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The Effects of Hypoxia and Hyperoxia*
764
AUSTIN I. FINK AND IRVING BARAS
2. Ashton, N., and Cook, C.: Effect of cortisone on healing of corneal wounds. Brit. J. Ophth., 35 :708 (Nov.) 1951. 3. Newell, F. W., and Dixon, J. M.: Effect of subconjunctival cortisone upon the immediate union of experimental corneal grafts. Am. J. Ophth., 34:977 (July) 1951. 4. Ragan, G., Howes, E. L., Plotz, C. M., et al.: Effect of ACTH and cortisone on connective tissue. Bull. New York Acad. Med., 26 :25l (Apr.) 1950. 5. Plotz, C. M., Blunt, J. W., Ragan, C.: Effect of pituitary adrenocorticotropic hormone (ACTH) on disseminated lupus erythematosus. Arch. Dermat. & Syph., 61:913 (June) 1950. 6. Bourne, G. H.: Effect of cortisone and vitamin C on wound healing. Internat. Rev. Vitamin Re search, 24:318, 1952. 7. Palmerton, E. S.: The effect of local cortisone on wound healing in rabbit corneas. Am. J. Ophth., 40:344-53 (Sept.) 1955. 8. Bangham, A. D.: Effect of cortisone on wound healing. Brit. J. Exper. Path., 32:77 (Apr.) 1951. 9. Maumenee, A. E.: The influence of donor recipient sensitization on corneal grafts. Am. J. Ophth., 34:142 (May) 1951. 10. Ragan, C, Howes, E. L., Plotz, C. M., Meyer, K., and Blunt, J. W.: Effect of cortisone on granu lation tissue in rabbits. Proc. Soc. Exper. Biol. & Med., 72:718, 1949. 11. Howes, E. L., Plotz, C. M., Blunt, J. W., Ragan, C.: Retardation of wound healing by cortisone, Surgery, 28:177 (Aug.) 1950. 12. Findlay, C. W., and Howes, E. L.: The combined effect of cortisone and partial protein depletion on wound healing. New Eng. J. Med., 246 :597 (Apr.) 1952. 13. Greene, R. W., Faloon, W. W., and Lozner, E. L.: Use of ACTH in preparing patients with idiopathic thrombocytopenic purpura for splenectomy. Am. J. Med. Sc, 226:203 (Aug.) 1953. 14. Grant, G.: Effect of cortisone on healing of tendons in rabbits. J. Bone & Joint Surg., 35-A:525 (Apr.) 1953. 15. Gonzalez, R. I.: Effect of cortisone of healing of tendons in rabbits. J. Bone & Joint Surg., 35-A: 525 (Apr.) 1953. 16. Ashton, N.: Cortisone in ocular conditions. Lancet, 2:919 (Nov.) 1951. 17. Cole, J. W., Orbison, J. L. Holden, W. D., et al.: A histological study of the effect of cortisone on wound healing per primum. Surg Gynec. & Obst., 93:321 (Sept.) 1951. 18. Kay, J. A., Odell, R. T., and Taylor, L. W.: Failure of cortisone to delay or prevent the healing of fractures in rats. J. Bone & Joint Surg., 34-A :665 (July) 1952. 19. Daily, L., Jr., and Daily, R. K.: Present status of corneal transplantation. Texas State M. J., 49: 706 (Sept.) 1953. 20. McDonald, P. R., Leopold, I. H., Vogel, A. W., and Mulberger, R. D.: Hydrocortisone (Com pound F) in ophthalmology. Arch. Ophth, 49 :400 (Apr.) 1953.
T H E EFFECTS OF HYPOXIA AND
HYPEROXIA*
U P O N T H E OXYGEN T E N S I O N I N T H E VITREOUS H U M O R OF T H E CAT A R L I N G T O N C.
K R A U S E , M.D.,
AND S E Y M O U R B.
GOREN,
B.A.
Memphis, Tennessee INTRODUCTION
It is obvious that oxygen is necessary for the maintenance of retinal life. The mecha nisms by which oxygen is transported to and metabolized by the retina, however, are * From the Division of Ophthalmology, Depart ment of Surgery, of The University of Chicago. This investigation was supported by the Douglas Smith Foundation for Medical Research and the Chicago Community Trust of Chicago. Presented at the Midwest Section of the Association for Research in Ophthalmology, March 17, 1956, at Chicago.
not so well known. The diffusion of oxygen through the vitreous humor is one factor in
retinal metabolism. It has been found that, under normal conditions, the oxygen tension in the anterior chamber of the eye is 40 to 50 mm. Hg, while that in the posterior chamber is 100 to 120 mm. Hg. 1 The pur pose of the present study was to determine the relationships between the oxygen ten sion in the vitreous humor of the adult cat and the environmental conditions of hypoxia and hyperoxia.
