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On The Determination Of Cracking Limits In Cryopreserved Cat Brains - Part 1

by Dr. Yuri Pichugin

(The following was compiled from several separate reports of variious phases of the work, the last received in May of 1998. Any flaws in editing or translation are our own. Reminder: There were no experiments done on live animals. The cats that were anaesthetized and then euthanized would have been euthanized in any case.)

I work with cat heads in order:

  1. to determine cracking limits;
  2. to study a mechanism of cryodamage by cracking;
  3. to compare an anti-cracking effectiveness of CPAs (glycerol, DMF, and another);
  4. to find the most effective procedure of CPA perfusion and reperfusion;
  5. to verify the most important results of the cat head experiments on sheep head and skulls; Also, to verify a correction of Rubinsky's equation and, maybe, to perfect it;
  6. to find the optimum CPA and method of cryopreservation and to verify it using cat brain slices;
  7. to perform more perfect calculations of cracking limits for human brains using the results of the determination of the cracking limits of the cat and sheep brains.

Transition from the work with sheep brains to the work with cat brains was conditioned by greater accessibility cats than sheep and a possibility to work with the cat brains in short order after death of the animals that it is especially necessary to work brain slice culture. The purposes of this work here reported was to study the effect of freeze-thawing modes on the morphological structure of the native cat brains and the cat brains perfused with cryoprotectant agents (CPAs) DMF (dimethyl formamide) and glycerol.

MATERIALS AND METHODS

General methods for all the cat brains are described here only. Cats (both cats and tom-cats) weighing 2.6-3.6 kg were used in the work. Firstly the animals were anesthetized by ether. Then 200 mg/kg hexanalum was injected intramuscularly. In 20-30 mins after that, intravenous injection (150 mg/kg) was performed for deep general anesthesia. Anesthetized cats were killed by rapid cutting of the throat with an extremely sharp knife, if their heads were not used for CPA perfusion. For CPA perfusion, anesthetized cats were killed by cutting of the jugular veins only. In 5 mins before that, heparin (700 units/kg) was injected into the jugular veins.

The washout cat heads from blood was performed by injection of 0.5 L of Tyrode's solution with heparin (10 units/ml) via the carotid arteries cannulated at the level of the second verterbra. A period of time from a moment of death of a sheep to the beginning of washout of head tissues from blood was practically absent. The rate of feeding washed solution was about 40 ml/min. The cat heads were then perfiised by low concentrations of DMF {20% DIVIF on Tyrode's solution) or 20% glycerol on T. s. All solutions were prepared using analytical grade chemicals. Temperature of the solutions was 25 C (room temperature). After perfusion, the cat heads were cut at the level of the second vertebra.

To obtain faster rates of freeze-thawing, all tissues and bones around the cat skulls were moved away. Then the heads or skulls were wrapped up in an aluminum foil and rapidly plunged directly into LN2. Temperature was measured by copper - constantan thermocouples. Thawing of the skulls was performed using a water bath at + 40 C. Washout of the whole brains from CPAs (reperfusion) after thawing was not performed. After thawing, the brain was removed for examination. Pieces of the frontal lobes of the brain were used for histological preparations. Pieces of the back part of cerebral hemispheres were used for determination of CPA concentrations. Determination of glycerol concentration in head tissues (the white and grey matter of brain, the mastication muscle, and the tongue) was carried out by the method previously described in The Immortalist 1994, v. 25, No. 8, p.5. (I'll write a special article on determination of CPA concentrations in tissues and cells.) The washout of cat brain pieces were not carried out. The tissues with CPA were fixed in 10% neutral formalin with addition of CPA.

RESULTS AND DISCUSSION

(Before description of freeze-warming modes, I give an information about average rates using Figs. 1,2 as an example. Figs. 1,2 concern my article in The Immortalist 1997, No. 3/4, p. 48-50 (about the sheep skulls). I am sorry, I have written in Figs. 1,2 "sheep heads" instead of "sheep skulls". Comparing the average rates in the Figs. with the ones in that article (p. 49), one can see that the average rates in Figs. 1,2 more correctly reflect the curve of freeze-warming. I'll therefore give an information about freeze-warming modes using merage rates on approximately linear regions of the curves of freeze-warming. All data on the freeze-warming modes were processed by PC (Pentium 133) and were presented in the form of the graphs. That is very helpful for my work.)

