X-Message-Number: 9460 Date: Sun, 12 Apr 1998 00:08:07 -0400 From: Mike Darwin <> Subject: Efficacy of CI Methods CRITIQUE AND COMMENTS ON "The Effect of Freeze-Thawing on the Structure of Glycerolized Brains of the Sheep by Mike Darwin Introduction: Problems and Caveats I would like to start with an inventory of the working materials at my disposal for evaluation of this research. These materials consist of 3 articles which appeared in The Immortalist #8,9 & 11, 1994, and three envelopes of photographs labeled "Pichugin Sheep # 1: Figures 15-32, 33- 59 and 86-100 supplied by Robert Ettinger. No text, key, or explanation accompanied these photos and the variety of material makes me question if they were really all from one animal: "Sheep Head #1." Furthermore, I have no idea what was done to this animal, although my working assumption is that this is from a glycerolized, and a glycerolized frozen- thawed brain (i.e., two animals at a minimum). An immediate problem is that figures 60 through 77 were NOT included in the original material(actual photographs) I received. Added handicaps are that while all these pictures are discussed in the text of the article in issue #11 of The Immortalist, only figures 62, 63, 71 and 75 are shown there and these figures are reproductions of very poor quality which makes evaluation difficult (and, in the case of photos which were not reproduced at all, or sent to me as photographic prints) impossible. In addition to lack of acceptable quality photos and appropriate information identifying the procedure the tissue in a given photo was subjected to, I have a number of other problems with the documentation and reporting of this study. To make my comments easily understood and referenced, I will set them out as discrete, numbered points: 1) Perfusion conditions were not adequately described. Reference is made to the "CI method", but the details of this method are not given. Specifically it is very important to know the following things: 2) How were the sheep prepared for this procedure? Were they anesthetized and cannulated in a beating heart state? Were these heads collected postmortem (i.e., after slaughter)? If the heads were removed after slaughter how was introduction of air into the vasculature prevented?. If the sheep were slaughtered, how were they killed? In the United States and most Western countries agricultural animals which are slaughtered for food purposes are first rendered unconscious by either a massive blow to the head using a pneumatically driven piston, or by electrocution by passage of high voltage/amperage alternating current through the head (brain). The animals are then hoisted into the air by their hind limbs and their throats cut: severing the carotids and jugulars which causes them to bleed-out and experience cardiorespiratory arrest. As is obvious from the above, such a procedure would result in serious pre-cryopreservation injury to the brain and would be a serious source of artifacts. To my knowledge all domestic animals slaughtered in the United States and Europe undergo this kind of stunning before dismemberment, with the sole exception being cattle prepared using the Kosher method (which utilizes exsanguination by rapid cutting of the throat with an extremely sharp knife). 3) What were the details of perfusate preparation? What was the exact composition of the perfusate and how was it prepared for perfusion: i.e., was it filtered, was the pH adjusted, etc. and, if so, how and to what values? 4) What perfusion pressures were used to carry out glycerol perfusion and/or attempts at fixative reperfusion? How did perfusion pressures vary over the course of the procedure? 4) What kind of perfusion circuit or equipment was used to carry out perfusion? What kind of pump was used? Were 20 micron or 40 micron filters incorporated in the circuit? What kind of equipment was used to measure temperature and where were the temperature probes placed (not only for perfusion but for cooling to, and rewarming from, -196 degrees centigrade)? 5) Was temperature data collected at regular intervals so that cooling curves and phase transitions during freezing can be determined and/or reproduced? This is of critical importance. 6) When tissue was removed (post-thaw) for immersion fixation did the fixative into which it was placed contain glycerol in approximately the same concentration as was present in the tissue or estimated to be present based on final venous effluent glycerol concentrations? 7) What was the exact formula of the fixative solution used, its method of preparation, its osmolality and the grade of chemicals used in its preparation? Of particular importance is the quality or "grade" of glutaraldehyde used. In particular, was the glutaraldehyde "Electron Microscopy" (EM) Grade"? 8) What exact protocol was used to prepare the tissue for electron microscopy? In particular, what agents were used for post fixation and staining and in what concentration (i.e., osmium, uranyl acetate, lead citrate, etc.) 9) How was tissue prepared for light microscopy and what stains were used? 10) What are the magnifications of the micrographs, both light and EM? It is virtually impossible to give a meaningful and rigorous evaluation without knowing the magnification of the pictures being examined (Although this can be guessed at in some situations where organelles or other structures of generally known size are present and thus provide reference for some estimate of evaluating the magnification.) 11) Light micrographs were supplied as poor quality prints in black and white. Color is an essential element in evaluating light microscopy as subtle color differences often reflect overlying layers of structure. Also, the way the tissue "takes up" the stain or appears after staining speaks not only to the visible structure of the tissue, but also to its molecular structure, i.e., alterations in histochemistry are often reflected in abnormal staining. 