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THE INFLUENCE OF GLYCEROL PERFUSION AND REPERFUSION

ON THE STRUCTURE OF SHEEP HEAD BRAIN TISSUE

( The report of the first and second stages of the work )

By Dr. Yuri Pichugin & Prof. Gennadi Zhegunov

(From The Immortalist August 1994)

"Click here for a text- only version of this page that will print out fast, so you can review it at your leisure."

MATERIALS AND METHODS

Washing out and perfusing the sheep heads were carried out according to your Instructions. Either sex sheep heads weighing 2,0 to 2,5 kg (brains 102 to 110 gm) were used for the work.

Three sheep heads were used by us for training tests before we assimilated the instructions. It was very important to cut sheep heads not above the second vertebra, if the atlas was implied the first.

The weight of the control sheep head was 2,1 kg, and that of the brain was 103 gm. One sheep head was used for analyzing glycerol distribution in head tissues. The weight of this head was 2,3 kg, and that of the glycerolized brain was 108 gm. The head for perfusion was weighing 2,0 kg and the weight of the brain from it was 101 gm.

Glycerol and D-mannitol were the analytical grade (Serva). According to the precise scientific standards, glycerol concentrations are given in weight percentages (w/w). 75 volume per cent of glycerol conform 82% W/W.

Perfusion was performed by portions with the mean rate of 50 ml per 80 sec. A period of time for filling a syringe was 60 sec. Reperfusion was carried out using 1 liter of Ringer's solution with the rate of 50 ml per 10 sec.

The method of measuring the final concentration of glycerol in the various head tissues. Tissue pieces (weight 2-3 gm, size 3x2x2 mm) were given a soak in 10 ml distilled water by being shaken periodically for twenty-four hours at 4 deg C. After centrifugation at 12,000 rpm for 30 min, refractive index and density of the supernatant were measured at 20 deg

Concentration of glycerol in the supernatant was determined using the calibration diagram of the dependence of refraction index and density of glycerol aqueous solutions on concentrations of glycerol. Glycerol concentration in the tissue was calculated as (concentration in the supernatant) to multiply by (the weight of the glycerolized tissue with the added volume of distilled water) to divide by (the weight of the glycerolized tissue). The error of measuring is 3-5% for this method.

The particles of the grey and white substance for biotests were isolated from the frontal lobes. Preparation of tissues for the light microscopic research was carried out by well-known methods (f.e. Bloom W., Fawcett D. A textbook of histology, Philadelphia, 1968%). Tissue pieces of grey and white substance were fixed in 10% neutral formalin. Pituitary pieces were fixed in Buen's solution. Staining of the nerve cells in the histologic slices was conducted by a method of Nissel, and the hypophysial cells were stained using methylyne blue. The magnification is 800 times.

Pieces of intact or reperfused tissues for the electron microscopic research were fixed in 2% buffered solution of glutaraldehyde on phosphate buffer (pH 7.2) at 4 deg C for 4 hr.

After washing with the phosphate buffer, the samples were postfixed with 1 % osmium tetraoxide on phosphate buffer at 4 deg C for 4 hours. The sample were dehydrated in alcohols of increasing strength (50 to 100 deg), embedded into epoxy resins (epon and araldite) and polymerized at 50 deg C for 4 hr. Ultrathin sections were made using an ultramicrotome UMTP-4, contrasted and examined under an electron microscope EVM- 1OOL. The magnification is a 7,000 - 10,000 times.

RESULT AND DISCUSSION

Distribution of glycerol in the head and brain tissues of a sheep:

Perfusion was carried out at 9-12 degrees Centigrade in order to obtain the data on the high temperature limit of the procedure. The head was exposed for 5 hours to 4-6 degrees centigrade for equilibration. The content of glycerol in grey substance was 57%, while in white substance it comprised 53%, and in the pituitary body it was 55%, in the mastication muscle -26%, and in the tongue it was only 14%.

