IV. EFFECTS OF CRYOPRESERVATION ON THE HISTOLOGY OF SELECTED TISSUES (Left Ventricle and Cerebral Cortex)
Left Ventricle
Figure 43: The myofibrils of each cardiac muscle cell are branched and contain a single nucleus. The branches interlock with those of adjacent fibers by adherens junctions which act to prevent scission of the cardiomycytes during the high-shear, forceful contractions of the heart. The muscle is richly supplied with mitochondria which are largely confined to the spaces between the fibrils. The fibrils are covered with a membrane, the Sarcolemma, which is frequently invaginated to form the Transverse tubules. These invaginations of the plasma membrane or sarcolemma, are called transverse tubules and they reach deep into the myofibrils and bring the action potential deep into the fibers. Specialized intercellular junctions, the Intercalated discs, facilitate rapid transmission of the electrical signals which initiate myocyte contraction. The myofibrils are formed by myosin and actin fibers aligned in a distinct pattern which is visible under light microscopy as the A-, H- and I- bands.
Yajima stain was used to prepare the Control (Figure 44), FGP and FIG cardiac tissue for light microscopy. The FGP cardiac muscle showed increased interstitial space, probably indicative of interstitial edema. In many areas the sarcolemma appeared to be separated from the cytoplasm of the myocyte and, occasionally, appeared to have disintegrated into debris in the interstitial spaces (Figure 45). The myofilaments appeared maximally relaxed with widened I-bands . The mitochondria were grossly swollen and contained numerous amorphous matrix densities. The sarcolemma was fragmented beneath an intact basement membrane and there was increased space between the capillary endothelium/basement membrane and intact areas of the sarcolemma of the cardiomyocytes. The cell nuclei were unremarkable.
Figure 44: Control-1, Left Ventricle, Yajima, 100x. Control cardiac muscle demonstrated crisp, well defined membranes and the normal density and pattern of myofibril structure. Capillary endothelium appeared intact and the capillary basement membrane was well anchored to adjacent myocytes and appeared intact.
Figure 45: FGP-1 Left Ventricle, Yajima, 100x. In the FGP animals the myocardium exhibited increased interstitial space (IIS) as well as the presence of debris in the IIS which appeared to be disrupted sarcolemma (yellow arrows). The capillary basement membrane was often observed to be separated from the sarcolemma of the adjacent myocytes and endothelial cell nuclei were sometimes observed devoid of plasma membranes or cytoplasm (red arrow).The occasional naked myocyte nucleus could also be observed (green arrow).
The same changes were also present in the FIG group with the added presence of a “ragged” or rough appearance of the myofibrils where they were silhouetted against interstitial space (Figure 46). There also appeared to be holes or spaces, possibly as a result of edema, in the fabric of the myofibrils that were not present in the myocardium of either the control, or the FGP animals.
Most surprising was the general absence of contraction band necrosis in the FIG group, possibly as a consequence of the protective effect of reasonably prompt post-cardiac arrest refrigeration. No microscopic evidence of fracturing, either gross or microscopic, was noted in the myocardium of either the FGP, or the FIG groups.
Figure 46: FIG-2 Left Ventricle, Yajima, 100x. Separation and fragmentation of the sarcolemma were observed in the FIG myocardium to a greater extent than that seen the in myocardium from the FGP animals (yellow arrow). Additionally, the fibers of myofibrils had a more ragged appearance and consistently displayed open spaces in the bands which were not seen in the myocardium of either the Control or the FGP animals (red arrows).
Figure 47: The myofibrils of both the FGP and FIG animals appeared maximally relaxed with a marked increase in the thickness of the I-band. Intact red blood cells (RBCs) were observed in the FIG animals and represent incomplete blood washout (red cell trapping) despite perfusion with large volumes of washout, cryoprotectant and fixative solution (~8-10 L) over a time course of ~140 minutes of perfusion.
Figure 48: The cerebral cortex consists of six distinct layers, beginning with the first layer, the Molecular Layer (Stratum zonale), which consists of finely branched medullated and non-medullated nerve fibers. The molecular layer is largely devoid of neuronal cell bodies. Those neuronal cell bodies which are present are the cells of Cajal which possess irregular cell bodies and typically have four or five dendrite that terminate within the molecular layer and a long nerve fiber process, or neuraxon, which runs parallel to the surface of the cortical convolutions.
The second layer of the cortex consists of a layer of small Pyramidal cells with the apices of the pyramids being directed towards the surface of the cortex. The apex of the small Pyramidal cells terminates in a dendron, which reaches into the molecular layer, giving off several collateral horizontal branches. The final branches in the molecular layer take a direction parallel to the surface. Smaller dendrites arise from the lateral and basal surfaces of these cells, but do not extend far from the body of the cell. The neuronal axon (neuraxon) always arises from the base of the small Pyramidal cells and passes towards the central white matter, thus forming one of the nerve-fibers of the white matter. In its path, the neuraxon gives off a number of collaterals at right angles, which are distributed to the adjacent grey matter.
The third cortical layer consists of Pyramidal neurons which are characterized by the presence of cells of the same type as those of the preceding layer, but of a larger size. The nerve-fiber process becomes a medullated fiber of the white matter.
The fourth layer is comprised of Polymorphous neurons which are irregular in outline and give off several dendrites which branch into the surrounding grey matter. The neuraxons of the Polymorphous neurons give off a number of collaterals, and then become a nerve-fiber of the central white matter. Scattered through these three layers are the cells of Golgi, whose neuraxon divides immediately with the divisions terminating in the immediate vicinity of the Polymorphous neuron cell-bodies. Some cells are also found in which the neuraxon, instead of extending into the white matter of the brain, passes towards the surface of the cortex; these are called cells of Martinotti.
The fifth cortical layer contains the largest pyramidal neurons which send outputs to the brain stem and spinal cord and comprise the the pyramidal tract. Layer 5 is particularly well-developed in the motor cortex.
Layer 6 consists of pyramidal neurons and neurons with spindle-shaped cell bodies. Most cortical outputs leading to the thalamus originate in layer 6, whereas most outputs to other subcortical nuclei originate in layer 5.
The cortical blood supply is via the pia mater which overlies the cerebral hemispheres.
Bodian stain was used to prepare the control, FGP, and FIG brain tissue samples for light microscopy. Three striking changes were apparent in FGP cerebral cortex histology: 1) marked dehydration of both cells and cell nuclei, 2) the presence of tears or cuts at intervals of 10 to 30 microns throughout the tissue on a variable basis (some areas were spared while others were heavily lesioned), and 3) the increased presence (over Control) of irregular, empty spaces in the neuropil as well as the occasional presence of large peri-capillary spaces (Figures 54,56, and 57). These changes were fairly uniform throughout both the molecular layer and the second layer of the cerebral cortex. Changes in the white matter paralleled those in the cortex, with the notable exception that dehydration appeared to be more pronounced (Figure 55).
