Transcardiac Perfusion

Safety Precautions

• You must complete required lab safety training before starting this procedure.

• You must complete required animal use training and listed on IACUC protocol before starting this procedure.

• If this is your first time doing this procedure, ask to be trained by an experienced lab member.  If you have not done this in a while, you should ask for a refresher.

• Also review the following material:

  • Kuwajima M, Mendenhall JM, Harris KM (2013) Large-Volume Reconstruction of Brain Tissue from High-Resolution Serial Section Images Acquired by SEM-Based Scanning Transmission Electron Microscopy. Methods Mol Biol (Nanoimaging: Methods and Protocols) 950:253-73. (PDF)

• Further reading:

  • Hayat MA (1981) Fixation for electron microscopy. Academic Press

  • Karnovsky MJ (1965) A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J Cell Biol 27:137A-138A. (PDF)

  • Tao-Cheng JH et al. (2007) Structural changes at synapses after delayed perfusion fixation in different regions of the mouse brain. J Comp Neurol 501:731-740. ()

  • Dehghani A et al. (2018) Nuclear expansion and pore opening are instant signs of neuronal hypoxia and can identify poorly fixed brains. Sci Rep 8:14770. ()

• Before starting, even if you have done this procedure before,

  • read this protocol entirely.

  • If you are using adult rats, review the instruction movie on the tracheotomy procedure.

  • review relevant Safety Data Sheets and Harris Lab SOP (also see below).

  • ensure you have all reagents and supplies listed below.

  • ensure all equipment is in good working order.

    • Isoflurane vaporizer must be serviced annually; check tubing, surgical instruments, etc.

  • have all waste containers ready (also see Clean-up).

  • plan your schedule well so that you wouldn’t be rushed to complete the procedure.

• Review SDS and Harris Lab SOP for the following hazardous chemicals used in this procedure:

  • Ethanol: flammable; irritant (eye)

  • Formaldehyde

  • Glutaraldehyde

  • Hydrochloric acid

  • Isoflurane

  • Sodium cacodylate: carcinogen; irritant (skin, eye); skin permeator

  • Sodium hydroxide

• Other hazards associated with this procedure include:

  • Physical: compressed gas cylinder (O₂/CO₂)

  • Sharps: Surgical scissors, scalpel, needles

  • Biological: Live rats/mice, blood, animal waste and soiled cage bedding

• The following Personal Protective Equipment is required for this procedure:

  • Lab coat

  • Nitrile gloves (double-layer required; regularly check for holes)

  • Eye goggles

  • Mask

  • Face shield

• Place a piece of absorbent sheet on the work surface before starting the procedure.  When done, discard into the “Solid Waste – No UA” bag

Reagents, Supplies, Equipment

Personnel Protective Equipment

• Eye goggles, face shield

• Gloves (nitrile)

• Lab coat

• Mask

• Plastic Apron (optional)

Assorted tubing, connectors, and clamps

• Barbed fittings from Cole-Parmer

• Luer kits from World Precision Instrument

• Silicon rubber tubing for anesthesia line

• Vacuum line for waste collection system

• IV line with clamp and flow regulator for perfusate lines

  • Perfusate lines should be transparent enough to be able to see air bubbles.

• Tygon tubing for general use

Air Pressure System

• Sphygmomanometer with a hand pump (cut off the cuff)

  • Use hand pump in the absence of an air pump.

• Air pump: Perfusion Two (Leica Biosystems 39471010)

  • This pump is designed to deliver constant air pressure at 300 mmHg. To be able to adjust pressure, we added a regulator (Air Logic R-7010-2B2B-W/K) between "Air Tank" and "Perfusion Pressure" switches (See Figures 2-4).

• Air filter (Parker Hannifin 9933-05-AAQ)

• 1-L glass bottle (Kimax, or equivalent) × 1

  • This can be substituted by a plastic air tank (e.g., Clippard AVT-PP-35).

• #6 two-hole stopper × 1

Oxygenation System

• O₂/CO₂ (95%/5%) cylinder with regulator and support

Anesthesia System

• Isoflurane (Animal Health International 19632158; stored at RT in well-ventilated area)

• Medical grade O₂ cylinder with regulator and support (Can be replaced by O₂/CO₂ or an oxygen concentrator)

• Large glass desiccator, wad of 4-5 Kimwipes, Pasteur pipet

• Matrx VIP3000 Isoflurane Vaporizer (Stoelting)

  • This apparatus must be serviced annually.

