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HYPERINSULINEMIC EUGLYCEMIC CLAMP PROTOCOL

By Elaine Reed,2014-07-09 09:33
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HYPERINSULINEMIC EUGLYCEMIC CLAMP PROTOCOL

HYPERINSULINEMIC EUGLYCEMIC CLAMP PROTOCOL

1) SURGERY

    Insertion of a catheter into the right jugular vein

    Before starting the surgery you need:

    Bring the mouse from the animal facilities, it doesn’t need to be fasted.

    - a catheter, solutions (heparin, NaCl, PVP), syringes (1 mL) filled with heparin, NaCl

    and PVP, anesthetics (isofluoran) and painkiller (Rimadyl), surgical instruments,

    thread and suture.

Catheters:

    Catheters should be set up a week ahead. See Figure 1.

    Micro-renathane MRE-025 catheter from AngTho’s AB...

    Size: ID: ? OD ?

    Drawing : length: ,;?; cm ; piece 1 & 2 (size & characteristics) ; bevel (for insertion

    into the jug. vein)

    Glue pieces 1 & 2. Piece 1 (cathet. HELIX MARK silicone tubing Ref: 60-441-42 ; ID

    0.64mm, OD 1.19mm. Piece 2???

    Solutions:

     - Heparin solution (0.5U/ml). Prepare 15 or 20ml

     - Sodium chloride (15 or 20ml in a tube)

     - Polyvinylpirolidone = PVP (7g/20ml water). Prepare 50ml and store it in a vial

    (wrap it with an aluminium foil).

     Syringes filled with different solutions

     - Before starting the surgery, set up on a rack 3 syringes:

     - 1ml syr (no needle) with NaCl (cf solutions, above) to prevent wound from drying.

     - 1ml syr with heparin solution (0.5U/ml) (cf solutions, above). This syr is:

    “MICRO-FINE U-100 INS from Becton Dickinson. (usually used for insul inj to

    diabetic patients. It has a needle which fits the diameter of the jug vein catheter.

    Fill up the catheter (no bubbles!!!) and leave the syringe connected to the

    catheter.

    - 1 ml syr with PVP (take 0.3ml of the stock solution, see above).

Anesthetics:

    Before starting the surgery inject Rimadyl (s.c). Rimadyl vial: 50mg/ml

    Inject: 0.5mg/100g b.w.

    For a mouse of 25g:

    Dilution of the Rimadyl: 200

    Inject: 125 l

    Isofluoran procedure.

    Put the mouse inside of the chamber and put some drop of isofluoran on the tissue is

    inside of the chamber. Wait until the mouse lost consciousness, it will happen rather

    quick so be alert, take the animal out and place the animal in the hood connected to the

    isofluoran pump. The pump should be set up to the maximum flow rate (~ 4.5) and the

    air concentration always higher than 100, range (100-200). Once you see that the

    mouse breathing rate slows down, set the pump to a flow rate of ~2.8 (this flow rate is

    working well for the mice we are using). Be careful when you move the mouse, he/she

    should be breathing always inside the hood if not it can wake up and start moving.

Important!!!. Once the animal is unconscious place a drop of Viscotears in each eye

    to avoid blindness.

If it is not possible to use Isofluoran use Ketamine/Xylazine:

    - Ketamine vial (= stock sol 50mg/ml).

    Injection of Ketam: 75mg/kg

    For a mouse of 25g inject 75x25/1000 = 1.85mg

    Dilution 5 of the stock sol (50mg/ml to 10mg/ml). Take 2ml of the stock sol and add

    8ml of NaCl.

    Volume to inject to a mouse of 25g: 1000x1.85/10 = 185l.

    - Xylazine vial (= stock sol 20mg/ml).

    Injection of Xylazine: 10mg/kg

    For a mouse of 25g inject 10x25/1000 = 0.25mg

    Dilution 10 of the stock sol (20mg/ml to 2ml/ml). Take 1ml of the stock sol and add

    9ml of NaCl.

    Volume to inject to a mouse of 25g: 1000x0.25/2 = 125l.

    - weigh the mouse and inject the anesth. (IP)

    - use 1cc syringe for each anesthetic with a needle (size: 26G)

    - when the mouse is anesthetized put her /him on the back on a heating pad (set the

     heating on 2).

Instruments, thread and suture

    *2 forceps,

    *microscissors

    *needle holder

    *catheter holder

    *thread ( black silk from Agnthos 6 0 USP …..)