Naturally, Your method of perfusion will certainly bring about differences in the equilibrium concentration of glycerol in brain tissues. These concentrations are determined by some factors, which may be not be completely and closely reproduced from one experiment to another. They are: the degree of passability of vessels, rate of perfusion, temperature of perfusate and head, rate of glycerol penetration into tissues, head dimensions, etc. The passability of the vessels, in turn, depends on the perfusion technique, the extent of head traumatization, and postmortem period. However, we believe we'll manage to improve the reproducibility of your method

While perfusing the head for conducting biotesting we have achieved the lower temperature limit of your procedure, i.e. 4-2 degrees Centigrade. The last fraction of perfusion contained 65% of glycerol, while the skull fluid of the meninx its content was 45% after 5-hour exposure at + 4 degrees Centigrade. Moreover, if the first perfusion at 15-10 degrees Centigrade results in a strong distortion of head and brain tissues, but by the end of perfusion the volume of tissues was nearly restored, while during glycerol perfusion at 4-2 degrees Centigrade the volume of the brain was restored by approximately 70% from the initial one during visual control through the skull window.

Even providing glycerol nicely penetrates into tissues, at first there is observed dehydration of cells, and then, when glycerol gradually penetrates into them, cells and tissues restore their volumes. If glycerol does not penetrate into the cells, or this process occurs very slowly, then only distortion of tissue is observed.

Reperfusion resulted in strong edema of tissues. The last fractions of reperfusion contained 3% of glycerol, i.e. it was practically eliminated. However, a moderate edema of tissues (nearly 10%) was maintained.

RESULTS OF THE BIOTESTS

Research under a light microscope:

The grey substance of the brain:

We have studied the external granular and the external and internal pyramidal layers of the ram brain grey substance. The outer (external) layer consists of pyramidal and stellate cells (Figs. 1, 4). The control pyramidal cells have regular shapes with sufficiently equable sides, and a large center-located nucleus. These cells have straight processes of axones and dendrites (Fig., 1).

Fig. 1 Click to view larger image.

Fig. 1a Click to view larger image.

Following perfusion and reperfusion of brain this layer of grey substance demonstrates the alterations in the shapes of neurons (Fig. 4). The cells do not become rounded but acquire angular and binding shapes instead. The bodies of the pyramidal neurons look somewhat shrunk, the processes of neurons and dendrite become of binding and irregular shapes. This may relate to a change in the topography of cells during perfusion and strong dehydration of tissue and subsequent reperfusion and tissue edema (Fig. 4). This Figure nicely displays a capillary with a partially exfoliated coat.

Fig. 4 Click to view larger image.

The cells of the external pyramidal layer of the grey substance are made up the medium-size neurons with round body boundaries, large nucleus and straight regular processes (Fig. 2). Following reperfusion we have observed a slight shrinking of neurons and the neural processes become irregular, though to a lesser extent as compared with external granular layer (Fig. 5).

The cells of the internal pyramidal layer consist of large pyramidal cells, and even giant cells are not infrequently come across (Fig. 3). Application of perfusion produces nearly a similar pattern, thought we are inclined to think, that the cells of this layer undergo slightly lesser changes (Fig. 6).

Conclusion: Perfusion results in a minor shrink-ing of the neuronal bodies and binding of the neural processes.

The white substance of the brain:

The structure of the white substance of the brain was investigated immediately below the grey substance at the depth of 8-10 mm into the brain (Figs. 7-10). Staining by the method of Nissel reveals mainly the nuclei of ganglia and a thin cytoplasmic layer around them. The capillaries, the dimensions of which are rather large, are nicely displayed.

Examination of the control samples has shown that the nuclei are located at random but they do observe a sufficiently regular distribution along the whole volume of the white substance. The vessels are of the normal structure, the capillary endothelium is not separated from the basic tissue, and blood cells are found inside the capillaries (Fig. , cross-section of the capillary; Fig. 8, longitudinal slice of the intact capillary).

Following reperfusion the topography of the nuclei localization is altered. They are joined into groups, and the distance between them is getting closer. The nuclei have slightly lesser dimensions, and they are shrunk (Fig. 9, 10). Here we can see the nucleus-free regions of the white substance. This may be indicative of the process of osmotic action of perfusion and reperfusion. Some vessels of the white substance are impaired, and those with the exfoliated endothelium are found sometimes (Fig. 10, large capillary with the one-sided exfoliation of the endothelium).

