X-Message-Number: 7658 Date: 06 Feb 97 16:47:15 EST From: Michael Darwin <> Subject: Fractures as a problem? Doug Skrecky writes: >I would like to suggest a possible solution to the problems that have >been occurring with replication of the Visser technique. It seems that >good viability requires that the rat hearts be retrieved from liquid >nitrogen promptly, while prolonged immersion is lethal. I strongly >suspect the problem is cracking and believe that an examination of the >hearts used in the experiments will back me up in this. Switching to a >two step fast then slow cooling rate should eliminate this problem. This is a thoughtful suggestion, but, unfortunately fracturing did not appear to be the problem in 3 of 4 of the Visser rat heart experiments. I make the following observations: 1) The 21st Century Medicine (21CM)staff (including myself) were granted extraordinary latitude in observing the entire procedure and in insuring that controls were done which were rigorous. We carefully observed the hearts after cryopreservation and did not note any fractures except during the last run where there was poor perfusion of the cryoprotective agent prior to freezing. In particular, I was able to examine the heart at distance of a few centimeters while it was being reperfused and the surface of the myocardium was crack free. 2) Sandra Russell, a researcher at 21CM (and a former EKG technician) was quite persistent in asking that the hearts be dissected and examined following thawing and reperfusion. She encountered considerable reluctance from Mrs. Visser, but Sandra persisted and was successful. The hearts were sectioned sagittally through the left ventricle. Two of the hearts revealed uniform blanching of the myocardium with accompanying edema. There were no signs of fractures visible to the naked eye, or with a loupe, in either the atria, the septum or the ventricular walls. One heart had epicardial blanching with evidence of failed endocardial reperfusion (see below). 3) Three of the four hearts reperfused to some degree following cryopreservation. Their appearance following freezing was the fairly normal "kidney bean" color typical or blood free hearts. Shortly after the start of reperfusion the myocardium began to blanch adjacent to the coronary vessels and the effluent perfusate from the right heart was noted to be pink-tinged. This is a _very_ typical finding after severe cryoinjury to the heart. The pink-tinge in the effluent is myoglobin being washed out of ruptured heart cells. This is a cell-level injury which has nothing to do with cracking. The same sort of injury is seen in red blood cells similarly treated except that the colored molecule released is hemoglobin, not myoglobin. 4) Similarly, in other studies, kidney and liver slices evaluated for viability after cryopreservation with 25% DMF exhibit protein loss from cell lysis and the bathing media takes on a typical "root beer float" appearance. 5) Other investigators, using no cryoprotectants or other cryoprotectants such as DMSO, have reported similar findings upon reperfusion following freezing of rat hearts to very low temperatures. Karow and Fahy in particular have extensively documented this effect. Further, they achieved recovery of contractility in rat hearts subjected to 20 minutes of freezing at -20 degrees C following cryoprotection with DMSO. 6) The injury I observed in the hearts cryopreserved with the Visser method was massive. The myocardium was globally compromised with lysis or major alteration in membrane permeability of the majority of the cardiac myocytes occuring as evidenced by onset of thaw rigor, blanching of the myocardium, and the appearance of myoglobin in the effluent following thawing. NOTE: the effluent was crystal clear long before the end of, and during the entire course of cryoprotective perfusion. 7) Variables between runs were so poorly quantified and so poorly controlled it is impossible to draw meaningful conclusions about those hearts that recovered following 60 seconds of immersion in LN2 on the Langendorff column (as reported by Zawacki/Ettinger). That was NOT the experiment I observed. There was no good measurement or control over "shroud" volume/composition, intracardiac temperature descent/rewarming, EKG acquisition, etc. In short, what I saw was not good science. Artifact was reported as evidence of electrophysiological recovery, and no paper strips were available for evaluation. This is unacceptable for an event that can occur in a fraction of a second. In conclusion, I don't think cracking was a limiting factor in this model. I think the most likely explanation for failure of the hearts to recover contractility was massive cryoinjury as a result of inadequate cryoprotection. Everything I observed in terms of outcome was consistent with the results seen by other investigators in hearts subjected to cooling to -196 degrees C in the absence of adequate cryoprotection. Lastly, I want to take this opportunity to congradulate and to thank Fred and Linda Chamberlain for what they did that weekend. Fred was passionately and unflinchingly insistent that the protocol be followed in terms of time, temperatures, and rate of cryoprotectant introduction. He and Linda also allowed the other investigators present (the 21CM staff and Paul Segall of Biotime) to observe the experiments first hand. This was _invaluable_. Such accountabilty and unflinching committment to getting at the truth took something rarely seen anywhere: REAL COURAGE. Whatever our differences have been, I congradulate Fred and Linda, and Alcor for this courage. I also thank them, and so should the rest of us. Mike Darwin Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=7658