Saber/poder en Foucault

For accomplishment of the glucose tolerance tests and GIP/Exendin-4 stimulation tests, two central venous catheters (Cavafix® Certo®, B.Braun) were surgically inserted into the external jugular vein under general anesthesia using a modified method of Moritz et al. (1989). After an intramuscular injection of 2 ml per 10 kg body weight (BW) Ketamin (Ursotamin®, Serumwerk Bernburg) and 0.5 ml per 10 kg BW Azaperon (Stresnil®, Janssen Pharmaceutica, Belgium) pigs could be prepared for surgery. The hair was extensively shaved around the neck and an indwelling catheter was placed into one of the ear veins. Surgical tolerance was reached and general anesthesia was maintained by an intravenous injection of Ketamin (Ursotamin®, Serumwerk Bernburg; dosage: 2 ml per 10 kg BW) and Xylazine (Xylazin®, 2%, WDT; dosage: 0.5 ml per 10 kg BW) as required. Metamizol (1 ml per 10 kg BW, Vetalgin®, Intervet) and Meloxicam (2 ml per 100 kg BW, Metacam®, Boehringer Ingelheim) were given intramuscularly for peri- and post-surgical analgesia. During surgery the inter digital reflex, nasal septum reflex and breathing pattern were controlled regularly. Following aseptic preparation of the surgical field a skin incision, five centimeters in length, was made followed by the exposure of the external jugular vein (Figure 3.1 A). Proximal and distal of the exposed vein a holding suture was placed to facilitate preparation of the intended site for vein cannulation (Figure 3.1 B). A venotomy was made and the catheters were inserted 10 to 15 cm into the vein depending on pig size (Figure 3.1 C). The objective was to position the catheters near the heart base (Figure 3.2). A proximal ligature was placed to stop blood reflux while a distal ligature was needed to fix the catheters (Figure 3.1 D). The incision was closed in two layers. External fixation of the catheters was carried out by a single suture to the skin (Figure 3.1 E). Further, the catheters were covered with sterile gauze and adhesive tape up to the withers level where they were coiled in a pouch to provide easy access (Figure 3.1F). An antibiotic (Cobactan® 2.5%, Intervet) was administered for three days (0.5 ml/10 kg BW) after catheter placement. The catheters were flushed with 250 IU Heparin/ml 0.9% isotonic NaCl solution (Heparin-Natrium, B. Braun, 0.9% NaCl, B. Braun) once daily. At the start of the

study period, all animals had fully recovered from the surgical procedure as evaluated by normal behavior and food intake.

A F E D C B A F E D C B

Figure 3.1 Placement of two central venous catheters:

(A) Exposition of the external jugular vein; (B) vein advanced by a proximal and distal holding suture; (C) placement of the first central venous catheter following venotomy;

(D) placement of the second central venous catheter and distal fixation; (E) skin suture and external fixation of the central venous catheter; (F) pouch for easy access covered with adhesive tape.

Figure 3.2 Latero-lateral thoracic radiograph for determination of catheter placement (arrow marks the end of the catheter near the heart base)

3.3.2.2 Oral glucose tolerance test (OGTT)

The OGTT was performed in 11-week-old non-restrained, freely moving animals. After an 18-h overnight fast, animals were fed 2 g glucose/kg body weight (BW) mixed with 50 g commercial pig fodder (Deuka primo care; Deuka, Düsseldorf, Germany). The meal was eaten from a bowl under supervision. Blood samples were obtained from the jugular vein catheter at -10, 0, 15, 30, 45, 60, 90 and 120 minutes relative to the glucose load. Serum glucose levels were determined using an AU 400 autoanalyzer (Olympus, Hamburg, Germany) while serum insulin levels were measured in duplicate using a porcine insulin radioimmunoassay (RIA) kit (Millipore, Billerica, USA) as described in 3.3.2.6.

