Companied by Hyperexcitability, Ataxia,Loss of Righting Ability, and a in The

CENTRAL NERVOUS SYSTEM LESIONS IN RATS EXPOSED TO OXYGEN AT HIGH PRESSURE

J. DOUGLAS BALENTINE, M.D.,* AND BRETT B. GUTSCHE, M.D.t From the Departments of Pathology and Anesthesiology, Duke University Medical Center, Durham, N. C. Spastic limb paralysis, as reported by Bean,1 occurs in rats exposed repeatedly to oxygen at high pressure (OHP). Such paralyses, affecting predominantly the forelimbs bilaterally, are usually preceded or ac- companied by hyperexcitability, ataxia, loss of righting ability, and a kangaroo-like posturing. In some cases the affected limbs are rigidly extended. Although these clinical manifestations, the delayed CNS signs of severe exposure to OHP, may be permanent,2 no consistent selective anatomic changes have been demonstrated in the central nervous system (CNS) to correlate with them. Reference has been made to areas of "anemic necrosis," softening and degeneration of white matter, and degeneration of fiber tracts in brains of rats with chronic motor disabilities induced by OHP,3 but their precise nature and distribution have not been defined. Other workers have reported finding no pathologic lesions in the CNS of rats similarly exposed to OHP.4 The purpose of the present study is to define the nature and distribution of CNS lesions found in rats exposed to repeated OHP until they developed severe paralysis. A correlation of the paralysis with damage to the cerebral peduncles was obtained. MATERIAL AND METHODS Chamber Operatiotts. A small animal hyperbaric chamber similar to that de- scribed by Schwartz and Breslau5 was used for all exposures. No more than 49 animals were exposed at one time. Preceding compression the chamber was flushed with high flows of oxygen (USP) to displace the ambient air present. Using a Pauling oxygen analyzer the method of flushing was checked by measuring the oxygen tension in samples from the outflow valve. The tension was always above 720 mm Hg. Pressurization was accomplished over a 3- to 4-minute period. During each exposure the chamber was ventilated with iO to T2 liters per minute of oxygen to prevent carbon dioxide accumulation. The CO2 tension was measured in outflow samples at various times during exposure using an Instrumentation Laboratory gas analyzer. At the time of this experiment the lowest value measurable on the instrument was io mm Hg and all of the determinations were below this. In similar experiments in Accepted for publication, July I9, I965. * Research Fellow in Neuropathology supported by Research Training Grant No. 2 T I-NB-5212 from the National Institute of Neurological Diseases and Blindness. t Research Fellow in Anesthesiology supported by Grant No. i F 2-NB-23, 984-OI from the National Institute of Neurological Diseases and Blindness. 107 io8 BALENTINE AND GUTSCHE VOL. 48, No. z the same chamber, using 48 rats of the same weight, this ventilation rate allowed an average accumulation of I.4 mm Hg of CO2 at the end of I hour, with no values above 5.4 mm Hg. Decompression was staged over a 5-minute period with stops being made as follows: at 30 pounds per square inch gauge (p. s. i. g.) for I minute; at I5 p. s. i. g. for I minute; at 7 p. s. i. g. for I minute; and at 4 p. s. i. g. for 2 minutes. Animals. White female rats of the Osborne-Mendel strain weighing ISO to 200 g were divided into 3 groups. All were anesthetized with sodium pentobarbital admin- istered intraperitoneally for tagging and weighing 24 hours prior to their first hyperbaric exposure. Group I consisted of 48 animals all of which were subjected in 2 groups of 24 for I hour per day to OHP at 57 p. s. i. g. on consecutive days until the development of severe paralysis or death. Nineteen rats developed paralysis. The remainder died before the onset of this endpoint as a result of the acute effects of severe exposure to OHP2; they, along with 5 of the paralyzed ones unsuited for study because of either poor fixation of incidental CNS lesions, were discarded. Two paralyzed rats exposed with group II were added to group I. Therefore, i6 animals with paralysis constituted the final basis for study in this group. They were sacrificed 4 to I20hours after the exposure that produced the paralysis. Group II consisted of 49 rats that were subjected simultaneously for I hour per day to OHP at 57 p. s. i.g. from I to 5consecutive days. Four animals without CNS motor signs were removed each day. The remainder either died and were discarded or developed paralysis and were included in group I. Therefore, the final basis for this group consisted of 20rats exposed in groups of 4 from I to 5 days without development of CNS motor signs. They were sacrificed arbitrarily 72 hours after their last exposure. Group III consisted of 6 normal unexposed rats that were sacrificed for histologic controls. Preparation of Tissues. Each animal