Conclusion: Following perfusion there occurs a change in the topography of oligodendryte localization. Some capillaries with the exfoliat-ed endothelium are found.

Pituitary body:

The anterior lobe of the pituitary body was the object of investigations. Investigation of the histologic slices have shown that the anterior lobe consists of the branchy cellular bands. The space between them is occupied by the capillaries, which are termed as sinusoids, and the width of which is great enough (Figs. 11 - 12). The endothelial undercover of the capillaries is surrounded by the basal membranes. A pericapillary space is observed between the edges of the epithelial bands and the basal membrane of the capillaries, which is filled by the tissue fluid. The intercellular space is not expanded. Following perfusion and reperfusion of the ram brain the capillary lumens are widened, while the intercellular and pericapillary spaces are expanded, thus testifying to a certain edema of the stromal hypophysial component. The secretory cells seem to be slightly swollen (Figs. 13, 14).

Conclusion: Reperfusion of the brain may result in the edema of the hypophysial intercellular space.

Electron microscopy:

The grey substance:

The pyramidal neurons of the grey substance have been investigated.

Fig. 15. A section of the body of the pyramidal neuron from the intact grey substance of the brain. There are observed the nuclei of the regular shapes, surrounded by a regular double coat; nucleolus in the centre of the nucleus; diffused and condensed chromatin. An increased vacuolization of the endoplasmic reticulum cysterns is evident, which relates, apparently, to the postmortem alterations in brain cells.

Fig. 16. A section of the body of the native pyramidal neuron. The nucleus and body of the neuron are of a typical structure. Mitochondria are small with the enlightened matrix and a minor amount of crysts. Mitochondria are also slightly swollen as well as the endoplasmic reticulum cysts, which is the first distinguishing feature of the beginning of the postmortem alterations. The cytoplasm displays a large amount of free ribosomes. A site of the neuropile is found below the neuron body, which demonstrates cross-sections of dendrites and myelinized fibers of the axon.The neuropile is of the native and nonedematic form. The axon contains the unchanged mitochondria.

Fig. 16 Click to view larger image.

Fig. 17. NeuroPile of the intact grey substance. Two large axons, cut in two, are clearly observed. They contain mitochondria, microtubules and microfilaments. Neuropile consists of the cross-cut neural processes of the neurons. Some of them a myelinic coat. Neuropile maintains its native shape and dimensions without any sign of the edema.

Following perfusion and reperfusion of the ram brain we have reported on the following alterations in the ultrastructure of the neurons.

Fig. 18. The perinuclear space is somewhat expanded. Chromatin starts to be exfoliated from the internal nucleated membrane. No changes are found in the nucleolus apparatus. Ribosomes and membranes in the endoplasmic reticulum remain unchanged, though the amount of the vesicular reticulum slightly increases, and they can be hardly distinguished from postmortem alterations.

Fig. 19. A section of the body of the pyramidal neuron and neuropile of the grey substance following reperfusion. At the top one can see a minor section of the nucleus with the expanded perinuclear space and exfoliated chromatin. The. cytoplasm of the neuron is slightly vacuolized. The intact Golgi apparatus and adjacent lysosome are nicely visuolized. The bottom left-hand corner of the photograph houses strongly edematic structures of the neuropile.

Fig. 20. A section of the body of the pyramidal neuron with the adjacent capillary. The cytoplasm of the neuron is characterized by the same features as those described for Figs.18 and 19. At the top of the left-hand corner the injured endothelial capillary is observed. The capillary is widely expanded.

Conclusion: On the whole, the fine structure of the neurons of the ram brain cortex is well maintained after perf usion and reperfusion. The impairments are found in the intercellular space of the neurons, where a considerable edema of the processes of the neurons, dendrites and microcapillaries is identified.

White substance:

Fig. 2 1. The photo displays cross-cut processes of the nervous cells and the sections of the glial cell hyaloplasm. The myelin coats of the fibers are not altered, they still house mitochondria, microfilaments and microtubules.

Fig. 22. The nucleus of the glial cell is centered at the photo, and its membrane is not injured. A large amount of the dark condensed chromatin closely applies to inner membrane of the nucleus. A normal-structured thin layer of the cytoplasm surrounds the nucleus. The myelinized and demyelinized nervous fibers are located at some distance from the nucleus.

