The purpose of these procedures is to perfuse the brain with methylene blue stain and paraformaldehyde fixative for blockface photography that reveals structural boundaries with a degree of detail equal to that obtained by photographing a conventional glass mounted section stained for Nissl substance, e.g., by cresyl violet stain. In brain atlas development we use this technique to map the boundaries of substructures from stained sections undistorted by cutting, floating and mounting sections on glass slides.

Using the techniques described in this section we obtain blockface sections as shown in Figs. 1 and 2; compare to blockface image of unstained brain in Fig. 3.

Figure 1: Two blockface photographs taken during sectioning of a macaque brain that was perfused with methylene blue and molybdate to fix the stain and with paraformaldehyde to fix the tissue. The brain was dissected and frozen immediately after perfusion and sectioned on a freezing microtome 5 hours later.

While cortical and nuclear boundaries are readily visualized immediately following perfusion (Fig. 1), they become more distinct if the specimen is immersion fixed in paraformaldehyde for a longer period of time. Leaving it in the fixative for an extended time appears to clear stain from the white matter without reducing the stain in gray matter. The brain shown in Fig. 2 was perfused with methylene blue, immersion fixed for 6 months and cryoprotected in successively more concentrated solutions of sucrose over a period of two weeks before it was sectioned.

Figure 2a: Blockface photograph of a macaque brain sectioned 6 months after perfusion staining with methylene blue followed by perfusion and immersion fixation with paraformaldehyde.

Figure 2b: Section from about the same level from a different animal that was cut, mounted and stained for Nissl substance with cresyl violet stain for comparison with the perfusion stained blockface in Fig. 2a.

Note that recipes are in amounts for total body perfusion and some are for two macaques, each with a body weight about 3kg.

Dissolve pre-weighed sodium phosphate (0.4M PB Stock in green-lidded plastic cup) through

Place 500ml buffer stock and 500ml distilled water in a 2L flask with a stir bar.

Heat with stirring in a chemical fume hood until clear checking temperature periodically.

Turn heat off when it reaches 60 degrees centigrade and allow to sit until clear. (This should

provide sufficient heat to disassociate the paraformaldehyde. If temperature exceeds 65

When solution is clear, take it off heater and cool to room temperature in a bucket of cold

Rinse the first flask and filter the solution through No. 1 Whatman filter paper.

Add 2 vials of 25g ammonium molybdate (preweighed in blue topped vials) to 1L distilled water.

Add 1g papaverine (1 green-topped preweighed vial) to about 100ml saline in small Ehrlenmeyer

Add papaverine solution to saline as it is filtering through No. 1 filter paper.

Introduction of fluids by placement of a catheter through the heart into the ascending aorta perfuses the entire body. To conserve perfusate, one can restrict perfusion to the upper limbs, upper thorax and head by clamping the descending aorta in the thorax.

Pentobarbital anesthesia IV to surgical level (.5ml (25mg/kg)/kg; 1/3 of dose at 3min intervals)

Palpate pulse of descending thoracic aorta against spine and clamp it (optional)

Incise pericardium: grasp and lift with toothed forceps; horizontal incision followed by incisions

Incise left ventricle close to apex (blunt scissors; to locate site of cardiac incision: rotate heart about 3/8 turn counterclockwise; grasp apex of heart with toothed forceps; make 5mm horizontal incision about 10 mm from apex near medial boundary of left ventricle (septum); if no impressive spurt, cut a little more... when you are in the ventricle the spurt is pronounced; insert cannula until you can confirm visually that it is in the aorta (the aorta is on the animal's right, pulmonary vein on its left and not as muscular as the aorta.)