studied was sacrificed by perfusion after being anesthetized with pentobarbital and given heparin intraperitoneally. The thoracic cavity was opened and a ig-gauge thin-wall scalp vein needle was inserted into the left ventricle. As much blood as possible was withdrawn. The right ventricle was then opened and the animal was perfused with IO to 20 CC of Io per cent heparinized formalin in saline followed by 5o to 8o cc of Susa solution. Fifteen to30 minutes following perfusion, the fixed brain was carefully dissected away from the skull. The portion of spinal column containing spinal cord was removed en bloc. All of the tissues were placed in io per cent phosphate buffered formalin for 3 to 5days. The spinal column was decalcified in 3.5 per cent formic acid for 24 to48 hours and divided into4 to5 mm serial slices. Two to4 mm serial coronal slices were made through the entire brain. All slices of brain and spinal cord were dehydrated in a 24-hour Technicon and embedded in paraffin. The slices from 3 experimental brains in group I were serially sectioned at io 1A and mounted on 35 mm film.6 The spinal columns of these animals were sectioned at every ioo M. Ten-I sections were taken from the other brains in group I and selected cases in groups II and III at least at every io00 through the brainstem and parts of the diencephalon and basal ganglia, and at every 250 to I,000g through the remaining areas. Serial sections were taken through selected lesions. In the other cases from groups II and III and from most spinal cords, one section was taken from each slice. The sections were stained with a combination of luxol fast blue-hematoxylin and eosin. Selected sections were stained with Holmes' silver (for axons) and cresyl violet (for Nissl substance). One experimental animal from group I and a control from group III were perfused with I5per cent formalinin 2 per cent ammonium bromide; their brains were removed and kept in the perfusing solution for 3 to 5days. Frozen sections cut at 20 j were made from an area of necrosis in the substantia nigra in the experimental Jan., I966 OXYGEN AT HIGH PRESSURE log brain and from the corresponding area in the control. These sections were stained with gold chloride (for astrocytes), silver carbonate (for other glial cells), and oil red 0 (for neutral fats and fatty acids). RESULTS The onset of paralysis in the I6 animals in group I occurred as follows: During the fourth exposure, 6 rats; the fifth, 3 rats; the sixth, 4 rats; the seventh, 2 rats; the eighth, o rats; and the ninth, i rat. All the rats in group I had central nervous system lesions which followed two morphologic patterns: (i) focal necrosis of individual neurons within certain nuclear groups (type A lesions), and (2) complete or partial necrosis of nuclear groups with frequent damage to myelin, axons and glia within the area of grey matter involved (type B lesion). As a rule, both types were bilateral and symmetrical. The cytologic changes in type A lesions were characterized by clump- ing of nuclear chromatin (Fig. i), pyknosis (Fig. 2), karyorrhexis (Figs. 3 to 5), and karyolysis; along with these nuclear changes there were eosinophilic homogenization (Figs. I to 5), shrinkage and fragmentation (Figs. 2 to 5) of the cytoplasm. Central and peripheral chromatolysis was rarely seen. Phagocytosis (Figs. 4 and 5) was observed in association with the cytologic changes. As neurons disap- peared there was glial cell proliferation, sometimes arranged in nodules (Fig. 6). Similar morphologic alterations characterized the type B lesions except that there was invariably a noticeable amount of pallor and separation of structures by cystic spaces (interpreted as edema) (Fig. 7); frequently myelin, axons and glia were damaged and removed as well as neurons. The magnitude of necrosis and repair was also con- siderably greater (Figs. 8 and 9). In some type B lesions neurons were affected with other structures being relatively spared, even when the neuronal necrosis was extensive. No precise time schedule can be given for the necrosis and phagocytosis because of the nature of the experimental endpoint. The time of sacrifice after the last exposure (4 to I20 hours) in the paralyzed rats had no influence on the distribution, type or stage of lesions, i.e., animals sacrificed 4 hours after paralysis showed as many lesions with a similar distribution and appearance as those sacrificed I20 hours after paralysis. The distribution and frequency of both types of lesions are given in Table I. The one animal used for special stains was omitted from the tabulation. Among those areas showing type A lesions, the dorso- and ventromedial neurons of the anterior horns of the cervical and lumbosacral regions of the spinal cord (Figs. io and ii), the nucleus of the spinal tract of cranial nerve V, the ventral cochlear nucleus (Fig. I2), I IO BALENTINE AND GUTSCHE Vol. 48, No. z

