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On The Determination Of Cracking Limits in Cryopreserved Cat Brains - Part 2

by Dr. Yuri Pichugin

HISTOLOGICAL EVALUATION OF CAT BRAIN EXPERIMENTS 3-6 AND 12

As reported previously, macrocracks were absent in the cat brains of the experiments 3-5 (freezing to -90 C) and so histological preparations were made in order to examine the presence of microcracks. Histological preparations of the experiments 6 and 12 (freezing to -196 C) were made to determine shape of microcracks and extent of cell damage of the cat brain tissues that were frozen with glycerol or DMF.

The microcracks were present in all the histological preparations of all the experiments 3-5 in spite of the absence of the macrocracks. However the shape of these microcracks differed from the shape of the microcracks of the untreated sheep brain tissue frozen to -196 C (The Immortalist 1996, v. 27, No. 7/8, p. 31) and the CPA-perfused sheep brain tissues frozen to -196 C (The Im. 1997, v. 28, No. 3/4, p. 48-50). Apparently, the microcracks which were formed in the cat brain tissue at freezing to -90 C did not result from thermo-mechanical tensions because this type of damage is characteristic for temperature below -90 C. They may be a result of a kind of ice crystallization. It is therefore better to use words "cavities" or "ice holes" for the name of "the microcracks from freezing to -90 C".

The third experiment. Freezing the glycerolized cat head to -90 C. The histological preparations of the brain tissue were stained by the Nissl method (Nl) and with hematoxylin and easin (h.-e.). All micrographs had a magnification 350x (h.-e.) and 700x (Nl).

The typical appearance of the cavities (or the ice holes) is shown in the micrographs 1,2 (Figs. 1,2 the white matter) and in Figs. 2,3 (the grey matter). The cavities in the white matter had a more round shape than the ones in the grey matter. The ice holes occupied approximately 20% of the grey matter and 40% of the white matter. Fig. 5 shows the typical appearance of the grey matter without the cavities. Figs. 6,7 demonstrate the stellate neurons. The neurons had irregular shapes and stained nuclei. The nucleoli were faintly seen. The pyramidal neurons have a like appearance (Figs. 8,9). Disintegrating nerve cells were very rarely met (Fig. 9).

The fourth experiment. Freezing the native cat head to -90 C.

The cavities in the white matter of the brain tissue had some greater sizes than the cavities in the white matter of the brain tissue of the experiment 3 (Fig. 10). The grey matter also contained the cavities (Figs. 11, 12). The ice holes occupied approximately 30% of the grey matter and 50% of the white matter. The neurons looked worse than the ones in the experiment 3. They more strongly uptake the stain. The nucleoli were not seen (Figs. 13, 14 the stellate neurons, Figs. 15, 16 the pyramidal neurons.)

The fifth experiment. Freezing the native cat skull to -90 C.

Distribution and sizes of the cavities in the brain tissue of this experiment were very like ones of the experiment 4. Figs. 17, 18 shows the typical cavities in the white matter. Figs. 19, 20 demonstrates the typical cavities in the grey matter. The neurons looked slightly worse than the ones in the experiment 4. A great number of the destroying neurons were found (Figs. 21, 22 the stellate neurons, Fig. 23 the pyramidal neurons).

The sixth experiment. Freezing the glycerolized cat head to -196 C.

The main feature of the histological picture of the cat brain tissue of the experiments 6 and 7 was a combination of the cavities with the microcracks from thermo-mechanical tensions. The white matter contained the cavities only (Figs. 24, 25) which looked as well as the cavities in the white matter of the experiment 3. The ice holes occupied approximately 40% of the white matter too. Fig. 26 shows the region of the white matter without the cavities. The grey matter had the typical microcracks side by side with the cavities (Figs. 27, 28). Usually, the microcracks were finished at the border of the grey matter with the white one (Fig. 29). The ice holes occupied approximately 20% of the grey matter. About 10% of the grey matter was occupied by the microcracks. Therefore this cat brain had only few microcracks inside. Fig. 30 demonstrates the region of the grey matter without the cracks and cavities. Some accumulations of fluid were observed near the neurons. The neurons looked very like the ones of the brain tissue of the experiment 3 (Figs. 31, 32 the stellate neurons, Figs. 33, 34 the pyramidal neurons).

The twelfth experiment. Freeze-warming the cat brain perfused with DMF.

