NEON METHODS

ENERGY EXPENDITURE

PHYSICAL ACTIVITY

FOOD INTAKE / FEEDING BEHAVIOR

BODY COMPOSITION

BLOOD HORMONES AND CYTOKINES

GLUCOSE METABOLISM

MOLECULAR METHODS

SURGICAL PROCEDURES

ENERGY EXPENDITURE

Indirect Calorimetry still is the leading method to quantify energy expenditure (EE). Substrate oxidation rate and respiratory quotient (RQ) are measured and utilized to establish energy balance of rodent models. Through continuous analysis of cage air samples for oxygen consumption and carbon dioxide production rates, EE and fuel preference can be calculated. We are using three types of indirect calorimetry systems:

TSE-LabMaster, Customized Indirect Calorimetry System combined with TSE Drinking and Feeding Monitor and TSE ActiMot
(www.TSE-Systems.com)

This is a novel custom made 32-cage mouse calorimetry system for the simultaneous and continuous quantification of food intake, fluid intake, locomotor activity, energy expenditure and respiratory quotient. This system was established in close collaboration with specialist engineers from the physiology measurement systems manufacturers from TSE (TSE GmbH, Bad Homburg, Germany). The instrument consists of a combination of food intake and fluid intake measurement weight sensors for automated online measurement, which are connected to four computer workstations. A grid of light beams and light beam sensors at two different height levels detects and records every ambulatory movement or rearing as well as climbing movement in every cage. Thereby we can measure all these parameters continuously, simultaneously and in a high number of no less than 30 mice to make meaningful conclusions and statistics easily and routinely feasible.

Indirect Calorimetry System
(self - construction, gas analyzer by ABB)

This calorimeter was established as a self-construction under the Supervision of Prof. S. Klaus before NEON started. It belongs to the animal facility of the German Institute of Human Nutrition in Potsdam. It is equipped with the gas analyzing system Advance Optima from ABB AG (Mannheim, Germany, formerly Hartmann & Braun). It consists of 6 cages and provides one measurement every six minutes per cage. Because of this high frequency measurement this calorimetry system is especially appropriate for investigation of acute treatment effects. In addition, this calorimetry system can be combined with biotelemetry to measure activity and body temperature at the same time (s. below) and it is appropriate for measurements in different environmental temperatures by usage of climatic exposure test cabinets.

Oxymax
(Columbus Instruments, Columbus, OH, USA)

This system has been established in our laboratory at the DIFE in Potsdam. It is an open-circuit indirect calorimeter consisting of calorimetric cages designed to study energy expenditure in mice. This equipment is suited to investigate phenotype effects on energy balance simultaneously in up to 16 mice.

Body Temperature Measurements

We can revert to several methods for measuring body temperature. Body core temperature can be measured in mice and rats by either applying biotelemetry (Mini Mitter Co., Inc., Bend, OR, USA, see below) or by using a rectal thermometer (Physitemp, Clifton, New Jersey). Peritoneal body surface temperature is measured by using an infrared thermometer (Fluke, Grossostheim, Germany). In addition, heat production in unconstrained mice and rats can be visualized by using a high resolution infrared camera (FLIR PM280, FLIR systems, Boston, MA, USA) that uses radiometry technology to convert infrared energy (radiant heat) into 12 bit dynamic range images.

Cold Exposure Studies

To further reveal deficits in energy homeostasis and/or body temperature regulation, mice or rats can be placed into a cold-temperature environment, and body core and surface temperature are subsequently measured. In combination with measurements of energy expenditure this also allows to determine the thermalneutral zone of an animal model.

PHYSICAL ACTIVITY

TSE ActiMot (www.TSE-Systems.de)

TSE ActiMot system (TSE GmbH, Bad Homburg, Germany)for small animals provides an extensive analysis of open field activity, including vertical and rotational behavior and stereotypic episodes. We are using this system in our Cincinnati labs as part of the TSE-LabMaster consisting of 32 cages. Detection of animal location is performed with infrared sensor pairs arranged in strips that can be combined to sensor rings ("X-Y frames"). They operate under any light condition, even in complete darkness thus reducing aversiveness of the test. Graphical evaluation includes a pattern of movement, an activity histogram and a graph displaying temporal distribution of the animal’s stay in the box. By combining indirect calorimetry with TSE Drinking and Feeding Monitor and TSE ActiMot an extensive characterization of parameters determining energy balance can be estimated (see TSE-LabMaster)

TSE InfraMot-Activity System
(www.TSE-Systems.de)

With TSE InfraMot (TSE GmbH, Bad Homburg, Germany) we are able to measure overall motion of animal in 30 cages. The InfraMot unit registers activity of one or more subjects by sensing the body-heat image, i.e. infrared radiation, and its spatial displacement over time. The sensor assembly is designed to be mounted on top of a home cage but it can also be configured for any other arena such as an operant cage or an open field. The convenience to use InfraMot with normal standard animal cages makes it a useful method for obtaining activity data. We are running this system in our labs in Potsdam optionally in combination with the TSE Drinking and Feeding Monitor thereby providing feeding data related to movement patterns.

