In vivo microdialysis for nonapeptides in rat brain--a practical guide

Microdialysis provides a direct approach to monitor changes in interneuronal communication by monitoring the fluctuation of local, extracellular concentrations of potential neurotransmitters/neuromodulators. The present article is based on more than 10 years experience in performing microdialysis experiments in freely moving animals with inexpensive self-made microdialysis probes and accessories for monitoring of intracerebral neuropeptide release. On the basis of this experience, we provide a guide for the construction of different types of microdialysis probes and their application. Furthermore, we give information about organizing and performing a microdialysis experiment that can easily be adapted to fit individual applications needs. Finally, on the basis of theoretical background information advantages as well as limitations of the microdialysis technique are discussed with the intent to provide help to potential users for designing an appropriate microdialysis experiment.     

Protection of mice against bacterial infection by oral administration of bacterial lipopolysaccharide

The effect of orally administered bacterial lipopolysaccharide (LPS) on host resistance against bacterial infections was studied. LPS orally given for 5 consecutive days prior to infection caused no apparent toxic effect and protected mice against Pseudomonas aeruginosa and Listeria monocytogenes infections.     

In vivo neurochemical monitoring by microdialysis and capillary separations

Microdialysis is valuable for studying the neurochemical changes underlying behavior. Recent advances include the application of the high-sensitivity methods of capillary electrophoresis and capillary liquid chromatography with mass spectrometry to dialysate analysis. These methods have improved temporal resolution, spatial resolution, multi-analyte capability and potential for compound discovery.     

In vivo microdialysis for determination of nasal liquid ion composition

Airway surface liquid (ASL) contains substances important in mucociliary clearance and airway defense. Little is known about substance concentrations in ASL because of its small volume and sampling difficulties. We used in vivo microdialysis (IVMD) to sample liquid lining the nasal cavity without net volume removal and incorporated into IVMD a potential difference (PD) electrode to assess airway integrity. The cystic fibrosis (CF) mouse nasal epithelia exhibit ion transport defects identical to those in CF human airways and, thus, are a good model for CF disease. We determined that nasal liquid [Na+] (107 +/- 4 mM normal; 111 +/- 9 mM CF) and [Cl-] (120 +/- 6 mM normal; 122 +/- 4 mM CF) did not differ between genotypes. The nasal liquid [K+] (8.7 +/- 0.4 mM) was significantly less in normal than in CF mice (16.6 +/- 4 mM). IVMD accurately samples nasal liquid for ionic composition. The ionic composition of nasal liquid in the normal and CF mice is similar.     

In vivo sampling with solid phase microextraction

This review discusses the most recent developments and future challenges in the application of solid phase microextraction (SPME) for sampling of live biological samples. The emphasis is placed on applications of fiber SPME for analysis of volatile emissions and drugs in biological fluids. The method development section highlights the main parameters that need to be considered in the case of in vivo experiments: extraction techniques, selection of extraction phases, calibration procedures, determination of free concentrations, and automation.     

In vivo sampling with solid phase microextraction

This review discusses the most recent developments and future challenges in the application of solid phase microextraction (SPME) for sampling of live biological samples. The emphasis is placed on applications of fiber SPME for analysis of volatile emissions and drugs in biological fluids. The method development section highlights the main parameters that need to be considered in the case of in vivo experiments: extraction techniques, selection of extraction phases, calibration procedures, determination of free concentrations, and automation.     

In vivo monitoring of cerebral agmatine by microdialysis and capillary electrophoresis

Agmatine is a putative neurotransmitter in the brain. Current analytical techniques do not allow the detection of agmatine in extracellular fluid, making it difficult to study its physiological role. However, a new method for in vivo monitoring agmatine in the brain was developed. Capillary zone electrophoresis and laser induced fluorescence detection (CZE-LIFD) was used to measure nanomolar concentrations of agmatine in submicroliter sample volumes. This analytical technique proved to detect 0.49 attomole of agmatine improving the sensitivity of previous analytical techniques. On the other hand, the hippocampus is a brain region well known for having a population of agmatine containing neurons. Therefore, intracerebral microdialysis was performed in the hippocampus and agmatine was extracted from the extracellular environment. Detectable amounts of agmatine were found in dialysates from probes located in the hippocampus but not from the probes located in the lateral ventricle. Furthermore, extracellular agmatine was calcium and impulse dependent and depolarization of hippocampal neurons increased extracellular agmatine concentration. The methods reported here are sensitive enough to study the physiological role of brain agmatine in freely moving animals.     

