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Vitrification Protocol Used on Dog

by Ben Best

On Thursday, February 10 the Cryonics Institute perfused the dog of one of our Members, Kevin Boyle. This was the second time we perfused the dog of one of our Members with Dr. Yuri Pichugin's Vitrification Mixture (VM).

Kevin had been ruefully watching the declining health of his beloved dog Thor and had come to the difficult decision to euthanize so as to optimize the conditions of cryopreservation. Thor had a pitiful limp due to worsening degenerative myelopathy in his hind legs. The once powerful dog was down to two-thirds his original weight. As Kevin monitored Thor's condition he struggled to decide when would be the best time to euthanize. Kevin didn't want to lose his companion too soon, but neither did he want to risk an untimely death which would reduce the chance for good cryopreservation.

When Thor's deterioration became unquestionable, Kevin finally bit the bullet. Thor, Kevin and Kevin's girlfriend Cheryl loaded into Kevin's van and drove to Michigan from Massachusetts. At 9 am that fateful Thursday morning I was met at the door of the CI Facility by a very sad-looking Kevin. Kevin and Cheryl spent their last hour together with Thor at CI. Cheryl too was obviously upset about Thor's condition and the prospect of euthanasia.

Shortly before 10 am we all drove to the veterinary clinic, which is conveniently located within a few blocks of the CI Facility. The veterinarian in charge of the case proved to be a very sensitive, compassionate and competent man. When I requested that an endotracheal tube be inserted immediately following euthanasia, he agreed without hesitation. (An endotracheal tube runs from the mouth into the trachea -- the tube leading to the lungs. Our ACDC Thumper is fitted to connect an endotracheal tube to the oxygen supply.)

CI Facilities manager Andy Zawacki parked his truck by the outside door of the room in which Thor was to be euthanized. Andy, Kevin, Cheryl, Thor, the veterinarian, the assistant veterinarian and I crowded into the small room.

Thor was given some heparin which was allowed to circulate for a few minutes. As Kevin embraced Thor for the last time he commented that he had never seen Thor shaking so badly -- as if in anticipation. (Andy speculated that Thor was sensing Kevin's upset rather than anticipating the impending event.) Yet Thor remained quiet, compliant and somewhat mournful as the veterinarian administered the injection to stop the heart.

Kevin clearly loved Thor deeply, and yet was able to maintain the discipline and rationality to do what he believed would be best for Thor by having him euthanized under controlled conditions which would allow for good cryopreservation. It is a stark contrast to many of our human cases. Yvan Bozzonetti's mother, our 68th patient, had deanimated in France where the authorities would not even allow administration of ice and would not release the body for three days. Although an S-A cryonics rescue team arrived fairly soon after the deanimation of our 67th patient in Florida, cancer had made the final moment difficult to predict. Other recent patients have been discovered hours or days after deanimating alone.

We at CI had been waiting for the opportunity to apply Dr. Pichugin's new vitrification protocol to human patients, yet we only succeeded in doing so for Thor and for another dog we perfused the previous August.

After the vet inserted the endotracheal tube into Thor we loaded Thor into a Ziegler shipping box on the back of Andy's truck, added ice and drove back to the CI Facility. We placed Thor in our portable ice bath, which contained two battery-operated bilge pumps (one 360 gallons per hour and one 1100 gph).

Cooling was slow at first, but once we got the ACDC Thumper in action, cooling was much faster. We only used the compression stroke because the dog was not shaved on the side. We tried to ensure that the compression stroke would not break the dog's ribs. Although the positioning was not optimal for cardiopulmonary support, the acceleration of cooling the Thumper provided seems to be an indication that blood circulation was occurring.

After two hours the temperature probe in the throat had dropped from 38.9ºC to 18.5ºC and the probe in the rectum had dropped from 39.0ºC to 26.8ºC. The 360 gph bilge pump was pumping water through a hose directly onto the head, which may have partly accounted for the greater cooling of the head. We wondered if we should have shaven the dog entirely beforehand to speed cooling. The dog we cryopreserved in August had been cooled in a quarter of the time (half an hour) by using cold washout solution. Thor was receiving oxygen from the Thumper, however, so the difference may have been more a matter of delay than anything else.