In the beginning of the work, the maximum cooling and warming rates of cat heads and skulls were found. Also, the present of macrocracks in those thawed cat brains was verified.

Here is a summary of the report:

 

1. 1 A native head (295 g) of a cat (3.3 kg) was frozen over the surface of LN2 and warmed at +40 C using an aqueous bath. The first thermocouple (tcl) which was located in the palate showed the cooling rates of 0.51 C/min from -5 C to -106 C, 0.43 C/min from -106 C to -145 C, and about 0.27 C/min to -196 C. The second thermocouple (tc2) which was located at a distance about 5 cm from the foremen of the second vertebra ( about in the middle of the brain) showed the cooling rates of 0.67 C/min from -5 C to -10 C, 0.51 C/min from -100 C to -145 C, and about 0.30 C/min to -196 C. The warming rates were 8.8 C/min from -196 C to -50 C for tcl and 8.4 C/min for tc2 in the same temperature region.

1.2 The same cat head was then frozen by a direct immersion into LN2 with the rates 27.0 C/min from -13 C to -116 C, 9.14 C/min from -116 C to -166 C, 2.05 C/min to -196 C for tcl and 20.3 C/min from -6 C to -151 C, 5.50 C/min from -151 C to -186 C for tc2. The head was warmed at +40 C with the rates 21.4 C/min from -196 C to -50 C, 0.94 C/min from -50 C to -18 C, 0.30 C/min to 0 C for tcl and 16.5 C/min from -196 C to -37 C, 1.0 C/min from -37 C to -7.5 C, 0. 14 C/min to 0 C for tc2. The brain of this cat head was very strongly cracked. It was not fixed in formalin and photographed.

2. Pilot experiments on determination of freeze-warming modes of a cat head perfused with 20% glycerol and its skull. The cat weighed about 4.0 kg, its head weighed 307 g, and its skull was 53 g. The thermocouple 1 and 2 had the same location as in the head of experiment 1. 1.

2.1  The head was frozen by immersion into LN2 with the rates 3.5 C/min from -12 C to -176 C for tcl and 4.6 C/min in the same temperature zone for tc2. The cat head was thawed at +40 C with the rates 14.1 C/min from -196 C to -80 C, 4.0 C/min to -24 C for tcl and 8.7 C/min from -196 C to -65 C, 3.0 C/min to -24 C for tc2. {These modes were presented in Fig. 3.4 which had been sent you. I am sorry, I have written in the Fig. 5 "a native cat head" instead of "a cat head perfused with 20% glycerol"}. The freeze-warming rates for the native cat head of experiment 1.2 were slower than the ones for the glycerolized head of this experiment.

2.2  The skull of the cat head was frozen only to -90 C by immersion into LN2 and thawed at +40 C. The thermocouple 1 was located on the bottom part of the skull under 1-2 mm of the tissue. Tc2 was located at a distance about 2.5 cm from the foremen of the atlas. The location of the probes was used for all the cat skulls later on.

2.2. 1  The cooling rates were 22.7 C/min from -4 C to -72 C for tcl and 24.7 C/min from -12 C to -65 C, 5.5 C/min from -65 C to -88 C ibr tc2. When temperature of tcl reached -72 C, the skull was rapidly lifted from LN2, then rapidly plunged into LN2, and again lifted from it so as not to reduce temperature in the brain lower -90 C. When temperature of tc2 reached -90 C, the skull was placed at the level over LN2, where temperature was - 90 Q and kept for 15-20 mins. Freezing the skull was performed into a high cylindrical vacuum flask ( high of the glass vessel is 1200 cm, its diameter is 100 cm) with the exactly determined temperature levels over the surface of LN2. The skull was warmed at +40 C. The warming rates were 37.6 C/min from -90 C to -36 C, 7.4 C/min from -36 C to -12 C for tcl and 15.3 C/min from -90 C to -37 C, 6.3 C/min from -37 C to -18 C for tc2.

