This is a remarkable statement from Ben Best, and one that perhaps speaks best as to why CI is not a cryonics organization being run on a rational, scientific,or evidence based basis. When Ben Best writes: “There is no incompatibility between DMSO and PEG. The PEG make the solution hyperoncotic as the expected. My big mistake, and it was a bad one, I acknowledge, is that most of the vitrification solution was ruined because I was not aware that PEG would come out of solution when placed in a freezer,” he is making a statement that has the following outright errors, misunderstandings or distortions in it:
First, DMSO and PEG are incompatible in that they cannot be used either safely or effectively under the conditions required to carry out cryoprotective perfusion in a clinical (or research) setting AS PRACTICED BY CI. The first fact to consider is that DMSO-PEG solutions will often undergo gel formation when cooled to temperatures above freezing if left under refrigeration long enough. This phenomenon has a variable time course and is akin to nucleation and freezing in supercooled solutions—such mixtures may remain clear for days, or undergo precipitation/gel formation within hours of cooling.
Second, the perfusate in question, VM-1, is designed to be administered at a SUBZERO temperature (-7 degrees C) in order to minimize toxicity. The final concentration of cryoprotectants in VM-1, a roughly equal mixture of DMSO and ethylene glycol (the latter is the principal ingredient in automotive antifreeze) and has a total concentration of these two agents of ~ 70%!
In the brain tissue slice experiments performed by CI’s researcher Dr. Yuri Pichugin who invented VM-1, this very high concentration of agent was not introduced until the temperature of the brain tissue was −20 degrees C! CI’s own protocol for human cryonics patients calls for the introduction of VM-1 at the lowest possible temperature that they can achieve (~ −7 degrees C), given that they have no heat exchanger in their patient perfusion circuit. The way CI attempts to get the temperature of the final pass of VM-1 below 0 degrees C, and as close to the ideal of −20 degrees C as possible, is by the expedient of placing bottles containing the perfusate into a standard household-type freezer. The pre-chilled bottles of perfusate are then loaded into picnic chests and the perfusate is dispensed from there.
This Mickey Mouse operation results in perfusate that is at some (variable) subzero temperature when it is pumped through the perfusion circuit and delivered to the patient. While CI case reports are chaotic and inconsistent—some report temperature data during perfusion (http://www.cryonics.org/reports/CI97.html), some do not (http://www.cryonics.org/reports/CI75.html) - it is clear that even with the practice of pre-cooling the VM-1 perfusate in a freezer before perfusing it, CI patients never (so far as I can determine from published case reports, see: http://www.cryonics.org/refs.html#cases) reached subzero temperatures of −7 degrees C throughout VM-1 administration and in fact rarely reach subzero temperatures at all. This despite what CI says in its own description of how its patients are to be perfused with VM-1:
I would also note that in the same document, it is stated that the positive research results achieved with VM-1 in rats were achieved only under these conditions:
*”To test the toxic effects of CI−VM−1 (with or without ice blockers) hippocampal slices were saturated with increasing concentrations of ethylene glycol at 0ºC and −7ºC before cooling to −20ºC for ten minutes of saturation with CI−VM−1 (with or without ice blockers). The DMSO in CI−VM−1 is less toxic at lower temperatures, and is least toxic when introduced at −20ºC. Adding the ethylene glycol first and cooling at 0.3ºC/minute ensured that the solution would not be frozen at −20ºC when the CI−VM−1 (with or without ice blockers) is introduced.
The results of the toxicity test were as follows:
86.1% viability +/- 5.8% for 55% concentration CI-VM-1 without ice blockers
89.6% viability +/- 6.2% for 52% concentration CI-VM-1 with ice blockers*
Refractive Index values only taken during CI−VM−1 perfusion
As you can see from the CI data above and below, patient temperatures never come anywhere near −7 degrees, let alone the −20 degrees C called out in either the original animal research, or in CI’s own publicly posted protocol for how cryoprotective perfusion is to be administered. In fact, it is necessary to look a number of case reports to even document that CI is perfusing its p
atients with VM-1 chilled in a mechanical freezer: “Perfusion with CI−VM−1 vitrification solution began at 3:04 A.M. The CI−VM−1 was at freezer temperature (about −20ºC) in contrast to the ethylene glycol, which was at refrigerator temperature (about 3ºC)” see: http://www.cryonics.org/reports/CI110.html
In fact, this patient was one of the very few who achieved any subzero temperature during cryoprotective perfusion with VM-1:
Since it is standard CI operating procedure (and a biological imperative to reduce toxicity) to pre-cool VM-1 in a freezer before use, and since PEG-VM-1 solutions invariably undergo gel formation/precipitation under such conditions, then how is it possible to say, as Ben Best does, “There is no incompatibility between DMSO and PEG”? In fact, there is, because PEG solutions with glycerol or ethylene glycol do NOT undergo this kind of transition—at least they didn’t in my laboratory. Even more to the point, Aschwin & Chana deWolf, two researchers who work with CI reported this phenomenon to Best some weeks or months (as I recall) before he decided to conduct this ad hoc experiment on Curtis Henderson. I know this because i was a party to the correspondence.
