Procedimiento Para La Valoración

The body weight of mice was recorded once a week from 3-15 weeks of age. Four- month-old mice were anesthetized with ether, weighed and bled from the retro-orbital sinus. After measurement of the NRL, the mice were killed by cervical dislocation. The weights and gross dimensions of the gastrointestinal tract (GIT) were measured according to the methods described by Ogiolda et al. (1998). Briefly, the abdominal cavity was opened, and the caudal end of the duodenum was located and cut with scissors (the duodenum was identified by the absence of mesentery, whereas jejunum/ileum are supported by mesentery). Then stomach and duodenum as well as the other segments of the GIT were separated from adjacent organs and from the mesentery, respectively, and placed on an ice-cold glass plate. After the lengths of duodenum, jejunum/ileum, caecum and colon were recorded, small longitudinal incisions were made, and the luminal contents were flushed out with PBS. The stomach was removed from the duodenum at the pylorus, opened along the large curvature, flattened on a piece of paper, and the outline of the surface area was drawn onto the paper for planimetric evaluation. All other internal organs and the carcass were weighed to the nearest mg and 0.1 g, respectively. The empty GIT was fixed in 10% PBS-buffered formalin in a glass cylinder. After an overnight fixation, all GIT segments were blotted dry and weighed. Then they were immersed for a week in formalin solution and routinely processed for histological analysis. For expression studies, some mice from all groups were sacrificed as described above. The luminal contents of the various GIT segments were removed, transferred into individual centrifuge tubes, solubilized in Laemmli buffer and analyzed immediately as described above. The empty GIT segments were frozen on dry ice and thereafter kept

in -80 °C freezer. Samples from the other organs were frozen on dry ice and stored at -80 °C for RNA and protein isolation or fixed in 4% paraformaldehyde for histological analysis.

3.10.2.2 Morphometry of duodenum

3.10.2.2.1 Tissue preparation and sampling

The duodenum of each animal was longitudinally opened, randomly spun, flatted on a sheet of paper and fixed in 10% PBS-buffered formalin. Vertical sections were created according to Baddeley et al. (1986). Briefly, tissue samples (about 0.8 cm in length) were taken from four equidistant locations from proximal to distal duodenum. For each sample, a pair of mutually perpendicular stripes (about 5 mm in length) was taken, dehydrated and embedded in plastic in order to minimize shrinkage of tissue (Gerrits & Horobin 1996) according to the following procedure:

- Dehydrate and clear the samples using an automate (Shandon) at RT for 20 h: 1 ´ washing solution, 2 h; 2 ´ 50% ethanol, each 1 h; 2 ´ 70% ethanol, each 1 h; 2 ´ 96% ethanol, each 2 h; 3 ´ 100% ethanol, each 2 h; 2 ´ xylene, each 2 h.

Washing solution:

Cacodylic acid sodium salt trihydrate (C2H6AsNaO2× 3H2O) 16.5 g

1 N HCl 6.23 ml

bidistilled water 1500 ml

The pH value was adjusted to 7.2, then 105 g of D (+)-Sucrose (C12H22O11) and 1.105 g CaCl2× 2H2O were added and resolved, pH was adjusted to 7.2. The solution was stored at RT and prepared freshly every month.

- Incubate with GMA-MMA solution [2-Hydroxyethyl methacrylate (GMA): Methyl methacrylate (MMA), 1:1] for 18-24 h at 4 °C with gentle agitation. - Incubate with solution A for 3-4 h (not longer than 4 h) at 4 °C with gentle

- The two tissue samples from the same location were placed on the bottom of a 12 ml embedding plastic cup, oriented perpendicular to surface, then the solution A containing 0.15% (v/v) N,N-dimethylaniline was poured into the cup. The cups were covered and placed in a pre-cooled water bath, which was subsequently placed at 4°C overnight. After the polymerization, the plastic-embedded tissue blocks were pulled out of the cups and stored in a -20°C freezer.

Solution A: Benbzoyl peroxide (with 25 % water) 3.38 g

MMA 200 ml

GMA 600 ml

Ethylene glycol monobutyl ether 160 ml Polyethylene glycol 400 20 ml

- 1.5 µm sections were cut normal to the horizontal plane, and PAS (Periodic-Acid- Schiff) staining was carried out as follows:

A. Incubate with 1% periodic acid solution for 15 min. B. 3 ´ rinsing with bidistilled water.

C. Incubate in fresh Schiff’s reagent in dark room for 80 min. D. Wash with running water for 30 min.

E. Dry on a 60°C stretching table.

F. Incubate with Mayer’s Haemalaun (hematoxylin) solution for 20 min. G. Wash with running water for 10 min.

H. Rinse shortly in 1% HCl-ethanol solution. I. Wash with running water for 10 min. J. Dry on a 60°C stretching table.

K. The sections were at last rinsed shortly in xylene and mounted with Eukitt mounting medium (O. Kindler GmbH & Co, Freiburg, Germany).