Fig. 23. A small capillary, housing the red blood cell inside its lumen, is located at the centre of the photo. The endothelial undercover of the capillary bears no significant changes, though certain vacuolization and swelling of the mitochondria are observed. Cross-cut nervous fibers are of the normal form.

Following perfusion and reperfusion of the brain we have observed stratification of the myelin coats of the axons and swelling of the inner space.

Fig. 24. A significant stratification of the myelin coats of the neuronal nervous processes is identified. The inner space of the nervous fibers is enlightened.

Fig. 25. A stratification of the myelin coats of the nervous processes and alteration of their inner structures are found.

Fig. 26. The central position is occupied by a glial cell. Its nucleus is slightly shrunk, chromatin is closely grouped together and separated of the internal membrane of the nucleus, and the perinuclear space is enlarged. A stratification of the myelin coats and their edema are detected. Vacuolization and swelling of the mitochondria are also found.

Conclusion: the white substance of the brain is damaged to a greater extent after reperfusion as compared with the grey one.

Pituitary body:

We have studied the anterior lobe of the pituitary body. The native cells contain large nuclei with a binding coat. Chromatin is closely grouped inside the nucleus, and located mainly along the periphery of the nucleus. Stroma comprises a major part of the pituitary body, consisting of a large amount of large capillaries and cells of the connective tissue, which fill in a major part of the volume of the intercellular substance of the setretory cells.

Fig. 27. The sites of various secretory cells are presented. A sufficiently thick layer of the intercellular substance is located among them. The cells are filled with secretory granules.

Fig. 28. Several types of secretory cells are presented. All of them stay in the active functional state and filled by the granules of the secretion. The fine structure is of the native type. The connective tissue bands are found between the groups of the secretory cells.

Fig. 29. A longitudinal section of the capillary is displayed. The endothelial undercover is not disturbed. A large pericapillary space is formed by fibroblasts. The granules of the secretion are detected within this space, further entering blood. The granules are also find in the capillary lumen.

Following perfusion and reperfusion the capillary network and intercellular space are getting damaged in the first place. The edema becomes evident.

Fig. 30. The edema of the secretory cells is observed, the inner structures are vacuolized, the nucleus is getting swollen and chromatin becomes loose. The granules of the secretion lose their clear-cut shapes.

Fig. 31. The capillary lumen is greatly widened. The endothelium is exfoliating. The edema of the pericapillary space is identified.

Fig. 32. A considerable expansion of the intercellular space occurs due to the swelling of the fibroblasts, which also significantly alter their fine structure. The organelles of the secretory cells are destructed.

Conclusion: The pituitary body is the most injured by the process of perfusion-reperfusion. The impairments are found both in the secretory cells themselves and in the stromal component of the gland.

Strong osmotic stresses are developed while implementing your method of perfusion and reperfusion. It is due to tissue stroma that the cells are not destroyed during this procedure.

We have found that during such a technique of perfusion-reperfusion red blood cells of both sheep and man are completely destroyed.

----------------------------------------------------

More recently we have received a brief initial report on results after freezing (without cryoprotectants, to isolate more variables), cooling to liquid nitrogen temperature, and rewarming. Slightly edited for English usage, this report says:

We have finished warming an intact sheep head on 27 June 1994. We did not find visible cracks of any tissues of the sheep head. Now samples of the brain tissues are being treated for the biotests. It is possible your super-slow procedure of freezing-warming may prevent tissues from visible cracks even without cryoprotectants.

Needless to say, we are gratified that our observation is confirmed so far. More important will be the results of the microscopic examination, and especially the last phase of the work, including perfusion, which may finally allow a better comparison of results of these procedures vs. others.

Dr. Yuri Pichugin is a researcher at the Institute for Problems of Cryobiology and Cryomedicine, Kharkov University; Prof. Gennadi Zhegunov is Chairman of the Department of Biology at Kharkov Medical University.

Following are a few of the 32 photos. The full set will be made available to interested parties.

