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V3000

Jugular Vein or Carotid Artery Catheterization
Jugular Vein or Carotid Artery Catheterization (please specify in your order).

$75.90 (external)

V3001

Cannulation of cerebral ventricle
Implantation of a cerebral ventricle cannula allows investigators to evaluate physiological responses following central administration of various compounds. Anesthetized mice are placed in a digital stereotaxic apparatus (0.001 mm accuracy, Cartesian Instruments) specifically designed for mice. The dorsal scalp will be shaved, wiped with a betadine solution, and then a small midline incision over the dorsal surface is made to allow access to the cranium. After the affixed centering scope is used to "zero" lambda and bregma landmarks, a single guide cannula (2.5 mm length, 26-gauge, Plastics One) is positioned 1.0 mm above the lateral ventricle (coordinates: 0.6 mm posterior to bregma, 1.5 mm lateral to midline, 1.4 mm below the surface of the skull) and fixed to the skull using two stainless steel screws and dental cement. The incision in the scalp is then closed with surgical thread. Animals are removed to a post-surgical warming bed, and then individually housed for several hours until fully awake. Animals will be allowed to recover from surgery for a minimum of 7 days prior to testing, during which time a 30-gauge dummy cannula is left inside the guide cannula to prevent blockage.

$92/per mouse

V3002

Jugular vein and carotid artery catheterization
Arterial catheterization allows investigators to sample arterial blood as required for adequate glucose clamping (Niswender et al. J. Biol. Chem, 1997, Halseth et al. Am. J. Physiol. 1999) or other infusion/sampling purposes (Rottman et al. Am. J. Physiol. 1999) Catheterization of the right jugular vein allows the infusion of hormones, substrates, and tracers into the systemic circulation. The jugular venous catheter can be used to sample venous blood in long-term experiments because the jugular vein catheter will work for almost a month. Arterial catheters are made from polyethylene tubing (PE-10) that is connected to silicone tubing (0.3 mm I.D., and 0.64 mm O.D.), 25 mm long. Jugular vein catheters are made from silicone tubing (0.3 mm I.D., and 0.64 mm O.D.). These catheters are connected to stainless steel tubes (0.3 mm I.D., 0.41 mm O.D., 15 mm) bent into an L shape. On the free end of the L shaped stainless steel tube a 20 mm piece of micro-renathane tubing (0.36 mm I.D., and 0.84 mm O.D.) is attached. The L shaped stainless steel tubes, attached to an arterial and a jugular vein catheter, are bundled together with silicone tubing (0.76 mm I.D. and 1.65 mm O.D.) and anchored with silastic medical adhesive (Silicone Type A). The catheters and the micro-renathane-stainless steel tubing will be heat sterilized. The mouse is anesthetized and its skin on the interscapula and ventral surface of the neck is depilated by plucking. The depilated area is sterilized with 10% povidone-iodine. A small longitudinal incision (about 5 mm) is made in the skin over where the anterior jugular, acromeodeltoid, and cephalic veins join together. The connective tissues surrounding this junction are carefully removed. Two thin threads of silk (6-0 Silk, Davis+Gech) are passed under the jugular vein below the level of the junction. They are separated by about 3 mm. The cephalic thread, placed just below the joint, is tied to prevent bleeding. A small incision is then made just below the ligature, and the catheter is pushed 13 mm into the lumen. The catheter is fixed with the second thread and the thread previously used to tie the jugular vein. The common carotid artery is separated from the vagus nerve and muscle, and then two thin threads of silk (6-0 Silk, Davis+Gech) are passed under the artery. The cephalic thread is tied to prevent bleeding and then the artery is clamped by small bulldog clamp. A small incision is made just below the ligature, and the catheter is inserted into the lumen. The clamp is taken off and the catheter is pushed in 10 mm. The catheter is fixed with a second thread and the thread previously used to prevent bleeding. A blunt needle (16 gauge) is carefully inserted through the incision on the interscapula and pushed subcutaneously until the end comes out through the incision in the neck. The catheters will be carefully seized and pulled slowly through the needle. The incisions in the skin are then sutured. The catheters are connected to the stainless steel tubes. The bent portion of the stainless steel tubing is implanted under the skin and the incision is sutured. The implanted catheter is flushed with saline containing 200 U heparin/ml and 1 mg ampicillin/ml. Then the micro-renathane tubing is closed with a stainless steel wire. The mouse is injected subcutaneously with 150 mg/kg ampicillin. The total duration of the operation is about 50 min. Animals are removed to a post-surgical warming bed, and monitored until fully awake. Postoperative body weight and food intake are measured daily.