Microdialysis sampling of cytokines

Microdialysis sampling is a well-known method for collection of low molecular weight hydrophilic analytes. Due to the success of this sampling technique for these analytes, many researchers have wanted to extend the use of this method to a wider range of analytes-particularly proteins and peptides. These analytes pose unique challenges during microdialysis sampling. The primary challenges are the reduced recovery across the dialysis semi-permeable membrane and the volume limitations/requirements for the typical immunoassay methods used for detection of proteins. This review covers the practical and theoretical aspects needed for in vivo microdialysis sampling of cytokines, which are a vitally important class of signaling proteins. In addition to the basics of the microdialysis method for sampling cytokines, the use of the microdialysis device as a localized cytokine delivery method is also described. Since relative recovery of cytokines is often low during microdialysis sampling, methods to improve the membrane recovery are discussed for in vitro and in vivo applications.     

In vivo lipolysis in adipose tissue from two anatomical locations measured by microdialysis

In this study, the difference in lipolytic response in inguinal subcutaneous and epididymal adipose tissues of male Sprague-Dawley rats was assessed in vivo by microdialysis. Probes were perfused with Ringer solution in which increasing concentrations of isoproterenol (10(-7) - 10(-4) mol/L) were added. Glycerol release, expressed as extracellular glycerol concentration, was used as lipolytic index. The effect of isoproterenol on local blood flow was investigated using the ethanol technique. No differences were found in the interstitial glycerol concentration between both adipose tissues under basal conditions. When isoproterenol was perfused, a dose-response increase in glycerol production was induced in both tissues. Interstitial glycerol concentration from epididymal adipose tissue was higher than that of inguinal subcutaneous depot at all isoproterenol concentrations. No vasodilatory effect of isoproterenol was found. These results suggest that epididymal adipose tissue is more responsive in vivo to beta-adrenergic lipolysis stimulation than is subcutaneous fat pad from the inguinal region.     

In vivo virulence properties of bacterial cytolethal-distending toxin

Multiple pathogenic Gram-negative bacteria produce cytolethal-distending toxins (CDTs). CDT is typically composed of three subunits: the catalytic subunit CdtB has DNase I-like activity, whereas CdtA and CdtC are binding proteins for delivering CdtB into target cells. Translocation of CdtB to the nucleus induces genotoxic effects on host DNA, triggering DNA repair cascades that lead to cell cycle arrest and eventual cell death. Several lines of evidence indicate that this toxin contributes to the pathogenicity of CDT-producing bacteria in vivo . Helicobacter hepaticus and Campylobacter jejuni CDTs are essential for persistent infection of the gastrointestinal tract and increase the severity of mucosal inflammation or liver disease in susceptible mouse strains. Haemophilus ducreyi CDT may contribute to the pathogenesis of chancroid in rabbits. Recently, H. hepaticus CDT has been shown to play a crucial role in promoting the progression of infectious hepatitis to pre-malignant, dysplastic lesions via activation of a pro-inflammatory NF-κB pathway and increased proliferation of hepatocytes, providing the first evidence that CDT has carcinogenic potential in vivo . Thus, both in vitro and in vivo data indicate that CDT is a bacterial virulence factor.     

In vivo and ex vivo regulation of bacterial virulence gene expression

Bacteria are remarkably adaptable organisms that are able to survive and multiply in diverse and sometimes hostile environments. Adaptability is determined by the complement of genetic information available to an organism and by the mechanisms that control gene expression. In general, gene products conferring a growth or survival advantage in a particular situation are expressed, while unnecessary or deleterious functions are not. Expression of virulence gene products that allow pathogenic bacteria to multiply on and within host cells and tissues are no exception to this rule. Being of little or no use to the bacterium except during specific stages of the infectious cycle, these accessory factors are nearly always subject to tight and coordinate regulation. As a result of recent advances, we are beginning to appreciate the complexities of the interactions between bacteria and their hosts. The ability to probe virulence gene regulation in vivo has broadened our perspectives on pathogenesis.     

The transport and inactivation kinetics of bacterial lipopolysaccharide influence its immunological potency in vivo

The extraordinary potency and pathological relevance of gram-negative bacterial LPSs have made them very popular experimental agonists, yet little is known about what happens to these stimulatory molecules within animal tissues. We tracked fluorescent and radiolabeled LPS from a s.c. inoculation site to its draining lymph nodes (DLN), blood, and liver. Although we found FITC-labeled LPS in DLN within minutes of injection, drainage of radiolabeled LPS continued for >6 wk. Within the DLN, most of the LPS was found in the subcapsular sinus or medulla, near or within lymphatic endothelial cells and CD169(+) macrophages. Whereas most of the LPS seemed to pass through the DLN without entering B cell follicles, by 24 h after injection a small amount of LPS was found in the paracortex. In wild-type mice, ≥70% of the injected radiolabeled LPS underwent inactivation by deacylation before it left the footpad; in animals that lacked acyloxyacyl hydrolase, the LPS-deacylating enzyme, prolonged drainage of fully acylated (active) LPS boosted polyclonal IgM and IgG3 Ab titers. LPS egress from a s.c. injection site thus occurred during many weeks and was mainly via lymphatic channels. Its immunological potency, as measured by its ability to stimulate polyclonal Ab production, was greatly influenced by the kinetics of both lymphatic drainage and enzymatic inactivation.     