Next up for discussions is the issue of “hyperonconicity.” Just as cells require a certain “tonicity” (electrolyte concentration) to maintain their normal volume, tissues with capillaries require a certain concentration (and type) of large (macro-) molecules (colloid) to avoid accumulating water between the cells and becoming swollen, or edematous. Hyperonconicity refers to any solution that has more ability to hold water in the circulatory system (circulating blood or perfusate) than would be the case under NORMAL conditions. The key word there is NORMAL. The macromolecules that comprise colloids can be thought of as molecular sponges that hold water in the capillaries and prevent it from accumulating in between cells as a result of the hydrostatic pressure of perfusion.
This water holding ability is quite complex and nuanced and depends upon the condition of the junctions between the cells in the capillary, the charge of the colloid, the unique chemical properties of the colloid (poorly understood), the configuration of the colloid molecule, and so on.
Onconicity and hyperonconicity are thus in actual practice, relative terms—relative to the condition of the capillary membrane. It is quite possible to have a markedly hyperoncotic perfusate and still have massive edema due to accumulation of water and of the colloid in between the cells! This is so because injured capillary membranes do not behave the same way as healthy or intact ones do—they leak! They leak colloid and with the colloid goes water. Simply cooling the organs (or bodies) of non-hibernating animals results in increased capillary permeability and the leakage of colloid and water into the spaces between cells. There is currently not a complete understanding of why this happens, or why some colloids do not leak as much in the cold as do others. In fact, only a very few species of colloid have been shown to leak less in hypothermia.
Capillary injury and consequent leakage of colloid from ischemia is vastly worse than that induced by hypothermia alone, and no colloid has been identified which is effective at inhibiting this leak, or even reducing it enough in clinical settings to meaningfully change outcome. In the setting of serious ischemic injury in the presence of high concentrations of cryoprotectant, NO COLLOID OR OTHER MOLECULAR SPECIES HAS BEEN SHOWN TO SIGNIFICANTLY REDUCE EDEMA—INCLUDING the PEGs OF VARIOUS MOLECULAR WEIGHTS. CI’s own research associates had reported this to CI prior to Ben’s decision to do an ad hoc experiment on a human patient with absolutely no prior laboratory animal or even bench testing of the perfusate. The only thing more unconscionable than such an uninformed and reckless action is the continued denial that it was such, and that his “mistake did not have the disastrous consequences implied by Mike Darwin.”
Here is what Ben Best says about the outcome for Curtis Henderson in terms of cryoprotective concentration at the end of perfusion:
″ The refractive index of the effluent was 1.366 after six liters of VM−1 had been perfused, and was 1.3586 at the end. Intermediate values were as low as 1.3586 and as high as 1.3651, but this was a small range with no trend, and is indicative of random variation. These values are well below the values of 1.416 for 60% VM−1 and 1.4275 for 70% VM−1 — and they showed no trend.”
Based on the reported refractive indexes of the venous effluent, I would estimate that Curtis Henderson had approximately 20% to 20% cryoprotectant in his brain—most of which was ethylene glycol. That would (again roughly estimating) equate to about 1.5M to 2.0 M glycerol in terms of colligative cryoprotective effect. I have not yet posted the electron micrographs (EMs) of the damage incurred when 3M glycerol is used as a cryoprotectant in the cat brain, but the histology is posted here: http://wp.me/p1sGcr-lt . I can tell you that the EM’s are vastly worse than are the light micrographs.
I have provided a detailed, and I believe accurate, scientific rebuttal to Ben Best’s claims. For onto a decade I have privately urged CI to either stop advertising that they are perfusing human patients under conditions which yield 86.1% viability +/- 5.8% (for a 55% concentration CI-VM-1 at − 20 deg C FOR TEN MINUTES, followed by cooling to and rewarming from −130 degrees C at 0.3 degrees C/min) brain tissue viability and ultrastructure, when in reality they are treating patients with 70% VM-1 delivered at +7 (or higher) to −7 deg C (rarely) over far longer periods of time (hours) and in the presence of ischemic insults that typically run to many hours, or even days!
