Depressed Metabolism

This is the fourth entry in a series about resuscitation of non-hibernating rodents from circulatory arrest at ultraprofound hypothermic and high subzero temperatures. Up to this point we have discussed the groundbreaking research in the early 1950s performed by Radoslav Andjus in resuscitating rats from body temperatures between 0 and 2 degrees C. Having determined that preferential heating of the heart improved chances of revival, Andjus perfected the technique a number of times, eventually obtaining a 100% resuscitation rate by use of microwave diathermy. Having established a technique that ensured a high percentage of recovery, he began to investigate other problems related to resuscitation from hypothermia, including the effects of repeated coolings to zero and the possibility of resuscitating rats cooled to subzero temperatures.

In his 1955 publication in the Journal of Physiology, Andjus briefly states that cooling to 0-2 degrees C was performed as described in earlier papers. Cooling to subzero temperatures required immersion of the animal into a bath of propylene glycol between -5 and -20 degrees C. The method of reanimation was microwave diathermy as described previously.

To study the effect of length of time at ultraprofound hypothermic temperatures, Andjus cooled six groups of ten rats to colonic temperatures of 0-1 degrees C, with each group kept cold for a different length of time before attempting reanimation. The “period of suspended animation” ranged from 60-70 minutes to 110-120 minutes in 10 minute increments. Andjus defined the period of suspended animation as that “spent below 15 degrees C prior to the application of heat.”

10 out of 10 animals recovered completely from the group that spent 60-70 minutes in hypothermic circulatory arrest, but the longer the period of suspended animation beyond that, the lower the recovery rate, with 6 animals recovering from 70-80 minutes, 4 from 80-90 minutes, 3 from 90-100 minutes, 1 from 100-110 minutes, and none from 110-120 minutes. Several delayed deaths also occurred in animals revived after 70-100 minutes of “suspended animation.”

Another series of rats was cooled to 0-0.5 degrees C for 60-70 minutes and resuscitated repeatedly. The results of this experiment were extremely interesting. Initially, a rat was cooled and resuscitated every other day for a total of 8 coolings within 16 days. The rat lost a lot of weight, was unable to regulate its body temperature, and died 18 days after the last cooling. The next rat was cooled repeatedly but had longer intervals between coolings and was allowed to regain its initial weight after the first cooling. Interestingly, this rat was able to tolerate a longer periods of suspended animation (80 minutes) after several coolings, and recovered fully from a total of 10 coolings over 43 days.

Other rats were then repeatedly cooled to zero and allowed to regain their initial weight before each cooling. Andjus noticed that the time to regain weight also decreased with successive coolings, noting that “one rat needed 11 days to regain its initial weight after the first cooling, 6 days after the second, and 1-3 days after the third to eight cooling. The means taken from the results obtained with a group of seventeen animals show the same tendency.”

This trend in improved recoverability after repeated coolings appeared to hold true across the board:

Further improvements in the recovery after reanimation were noted in repeatedly cooled rats. For a few hours after the first reanimation and artificial rewarming to 37 degrees C the rat is not able to maintain its normal body temperature in a cold environment. When left in a refrigerator at 0 to +3 degrees C the reanimated rat steadily cools down. By contrast, a number of rats reanimated for the sixth to eighth time were perfectly capable of maintaining their normal body temperature in the refrigerator.

It was also noted that rats reanimated for the first time, and having just resumed their heart beat and respiration, with a body temperature of 15 degrees C (see Andjus & Smith, 1955) were not capable of spontaneous rewarming to 37 degrees C when left at room temperature (21-23 degrees C), and died after a few hours if the rewarming was not completed artificially. By contrast, a number of rats reanimated for the fifth to eighth time spontaneously regained their normal body temperature when left on the bench with colonic temperatures of 15 degrees C.

In addition, one rat cooled for the third time and another for the sixth time tolerated 120 minutes of suspended animation with full subsequent recovery. None of the ten control rats even recovered spontaneous respiration after cooling the first time to zero for 120 minutes.

Finally, Andjus investigated the recovery of rats from subzero temperatures. Some rats were “supercooled,” while others were allowed to undergo ice crystallization. In supercooled rats, both subcutaneous and colonic temperatures dropped steadily to temperatures as low as -5.7 degrees C subcutaneous and -3.3 degrees C colonic and for as long as 40 minutes in the subzero range. Andjus reported that “all rats supercooled without  crystallization were reanimated, recovered completely, and resumed growth.”

Animals that underwent crystallization did not fare so well. Eight of nine recovered heart beat and spontaneous breathing, but all died during rewarming or within 24 hours post-reanimation.

These experiments mark the beginning of investigation into the effects of hypothermic temperatures on mammalian physiology in a number of laboratories. More details about the freezing and supercooling experiments will be discussed in subsequent posts in this series, as well as the ongoing experiments of Radoslav Andjus and Audrey Smith.