This experiment was chosen for histological evaluation as the best of the experiments 10-12 with the use of DMF as cryoprotectant agent (CPA). Morphology of the brain tissue was very like morphology of the brain tissue of the experiment 6. There were the like appearance and a number of the cavities in the white matter (Fig. 35). The grey matter also had the typical microcracks (Figs. 36, 37). However they were some more often met and had some greater sizes (Figs. 38, 39). Therefore this cat brain contained a greater number of the macrocracks than the brain of the experiment 6. But in contrast to that experiment, the neurons practically have no accumulation of fluid near their bodies (Fig. 40 and 36-39). The structure of the neurons was also like the one of the experiment 6.

Thus, the cat brain tissues frozen to -90 C contained only the cavities which did not convert into any macrocracks or macroruptures. The cat brain tissues frozen to -196 C contained not only the cavities but also the microcracks which converted into few macrocracks. Glycerol and DMF even in low concentrations favoured some better cryopreservation of the neurons however they looked pretty bad.

A comparison of the results of the cat experiments 3-5 with the freezing of the native sheep head to -90 C (The Immortalist, 1996, v. 27, No. 7/8, p. 31-32) shows that, likely, sheep's brain tissues are more cryoresistant than cat's brain tissues because they did not contain the cavities after freezing. However the sheep brain was frozen with the slower rate 20 C/hour (0.33 C/min) than the cat brains. Only in the cat experiment 4, the cooling rate was nearer that rate (1.3 C/min from -6 to -64 C and 0.33 from -64 to -90 C).

Comparing the cat experiments 6 and 12 with freezing the sheep brains using the faster rate (about 10 C/min, The Immortalist 1997, v. 28, No. 3/4, p. 48-50), one can note that the sheep brain tissues did not contained any cavities or ice holes but had the stronger damages by a great number of the micro- and macrocracks. Maybe, there were different rates of growth of the microcracks for sheep and cat brain tissues. The sheep brain tissues could have a faster rate of growth of the microcracks because they cut the neurons. The cat brain tissues could have a slower rate of growth of the microcracks and cavities because the cut neurons were very rarely met. The neurons of the cat brains could be moved aside by the slowly growing microcracks or cavities. Apparently, a deformation of the frozen cat brain tissues was more plastic than for the frozen sheep ones.

FREEZE-WARMING CAT BRAINS AT THE RATE 1 C/MIN AT THE ZONE FROM -90 C TO -196 C.

Using Rubinsky's equation (Cryobiology, v. 17, pp. 66-73 (1980)), the rate of freeze-warming at which cracking of the cat brains should not occur have been calculated like the calculation for a human brain (The Immortalist 1997, v. 28, No. 3/4, p. 50). {In the article in The Immortalist, all the radiuses (R) have to be in square. I had sent so, but, maybe, during a reformation of my e-mail message for the article "square" as 2 over R has been lost.}

An average weight of the cat brain was 21 g. The radius of the brain sphere was 1.71 cm. The rate, faster which strong cracking of the cat brain may be, is 27 C/min. The rate, slower which no cracking of the cat brain may be, is 2.7 C/min. We chose the rate 1 C/min at which cracking of the cat brain should not occur according to Rubinsky's equation. However, cracking occurred.

A numeration of experiment and photos was continued from the part 1 of the report.

13. Freezing a glycerolized cat skull (46.6 g).

For this experiment and the experiment 14, freeze-warming was carried out into the high cylindrical vacuum flask (see the experiment 2.2.1).

The cooling rates were 37.5 C/min from 0 C to -30 C (0 - 30), 27.3 C/min (30 - 80); 1.5-1.0 C/min (90 -196) for all the subsequent experiments for the thermocouple 1 (tc1) and 12.8 C/min (5 - 80); 1.0 C/min (90 - 196) for all the subsequent experiments for tc2. The warming rates were 1.5-1.0 C/min (196 - 80) for all the subsequent experiments, 11.3 (80 - 0) C/min for tc1 and 1.0 C/min (196 - 80) for all the subsequent experiments, 5.3 C/min (80 - 5) for tc2. The cat brain weighed 18 g.

The photos 11.1 - 11.3 show the brain of the cat skull after warming and fixation. There are no macrocracks on the upper surface of the brain (Ph. 11.1). The lower surface had a large crack on the left lower part of the brain and a small crack on the left upper part of the brain (Ph. 11.2). However the cracks were superficial and did not carry inside the brain as it is seen in the photo 11.3 (the left upper piece is the lower part). There were no macrocracks inside this brain (Ph. 11.3).