    *suture (vicryl 4/0 with needle: FS-2S 19.0mm 3/8c, from ETHICON)

     - Surgery strictu senso

    - Incisions & positioning of threads

     * Incision and positioning of threads (see drawing) Attach drawing to the figures file

    * Once the incision is done look for the jugular vein, clean the area under the vein and position the threads. The first one should surround the jugular vein plus the peripheral veins ending in the jugular vein. After getting this previous step move gently the thread towards the neck, enough to have space for the incision between both threads. Finally tie up the vein to avoid bleeding, make two knots. The second thread should be place as well surrounding the jugular vein and the closest you can to the muscle wall into the one the jugular vein goes. Don’t tie up the thread yet but leave it ready for it

    just in case the mouse start bleeding after the catheter is inserted in the vein (most probably it will happen this way).

    * The cut should be done carefully and without cutting the whole vein circumference. Try to perform a small incision but enough to be sure that the catheter can get into the vein. Make the incision while pulling gently the thread that is closer to you and already tied up. In this way the vein will be flatter and the bleeding will be less severe. * Insertion of the catheter connected to the syringe with the heparin solution. Be careful and check that there is not air bubbles in the syringe. When the cath is in the vessel inject about 50 l of hep and withdraw blood to check, if it works (the cath is in

    the heart). Very important to verify that you can withdraw blood, if you don’t, they

    are much higher chances that your CLAMP goes wrong the following week.

     * Inject about 50 l of PVP and then close the end of the cath (knot).

     * Ligations (2) to secure the catheter on the piece 1 shown in Figure 1.

    * Tunnel and exteriorise the catheter etc… Make a skin incision between both ears on

    the top of the head, a place where the mouse has a difficult access so it can not pull the

    catheter out. From there make room in the back of the mouse underneath the skin to

    place the catheter. Afterward, take the forceps surrounding the left ear of the animal

    and take it until show up in the ventral incision, now you can grip the catheter with the

    forceps and pull out from the new incision on top of the head. Be careful; don’t pull

    too much just until you see the piece 2 coming from the incision on the head.

    * Now it is time to prepare the end of the catheter. Place a cylinder from a PCR tube

    where the distal knot of the catheter is (it should be at the very end of the catheter ~1-2

    cm from the end) and them fix it with the special “gum”. Then place the catheter under

    the skin on the back of the mouse.

     * close the incision with a suture (vicryl 4/0 with needle, see above)

     - Recovery

     Leave the mouse on the heating pad until he/she wakes up.

    In the surgery notebook:

    Note: date, anaesthetics, mouse weight, strain, ID, and comments about your perception of the surgery, very important for later assessment of the rate of success.

    A day after the surgery reinjects Rimadyl s.c. (0.5mg/100g). See above. Every day after surgery it should be recorded in the animal lab book the mice status until the day of the CLAMP, them you should report the experimental procedure and the way the mice were sacrificed

    2) CLAMP (hyperinsulinemia & euglycemia) + 2 Deoxyglucose uptake in individual tissues)

    Can be performed 3-4 days post-surgery (minimum, best 5-7 days) or a week later (the cath is still patent).

    Equipment:

    *2 pumps

    - pump #1 (Harvard, ) for insulin infusion. Set the flow rate on 3l/min

    - pump #2 (Harvard) for 30 % glucose infusion (= non radioactive glucose = cold glucose) *2 syringes (5ml) for insulin & 30% glucose infusions.

    *tubings to connect the infusion syringes to the jugular vein

Set up the insulin and glucose infusions

    1) Set up the insulin infusion and the bolus of insulin

    - a) Insulin infusion

    The insulin infusate should be prepared from a stock solution 0.4 U/ml

    Set up a stock solution: 0.4U/ml

    Take the vial of insulin (Actrapid NOVO) which is stored in the refrigerator. It should be

    diluted 250 times (from 100U/ml to 0.4 U/ml). Take 50l (Actrapid:100U/ml) and add

    12.5ml of NaCl + BSA ( 2-3%) and aliquot the sol into 1.5ml tubes and store them in the

    freezer.

    Set up the insulin infusate from the stock solution (0.4U/ml)

    During the clamp the insulin infusion rate is: 10mU/min/kg and the flow rate of the

    pump is set on 3l/min.

    How to calculate the volume to pipet (from the stock solution) in order to infuse

    10mU/min/kg?