TABLE I

DISTRIBUTION OF CNS LESIONS IN RATS EXPOSED TO OXYGEN AT HIGH PRESSURE Number of rats Type of affected * General area Specific area lesions (Total, I5 rats) Spinal cord Ventromedial grey A I5 (cervical) Ventrolateral grey (dorsal) A 6 Pericentral grey B I Spinal cord Ventromedial grey A 5 (thoracic) Intermediate grey, medial A 3 Intermediate grey, lateral A 2 Spinal cord Ventromedial grey A is (lumbosacral) Ventrolateral grey A I Cranial nerves III Oculomotor nucleus B 3 V Nucleus of spinal tract of V A 12 VIII Ventral cochlear nucleus A I4 Lateral vestibular nucleus A 5 X Nucleus ambiguus A 2 IX and I XI Spinal accessory nucleus A XII Hypoglossal nucleus A Medulla Reticular substance A 3 Inferior olive A 2 Pons Superior olivary complex A I. Pontine nuclei A 2 Pontine nuclei B 2 Reticular substance A 2 Midbrain Substantia nigra B '4 Posterior colliculus A 2 Cerebellum Purkinje cells A 5 Cortex B I Dentate nucleus A I Thalamus Dorsolateral nuclei B 4 Dorsomedial nuclei B 2 Nucleus entopeduncularis B 6 Basal ganglia Globus pallidus B II Amygdala B 6 Rhinencephalon Areas 27, 28, 49 of Kreig B 6 Medial parolfactory area B 4 Medial septal nucleus B I Hippocampus A I Cerebral peduncles Above rostral substantia nigra Damage to myelin 8 Fornix Commissure Damage to myelin I * For description of types A and B lesions, see text. and the superior olivary complex were consistently involved. As for type B lesions, involvement of the substantia nigra (Fig. I3) and globus pallidus (Fig. I4) was constant. There was no injury to white matter that was not within necrotic nuclear areas or not continuous with such areas. The infero-medial Jan., I966 OXYGEN AT HIGH PRESSURE I I I portions of the cerebral peduncles were damaged in 8 cases (Fig. I5). The damage, breaking up and phagocytosis of myelin, was always continuous with extensive necrosis of the substantia nigra. The axons re- mained intact. In one animal the fornix was likewise damaged in an area of necrosis of the medial septal nucleus. The rhinencephalon was involved in 8 animals. Relatively small focal areas of type B necrosis were found in 6 cases in the retro-hippocampal region of the telencephalic cortex corresponding to areas 27, 28 and 49 of Kreig.7 The medial parolfactory area in the anterior cingulate region demonstrated the same type of lesions in 4 rats (Fig. I6). The hippocampus was involved in i case, with necrosis of segments of the lamina granularis of the dentate gyrus and of the pyramidal layer of Ammon's horn. The pattern of necrosis was similar to that of the type A lesions except that large numbers of neurons in segments rather than at random were affected. Dorsal or ventral nuclei of the thalamus were affected (type B lesions) in 7 animals (Figs. I7 and i8). The cerebellar Purkinje cells were involved in some cases, but only focally. The type A lesion, involving Purkinje cells at random in the midline and hemispheres, was the usual finding. In one case a type B lesion was found involving a large part of the cortex in the vermis. Interpretation of type A changes found in the granular cell layer was guarded since there were similar changes in controls. Neuronophagia of granular cells seen in a few experimental cases, and not in controls, indicated that they were involved. This area, however, was not listed in the tabulations for the reasons given above. In the group II rats type A lesions were seen in 6 animals, the earliest after 3 exposures. Only i animal, exposed 5 times, revealed type B lesions, indicating a closer time relation with the onset of paralysis in this type. In group III glial nodules were found in several animals, and this group served as a control for incidental lesions as well as for histologic orientation. DIscuSSION It is well known