Cryonics, January 2014

Isolation of the Brain for Human Cryopreservation

by Chana Phaedra

INTRODUCTION

There are some generally unacknowledged advantages to cryopreserving just the brain rather than the entire head as is customary with Alcor’s “neuropreservation” option. Such advantages include the ability to supplement cryoprotective perfusion with subsequent immersion of the brain in cryoprotectant to compensate for ischemia-induced perfusion impairment. Indeed, in cases of long-term ischemia, where perfusion is not possible, cryoprotection of the isolated brain by diffusion represents the only option for mitigating ice crystal formation.

The current protocol in such cases is to carry out what is known as a “straight freeze,” or cryopreservation of the patient without administration of a cryoprotectant. As the name implies, a straight freeze results in complete freezing (i.e., ice crystal formation throughout all tissues of the patient). And while we do not yet know how difficult the damage caused by a straight freeze will be to interpret and repair, the conservative approach is to prevent as much damage from occurring as possible, thus making it easier for future technologies to infer the original state from the damaged state.

In cases where the ischemic period is too long to conduct cryoprotective perfusion (>48 hours), but not long enough for “information-theoretic death” to occur, cryoprotective diffusion of the brain at low temperature represents an interesting theoretical alternative for a straight freeze. Furthermore, because chemical fixatives penetrate tissues more quickly than cryoprotectants, the addition of diffusion fixation to stabilize the brain prior to cryoprotection may also be an option for severely compromised patients.

Lastly, it cannot be denied that there are space-saving benefits to storing brains rather than entire heads. A mold may easily be designed to provide adequate support for the brain and also to minimize the amount of storage space required. It has been argued that this would result in a roughly 50% decrease in volume associated costs for long-term care, which would translate into lower cryopreservation minimums. Additional space- and cost-savings may be realized if whole-body members who would otherwise face a straight freeze opt for cryopreservation of the isolated brain under such circumstances.

So, with those arguments in mind, let’s get down to the nitty-gritty. What is the best procedure for removal of the human brain for subsequent cryopreservation?

To answer this question, I have drawn from three primary sources. First, ubiquitously available video demonstrations of dissections of the head and removal of the brain using human cadavers; second, a personal friend who is a forensic medical examiner; and third, literature in brain removal for human plastination.

While all three approaches have the same ultimate goal—to remove the brain—they vary somewhat according to the reasons for removal. In general, removal of the brain for autopsy and anatomical instruction is performed quickly but with little regard for avoiding physical trauma to tissues. For purposes of plastination, however, in which the goal is to preserve specimens in as close to their natural conformation as possible, greater care is taken to minimize damage from surgical instruments and the physical force of removal. A combination of approaches aimed at minimizing physical trauma and duration of the procedure would constitute an optimal method for removal of the brain for cryopreservation.

MATERIALS

In addition to basic surgical instruments such as scalpels and forceps, the devices used for removal of the brain range from basic hand- and power-tools to specialized surgical instruments. The most necessary are: (1) a circular or an oscillating saw, such as a Stryker bone saw; (2) a cross-chisel, aka the “Virchow skull breaker;” and (3) a brain knife, designed to cleanly slice brain tissue and to sever nerves in hard-to-reach areas. While it is true that the “fresh” (as opposed to fixed) room-temperature brain is unable to maintain its shape upon removal from the cranium, it is important to remember that in cryonics scenarios the brain would only be extracted at low above-freezing temperatures, which “firm up” the tissue and provide stability. In good cases, additional stability may come from dehydration as a result of cryoprotective perfusion prior to removal. Additionally, a cranium-shaped vessel may be designed which will support the brain properly after removal and help to maintain its form during long-term care. Methods It is important to remember that all surgical procedures are carried out with the patient’s cooled body and cephalon packed in water ice to maintain the patient at or below the target temperature for surgery. Temperature is monitored continuously using thermocouples attached to data acquisition devices. Additionally, cold saline or other chilled physiological solution should be used in conjunction with any heat-producing instruments, such as the Stryker saw. Keep squirt bottles of saline on hand and packed in ice when not in use. Spray the instrument blade with chilled solution as it cuts through bone to reduce heat and minimize dust.

In removing the brain, the skull is first exposed by separation of the scalp, using a scalpel, circumferentially around the cranium at a level about one finger’s width above the supraorbital margin and the ears. The use of a piece of string around the head is useful in maintaining the cut at the appropriate level from start to finish. A periosteal elevator is used to separate the loose areolar tissue of the scalp from the cranium and the tissue is then removed, exposing the cranium. The cap of the skull is cut along the same circumferential line as the scalp with the aid of a circular saw or an oscillating saw such as a Stryker bone saw. Because skull thickness varies, caution must be exercised. The use of a small diameter blade (about 5 cm) in concert with a plate limiting the depth of the cut to 0.4 cm is helpful in preventing the blade from traumatizing the brain, but it cannot be relied upon completely. Ultimately, the depth of the cut must be carefully regulated by hand to prevent damage to underlying structures. Once the cut is complete, the cranial cap may be removed with careful use of a crosschisel, or “Virchow skull breaker.” This is accomplished by wedging it between the skull and the dura at approximately 2 cm intervals around the circumference of the cap. The goal is to separate the adhesion of the dura from the skull without using the brain as leverage. Once this is done, the cap may be pulled away to expose the dorsal surface of the brain.

crosschisel A crosschisel, aka “the Virchow skull breaker.”