    Weigh the mouse:

    For a mouse of -10g the insulin infusion rate is 0.033mU/l/min

     - 25g is 0.083 mU/l/min

     - 30g is 0.1 mU/l/min

     - 35g is 0.12 mU/l/min

     - 40g is 0.13

     - 45g is 0.17

     - 50g is 0.20

    - Insulin infusate for a mouse of 25g:

    The final volume of the infusate is 2000l and he/she should get 0.083 mU/l/min

    (see above)

    Take the insulin stock solution (0.4mU/l) stored in the freezer. The volume of

    insulin(x l) to pipet is obtained as follows:

    0.083/0.4 = x l of the stock sol./2000l (final volume). In that case xl=415

    Pipet 415l of the stock sol and add 1585 l of saline)r a mouse of 40g

    0.13/0.4 = x l of the stock sol./2000l (final volume). Pipet 650l of the stock sol

    and add 1400 l of saline.

    Etc …

    Put the insulin infusate (2ml) into a 5ml syringe and fill up the infusion tubing. No

    bubble!!!. Fix the syringe on the pump #1 (Harvard). Set the flow rate on 100l/min

    for 2min in order to be sure that there will be no delay when starting the insulin

    infusion (time zero of the clamp). Don’t forget to reset the pump on 3l/min!!!

    - b) Bolus of insulin:

    In order to quickly reach a steady state during the insulin infusion (hyperinsulinemia)

    a bolus of insulin should be injected just before starting the clamp.

    Take 90l of the stock solution (0.4mU/l) which has been defrost for preparing the

    insulin infusion. Add 510l of NaCl

    Put the bolus into a 1ml syringe (“MICRO-FINE”)

    2) Set up the non radioactive glucose

    Fill up a 5ml syringe with 30% glucose and fill up the infusion tubing. No

    bubble!!!. Fix the syringe on the pump #2 (Harvard). Set the flow rate on 100l/min

    for 2min in order to be sure that there will be no delay when starting the glucose

    infusion (time zero of the clamp). Reset the pump on 2.5l/min!!!

    On a rack prepare a 1ml syringe filled up with the heparin solution (0.5U/ml)

     14Set up the 2 C- deoxyglucose (2DOG)injection

    It could be prepared during the clamp

    Pipet 15 l (= 3Ci) of 2DOG (stored in the freezer) and add 50l ofNaCl. Put the total

    volume into a 1ml syringe (“MICRO-FINE”). Write it down in the right isotope sheet (on the wall close to the door) 14Set up tubes for processsing the 2 C- deoxyglucose (blood samples)

    It could be prepared during the clamp

    After the 2-DOG inj, blood samples should be processed at different time-points: 3, 6, 10, 15,

    20, 30, 40 and 60min. Figure 2

To process them set up 8 tubes with 100l of Zn SO4 (0.3N). After the 2DOG inj 20l of

    blood and 100 l of Ba(OH)(0.3N) will be added (see further down parag. 2DOG injection) 2

    Figure 2 14Set up tubes for processsing the 2 C- deoxyglucose uptake in the tissues

    It could be prepared during the clamp

    Each individual tissue is going to be hydrolyzed with NaOH (1N). Pipet 0.5ml of NaOH into a tube of 1.5ml. Label EDL , SOLEUS etc. Figure3

    Clamp strictu senso

    - Put him/her in a special “beaker”

    - Draw blood from the tail :

    *a drop for measuring the BASAL glucose concentration. It should be measured twice at

    5-10 min time interval.

    * 20l of blood with an heparinized capillary. This sample is put in the refrigerator and it should be centrifuged ASAP at 12000g for 2min. The plasma is pipetted and stored in the freezer for BASAL insulin assay.

    *Pull out the jugular vein catheter which is underneath the skin

    - Flush the catheter with 50 l of heparin solution (0.5U/ml) with 1ml syringe. No

    bubbles!!!!

    - Inject 50l of insulin (= bolus). No bubbles!!!

    - Flush the cath with 50 l of heparin solution (0.5U/ml). No bubbles!!!

    Connect the catheter with the pumps delivery tubing system.

    *Time zero (timer) and turn on both insulin infusion pump (3l/min) and glucose infusion

    pump (2.5l/min)

    *Time 5min: draw a drop of blood for measuring glucose concentration. If the blood glucose level drops, increase the 30% glucose infusion rate to 3l/min in order to go back to the

    BASAL glucose concentration (euglycemia)

    Draw blood every 5-10 min over the first 30-35min

    See a typical chart for the stepwise increment of the glucose infusion rate. Add to the Figures file

    Report in the notebook at each time-point: blood glucose concentrations (basal and during the clamp) and the glucose infusion rates. The glucose infusion rate is going to change over the first 40 min

    It takes about 45 min to reach a steady blood glucose level. It is very important to understand the principle of the euglycemic-hyperinsulinemic CLAMP in order to decide the glucose infusion rate and reach the steady state. If you have doubts ask the people in the lab with experience performing this technique.