2.2.2  A duplication of the experiment 2.2.1 with the same cat skull. The freezing rates were 73.8 C/min from -5 C to -36 C, 21.1 C/min from -36 to -97 C for tcl and about 2.0 C/min from -5 C to -12 Q 33.3 C/min from -12 C to -37 Q 62.2 C/min from -37 C to -65 Q 27.0 C from -65 C to -80 Q 6.3 C from -80 C to -90 C 1br tc2. The warming rates were 23.8 C/min from -90 C to -24 Q 4.8 C/min from -24 C to -4 C for tcl and 20.7 C/min from -90 C to -30 Q 5.7 C/min from -30 C to -18 Q 1.24 C/min from -18 C to 0 C for tc2. The duplication showed a rather bad reproduction of the freeze-warming modes using a manual control but not an automatic programmed control. {To reproduce by hands freezing modes of rat hearts is still more difficult because a rat heart weighs about 1 g.)

2.3  The same skull was also two times frozen to -196 C and warmed.

2.3.1  The cooling rates were 108.0 C/min from -5 C to -50 C, 22.5 C/min from -50 C to -176 C, 5.0 C/min from -176 C to -196 C for tcl and 11.1 C/min from -5 C to -18 C, 53.9 C/min from -18 C to -123 C, 24.4 C/min from -123 C to -176 C, 6.10 C/min from -176 C to -196 C for tc2. The warming rates were 114.0 C/min from -196 C to -80 C, 30.0 C/min from -80 C to -42 C, 8.72 C/min from -42 C to -6 C for tcl and 49.1 C/min from -196 C to -65 C , 20.4 C/min from -65 C to -43 C, 5.28 C/min from -43 C to -10 C for tc2.

2.3.2   A duplication of experiment 2.3. 1. The cooling rates were 26.2 C/min from -4 C to -176 Q 5.76 C/min from -176 C to -50 C, 9.5 C/min from -50 C to -12 C 1br tcl and 44.5 C/min from -196 C to -50 C, 7.7 C/min from -50 C to -18 C for tc2. The method of the rapid immersion of the skull into LN2 had a rather good reproduction. The brain of this cat head was very strongly cracked as well as the first cat brain ( the experiment 1). It was not fixed in formalin and photographed.

3. Freezing a glycerolized cat head to -90 C. The glycerolized head (274 g) of the cat (3.8 kg) was frozen over the surface of LN2 to -90 C and warmed at +40 C. The cooling rates were 3.0 C/min from -16 C to -80 C [(16 - 80) - used as an abridgement below] and 0.9 C/min (80 - 90) for both tcl and tc2. The warming rates were 3.8 C/min (90 - 32), 1.3 C/min (32 - 18), 0.38 C/min (18 - 0) for tcl and 3.3 C/min (90 - 37), 1.2 C/m in (37 - 18), 0.25 C/min (18 - 0) for tc2. The photos 1. 1 - 1. 3 show the brain of the cat head after warming and fixation.

Photos below - 1 shows the upper surface of a cat brain, a photo .2 shows the lower surface of the cat brain, and a photo .3 demonstrates the brain cutting into 2-3 longitudinal sections for determination macrocracks inside brains. All brains are on a Petri cup. The back parts of cerebral hemispheres from CPA-perfused brains were cut after thawing for determination of CPA concentrations. Then the brains were fixed in 10% formalin. After fixation, the brains were photographed (the top and the bottom), and pieces of the frontal lobes were cut for histological preparations. Only after that, brains were cut into the sections which were also photographed. The top of the photos is that where the numbers of photos had been written.

The photo 1. 1 shows the top of the glycerolized brain on its front side. There are no macrocracks. The large central fissure between cerebral hemispheres was very well observed. The photo 1.2 shows the bottom part of the brain on the front side too. There are no macrocracks too. The back parts of the hemispheres were taken for determination of CPA concentration.