  • For Central Texas area: William Connery, Handlebar Anesthesia, (512) 423-7668, handlebaranesthesia@yahoo.com

• A small animal ventilator (Harvard Apparatus Model 683, or equivalent)

• Nose cone (e.g., Stoelting; Kent Scientific)

• endotracheal tube (a modified 16-gauge hypodermic needle) with Y-connector

• Optional: Plexiglas anesthesia induction chamber (e.g., Stoelting; Kent Scientific)

Perfusate System

• Deep water bath (e.g., VWR Digital Unstirred Water Bath, L×W×D = 12⁷/₈"×11¹³/₁₆"×5²⁹/₃₂"; VWR 89032-216)

• Lead donuts × 2

• #6 stoppers: three-hole × 1, two hole × 1

• 3-way valve × 1

• Flow regulator × 1

• 13-gauge needle with 60° bevel with cork disk ½ inch from tip (to restrict depth of penetration; use a16-gauge for juvenile rats)

• Air stone × 1

Waste Collection System

• 4-L vacuum flask (preferably plastic-coated) × 1

• #11½ one-hole stopper × 1 

• Vacuum line filter (Whatman VACU-GUARD, VWR 28137-858), 1 per day of perfusions

Dissection/surgical instruments

Scissors and knife etc. should be sharpened periodically.

• Stainless steel dissection tray with Styrofoam board cut to fit snugly lengthwise in the tray with ~1-inch space on one side. 

• Pins to secure animal to Styrofoam board

• Scalpel and blades

• Assorted dissecting scissors

• Forceps

• Hemostats

• Retractors

• Knife (to decapitate rat)

• Bone Rongers

• Spatulas

• Silk suture (or cotton tread from fabric store can be used instead)

• Gauze pads

• Applicator sticks

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For Preparation of Perfusates

All reagents are stored at RT unless otherwise noted.

• Calcium chloride dihydrate (CaCl₂·2H₂O; Sigma-Aldrich 223506)

• d-Glucose (Sigma-Aldrich G7528)

• Formaldehyde (20% aqueous solution in 100 ml bottles; Ladd Research 20304)

• Glutaraldehyde (50% aquieous solution in 100 ml bottles; Ladd Research 20211; stored at 4°C)

• Hydrochloric acid (HCl; 1 M aqueous solution; for adjusting pH)

• Magnesium sulfate heptahydrate (MgSO₄·7H₂O; Sigma-Aldrich M5921)

• Potassium chloride (KCl; Sigma-Aldrich P9333)

• Sodium bicarbonate (NaHCO₃; Sigma-Aldrich S6297)

• Sodium cacodylate trihydrate (Ladd Research 20305)

• Sodium carbonate (Na₂CO₃; Sigma-Aldrich 223530)

• Sodium chloride (NaCl; Sigma-Aldrich S7653)

• Sodium hydroxide (NaOH; 1 M aqueous solution; for adjusting pH)

• Purified water

  • double-distilled, ASTM type I, WFI (water for injection), or equivalent; e.g., Fisher 91-502-5

  • ASTM type I water can be made with a properly maintained water filtration system (e.g., Milli-Q® Type 1 Ultrapure Water System )

• 2- or 4-L glass beaker × 2

• Magnetic stirrer × 2

• 100-ml glass beaker and a disposable pipet (for dissolving sodium carbonate [Na₂CO₃])

• 1- or 2-L graduated cylinder (depending on the final volume)

• pH meter (and calibration standards)

• 1- or 2-L glass bottle (Kimax, or equivalent) × 2

• Vacuum filtration system (pore size = 0.22 µm; e.g., VWR 10040-468 – this one screws onto Kimax glass bottles)

• WESCOR VAPRO 5520 Osmometer (with calibration standards, filter paper disks, pipet, and pipet tips)

Record Keeping

• Timer (in count-up mode)

• Worksheet

Perfusion Apparatus Assembly

The perfusion apparatus consists of five sub-systems (Fig. 2): 1) air pressure (Green), 2) perfusate (Blue), 3) oxygenation (Red), 4) anesthesia (Pink), and 5) waste collection (Brown).

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Air Pressure System

The air pressure system (Figs. 2 and 3) consists of a modified "Perfusion Two" air pump, air tank, and a modified sphygmomanometer.