14. A duplication of experiment 13.

The glycerol perfusion of the cat head of this experiment was performed unsuccessfully. However this was cleared up after thawing the skull and the determination of glycerol concentrations in the brain tissue. The content of glycerol was two time lower than in the experiment 13. The cat skull weighed 53.5 g and its brain weighed 20.2 g. The cooling rates were 34.3 C/min (0 - 30), 25.2 C/min (30 - 80) for tc1 and 10.3 C/min (5 - 80) for tc2. The warming rates were 27.8 C/min (80 - 0) for tc1 and 10.0 C/min (80 - 5) for tc2.

The photos 12.1 - 12.3 show the fixed brain. The brain was well washed out from blood. The upper surface of the brain has no macrocracks and look well (Ph. 12.1). The lower surface had a small crack on the middle left part of the brain (Ph. 12.2). 6-8 macrocracks were found inside the brain (Ph. 12.3). Most likely, the greater number of the cracks resulted from the lower glycerol concentration. But also another cause may be. So, in time of moving dawn or up a container with a cat skull into the high cylindrical vacuum flask, joggles of the skull against the walls of the flask can initiate the macrocracks during freeze-warming at the zone from -90 C to -196 C. To verify the presumption, a new chamber for controlled freeze and warming was used. Cat heads or skulls were not moved in the chamber. The following three cat experiments 15-17 were performed using the freezing chamber.

{It had been made in March 1997 but had not been used in that time because of greater consumption of LN2 ( 20-30 liters of LN2 per a experiment) as compared with the old chamber (the vacuum flask had a consumption of 5-7 liters of LN2 per the like experiment). We had difficulties with LN2 in that time, though to work using the new chamber was easier than using the old one. The staple of the new chamber is a chemical glass vessel (volume 600 ml) with a copper coil for warming. I have sent you the photo where I have been photographed with the chamber.}

15. Freezing a cat head (372 g) perfused with DMF.

The cooling rates were 36.2 C/min (0 - 30), 25.2 C/min (30 - 80) for tc1 and 7.1 C/min (5 - 80) for tc2. The warming rates were 10.8 C/min (80 - 18), 7.5 C/min (18 - 0) for tc1 and 3.4 C/min (80 - 5) for tc2. The brain of the head weighed 23,0 g.

The photos 13.1 - 13.3 show the fixed brain. The upper and lower surfaces of the brain had no macrocracks and looked well (Phs. 13.1-13.2). However there were 10-12 cracks inside the brain (Ph. 13.3 mainly, in the upper part of brain; it is the upper right piece). The cracks were of smaller sizes than the cracks inside the brain of the experiment 14.

16. Freezing a glycerolized cat head (390 g). A duplication of experiment 13.

The glycerol perfusion of the cat head of this experiment was performed in norm. The cat brain weighed 18 g. The cooling rates were 1.5-1.0 C/min (0 -196) for tc1 and 1.0 C/min (5 - 196) for tc2. The warming rates were 1.5-1.0 C/min (196 - 0) for tc1 and 1.0 C/min (196 - 5) for tc2. The cat brain weighed 20.4 g.

The photos 14.1 - 14.3 demonstrate the fixed brain. The upper and lower surfaces of the brain did not contained macrocracks (Phs. 14.1-14.2). But 8-10 macrocracks were found inside the brain (Ph. 14.3). Four of them had greater sizes than the cracks inside the brain of the experiment 15 (with DMF). In comparison with the experiment 13 (with glycerol), the brain had greater numbers of cracks. But the brain was better cryopreserved as comparison with the experiment 14 (with lower glycerol concentration).

17. Freezing a cat head (485 g) perfused with DMF. A duplication of experiment 15.

The rates of freeze-warming were as the same as in the experiment 16 (about 1 C/min). The brain of the head weighed 23,0 g. The upper and lower surfaces of the brain had no macrocracks (Phs. 15.1 - 15.2). However 10-12 cracks were found inside the brain (Ph. 15.3). In the main, the cracks were met in the upper part of brain (it is the upper right piece, Ph. 15.3) as well as in the experiment 13. But some of the cracks were greater sizes than the cracks inside the brain of that experiment 13.

In general, the results of the experiments 13 (with glycerol) and 15 (with DMF) were well reproduced in the experiments 16 and 17. Thus, the joggles did not influence on degree of cracking of deeply frozen cat brains. Even the results of the experiments 13 (with the joggles) were slightly better than the results of the experiments 16 (without the joggles). Histological preparations of all the experiments 13-17 were not made because all the cat brains had the macrocracks.

In conclusion:

1. No advantage of DMF over glycerol was found in these cat experiments as well as in the sheep experiments.

2. Rubinsky's equation needs improvement.


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