PROTOCOL FOR RAT OCCLUSION/REPERFUSION STUDY

In Vivo Study:

Pre-Surgical Procedure:

    1. Weigh rat
    2. Anesthetize with ketamine/xylazine cocktail (100mg/kg ketamine:10mg/kg xylazine) via intraperitoneal injection.
    3. Shave left side of chest for left thoracotomy procedure.
    4. Intubate rat with otoscope, guide guidewire into trachea.
    5. Use guidewire to place intubation tube into trachea. BE CAREFUL TO NOT PUSH TO STRONG AND PUNCTURE TRACHEA.
    6. Check placement of tube via condensation on metal instrument or movement of string.
    7. Connect tube to respirator and secure tightly.

Checklist of Instruments for Rat Occlusion/Reperfusion Surgery

Number

Instrument

1

Large micro needle drivers

1

Small micro needle drivers

1

Classic smaller needle drivers

1

Large rodent spreaders

1

Small rodent spreaders

1

Small curved blunt scissors

1

Small metzenbaums (metz)

1

Small curved sharp scissors

1

Suture scissors

1

Microscissors

2

Blunt small forceps

1

Small rat’s tooth forceps

2

Small sharp microforceps

2

Straight mosquitoes

20

Small Q-tips

10-20 pieces

Small gauze (2x2)

1

Tubing for occlusion

1

Small drape

 

Items need to be autoclaved for reperfusion periods longer than 3 hours.

Surgical Procedure:
    1. Make skin incision with #10 surgical blade over heart.
    2. Blunt dissect through muscle until ribs are visible.
    3. Cut in between ribs: where left atrium and top of left ventricle is most visible.
    4. Gently tease away pericardium: better to tease away over ventricle and not atrium so not to puncture the atrium.
    5. Push lungs, fat pad, and atrium aside with cotton swabs.
    6. Place suture (6-0 silk on taper) close to base of the anterior section of LV and exit close to the septum-circumferential to long axis of LV.
    7. Place the two ends of the suture through a polyvinyl tube and push tube down so that the region becomes pale compared to rest of LV and branching of veins and arteries should be more apparent.-this verifies that occlusion of the artery has taken place.
    8. Clamp securely with a hemostat.
    9. Monitor rat through occlusion time period-30 Minutes
    10. After 30 minutes of occlusion, slowly unclamp hemostat and remove tubing.
    11. Reperfusion times vary as follows:

    RATS NEEDED FOR I/R EXPERIMENTS FOR DIEGO (30 MIN ISCHEMIA)

    REPERFUSION TIME

    # OF RATS DONE

    # OF RATS NEEDED FOR N=3

    5 MIN

    2

    1

    10 MIN

    2

    1

    20 MIN (20-25MIN)

    2

    1

    30 MIN

    1

    2

    1 HOUR

    3

    3

    2 HOURS

    2

    1

    3 HOURS

    0

    3

    6 HOURS (5-6HOURS)

    2

    1

    24 HOURS

    1

    2

    3 DAYS

    2

    1

    5 DAYS

    0

    3

    7 DAYS

    0

    3

    10 DAYS

    5

    0

    CONTROLS (NO I/R)

    ?

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  1. After reperfusion, clamp aorta and inject approximately 4 mL of cardiopeligic solution into the LV.
  2. Excise heart:

Heart Tissue:
    1. Dissect non-heart material.
    2. Dissect aorta, atrium, and right ventricle.
    3. Take an RV, LV, and whole heart weight (RV + LV + atria and aorta)
    4. Cut off RV and place snap tube.
    5. Divide base portion into LV normal tissue (LVN), region not subjected to ischemia and LV damaged tissue (LVD), region of LV that was subjected to ischemia-reperfusion injury and place portions in snap tube.
    6. Submerge punctured snap tubes in liquid nitrogen to freeze.

Place RV, LVN, and LVD into separate test tubes and place in -80°C.