that both man and animal show marked variability in their responses to oxygen at high pressure. This was re-emphasized by the great variability in the time required to develop paralysis in the present study. It is clear, however, that, under the conditions of this experiment, selective CNS lesions in rats were consistently present by the onset of severe paralysis. A limited number of lesions, especially type A, preceded paralysis by several hours. The paralyses were probably related to damage in the cerebral pe- I I 2 BALENTINE AND GUTSCHE Vol. 48, No. z duncles as they coursed under the substantia nigra. Clear evidence of this was found in 8 animals. It is not known whether or not this damage was primary or secondary to local tissue changes associated with necrosis of the substantia nigra. Although there was type A necrosis in the ventrolateral grey matter of some spinal cords at the level of the cervical enlargment this would not explain the spastic paralysis. There is no simple explanation for the selective distribution and variation in morphologic appearances of the lesions. Selective vulnerabilities of various regions to hypoxia have been attributed to peculiar- ities and paucities of blood supply or to variation in tissue susceptibilities as determined by differences in composition and metabolic demand.8 These factors undoubtedly apply to the selective vulnerabilities to repeated OHP. Since no transition between the two types of lesions was observed and the distribution of each was distinctive, it is probable that they were not related in a time sequence, but that their differences were related to the same factors that applied to regional vulnerabilities. Postulating differences in etiology for the two types seems unwarranted, in view of the morphologic variation encountered in hypoxic injuries to the CNS. The type B lesions, with damage to axons, myelin and glia as well as neurons, suggest the possibility of ischemic necrosis. An experimental model of generalized anoxic-ischemia produced in rats by Levine 9 offers a good comparison between the effects of generalized cerebral ischemia and the conditions of hyperbaric oxygen. The globus pallidus, thalamus and substantia nigra are involved in both instances. However, the most frequent lesion produced in anoxic-ischemia, necrosis of the hippocampus, was observed only once in this study. The high incidence of necrosis of the neocortex and corpus striatum in the former was in contrast to a lack of involvement of these areas in the latter. This comparison suggests that the lesions produced by OHP are not a result of generalized cerebral ischemia. There is evidence produced by Jacobson, Harper and McDowall 10 that blood flow and oxygen uptake in the cerebral cortex may be reduced by as much as 2I per cent and 38 per cent respectively at 2 atmospheres of OHP. CO2 accumulation was reduced, however, in their experiment by controlled ventilation. In animals under hyperbaric oxygen without controlled ventilation, CO2 accumulation would be ex- pected to occur and to counteract to some degree the vasoconstriction produced by oxygen.2 Regardless of the balance between the cerebral vascular effects of 02 and C02, oxygen tension in the cerebral cortex and cerebral spinal fluid in rats increases in almost linear manner with in- creasing atmospheres of oxygen.1' The increase is augmented by adding Jail., I966 OXYGEN AT HIGH PRESSURE II 3 C02 to the oxygen. Pertinent facts relating to the regional blood flow and oxygen tension in the areas of the CNS damaged by OHP are, however, not available, and although generalized ischemia is unlikely, regional ischemia remains a possible explanation for the occurrence of CNS lesions. Regional ischemia could be produced by focal vasoconstriction