Rodent Lab 3:  Anesthesia and Blood Collection

Practice the procedures that you learned last week after you have completed this lab's assignments.

1. Assess the physical condition of both of your rodents first!
   
   1. Evaluate the rodent's health:
      • What is its attitude-  friendly, frightened, alert...?
      • What is its general appearance-  well-cared for, unkempt...?
      • Are there any abnormalities-  fight wounds, torn pinna, chromodacryorrhea...?
      • Weigh your rodent-  has its weight changed?  how much?

2. Record the results of your assessment and weight on the...

• Cage card
• Procedure Assessment Form for the animal that you will anesthetize
  

3. If your rodent is anything other than not remarkable, notify your supervisor.

 

2. You will collect blood by several different methods
   1. Mouse:  retro-orbital venous plexus (and tail vein if time allows)
   2. Rat:  cardiac puncture (and lateral saphenous vein if time allows)

    

3. Small rodent anesthesia
   
   
   1. Materials needed:

      (2)  1.00 ml syringes
      (1)  25G x 5/8" needle
      (1)  Lab kit
      
      

   2. Administration of anesthetic  (read Tranquilization, Anesthesia and Analgesia)

      Assess your rodent and write the information on a Procedure Assessment Form
      
      The anesthetic used for the rats and mice at Medaille College is a "cocktail" (mixture) of ketamine and xylazine.  We use the concentration of xylazine to calculate the dose of the anesthetic:

      Anesthetic:	Ketamine/xylazine  (K/X)
      Dosage for mice:	10 mg/kg xylazine IP
      Dosage for rats:	5 mg/kg xylazine IM
      Concentration:	9.1 mg/ml of xylazine

Calculation of the dose:

Weight the rodent and convert its weight to kilograms
Calculate the dose using the values above

Dose  =  weight  x  dosage  x  1 / concentration

Example:  

Mouse weighs 25.6 grams 25.6 g x   1 kg     x  10 mg  x   1 ml     =  ?               1000 g       1 kg       9.1 mg The calculated volume dose of anesthetic is 0.028 ml.  However, this must be rounded to 0.03 ml, because a 1.00 ml syringe can only measure accurately to the nearest 0.01 ml and this will be the actual volume dose. The dose weight must now be corrected for rounding: 0.03 ml x 9.1 mg   =  0.273 mg                  1 ml and the dose information should be written on the record as: 0.273 mg (0.03 ml) K/X IP lower (R) quadrant

Calculation of K/X for my rodent:

I am anesthetizing Rat / Mouse _____ from Cage_____

Calculation:

 

 

Properly complete this "cage card" and have an instructor approve it and the Procedure Assessment Form before drawing up the anesthetic:

Date	Procedures	Name

4. Collection of blood by puncture of the retro-orbital venous plexus

Materials needed:

(1)  Anesthetized mouse
(1)  Insulating pad
(1)  Heparinized Natelson tube
(1)  Clay sealant

Procedure:

• Place the mouse in lateral recumbency
• Assess the depth of anesthesia using the pedal reflex (slight movement is acceptable)
• Place your index finger and thumb on either side of the orbital socket and stretch the skin so that the eye bulges out (becomes opaque)
• Place the narrow end of the Natelson tube into the medial canthus of the eye
• Direct the tube between the eyelids and behind the eye
• With pressure, twist the tube until it enters the venous plexus
• If blood does not appear immediately, withdraw the tube slightly so that it is not occluded by the eye socket
• Tilt the tube downward to allow the blood to flow into the tube
• Fill the tube with approximately 1 cm (1/4 inch) of blood
• Remove the tube with your finger covering the distal end of the tube and seal it in the clay
• Do not wipe blood from the eye-  close the eye and allow the blood to clot (wiping the eye will tear the clot off and cause more bleeding to occur)

Recovery:

• Place the mouse on a heating pad until the end of lab or the mouse can walk
• Return the mouse to a cage with only same sex anesthetized mice
• Complete Procedure Assessment Form and leave it on the clipboard in the lab.

Record on the cage card (you should previously have recorded the results of the P.E. and administration of the anesthetic):

Collected 1.2 cm blood in Natelson tube via (R) retro-orbital venous plexus

PostProcedure Assessment:   return the next class day to-

• Assess the mouse's condition  (and report any problems)
• Complete the PostProcedure Assessment Form 
• Return the mouse to its original cage

 

5. Collection of blood via cardiac puncture
   (This is a terminal procedure when used for blood collection, but the intracardiac (IC)  injection is an important technique in the small animal practice for emergency drug administration and euthanasia of very small or very ill animals.  This will be your only opportunity to practice this technique during your education at Medaille College.)

