This is the seventh entry in a series about resuscitation of non-hibernating rodents from circulatory arrest at ultraprofound hypothermic and high subzero temperatures. After spending a few years on perfecting on Andjus’ technique for resuscitating rodents (rats and hamsters) from ultraprofound hypothermic and high subzero temperatures, Audrey Smith upped the ante and attempted the same feat in larger mammals. In her 1957 publication, “Problems in the resuscitation of mammals from body temperatures below 0 degrees C,” she detailed the results of such experiments performed on Dutch rabbits and small primates of the species Galago crassicaudatus agisymbanus.
Smith used a modified version of the closed vessel technique to anesthetize and initiate cooling in the rabbits, then placed them in ice water for further cooling. Respiration ceased between 13 and 21 degrees C and the heart stopped beating a couple of minutes later when temperatures were 3 or 4 degrees lower, at which point the rabbits were immersed in -5 degrees C baths. Due to larger body mass, it took much longer for deep body temperature in rabbits to drop from 15 to 10 degrees C than it had in hamsters, though the rabbits’ extremities froze quickly. Smith wished to avoid this discrepancy, so she attempted to speed cooling by injecting a cold, creamy saline and serum mixture into the stomach and rectum. Cooling was certainly faster, but unfortunately the gastric mucous membrane was damaged and sometimes the stomach ruptured, forcing her to abandon this method. Further investigation finally led her to determine that thoroughly wetting the undercoat to remove all insulating air from the fur and vigorously stirring the -5 degree bath led to a a fall in deep body temperature to the freezing point of plasma within 20 to 40 minutes of extremities freezing. Galagos were cooled similarly. Their extremities had been freezing for around 40 minutes by the time internal organs began to freeze.
James Lovelock built a larger microwave diathermy apparatus for Smith’s rabbit and primate experiments on the assumption that larger body masses simply needed more magnetronic power. Initial warming attempts resulted in severe superficial burns before the rest of the animal had been thawed. Compensating for this effect resulted in the next few animals’ visceras being cooked. Finally, a technique was determined for warming from -0.6 degrees to 10 or 15 degrees C within a minute.
Fifteen rabbits and two galagos underwent this treatment and resumed heartbeat and pink mucous membrane coloring when temperature reached around 15 degrees C. Between 20 and 30 degrees C they began breathing and diathermy and artificial respiration was stopped while gentle warming was continued under a heat lamp or in an incubator, while a few rabbits were left at room temperature. Smith describes the results:
Muscle tone improved and the animals made spontanous movements. Some of them, including the two galagos, sat up and moved around. Within about an hour, however, the reanimated rabbits and galagos all collapsed and died. At post mortem the only obvious lesion was a severe haemorrhage in the upper part of the stomach. This is the part of the stomach which secretes hydrochloric acid.
Smith had noticed similar lesions in the stomachs of hamsters she had frozen which had died shortly after resuscitation, also from the acid-secreting portion of the stomach. She theorized that lowering body temperature disabled the function of mucous-secreting cells (which protect the stomach from acid) by increasing their permeability to hydrochloric acid and causing the acid in the stomach to diffuse and injure blood vessels. Smith tested this theory by neutralizing stomach acid with sodium bicarbonate during cooling but before freezing. This time, after resuscitation, there was no sign of gastric hemorrhage. Sadly, the rabbits undergoing this treatment still did not live more than 4 hours, and two galagos which seemed to make an excellent recovery died within 24 hours. Though their stomachs were normal, these animals were found to suffer from pulmonary edema and one had bloody fluid in the duodenum and jejunum.
Other topics investigated and reported within her manuscript were the effects of freezing on the hamster placenta and studies on the isolated heart. Observations made on the placentas of hamsters frozen on the 9th, 10th, and 11th days after fertilization of the egg (when the hamster placenta undergoes rapid growth and freezing disrupts foetal development) indicated that bleeding may also be induced by circulatory disorders. Smith speculated that it may be due to derangement of cardiac muscle tissue itself.
This compelled her to experiment on isolated hamster hearts. Interestingly, although whole hamsters did not survive freezing for 3 hours, isolated hamster hearts resumed beating for several hours when perfused in vitro after freezing for 3 hours. She also found that the isolated rat heart recovered completely after freezing at -2 degrees C for 1 hour, but failed to recover from temperatures below -5 degrees C. Further investigations involving perfusion of hamster hearts with glycerol led to resumed beating of hearts after lowering to -20 degrees C, many of which established a regular beat. These results indicated that the heart may not have been the limiting factor in resuscitating whole animals from subzero temperatures, and that improved methods of cryoprotection might be developed for resuscitation of whole animals from subzero temperatures.