Other than the above changes, both gray and white matter histology appeared remarkably intact, and only careful inspection could distinguish it from control (Figures 52, 58, 59 and 60). The neuropil appeared normal (aside from the aforementioned holes and tears) and many long axons and collaterals could be observed traversing the field. Cell membranes appeared crisp, and apart from appearing dehydrated, neuronal architecture appeared comparable to control. Similarly, staining was comparable to that observed in Control cerebral cortex. Cell-to-cell connections appeared largely intact.
The histological appearance of FIG brain differed from that of FGP animals in that ischemic changes such as the presence of pyknotic and fractured nuclei were much in evidence and cavities and tears in the neuropil appeared somewhat more frequently. The white matter of the FIG animals presented a macerated appearance, in addition to exhibiting the rips or tears observed in the white matter of the FGP brains (Figure 61).
Both FGP and FIG brains presented occasional evidence of microscopic fractures.
Figure 49: Control-1, 1st (molecular) cell layer, cerebral cortex, Bodian, 40x. Cells of Cajal (N) and a dense weave of axons (A) are visible. The tissue is perforated by numerous capillaries (C) and a small venue containing many red blood cells (RBCs).
Figure 50: Control-1, 2nd cell layer, cerebral cortex, Bodian, 40x, showing a pyramidal neuron (N, lower left) multiple capillaries (C) and the interwoven connections of dendrites that comprise the neuropil.
Figure 51: Control-1, white matter, cerebral cortex, Bodian, 40x. Myelinated axons (MA) appear both in cross section (yellow arrows) and laterally (green arrows). Unmyelinated axons are present inside the black circle. Glial cell nuclei (GN) are scattered throughout the tissue.
Figure 52: FGP-1, Cerebral Cortex, 1st cell layer, Bodian, 40x. Two large capillaries (LC) are present, one with a red blood cell present (right). Neurons (N, cells of Cajal) are present in normal density and the neuropil appears intact. This section appears indistinguishable from that of the Control animal.
Figure 53: FGP-1, Cerebral Cortex, 2nd cell layer, Bodian, 40x. This area of FGP cerebral cortex shows injury typical of that seen in both FGP and FIG animals. There are a number of large tears in the neuropil (red arrows) approximately 10 to 30 microns across. A pyramidal neuron is present in the lower left of the micrograph and it appears somewhat dehydrated. There are a number of naked glial cell nuclei (yellow arrows), as well some nuclei with what appears to adherent cytoplasm visible at the margins of the tears in the neuropil.
Figure 54: FGP-1, Cerebral Cortex, 2nd cell layer, Bodian, 40x. In this area of the 2nd layer of the cerebral cortex the neuropil presents a somewhat “moth eaten” appearance, with numerous tears and vacuoles in evidence (red arrows). One large tear appears to be a pericapillary ice hole (yellow arrow).
Figure 55: FGP-3, Cerebral Cortex, white matter, Bodian, 100x. There are numerous open spaces in the white matter that appear to be ice holes (red arrows). The density of the tissue appears markedly increased over that of the Control white matter, possibly as a result of glycerol-induced dehydration. This apparent dehydration is also evident in the increased density of the axoplasm seen in the myelinated axons (green arrows).
Figure 56: FIG-3, Cerebral Cortex, 1st cell layer, Bodian, 40x. Extraordinarily normal appearing Molecular layer of the FIG cerebral cortex. The neuropil appears intact with the exception of what appear to be scattered tears or ice holes (red arrows).
Figure 57: FIG-2, Cerebral Cortex, 1st cell layer, Bodian, 40x. Large tears are evident (red arrows) and naked glial cell nuclei and fragmented cytoplasm are apparent (nn). Several intact capillaries are in evidence (C) as well as what appears to be two capillaries that have been separated from the neuropil and appear largely surrounded by open (pericapillary) space (green arrows). A mass of debris appears to occupy some of the luminal space of what appears to have been a capillary (Cd).
Figure 58: FIG-2, Cerebral Cortex, 2nd cell layer, Bodian, 40x. Remarkably intact neuropil with several capillaries, including several capillaries sectioned oblique to the plane of the tissue (OC). A neuron (N) with what appears to be a crisp plasma membrane is present at the upper right of the micrograph.
Figure 59: FIG-2, Cerebral Cortex, 2nd cell layer, Bodian, 40x.Normal appearing cerebral cortex in an FIG animal. There are multiple intact neurons with normal appearing dendrites (D) and axons (A). An intact large capillary (LC) is present and appears free of red cells.
Figure 60: FIG-2, Cerebral Cortex, 2nd cell layer, neuropil, Bodian, 100x. Normal appearing layer 2 of the cerebral cortex with intact neurons (N), axons (A), and neuropil. A capillary (C)with intact endothelial cells and an endothelial cell nucleus (EN) is also visible (left, center).
Figure 61: FIG-2, Cerebral Cortex, white matter, Bodian, 40x. Severely injured white matter typical of that seen in FIG animals. The tissue presents a macerated appearance (black circles) with numerous rips and tears, possibly as a result of ice formation (red arrows). The capillaries (C) are separated from the tissue parenchyma (yellow arrow) and what appears to be a naked endothelial cell nuclei projected into the intraluminal space of one capillary (green arrow).
Histology was evaluated in two animals each from the FIG and FIGP groups, and in one control animal. Only brain histology was evaluated in the straight-frozen control animal.
The histological appearance of the liver in all three groups of animals was one of profound injury. Even in the FGP group, the cellular integrity of the liver appeared grossly disrupted. In liver tissue prepared using Yajima stain, the sinusoids and spaces of Disse were filled with flocculent debris, and it was often difficult or impossible to discern cell membranes (Figures 30-32). The collagenous supporting structures of the bile canaliculi were in evidence and the nuclei of the hepatocytes appeared to have survived with few alterations evident at the light level, although frequent pyknotic nuclei were noted in the FIGP group (Figures 31 & 34). Indeed, the nuclei often appeared to be floating in a sea of amorphous material (Figure 34). Not surprisingly, the density of staining of the cytoplasmic material was noticeably reduced over that of the fixative-perfused control. Few intact capillaries were noted.
FGP liver tissue prepared with PAS stain exhibited a similar degree of disruption (Figure 32). However, quite remarkably, the borders of the hepatocytes were defined by a clear margin between glycogen granule containing cytoplasm and non-glycogen containing membrane or other material (membrane debris?) which failed to stain with Yajima due to gross physical disruption, or altered tissue chemistry (Figure 35).
The cords of hepatocytes comprise the hepatic parenchyma. In section, the hepatic cords appear as linear ropes (or cords) of hepatocytes. Viewed 3-dimensionaly, the cords consist of intricately folded branching and connected planes of cells which extend parallel to the long axis of the lobule and radiate out from the its center. The hepatocytes in each cord are attached to each other wherever they come into contact, as well as to the sinusoids at either end of the lobular pyramid. The sinusoids are vascular spaces lined by fenestrated endothelium that has no basement membrane, thus allowing the plasma to pass over the large surface area sheets of hepatocytes for detoxification. The sinusoid endothelium stands off from the underlying hepatocytes allowing space for the plasma to interact with the hepatocytes and Kupffer cells (the space of Disse).
Bile canaliculi, formed by apical surfaces of adjacent hepatocytes, form a network of tiny passages contained within each hepatic cord.