12) How thick were the sections cut from the brain for fixation after cryopreservation? Was the fixative warm or cold and/or was the tissue refrigerated during fixation, and, if so, at what temperature? General Evaluation of The Research Given the caveats above it is difficult for me to evaluate how meaningful this work is. I am left to make "default" conclusions such as assuming tissue loaded with hyperosmolar cryoprotectant (glycerol) was cut and plunged into fixative presumably at room temperature? In the absence of information to the contrary I would also assume that fixative was very hypo-osmolar with respect to the tissue: for instance, even normal Trump's storage fixative or Karnofsky's has an osmolality of under 2000 mOsm. Further, if the tissue was fixed in glycerol-containing fixative, how was the glycerol removed prior to removal of the fixative by buffer, solvent washing to remove water, and post fixation staining? (The same questions also apply for histological preparation procedures). Because the light micrographs are black and white, have no magnifications listed and are of such poor quality, I will keep my comments brief. My general impression of both the published photos and of the micrographs sent to me is one of marked injury. It is impossible for me to sort out the source of the injury but the following remarks are generally applicable: There is evidence of massive cellular dehydration. There is a great deal of free-space and apparent disruption of the neuropil. While the overall structure of the tissue is discernible, the disruptions here are about the worst I've seen for frozen-thawed cryoprotected brain. In particular, they are far worse than Bodian stained and Nissl stained light micrographs produced by Cryovita/Alcor in the mid-80's using controlled 4M glycerol perfusion In those micrographs evidence of dehydration was much less apparent and the fine structure of the neuropil was evident even in animals subjected to 30 minutes of warm ischemia followed by 24 hours on ice (with blood present). My *impression* is that there is a lot of loss of structure in many of these light level micrographs; this was not seen in the work done with cats in the mid-80's. On a more favorable note I see no evidence of fractures (which are rather distinctive in their appearance) although I do see what appears to be tears or ice holes in figure 37 &38. Visible disruption and light-level debris are also seen in figure 44 Evaluation of Specific Electron Micrographs Figures 15 & 16 show cell nuclei and cytoplasm. The nuclei appear to show postmortem changes compounded perhaps by cryoprotective perfusion and or cryopreservation injury. There is clumping of the chromatin and large losses of chromatin which are especially apparent in Figure 16. These two micrographs also show very typical changes in intracellular organelle structure: the mitochondria are "blown" as evidenced by extensive swelling and partial obliteration of the cristae. There is vacoulation of the cytoplasm, loss of ground substance, and alteration (disorganization) of the ground substance. On the positive side the nuclear membrane is visible and there is visible plasmalemma near the bottom of the field and in the upper right hand corner of the field. Figure 17 shows several debris filled cavities (some containing blebs or vesicles) which in at least one case appears to be the remnants of a mitochondrion. These open, debris strewn areas may be the locations of capillaries. There is some nicely dense myelin present, but many of the axons contain what appears to be debris, are empty, or contain what appears to be shrunken axoplasm presumably as a result of dehydration. Some of the vacuoles contain debris suggestive of organelle ultrastructure Figure 19 has many of the same changes observed in the previous micrographs. Clearly some of the vacuoles are the remnants of massively swollen mitochondria. There is a large, debris strewn lacunae-like area near the upper right of the field the origin of which I cannot identify. The lower right of the field shows a large open space occupied with membranous material organized into blebs. Figure 20 shows large scale disruption of cytoplasm,with a large open space with remnants of intracellular organelles projecting into it. Some of the plasma membrane of the cell occupying the upper left 2/3rds of the micrograph is evident. My impression of this shot is that it is at a magnification of 5K or less, although due to the dilation of the mitochondria it is hard to tell. The myelin is mildly compromised but looks reasonably intact, however the axoplasm is extremely shrunken and appears to have lost a lot of ground substance given its low density and its obvious dehydration. Figure 21 looks good at first glance. This is because the myelin is well preserved here and nicely dense and most of the axons in this field are myelinated. However, as is typical of most of the micrographs the field is strewn with vacuoles and intracellular organelles with varying degrees of disruption. Figure 22 has two positives to remark on: a normal (non- ischemic) appearing nucleus with good density, and a lot of good myelin. The neuropil looks very bad overall and of course, the typical injury to cell organelles seen in the previous micrographs is evident here as well. Figure 29 appears to be pituitary tissue. There is a capillary occupying the left of the field with two small blebs present in the lumen. There is some partially intact endothelium, however most of the endothelial cell on the upper left is missing and there is a naked endothelial cell nucleus still attached to the basement membrane. The membrane of this nucleus looks intact. Figures 30 &31 show much less well preserved pituitary. The nuclei are missing most of the chromatin and the intracellular structure has been reorganized into membranous vacuoles as is the case with almost all of the micrographs. Figures 45,46, &47 are pictures of almost total destruction. The nuclear membrane of the cell is visible in the upper right of the picture but the nucleoplasm is abnormal with clumping and loss of ground substance. There is massive vacoulation and some of the most dilated mitochondrion I've ever seen (assuming that's what they are). Figure 48 looks a little better: the nucleus is more normal in appearance but the cytoplasm is mostly debris and the plasma membrane is not continuous