   Photo 1.1

  Photo 1.2

The photo 1.3 demonstrates the three longitudinal sections of the brain without any macrocracks and macroruptures. The part of the front lobes was taken ibr the histological examination of microcracks. The "cavities", which are observed on the left piece, are not cracks. The macrocracks have the characteristic appearance (see below). That is the convolution and the other bounds of the macrostructures of the brain. On the right piece, the white spots (like white colouring) may be observed. However that is not any colouring but is patches of light which were resulted from a photo-flash. The photos 1. 1-1. 3 showed one of the best cryopreserved brains.

  Photo 1.3

4. Freezing a native head (314 g) of a cat (3.8 kg) to -90 C.

The head was washed out from blood but was not perfused with any cryoprotective agent. The freezing was carried out by immersion of the head into methylcellosolve bath cooled to -90 C. Thawing of the head was performed using the water bath at +40 C. Tc2 was used in this experiment only. The cooling rates were 1.3 C/min (8 - 64) and 0.33 C/min (64 - 90). The warming rates were 3.8 C/min (90 - 19), 1.1 C/min (19 - 10), and 0.2 C/min (10 - 0).

The photos 2.1 - 2.3 display the fixed brain of the cat head. This brain (the photo 2. 1) has the dura matter which was usually ablated. The photo 2.2 shows the lower part of the brain on the side of the back part. In all the cases, the cat brain was taken out from its skulls without its cerebellum because the cat cerebellum was located into the special compartent of its skull in contrast to the sheep cerebellum. Therefore to take out the cat brain together with its cerebellum from its skull is very complicated and difficult. A foremen from the tip of the thermocouple 2 is seen in the photo 2.2. The photo 2.3 shows that macrocracks are absent inside the brain as well as on the upper and lower surfaces. On the upper left section, the central fissure was observed but it is not a crack. The brain even without glycerol was well cryopreserved as well as the glycerolized brain in the experiment 3.

5. To test the presence or absence of macrocracks in harder conditions, a cat brain was frozen using faster rates but to -90 C too. The cat weighed 2.2 kg. Its skull weighed 49 g. The native skull was frozen using direct immersion into LN2 but to -90 C as the same way as the skull in experiments 2.2. The cooling rates were 32.4 C/min (0 - 80), for tcl and 22.6 C/min (10 - 50), 8.6 C/min (50 - 78), 1.2 C/min (78 - 90) for tc2. The warming rates were 27. 0 C/min (90 - 65), 19.1 C/min (65 - 24), 6.3 C/min (24 - 0) for tcl and 13.0 C/min (90 - 18), 2.8 C/min (18 -4) for tc2. The rates of this experiment were approximately comparable with the rates of experiments 2.2. However the rates were much faster than the rates of experiment 3 and 4.

The photos 3.1-3.3 demonstrate this fixed brain without evident macrocracks. However the lower slice of the brain in the photo 3.3 possibIly has a rupture as prolongation of the central fissure. The final conclusion about cracking may be made only after examination of the histological preparations of this brain. The brain was not washed out from blood but its blood vessels were not observed because haemoglobin lost its colour during 1-1.5 month of keeping in formalin.

6. Freezing a glycerolized head (328 g) of a cat (3.2 kg) to -196 C. The cooling rates were 4.0 C/min (24 - 183), 0.73 C/min (183 - 196) for tcl and 4.7 C/min (16 - 183), 0.65 C/min (183 - 196) for tc2. The warming rates were 9.3 C/min (196 -42), 3.5 C/min (42 - 30), 0.84 C/min (30 - 0) for tcl and 8.0 C/min (196 - 50), 4.0 C/min (50 - 37), 0.71 C/min (37 - 0) for tc2. The rates were wry similar to the rates of experiment 2. 1.