THE EFFECT OF FREEZE-THAWING [WITHOUT PERFUSION]

ON THE STRUCTURE OF HEAD BRAIN TISSUES OF A SHEEP

(THE REPORT OF THE THIRD STAGE OF THE WORK)

By Yuri Pichugin, Ph.D., & Gennadi Zhegunov, Ph.D.

[Ed. note: This stage, like the previous ones, was intended to help isolate some of the variables, this time by freezing and thawing according to the Cryonics Institute procedures used, but WITHOUT cryoprotective perfusion. The last stage will fully duplicate (and extend) the Cl work, including perfusion. This report is slightly edited, and only some of the micrographs are shown (following the report). The full set is available to interested and competent parties.]

We have also received a very brief initial report of the fourth stage. Again, no cracking was observed on the naked eye level, confirming the Cl report, and in contrast to results of other procedures, on human patients, which showed cracking at all levels including naked eye. It was also reported that washout of the glycerol was not successful.

The final 4th stage report, including micrographs, and including assessment of glycerol content in the brain tissues, should allow us to decide whether the Cl procedure produced offsetting damage at finer levels, or whether it has on-balance advantages.]

Freezing and thawing sheep heads were carried out according to your instructions.

One sheep head weighing 2,2 kg was used for elaboration and testing of the freeze-thaw mode without biological analysis. We have to spend a lot of time and efforts for elaborating the mode of freeze-thawing in our existing conditions. We have to discard one freezing chamber due to a large consumption of liquid nitrogen and complicated control procedure. Then we refused fiber-glass (glass wool) because 'of difficulties of a work with this material. It is hard to reconstruct a uniform, precise thickness of a layer of insulation using glass wool. A foam plastic box was used as an insulator. The thicknesses of all walls, the bottom and the cover were 5 centimeters. A gradient (difference) of temperatures between the freezing chamber and inside of the foam plastic box was 50-70 deg C to make a cooling rate of ai'slieep head is equal to 0,9 + and - 0,2 (from 0,7 to 1, 1) deg C per hour. A gradient of temperatures between a surface and inside (in the upper portion of the palate) of the sheep head was 4 + and - 1 (3-5) deg C.

On reaching temperature inside the head - 140 deg C, the box was plunged into liquid nitrogen. Then it was kept for 3 days to reach - 196 deg C inside the head.

Thawing the head was reverse of the cooling and freezing process. On reaching temperature inside the head - 50 deg C, the box was kept at room temperature. When temperature of the head was - 30 deg C it was taken out the box and was put in a refrigerator with + 4 deg C.

Visible cracks were not on tissues of the head used for elaborating the mode of freeze-thawing. However, we did not want to inform you about that because we have to be convinced of that using the exact duplication of your rate of freeze-thawing and using a control intact (the second) sheep head. The control head weighed 2,0 kg, brain 101 g.

To avoid postmortem alterations, head brain tissues were taken for biotesting at + 4 deg C.

THE BIOTESTS: Visible examination of tissues of the control sheep head did not bring to light any cracks too. But tissues became more soft, thin, amorphous, especially the brain. This is a result of damage of the tissue stroma by ice. Hair did not pull out from the skin easily. The cornea did not crack too. Soft tissues did not separate from more hard tissues. All of these facts demonstrate that thermo-mechanical tensions (TMT) of tissues were not at such superslow rate of freeze-thawing. TMT are basic cause of macrocracks.

RESEARCH UNDER A LIGHT MICROSCOPE: Numerous microcracks were seen under a light microscope both in the grey substance of the brain and in the white one. Distribution of cracks in the brain tissues had mosaic disposition. But a number of regions with microcracks were more than ones without cracks. Dimensions of regions both with microcracks and without ones were 0,1 - 1,0 square millimeters.

THE GREY SUBSTANCE OF THE BRAIN: The grey substance was strongly damaged by ice crystals. Here (e.g.Figs. 33,34 [34 at right]) we can see both a lot of microcracks and ruptures of tissues, and vessels (Fig. 35. A rupture of a vessel). Neurons look rather dense, good absorbed the stain that d e m o n s t r a t e s existence of damages in cells. Neuronal bodies lost their clear contours that shows existence of injures in cellular membranes. Nuclei of cells are very bad observed. Although nuclei were very good seen together with their nucleoloi in intact cells of native tissues. Hence nucleus membranes were damaged too. Neuropile looks rather swelled.