* Time 1h : inject 2 DOG while running clamp (once the mouse has reached a steady

    blood glucose level)

    2DOG injection

    Disconnect the infusion tubing from the jug cath and inject the total volume (120l) of 2DOG 14previously set up (see above: Set up the 2 C- deoxyglucose (2DOG)injection). Flush with

    0.5 ml of heparin solut (0.5U/ml) and reconnect the infusion tubing. Reset the timer to zero Draw blood from the tail vein at different time-points: 3, 6, 10, 15, 20, 30, 40 and 60min. At each time point get:

    *one drop for measuring the blood glucose concentration

    *25-30 l of blood with a heparinized capillary. Put the blood in a eppendorf tube and pipet 20l of blood and put it in the 1.5ml tube containing 100 l of ZnSO4 (0.3N) which has been

    14set up (see above: Set up tubes for processsing the 2 C- deoxyglucose samples). Add 100l

    of Ba(OH) (0.3N) to precipitate the proteins. Vortex and leave the sample on the rack. At 2

    completion of the experiment (time 60min) the samples can be centrifuged (3min, 12000g) At time 60 min draw an extra sample of bood (20l) for measuring the insulin concentration

    during the clamp (=HYPERINSULINEMIA). See Figure 2.

    Then, sacrificed the mouse with a pentobarbital inj through the jugular vein Dissect the tissues and process them as follows:

    *Tissue sample processing: see the protocol (Figure 3)

    Each tissue after being dissected and clean (VERY careful with muscle dissection, specially white gastrocnemius: and with BAT make sure to take out all the peripheral white adipose tissue surrounding BAT) is weighed and transferred in the tube already set up with 0.5ml of 14NaOH (see above: Set up tubes for processsing the 2 C- deoxyglucose uptake in the

    tissues)

    To be consistent with the dissections be sure you see and learn from the people with dissection experience. Very important to perform the dissection in the same way and take the same part of each tissue in order to minimize variability.

    Put the tubes in the “Thermo-mixer” which is set on 60? C and shaking (950-1000).

    One hour later, add 0.5ml of HCl (1M) to neutralize NaOH (1M)

    Vortex

    Take 0.2ml of homogenate etc. Details are reported on the drawing (“Tissues processing for 2DOG uptake”). Figure 3

Calculations

    2 DOG uptake in individual tissues (see Excel template). Units: ng of glucose/min/mg tissue

    CLAMP PERFORMED ON OB/OB MICE

    Insulin infusion rate: 75 mU/min/kg (instead of 10)

    - Insulin infusate (can be prepared extempo. or stored in the freezer): 1.2 U/ml

    For a mouse of 50g (that’s the weight most of the time)

    To get a concentration of 1.2 U/ml, take 24l of the insulin (Novo in the refrigerator =

    100mU/l) and add 2ml of NaCl + BSA (2-3%). Dilution: 83

    Insulin flow rate (Harvard pump # 1): 3l/min

    The mouse will get: 1.2mUx3l= 3.6mU/min/50g = 72 mU/min/kg

    - Bolus of insulin: 40mU

     Take 0.1ml of the stock solution (0.4U/ml) and inject (= 40mU) through the jug vein

    - Glucose infusion

    Set the Harvard pump #2 on 1l/min at time zero

    See a typical chart for the stepwise increment of the glucose inf. rate. Add to the Figures file

    GTR (BASAL STATE + CLAMP HYERINSULIUNEMIA-

    EUGLYCEMIA )

SOLUTIONS

    3Radioactive glucose (3-H glucose) 3 - 3-H glucose infusate:

    Pipet 55l (=55Ci) of the stock solution (Amersham vial in the refrigerator) and put it in a counting vial or a 14ml tube.

    Add 2ml of NaCl and take 1.950ml into a 5ml syringe It does not have to be precise. Pipet the rest ( 0.05ml) and put it in an Eppendorf tube (1,5ml). It will be use later on for 3measuring the infusion rate of tracer (cpm of 3-H glucose infused /min)

    Fill up the infusion tubing (no bubbles!!!). Be careful this is a radioactive stuff. Collect the drops in a tube.