or by oxygen bubbling with cerebral embolization. There is strong evidence against the latter. Rapid decompression from 20- to 40-minute exposures to oxygen at 3 to 6 atmospheres causes no untoward symptoms in man or guinea pigs.2 It is unlikely that the relatively slow decompression of oxygen used in this experiment and by other workers 12 would produce any harmful effects. More importantly, the lack of involvement of the white matter apart from grey matter in the OHP-induced lesions is in considerable contrast with the type of CNS lesions produced by gas embolization.'3"4 Besides ischemia one must consider the potential role of hypoxia. There are two possible mechanisms for the development of hypoxia in- dependently of ischemia under the conditions of the experiment. Pul- monary damage is a known feature of oxygen toxicity and occurs at shorter exposures than used in this study.12 The pathologic alterations are those of acute pulmonary edema and congestion with or without the formation of hyaline membranes. Alveolar hyperplasia may be induced. Damage to the alveolar membranes is considered to be the primary in- sult. It would seem likely that pulmonary function could be chronically altered with repeated exposures, as in the present experiment. Although this may be true, clinical observations of the rats failed to reveal signs of chronic hypoxia. Animals with clinical pulmonary difficulties usually either completely recovered within a few minutes after exposure, or died. Lungs from 7 of the experimental rats with CNS lesions in the present investigation were examined histologically, and 5 were normal, sug- gesting a lack of correlation between CNS and pulmonary lesions. In addition to pulmonary damage, the problem of convulsions must be considered. In this laboratory, the onset of convulsions prior to the development of paralysis seems to be a general rule in untreated rats exposed to OHP.15 They are grand mal in type and are usually continuous. They are not associated with hypoglycemia.2 Most animals are in status epilecticus for IO to 30 minutes on one or more exposures prior to the onset of paralysis. Although this correlation exists, the development of paralysis occurs in the absence of convulsions.1 It has been shown that anesthetic dosages of pentobarbital, which prevent convulsions, actually enhance the development of delayed CNS signs.', Severe paralysis has been produced in anesthetized rats with one 30- II 4 BALENTINE AND GUTSCHE VoI. 48, No. -r minute exposure to 98 per cent 02 and 2 per cent C02 at 57 p. s. i. g. in the absence of convulsions.17 Gutsche, Balentine, North and Stephens18 have shown that sedative dosages of pentobarbital and other CNS de- pressants protect female rats from the CNS signs of OHP. Attempts in this laboratory to produce paralysis in unanesthetized rats without convulsions by repeated i-hour exposures to OHP have generally been unsuccessful. Thus, the conditions required to produce signs without convulsions seem to indicate that convulsions do play a role in the pathogenesis of CNS damage, a role that may be shared by anesthesia. It is improbable that the role is related to hypoxia in view of the high P02 produced in the lungs and brain 11 by OHP. A strong point against hypoxia is found in the distribution of the lesions in this study. CNS lesions of hypoxia are similar to those of generalized cerebral ischemia.'9'20 In addition to the differences discussed in relation to anoxic- ischemia and OHP, experimental hypoxia produced in rats by exposure to low oxygen tension 21 reveals a contrast in the distribution of cranial nerve involvement with the oxygen induced lesions. In hypoxia the motor nuclei of cranial nerves III, IV, V, VII, and XII were frequently found to be involved. In