This isn’t about elegance of writing, it’s about facts, most of which are derived from CI’s own website.
This is a remarkable statement from Ben Best, and one that perhaps speaks best as to why CI is not a cryonics organization being run on a rational, scientific,or evidence based basis. When Ben Best writes: “There is no incompatibility between DMSO and PEG. The PEG make the solution hyperoncotic as the expected. My big mistake, and it was a bad one, I acknowledge, is that most of the vitrification solution was ruined because I was not aware that PEG would come out of solution when placed in a freezer,” he is making a statement that has the following outright errors, misunderstandings or distortions in it:
First, DMSO and PEG are incompatible in that they cannot be used either safely or effectively under the conditions required to carry out cryoprotective perfusion in a clinical (or research) setting AS PRACTICED BY CI. The first fact to consider is that DMSO-PEG solutions will often undergo gel formation when cooled to temperatures above freezing if left under refrigeration long enough. This phenomenon has a variable time course and is akin to nucleation and freezing in supercooled solutions—such mixtures may remain clear for days, or undergo precipitation/gel formation within hours of cooling.
Second, the perfusate in question, VM-1, is designed to be administered at a SUBZERO temperature (-7 degrees C) in order to minimize toxicity. The final concentration of cryoprotectants in VM-1, a roughly equal mixture of DMSO and ethylene glycol (the latter is the principal ingredient in automotive antifreeze) and has a total concentration of these two agents of ~ 70%!
In the brain tissue slice experiments performed by CI’s researcher Dr. Yuri Pichugin who invented VM-1, this very high concentration of agent was not introduced until the temperature of the brain tissue was −20 degrees C! CI’s own protocol for human cryonics patients calls for the introduction of VM-1 at the lowest possible temperature that they can achieve (~ −7 degrees C), given that they have no heat exchanger in their patient perfusion circuit. The way CI attempts to get the temperature of the final pass of VM-1 below 0 degrees C, and as close to the ideal of −20 degrees C as possible, is by the expedient of placing bottles containing the perfusate into a standard household-type freezer. The pre-chilled bottles of perfusate are then loaded into picnic chests and the perfusate is dispensed from there.
Continued....
This Mickey Mouse operation results in perfusate that is at some (variable) subzero temperature when it is pumped through the perfusion circuit and delivered to the patient. While CI case reports are chaotic and inconsistent—some report temperature data during perfusion (http://www.cryonics.org/reports/CI97.html), some do not (http://www.cryonics.org/reports/CI75.html) - it is clear that even with the practice of pre-cooling the VM-1 perfusate in a freezer before perfusing it, CI patients never (so far as I can determine from published case reports, see: http://www.cryonics.org/refs.html#cases) reached subzero temperatures of −7 degrees C throughout VM-1 administration and in fact rarely reach subzero temperatures at all. This despite what CI says in its own description of how its patients are to be perfused with VM-1:
http://www.cryonics.org/research/CI-VM-1.html
“The Cryonics Institute protocol for perfusing the heads (brains) of cryonics patients is a 4-stage stepped open circuit perfusion:
(1) blood washout with carrier solution (4ºC) (2) 10% Ethylene Glycol (4ºC) (3) 30% Ethylene Glycol (4ºC) (4) 70% CI−VM−1 (−7ºC)”
I would also note that in the same document, it is stated that the positive research results achieved with VM-1 in rats were achieved only under these conditions:
*”To test the toxic effects of CI−VM−1 (with or without ice blockers) hippocampal slices were saturated with increasing concentrations of ethylene glycol at 0ºC and −7ºC before cooling to −20ºC for ten minutes of saturation with CI−VM−1 (with or without ice blockers). The DMSO in CI−VM−1 is less toxic at lower temperatures, and is least toxic when introduced at −20ºC. Adding the ethylene glycol first and cooling at 0.3ºC/minute ensured that the solution would not be frozen at −20ºC when the CI−VM−1 (with or without ice blockers) is introduced. The results of the toxicity test were as follows:
86.1% viability +/- 5.8% for 55% concentration CI-VM-1 without ice blockers 89.6% viability +/- 6.2% for 52% concentration CI-VM-1 with ice blockers*
Refractive Index values only taken during CI−VM−1 perfusion
CI Patient 97: http://www.cryonics.org/reports/CI97.htm
TIME (AM) TEMP (ºC) Flow rate(liters/minute) Pressure mm Hg RJVRI LJVRI 1:11 3.2 1.13 127
1:14 3.8 1.06 131
1:20 5.5 1.36 120 1.3976
1:26 7.0 1.07 117 1.3986
1:30 5.6 1.32 103 1.4017 1.4167 1:35 4.9 1.4048
1:37 4.1 1.4258 1.4242 1:40 3.5 1.4043 1.4183 1:45 2.5 1.4137 1.4209 1:47 2.0 1.4153 1.4224 1:50 1.6 1.15 139 1.4207 1.4236 1:52 Upper Body Perfusion Halted
2:00 Lower Body Perfusion Begun
2:00 0.5 0.42 121
2:03 0.5 0.32 136
2:05 0.5 0.32 134
2:10 0.5 0.31 143
2:13 0.5 0.40 200
2:15 0.5 0.46 185
2:20 0.5 0.46 175
2:25 0.5 0.48 191
2:33 0.5 0.48 174
Lower Body Perfusion Halted
Dry Ice Slurry Added to Head
2:37 −2.0
Refractive Index values taken during CI−VM−1 perfusion CI Patient 91: http://www.cryonics.org/reports/CI91.html
TIME (am) TEMP (ºC) RJVRI RBHRI LBHRI 9:35 7.0 1.4084
9:38 5.4 1.3655 9:40 4.2 1.4169
9:42 3.7 1.4198
9:46 2.1 1.4041 9:48 1.7 1.4138
9:50 1.8 1.4194
9:53 1.5 1.3721 9:55 1.1 1.4239
9:57 0.6 1.4206
10:00 0.4 1.3809 10:02 0.4 1.3830 10:07 0.7 1.4229
10:09 0.7 1.4233
10:11 0.6 1.3959 10:15 0.6 1.3971 10:16 0.8 1.4046
Continued....
As you can see from the CI data above and below, patient temperatures never come anywhere near −7 degrees, let alone the −20 degrees C called out in either the original animal research, or in CI’s own publicly posted protocol for how cryoprotective perfusion is to be administered. In fact, it is necessary to look a number of case reports to even document that CI is perfusing its p atients with VM-1 chilled in a mechanical freezer: “Perfusion with CI−VM−1 vitrification solution began at 3:04 A.M. The CI−VM−1 was at freezer temperature (about −20ºC) in contrast to the ethylene glycol, which was at refrigerator temperature (about 3ºC)” see: http://www.cryonics.org/reports/CI110.html In fact, this patient was one of the very few who achieved any subzero temperature during cryoprotective perfusion with VM-1:
Refractive Index values only taken during CI−VM−1 perfusion CI Patient 110: http://www.cryonics.org/reports/CI110.html
TIME (AM) Nasopharyngeal temperature (ºC) Flow rate(liters/minute) Pressure mm Hg RJVRI 3:07 8.25 1.07 102
3:08 6.9 1.06 101
3:09 5.3 1.07 100 1.3700 3:11 3.6 1.3769 3:16 4.3 1.39 101 1.3670 3:19 2.0 1.37 3:20 0.8 1.00 1.62 1.367 3:20 Perfusion Halted/Surgery
3:30 0.4 0.35 134 1.4166 3:33 −1.4 0.29 135
3:37 −2.6 0.26 120 1.42 3:40 −3.6 0.24 111 1.424 3:41 −1.4 0.29 135
3:43 −3.7 0.26 127 1.42 3:40 −3.0 0.28 126 1.454 3:45 −3.7 0.26 118
3:48 −3.9 0.28 128 1.4346 3:53 −5.3 0.28 125 1.4281 3:57 −5.6 0.27 122 1.4285 4:00 −5.8 0.26 120 1.4296 4:03 −5.8 0.26 117 1.4276 4:05 −5.8 0.26 117 1.4276 4:10 −5.7 0.26 115 1.4284 4:15 −4.6 0.26 114 1.4284 4:20 −3.8 0.26 109 1.4250 4:23 −3.0 0.27 86 1.4181 4:07 −2.3 0.34 82 1.4204
Since it is standard CI operating procedure (and a biological imperative to reduce toxicity) to pre-cool VM-1 in a freezer before use, and since PEG-VM-1 solutions invariably undergo gel formation/precipitation under such conditions, then how is it possible to say, as Ben Best does, “There is no incompatibility between DMSO and PEG”? In fact, there is, because PEG solutions with glycerol or ethylene glycol do NOT undergo this kind of transition—at least they didn’t in my laboratory. Even more to the point, Aschwin & Chana deWolf, two researchers who work with CI reported this phenomenon to Best some weeks or months (as I recall) before he decided to conduct this ad hoc experiment on Curtis Henderson. I know this because i was a party to the correspondence.