The photos 4.1-4.3 display the fixed brain of this experiment. The photos 3.1-3.2 show that the brain was wry well washed out from blood and has no macrocracks on its surface. However the lack of the cracks on the surface of the brain does not signify the absence of the macrocracks inside the brain. So the photo 3.3 demonstrates a few macrocracks on the brain sections. The cracks are shown by the arrows. The top part of the brain was broken down into the two pieces (hemispheres) along the central fissure (on the lower part of the photo 3.3). On the upper left part of the photo, the middle part of the brain has a large crack ( or a rupture) as prolongation of the central fissure. The section was almost broken down into two pieces. On the upper right part of the photo, the lower part of the brain also contains the same rupture. Thus, the given mode of freeze-warming does not cryopreserved the glycerolized cat brain from macrocracks.

    Photo 3.3

7. Freezing a native skull (77 g) of a cat (3.7 kg) to -196 C. The cooling rates were 33.1 C/min (0 - 88), 14.6 C/min (88 - 144), 9.6 C/min (144 - 176), 2.1 C/min (176 - 196) for tcl and 32.0 C/min (12 123), 13.8 C/min (123 - 176), 2.7 C/min (176 - 196) for tc2. The warming rates were 133 C/min (196 - 115), 50.9 C/min (115 - 58), 21.5 C/min (58 24), 4.9 C/min (24 - 0) for tcl and 33.1 C/min (196 - 56), 12.4 C/min (56 - 24), 4.7 C/min (24 - 12) for tc2. The rates were in general slightly slower than the ones of the experiment 2.3 because the weight of this skull (77 g) was less than that skull (53 g).

The photos 5.1 - 5.3 show the brain after thawing and fixation. The photo 5.1 displays that hemoglobin of the unwashed brain have lost its colour 30-50 per cent. There are no macrocracks on the upper surface of the brain. On the lower surface, the small crack is present in the right part of the brain. The photo 3.3 demonstrates the obvious macrocracks, but the brain does not break down into pieces. Also, the cracks are located across the central fissure but not along it.

8. Freeze - warming a native skull (47.4 g) of a cat (2.7 kg) with slower rates at the temperature zone from -90 C to -196 C.

The freeze-warming of the skull was performed into the high vacuum flask (see experiments 2.2). In the beginning of freezing, the skull was rapidly immersed into LN2 1br about a minute. Then the skull was lifted from LN2 and again plunged into LN2 as long as tc2 showed -80 C. After that, the skull was kept at - 90 C over LN2 for few minutes and then frozen with a slow rate to -196 C. The cooling rates were 84 C/min (15 - 88), 8.0 C/min (88 - 65, warming), 17 C/min (65 - 100), 8.0 C/min (100 - 90, warming), 0.2 C/min (90 - 107), 0.3 C/min (107 - 166), 0.87 C/min (166 - 196) for tc1 and 18.2 C/min (5 - 50), 5.4 C/min (50 - 56), 17.1 C/min (56 -78), 5.0 C/min (78 - 88), 0.19 C/min (88 - 107), 0.28 C/min (107 - 166), 0.87 C/min (166 - 196) for tc2. The warming rates were 0.96 C/min (196 - 175), 0.32 C/min (175 - 80) for tcl and tc2. After warming the skull to -80 C over LN2, it was warming using a water bath at +40 C with the rates 48 C/min (80 - 50), 13.1 C/min (50 - 18) for tcl and 35 C/min (80 - 65), 11.0 C/min (65 - 18) for tc2.

The photos 6.1 - 6.3 show this fixed brain with visible blood vessels but dissociating hemoglobin. The upper surface of the brain has no macrocracks (Ph. 6. 1). The photo 6.2 shows the back part of the brain with a foremen from tc2 but without macrocracks. However few cracks were found inside the brain (Ph. 6.3). This brain looked slightly better than the brain of the experiment 5, although even the such slow rates at the zone (196 - 90) cannot preserve the native brain against the macrocracks.

   Photo 6.1

 

9. Freeze-warming a native head (212 g) of a cat (2.5 kg) with slower rates at the zone from o C to -90 C.

The cooling rates were 6.2 C/min (18 - 144), 2.27 C/min (144 - 181), 0.39 C/min (181 - 196) 1br tcl and 6.9 C/min (18 -148), 1.83 C/min (148 - 181), 0.35 C/min (181 - 196) ibr tc2. The warming rates were 16.0 C/min (196 - 106), 7.4 C/min (106 - 50), 2.8 C/min (50 - 12) for tcl and 14.1 C/min (196 - 100), 7.4 C/min (100 - 50), 2.78 C/m in (50 - 12) for tc2.