Modification of Perfusion Two pump

Perfusion Two pump is designed to generate a constant pressure at 300 mmHg. Because this procedure requires the pressure to be varied from 180 to 80 mmHg (or lower), we have added a small regulator to be able to control the output pressure. Figure 4 shows the inside of the pump after modification: the regulator is placed such that the compressed air flows into it from the Air Tank switch and out to the Perfusion Pressure switch. This configuration allows the modified pump to be used in a way analogous to a cylindar of compressed air with a 2-stage regulator.

Air tank and a modified sphygmomanometer

The air tank (1-L Kimax bottle; 5 in Fig. 3) is sealed with a #6 stopper.  A Y- (or T-) connector is attached to a hole in the stopper, to which two pieces of tubing are attached to connect the air tank to #2 and #3 ports of the Perfusion Two air pump (2 and 3 in Fig. 3, respectively).  Between the #3 port and the air tank, an in-line air filter (4 in Fig. 3) is connected to prevent compressor oil from getting into the perfusate bottles. #1 port of the air pump (1 in Fig. 3) is connected to a hand pump (the bulb detached from the cuff of a sphygmomanometer), the sphygmomanometer (7 in Fig. 4), and perfusate bottles (see below). If the "Perfusion Two" pump is not available, simply connect a hand pump and sphygmomanometer to the perfusate system.

Perfusate System

The perfusate system (Figs. 2, 5-8) consists of a water bath, two 2-L bottles (one for KRC and the other for the fixative), perfusion needle, assortment of tubing, connectors, clamps, and valves.  

Lead donuts (5 in Fig. 5) are placed around the necks of the bottles to keep them from floating and tipping over in the water bath as the fluid in them is exhausted.  The fixative and the KRC bottles are sealed with #6 stoppers (1 in Figs. 6 and 7).  The holes are used to connect the bottles with (1) air pressure system, (2) oxygenation system (KRC only), and (3) a 3-way switch and tubing leading to the perfusion needle.  KRC and fixative are driven during perfusion by the air pressure system (see above).  It is very important that the stoppers and the inside of the neck of the bottles be completely dry before inserting the stoppers to prevent the stoppers from blowing out under pressure.  When the apparatus is assembled prior to each perfusion, it should be tested for the pressure of up to 200 mmHg to ensure the connections throughout the system will withstand pressure and not blow apart.  Superglue is used to secure joints between different sized tubing.  

Assembling the Apparatus

Connecting the perfusate bottles to the air pressure system:

Attach the long end of Y-connector into one of the three holes in the stoppers (2 in Figs. 6 and 7).  Use a short piece of Tygon tubing on one side of Y-connector (2a in Figs. 6 and 7) to connect the two perfusate bottles.   On one of the stoppers, another piece of tubing (2b in Fig. 6) is attached to the unused side of the Y-connector to connect the sphygmomanometer (6 in Fig. 5), hand pump (7 in Fig. 5), and the #1 port of Perfusion Two air pump (8 in Fig. 5 connects to 1 in Fig. 3) via a 4-way connector.  On the other stopper, a short piece of tubing is attached to the unused side of the Y-connector for use as a pressure relief vent while KRC and fixative are gassed with O₂/CO₂ (2b in Fig. 7 and 4 in Fig. 5).  This pressure relief tubing is folded over and clamped with a tubing clamp when pressure is applied by the air pump during the perfusion.  

Connecting the perfusate bottles to the perfusion needle:

Through the second hole of the bottle stoppers, run a piece of Tygon tubing (3 in Figs. 5-7; must be long enough to reach the bottom of the bottles), which is then connected to the three-way valve (1 in Fig. 8).  If the tubing is curled too much and its end is raised from bottom of the bottle, an elbow connector can be attached (Figs. 6 and 7). In this case, the unused end of the connector should be beveled and pointed towards the bottom to facilitate perfusate flow. The third connector on the three-way valve is connected via a luer-lock connector to IV tubing, to which the perfusion needle (4 in Fig. 8) is attached.  A flow regulator (3 in Fig. 8) and clamp (2 in Fig. 8) is installed on this segment of the tubing.  The length of the IV tubing from the bottles to valve and then the needle should be kept as short as practical: the longer the distance between the water bath and the animal the greater the drop in temperature of the perfusates.  Adjust the temperature of the water bath in order to deliver fixative at 37°C at tip of the needle.  A cork disk (cut from a small cork stopper; 5 in Fig. 8) is attached to the needle with superglue to prevent it from piercing through the heart.