TSE Running Wheels
(www.TSE-Systems.de)

As opposed to the small space available in a mouse or rat home cage, equipping mouse home cages with running wheels allows voluntary exercise and provides a method to measure physical activity in long-term experiments. In our laboratories in Potsdam we are using up to 32 running wheels that are controlled by a single PC (TSE GmbH, Bad Homburg, Germany). By online registration of running wheel usage after a certain treatment or in a certain animal model we collect information on changes in running habits (time, duration, intensity) and in drive for spontaneous physical activity. Basically, movement is monitored with a rotation sensor which records direction, time duration and number of revolutions. Results protocols are generated listing clockwise and counter-clockwise rotations in user-defined analysis intervals together with changes of directions.

Biotelemetry

Implantation of transponders into the abdominal cavity of rodents (telemetry system) allows for measurement of body core temperature and gross locomotor activity (Mini Mitter Co., Inc., Bend, OR, USA). The normal cage of the animal carrying the transponder is placed on a receiver collecting data about frequency (body temperature) and localization (activity) of the transponder signal. This telemetry system can be used in combination with the self-constructed open circuit calorimeter situated in our animal facility in Potsdam and thereby provides data connecting changes in energy expenditure with changes in body temperature regulation and physical activity. Using this biotelemetry system we collaborate with the research group of Energy Metabolism at the German Institute of Human Nutrition leaded by Prof. Susanne Klaus.

FOOD INTAKE

TSE Drinking and Feeding Monitor
(www.TSE-Systems.de)

This system enables us to measure feeding and drinking behavior online over several days or weeks in mice and rats (TSE GmbH, Bad Homburg, Germany). Circadian rhythms of feeding and drinking and their impairment by special treatments (acutely or in the long-term) can be obtained. Feeding baskets and drinking bottles are attached to a scale, which monitors weight changes due to withdrawal of food or water, by the laboratory animal. Both of our labs endue this system provided by TSE. In Cincinnati the drinking and feeding monitoring system is combined with an indirect calorimetry system (see TSE-LabMaster).

BODY COMPOSITION

EchoMRI, NMR technology
(www.echomri.com)

Echo Medical Systems (Echo) is a Houston-based company that develops manufactures, and markets NMR-MRI and CT-based whole body composition and tissue characterization equipment for use by metabolic research laboratories, the pharmaceutical industry and hospitals, and other medical facilities. EchoMRI™ Quantitative Magnetic Resonance Body Composition Analyzers for animals and humans take direct measurements of total body fat, lean mass, free water, and total body water. The measurement principle depends on the density of hydrogen nuclei and the physical state of the tissue. More rapid, accurate, and precise than other methods, EchoMRI™ technology allows for fast measurements (1min for mice and rats) in vivo without anesthesia or sedation. Unlike DEXA, Echo measurements are radiation-free, do not require the subject to remain still, and in a peer-reviewed study yielded 24 times DEXA's precision facilitating convenient, low-stress repeated tracking of small changes in body composition. Moreover, Echo instruments measure fat and lean mass as an independent characteristics, unlike DEXA, which measures the fat to lean ratio and is, therefore, prone to error propagation from lean to fat. The systems are much less expensive than conventional MRI, are completely silent, and do not require advanced training to use. For further information on EchoMRI systems, please see this article on bone and whole body composition analysis in live mice and visit Echo’s website at www.EchoMRI.com.

Dual-energy-x-ray absorptiometry (Lunar PIXImus)

Another option for the exact measurement of body composition is the DEXA-scan device Lunar PIXImus (Lunar Corporation, Madison, WI, USA), which was specifically designed for small mammals. Besides fat and lean mass dual-energy-x-ray absorptiometry also quantifies bone density. One measurement takes about 5-10 minutes and requires anaesthetization of each animal. The software allows the exclusion of certain arbitrary regions from the analysis to limit the measurement to specific body parts if required by the experimental design. The PIXImus belongs to the research group of Energy Metabolism at the German Institute of Human Nutrition lead by Prof. Susanne Klaus. Both groups closely collaborate.

Tissue Fat Content

To quantify the fat content of specific tissues, we homogenize the tissue, and quantify triglycerides by using the Infinity Triglyceride reagent (Thermo Electron, Pittsburgh, Pennsylvania, USA). To normalize triglyceride contents amongst animals/ groups, protein concentrations or DNA content of the homogenates are being quantified using enzymatic (Bradford) or spectrophotometric methods, respectively.