Figure 28: Control-1 Liver, Yajima, 100x. Liver sections from the Control animal demonstrated normal morphology as can be seen in the image above.
Figure 29: Control-1 Liver, PAS, 100x. Liver sections were prepared with both Yajima and PAS stain in order to allow visualization of structures that neither stain discloses alone; in this case, most importantly, the presence or glycogen granules in the hepatocytes of the Control animal. Note the presence of normal intralobular architecture with crisp cell membranes in evidence, normal appearing sinusoid spaces, and residual sinusoidal red blood cells (RBCs) not washed out during fixative perfusion.
Figure 30: FGP-1 Liver, Yajima, 100x. The livers of FGP animals demonstrated extensive histological disruption. The sinusoids were all but obliterated and appeared filled with debris (ds) and the cytoplasm was extensively vacuolated (v).
Figure 31: FIG-2 Liver, Yajima, 100x. As was the case with the FGP animals, the sinusoids were barely discernable and appeared filled with cellular debris (cd). In addition to extensive cytoplasmic (cv) and nuclear vacuolization (nv), pyknotic nuclei (pn) were also present. Cell membranes were difficult to discern and in many areas, frank cell lysis appears to have occurred with flocculent cellular debris (cd) appearing to fill the sinusoids.
Figure 33: FIG-2, Liver, Yajima, 100x, well preserved area. While the bulk of the hepatic parenchyma exhibited the severe injury seen in Figures 30-32, there were frequently observed islands of comparatively well preserved tissue visible in both the FGP and FIGP sections suggesting that freezing injury is occurring non-homogenously.
Figure 34: FIG-2, Liver, PAS, 100x, necrotic area. There were patchy areas of frank necrosis visible in the livers of the FIGP animals that were not present in the livers of the FGP animals. This area, adjacent to a central vein, shows extensive cell lysis with heavy vacuolization of the cytoplasm (v) and many pyknotic nuclei (pn) in evidence.
Figure 35: FIG-2, Liver, PAS, 100x. Note the presence of a few scattered glycogen granules (GG). Interestingly, in this comparatively well preserved area of FIGP liver it is possible to see some remaining deposits of glycogen that were not consumed during the long post-arrest ischemic interval. The absence of pyknotic nuclei and the relative absence of large intracellular vacuoles is also remarkable.
Figure 36: The functional unit of the kidney is the nephron, consisting of the glomerulus and the uriniferous tubule ( the renal corpuscle: a).The capillary tuft of the nephron, the glomerulus, is enclosed within a double cell layered structure; Bowman’s capsule. Bowman’s capsule and the capillary tuft it encloses comprise the glomerulus. Bowman’s capsule and the glomerular capillary tuft constitute the renal (or Malpighian) corpuscle (b).
Bowman’s capsule opens into the proximal convoluted tubule which leads to the loop of Henle. The loop of Henle leads to the distal convoluted tubule which then leads to the collecting duct.
The inner layer of Bowman’s capsule is the visceral layer. It consists of cells called podocytes. The outer layer of Bowman’s capsule is the parietal layer. The pedicels are the foot processes on the podocytes.
The juxtaglomerular cells secrete renin which is ultimately metabolized into angiotensin II, a potent vasoconstrictor critical to maintaining normotension. The macula densa are specialized cells in the distal convoluted tubule that are responsible for sodium, and thus fluid regulation. The juxtaglomerular cells and macula densa make up the juxtaglomerular apparatus.
PAS stain was used to prepare the control, FGP and FIGP renal tissue for light microscopy. The histological appearance of FGP renal tissue was surprisingly good (Figures 329, 40 & 41). The glomeruli and tubules appeared grossly intact and stain uptake was normal. However, a number of alterations from the appearance of the control were apparent. The capillary tuft of the glomeruli appeared swollen and the normal space between the capillary tuft and Bowman’s capsule was absent. There was also marked interstitial edema, and marked cellular edema as evidenced by the obliteration of the tubule lumen by cellular edema.
By contrast, the renal cortex of the FIGP animals, when compared to either the control or the FGP group, showed a profound loss of detail, absent intercellular space, and altered staining (Figures 40 & 42). The tissue appeared frankly necrotic, with numerous pyknotic nuclei and numerous large vacuoles which peppered the cells. One striking difference between FGP and FIGP renal cortex was that the capillaries, which were largely obliterated in the FGP animals, were consistently spared in the FIGP animals. Indeed, the only extracellular space in evidence in this preparation was the narrowed lumen of the capillaries, grossly reduced in size apparently as a consequence of cellular edema.
Both ischemic and non-ischemic sections showed occasional evidence of fracturing, with fractures crossing and severing tubule cells and glomeruli (Figure 41).
Figure 37: Control, Renal Cortex, PAS 40x. Three glumeruli are present (G) adjacent to crisp, well defined proximal (P) and distal (D) convoluted tubules. The intertubular capillaries (C) show normal diameter with lumens free of red cells or debris. There is normal capsular space between Bowman’s capsule (BC, yellow arrows) and the glomerular capillary tuft and vascular pole (VP) are also normal in appearance.
Figure 38: Control-1, PAS 40x. Collecting ducts (CD), distal tubules (D) and a glomerulus (G) are present in 6this micrograph of renal apical column. At right, a glomerulus is present with normal Bowman’s space (BS) and the macula densa (MD) in evidence.
Figure 39: FGP-1, Renal Cortex, PAS, 40x. The intertubular space (ITS) is great expanded and the tubule cells are heavily vacoulated (V) and lack definition. The intratubular space (IS) is no longer in evidence and the architecture of the glomerluar capillary tuft (GT) is radically altered and there is an absence of the normal architecture of Bowman’s space (yellow arrows). The intertubular capillaries appear to have been reduced to debris (D) visible in the intertubular spaces.
Figure 40: FIG-2 Renal Cortex, PAS, 40x. There is massive cellular edema present with almost complete obliteration of Bowman’s space. The tubule (T) lumens are no longer visible and the tubule cells are extensively vacoulated with many pyknotic nuceli in evidence. Individual tubular cell membranes are impossible to resolve. The afferent glomerular arteriole (AA) appears intact (red arrow).
Figure 41: FIG-2 Renal Cortex, fracture present (arrows), PAS, 40x. Two renal tubules, possibly a proximal and distal convoluted tubule (T) are dissected by a fracture as is the macula densa (MD) of the glomerulus (G). Remarkably, there is still a small amount of intertubular space present in this micrograph.
Figure 42: FIG-2 Renal Cortex, PAS 40x. vacuolization (black arrows) and extensive vacuolization (blue arrows) accompanied by necrotic changes, such as the frequent presence of pyknotic nuclei (red arrows).