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Connecting the perfusate bottles to the oxygenation system:

Through third hole in the stoppers, run a piece of Tygon tubing (4 in Fig. 6; long enough to reach the bottom of the bottle).  Attach air stones (2 in Fig. 5 and 5 in Fig. 6) on the bottle side of the tubing.  The other side of tubing is connected to O₂/CO₂ cylinder-regulator, with a clamp in between (1 in Fig. 5; clamp not shown).

Connecting the oxygenation system to the anesthesia system:

The anesthesia system (Figs. 2, 9, and 10) consists of an isoflurane vaporizer driven by an O₂ cylinder (or the O₂/CO₂ cylinder from above), a nose cone, endotracheal tube, and a ventilator to ventilate the anesthetized animal during the dissection and the initial stages of perfusion.  The vaporizer and ventilator will not be used for mice or juvenile rats.  A large glass desiccator is used to anesthetize the animal prior to putting on the nose cone (an optional small Plexiglas chamber connected to the vaporizer can be used for this purpose).  For juvenile rats and mice, a 15-ml conical tube with gauze pads containing ~1 ml isoflurane is used as the nose cone.

Connect O₂ regulator to the O₂ input on the back panel of vaporizer (behind the O₂ flow control; 1 in Fig. 9).  Connect the isoflurane output (2 in Fig. 9) to a Y-connector.  One side of the Y-connector is attached to a piece of silicone rubber tubing (3 in Fig. 9) leading to the nose cone (1 in Fig. 10).  Use another piece of silicone rubber tubing to connect the other side of the Y-connector to air intake of the ventilator (4 in Fig. 9).  Output from the ventilator (5 in Fig. 9 and 2 in Fig. 10) is connected to the endotracheal tube (3 in Fig. 10; a blunted and polished 16-gauge needle) via a Y-connector.  Exhaust line from the endotracheal tube (4 in Fig. 8) connects back to the exhaust port of the ventilator (6 in Fig. 9).  Make sure to label the exhaust line.

Waste Collection System

The waste collection system (Figs. 2, 10 and 11) is used to remove blood and perfusate from the dissection tray as it accumulates, and consists of a plastic-coated 4-L vacuum flask attached to vacuum line.  A #11½ one-hole stopper is used to seal the flask (Fig. 11).  Through the hole, attach a short segment of a plastic serological pipet (long enough to pass the vacuum port of the flask), to which Tygon tubing is connected (1 in Fig. 9).  Place the other end of this tubing to the dissection tray (5 in Fig. 10).  The flask is connected to vacuum line via heavy rubber vacuum tube (3 in Fig. 11).  An in-line filter (2 in Fig. 11) is placed to prevent waste liquid from getting into vacuum line.  Replace the in-line filter after each day of perfusions, or whenever it gets wet and occluded.  After the procedure contents of the vacuum flask are transferred to appropriate disposal containers in accordance with local institutional policies.

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Test-run

A newly assembled perfusion apparatus (air pressure, perfusate, oxygenation, and waste subsystems) needs to be tested for:

• air pump operation

• air and fluid leaks

• air pressure up to 200 mmHg

• perfusate temperature (37°C at the needle tip)

• waste suction line

This test does not involve the anesthesia system. The isoflurane vaporizer should be tested by a qualified vendor at least once a year.

Test Procedure

1. Fully assemble the apparatus with the bottles filled with RO water. Place them in water bath for at least 30 min before starting. Get a thermometer.

2. 3-way switch should have the fixative line in OFF position (1 in Fig. 8). Close all clamps (see Fig. 2). Perfusate line should be closed at this point. On the Perfusion Two pump, turn off Air Tank and Perfusion Pressure switches.

3. Do you know how to operate the regulator on O₂/CO₂ tank?

   1. If yes, proceed to the next step.

   2. If not, you should be trained for it ASAP.

4. Open air vent (clamp 2 in Fig. 2) and O₂/CO₂ line (clamp 3 in Fig. 2). Slowly open O₂/CO₂ regulator. Do you see bubbles coming out of the air stone?

   1. If yes, make sure you have enough O₂/CO₂ for the next round of perfusion and close the regulator. Clamp O₂/CO₂ line (clamp 3 in Fig. 2). O₂/CO₂ tank and its regulator can be removed at this point. Proceed to the next step.

   2. If no, check O₂/CO₂ line to see it it is blocked or leaking.

5. Close air vent (clamp 2 in Fig. 2). Open clamp 1 in Fig 2.

6. Open vacuum line. Is suction on?

   1. If yes, proceed to the next step.

   2. If no, check to see if the waste suction line is blocked. Also check the in-line filter (2 in Fig. 11).

7. Turn on the Perfusion Two pump. Do you hear low buzzing noise of air compressor coming from the pump? It should stop when pressure in the air tank reaches ~300 mmHg (although this is not measured by the manometer).