Insert conical cannula through ventricle into aorta: identify aorta first and feel tip of connector as it

Incise right flank and diaphragm to facilitate flow of fluids from atrium away from thorax

Start pump (setting ~2.0); if lungs clear very fast and pressure is very low the connector is probably

either in the right ventricle pumping through the lungs or in the mitral valve of the left ventricle

pumping in reverse through the lungs; reduce pump speed and reset into aorta; if pressure is

much above 100mmHg and the tongue and mucous membranes start to clear you are probably

When clear saline is flowing from the right atrium or 1000ml have passed, slow pump, clamp saline

tube, unclamp methylene blue tube, deal with any bubbles and set pump speed back to ~2

When almost all of the methylene blue has passed, slow pump speed; clamp methylene blue tubing;

When the tubing is clear of methylene blue, slow pump, clamp tubing near outlet from saline bottle; pour out remaining saline and pour 500ml molybdate into the bottle; bleed off bubbles; return pump setting to ~2

When molybdate has almost emptied, fill the same bottle with paraformaldehyde to the 2L level (no need to remove remaining molybdate)

When most of the paraformaldehyde has passed, stop the pump; remove all clamps and the cardiac connector

Open as much of cranium as possible without disturbing the external auditory canals and inferior orbital ridges, which will be needed for stereotaxy; also leave enough of the cranial vault to give something to hold later when dissecting the bone around the flocculus; include in removal the area likely to be used for registration rods (5mm anterior to external auditory canals); leave dura intact; in this animal, occipital bone removed starting from foramen magnum; forward cranium entered laterally ~3mm rostral to external auditory canals (large rongeurs); temporal bones and lateral parts of frontal bones removed leaving a peninsula of frontal bone attached to brows.

Make a nest of cling wrap in jar of paraformaldehyde for transport; place specimen there; add more cling wrap; close with jar lid tightly; wrap tightly 3 or more times with parafilm; apply tape to jar; write animal ID, date, and Bowden on the tape;

When time for transport: place in black plastic bag and tie it; place in a second black bag and tie it;

In cases requiring perfusion of the brain without exposure of the remainder of the body to perfusate, one can perfuse through the carotid arteries. This has the advantage that one can perfuse the brain with fixative and still obtain tissues from other organs without exposing them to fixation. Compared with total body perfusion, it has the theoretical disadvantage that, if the circle of Willis is not patent, parts of the brain served by the vertebral artery may be less well fixed than those served by the carotid arteries. We have not found this to be a problem in the five or more cases where we have applied the approach.

30cc syringes (4) with perfusate to clear the vessels of blood: 0.9% NaCl; to prevent clotting:

heparin (1000U/L); to prevent reflex arterial constriction: papaverine-HCl (.05%)

Animal Y-tubing (single tube from pump to Y-connector that splits flow to two tubes connect to

#14 angiocath needles (3) with Luer lock hubs (2); smaller angiocaths (3) in case #14 too large for

Lay out instruments: small scissors, small forceps, extra clamps, chest retractor, silastic vessel retraction strings, syringes of heparinized saline, angiocaths, animal Y-tubing.

Anesthetize to surgical level with pentobarbital (.5ml (25mg/kg)/kg; 1/3 of dose at 3min intervals)

Make a longitudinal incision from 2-5 cm behind mentum to sternal notch (scalpel with #10 blade)

Dissect between the sternohyoid and sternomastoid muscles to expose the carotid vascular compartments bilaterally (dissection scissors, blunt forceps; straight clamps and/or retractor) this may require section of the omohyoid muscle.

Dissect the common carotid artery in each compartment to separate it from the internal jugular vein and vagus nerve for a length of 2-3cm;[1] dissect the artery from the vein for 2-3cm;[2] repeat on the other side.

Dissect an opening under the artery; work the angle clamp through it and drag back a silastic suture so that it runs under and perpendicular to the artery; use it to elevate the artery and use the dissection scissors to complete freeing it from its attachments in the neurovascular compartment; place two silk sutures around the artery, make a half-tie in each; place one at the cranial end and the other to the cardiac end of the exposed section of artery; remove the silastic vessel retractor; repeat on the other side.