the hyperbaric oxygen setting sensory nuclei were predominantly effected. A likely common denominator between convulsions and anesthesia in the potentiation of the CNS effects of OHP is the interference with ventilation resulting in CO2 accumulation. It is of interest that when CO2 is added to OHP the delayed CNS signs are enhanced.'0 Although this enhancing effect of CO2 iS generally attributed to increased oxygen flow, there is evidence that breathing high concentrations of CO2 causes CNS damage. Several investigators22'23 have demonstrated lesions in the thalamus, midbrain and spinal cord of rats exposed to high concentrations of C02 at ambient pressure. Exposure to high concentrations of C02 has been shown to cause an increase in cerebral PCO2 and a decrease in pH.24 It seems likely, therefore, that CO2 is an important factor to be dealt with in interpreting effects of OHP. Even in the absence of conditions which would tend to cause C02 accumulation through impaired ventilation, there is some suggestion that tissue CO2 increases in exposure to OHP because of an interference with C02 transport imposed by a diminished reduction of oxyhemo- globin.2 Despite the possible changes in tissue C02 in vivo, OHP has been shown to have a direct toxic effect on cellular metabolism in the brain and other organs in in vitro systems, where pH and other factors are controlled.25'26 Because of the complexities in the functional aspects of OHP, it is im- possible to attribute the CNS lesions found in this study to any one factor. It is possible that regional blood flow is altered in such a way Jan., I966 OXYGEN AT HIGH PRESSURE 115 as to render certain areas of the CNS ischemic while others are ade- quately perfused and have an increase in oxygen tension. It is also possible that OHP alone may initiate neuronal injury which becomes irreversible in the presence of a critical degree of acidosis, contributed to by an increase in tissue C02. Whether or not CO2 alone is primarily important in producing the lesions is unknown. SUMMARY Adult female rats were paralyzed by repeated exposures to oxygen at high pressure (OHP) and their central nervous systems were studied histologically in detail. Two patterns of selective CNS lesions were found and designated as types A and B. Type A lesions were characterized by focal individual neuronal necrosis within generally preserved nuclear groups. Type B lesions appeared as large areas of neuronal necrosis, frequently involving entire nuclear groups, in which there was often damage to axons, myelin and glia. The distribution and frequency of the lesions were plotted in I5 animals. Among areas showing type A lesions the spinal cord, superior olivary complex, ventral cochlear nuclei, and nuclei of the spinal tract of cranial nerve V were consistently involved; whereas among areas showing type B lesions the substantia nigra and globus pallidus were constantly affected. Type B lesions were frequently found in the rhinencephalon; the neocortex, however, was completely spared. The lesions differed in distribution or type from those of generalized cerebral ischemia, hypoxia and gas embolization. Damage to myelin in the cerebral peduncles was frequent and was considered to be the most reasonable explanation for the severe spastic paralyses. The mechanism of the lesions could not be determined, but various possibilities were discussed in terms of known physiologic principles relating to oxygen at high pressure. REFERENCES I. BEAN, J. W., and SIEGFRED, E. C. Transient and permanent after-effects of exposure to oxygen at high pressure. Amer. J. Physiol., 1945, I43, 656-665. 2. BEAN, J. W. Effects of oxygen at increased pressure. Physiol. Rev., I945, 25, I-I47. 