Dr. Pichugin performed the surgery, with Andy and me providing support. The chest was opened, the descending aorta was clamped and the aorta was cannulated. The superior vena cava was cut for drainage. Thus, we perfused the whole upper body including the forelegs, head and brain. With a perfusion pressure of about 70-80 mm Hg and a flow rate of about one liter per minute, the throat temperature dropped nearly 10ºC to approximately 10ºC in less than 20 minutes.

Yuri then drilled a burr hole into the dog's skull. Burr holes have long been used in cryonics and were sensationalized by the media with the revelation that holes had been drilled in Ted William's head -- as if this were a careless & abusive mutilation. But burr holes do not injure the brain and they provide a means of monitoring possible edema or dehydration as well as allow for evaluation of perfusion. Burr holes are a standard life-saving medical procedure to relieve pressure due to bleeding in the brain. The holes are drilled as standard procedure in all hospital neurosurgical operations. As the perfusion continued Yuri sampled the burr hole as a means of determining when perfusion was complete.

Over the space of an hour and eight minutes Yuri increased concentration of his cold vitrification mixture perfusate until he was perfusing at full strength (70%) and perfusion pressure was 110 mm Hg. Examining samples from the burr hole with a refractometer he determined that the samples had a refractive index matching that of the perfusate and that the brain must be fully saturated with vitrification mixture. Throat temperature at the end of perfusion was -2.3ºC.

PERFUSION DATA m-RPS-2 is modified Renal Perfusion Solution EG is Ethylene Glycol VM-1 is Dr. Pichugin's Vitrification Mixture TEMPERATURE refers to the temperature of administered perfusate
name/percent liters degrees Celcius liters/minute mm Hg
 1.0 70-80
10% EG
 1.0 80-90
30% EG
 1.0 90-100
70% VM-1
 1.0-0.7 100-120
70% VM-1
 0.7-0.7 100-120
70% VM-1
 0.5 100-120

Thor was then placed on a board and moved to our cooling box. A couple of inches of liquid nitrogen was placed in the bottom of the box and the dog on his board were placed in the box just above the liquid nitrogen, supported by metal blocks. A fan in the side of the box circulated nitrogen vapor at a fairly uniform rate. Two ordinary fans were used on the cooling-box fan motor to keep it from overheating. Probes at the top of the board, the top of the dog and six inches above the dog all eventually recorded temperatures close to -180ºC.

In about nine hours we dropped throat temperature to just below -130ºC and rectal temperature to about -60ºC. Dr. Pichugin estimates the glass-transition temperature of his vitrification mixture to be between -110ºC and -130ºC. It is important to cool as rapidly as possible prior to solidification in order to minimize the possibility of ice formation. But below the glass transition temperature it is important to cool very slowly to minimize thermal stress in the solid which can lead to cracking.

We turned-off the fan and stabilized the temperature for the next eight hours until rectal temperature reached -112.6ºC. Then the dog was removed, Yuri and Andy examined the burr hole and found that the brain region through the hole showed no evidence of visible ice crystals. The dissected head tissues (the skin and muscles) around the burr hole was vitrified. The refractive index of the last portion of fluid from the burr hole during perfusion of the dog may be another indication that the brain was saturated with vitrification mixture completely. The use of the CI vitrification protocol for the big dog was successful. Previously Yuri had been able to vitrify the brain of rats and sheep using the CI vitrification protocol. Then the dog was removed, Yuri sampled the burr hole, the dog was placed in a sleeping bag and ropes tied to the board were attached to cranks above the cooling box to allow control of elevation of the board. Thereafter, the fan was not used and cooling was very slow -- about 10ºC per day -- until liquid nitrogen temperature was reached.

In conclusion, although many new things were tried with Thor, we felt there was reason to believe these would result in greatly improved cryopreservation. We believe that we achieved this, and Thor received optimal treatment, resulting in an extrememly successful cryopreservation.