The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

This protocol outlines the steps required to produce a robust model of infectious disease and colitis, as well as the methods used to characterize Citrobacter rodentium infection in mice. C. rodentium is a gram negative, murine specific bacterial pathogen that is closely related to the clinically important human pathogens enteropathogenic E. coli and enterohemorrhagic E. coli. Upon infection with C. rodentium, immunocompetent mice suffer from modest and transient weight loss and diarrhea. Histologically, intestinal crypt elongation, immune cell infiltration, and goblet cell depletion are observed. Clearance of infection is achieved after 3 to 4 weeks. Measurement of intestinal epithelial barrier integrity, bacterial load, and histological damage at different time points after infection, allow the characterization of mouse strains susceptible to infection.The virulence mechanisms by which bacterial pathogens colonize the intestinal tract of their hosts, as well as specific host responses that defend against such infections are poorly understood. Therefore the C. rodentium model of enteric bacterial infection serves as a valuable tool to aid in our understanding of these processes. Enteric bacteria have also been linked to Inflammatory Bowel Diseases (IBDs). It has been hypothesized that the maladaptive chronic inflammatory responses seen in IBD patients develop in genetically susceptible individuals following abnormal exposure of the intestinal mucosal immune system to enteric bacteria. Therefore, the study of models of infectious colitis offers significant potential for defining potentially pathogenic host responses to enteric bacteria. C. rodentium induced colitis is one such rare model that allows for the analysis of host responses to enteric bacteria, furthering our understanding of potential mechanisms of IBD pathogenesis; essential in the development of novel preventative and therapeutic treatments.     

Plasma appearance and tissue accumulation of non-esterified, free astaxanthin in C57BL/6 mice after oral dosing of a disodium disuccinate diester of astaxanthin (Heptax)

Oral bioavailability of natural and synthetic carotenoids is generally poor in rodents, and this has limited the ability to test these antioxidant compounds in well-defined rodent models of human disease. Various strategies have been employed, with variable success, to increase the percentage of the total oral dose absorbed by the rodent GI tract. In the current study, a novel carotenoid derivative (the disodium disuccinate diester of astaxanthin; Heptax) was administered by oral gavage in a lipophilic emulsion to C57BL/6 mice. Plasma appearance and tissue accumulation of non-esterified, free astaxanthin was studied by HPLC over 72 h after single- and multiple-dose regimens. One-time dosing of Heptax in emulsion at 500 mg/kg resulted in significant appearance of free astaxanthin in plasma (Cmax=0.2 mg/l; 381 nM) and accumulation in solid organs (e.g. liver Cmax=0.9 mg/l; 1735 nM), levels not previously reported after single carotenoid doses in rodents. At each point in the concentration/time curve (AUC), free astaxanthin levels in liver were greater than the corresponding concentration in plasma, suggesting concentrative uptake by the liver. As the ED50 as an antioxidant for non-esterified, free astaxanthin in model systems is approximately 200 nM, the current results suggest that hepatoprotection against oxidative insults may be achieved after a single dose of Heptax in these animals. In humans, where the bioavailability of oral carotenoids ranges from 40 to 60% of the total dose when given in lipophilic vehicle, much smaller oral doses may be utilized for therapeutic benefit in a particular clinical application.     

Statistical Comparison of Carcinogenic Effects and Dose–Response Relationships in Rats and Mice for 2,4-Toluene Diamine to those Ascribed to Toluene Diisocyanate

The U.S. National Toxicology Program (NTP) conducted 2-year bioassays of commercial grade toluene diisocyanate (TDI) (80% 2,4-TDI and 20% 2,6-TDI) and 2,4-toluene diamine (TDA) and concluded that both were carcinogenic in rodents. In the TDI study, there was an unproven but likely formation of TDA either because of flawed test-substance handling and storage conditions and/or the atypical exposure conditions employed. Although the carcinogenic responses in both studies were qualitatively similar, several statistical analyses were performed to substantiate this possibility more rigorously. Seven different statistical approaches combine to yield a robust and consistent conclusion that, if only a small fraction (approximately 5%) of the dose of TDI were hydrolyzed to TDA in the TDI study, then that would be sufficient to explain the observed carcinogenic responses in the TDI study.     

Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 μl of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.     

Role of the rat liver in the disposal of a glucose gavage

An oral gavage of either 3, 1 or 0.1 mmoles of glucose was given to rats under standard feeding conditions or food deprived for 24 hr. The blood flow of the portal and suprahepatic veins as well as the hepatic balances for glucose, lactate, alanine and pyruvate were estimated.In fed rats, after the administration of an oral 3 mmoles load, the liver actually released 310 µmoles of glucose and 90 of lactate, amounts that could be accounted for by the uptake of alanine (148 µmoles) and small loss of glycogen (275 µmoles of glycosyl residues). In starved rats, however, the liver took a very high proportion (c. 71%) of the glucose absorbed, both as glucose (780 µmoles), lactate and pyruvate (892 µmoles) or alanine (134 µmoles). The synthesis of glycogen was considerably limited, accounting for only 205 µmoles, and leaving practically one mmol of glucose equivalent energy available for liver function and the synthesis of other compounds. Practically all glycogen was synthesized directly from glucose, since the synthesis from 3 C carriers was less than a 5%. Smaller gavages (1 or 0.1 mmoles) resulted in a much lower liver uptake activity.The strikingly different activity of the liver with respect to the available glucose and 3 C fragments could not be explained alone by the circulating levels of these compounds, suggesting a very deep influence of the intestine in hepatic function. The liver plays a very passive role in fed animals, with a very small involvement in the disposal of a glucose load, whereas it takes on an important role when the overall availability of energy is diminished.     