   1. If yes, proceed to the next step. 

   2. If no, make sure the power cable is connected. Still doesn't work? You might have to replace the fuse next to the main switch.

8. Check for air leaks. If no leaks, hold for several min to see if the stopper on the air tank bottle remain closed.

   1. If still intact, proceed to the next step.

   2. If it blows out, turn off the pump and re-assemble the air tank.

9. On the pump, turn on Air Tank switch, and then Perfusion Pressure switch. Open the perfusate lines (clamps 4 and 5 in Fig. 2; 2 and 3 in Fig. 8). Check for leaks along the KRC line. Does water from KRC bottle come out at needle tip?

   1. If yes, proceed to the next step.

   2. If no, check KRC line for any blockage. Also, turn the regulator knob clockwise several times.

10. Is the waste suction line picking up water released from the needle?

    1. If yes, proceed to the next step.

    2. If no, check to see if the waste suction line is blocked. Also check the in-line filter (2 in Fig. 11).

11. Check the temperature of water at the needle tip. Is it 37°C?

    1. If yes, proceed to the next step.

    2. If no, close the perfusate line, adjust the temperature of the water bath, wait for 30 min to equibrate before testing again.

12. With water running, turn the regulator knob on the pump. Clockwise to increase, counter-clockwise to decrease. Does the pressure change?

    1. If yes, get yourself familiar with how many turns translate into pressure changes. Proceed to the next step.

    2. If not, check the regulator's connections (see Fig. 4).

13. Turn the regulator clockwise until the pressure reaches 200 mmHg. Close the flow regulator (3 in Fig. 8). Hold for several min to see if the stoppers on the KRC and fixative bottles remain closed.

    1. If still intact, proceed to the next step.

    2. If it blows out, turn off the pump. Wipe dry the stoppers and the mouth of the bottles. Replace the stoppers.

14. Turn the 3-way switch so that the KRC line is in OFF position. Open the flow regulator (3 in Fig. 8). Reduce the pressure to 180 mmHg. Check for leaks along the fixative line. Does water from fixative bottle come out at needle tip?

    1. If yes, proceed to the next step.

    2. If no, check fixative line for any blockage.

15. Turn off the pump. Release pressure by opening vent on the hand pump (7 in Fig. 5).

16. Congrtulations! Test is now complete and your apparatus is ready for action. Proceed to cleanup.

    1. Remove O₂/CO₂ tank and its regulator.

    2. Empty the bottles and replace the stoppers.

    3. Close the hand pump vent.

    4. Turn on the pump again to run air through the perfusate line.

    5. Turn off the pump. Remove air pressure line from Port #1 of the pump (1 in Fig. 3).

    6. Remove the needle from perfusate line.

    7. Remove the stoppers and perfusate lines from the bottles and pump to hang dry. Keep all clamps open.

    8. Clean the bottles and store all equipment.

    9. Water collected in the vacuum flask should be placed in an appropriate waste container, unless the flask is new/clean.

Preparation of Perfusate Solutions

Prefix perfusate (Krebs-Ringer Carbicarb buffer, or KRC)

Oxygenated "Krebs-Ringer Carbicarb (KRC)" buffer is used to flush blood cells prior to fixative perfusion.  It is prepared from dry reagents as described here on the day before the procedure and stored at RT overnight.  Make a fresh batch before each day of perfusions.  One-liter of KRC should be sufficient for up to eight mice or juvenile rats (P45 and younger), or 2 L for up to four adult rats (P46 and older). Use KRC Reagent Calculator to figure out the amounts of reagents needed.

*IMPORTANT: DO NOT directly add Na₂CO₃ (sodium carbonate) as a dry solid reagent!!  Dissolve Na₂CO₃ in ~50 ml of water separately from the other reagents.  When the other reagents have dissolved, slowly (1-2 ml at a time) add the Na₂CO₃ solution with continuous mixing.  As Na₂CO₃ is added, it will be necessary to adjust the pH between 7.5 and 8 with 1M HCl to prevent precipitation.  After all of the Na₂CO₃ has been added, allow the solution to mix for several minutes before adjusting the pH to 7.35-7.40, then bring to the final volume with purified water.  Filter through a vacuum filtration system into a 1- or 2-L bottle.