$136.40

V3003

Glucose Tolerance Test (Oral, i.p., Intravenous, or gastric catheter)
Oral glucose tolerance tests are performed on conscious mice with catheters chronically implanted directly in the stomach and the carotid artery. Intravenous glucose tolerance tests are performed on conscious mice with catheters chronically implanted in the jugular vein and carotid artery. Glucose will be given at 1g/kg or 2g/kg. These doses lead to peak blood glucose levels of 250 mg/dl to 400 mg/dl in wild type C57/bl/6 mice.

oral/ip: $36.30; With catheters: consult with Director

V3004

Glucose turnover
A primed (2 µCi) continuous infusion of [3-3H]glucose (0.4 µCi/min) is used to assess the rates of glucose appearance (Ra) and disappearance (Rd). Tracer is infused to allow a steady state to be reached then blood samples are taken to assess arterial glucose specific activity. Ra will be estimated as the ratio of the rate of infusion of [3-3H]glucose and the steady state plasma [3H] glucose specific activity (dpm/mg). Under steady state conditions, the rate of glucose disappearance (Rd) equals the rate of glucose appearance. The rate of glucose clearance is calculated by dividing the Rd by the arterial glucose concentration. Application of this technique is described by Niswender et al. J. Biol. Chem. 1997, and She et al. Mol. Cell. Biol. 2000.

Consult with Director - included with V3005

V3005

Hyperinsulinemic clamp
The hyperinsulinemic clamp is used to measure insulin action in vivo. Hyperinsulinemic clamps are performed on conscious mice with catheters chronically implanted in the jugular vein and carotid artery. A continuous infusion of insulin is given. Glucose levels are monitored in arterial samples every 5-10 min using an Accuchek glucose analyzer that allows the analysis of glucose with only 1 µl of blood. Glucose is infused in the jugular vein catheter at rates necessary to achieve the desired glucose level, based on feedback from arterial glucose measurements. These methods allow assessment of the responsiveness of the body to insulin. Blood from a donor animal is infused to maintain blood volume. By combining this technique with the tracer method one can also examine the impact of insulin on suppression of endogenous glucose production. (1, 2) 1. Ayala JE, Bracy DP, Malabanan C, James FD, Ansari T, Fueger PT, et al. Hyperinsulinemic-euglycemic Clamps in Conscious, Unrestrained Mice. J Vis Exp. 2011:e3188. 2. Halseth AE, Bracy DP, Wasserman DH. Overexpression of hexokinase II increases insulin and exercise-stimulated muscle glucose uptake in vivo. Am J Physiol. 1999 1/1999;276(1 Pt 1):E70-E7.

$639/mouse with [3-3H] glucose + [14C] deoxyglucose (includes surgery and analysis and hormones)

V3006

Hyperglycemic clamp
The responsiveness of the pancreas to glucose is assessed using the hyperglycemic clamp. Hyperglycemic clamps are performed on conscious mice with catheters chronically implanted in the jugular vein and carotid artery. Our clamps include 11 arterial insulin measurements and 3 arterial c-peptide measures. A defined hyperglycemic stimulus is created using a primed variable glucose infusion to raise the glucose level to twice basal for 120 min. An established priming algorithm is used to elevate glucose quickly. Mice with extra copies of the glucokinase gene locus were demonstrated to have a blunted insulin response to hyperglycemia using this technique (Niswender et al. J. Biol. Chem. 1997).

$598 (includes surgery, insulin + c-peptide measurements and analysis)

V3007

Gluconeogenesis & glycogenolysis (from hepatic 14C-UDPglucose and PEP)
The contribution of gluconeogenesis to the rate of glucose appearance is estimated from the specific activities of 14C-labeled hepatic uridine diphosphoglucose (this is assumed to reflect the specific activity of hepatic glucose 6-phosphate), and hepatic phosphoenolpyruvate (PEP) following the infusion of [U-14C]lactate. [3-3H]glucose is used to measure the rate of glucose appearance. Gluconeogenesis is equal to the rate of glucose appearance x [14C]UDP-glucose specific activity/[14C]PEP specific activity x 2. Glycogenolysis is equal to the difference between rates of glucose appearance and gluconeogenesis. The contribution of the kidney to these measurements is assumed to be small.