OXYGEN TENSION IN VITREOUS T h e determination of oxygen tensions in the inner cavities of the eye is pertinent to the better understanding of the relationships between oxygen tensions in the environ mental atmosphere and disease processes of the eye. Perhaps the most dramatic ocular disease caused by excess oxygen administra tion is retrolental fibroplasia. Patz 2 concluded that the eye is susceptible to this damage only when the retina is in completely vascularized. This, of course, ex plains the high incidence of retrolental fibroplasia in premature infants who are ad ministered large quantities of oxygen for prolonged periods of time. Since the vitreous humor derives much of its oxygen by diffusion from the retinal vessels, the oxygen tension in the vitreous humor is of interest in the study of this dis ease process. Although retrolental fibro plasia is not incurred by the adult, it is of equal interest to know how much oxygen is present in the vitreous humor of the fully vascularized eye under conditions of hypoxia and hyperoxia. METHOD
The polarographic method, similar to that used by Davies and Brink 3 and Montgomery and Horwitz, 4 was utilized for measuring the oxygen tension in the vitreous humor of the cat under conditions of hypoxia and hyperoxia. Platinum electrodes were used because, in this type of circuit, platinum is capable of reducing the oxygen in the tissue being studied, thereby creating a current which can be measured with a galvanometer. T h e galvanometer serves as an ammeter whose deflections are directly proportional to the oxygen tension in the tissue or solution be ing studied. The electrodes were prepared of soda-lime glass tubing having a 3.0-mm. O.D. and a 1.5-mm. I.D., a 2.0-cm. strip of platinum wire having a diameter of 0.02 to 0.03 mm., and mercury. A recessed electrode was not used in this experiment because of the in
765
creased recovery time necessary when using such an electrode; an open type of electrode was not used because only relative values could be obtained with it. This is in agree ment with the results obtained by Davies and Brink. 3 Consequently, 1.0 mm. of platinum wire was allowed to project beyond the edge of the glass tubing. Such an electrode was found to be the most favorable for this experiment. A 5.5 to 6.5 cm. column of mercury was placed in the open end of the glass tubing. No. 18 insulated copper wire was inserted into the mercury, and the completed elec trode was stored in a solution of 0.9-percent sodium chloride. T h e electrodes were re moved from this solution only when in use. T h e saline was renewed every 48 hours. T h e electrical circuit is illustrated in F i g u r e 1. T h e main switch ( E ) was kept closed throughout all determinations and the zero adjustment of the string galvanometer was controlled by the 500-ohm variable re sistance ( H ) . T h e sensitivity of the galva nometer was 2.5 X 10~8 amperes. Through out all experiments the voltage was kept at
Fig. 1 (Krause and Goren). Diagram of electric circuit. (A) Calomel half-cell containing 0.9-per cent NaCl (anode). (B) Tissue electrode (cathode). (C) Variable resistance of 10,000 ohms. (D) Fixed resistance of 40,000 ohms. (E) Main switch. (F) 1.5-volt dry cell. (G) Galvanometer (Rubicon No. 3418). (H) Variable resistance of 500 ohms. (J) Fixed resistance of 2,000,000 ohms. (K) Aryton shunt (Rubicon No. 1243). (L) Electrode switch. (V) Voltmeter.