Place the silastic vessel retractor around the artery at the cranial end; wrap it around twice so that when it is retracted it stops blood flow; triple-tie off the artery at the cardiac end using the silk suture there;[3]^,[4] with an assistant (or clamp to skin) occluding the cranial end by traction on the silastic vessel retractor, pinch the artery gently with the forceps about 3mm in the cranial direction from the silk tie that is occluding the artery; at the tip of the forceps, on the cardiac side, make an incision of 10-20% of the artery’s diameter perpendicular to the long axis of the artery[5] (pointed scalpel, i.e., #11 blade).

Note the location of the bifurcation of the carotid; insert the catheter introducer into the incision with the point in the cranial direction; once it is in, use it to guide the insertion of the intracath needle into the vessel;[6] insert the needle about 5mm and remove the introducer; advance the intracath 2-3cm; being careful not to get the tip within 5-10mm of the carotid bifurcation;[7] clamp the artery to the needle with a bull-dog clamp; triple tie the suture at the cranial end onto the vessel about 1cm cranial to the site of needle entry; grasp one end from each of the silk sutures and cross-tie them to reduce the likelihood that the needle will pull out[8]; remove the silastic vessel retractor; inject about 25 ml of the saline/heparin/papaverine solution; repeat on the other side.

Clamp carotids and jugulars on the cardiac side of the tied off arteries; section the carotids and jugulars being careful not to disturb the implants; perform cephalectomy by severing the remaining soft tissues (#10 scalpels) and spinal column (bone shears); cutaneous incisions low on neck to avoid loss of fluid through facial vessels during perfusion; cushion head in polypad; transfer to fume hood for perfusion.

Attach the animal Y-tubing to the pump tube being careful to have no bubbles in the line.

Start pump (setting ~2.0 delivers 64ml/min with an open line pressure through two 14 gauge

intracaths of 20mmHg). Clamp one limb of the Y-tubing so that perfusate flows only through one

carotid. After two minutes unclamp the limb of the Y and clamp the other. Alternate every two

When clear saline is flowing from neck vessels or 400ml have passed, slow pump, clamp saline tube, unclamp methylene blue tube, deal with any bubbles and set pump speed back to ~2

When almost all of the methylene blue has passed, slow pump speed; clamp methylene blue tubing; unclamp saline; deal with bubbles; and reset pump speed to ~2

When molybdate has almost emptied, fill the same bottle with paraformaldehyde to the 2L level (no need to remove remaining molybdate)

When most of the paraformaldehyde has passed, stop the pump; remove all clamps and the cardiac connector

Make a nest of cotton balls in jar of paraformaldehyde for transport; place specimen there; add more cotton balls; close with jar lid tightly; wrap tightly 3 or more times with parafilm; apply tape to jar; write animal ID, date, and investigator’s name on the tape;

When time for transport: place in black plastic bag and tie it; place in a second black bag and tie it;

The cut surface of the fresh unperfused brain (not shown) shows remarkable detail and may be useful in applications where one can use the Vibratome to section the brain.

Figure 3: Blockface photograph of an unperfused, frozen macaque brain (shown here) reveals less detail than blockface of fresh unperfused brain (not shown) or the frozen brain perfused with methylene blue (Figs. 1,2) potassium dichromate (Figs. 4,5).

Perfusion staining with methylene blue without molybdate produced a specimen in which the color was very unstable. The surface of a block showed little gray-white contrast when first cut, then became gradually darker blue as it stood on the microtome stage exposed to air and warming. As one took sections into the block the coloration varied from absent to dark depending on the time interval between cuts.

Perfusion staining with methylene blue following perfusion with heparinized saline lacking papaverine produced a specimen in which much of the brain was well stained, but isolated areas were totally unstained. We believe that without the papaverine some arterioles constrict and block the circulation of perfusate into the vascular beds that they would otherwise supply.