II. Materials and Methods
IV. Gross Effects of Cooling to and Rewarming From -196°C
The immediate goal of human cryopreservation is to use current cryobiological techniques to preserve the brain structures which encode personal identity adequately enough to allow for resuscitation or reconstruction of the individual should molecular nanotechnology be realized (1,2). Aside from two previous isolated efforts (3,4) there has been virtually no systematic effort to examine the fidelity of histological, ultrastructural, or even gross structural preservation of the brain following cryopreservation in either an animal or human model. While there is a substantial amount of indirect and fragmentary evidence in the cryobiological literature documenting varying degrees of structural preservation in a wide range of mammalian tissues (5,6,7), there is little data of direct relevance to cryonics. In particular, the focus of contemporary cryobiology has been on developing cryopreservation techniques for currently transplantable organs, and this has necessarily excluded extensive cryobiological investigation of the brain, the organ of critical importance to human identity and mentation.
The principal objective of this pilot study was to survey the effects of glycerolization, freezing to liquid nitrogen temperature, and rewarming on the physiology, gross structure, histology, and ultrastructure of both the ischemic and non-ischemic adult cats using a preparation protocol similar to the one then in use on human cryopreservation patients. The non-ischemic group was given the designation Feline Glycerol Perfusion (FGP) and the ischemic group was referred to as Feline Ischemic Glycerol Perfusion (FIGP).
The work described in this paper was carried out over a 19-month period from January, 1982 through July, 1983. The perfusate employed in this study was one which was being used in human cryopreservation operations at that time, the composition of which is given in Table I.
The principal cryoprotectant was glycerol.
Nine adult cats weighing between 3.4 and 6.0 kg were used in this study. The animals were divided evenly into a non-ischemic and a 24-hour mixed warm/cold ischemic group. All animals received humane care in compliance with the “Principles of Laboratory Animal Care” formulated by the National Society for Medical Research and the “Guide for the Care and Use of Laboratory Animals” prepared by the National Institutes of Health (NIH Publication No. 80-23, revised 1978). Anesthesia in both groups was secured by the intraperitoneal administration of 40 mg/kg of sodium pentobarbital. The animals were then intubated and placed on a pressure-cycled ventilator. The EKG was monitored throughout the procedure until cardiac arrest occurred. Rectal and esophageal temperatures were continuously monitored during perfusion using YSI type 401 thermistor probes.
Following placement of temperature probes, an IV was established in the medial foreleg vein and a drip of Lactated Ringer’s was begun to maintain the patency of the IV and support circulating volume during surgery. Premedication (prior to perfusion) consisted of the IV administration of 1 mg/kg of metubine iodide to inhibit shivering during external and extracorporeal cooling and 420 IU/kg sodium heparin as an anticoagulant. Two 0.77 mm I.D. Argyle Medicut 15″ Sentinel line catheters with Pharmaseal K-69 stopcocks attached to the luer fittings of the catheters were placed in the right femoral artery and vein. The catheters were connected to Gould Model P23Db pressure transducers and arterial and venous pressures were monitored throughout the course of perfusion.
Following placement of the monitoring catheters, the animals were transferred to a tub of crushed ice and positioned for surgery. The chest was shaved and a median sternotomy was performed. The aortic root was cleared of fat and a purse-string suture was placed, through which a 14-gauge Angiocath was introduced. The Angiocath, which served as the arterial perfusion cannula, was snared in place, connected to the extracorporeal circuit and cleared of air. The pericardium was opened and tented to expose the right atrium. A purse-string suture was placed in the apex of the right atrium and a USCI type 1967 16 fr. venous cannula was introduced and snared in place. Back-ties were used on both the arterial and venous cannulae to secure them and prevent accidental dislodgment during the course of perfusion. Placement of cannulae is shown in Figure 1.
Extracorporeal Circuit
Figure 2: Cryoprotective perfusion apparatus: RR = recirculating reservoir, PMC = arterial pressure monitor and controller, MBD = micro-bubble detector, US = ultrasonic sensor, ADC = arterial drip chamber, D/0 = dialyzer/oxygenator, RP = cryoprotective ramp pump, HEX = arterial heat exchanger, 40 MFH = 40 micron filter holder, PT = arterial pressure transducer, CR = glycerol concentrate reservoir, EKG = electrocardiograph, TT = thermistor thermometer, TS = thermistor switch box, IB = ice bath, EC = electrocautery, APD = arterial pressure display.
The extracorporeal circuit (Figures 2&3) was of composed of 1/4″ and 3/8″ medical grade polyvinyl chloride tubing. The circuit consisted of two sections: a recirculating loop to which the animal was connected and a glycerol addition system. The recirculating system consisted of a 10 liter polyethylene reservoir positioned atop a magnetic stirrer, an arterial (recirculating) roller pump, an Erika HPF-200 hemodialyzer which was used as a hollow fiber oxygenator (8) (or alternatively, a Sci-Med Kolobow membrane oxygenator), a Travenol Miniprime pediatric heat exchanger, and a 40-micron Pall LP 1440 pediatric blood filter. The recirculating reservoir was continuously stirred with a 2″ Teflon-coated magnetic stir bar driven by a Corning PC 353 magnetic stirrer. Temperature was continuously monitored in the arterial line approximately 15.2 cm from the arterial cannula using a Sarns in-line thermistor temperature probe and YSI 42SL remote sensing thermometer. Glycerol concentrate was continuously added to the recirculating system using a Drake-Willock dual raceway hemodialysis pump, while venous perfusate was concurrently withdrawn from the circuit and discarded using a second raceway in the same pump head.
Figure 3: Schematic of cryoprotective perfusion circuit.
Storage and Reuse of the Extracorporeal Circuit
After use the circuit was flushed extensively with filtered tap and distilled water, and then flushed and filled with 3% formaldehyde in distilled water to prevent bacterial overgrowth. Prior to use the circuit was again thoroughly flushed with filtered tap water, and then with filtered distilled water (including both blood and gas sides of the hollow fiber dialyzer; Kolobow oxygenators were not re-used). At the end of the distilled water flush, a test for the presence of residual formaldehyde was performed using Schiff’s Reagent. Prior to loading of the perfusate, the circuit was rinsed with 10 liters of clinical grade normal saline to remove any particulates and prevent osmotic dilution of the base perfusate.
Pall filters and arterial cannula were not re-used. The circuit was replaced after a maximum of three uses.
Preparation of Control Animals
Two control animals were prepared as per the above. However, the animals were subjected to fixation after induction of anesthesia and placement of cannulae. Fixation was achieved by first perfusing the animals with 500 mL of bicarbonate-buffered Lactated Ringer’s containing 50 g/l hydroxyethyl starch (HES) with an average molecular weight of 400,000 to 500,000 supplied by McGaw Pharmaceuticals of Irvine, Ca (pH adjusted to 7.4) to displace blood and facilitate good distribution of fixative, followed immediately by perfusion of 1 liter of modified Karnovsky’s fixative (Composition given in Table I). Buffered Ringers-HES perfusate and Karnovsky’s solution were filtered through 0.2 micron filters and delivered with the same extracorporeal circuit described above.
Immediately following fixative perfusion the animals were dissected and 4-5 mm thick coronal sections of organs were cut, placed in glass screw-cap bottles, and transported, as detailed below, for light or electron microscopy.