In vivo cytogenetic activity of diphenylhydantoin in mice

Diphenylhydantoin was tested in vivo in mice using a variety of cytogenetic endpoints to evaluate its genotoxicity. Injected doses of 125, 250 and 500 mg/kg failed to increase the number of chromosome aberrations in marrow cells at 17 h post-treatment, and 37.5, 75 and 150 mg/kg doses were likewise ineffective at 36 h. SCEs were significantly increased by doses of 125 mg/kg (but not 250 mg) after 23 h and modestly, in relation to dose, at 42 h. No increase in the number of micronuclei among marrow PCEs was seen following single i.v injections ranging from 0.1 to 20 mg/kg. Three daily i.p. injections of doses up to 70 mg/kg also failed to increase the number of micronuclei in either marrow or peripheral blood PCEs. Some cytotoxic effect was evident following relatively high doses.     

Mucociliary transport determined by in vivo microdialysis in the airways of normal and CF mice

We report a novel method to measure mucociliary transport (MCT) in both the upper and lower airways of normal and CF mice. The in vivo microdialysis technique involves placing a small quantity of dye on the airway surface and a microdialysis probe a defined distance from the site of dye deposition. The dye is transported toward the probe by ciliary transport and, upon reaching the microdialysis probe, diffuses across the dialysis membrane and is collected in the dialysate leaving the probe. The rate of MCT is calculated from the length of time from dye deposition to recovery. The rate of tracheal MCT in normal mice was 2.2 +/- 0.45 (SE) mm/min (n = 6), a value similar to that in reports using other techniques. MCT in CF mice was not different (2.3 +/- 0.29, n = 6), consistent with previous observations suggesting that tracheal ion transport properties are not different between CF and normal mice. The rate of MCT in the nasal cavity of normal mice was slower than in the trachea (1.3 +/- 0.26, n = 4). MCT in the CF mouse nasal cavity (1.4 +/- 0.31, n = 8), a region in which the CF mouse exhibits bioelectric properties similar to the human CF patient, was, again, not different from the normal mouse, perhaps reflecting copious gland secretion offsetting Na(+) and liquid hyperabsorption. In conclusion, we have developed a versatile, simple in vivo method to measure MCT in both upper and lower airways of mice and larger animals.     

Production of cytokines after lipopolysaccharide stimulation of murine spleen cells during lymphoma development in AKR mice

Summary Injection of syngeneic lymphoma cells in AKR mice resulted in an important increase of splenic natural killer (NK) activity in the early days following the graft. Modifications of the production of different types of cytokine: interferon, interleukins 1 and 2, tumor necrosis factor (IFN, IL-1, IL-2, TNF), involved in the regulation of NK activity, were investigated in short-term cultures of total, adherent and non-adherent fractionated spleen cells, using lipopolysaccharide as the triggering or amplifying agent.Upon stimulation with lipopolysaccharide, splenocytes from lymphoma-grafted mice released a large amount of interferon as compared to controls with a maximum level 1 day after the graft. Equal amounts of IFN- and IFN-/ were detected. Treatment of spleen cells prior to culture with anti-(asialo-GM1) or anti-(Thy-1.1) antibodies reduced interferon production by 80% and 50% respectively. This finding indicates that (a) the IFN- is produced by Thy-1-positive cells and (b) the production of IFN- by these cells is at least partially under the control of asialo-GM1-positive cells. We also showed that non-adherent fractionated spleen cells from lymphoma-grafted mice produced IL-1 and IL-2. IL-1 was released by asialo-GM1-positive cells and IL-2 by Thy-1-positive cells. Adherent cells released only IL-1. In contrast, total cells released smaller amounts of IL-1 and IL-2, suggesting a reciprocal inhibition between subpopulations of non-adherent and adherent cells. A high level of TNF production by adherent cells was observed only 4 days after the graft. These results indicate that graft of lymphoma cells entails important modifications of spleen cell populations releasing different types of cytokines implicated in NK activation.