3. BEAN, J. W. Alterations in C.N.S. associated with chronic motor disabilities induced by 02 at high pressure. Proc. Soc. Exp. Biol. Med., I945, 58, 20-2I. 4. EDSTROM, J.-E., and R6CKERT, H. The effect of oxygen at high pressure on the histology of the central nervous system and sympathetic and endocrine cells. Acta. Physiol. Scand., I962, 55, 255-263. 5. SCHWARTZ, S. I., and BRESLAU, R. C. The small animal chamber. Ann. N. Y. Acad. Sci., i965, II7, 865-874. 6. PICKETT, J. P.; GREENE, W. B., and SOMMER, J. R. Improved film strip technique for the laboratory. Arcli. Path. (Chicago), I964, 77, 429-433. I I6 BALENTINE AND GUTSCHE Vol. 48, No. z 7. ZEMAN, W., and INNES, J.R.M. Craigie's Neuroanatomy of the Rat. Academic Press, New York and London, I963, 230 PP. 8. SCHADE', J. P., and MCMENEMEY, W. H. (eds.). Selective Vulnerability of the Brain in Hypoxaemia. F. A. Davis Co., Philadelphia, I963, 395 PP. 9. LEVINE, S. Anoxic-ischemic encephalopathy in rats. Amer. J. Path., I960, 36, I-I7. IO. JACOBSON, I.; HARPER, A. M., and MCDOWALL, D. G. The effects of oxygen at I and 2 atmospheres on the blood flow and oxygen uptake of the cerebral cortex. Surg. Gynec. Obstet., I964, II9, 737-742. II. JAMIESON, D., and VAN DEN BRENK, H.A.S. Measurement of oxygen tensions in cerebral tissues of rats exposed to high pressures of oxygen. J. Appl. Physiol., I963, I8, 869-876. I2. VAN DEN BRENK, H.A.S., and JAMIESON, D. Pulmonary damage due to high pressure oxygen breathing in rats. I. Lung weight, histological and radiological studies. Aust. J. Exp. Biol. Med. Sci., I962, 40, 37-50. 13. HAYMAKER, W., and JOHNSTON, A. D. Pathology of decompression sickness. Milit. Med., I955, II7, 285-306. I4. SCHOLZ, W., and WECHSLER, W. Nekrotisierende und Entmarkungsvorginge bei cerebraler Gasembolie. Acta Neuropath. (Berlin), I961, I, 85-Ioo. i5. BALENTINE, J. D., and GUTSCHE, B. B. Unpublished data. I6. VAN DEN BRENK, H.A.S., and JAMIESON, D. Potentiation by anaesthetics of brain damage due to breathing high-pressure oxygen in mammals. Nature (London), I962, 194, 777-778. I7. BALENTINE, J. D. Unpublished data. I8. GUTSCHE, B. B.; BALENTINE, J. D.; NORTH, W. C., and STEPHENS, C. R. Alterations of hyperbaric oxygen toxicity by drugs. Submitted for publication, I965. I9. HICKS, S. P. Brain metabolism in vivo. Arch. Path. (Chicago), I950, 49, III- I37. 20. HOFF, E. C.; GRENELL, R. G., and FULTON, J. F. Histopathology of the central nervous system after exposure to high altitudes, hypoglycemia and other conditions associated with central anoxia. Medicine (Bait.), I945, 24, I6I-2I7. 2I. YANT, W. P.; CHORNYAK, J.; SCHRENK, H. H.; PATTY, F. A., and SAYER, R. R. Studies in asphyxia. U.S. Public Health Bulletin, I934, 2II, i-6I. 22. STEPHENS, W. M. The central nervous system changes resulting from increased concentrations of carbon dioxide. J. Neuropath. & Clin. Neurol., I95I, I, 88-97. 23. MEESEN, H. Chronic carbon dioxide poisoning. Experimental studies. Arch. Path. (Chicago), I948, 45, 36-40. 24. SCHAEFER, K. E. The Effects of CO2 and Electrolyte Shifts on the Central Nervous System. In: Selective Vulnerability of the Brain in Hypoxaemia. SCHADE, J. P., and MCMENEMEY, W. D. (eds.). F. A. Davis Co., Philadelphia, I963, IOI-I23. 25. HAUGAARD, N. Poisoning of cellular reactions by oxygen. Ann. N. Y. Acad. Sci., I965, II7, 736-744. 26. THOMAS, J. J., and NEPTUNE, E. M., JR. Chemical Mechanisms in Oxygen Toxicity. In: Proceedings Second Symposium on Underwater Physiology. LAMBERTSEN, C. J., and GREENBAUM, L. J. (eds.). National Academy of Sciences-National Research Council, Washington, D. C., Publication ii8i, I963, I39-I51. The authors wish to extend their appreciation to Doctors F. Stephen Vogel and Gordon K. Klintworth for their assistance in preparation of this manuscript. Jan., 1966 OXYGEN AT HIGH PRESSURE I"7

