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.

Cerebral Cortex

 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).

END OF PART 3

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.

Liver

      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).

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 32: FGP-1, Liver, PAS, 100x. The intensely red-stained granules present in the cytoplasm of the hepatocytes are glycogen deposits selectively stained by PAS. There is extensive cytoplasmic (cv) and nuclear vacuolization (nv) and the sinusoids appear filled with flocculent cellular debris (d). Indeed, it is only possible to discern the outlines of the original individual hepatocytes from the pattern of the intracellular glycogen granules disclosed by the PAS stain.

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.

 Kidney

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).

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).

END OF PART 2

I.    Introduction                                  

II.   Materials and Methods

III   Effects of Glycerolization

IV.  Gross Effects of Cooling to and Rewarming From -196°C

I. INTRODUCTION

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.

II. MATERIALS AND METHODS

Pre-perfusion Procedures

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.

Surgical Protocol

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.

Figure 1: Vascular access for extracorporeal perfusion was via median sternotomy. The arterial cannula consisted of a 14-gauge  Angiocath (AC) which was placed in the aortic root (AR) and secured in place with a purse string suture. A USCI  type  1967 16 fr. venous cannula (VC) was placed in the right atrium (RA) and snared in place using 0-silk ligature and a length of Red Robinson urinary catheter (snare). The chest wound was kept open using a Weitlander retractor. The left ventricle (LV) was not vented.

  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

Fixative Perfusion

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

 TABLE I

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

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.

Figure 5: pH of non-ischemic Δ•▪*(FGP) and ischemic ●●● ᴑ  (FIGP) cats during cryoprotective perfusion. The FIGP animals were, as expected, profoundly acidotic with the initial arterial pH being between 6.5 and 6.6.

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:

Vp

Mc = ——— Mp

Vc + Vp

where

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.

Cooling to -79°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.

Preparation of Tissue Samples For Microscopy

Fixation

 TABLE II.

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.

III. EFFECTS OF GLYCEROLIZATION

 Perfusion of FGP Animals

Blood  washout  was  rapid and complete in the  FGP  animals  and vascular  resistance  decreased  markedly  following  blood   washout.  Vascular  resistance increased steadily as the glycerol  concentration increased,  probably  as a result of the increasing viscosity  of  the perfusate.

Within   approximately  5  minutes  of  the  beginning   of   the cryoprotective ramp, bilateral ocular flaccidity was noted in the  FGP animals.   As  the perfusion proceeded, ocular  flaccidity  progressed until  the  eyes had lost approximately 30% to 50%  of  their  volume.

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.

TABLE III.

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.

Figure 16: The eye of an FGP animal following cryopreservation. The cornea has  become concave due to the glycerol-induced osmotic evacuation of the aqueous humor. The vitreous humor is completely obscured by the lens which has become white and opaque as a result of the precipitation of the crystallin proteins in the lens.

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).

IV. GROSS EFFECTS OF COOLING TO AND REWARMING FROM -196°C

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.

END OF PART 1