Straight Frozen Non-ischemic Control
One animal was subjected to straight freezing (i.e., not treated with cryoprotectant). Following induction of anesthesia and intubation the animal was supported on a ventilator while being externally cooled in a crushed ice-water bath. When the EKG documented profound bradycardia at 26°C, the animal was disconnected from the ventilator, placed in a plastic bag, submerged in an isopropanol cooling bath at -10°C, and chilled to dry ice and liquid nitrogen temperature per the same protocol used for the other two experimental groups as described below.
Preparation of FGP Animals
Following placement of cannulae, FGP animals were subjected to total body washout (TBW) by open-circuit perfusion of 500 mL of glycerol-free perfusate. The extracorporeal circuit was then closed and constant-rate addition of glycerol-containing perfusate was begun.
Cryoprotective perfusion continued until the target concentration of glycerol was reached or the supply of glycerol-concentrate perfusate was exhausted.
Preparation of FIGP Animals
In the FIGP animals, ventilator support was discontinued following anesthesia and administration of Metubine. The endotracheal tube was clamped and the ischemic episode was considered to have begun when cardiac arrest was documented by absent EKG.
After the start of the ischemic episode the animals were allowed to remain on the operating table at room temperature ( 22°C to 25°C) for a 30 minute period to simulate the typical interval between pronouncement of legal death in a clinical environment and the start of external cooling at that time. During the 30 minute normothermic ischemic interval the femoral cut-down was performed and monitoring lines were placed in the right femoral artery and vein as per the FGP animals. Prior to placement, the monitoring catheters were irrigated with normal saline, and following placement the catheters were filled with 1000 unit/mL of sodium heparin to guard against clot obstruction of the catheter during the post-arrest ischemic period.
Figure 4: Typical cooling curve of FIGP animals to ~1°C following cardiac arrest.
After the 30 minute normothermic ischemic period the animals were placed in a 1-mil polyethylene bag, transferred to an insulated container in which a bed of crushed ice had been laid down, and covered over with ice. A typical cooling curve for a FIGP animal is presented in Figure 4. FIGP animals were stored on ice in this fashion for a period of 24 hours, after which time they were removed from the container and prepared for perfusion using the surgical and perfusion protocol described above.
Perfusate
Perfusate Composition
Component mM
Potassium Chloride 2.8
Dibasic Potassium Phosphate 5.9
Sodium Bicarbonate 10.0
Sodium Glycerophosphate 27.0
Magnesium Chloride 4.3
Dextrose 11.0
Mannitol 118.0
Hydroxyethyl Starch 50 g/l
The perfusate was an intracellular formulation which employed sodium glycerophosphate as the impermeant species and hydroxyethyl starch (HES)(av. MW 400,000 - 500,000) as the colloid. The composition of the base perfusate is given in Table I. The pH of the perfusate was adjusted to 7.6 with potassium hydroxide. A pH above 7.7, which would have been “appropriate” to the degree of hypothermia experienced during cryoprotective perfusion (9), was not achievable with this mixture owing to problems with complexing of magnesium and calcium with the phosphate buffer, resulting in an insoluble precipitate.
Perfusate components were reagent or USP grade and were dissolved in USP grade water for injection. Perfusate was pre-filtered through a Whatman GFB glass filter (a necessary step to remove precipitate) and then passed through a Pall 0.2 micron filter prior to loading into the extracorporeal circuit.
Perfusion of both groups of animals was begun by carrying out a total body washout (TBW) with the base perfusate in the absence of any cryoprotective agent. In the FGP group washout was achieved within 2 – 3 minutes of the start of open circuit asanguineous perfusion at a flow rate of 160 to 200 mL/min and an average perfusion pressure of 40 mm Hg. TBW in the FGP group was considered complete when the hematocrit was unreadable and the venous effluent was clear. This typically was achieved after perfusion of 500 mL of perfusate.
Complete blood washout in the FIGP group was virtually impossible to achieve (see “Results” below). A decision was made prior to the start of this study (based on previous clinical experience with ischemic human cryopreservation patients) not to allow the arterial pressure to exceed 60 mm Hg for any significant period of time. Consequently, peak flow rates obtained during both total body washout and subsequent glycerol perfusion in the FIGP group were in the range of 50-60 mL/min at a mean arterial pressure of 50 mm Hg.
Due to the presence of massive intravascular clotting in the FIGP animals it was necessary to delay placement of the atrial (venous) cannula (lest the drainage holes become plugged with clots) until the large clots present in the right heart and the superior and inferior vena cava had been expressed through the atriotomy. The chest was kept relatively clear of fluid/clots by active suction during this interval. Removal of large clots and reasonable clearing of the effluent was usually achieved in the FIGP group after 15 minutes of open circuit asanguineous perfusion, following which the circuit was closed and the introduction of glycerol was begun.
The arterial pO2 of animals in both the FGP and FIGP groups was kept between 600 mm Hg and 760 mm Hg throughout TBW and subsequent glycerol perfusion. Arterial pH in the FGP animals was between 7.1 and 7.7 and was largely a function of the degree of diligence with which addition of buffer was pursued. Arterial pH in the FIGP group was 6.5 to 7.3. Two of the FIGP animals were not subjected to active buffering during perfusion and as a consequence recovery of pH to more normal values from the acidosis of ischemia (starting pH for FIGP animals was typically 6.5 to 6.6) was not as pronounced (Figure 5).
Figure 6: Calculated versus actual increase in arterial and venous glycerol concentration in the FGP animals. Arrow indicates actual time of termination of perfusion.
Introduction of glycerol was by constant rate addition of base perfusate formulation made up with 6M glycerol to a recirculating reservoir containing 3 liters of glycerol-free base perfusate. The target terminal tissue glycerol concentration was 3M and the target time course for introduction was 2 hours. The volume of 6M glycerol concentrate required to reach a terminal concentration in the recirculating system (and thus presumably in the animal) was calculated as follows:
Mc = ——— Mp
Vc + Vp
Mc = Molarity of glycerol in animal and circuit.
Mp = Molarity of glycerol concentrate.
Vc = Volume of circuit and exchangeable volume of animal.*
Vp = Volume of perfusate added.
* Assumes an exchangeable water volume of 60% of the pre-perfusion weight of the animal.
Glycerolization of the FGP animals was carried out at 10°C to 12°C. Initial perfusion of FIGP animals was at 4°C to 5°C with warming (facilitated by TBW with warmer perfusate and removal of surface ice packs) to 10°-12°C for cryoprotectant introduction. The lower TBW temperature of the FIGP animals was a consequence of the animals having been refrigerated on ice for the 24 hours preceding perfusion.
Following termination of the cryoprotective ramp, the animals were removed from bypass, the aortic cannula was left in place to facilitate prompt reperfusion upon rewarming, and the venous cannula was removed and the right atrium closed. The chest wound was loosely closed using surgical staples.
Concurrent with closure of the chest wound, a burr hole craniotomy 3 to 5 mm in diameter was made in the right parietal bone of all animals using a high speed Dremel “hobby” drill. The purpose of the burr hole was to allow for post-perfusion evaluation of cerebralvolume, assess the degree of blood washout in the ischemic animals and facilitate rapid expansion of the burr hole on re-warming to allow for the visual evaluation of post-thaw reperfusion (using dye).