Consult with Director

V3008

Glycogen synthesis
Using [U-14C]glucose, the incorporation of the carbon of glucose into glycogen can be measured. If the ratio of [14C]UDP-glucose to blood glucose specific activity is assessed the fraction of glycogen formation from direct and indirect pathways can be calculated.

Consult with Director

V3009

Amino acid kinetics
The turnover of phenyalanine (3H ring 2,3,4,5,6 phenylalanine) , glutamine (U-14C-glutamine) and leucine (1-14C-leucine) is assessed by a primed continuous infusion of their respective isotopes for 2 hours (0.2-0.4 µCi/min). Blood samples (20 µl) are taken after a steady state is reached to assess plasma amino acid specific activity. Blood samples are mixed with an equal volume of 6% sulfosalicylic acid. Incorporation of tracer in tissue protein is used to assess tissue specific protein synthesis.

Consult with Director

V3010

Tissue specific glucose uptake
Tissue specific glucose uptake is assessed by measuring the tissue specific uptake of [2- 3H]-deoxyglucose([2-3H]DG). [2-3H]DG is infused (0.2 µCi/min) for 40 minutes or injected (12 µCi) . Arterial plasma samples are taken to determine the time course of [2-3H]DG during the 40 min period. [2-3H]DG is transported into cells and phosphorylated to yield [2-3H]DG-6-phosphate which is trapped in muscle. After 40 min mice are anesthetized with an intravenous infusion of pentobarbital and tissues of interest are rapidly removed and frozen in liquid nitrogen. This method has been applied during insulin- and exercise-stimulated conditions (Halseth et al. Am. J. Physiol. 1999).

Consult with Director

V3011

Tissue specific fatty acid uptake
Tissue fatty acid uptake is assessed by measuring tissue-specific incorporation of circulating 125I-BMIPP (Rottman et al. Am. J. Physiol. 2002). The beta-methyl modification of the long-chain fatty acid BMIPP (beta-methyl-p-iodophenylpentadecanoic acid) causes terminal trapping in the TCA cycle. Studies in man and a variety of small animal models, including rodents, have shown that BMIPP uptake and metabolism closely tracks that of endogenous long-chain fatty acids in a variety of normal and pathophysiologic states. BMIPP is dissolved in proprionic acid, and incorporation of 125I is catalyzed with CuSO4. After extraction, the purified 125I-BMIPP is dissolved in ursodeoxycholic acid, filtered, and adjusted to defined activity. This stable compound is suitable for direct intravascular injection. Serum levels are stable in tracer amounts after injection, and tissue incorporation is measured by gamma counting of freeze-clamped samples in protocols compatible with the simultaneous assay of, for example, [2-3H]DG.

Consult with Director

V3012

Indirect calorimetry / energy expenditure in the Promethion
Whole body VO2 and VCO2 is measured continuously in conscious mice using a Promethion system (Sable Systems Int). The system is sensitive enough to measure small changes in VO2, VCO2 and RQ. They can be used to measure resting or exercising (running wheels or treadmill) gas exchange and energy expenditure. The Promethion is very advanced, allowing for measurement and control of food or water intake. In the Promethion the animals are housed in regular home cages with normal bedding. Activity is measured using beam breaks and converted to pedestrian locomotion. Food and water intake are very accurately quantified. The system integrates all of these data to monitor behaviors and patterns. Body weight and composition is included and measured before and after the metabolic cages. All mice remain in the Promethion cages for 5 days with continuous data collection, allowing for acclimation while also providing extensive data. Full data is reported to the investigator in excel format.

$96.80

V3013

Exercise capacity/Exercise Stress Test (metabolic response to exercise)
Exercise is an integrated measure of fitness. Abnormal exercise capacity and decreased activity are a hallmark of most severe cardiovascular and metabolic diseases, and changes in exercise capacity are sensitive and early markers of cardiac and metabolic dysfunction. Thus abnormalities can be revealed with exercise that may not otherwise be manifested. Gas exchange techniques can be used during treadmill exercise in the mouse to describe the metabolic cost of exercise. Substrate fluxes and metabolism can be assessed isotopically during exercise in chronically catheterized mice (Halseth et al. Am. J. Physiol. 1999). Treadmill exercise can be used to quantify the capacity of a mouse for either endurance or high intensity exercise. Peak exercise capacity and VO2 max will be measured using a closed gas exchange treadmill. Acclimated mice will exercise will at 10 m/min, 0° grade, increased to 14 m/min, 3 minutes later and then increased by 4 m/min a every 3 min thereafter up to 46 m/min or until mouse is exhausted. Exhaustion is defined as the mouse sitting on the shock pad and unable to get off. Time to exhaustion and speed at exhaustion is recorded.