Fig. 34 Click image to view it larger.

THE WHITE SUBSTANCE OF THE BRAIN: It was strongly damaged by ice too. Figs. 36, 37 display micro-cracks and microruptures of tissues. Nuclei of oligodendrytes are rather dense too. Although there are small regions without cracks are observed under a light microscope as seeming slightly injured (Fig. 38 [next page]).

PITUITARY BODY: Figs. 39, 40, 41 display regions of the intact pituitary tissue. We have done this work over again in order to stain the tissue of hypophysis by a method of Homory.

That is more adequate staining of secretory tissues than using methylyne blue. These Figures nicely display groups of secretory cells around vessels. Nuclei and bodies of cells are good observed. Acini and vessels have clear contours too. Fig. 41 displays besides acidi of the anterior lobe of hypophysis, cells of the intermediate lobe occupying the half of the photo.

After freeze-thawing, microcracks; and ruptures of hypophysis tissues are observed too (Figs. 42, 43, 44). Acidi lost the clear structure and contours. Nuclei and contours of cells are bad observed. Vessels look expanded with injured walls (Fig. 44). (A remark. Perhaps, it is not easy for nonspecialists in this field to distinguish unstaining places stroma in the intact hypophysis from cracks of the freeze-thawing tissue).

RESEARCH UNDER AN ELECTRON MICROSCOPE: Very extensive and heavy damages of ultrastructure of freeze-thawing, tissues of the brain were found. Here we show the typical pictures of the damages of the ultrastructure.

THE GREY SUBSTANCE: Fig. 45 [right] displays a region of the body and nucleus of a pyramidal neuron. The nucleus have an irregular, angular shapes and loose contours. There are ruptures and exfoliations of the bilayer of the nuclear membrane. Chromatin exfoliates from the membrane of the nucleus. The regions of very condensed chromatin are observed. The cell has very loose contours because its membrane is very hardly destroyed. The cytoplasm is strongly vacuolized and has nearly a homogeneous state where cellular organelles are hardly distinguished. The mitochondria can still be distinguished by the presence of the vacuolized cristae.

Fig. 45 Click to view larger image.

Fig. 46. Neuropile of the freeze-thawing grey substance. It is swelled, has signs of dystrophic changes and damages of subcellular structures. Membranes of dendrites are damaged too. A part of inner contents of the dendrites went out. Cristae of mitochondria. are very vacuolized.

Fig. 47. A region of neuropile and a longitudinal slice of a large region of an axon. The membrane of the axon is very injured. There are cross-cutting dendrites inside the axon.

Fig. 48. A glial cell of the grey substance with strong damage.

Fig. 49. A capillary with a destroyed membrane. The bottom part of the Fig. houses the completely destroyed endothelium and the basal membrane of the capillary. The content of the endothelial cell occupies the lumen of the capillary. There are regions of myelinized and demyelinized nervous fibres there. The dark spot is observed at the top of the left part of the lumen of the capillary. It is a track from a large ice crystal.

THE WHITE SUBSTANCE OF THE BRAIN: A white substance of a head brain of a sheep contains large myelinized branches of neurons (axons) on the whole. First of all myelinized membranes are damaged. They are strongly exfoliated, disordered. Rough ruptures of membranes are seen in many places. Inner structures of axons are damage too.

Fig. 50 Rough injures of a myelinized membrane by ice are observed in the center of the photo. Tracks of ice crystals remained in the appearance of the dark rounded spots.

Fig. 51 A longitudinal slice of an axon.

Fig. 52 Cross-cutting axons. There is a enlightened region without some kind of structures. Apparently the structures of the cell have been displaced by an ice crystal.

Fig. 53 There is a nucleus of an oligodendrite. Its nuclear membrane has signs of injures. Contents of the cell are strongly vacuolized and destroyed.

Fig. 54 An injured capillary. It was tom away from the main substance of the brain. The pericapillary space is strongly expended.