or even in evidence most of the time. Massively swollen mitochondria are present as are dilated axons in varying states of destruction. Figure 49 presents a debris strewn field for the lower 1/3rd of the picture. There is discontinuous plasma membrane which trails off into disorganized debris toward the right middle of the field. Nuclei do not appear to have membranes. There is a single myelinated axon in an area of debris strewn open space which contains very abnormal looking axoplasm. Figure 50 shows massive disruption of myelin. Some axons appear dilated and filled with debris while others have electron dense apparently dehydrated axoplasm. Figure 52 shows massive disruption. The lower left 1/3rd of the field is more or less amorphous debris (myelin and cytoplasmic remains?). The myelin is unraveled and (lower right) disintegrating. Figure 53 shows a disrupted nucleus with clumping and possible loss of chromatin, loss of the nuclear membrane at about the 3 o'clock position, a ruptured, empty axon on the lower edge of the frame, and, slightly to the left, generalized intracellular chaos. Figure 86-90 are more of the same: massive disruption of cytoplasm, large cavities which may be ice holes, "empty" axons, severely damaged myelin, and so on. Figures 92, 93, 94,& 95, appear to be from frozen thawed tissue. Aside from some reasonably intact looking nuclei in Figure 95, this is some of most severely injured tissue I have ever seen. This looks more like tissue homogenate than organized tissue. The only clue as to what kind of tissue this was is the presence of badly compromised myelin. I could have spent a lot more time going over every micrograph, but it hardly seems productive since it is so consistent in appearance. General Conclusions In his report entitled "Cryoconservation of sheep heads by the Cryonics Institute's method" Pichugin states that glycerolized frozen-thawed brains demonstrate (sic)"On the whole ultrastructure of the tissues may be qualified as relatively good." He also states (sic): "Moreover there were excellent histological cryopreservation of the thawed, glycerolized brain tissues and the good ultrastructure of these tissues." Similar remarks are made in earlier reports. Both the published photos in The Immortalist and the copies of the micrographs (prints) I received do not support any of these conclusions. In sharp contrast to previous work by Cryovita/Alcor and by Fahy, et al the light-level injury was far worse in these brains than was seen in either the beating heart perfused animals or the 24-hour warm/cold ischemia animals done in the mid-1980's. The light level results are markedly worse than those obtained by Fahy using 6M glycerol; in fact there is no comparison. What I find most surprising is Picghugin's evaluation of the EM results. This tissue is grossly disrupted on every level. A first year neurophysiology graduate student would be able to tell this as would someone who was briefly oriented to what normal brain ultrastructure looks like. Particularly disturbing is the absence of control photos showing normal brain architecture in the absence of ischemia, cryoprotective treatment, or freezing and thawing. The notion that this kind of injury is compatible with any resumption of functional indices of organized brain activity/metabolism is completely unsupported by even the best of the micrographs. This is further confirmed by the inability of the investigators to reperfuse the brains after thawing. In my opinion, the only positive finding in this study is the absence of fracturing and its probable relationship to slow rates of cooling and rewarming. Caveats On These Comments It should be noted that much of the injury apparent here could have resulted from osmotic stresses induced during fixation. Additionally, the inability to reperfuse the brain which necessitated the use of immersion fixation is another possible complicating factor. It is well established that brain does not fix well by immersion and that the rate of penetration of fixative is typically on the order of 1 mm per day into tissue blocks. This may not have been a significant confounding problem if small blocks were cut at near 0 degrees centigrade, fixation was carried out at near 0 degrees centigrade, and samples were cut from the outside, and presumably well (and more rapidly) fixed part of the tissue block. The usual caveats apply about "stirring" or disruption of ultrastructure as a result of thawing, however this would not account for poor results observed in glycerolized (unfrozen tissue) although I have no way of telling which tissue was frozen and which was merely glycerolized. Finally, glycerol may well interfere with fixation and certainly interferes with osmication and staining with uranyl acetate and lead citrate (our own results). Thus, if the tissue was not deglycerolized thoroughly prior to osmication this may resulted in poor fixation of myelin and cell membranes (however, this is not my impression from these micrographs: for instance, the myelin, even when disrupted, appears well osmicated). Final Comments Thank you for the opportunity to review this interesting pilot study. I feel this work has real merit in that it: a) Provides some level of real-world feedback about the efficacy of current cryopreservation techniques (very poor). Fuller appreciation of the significance of this work will come when other laboratories and/or these investigators control for potentially artifact-inducing variables (osmotic shock, lack of perfusion fixation, etc.). b) The absence of macroscopic and/or microscopic fractures is also of significance and the reason for this needs to be understood (for instance separating the variables of cryoprotectant perfusion from cooling/warming rates). Unfortunately, I cannot agree with the authors' comments that this work demonstrates good histological or ultrastructural preservation using the CI method. In fact, it is my considered opinion that the reverse is the case: the ultrastructural and histological preservation are very poor and rank amongst the worst I have seen in any cryoprotected tissue. Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=9460