Pancreatic Intravital Imaging Window Installation in Mouse Model: An Integrated Pancreatic Imaging Window for Time-lapse In Vivo Imaging of Murine Pancreas Microstructure

Protocolo

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Surgery

1. Prepare a sterile surgical platform and sterilize the surfaces with 70% ethanol.|
   NOTE: For longitudinal imaging sessions, aseptic technique is essential.
2. Anesthetize mice with a mixture of tiletamine/zolazepam (30 mg/kg) and xylazine (10 mg/kg).
   NOTE: Use of tiletamine/zolazepam is recommended instead of ketamine because of its adverse effect of hyperglycemia. Optimal anesthesia should be selected for the purpose of the experiment.
3. Monitor the body temperature using a rectal probe with a homeothermic controlled heating pad.
4. Shave the left flank of the mouse and apply three rounds of alternating alcohol and iodine-based scrub.
   NOTE: If depilatory cream is used, do not leave longer than 1 minute to avoid chemical burn.
5. Make a 1.5 cm incision on the left flank of the mouse and dissect the skin and muscle.
6. Perform a purse-string suture with a black or nylon 4-0 suture in the incision margin.
7. Use a micro retractor on the incision and gently expose the spleen.
8. Carefully pool the spleen with ring forceps and identify the pancreas.
9. Place the window at the flank of the mouse and pass the spleen and pancreas through the open space of the window. Manipulation of the pancreas has to be very gentle as it can cause bleeding or pancreatitis.
   NOTE: Custom design the pancreatic intravital imaging window to seclude the pancreas from the bowel in the abdominal cavity (Figure 1A, B).
10. Gently place the pancreas on the plate of the imaging window; the spleen will be placed on the open space of the window.
11. Apply drops of N-butyl cyanoacrylate glue on the margin of the imaging window.
    1. To minimize the amount of the applied glue, use a 31 G catheter needle for the application. If the amount of the drop is large, then the tissue will unintentionally adhere to the window or cover glass.
12. Gently apply a 12 mm round cover glass to the margin of the imaging window.
13. Pull the suture loop to fit into the lateral groove of the window and tie it three times.
14. Cut the maximal proximal site of the knot to prevent the interruption of tight stitches when these mice are awake.
15. Let mice recover from anesthesia (2-3 hours) and inject ketoprofen (5 mg/kg, subcutaneous in the loose skin at the base of neck or hip of the animal) for pain relief. Reassess for signs of pain every 12 hours and ketoprofen should be considered every 24 hours for 1-3 days.
    NOTE: Analgesia influences insulin secretion in response to glucose. The choice and timing of analgesia must be individualized for the experimental purpose.
16. Accommodate the mouse in the cage with sufficient bedding to avoid the contact of the window to the cage. Avoid housing the house with the mice without the window surgery.

Resultados

Figure 1. Design and photograph of the pancreatic intravital imaging window. (A) A 3D and cross-sectional view of the pancreatic intravital imaging window. (B) Anterior and posterior photograph of the pancreatic imaging window.

Materiais

Name	Company	Catalog Number	Comments
Cover glasses circular	Marienfeld	111520	Cover glass for pancreatic imaging window
IRIS Scissor	JEUNGDO BIO & PLANT CO, LTD	S-1107-10	This product can be replaced with the product from other company
Loctite 401	Henkel	401	N-butyl cyanoacrylate glue
Micro Needle holder	JEUNGDO BIO & PLANT CO, LTD	H-1126-10	This product can be replaced with the product from other company
Micro rectractor	JEUNGDO BIO & PLANT CO, LTD	17004-03	This product can be replaced with the product from other company
Microforceps	JEUNGDO BIO & PLANT CO, LTD	F-1034	This product can be replaced with the product from other company
Nylon 4-0	AILEE	NB434	Non-Absorbable Suture
Omnican N 100 30G	B BRAUN	FT9172220S	For Vascular Catheter, Use only Needle part
Pancreatic imaging window	Geumto Engineering	Custom order	Pancreatic imaging window - custom order
Physiosuite	Kent Scientific	PS-02	Homeothermic temperature controller
Ring Forceps	JEUNGDO BIO & PLANT CO, LTD	F-1090-3	This product can be replaced with the product from other company
Rompun	Bayer	Rompun	Anesthetic agent
Zoletil	Virbac	Zoletil 100	Anesthetic agent
BALB/C Nude	OrientBio	BALB/C Nude	BALB/C Nude