It will now be easy to see that the brain is covered by the outermost meningeal layer, the dura mater. The dura is a thick, elastic membrane encasing the brain that must be removed to observe and access the brain itself. To do so, use surgical scissors to cut the dura along the cut of the skull. The dura may then be reflected upward toward the midline of the brain, where it reaches into the depths of the medial longitudinal fissure. This arched fold of dura mater is known as the falx cerebri and facilitates stability of the two brain hemispheres. Along this plane, the dura is attached to the skull anteriorly at the ethmoid bone. Additionally, several small veins exit the dura along the midline. To remove the dura from the longitudinal fissure, one must sever the anterior attachment and cut away the venous structures of the sagittal sinus until reaching the area of the occipital lobe, where the falx cerebri meets and connects with dura at yet another orientation known as the tentorium cerebelli. There are lots of dural reflections in this area, making separation difficult. Just remove as much dura as possible.

brain anatomy

Image: An illustration of the anatomical locations of the falx cerebri, tentorium cerebelli, olfactory nerves, optic nerves, and twelve cranial nerves.

The soft, filmy layer of arachnoid mater covering the cortex is now visible. During normal physiology, cerebrospinal fluid flows underneath the arachnoid. Interestingly, injection of formalin in the subarachnoid space 20-24 hours prior to brain removal is sometimes recommended in the plastination literature, through “injection” (perfusion) of formalin prior to removal and diffusion fixation. Both methods result in shorter time to completion of fixation than diffusion fixation alone.

Now a “wedge” cut is made in the skull on either side of the occipital bone down to the foramen magnum, which is the hole at the base of the skull through which the spinal cord exits the vault of the skull. Removal of the resulting wedge of bone reveals the cerebellum, covered in dura which must again be carefully removed.

Dura also covers the spinal cord, and if we would dissect the back of the head and neck we would find that it is continuous with the cranial dura. It would take longer, but it is entirely possible to do this dissection in order to remove the entire brain and a portion of the spinal cord carefully and completely. Where a more extensive dissection is not advisable, the spinal cord is severed as low as possible given its accessibility.

To remove the brain several nerves must be severed, including the olfactory nerve, optic nerve, cranial nerves, and (if removing some portion of the spinal cord) the spinal nerves. Again, depending on how much anatomical knowledge the practitioner has, and how much time one is willing to expend, these may be made more accessible and cut cleanly in order to remove the brain (and spinal cord) in one continuous piece.

Removal of the brain is performed manually, with one hand supporting the occipital lobes while the other is used to free the brain from the cranial fossae (i.e., depressions in the cranial vault which house the various lobes of the brain). In this way, one may pull the anterior brain (frontal lobes) upward lightly to elevate and then sever the olfactory nerves from the frontal base of the skull. Next the optic nerves may be cut, followed by the internal carotid arteries and the oculomotor nerves (i.e., cranial nerve III). Then the fingers may be worked underneath the temporal lobes in order to free them from their cavities, making the tentorium visible.

Both sides of the tentorium may be cut in a mediolateral direction along the petrous portions of the temporal bone using a long, pointed knife. The cerebellum can then be gently pushed back and away from the temporal lobes enough to observe the spinal cord where it passes through the foramen magnum. The spinal cord is then transected as far back as possible in the spinal canal. Supporting the cerebellum, the brain and remaining spinal cord can now be gently lifted out of the cranium in one piece.

The greatest potential risk for trauma to the brain is that of damaging the underlying tissues when cutting through bone, particularly when using a circular or oscillating power saw. A depth-limiting blade may be used to reduce this risk, but ultimately cutting through the bone closest to the brain must be performed manually and with great care. Additionally, it is important to use gentle but controlled force when manipulating the brain with the hands so as not to rip or tear nerves or other structures.

LONG-TERM CARE OF THE ISOLATED BRAIN

Congratulations, you’ve just removed the brain of a cryonics patient. Now what?

As discussed in the introduction, the brain in question may or may not be cryoprotected yet, depending on the particular circumstances of the individual case. If the patient suffered ischemic injury and full or supplemental cryoprotection is required, the brain should be transferred to a container of cryoprotectant solution and the appropriate protocol for diffusion cryoprotection followed. Generally, stepped increases in cryoprotectant concentration at low temperature over a period of months is necessary for equilibration of the entire brain prior to cryopreservation. Alternatively, brain ultrastructure may be stabilized by immersion of the brain in a fixative solution prior to cryoprotection. Either way, the brain may be suspended in solution by a basilar artery in order to prevent compression and deformation of the brain as might occur if it is placed on a flat surface.

brain anatomy

Image: The basilar artery is formed where the two vertebral arteries join together at the base of the skull. It can be used to suspend the brain in solution during diffusion protocols to prevent compression and deformation.

Once cryoprotection is complete, the brain may finally be transferred to a cooling box for cryogenic cooling prior to long term care. As mentioned, the procurement of a mold to support and protect the brain is advisable and should bring about significant cost-savings, too. Exactly such a device, called a “hedgehog mold,” is described in the early human plastination literature which was used to maintain the shape of the isolated brain for several hours prior to fixation. Obviously, there is nothing preventing us from developing something similar to encase cryonics patients’ brains prior to immersion in liquid nitrogen for long-term care.

CONCLUSION

It may be easier than we think to provide cryonics patients with better alternatives to a straight freeze. With practice, removal of the human brain from the cranium can be done quickly (within 30 minutes) and with little physical trauma. The equipment required to carry out such a procedure is minimal, relatively inexpensive, and easy to use. Unlike more intricate surgeries like cannulation of vessels for perfusion, brain extraction requires little more than adequate knowledge of cerebral anatomy and the ability to use surgical tools in a controlled fashion.

Lastly, removing and cryopreserving a brain would help reduce negative public perception of neuropreservation. Most cryonicists have experienced the disgust that the image of a cryopreserved head seems to conjure up for many people. We may benefit significantly from the less visceral responses to be expected when discussing the preservation of isolated brains.