The photos 7.1-7.3 show this fixed brain with the clearly visible blood vessels. Hemoglobin was well remained during two weeks from the beginning of brain fixation to a moment of the photographing. The morphological structure of this brain is very like the one of the brain in the experiment 8. Thus, the freeze-warming modes no visible influence on the native brain in comparison with that experiment.

10. Freeze-warming a cat head (400 g) perfused with DMF.

The thermocouple 2 was used for this experiment only. The cooling rates were 4.6 C/min (0 - 18), 18.2 C/min (18 - 128), 6.58 C/min (128 - 177), and 1.12 C/min (177 -196). The warming rates were 15.2 C/min (196 - 73),  6.7 C/min (73 - 43), and 2.6 C/min (43 - 10). The rates of this experiment were faster than the ones of the like experiment 6 with glycerol.

The photos 8.1-8.3 show the fixed brain. There were no macrocracks on the upper surface of the brain (Ph. 8. 1). The photo 8.2 displays mainly the back part of the brain with the macrocracks, but the lower surface has no visible cracks. The middle part of the brain had more numbers of the macrocracks than the one of the glycerolized brain of the experiment 6. Thus, this brain looked worse than that brain in general.

11. Freeze-warming a cat skull (61,4 g) perfused with DMF.

The cooling rates were 10.8 C/min (24 - 50), 21.3 C/min (50 -178), 3.13 C/min (178 - 196) for tcl and 17 C/min (6 - 176), 3.85 C/min (176 - 196) for tc2. The warming rates were 57 C/min (196 - 133), 22.7 C/min (133 - 80), 13.1 C/min (80 - 42), 4.6 C/min (42 - 12) for tcl and 40 C/min (196 - 162), 23.5 C/min (162 - 78), 13.5 C/min (78 - 50), 4.7 C/min (50 - 18) for tc2.

The photos 9.1-9.3 show the fixed brain. This CPA-perfused brain looked (in some reason, ?) worse than even the native brain in the like experiment 7. So even the upper surface of the brain had a large crack across the central fissure (Ph. 9. 1). The lower surface had few macrocracks too (Ph. 9.2). A slice of the brain broke down into pieces (P h. 9.3).

   Photo 9.3

12. Freeze-warming a cat skull (61,8 g) perfused with DMF and warming it with slower rates.

The cooling rates were 44 C/min (5 - 80), 19.9 C/min (80 - 176), 4.5 C/min (176 - 196) for tcl and 35 C/m in (24 - 176), 2.3 C/min (176 - 196) for tc2. The rates were faster than the rates in the experiment 11. The warming rates were 0.25 C/min (196 - 155), 0.5 C/min (155 - 133), 1.0 C/min (133 - 72) 1br both tcl and tc2. Then the skull was warming on air at +24 C with the rates 5.34 C/min (72 - 42), 2,56 C/min (42- 10) for tcl and 4.15 C/min (72 - 73), 2.03 C/min (37 - 10) for tc2.

This brain looked rather better than the brain of preceding experiment (Phs. 10. 1-10.3). The upper surface of the brain had no macrocracks (Ph. 10.1). The lower surface had a crack only (Ph. 10.2). The middle part of the brain contained macrocracks (Ph. 10.3) like the brain of the experiment 8 (Ph. 6.3). The slower warming made somewhat better cryopresenation of the brain.

Thus, some over-usual, remarkable properties of DMF have not been found in the present for the hard conditions of brain cryopresentation (low CPA concentrations and fast rates). The next series of experiments will be devoted to comparison of cryoprotective activity of DMF with one of glycerol for easier conditions of brain cryopreservation (slower rates and higher CPA concentrations).

 

Part two of Dr. Pichugin's report here


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