The rectal thermistor probe used to monitor core temperature during perfusion was replaced by a copper/constantan thermocouple at the conclusion of perfusion for monitoring of the core temperature during cooling to -79°C and -196°C.
Figure 7: Representative cooling curve (esophageal and rectal temperatures) of FGP and FIGP animals from ~ 10°C to ~ -79°C. The ragged curve with sharp temperature excursions and rebounds is an artifact of the manual control of temperature descent via the addition of chunks of dry ice.
Cooling to -79°C was carried out by placing the animals within two 1 mil polyethylene bags and submerging them in an isopropanol bath which had been pre-cooled to -10°C. Bath temperature was slowly reduced to -79°C by the periodic addition of dry ice. A typical cooling curve obtained in this fashion is shown in Figure 7. Cooling was at a rate of approximately 4°C per hour.
Cooling to and Storage at -196°C
Figure 8: Animals were cooled to -196°C by immersion in liquid nitrogen (LN2) vapor in a Linde LR-40 cryogenic dewar. When a core temperature of ~-180 to -185°C was reached, the animals were immersed in LN2.
Following cooling to -79°C, the plastic bags used to protect the animals from alcohol were removed, the animals were placed inside nylon bags with draw-string closures and were then positioned atop a 6″ high aluminum platform in an MVE TA-60 cryogenic dewar to which 2″-3″ of liquid nitrogen had been added. Over a period of approximately 15 hours the liquid nitrogen level was gradually raised until the animal was submerged. A typical cooling curve to liquid nitrogen temperature for animals in this study is shown in Figure 8. Cooling rates to liquid nitrogen temperature were approximately 0.178°C per hour. After cool-down animals were maintained in liquid nitrogen for a period of 6-8 months until being removed and re-warmed for gross structural, histological, and ultrastructural evaluation.
Re-warming
Figure 9: Rewarming of all animals was accomplished by removing the animals from LN2 and placing them in a pre-cooled box insulated with 15.2 cm of polyurethane (isocyothianate) foam to which 1.5 L of LN2 (~2 cm on the bottom of the box) of LN2 had been added. When the core temperature of the animals reached -20°C the animals were transferred to a mechanical refrigerator at 3.4°C.
The animals in both groups were re-warmed to -2°C to -3°C by removing them from liquid nitrogen and placing them in a pre-cooled box insulated on all sides with a 10.2 cm thickness of Styrofoam and containing a small quantity of liquid nitrogen. The animals were then allowed to re-warm to approximately -20°C, at which time they were transferred to a mechanical refrigerator at a temperature of 8°C. When the core temperature of the animals had reached -2°C to -3°C the animals were removed to a bed of crushed ice for dissection, examination and tissue collection for light and electron microscopy. A typical re-warming curve is presented in Figure 9.
Modification of Protocol Due To Tissue Fracturing
After the completion of the first phase of this study (perfusion and cooling to liquid nitrogen temperature) the authors had the opportunity to evaluate the gross and histological condition of the remains of three human cryopreservation patients who were removed from cryogenic storage and converted to neuropreservation (thus allowing for post-arrest dissection of the body, excluding the head) (10). The results of this study confirmed previous, preliminary, data indicative of gross fracturing of organs and tissues in animals cooled to and re-warmed from -196°C. These findings led us to abandon our plans to reperfuse the animals in this study with oxygenated, substrate-containing perfusate (to have been followed by fixative perfusion for histological and ultrastructural evaluation) which was to be have been undertaken in an attempt to assess post-thaw viability by evaluation of post-thaw oxygen consumption, glucose uptake, and tissue-specific enzyme release.
Re-warming and examination of the first animal in the study confirmed the presence of gross fractures in all organ systems. The scope and severity of these fractures resulted in disruption of the circulatory system, thus precluding any attempt at reperfusion as was originally planned.
Samples of four organs were collected for subsequent histological and ultrastructural examination: brain, heart, liver and kidney. Dissection to obtain the tissue samples was begun as soon as the animals were transferred to crushed ice. The brain was the first organ removed for sampling. The burr hole created at the start of perfusion was rapidly extended to a full craniotomy using rongeurs (Figure 14). The brain was then removed en bloc to a shallow pan containing iced, modified Karnovsky’s fixative containing 25% w/v glycerol (see Table II for composition) sufficient to cover it. Slicing of the brain into 5 mm thick sections was carried out with the brain submerged in fixative in this manner. At the conclusion of slicing a 1 mm section of tissue was excised from the visual cortex and fixed in a separate container for electron microscopy. During final sample preparation for electron microscopy care was taken to avoid the cut edges of the tissue block in preparing the Epon embedded sections.
Figure 10: The sagitally sectioned (5 mm thickness) brains of the animals were placed in a perforated basket immersed in Karnofsky’s fixative. This assembly was placed atop a magnetic stirring table and the fixative was gently stirred with a magnetic stirring bar.
The sliced brain was then placed in 350 ml of Karnovsky’s containing 25%w/v glycerol in a special stirring apparatus which is illustrated in Figure 10. This fixation/de-glycerolization apparatus consisted of two plastic containers nested inside of each other atop a magnetic stirrer. The inner container was perforated with numerous 3 mm holes and acted to protect the brain slices from the stir bar which continuously circulated the fixative over the slices. The stirring reduced the likelihood of delayed or poor fixation due to overlap of slices or stable zones of tissue water stratification. (The latter was a very real possibility owing to the high viscosity of the 25%w/v glycerol-containing Karnovsky’s.)
De-glycerolization of Samples
Figure 11: Following fixation, the tissues slices of all organs evaluated by microscopy were serially de-glycerolized using the scheme shown above. When all of the glycerol was unloaded from the tissues they were shipped in modified Karnovsky’s to outside laboratories for histological and electron microscopic imaging.
To avoid osmotic shock all tissue samples were initially immersed in Karnovsky’s containing 25%w/v glycerol at room temperature and were subsequently de-glycerolized prior to staining and embedding by stepwise incubation in Karnovsky’s containing decreasing concentrations of glycerol (see Figure 11 for the de-glycerolization protocol).
Figure 12: Fixation and de-glycerolization set up employed to prepare tissues for subsequent microscopic examination. Karnofsky’s fixative (A) was added to the tissue slice fixation apparatus (B) and the tissue slices were then subjected to serial immersion in fixative bathing media containing progressively lower concentrations of glycerol (C) (see Figure 11).
To prepare tissue sections from heart, liver, and kidney for microscopy, the organs were first removed en bloc to a beaker containing an amount of ice-cold fixative containing 25% w/v glycerol sufficient to cover the organ. The organ was then removed to a room temperature work surface at where 0.5 mm sections were made with a Stadie-Riggs microtome. The microtome and blade were pre-wetted with fixative, and cut sections were irrigated from the microtome chamber into a beaker containing 200 ml of room-temperature fixative using a plastic squeeze-type laboratory rinse bottle containing fixative solution. Sections were deglycerolized using the same procedure previously detailed for the other slices.
