TITLE: Pressure measurement in intact mouse fundus 
AUTHORS: Maryrose Sullivan, Vivian Cristofaro

[DESCRIPTION]
Each region of the stomach has distinct functional characteristics to ensure coordinated gastric storage and emptying. Inhibitory and excitatory enteric neurotransmission plays an important role in the adaptive relaxation of the fundus and the emptying of its contents. This protocol describes a procedure to examine the functional and mechanical properties of this specific region of the stomach by measuring intralumenal pressure in the isolated intact fundus. This method can be used to explore the motor function of the fundus under a variety of experimental conditions or disease states while largely preserving its three-dimensional shape. 

[STEPS]
SECTION: Isolation of Gastric Fundus
1. Euthanize mice according to IACUC approved method (i.e., CO2 asphyxiation).
SECTION: Isolation of Gastric Fundus
2. After abdominal incision, excise the stomach by transecting the lower esophagus and the pylorus.
SECTION: Isolation of Gastric Fundus
3. Quickly place stomach in Sylgard-coated dissecting dish filled with cold Krebs solution and continuously bubbled with carbogen (95% O2 and 5% CO2).
Kreb's Solution:
NaCl 120mM
KCl 5.9mM
NaHCO3 25mM
Na2H3PO4 1.2mM
MgCl2 • 6H2O 1.2mM
CaCl2 2.5mM
dextrose 11.5mM
. 
SECTION: Isolation of Gastric Fundus
4. Isolate the fundic portion of the stomach from the remaining regions by cutting through the corpus below the gastroesophageal junction, leaving about 3mm of corporal tissue attached to the fundus.
SECTION: Isolation of Gastric Fundus
5. Gently remove the stomach contents.
SECTION: Isolation of Gastric Fundus
6. Immobilize the fundus on the bottom of the dissecting dish using insect pins inserted into the edges of the corpus.
SECTION: Isolation of Gastric Fundus
7. With the aid of a stereoscope, insert a 4F double lumen catheter into the esophageal stump and through the gastroesophageal junction, taking care not to evert the mucosa. Secure in place with suture. Flush both lumens with Kreb's solution to ensure that air bubbles have been removed.
SECTION: Isolation of Gastric Fundus
8. Close the distal edges of the fundus with a horizontal mattress suture using 8-0 silk (8-0 ophthalmic silk, cat# 2023, Mani Inc., Japan), keeping the mucosal surfaces in apposition and using the reflected edge of the corpus to support the stitches. The boundary between the fundus and body can be identified by the difference in mucosal color. 
SECTION: Isolation of Gastric Fundus
9. Close the region below the base of the esophagus with a purse-string suture.
SECTION: Isolation of Gastric Fundus
10. Test for fluid leakage: Attach a 1mL syringe to the catheter and slowly infuse Kreb's solution into the fundic lumen. Verify the absence of bubbles from the suture line.
SECTION: Measurement of pressure in the fundus
12. Attach one lumen of the catheter to a calibrated pressure transducer (PX260, Edwards Lifesciences, Irvine, CA) and the other lumen to an infusion pump (PHD Ultra syringe pump, cat# 70-3007 or 70-3005, Harvard Apparatus, USA). Monitor intraluminal pressure continuously using a Data-Q acquisition system driven by WINDAQ software (DATAQ Instruments, Inc. USA).  Many commercially available acquisition systems are adequate for this purpose.    
SECTION: Measurement of pressure in the fundus
11. Position the intact fundus between platinum electrodes in a 5ml organ bath (cat# 158505, Radnoti LLC, Covina, CA) that has been filled with Kreb's solution, maintained at 37°C and continuously aerated with carbogen.        
SECTION: Generation of nerve-mediated relaxation responses in contracted fundus
14. After equilibration,  slowly infuse Kreb's solution into fundus at 10μl/minwhile continuously measuring intralumenal pressure.
SECTION: Generation of nerve-mediated relaxation responses in contracted fundus
15. With 100μl instilled into the lumen, pre-contract the fundus with prostaglandin F2α (10μM) under non-adrenergic, non-cholinergic conditions (i.e., in the presence of atropine 1μM, and guanethidine 10μM). 
SECTION: Generation of nerve-mediated relaxation responses in contracted fundus
16. Once the isovolumetric pressure in response to PGF2α reaches a plateau, generate nerve-mediated relaxation responses using electrical field stimulation (EFS, 30V, 0.5ms pulse duration, 10 seconds) over a range of frequencies (2-30Hz) delivered by an electronic stimulator (RADSTIM Stimulator, cat# RS1000, Radnoti, Covina, CA, USA).
SECTION: Generation of nerve-mediated relaxation responses in contracted fundus
17. Post-junctional relaxation responses can be examined by exposing the fundus to a nitric oxide donor such as sodium nitroprusside (SNP 10μM).
SECTION: Generation of nerve-mediated relaxation responses in contracted fundus
18. Withdraw the infused fluid from the lumen and wash the tissue by changing the Kreb's solution in the organ bath 2-3 times over 15 minutes. 
SECTION: Evaluation of nitric oxide-mediated relaxation in the fundus
19. To evaluate the contribution of nitric oxide to the relaxation response, add a nitric oxide inhibitor (Nω-nitro-L-arginine methyl ester, 10μM or Nω-nitro-L-arginine, 10μM) to the organ bath for 15 minutes prior to evoking neurogenic relaxation responses as above (repeat steps 14-18).
SECTION: Pressure measurement in response to contractile stimuli
20. Slowly infuse Kreb's solution into fundus at 10μl/min while continuously measuring intralumenal pressure.    
SECTION: Measure weight of fundus
22. At the end of the experiment, remove the catheter, open the fundus along the suture line and weigh the tissue after blotting excess liquid on filter paper.
SECTION: Measurement of pressure in the fundus
13. Equilibrate tissue for 45-60 minutes.  
SECTION: Pressure measurement in response to contractile stimuli
21. To evaluate receptor-independent contractile efficiency of the intact fundus, expose the preparation to high extracellular potassium by replacing the solution in the organ bath with a modified Kreb's solution in which the concentration of KCl is increased to 120mM, with a proportional reduction in the concentration of NaCl.