Materials needed:

(1)  Anesthetized rat
(1)  3.0 ml syringe
(1)  22G x 3/4" needle
(1)  Insulating pad
(1)  Lab kit

Procedure:

• Place the rat in dorsal recumbency
• Ensure that there is NO pedal reflex
• Insertion site:  draw an imaginary line down the sternum perpendicular to the rat's elbow (between ribs 5 & 6). Move slightly to the left of the sternum-  this is the insertion site
• Preparation-
  • Prepare a cotton ball saturated with alcohol for disinfection
  • Prepare your needle and syringe by breaking the seal of the plunger
  • Hold the rat's thorax between your thumb and middle finger
  • Palpate the thorax with your finger to find the strongest heartbeat
  • Swab the thoracic area with the alcohol swab...then don't touch it!
• Insertion-
  • ONLY MOVE YOUR NEEDLE IN AND OUT!!  Never move the needle side-to-side:  you may lacerate the heart if your needle is moved laterally!!
  • With your dominant hand, hold the syringe like a pencil 
  • Insert the needle through the thoracic wall between ribs 5 & 6 (at the site of the strongest heartbeat) with the needle angled toward the midline 
  • If you see blood percolating into the hub of the syringe, gently aspirate to collect 0.2 ml of blood
  • If no blood is seen-
    • Insert the needle into its hub and gently aspirate
    • While gently aspirating, slowly withdraw the needle
    • If still no blood is obtained, remove the needle and apply gentle pressure to the insertion site  (the needle must be replaced with a new sterile needle)
  • Recap the needle securely into the syringe tray
    
    
• Cardiac puncture can also be performed with the rat in lateral recumbency-
  • Place the rat on its right side
  • Feel for the strongest heartbeat (under the left elbow)
  • Insert the needle at an 80 degree angle (not quite perpendicular)
  • Complete collection as described above
    
    
• Recovery:
  • Place the rat on a heating pad until the end of lab or the rat can walk
  • Return the rat to a cage with only same sex anesthetized rat
  • Complete Procedure Assessment Form and leave it on the clipboard in the lab.

  Record on the cage card (you should previously have recorded the results of the P.E. and administration of the anesthetic):

  Collected 0.2 ml blood by cardiac puncture

  PostProcedure Assessment:   return the next class day to-

  • Assess the rat's condition  (and report any problems)
  • Complete the PostProcedure Assessment Form 
  • Return the rat to its original cage

 

6. Collection of blood from the lateral saphenous vein

   Materials needed:

   (1)  Unanesthetized rodent
   (1)  25G hypodermic needle
   (1)  Clippers or scalpel blade
   (1)  Heparinized microcapillary tube
   (1)  Lab Kit

Restraint-

• Insert the rodent's head into a restraining device
• Extend a hind leg and immobilize it
• Shave the fur off of the lateral surface of the leg

Procedure-

• Swab the area with an alcohol swab
• Use a sterile 25G hypodermic needle to puncture the saphenous vein as it crosses the lateral surface of the leg at the level of the patella
• A drop of blood should be visible at the puncture site
• Hold a capillary tube against the drop of blood to fill it.
• Place your finger over the opposite end of the tube and withdraw it 
• Holding the lower portion of the tube, press it into the clay

To ensure hemostasis (clotting of blood), relax your hold on the rodent's leg and apply direct pressure with a dry cotton ball

Record the procedure on the rodent's cage card and return it to its original cage

7. Collection of blood from the lateral tail vein

Materials needed:

(1)  Unanesthetized rodent
(1)  Cage lid or other restraining device
(1)  27G x 1/2" hypodermic needle
(1)  Heparinized microcapillary tube

Procedure: 

• Apply lidocaine topical anesthetic, wait 1 minute and wipe with an alcohol swab
• Thread the hypodermic needle cranially into the lateral tail vein
• When blood appears, insert the capillary tube into the hub of the needle
• If blood does not appear, slowly pull the needle out of the vein.  You may have passed through the vein and may enter it while withdrawing.  As soon as blood appears, stop withdrawing the needle and collect blood
• To ensure hemostasis, place a dry cotton ball over the needle prior to removing the needle from the vein and then apply pressure until bleeding ceases