirmed using histology, electron microscopy, flow cytometry, and real time RT-PCR. Cecal samples were analyzed at various time points to determine the effects of Paneth cell depletion with and without Klebsiella gavage on the microbiome. Deficient Paneth cell function induced significant compositional changes in the cecal microbiome with a significant increase in Enterobacteriacae species. Further, the bloom of Enterobacteriaceae species that occurs is phenotypically similar to what is seen in human NEC. This further strengthens our understanding of the importance of Paneth cells to intestinal homeostasis in the immature intestine.

Topics: Animals; Animals, Newborn; Autophagosomes; Cecum; Cytokines; Diphtheria Toxin; Disease Models, Animal; Dithizone; Enterobacteriaceae; Enterocolitis, Necrotizing; Gastrointestinal Microbiome; Klebsiella pneumoniae; Mice; Mice, Inbred C57BL; Muramidase; Paneth Cells

Necrotizing enterocolitis (NEC) is a leading cause of morbidity and mortality in premature infants. During NEC pathogenesis, bacteria are able to penetrate innate immune defenses and invade the intestinal epithelial layer, causing subsequent inflammation and tissue necrosis. Normally, Paneth cells appear in the intestinal crypts during the first trimester of human pregnancy. Paneth cells constitute a major component of the innate immune system by producing multiple antimicrobial peptides and proinflammatory mediators. To better understand the possible role of Paneth cell disruption in NEC, we quantified the number of Paneth cells present in infants with NEC and found that they were significantly decreased compared with age-matched controls. We were able to model this loss in the intestine of postnatal day (P)14-P16 (immature) mice by treating them with the zinc chelator dithizone. Intestines from dithizone-treated animals retained approximately half the number of Paneth cells compared with controls. Furthermore, by combining dithizone treatment with exposure to Klebsiella pneumoniae, we were able to induce intestinal injury and inflammatory induction that resembles human NEC. Additionally, this novel Paneth cell ablation model produces NEC-like pathology that is consistent with other currently used animal models, but this technique is simpler to use, can be used in older animals that have been dam fed, and represents a novel line of investigation to study NEC pathogenesis and treatment.

Topics: Aging; Animals; Cell Differentiation; Cytoplasmic Granules; Disease Models, Animal; Dithizone; Enteral Nutrition; Enterocolitis, Necrotizing; Humans; Infant, Newborn; Inflammation; Intestine, Small; Klebsiella pneumoniae; Mice; Paneth Cells; Zinc

Insulin is an anabolic hormone with a broad spectrum of action. When linked with the specific receptors of the cell membrane insulin causes the changes in the whole plasmatic net of the cell and favourable conditions are created for complex biochemical processes. The goal of the research is to study the metabolic effect of insulin on the emotional state of the anxiety according to the animal model (rats). In order to reach the aim passive avoidance test ("punished water drinking") was used in an intact animal model as well as in the one with the experimental diabetes mellitus. Pharmacological substances: insulin and dithizone were applied. After insulin injection the intensification of anxiety state in central neural system is connected to the activation of the enzyme (thirozin-amino-transpherasa), which in its turn intensifies formation of amino acids (glutamine). The latter through either synaptic or outer-synaptic ways effects hippocampus and neurons of the brain tunic causing the increase of excitation and consequently intensifies the anxiety state. It was confirmed that exogenic introduction of insulin causes activation of innate behaviours, in particular the generation of anxiety emotion, that should be considered by an endocrinologist within the whole treatment period of insulin-dependent diabetic patients.

Topics: Amino Acids; Animals; Anxiety; Behavior, Animal; Diabetes Mellitus, Experimental; Disease Models, Animal; Dithizone; Emotions; Hippocampus; Humans; Insulin; Insulin-Secreting Cells; Male; Rats; Rats, Wistar

Zinc has been proven to be anticonvulsant in several studies which indicate that diphenylthiocarbazone (dithizone) and diethyldithiocarbamate (DEDTC), zinc chelating agents, enhance seizure activities. There is also evidence that nitric oxide (NO) generators increase zinc concentration in the brain. On the other hand, the increased level of NO in the nervous system and the consequently increased seizure threshold in cholestatic mice have been well studied. Thus, it could be hypothesized that one of the reasons for the increased seizure threshold in cholestasis is partly the enhanced endogenous zinc concentration, at least in part, due to the overproduction of NO. In this study, we examined the hypothesis that zinc chelating agents might decrease seizure activity to its pre-cholestatic level in bile duct-ligated (BDL) mice. Mice were intra-peritoneally injected with dithizone and diethyldithiocarbamate (DEDTC) before the induction of seizure by pentylenetetrazole (PTZ) and then the seizure activity was recorded. Dose response (dithizone: 5, 30, 100 and 200mg/kg; DEDTC: 25, 50 and 100mg/kg) and time course (only for dithizone: 15, 30, 60 and 120 min) studies were performed first. Then, the effects of cholestasis, with and without dithizone injection, on seizure activity were assessed. Proconvulsant effect of dithizone and DEDTC was proved to be dose dependent although time interval between dithizone and PTZ injections did not play any significant role in the seizure activity. Cholestasis decreased seizure activity and increased lag phase before seizure and both effects were decreased by dithizone injection. It is elicited that zinc may mediate the cholestasis-induced decrement in seizure activity.

Topics: Animals; Bile Ducts; Chelating Agents; Cholestasis; Disease Models, Animal; Dithizone; Ditiocarb; Dose-Response Relationship, Drug; Male; Mice; Pentylenetetrazole; Seizures; Severity of Illness Index; Statistics, Nonparametric; Time Factors

Paneth cells are located at the bases of intestinal crypts, and their cytoplasmic granules contain large amounts of zinc. We previously showed that administration of diphenylthiocarbazone (dithizone), a zinc chelater, to rats induced the selective death of Paneth cells. This was followed by a transient wave of epithelial cell proliferation in the entire crypts. In the study described here, we again applied this experimental model in an attempt to identify novel growth-promoting factors in the small intestine. Male Wistar rats were injected with dithizone and killed 6 hr later. Messenger RNAs (mRNAs) were extracted from the terminal ileum for the construction of a cDNA library. This library was then transfected into the human intestinal cell line Caco-2, and the cells that continued to grow in the medium containing only 1% FBS were cloned. One of the cDNA sequences identified from those transfection experiments was the full-length rat thioredoxin (TRX) gene. To confirm the growth-promoting effect of this cDNA, we transfected it into Caco-2 cells again. These clones proliferated in the medium containing only 1% FBS, while the control clones failed to show any growth. Transient oxidative stress exerted by the addition of oxidative reagents diamide and hydrogen peroxide partially suppressed the growth of TRX-transfected cells. Northern hybridization analysis revealed that TRX expression in rat ileum after dithizone treatment was altered in accordance with intestinal epithelial regeneration in the crypts. Single-cell RT-PCR also showed TRX mRNA expression in Paneth cells. These studies identify rat thioredoxin as a growth-promoting factor for intestinal epithelial cells.

Topics: Animals; Base Sequence; Blotting, Northern; Caco-2 Cells; Cell Division; Culture Media; Disease Models, Animal; Dithizone; DNA, Complementary; Humans; Ileum; Male; Molecular Sequence Data; Paneth Cells; Random Allocation; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sensitivity and Specificity; Thioredoxins