3.3.2.3 Intavenous glucose tolerance test (IVGTT)

The IVGTT was performed in 11-week-old and 5-month-old pigs (22.5 ± 1.5 weeks). After an 18-h overnight fast, a bolus injection of concentrated 50% glucose solution (0.5 g glucose/kg BW) was administered through one marked central venous catheter. Blood was collected at -10, 0, 1, 3, 5, 10, 20, 30, 40, 60

and 90 minutes relative to the glucose load in 11-week-old pigs and at -10, 0, 1, 3, 5, 7, 10, 15, 20, 30, 40, 50, 60 and 90 minutes relative to the glucose load in 5-month-old pigs. For blood sampling the second catheter was used to avoid a contamination with the applied glucose. Serum glucose and serum insulin levels were determined as described above (see 3.3.2.2).

3.3.2.4 GIP/Exendin-4 concentration test

To determine the amount of GIP or Exendin-4 administrated in combination with glucose leading to a distinct higher insulin secretion compared to glucose administration only a GIP/Exendin-4 concentration test was performed in 11- week-old non-transgenic pigs. Three different concentrations of synthetic porcine GIP (40, 80 or 160 pmol/kg BW; Bachem, Weil am Rhein, Germany) and synthetic Exendin-4 (20, 40 and 60 pmol/kg BW; Bachem) were tested. Animals were studied on three occasions separated by at least 24 hours. After an 18-h fasting period a bolus of concentrated 50% glucose solution (0.5 g glucose/kg BW) was administered intravenously through one marked central venous catheter. Immediately after glucose administration, synthetic porcine GIP (40, 80 or 160 pmol/kg BW) or synthetic Exendin-4 (20, 40 and 60 pmol/kg BW) mixed with 1% of porcine serum albumin (Sigma-Aldrich, Taufkirchen, Germany) was applied intravenously. Blood samples were collected at -10, 0, 1, 3, 5, 7, 10, 15, 20, 30, 40, 50 and 60 minutes relative to the glucose load through the second catheter. Serum glucose levels and serum insulin levels were measured as described above (see 3.3.2.2).

3.3.2.5 GIP/Exendin-4 stimulation test

The GIP/Exendin-4 stimulation test was performed in 11-week-old GIPRdn transgenic and control animals. Following an 18-h fasting period, 0.5 g glucose/kg BW were administered intravenously as a bolus of concentrated 50% glucose solution through the central venous catheter. Immediately after glucose administration, 80 pmol/kg BW of synthetic porcine GIP (Bachem) or 40 pmol/kg BW of synthetic Exendin-4 (Bachem) mixed with 1% of porcine serum albumin (Sigma-Aldrich) were administered intravenously. Blood samples were collected at -10, 0, 1, 3, 5, 7, 10, 15, 20, 30, 40, 50 and 60

minutes relative to the glucose load. Serum glucose levels and serum insulin levels were measured as described above (see 3.3.2.2).

3.3.2.6 Determination of serum insulin levels by radioimmunoassay (RIA) A porcine insulin RIA kit (Millipore, Billerica, USA) was used to analyze serum insulin levels during an OGTT, IVGTT and GIP/Exendin-4 concentration and stimulation test according to the manufacturer’s instructions. In this RIA, a fixed concentration of labeled tracer antigen (125I labeled insulin) was incubated with a constant dilution of anti-porcine insulin antiserum such that the concentration of antigen binding sites on the antibody was limited. The addition of unlabeled insulin in the form of a serum sample resulted in competition between labeled tracer and unlabeled insulin for the limited and constant number of binding sites on the antibody. Thus, the amount of tracer bound to antibody decreased as the concentration of unlabeled antigen increased. This was measured after separating antibody-bound from free tracer and counting the antibody-bound fraction in a γ-counter. A calibration or standard curve was set up with increasing concentrations of standard unlabeled insulin and from this curve the amount of insulin in unknown samples was calculated. All samples were measured in duplicate. Only duplicates with a coefficient of variance (CV) less than 10% were accepted.

Table 3.6 Assay flow chart of the RIA

Step 1 Add assay buffer

Step 2-3 Add insulin standard/ QC or sample of unknown IC Step 4 Add 125I-Insulin Tracer

Step 5 Add porcine insulin antibody

DAY 1

Step 6 Vortex, cover and incubate 20-24 h at 4°C Step 7 Add precipitating reagent

Step 8 Vortex and incubate 20 min at 4°C

DAY 2

Step 9-11 Centrifuge at 4°C for 20 min, decant and count pellets using a γ-counter

3.3.3 Morphological characterization of GIPRdn transgenic pigs