Intestinal handling of a glucose gavage by the rat

An oral gavage of either 3, 1 or 0.1 mmoles of ¹⁴C-labelled glucose was given to rats under standard feeding conditions or food deprived for 24 hr. The fate of the glucose label was determined at 10, 15, 30 and 60 min after gavage; at 60 min 40% of the glucose was absorbed in fed rats (60% in food deprived). The portal vein blood flows were determined and the levels of glucose, lactate, alanine and pyruvate, and their radioactivity, as well as that of CO, were measured in both portal and arterial blood.The net computed glucose and 3-carbon carriers (lactate, alanine and pyruvate) actually released into the portal system by the intestine was lower than the amount of glucose taken up from the intestinal lumen in one hour. Oxidation to ¹⁴CO₂ accounted for a 12–15% of the absorbed glucose. The size of the gavage deeply affected the proportion of glucose released into the portal blood (c. 50% with a 3 mmoles gavage and practically nil with a 0.1 mmoles gavage), but it affected much less the generation of lactate and other 3 C carriers. In fed rats, the net intestinal balance of non-radioactive glucose was negative, and that of lactate positive; when radioactive glucose was considered, the pattern was inverted. In starved rats, both glucose and lactate were released in large proportions by the intestine, but alanine efflux was lower.It can be concluded that the intestine consumes a considerable proportion of glucose in the fed state. Glucose handling by the intestine is compartmentalized in two functional circuits: glucose is taken up from the arterial blood and used for intestinal metabolism and lactate production, luminal glucose is absorbed mainly unaltered and transferred to the portal blood. Thus, the generation of lactate is mainly related to the availability of arterial glucose. In addition to the release of the ingested glucose as 3 C carriers or glucose, an extraportal pathway for glucose transfer into the bloodstream is postulated.     

Systemic and Local Drug Delivery for Treating Diseases of the Central Nervous System in Rodent Models

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution in assessing therapeutic efficacy. Between the two major classifications of administration, local vs. systemic, systemic delivery approaches are often preferred due to ease of administration. However, systemic delivery may result in suboptimal drug concentration being achieved in the CNS, and lead to erroneous conclusions regarding agent efficacy. Local drug delivery methods are more invasive, but may be necessary to achieve therapeutic CNS drug levels. Here, we demonstrate proper technique for three routes of systemic drug delivery: intravenous injection, intraperitoneal injection, and oral gavage. In addition, we show a method for local delivery to the brain: convection-enhanced delivery (CED). The use of fluorescently-labeled compounds is included for in vivo imaging and verification of proper drug administration. The methods are presented using murine models, but can easily be adapted for use in rats.     

Method for detecting hemodynamic alterations following a single gavage in rats

It is known that administering a gavage to rodents evokes a cardiac reflex, due to gastrointestinal stimulation. Consequently, it is difficult to evaluate changes in hemodynamics after a single oral dose of a pungent or astringent, which alters the circulation by increasing sympathetic activity. In the present study, we developed a method for administering a gavage without significantly affecting hemodynamics measurements. We marked a gastric tube at 10 cm from the tip, to mark the distance from the oral cavity to the stomach body of Wistar male rats. Rats were intubated under urethane anesthesia.After 10–15 min of stabilization, we measured the mean blood pressure (MBP), heart rate (HR), and blood flow (BF) in the cremaster arteriole under two different conditions; condition 1: a pointed gastric tube, room temperature distilled water, and injected at normal speed (approximately 3 ml/min); condition 2: a rounded gastric tube, 37°C distilled water, and injection at 1.0 ml/min. Under condition 1, we observed striking hemodynamic alterations, due to the somatic afferent reflex. In contrast, under condition 2, these hemodynamic changes were nearly eliminated. In addition, we could clearly detect hemodynamic changes in rats after a single gavage treatment of pungent (capsaicin) or astringent (cinnamtannin A2). We observed transient increases in the HR and MBP soon after treatment with capsaicin. Moreover, cremasteric BF was elevated with cinnamtannin A2. These results confirmed the utility of the gavage method developed in this study.     

Determination of Vitamin D-dependent calcium absorption by 45Ca gavage in the rat

Precise determination of Vitamin D-dependent intestinal calcium absorption in longitudinal studies is problematic. We have assessed Vitamin D-dependent intestinal calcium absorption by ⁴⁵Ca gavage. Rats were gavaged with a 1 mL solution containing ⁴⁵Ca (CaCl₂, 9.3 MBq/mL) maintained at 37 °C. Total Ca concentration of the gavage fluid was optimised by comparing the absorption curves for fluids made up to 0.025, 2.025, 4.025 and 40.025 mmol/L with ⁴⁰CaCl₂. The effect of varying dietary Ca on fractional Ca absorption was determined in rats fed semi-synthetic diets containing either 0.05%, 0.2%, 0.4% or 1.0% Ca for 50 days. Serum 1,25 dihydroxyvitamin D (1,25D) was determined by radioimmunoassay. Total gavage Ca of 0.025 mmol/L achieved the highest peak fractional absorption and was adopted for all future experiments. Fifty days after allocation to the diets both fractional Ca absorption and 1,25D were highest in rats fed 0.05% Ca and lowest in those fed 1.0% Ca (absorption, P < 0.05 and 1,25D, P < 0.05). There was a strong logarithmic relationship between 1,25D and fractional Ca absorption (R² 0.69, P < 0.001). Weekly repetition of the procedure did not cause a fall in haematocrit over 7 weeks. Radiocalcium (⁴⁵Ca) absorption by gavage provides a simple measure of Vitamin D-dependent Ca absorption for repetitive use in longitudinal studies.