$61.60

V3014

Spontaneous exercise activity
Spontaneous exercise activity is measured using a recording wheel placed in the cage during a 48 h period. The light dark cycle will be stringently controlled to minimize diurnal variations and training effects will be minimized by placing an identical wheel in the cage for the 24 hrs preceding the test measurement. Variables recorded include total distance traveled, peak speed and exercise duration.

Consult with Director - included with V3012

V3015

Food Consumption
Food consumption is assessed using an automated feeding apparatus that continually measure feeding behavior in an unobtrusive manner by allowing animals free access to food cups that are mounted on balances. The apparatus currently is capable of measuring and time-stamping individual weights from 16 balances simultaneously every 30 seconds and downloading the data directly to a computer for subsequent analysis. Therefore, cumulative food consumed and the time at which feeding bouts occur are continuously monitored. All feeding studies are done after the animal has acclimatized to the facility for at least 24 hours.

$4.20/day- included with V3012

V3016

Exploratory locomotor activity
Exploratory locomotor activity

Consult with Director- included with V3012

V3017

Assess real time imaging of cellular metabolic events
Assess real time imaging of cellular metabolic events will be done.

Consult with Director

V3018

In vivo optical imaging of gene expression
In vivo optical imaging of gene expression will be done.

Consult with Director

V3030

In vitro Morphology, Morphometrics and Histology (isolated heart)
A limited necropsy is conducted, noting gross observations, and removing and weighing the heart and lungs separately. After fixation, the heart is sectioned in a standard four-chamber view. Digital photographs on each heart is recorded and archived in a web-accessible format. Chamber and mural dimensions will be measured. The fixed hearts will be maintained in a physical archive, while one slice will be paraffin embedded. 4-5 sections will be cut and prepared for H& E and Masson trichrome. Digital photomicrographs will be recorded and archived together with summary evaluations. A cardiologist with special expertise in mouse cardiac development and histology reviews all gross and microscopic sections.

Consult with Director

V3031

Echocardiography, in vivo morphology, systolic and diastolic function; Stress echocardiography
Echocardiography is a non-invasive technique that can detect the presence of localized or generalized hypertrophy or thinning of the myocardium of the left ventricle (LV) and the presence of regional or global wall motion abnormalities associated with systolic dysfunction. The application of transmitral Doppler analysis allows the detection of abnormal filling patterns associated with LV diastolic dysfunction. The core employs two echocardiography systems, one for high resolution imaging and a high throughput lower resolution imaging system. Echocardiography can be performed on conscious as well as anesthetized mice. In addition to imaging under basal conditions, the mice may undergo stress testing following the administration of dobutamine. (1) Low-Resolution Echocardiography. A 15 MHz linear-array transducer (Sonos 5500, Agilent) is used for high throughput echocardiography in conscious mice. Measurements include LV wall thickness in the intraventricular septum and posterior wall, diastolic and systolic LV diameter, and heart rate. Fractional shortening and ejection fraction are computed as a measure of systolic function. This system also allows for transmitral Doppler analysis that can detect abnormal filling patterns associated with LV diastolic dysfunction. (2) High-Resolution Echocardiography. The Vevo 2100 Imaging System (VisualSonics) utilizes a ultra-high frequency 18-38 MHz linear-array transducer with a digital ultrasound system for superior imaging of mouse cardiac morphology and function as well as Doppler analysis. Measurements include those described for the low-resolution echo. In addition, the VevoStrain analysis software provides a highly sensitive speckle-tracking based echocardiographic imaging technique that offers quantitation of the velocity of the walls, displacement, strain, strain rate, and time to peak analysis.