3.10.2.2.2 Morphometric analysis

Light microscopic morphometric evaluation was performed on a semiautomated image analysis system as described above. A 10 ´ objective was used, providing a 350 ´ final linear magnification. A cycloid test system consisting of 35 cycloid arcs and 70 test points (Fig. 7B in Baddeley et al. 1986) was photocopied on a transparent

sheet and superimposed over each test field displayed on the monitor screen (Figure 3.4). The vertical axis of the test system was aligned with the vertical direction on the section. Point counting was performed according to Baddeley et al. (1986). The number (P) of test points which hit the different layers of duodenum, and the number (I) of intersection points between cycloid arcs and villous surface were counted. Each vertical section was completely evaluated. With these data, the following parameters were calculated:

Fractional volumes of the 3 layers in duodenum (%):

Vv(x/duo) = 100 ´å P(x) / å P(duo) Total volumes of the 3 layers of duodenum (cm3_):

V(x) = Vv(x/duo)´ V(duo) Villous surface area density (cm-1_):

Sv = 2 (p/l) ´ M ´å I(muc) / å P(muc)

ve

rti

ca

l

1 CYCLOID ARC = — x (FRAME W IDTH) T(10 x 7)₁₀1

ve rti ca l Muc Sub Mus

Figure 3.4 Schematic representation of the cycloid test system (left panel) super- imposed over a test field of a vertical section of the duodenum (right panel). Muc, mucosa; Sub, submucosa; Mus, muscularis.

Total villous surface area (ViSA) of duodenum (cm2_): ViSA = Sv ´ V(muc) where

x = one of the three layers of the duodenum: mucosa (muc), submucosa (sub) or muscularis (mus).

å P(x) = total number of test points which hit the muc, sub or mus layer. å P(duo) = total number of test points which hit the total duodenal wall.

p/l = the ratio of test points to test curve length = 70/(35 x 1.2 cm) = 1/0.6 (cm-1_). M = the final linear magnification (x 350).

å I(muc) = total number of intersection points between cycloid arcs and villous surface. å P(muc) = total number of test points which hit the mucosal wall.

V(duo) = W(duo) (g) / 1.06 g/cm3.

W(duo) = the weight of duodenum before fixation.

1.06 g/cm3 _{= the mean specific gravity of the murine small intestine determined using the fluid} displacement method (Ogiolda et al. 1998).

3.10.2.3 Scanning electron microscopy

Duodenum samples were taken from one- and two-month-old transgenic and control mice, opened longitudinally, washed three times in 0.9 % NaCl solution and fixed in 1 % glutaraldehyde solution. The specimens were then washed in PBS (pH 7.4), dehydrated in ascending concentrations of acetone, dried with a CPD-030 critical- point dryer (BAL-TEC, Schalksmuehle, Germany), and sputter coated (Balzers Union SCD-040, Balzers, Wiesbaden, Germany) with gold-palladium, mounted on a stub and then examined with a scanning electron microscope (DSM-950, Carl Zeiss, Oberkochen, Germany).

Blood was extracted from the tail of 12 h-fasted animals and again after a period of 4 hours after the animals had free access to food. Glucose blood levels were measured with the Medisense Precision QIDTM System (MediSense, Taufkirchen, Germany).

3.10.2.5 Serum glucagon levels

Serum glucagon levels were measured using serum from non-fasted mice with a commercial glucagon RIA kit (BioTrend GmbH, Cologne, Germany).

3.10.2.6 Glucose tolerance test

12 h-fasted animals received orally 250 µl of 1 M glucose solution/30 g body weight. Blood was collected from the tail vain immediately prior to glucose administration and after 10, 30, 60, 90 and 120 min. Blood glucose levels were measured with the Medisense Precision QIDTM System.

3.10.2.7 Serum insulin levels

Blood was collected by bleeding from the retro-orbital sinus from 12 h-fasted animals and again after a period of 4 h after the animals had free access to food. Insulin serum levels were measured with a commercial insulin RIA kit (Insulin-CT, CIS Bio International, Gif Sur Yvette Cedex, France).