Perfusion staining of one baboon specimen with cresyl violet failed to reveal more detail than perfusion with no stain. Cresyl violet failed to pass the blood brain barrier. The vasculature was nicely defined, so perfusion with this stain might produce a specimen suitable for mapping vessels, but it was of no use for defining nuclear boundaries.

Perfusion staining of one longtailed macaque specimen with potassium dichromate following, as nearly as possible, the method used by Clarke, R. and E. Henderson ("Investigation of the Central Nervous System." The Johns Hopkins Hospital Reports Special Volume: 163-172, 1920) provided good nuclear detail (Figs. 4, 5). The gray-white contrast was not as pronounced as with methylene blue, but the method is simpler (fewer steps) and less time-consuming (no need to wait weeks or months for white matter to clear). Thus, it may be a preferable technique for some purposes.

Figure 4: Blockface photograph of block from a macaque brain perfused with 2% potassium dichromate and 5% formalin in 0.9% NaCl and immersion fixed in the same solution for 72 hours.

Figure 5: Blockface photograph of a coronal section about 1mm deep to the surface of the block of macaque brain perfused and immersed as described in legend to Fig. 4.. Gray-white boundaries, particularly between subcortical nuclei, are more distinct than in unperfused brain (Fig. 3), but gray-white contrast is not as pronounced as with the methylene blue stain (Fig. 2) and some ‘white areas’, such as internal capsule, show considerably less contrast.

Notes from Clarke and Henderson (1920; see above) on preparation of brain for potassium dichromate stain: "As a preliminary, the brain must be injected. A cannula is introduced into the thoracic aorta, the right side of the heart opened and a warm solution of 5 per cent formalin and 2 per cent potassium bichromate injected from a bottle raised from 1 to 2 meter (p. 89). It is advisable to introduce ear cones before the injection, as with formalin the meatus becomes rigid" (p. 90). "Heads are kept till sufficiently stained and hardened in formalin 5 per cent and potassium bichromate 2 per cent. They are frozen in carbonic acid snow...." (powdered dry ice) (p. 25).."...the brain may be embedded in gelatine, 15 per cent, and glycerine, 5 per cent, and frozen, or it may be embedded and cut in celloidin...." "Brains hardened in formalin and bichromate shrink very little." (p. 99).

[1] The carotid artery pulsates and is the more medial of the two vessels. Hints: lift tissue with forceps as close as possible to where you want to insert the angiocath and spread the scissors for dissection. Don’t lift tissue so far that you lose sight of the structure you’re trying to expose, usually less than 3mm will suffice. Don’t spread the scissors wider than a few millimeters; many small spreads are more effective than a few large ones. Lift and snip connective tissue always envisioning penetration into the compartment where the artery runs. Once you have snipped your way in, grasp the edge of the opening with the forceps and blunt dissect your way in a millimeter or two. Incise that much of the sheath, then regrasp the edge with the forceps, blunt dissect and clip again. Never clip more tissue than you can see through clearly enough to be sure that it does not contain a blood vessel.

[2] Again grasp connective tissue or a vessel very gently and, working very close to the forceps, tease the vessels apart with the dissection scissors with very small openings of the scissor blades.

[3] Be careful not to trap tissue other than the artery in the suture loop, and tie it tightly enough to prevent blood flow past the suture.

[4] For the following steps the surgeon, if right handed, should stand at the side of the table to the animal’s right; if left handed, to the animal’s left.

[5] To avoid accidentally cutting through the artery, have the sharp edge of the blade face upward.

[6] The catheter introducer will insert most easily if the pointed head is maintained almost parallel to the long axis of the artery; grasp the hub of the intracath needle from above, so that it can also be introduced parallel to the artery’s long axis.

[7] The needle may advance more easily if the underside of the vessel is supported with forceps as it is inserted; also, make sure the vessel is straight as you loosen the silastic vessel retractor to let the needle pass.

[8] Use surgeon’s tie, i.e., double tie the first loop so it doesn’t slip as you make the second loop