Continued....
Next up for discussions is the issue of “hyperonconicity.” Just as cells require a certain “tonicity” (electrolyte concentration) to maintain their normal volume, tissues with capillaries require a certain concentration (and type) of large (macro-) molecules (colloid) to avoid accumulating water between the cells and becoming swollen, or edematous. Hyperonconicity refers to any solution that has more ability to hold water in the circulatory system (circulating blood or perfusate) than would be the case under NORMAL conditions. The key word there is NORMAL. The macromolecules that comprise colloids can be thought of as molecular sponges that hold water in the capillaries and prevent it from accumulating in between cells as a result of the hydrostatic pressure of perfusion.
This water holding ability is quite complex and nuanced and depends upon the condition of the junctions between the cells in the capillary, the charge of the colloid, the unique chemical properties of the colloid (poorly understood), the configuration of the colloid molecule, and so on.
Onconicity and hyperonconicity are thus in actual practice, relative terms—relative to the condition of the capillary membrane. It is quite possible to have a markedly hyperoncotic perfusate and still have massive edema due to accumulation of water and of the colloid in between the cells! This is so because injured capillary membranes do not behave the same way as healthy or intact ones do—they leak! They leak colloid and with the colloid goes water. Simply cooling the organs (or bodies) of non-hibernating animals results in increased capillary permeability and the leakage of colloid and water into the spaces between cells. There is currently not a complete understanding of why this happens, or why some colloids do not leak as much in the cold as do others. In fact, only a very few species of colloid have been shown to leak less in hypothermia.
Capillary injury and consequent leakage of colloid from ischemia is vastly worse than that induced by hypothermia alone, and no colloid has been identified which is effective at inhibiting this leak, or even reducing it enough in clinical settings to meaningfully change outcome. In the setting of serious ischemic injury in the presence of high concentrations of cryoprotectant, NO COLLOID OR OTHER MOLECULAR SPECIES HAS BEEN SHOWN TO SIGNIFICANTLY REDUCE EDEMA—INCLUDING the PEGs OF VARIOUS MOLECULAR WEIGHTS. CI’s own research associates had reported this to CI prior to Ben’s decision to do an ad hoc experiment on a human patient with absolutely no prior laboratory animal or even bench testing of the perfusate. The only thing more unconscionable than such an uninformed and reckless action is the continued denial that it was such, and that his “mistake did not have the disastrous consequences implied by Mike Darwin.”
Here is what Ben Best says about the outcome for Curtis Henderson in terms of cryoprotective concentration at the end of perfusion:
″ The refractive index of the effluent was 1.366 after six liters of VM−1 had been perfused, and was 1.3586 at the end. Intermediate values were as low as 1.3586 and as high as 1.3651, but this was a small range with no trend, and is indicative of random variation. These values are well below the values of 1.416 for 60% VM−1 and 1.4275 for 70% VM−1 — and they showed no trend.”
Based on the reported refractive indexes of the venous effluent, I would estimate that Curtis Henderson had approximately 20% to 20% cryoprotectant in his brain—most of which was ethylene glycol. That would (again roughly estimating) equate to about 1.5M to 2.0 M glycerol in terms of colligative cryoprotective effect. I have not yet posted the electron micrographs (EMs) of the damage incurred when 3M glycerol is used as a cryoprotectant in the cat brain, but the histology is posted here: http://wp.me/p1sGcr-lt . I can tell you that the EM’s are vastly worse than are the light micrographs.
I have provided a detailed, and I believe accurate, scientific rebuttal to Ben Best’s claims. For onto a decade I have privately urged CI to either stop advertising that they are perfusing human patients under conditions which yield 86.1% viability +/- 5.8% (for a 55% concentration CI-VM-1 at − 20 deg C FOR TEN MINUTES, followed by cooling to and rewarming from −130 degrees C at 0.3 degrees C/min) brain tissue viability and ultrastructure, when in reality they are treating patients with 70% VM-1 delivered at +7 (or higher) to −7 deg C (rarely) over far longer periods of time (hours) and in the presence of ischemic insults that typically run to many hours, or even days!
This isn’t about elegance of writing, it’s about facts, most of which are derived from CI’s own website.