$27.60 low resolution; $72.00 high resolution using VEVO 2100

V3032

Telemetry (in vivo chronic arterial blood pressure measurement)
Because of difficulties in making physiological measurements in anesthetized mice, a commercially-available system for recording mean arterial blood pressure, heart rate, systolic, diastolic, and mean pressure are used. The primary probe is the PA-C10 which is completely implantable, reducing animal stress, and ensuring the most reliable data. Probes to measure ECG and blood glucose are also available.upon request. This service includes probe implantation, probe removal, data acquisition, and a partial probe refurbishment fee.

$593 per mouse for two week experiment.

V3033

Blood pressure measurements
Blood pressure (BP) represents an integrated measure of overall cardiovascular function, and is affected by stroke volume, heart rate, inotropic state, and vascular tone. Abnormalities of BP regulation (primarily hypertension, but also hypotension) are associated with major cardiovascular morbidity and mortality, and are epidemiologically associated with diabetes and obesity. Three complementary technologies are available for the measurement of BP: 1) non-invasive tail-cuff plethysmography, 2) direct arterial measurement by intracarotid catheterization, and 3) telemetry via implanted catheter. (1) Tail-cuff Plethysmography. Plethysmography (tail-cuff) is performed using a tail-cuff BP apparatus (BP-2000, Visitech Systems, Inc.). This technology is non-invasive and there is good concordance with the direct BP measurements described below. (2) Carotid Catheterization. Direct arterial measurements are obtained via a chronically placed catheter in the carotid artery. The catheter is connected to a TXD-310 transducer and BP measured using a Digi-Med BPA 400 (Micromed). Experiments using this approach are typically coordinated with metabolic measurements. A dual catheter approach (arterial and venous) allows for BP measurements in response to specific pharmacological infusions in the awake or anesthetized state; and enables evaluation of both peripheral and central mechanisms of BP regulation. (3) Telemetry. Telemetered direct BP measurement is performed using an implanted micro-miniature device (PA-C10, DSI) implanted subcutaneously with the catheter typically placed in the right carotid artery. The mouse is housed individually in a cage placed over the receiver platform and BP data digitally recorded via the DATAquest A.R.T. system (DSI). Advantages of this approach include the ability to continuously record BP over a period of weeks, to assess the diurnal range in BP, and stress artifact induced by animal handling is avoided.

Tail-cuff Plethysmography-$8.40; Carotid Catheterization-$81.60; Carotid and jugular vein catheterization-$129.60; Telemetry-$593

V3034

Vascular morphology
A variety of tissues (heart, aorta, kidney, brain) can be processed in four µm sections. Aortic sections are examined for wall thickening, perivascular fibrosis, and fibrin deposition. The inner border, the lumen outer border, the tunica media are traced in each arterial image with Masson's trichrome stain and imaged at a magnification of 200X. The lumen ratio (the medial thickness to internal diameter and the area fibrosis (collagen deposition stained with aniline blue) surrounding blood vessels are calculated and compared. Perivascular fibrosis is determined as the ratio of the area of fibrosis surrounding the vessel wall to the total vessel area.

Consult with Director

V3036

Tail vein injections
Tail vein injections are done in the conscious mouse.

$7.20

V3050

Insulin
30 µl plasma for duplicate analysis. 5-day double antibody assay w/dilution procedure. Detection range: 0.01- 0.5 or 0.1-10 ng/ml. Interspecies or human specific assays are available.

$9.49/sample

V3051

Glucagon
60 µl plasma for samples with basal glucagon concentrations (~50 pg/ml). 30 µl plasma for concentrations >100 pg/ml. Duplicate analysis in plasma preserved in EDTA/Trasylol. 5-day double antibody assay. Detection range: 10-200 pg/ml.

$8.04/sample

V3052

Corticosterone
15 µl plasma for duplicate analysis. 1-day double antibody assay. Detection range: 25-1000 ng/ml.

$25.75/sample

V3053

Catecholamines
Epinephrine and Norepinephrine (HPLC) 50 µl plasma preserved in EGTA-glutathione 1-day assay. Detection range: 50-2000 pg/ml.

$10.75/sample

V3054

Leptin
Available as a Luminex assay

$16.33/sample

V3055

C-Peptide
Available as a Luminex assay

$38.56/sample

V3056

Growth Hormone (GH)
Available as Luminex assay.

$33.29/sample

V3058

TSH
TSH is assayed in a double-antibody RIA format. 55 micro liters of plasma is required for duplicate analysis. It involves a dilution step and is a 5-day double antibody procedure. The lower limit of the assay is 0.4ng/ml.