PITUITARY BODY: On the whole, the same picture of damage are observed in the hypophysis (Fig. 55-59). Secretory granules were damage too. Many granules became the looses and the enlightened. They have lost the regular shapes. Some of them were completely destroyed.

About the 1st part of the 4th stage. We have finished thawing of the glycerolized sheep head on 26th July. We did not observe any visible cracks of any tissues of the head. Unfortunately, we had no success of washing out tissues from grycerol. We'll inform you about this in detail later on. We have taken the glycerolized pieces of the brain tissues for biotests; and for the examination of glycerol concentrations.

THE EFFECT OF FREEZE - THAWING

ON THE STRUCTURE OF GLYCEROLIZED

BRAIN TISSUES OF THE SHEEP

(THE REPORT OF THE FIRST PART OF THE FOURTH STAGE OF WORK)

By Yu. Pichugin, Ph.D.; Prof. G. Zhegunov

A few words have been changed to correct apparent typos. Some sentences or phrases have been paraphrased, in brackets, to make the meaning a bit clearer or the English a bit smoother.

Materials and methods have been described earlier (see The Immortalist, 1994, N8, N9).

The mode of freeze-thawing was nicely reproduced. The sheep head for this stage weighed 2,2 kg. It was reported that we failed to succeed in washing tissues of the sheep from glycerol. At first we carefully supercharged Ringer's solution with dye (methylene blue) both into the left carotid artery and into the right one. However solution only soaked the layers of tissues, where the head had been cut off. The tissues were swelling [swollen]. Perfusate did not enter inside the head neither through blood vessel of the brain nor through facial ones. As the pressure was increasing [increased] the solution rapidly passed only through the tissues around the cut again.

Perfusate flowed out not only from the jugular veins but from the tissues themselves. Attempts to stop the leaks were not successful. Apparently, the layers of the tissues, where the head had been cut off, had not been soaked by glycerol during perfusing [perfusion] due to the damage of the vascular system during cutting off the sheep head. Probably, as a result of this, the tissues were damaged during freeze-thawing. We did not conduct an [any] additional saturation of the tissues.

Reperfusion was carried out at + 4 deg C to avoid postmortem alterations. Concentrated solutions of glycerol are very viscous at such temperature. They resisted a flow of perfusate and did not let it pass inside the head. Maybe this complicated the situation. After 30 minute period of unsuccessful attempts of reperfusion, we took the glycerolized pieces of the head and brain tissues for the examination of glycerol concentrations and the brain tissues pieces for biotests.

The content of glycerol in grey substance was 45%, in the white substance it was 42%, in the mastication muscle it was 31 %, and in the tongue - 37%. Thus, these results differ from those, which have been obtained for the perfused head in the study of distribution of glycerol in the head and brain tissues of the sheep (see The Immortalist 1994, N8, p. 6). The volume of the brain was reduced by approximately 15-20% from the initial one as it was seen through the skull window.

THE BIOTESTS: VISIBLE EXAMINATION OF THE THAWED GLYCEROLIZED SHEEP HEAD DID NOT SHED ANY LIGHT ONTO ANY CRACKS AS WELL AS IN THE CASE OF THE THAWED NON-GLYCEROLIZED ONE. [THERE WERE NO CRACKS AT THE NAKED-EYE LEVEL.] However [Furthermore] the tissues did not become softer, thinner, and amorphous in comparison with intact sheep head tissues. They even became rather more dense.

RESEARCH UNDER A LIGHT MICROSCOPE. ANY MICROCRACKS AND MICRORUPTURES OF THE BRAIN TISSUES AND PITUITARY BODY WERE ABSENT IN THE ALL BIOLOGICAL SAMPLES STUDIED BY US. [THERE WERE NO CRACKS AT THE LIGHT MICROSCOPE LEVEL.] MOREOVER, THE STRUCTURE OF THE THAWED GLYCEROLIZED TISSUES WAS VERY GOOD.

However, a certain dehydration of cells and tissues, as well as their shrinking, are to be reported.

Fig, 60. A group of pyramidal cells of the grey substance. The cells are somewhat shrunk due to dehydration. The cells are surrounded by the enlightened space, which is filled, apparently, by the fluids from the dehydrated cells. Blood vessels bear no symptoms of mechanical injury.