    Fix the syringe on pump#1 and set the flow rate on 100l/min for 2min in order to be sure

    that there will be no delay when starting the infusion (time zero). Then reset the pump on 3l/min!!! 3 - Bolus of 3-H glucose:

    Pipet 2.5l (= 2.5Ci) of the stock solution (Amersham vial in the refrigerator) and put it in a 1.5m tube

    Add 50l of NaCl. Take the whole volume into 1ml syringe (“MICRO-FINE”).

Insulin infusate and bolus of insulin

    * Set up the insulin infusate (prepared with NaCl instead of hepar solut) and fix the syringe on pump#1 (see clamp HYPERINS-EUGLYCEM). Don’t connect it to the

    infusion tubing!!!!

    *The bolus of insulin (see clamp HYPERINS-EUGLYCEM) can be set up during the GTR (basal state)

Set up the non radioactive glucose (see clamp HYPERINS-EUGLYCEM)

    * Set up the non radioactive infusate and fix the syringe on pump#2 (see clamp HYPERINS-EUGLYCEM for the setting of the flow rate). Don’t connect the syringe to

    the infusion tubing!!!!

GTR (BASAL STATE)

    Animal handling (see clamp hyperinsul-euglycemia)

    Flush the jugular catheter (see clamp hyperinsul-euglycemia) 3Give the bolus of 3-H glucose and flush the catheter with 0.1ml of NaCl 3Connect the tubing to the jug vein catheter and start the 3-H glucose infusion (turn on the

    pump#1) The infusion rate is 0.09Ci/min

    Time zero

    Note that the insulin and non radioactive glucose (30%glucose) infusion pumps are running but the syringes are not connected to the infusion tubing.

    Time 65, 70 and 75min draw blood from the tail vein and process it as follows:

     - one drop for measurement of glucose concentration (mM)

     - 25l of blood collected with a heparinized micropipet (see fig for processing the

     sample). After processing it gives the cpm/ml of blood. Figure 4

    The ratio: cpm/mM is called specific activity

     - take about 25l of blood for insulin assay (basal). Spin the blood and store the plasma in the freezer.

GTR (CLAMP HYERINSULIUNEMIA-EUGLYCEMIA) 3The 3-H glucose infusion pump runs during the clamp (don’t stop it!!!)

    - Give the bolus of insulin (see clamp hyperinsul-euglycemia)

    - Start the insulin infusion (see clamp hyperinsul-euglycemia): connect the syringe to the infusion tubing.

    -Reset the timer: time zero

    Adjust the infusion rate of 30% glucose (see clamp hyperinsul-euglycemia) At steady state 1h later, draw blood from the tail vein at 65, 70 and 75min and process it as described above (BASAL STATE). . Figure 4.

    - take about 25l of blood for insulin assay (clamp). Spin the blood and store the plasma in the freezer

     3- At completion of the clamp, pipet 10l (should be weighed) of the 3-H glucose infusate

    and put it in a counting vial. Should be done in duplicate

    CALCULATIONS

    See Figure 4 and add template to the protocol or folder in the network.

    GTR = tracer infusion rate (cpm infused/min)/ specific activity at steady state (cpm/mM glucose)

    GTR : moles of glucose /min/ Kg body weight

* Calculation of cpm infused/min

    3 - cpm from 10l (= 10g) of the 3-H glucose infusate should be expressed in cpm per 3l (that’s the tracer infusion rate). See figure 4

    * Calculation of the specific activity at steady state (BASAL & CLAMP)

    The specific activity is given by the ratio: cpm per ml of blood/moles of

    glucose per ml of blood. Note that the glucosemeter gives the concentration of glucose in mM (= mmoles/l of blood)

     - cpm/ml of blood are calculated as follows:

     After processing 20l of blood with Ba(OH)2 & ZnSO4, (See figure 4) only 120

    l of supernatant from a total volume of 220l have been counted (cpm). This cpm value

    should be multiplied by 1.83 in order to get the amount of radioactivity in a total volume of 220l. This latter value (cpm) should be multiplied by 50 in order to get the amount of radioactivity in a total volume of 1ml of blood. In summary just multiply cpm

    (from 120l of supernatant) by 91.6 (1.83x50= 91.6) to get cpm/ml of blood

    - concentration of glucose from the glucosemeter (mM) should

    be expressed in moles of glucose per ml of blood.

*Calculation of the GTR (BASAL & CLAMP):

    cpm infused per min/cpm per moles of glucose = moles

    glucose/min. It should be expressed /Kg body weight.

    * Calculation of the hepatic glucose production at completion of the CLAMP:

     GTR clamp (moles of glucose per min/kg body weight) minus the amount of 30% glucose infused (moles of glucose per min/kg

    body weight) at steady state.

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