BLOOD HORMONES AND CYTOKINES

Blood Chemistry

A wide variety of blood chemistry parameters, such as cholesterol, free fatty acids, triglycerides, glucose, ketone bodies as well as diagnostic parameters like ASL/AST and others are routinely measured in our laboratory, using reagents from various suppliers including Thermo Electron (Pittsburgh, Pennsylvania, USA), Wako (Neuss, Germany), Sigma-Aldrich (USA), and others.

Immunoassays (ELISA, FIA, RIA)

For the quantification of hormones and circulating metabolic factors in human and rodent plasma or serum, we established both the use of radioimmunoassays and non-radioactive Enzyme-Linked ImmunoSorbent Assays (ELISAs). Our research includes the analysis of ghrelin (total, active), leptin, insulin, GLP-1, PYY (total, 3-36), IGF-1, and others, using kits supplied mainly by DSL (Diagnostic Systems Laboratories, Inc., Houston, TX, USA), Linco Research (St. Charles, Missouri, USA), and PhoenixPeptides (Burlingame, California, USA)

Luminex/Lincoplex MultiAnalyte Assays

For the combined measurement of several hormones or cytokines from very small samples of plasma or serum, we are applying the new Luminex technology. The Luminex 100TM IS instrument (Luminex Corporation, Austin, TX, USA) measures multiple analytes simultaneously in a single reaction vessel. Our laboratory succesfully applied Lincoplex Adipokine Panels, Endocrine Panels, Adiponectin Single Plex & Cytokine Panels, and others, to quantify analytes in mouse, rat or human plasma/serum using volumes as low as 10 micro litre.

GLUCOSE METABOLISM

We established glucose tolerance and insulin tolerance tests for investigating changes in glucose homeostasis. In addition, we are currently establishing hyperinsulinemic-euglycemic clamps for evaluation of insulin sensitivity in rats and mice.

MOLECULAR METHODS

For investigating functional interrelationships, our laboratory relies on a wide range of standard techniques in cellular and molecular biology. We routinely use real-time PCR to quantify RNA expression of a wide range of targets in multiple tissues. In addition, we use SDS page with western blotting to quantify protein levels and thereby verify gene expression changes. In-situ hybridization is routinely used to visualize gene expression of selected targets, e.g. NPY, AGRP, POMC, or Bsx, in distinctive brain regions of mice and rats. We furthermore apply immunohistochemistry in brain slices and peripheral tissue slices to (co-)localize and quantify protein levels, or study neuronal activity by counting of c-fos positive cells. Tissue pathology is analysed by using H&E stains of paraffin-embedded tissues, lipid content is measured by oil-red staining slices of frozen-tissues.

We also established enzyme kinetic assays to measure the activity of a selected protein. One example is the quantification of fatty acid synthase (FAS) activity in liver and fat tissue by measuring NADPH depletion in a sample using spectrophotometry. To investigate changes in cholesterol metabolism we reestablished a method to evaluate cholesterol distribution among the various classes of lipoproteins. To generate a lipoprotein profile requires plasma samples to be run by fast phase liquid chromatography gel filtration. Afterwards, each fraction of lipoproteins is analyzed for cholesterol by using a colorimetric assay. In addition to in vitro studies, our lab also recently began to perform ex vivo experiments. One approach is to incubate extracted fat cells with the radioactive FAS substrate acetate and measure incorporation of radioactivity into triglycerides to evaluate FAS activity in a “close to in vivo” condition. A number of different ex vivo activity assays are currently being employed in our laboratory.

SURGICAL PROCEDURES

To deliver drugs into the lateral or 3rd ventricle of the brain, our laboratory established intracerebroventricular (ICV) cannula implantation in mice and rats. For peripheral treatments, in addition to injections we also routinely implant osmotic minipumps (Alzet, subcutaneous or peritoneal). We also successfully established the chronic delivery of compounds into the brain by surgical implantation of a minipump connected via a small polypropylen tube to an icv cannula. To study the interaction of the CNS with hepatic metabolism and specifically the impact of the vagus nerve, we managed to employ a surgical procedure in both rats and mice to specifically cut the hepatic branch of the vagus. This technique was established according to recently published procedures for vagotomy. To study the role of gender, and sex hormones, our laboratory performs ovarectomy or orchidectomy surgeries (removal of ovaries, or testes, respectively), with or without the concomitant supplementation of sex hormones. In addition, we perform adrenalectomy (removal of adrenals) and hypophysectomy (removal of pituitary) for investigating endocrine characteristics of rodent models. Sham operations serve to control for surgery related side effects.