Osmication and Further Processing
At the conclusion of de-glycerolization of the specimens all tissues were separated into two groups; tissues to be evaluated by light microscopy, and those to be examined with transmission electron microscopy. Tissues for light microscopy were shipped in glycerol-free modified Karnovsky’s solution to American Histolabs, Inc. in Rockville, MD for paraffin embedding, sectioning, mounting, and staining.
Tissues for electron microscopy were transported to the facilities of the University of California at San Diego in glycerol-free Karnovsky’s at 1° to 2°C for osmication, Epon embedding, and EM preparation of micrographs by Dr. Paul Farnsworth.
Due to concerns about the osmication and preparation of the material processed for electron microscopy by Farnsworth, tissues from the same animals were also submitted for electron microscopy to Electronucleonics of Silver Spring, Maryland.
Gross examination of the eyes revealed that initial water loss was primarily from the aqueous humor, with more significant losses from the posterior chamber of the eyes apparently not occurring until later in the course of perfusion. Within 15 minutes of the start of glycerolization the corneal surface became dimpled and irregular and the eyes had developed a “caved-in” appearance.
Dehydration was also apparent in the skin and skeletal muscles and was evidenced by a marked decrease in limb girth, profound muscular rigidity, cutaneous wrinkling (Figure 11), and a “waxy-leathery” appearance and texture to both cut skin and skeletal muscle.
Tissue water evaluations conducted on ileum, kidney, liver, lung, and skeletal muscle confirmed and extended the gross observations.
Figure 13: Cutaneous dehydration following glycerol perfusion is evidenced by washboard wrinkling of the thoraco-abdominal skin (CD). The ruffled appearance of the fur on the right foreleg (RF) is also an artifact of cutaneous dehydration. The sternotomy wound, venous cannula and the Weitlaner retractor (R) and the retractor blade (RB) holding open the chest wound are visible at the upper left of the photo.
Preliminary observation suggest that water loss was in the range of 30% to 40% in most tissues. As can be seen in Table III, total body water losses attributable to dehydration, while typically not as profound, were still in the range of 18% to 34%. The gross appearance of the heart suggested a similar degree of dehydration, as evidenced by modest shrinkage and the development of a “pebbly” surface texture and a somewhat translucent or “waxy” appearance.
Animal Pre-Perfusion Post-Perfusion Kg./ % Lost As
# Weight Kg. Weight Water Dehydration
FGP-1 4.1 3.6 2.46 18
FIGP-1 3.4 3.0 2.04 18
FIGP-2 3.4 3.2 2.04 9
FIGP-3 4.32 3.57 2.59 29
Figure 14: Cerebrocortical dehydration as a result of 4M glycerol perfusion. The cortical surface (CS) is retracted ~5-8 mm below the margin of the cranial bone (CB).
Examination of the cerebral hemispheres through the burr hole (Figure 14) and of the brain in the brain brainpan (Figure 19) revealed an estimated 30% to 50% reduction in cerebral volume, presumably as a result of osmotic dehydration secondary to glycerolization. The cortices also had the “waxy” amber appearance previously observed as characteristic of glycerolized brains.
The gross appearance of the kidneys, spleen, mesenteric and subcutaneous fat, pancreas, and reproductive organs (where present) were unremarkable. The ileum and mesentery appeared somewhat dehydrated, but did not exhibit the waxy appearance that was characteristic of muscle, skin, and brain.
Figure 15: Oxygen consumption was not apparently affected by glycerolization as can be seen in the data above from the perfusions of FGP-5 and FGP-5.
Oxygen consumption (determined by measuring the arterial/venous difference) throughout perfusion was fairly constant and did not appear to be significantly impacted by glycerolization, as can be seen Figure 12.
Perfusion of FIGP Animals
As previously noted, the ischemic animals had far lower flow rates at the same perfusion pressure as FGP animals and demonstrated incomplete blood washout. Intravascular clotting was serious a barrier to adequate perfusion. Post-thaw dissection demonstrated multiple infarcted areas in virtually all organ systems; areas where blood washout and glycerolization were incomplete or absent. In contrast to the even color and texture changes observed in the FGP animals, the skin of the FIGP animals developed multiple, patchy, non-perfused areas which were clearly outlined by surrounding, dehydrated, amber-colored glycerolized areas.
External and internal examination of the brain and spinal cord revealed surprisingly good blood washout of the central nervous system. While grossly visible infarcted areas were noted, these were relatively few and were generally no larger than 2 mm to 3 mm in diameter. With few exceptions, the pial vessels were free of blood and appeared empty of gross emboli. One striking difference which was consistently observed in FIGP animals was a far less profound reduction in brain volume during glycerolization (Figure 17). This may have been due to a number of factors: lower flow rates, higher perfusion pressures, and the increased capillary permeability and perhaps increased cellular permeability to glycerol.
Whereas edema was virtually never a problem during glycerolization of FGP animals, edema was universal in the FIGP animals after as little as 30 minutes of perfusion. In the central nervous system this edema was evidenced by a “rebound” from initial cerebral shrinkage to frank cerebral edema, with the cortices, restrained by the dura, often abutting or slightly projecting into the burr hole. Marked edema of the nictating membranes, the lung, the intestines, and the pancreas was also a uniform finding at the conclusion of cryoprotective perfusion. The development of edema in the central nervous system sometimes closely paralleled the beginning of “rebound” of ocular volume and the development of ocular turgor and frank ocular edema.
Figure 17: The appearance of the brain of an FIGP animal following cryoprotective perfusion as seen through a craniotomy performed over the right temporal lobe. The cortical surface (CS) is retracted ~3-5 mm from the cranial bone (CB) and appears
In contrast to the relatively good blood washout observed in the brain, the kidneys of FIGP animals had a very dark and mottled appearance. While some areas (an estimated 20% of the cortical surface) appeared to be blood-free, most of the organ remained blood-filled throughout perfusion. Smears of vascular fluid made from renal biopsies which were collected at the conclusion of perfusion (for tissue water determinations) revealed the presence of many free and irregularly clumped groups of crenated and normal-appearing red cells, further evidence of the incompleteness of blood washout. Microscopic examination of recirculating perfusate revealed some free, and a few clumped red cells. However, the concentration was low, and the perfusate microhematocrit was unreadable at the termination of perfusion (i.e., less than 1%).
The liver of FIGP animals appeared uniformly blood-filled throughout perfusion, and did not exhibit even the partial blood washout evidenced by the kidneys. However, despite the absence of any grossly apparent blood washout, tissue water evaluations in one FIGP animal were indicative of osmotic dehydration and thus of some perfusion.
The mesenteric, pancreatic, splanchnic, and other small abdominal vessels were largely free of blood by the conclusion of perfusion. However, blood-filled vessels were not uncommon, and examination during perfusion of mesenteric vessels performed with an ophthalmoscope at 20x magnification revealed stasis in many smaller vessels, and irregularly shaped small clots or agglutinated masses of red cells in most of the mesenteric vessels. Nevertheless, despite the presence of massive intravascular clotting, perfusion was possible, and significant amounts of tissue water appear to have been exchanged for glycerol.