[Illustrations follow] Ii I8 BALENTINE AND GUTSCHE Vol. 48, No. I

LEGENDS FOR FIGURES Photographs were prepared from sections stained with luxol fast blue-hematoxylin and eosin. FIG. i. Anterior horn cell of spinal cord. The nuclear chromatin in the area of the nucleolus (identified in a different focal plane) is clumped and there is beginning eosinophilic homogenization of the cytoplasm. X I,337. FIG. 2. Purkinje cell of cerebellum. The nucleus exhibits pyknosis and there is eosinophilic homogenization and shrinkage of the cytoplasm. X I,337. FIG. 3. Neuron, spinal nucleus of cranial nerve V. Evident are karyorrhexis, and shrinkage and eosinophilic homogenization of the cytoplasm. X I,337. FIG. 4. Neuron of spinal nucleus of cranial nerve V. Early phagocytosis accompanies beginning fragmentation of the shrunken, eosinophilic and homogenized cytoplasm. Karyorrhexis is apparent. X I,337. Jan., I966 i''''''OXYGEN AT HIGH- PRESSURE_.-:~~~~~~~~~~~~I I9

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4 I20 BALENTINE AND GUTSCHE Vol. 48, No. z

FIG. 5. Anterior horn cell of spinal cord. Advanced phagocytosis is shown. X I,337. FIG. 6. Anterior horn cell of spinal cord. Glial nodule. X 785. FIG. 7. Type B lesion. Pallor and cystic changes, interpreted as edema, are associated with necrosis of neurons in the dorsolateral thalamus. X 6o. FIG. 8. Type B lesion. Phagocytosis is apparent at the margin of a necrotic substantia nigra. X 228. Jan., I966 OXYGEN AT HIGH PRESSURE I 2 I

:zl

8 I 2 2 BALENTINE AND GUTSCHE Vol. 48, No. I

FIG. 9. Type B lesion. Phagocytosis and gliosis are evident in the necrotic substantia nigra. X 228. FIG. IO. A low power view at the level of the cervical enlargement demonstrates the characteristic distribution of the type A lesions in the spinal cord. A lack of neurons medially may be compared to those laterally in the ventral horn. The arrow identifies an involved neuron with cytologic changes similar to those shown in Figure 2. X 66. FIG. I I. Necrosis (arrows) with phagocytosis appear in the spinal accessory nucleus at the upper cervical cord (type A lesion). X 228. FIG. I2. Ventral cochlear nucleus. Random necrosis of neurons characterizes the type A lesions. The arrows identify some of the involved neurons. X 228. Jan., I966 OXYGEN AT HIGH PRESSURE I23

12 I24 BALENTINE AND GUTSCHE Vol. 48, No. I

FIG. I3. Type B lesions. Bilateral necrosis of the substantia nigra (arrows). X 8. FIG. I4. Type B lesions. Bilateral necrosis of the globus pallidus (arrows). The lesion on the left extends into the amygdala. Small pale staining patches in the hypothalamus and fornix are not associated with necrosis. Similar patches were seen in controls and have been interpreted as perfusion artifacts. X 8. FIG. I5. Bilateral damage to the rostral cerebral peduncles (arrows). Immediately caudad to this was extensive bilateral necrosis of the substantia nigra. Small patches of pallor in the thalamus were not associated with necrosis and are interpreted as perfusion artifacts; (see legend for Figure I4). X I3. Jan., r966 OXYGEN AT HIGH PRESSURE I25

15 r26 BALENTINE AND GUTSCHE Vol. 48, No. r

FIG. I6. Type B lesions. Bilateral necrosis of the medial parolfactory area. The process is more prominent on the left (arrows). X 9. FIG. I7. Type B lesions. Bilateral necrosis of the dorsomedial thalamus (arrows). X 6.5. FIG. i8. Type B lesions. Bilateral necrosis of the nucleus entopenduncularis (arrows). Also involved are the dorsomedial thalamic nuclei. x 6.5. Jan., IY966 OXYGEN AT HIGH PRESSURE I27