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 

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 

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 

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 

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