One immediately apparent difference between the FGP and FIGP animals was the accumulation in the lumen of the ileum of large amounts of perfusate or perfusate ultrafiltrate by the ischemic animals. Within approximately 10 minutes of the start of reperfusion, the ileum of the ischemic animals that had been laparotomized was noticed to be accumulating fluid. By the end of perfusion, the stomach and the small and large bowel had become massively distended with perfusate. Figure 14 shows both FIGP and FGP ileum at the conclusion of glycerol perfusion. As can be clearly seen, the FIGP intestine is markedly distended. Gross examination of the gut wall was indicative of tissue-wall edema as well as intraluminal accumulation of fluid. Often by the end of perfusion, the gut had become so edematous and distended with perfusate that it was impossible to completely close the laparotomy incision. Similarly, gross examination of gastric mucosa revealed severe erosion with the mucosa being very friable and frankly hemorrhagic.
Escape of perfusate/stomach contents from the mouth (purging) which occurs during perfusion in ischemically injured human suspension patients did not occur, perhaps due to greater post-arrest competence of the gastroesophageal valve in the cat.
Oxygen consumption in the two ischemic cats in which it was measured was dramatically impacted, being only 30% to 50% of control and deteriorating throughout the course of perfusion (Figure 12).
The most striking change noted upon thawing of the animals was the presence of multiple fractures in all organ systems. As had been previously noted in human cryopreservation patients, fracturing was most pronounced in delicate, high flow organs which are poorly fiber-reinforced. An exception to this was the large arteries such as the aorta, which were heavily fractured.
Fractures were most serious in the brain, spleen, pancreas, and kidney. In these organs fractures would often completely divide or sever the organ into one or more discrete pieces. Tougher, more fiber-reinforced tissues such as myocardium, skeletal muscle, and skin were less affected by fracturing; there were fewer fractures and they were smaller and less frequently penetrated the full thickness of the organ.
Figure 18: All of the animals in the study exhibited fractures of the white matter that transected the brain between the cerebellum and the cerebral cortices. Similarly, the spinal cord was invariable severed by fractures in several locations and exhibited the appearance of a broken candle stick. The yellow box encloses a sampling area used to determine brain water content.
Figure 19: Deep fracture of the left occipital cortex. Note the absence oif fracturing in the adjacent skeletal muscle (M) observed in FGP-1. Note that the brain appears shrunken and retracted in the brainpan.
Figure 20: Appearance of the brain after removal from the brainpan. There is a massive fracture of thew right frontal=temporal cortex which penetrates the full thickness of the cerebral hemisphere to expose the right cerebral ventricle observed in FIGP-2. The cortex appears buff colored and gives the appearance of being incompletely washed out of blood.
Figure 21: Typical fracture sites in the brain (arrows and yellow shading). The olfactory cortices and the brainstem were invariably completely severed by fractures.
In both FGP and FIGP animals the brain was particularly affected by fracturing (Figures 18, 19 & 22) and it was not uncommon to find fractures in the cerebral hemispheres penetrating through to the ventricles as seen in Figure 20, or to find most of both cerebral hemispheres and the mid-brain completely severed from the cerebellum by a fracture (Figure 18). Similarly, the cerebellum was uniformly severed from the medulla at the foramen magnum as were the olfactory lobes, which were usually retained within the olfactory fossa with severing fractures having occurred at about the level of the transverse ridge. The spinal cord was invariably transversely fractured at intervals of 5 mm to 15 mm over its entire length (Figure 21). Bisecting CNS fractures were most often observed to occur transversely rather than longitudinally. In general, roughly cylindrical structures such as arteries, cerebral hemispheres, spinal cord, lungs, and so on are completely severed only by transverse fractures. Longitudinal fractures tend to be shorter in length and shallower in depth, although there were numerous exceptions to this generalization.
Figure 22: Crisp olfactory lobe fracture which also partially penetrated the pia matter in FGG-4.
In ischemic animals the kidney was usually grossly fractured in one or two locations (Figure 25). By contrast, the well-perfused kidneys of the non-ischemic FGP group exhibited multiple fractures, as can be seen in Figure 24. A similar pattern was observed in other organ systems as well; the non-ischemic animals experienced greater fracturing injury than the ischemic animals, presumably as a result of the higher terminal glycerol concentrations achieved in the non-ischemic group.
Figure 23: Appearance of a fractured kidney before removal of the renal capsule. The renal capsule has only one fracture, however when the capsule is removed, the extensive fracturing of the renal cortex and medulla become evident (Figure 24, below).
Figure 24: Fractured renal cortex from FGP-1 after removal of the renal capsule. The renal cortex is extensively fractured, the renal medulla slightly less so. Note the uniform, tan/light brown color of the cortex indicating complete blood washout and the absence of red cell trapping.
Cannulae and attached stopcocks where they were externalized on the animals were also frequently fractured. In particular, the polyethylene pressure-monitoring catheters were usually fractured into many small pieces. The extensive fracture damage occurring in cannulae, stopcocks, and catheters was almost certainly a result of handling the animals after cooling to deep subzero temperatures, as this kind of fracturing was not observed in these items upon cooling to liquid nitrogen temperature (even at moderate rates). It is also possible that repeated transfer of the animals after cooling to liquid nitrogen temperature may have contributed to fracturing of tissues, although the occurrence of fractures in organs and bulk quantities of water-cryoprotectant solutions in the absence of handling is well documented in the literature (12, 13).
There were subtle post-thaw alterations in the appearance of the tissues of all three groups of animals. There was little if any fluid present in the vasculature and yet the tissues exhibited oozing and “drip” (similar to that observed in the muscle of frozen-thawed meat and seafood) when cut. This was most pronounced in the straight-frozen animal. The tissues (especially in the ischemic group) also had a somewhat pulpy texture on handling as contrasted with that of unfrozen, glycerolized tissues (i.e., those handled during pre-freezing sampling for water content). This was most in evidence by the accumulation during the course of dissection of small particles of what appeared to be tissue substance with a starchy appearance and an oily texture on gloves and instruments . This phenomenon was never observed when handling fresh tissue or glycerolized tissue prior to freezing and thawing.
There were marked differences in the color of the tissues between the three groups of animals as well. This was most pronounced in the straight-frozen control where the color of almost every organ and tissue examined had undergone change. Typically the color of tissues in the straight-frozen animal was darker, and white or translucent tissues such as the brain or mesentery were discolored with hemoglobin released from lysed red cells.
Figure 25: The (ventral) dependent and dorsal (less dependent) surfaces of the right kidney from FIGP-1. There is extensive mottling evidencing incomplete blood washout despite perfusion with many liters of CPA solution. Fracturing is much less extensive than that observed in FGP animals not subjected to prolonged periods of post-arrest ischemia. Note the pink colored “drip” from the organ that is present on sectioning board.
Figure 26: Appearance of the kidney from FIGP-1 shown above on cross-section. The renal medulla appears congested and blood filled.
The FGP and FIGP groups did not experience the profound post-thaw changes in tissue color experienced by the straight-frozen controls, although the livers and kidneys of the FIGP animals appeared very dark, even when contrasted with their pre-perfusion color as observed in those animals laparotomized for tissue water evaluation.