Fig. 61. Large pyramidal cells of the grey substance. Similarly to Fig. 60, the cells are intact, though dehydrated, due to which they have slightly altered their shapes: they look somewhat "baggy". The enlightened areas are found in the vicinity of the neuron bodies, which were occupied, apparently, by the cells with complete volumes prior to glycerolization.

Fig. 62. A group of large pyramidal neurons. The area of the grey substance, in which nerve cells and their processes underwent the smallest changes. In this case the cells are poorly dehydrated, cell nuclei are clearly visualized. The processes bear almost no impairments.

Fig. 62 Click to view larger image.

Fig. 62a Click to view larger image.

It should be noted that Figs. 60-62 nicely display the network of the nervous processes of the /pyramidal neurons. Both axons and dendrites have no ruptures or other mechanical injuries.

Figs. 63-65 demonstrate the white substance of the sheep brain. We failed to identify any injuries in all samples, which may relate to formation of ice crystals or their mechanical action. Following freezing down to -196 degrees C under the above conditions the normal structure of the tissue is maintained. The Figures display slightly dehydrated nuclei of oligodendrites. Blood vessels are well preserved, though an increased density of the endothelial cells, covering the surface of the vessels, is to be reported.

Fig. 63a Click to view larger image.

The pituitary body is located closer to the carotid arteries as compared with brain tissues. However, we have found that it has just started being washed from glycerol. A certain amol4nt of perfusate still remains in the vessels of the pituitary body. Thus, Figs. 66-68 testify to the fact the capillary network and intercellular space remain swollen. Still, the secretory cells stay in a rather dehydrated state. The anterior lobe of the pituitary body is also shown in these Figures. One can see that cracks or ruptures, produced by ice crystals, are missing.

ELECTRON MICROSCOPY, GREY SUBSTANCE

Fig. 69 shows a section of the nerve cell. It is clear that the structure of the cytoplasmic organelles does not experience drastic alterations, though an increased vacuolization of the cytoplasm and some changes in the mitochondrial crysts are found. The nucleus bears no injuries, chromatin is regularly distributed in karyoplasm.

Fig. 70. A section of the intracellular contents of the grey substance. The processes of the nerve cells are somewhat vacuolized and enlightened. The axon myelin coat is slightly shrunk. No significant impairments are identified in the cytoplasm of the oligodendrite. A Golgi apparatus is clearly manifested, which maintains a nearly native structure.

Fig. 71. Here one can see a section (part, portion) of oligodendrite, getting in contact with a capillary. A nice integrity of the endothelial cell fine structure should be reported, which cover the walls of the capillary. A certain exfoliation of the axon myelin coat is reported.

Fig. 71 Click to view larger image.

WHITE SUBSTANCE

Figs. 72-74 display a relatively nice integrity of the fine structure of the nerve processes and cell structures of oligodendrites following the above exposure. Though vacuolization of the fine structures should certainly be reported, which are difficult to be distinguished from postmortem alterations, and mitochondrial crysts are damaged. The matrix of the axon and dendrite is enlightened. Particular attention is to be paid to the unfavorable action of the above exposure (treatment) on the fine structure of the myelin fiber. The most significant alterations are detected in the myelin.

Fig. 72 Click to view larger image.

PITUITARY BODY

Fig. 75 shows the neuropil and sections of three secretory cells of the hypophyseal anterior lob

Fig. 75 Click to view larger image.

A fine survival of the cell plasma membrane is reported. Large vacuoles may be found inside some cells. The neuropil is slightly expanded, though the fine structures of the cells are nicely preserved. The secretory granules are detected in the intercellular space, which is also characteristic of the normal tissue. The nuclei of the secretory cells are slightly shrunk, and chromatin is condensed.

Fig. 76. shows the field of contact of three secretory cells. The observed alterations are similar to those displayed in Fig. 75. At the bottom of the Figure the wall and lumen of the capillary are shown, which maintain their structures intact.

Fig. 77. the field of contact of two secretory cells. The secretory granules are nor damaged. Intracellular vacuolization and matrix enlightening are reported. However, the membranes aren't damaged, and the neuropil maintains its structure.

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