766
ARLINGTON C. KRAUSE AND SEYMOUR B. GOREN
0.7 V. Higher voltages did not give an ap preciable deflection of the galvanometer, while lower voltages decreased the accuracy and the sensitivity of the galvanometer be cause of the increased current flow. The electrodes were calibrated by measur ing the current created by the reduction of oxygen in standard saline solutions of known oxygen tension and temperature. They were calibrated with 0.9-percent solution of so dium chloride equilibrated with 20.9-percent oxygen and then with the same solution equilibrated with 100-percent oxygen at con stant temperatures. In order to equilibrate 0.9-percent solution of sodium chloride with 100-percent oxygen, pure oxygen was bub bled into the solution for 45 minutes before readings were taken and discontinued there after for only 20 seconds of each minute (that is, 10 seconds before the switch was closed and 10 seconds necessary for the de termination of a reading). Experimental data revealed that the saline solution was equilibrated with the desired oxygen tension after 45 minutes (not tabled). The bubbling was stopped 10 seconds prior to closing the switch so as to allow the solution to stop swirling. This precaution was taken because agitation of the solution produced increased and inconstant deflections of the galvanome ter as a result of convection currents in creasing the rate of oxygen supply to the electrode. In accordance with the work of Montgomery and Horwitz,4 it was found that errors caused by excessive reduction of oxygen by prolonged current flow and from the period swing of the galvanometer were avoided by reading the galvanometer 10 seconds after closing the electrode switch. For calibration, the electrodes were dipped into a 150-ml. beaker which contained 100 ml. of 0.9-percent sodium chloride solution. The calomel half-cell was placed in the same beaker as the electrode, thereby com pleting the circuit. Care was taken so that no air bubbles were trapped in the calomel halfcell. Determinations were made at 110-second intervals to allow sufficient time for the
electrode to recover from its previous use. All readings were recorded 10 seconds after the electrode switch was closed. Multiple determinations were made with 0.9-percent solution of sodium chloride at 14°C, 24°C, 34°C, and 37°C. equilibrated with 20.9-percent and 100-percent oxygen respectively as shown in Figure 2. Increased temperatures up to 60° C. resulted in in creased galvanometric deflections because of the greater diffusibility of oxygen at higher temperatures. From 60°C. to 85°C, how ever, the readings decreased as a result of the increased temperature agitating the mole cules of oxygen to a point where they were forced out of solution (not tabled). Cats weighing approximately four kg. were anesthetized with an intraperitoneal in oculation of Nembutal-Sodium.® They were then placed in an Isolette incubator and the desired oxygen tension was maintained for 45 minutes. After 45 minutes, the oxygen tension in the vitreous humor had reached a plateau. It was therefore assumed that the oxygen tension in the tissue being studied was at equilibrium with the oxygen tension in the environmental atmosphere. A Beckman oxygen analyzer, Model D, was used to measure the oxygen tension in the circulating air in the incubator. Blood on the tip of the electrode caused inaccurate
o
to
-to
60
80
PERCENT OWSEN IN ENVIRONMENTAL ATMOSPHERE
Fig. 2 (Krause and Goren). Temperature cor rection curves. (A) 37°C. (B) 34°C. (C) 24°C. (D) 14°C.
OXYGEN TENSION IN VITREOUS
and variable results because the small sur face to volume ratio of a droplet of blood prohibited it from giving off oxygen as easily as the tissue cells could. Therefore, bloodless surgery was performed. The cornea was removed and the aqueous humor allowed to drain. Forceps were used to retract the lens, and the electrode was in serted into the vitreous humor. Absorbent cotton soaked in 0.9-percent saline was used to make contact between the eye and the calomel half-cell, thereby completing the elec trical circuit. Temperature corrections were extrapolated from the experimental data shown in Figure 2. RESULTS
The oxygen tension in the vitreous humor of the cat under conditions of hypoxia and hyperoxia are tabulated in Table 1 and graphed in Figure 3. The ideal equation of the curve was found to have an exponential form. Under normal physiologic conditions, the average oxygen tension in the vitreous humor was 53 mm. Hg. When the animal was submitted to moderate hypoxia, such as 15-percent oxygen in the inspired air, the average oxygen tension in the vitreous hu
767
rt
o
UJ
g 180 > ? 135
£ o
I
30
45
-+-
0
200
■+■
WO
600
H 600
OXYGEN TENSION (MM. H&) IN ENVIRONMENTAL ATM05PHERE
Fig. 3 (Krause and Goren). The exponential curve expressing the relationship between the oxy gen tension in the vitreous humor and that in the environmental atmosphere.
mor was 28 mm. Hg. The maximum oxygen tension attained in the tissue being studied was 172 to 177 mm. Hg. Oxygen tensions in the environmental atmosphere above 610 mm. Hg failed to cause any significant in crease in the oxygen tension in the vitreous humor. Another series of experiments were per formed in which cats were first placed in an
TABLE 1 OXYGEN TENSION IN VITREOUS OF CAT Oxygen Tension (mm. Hg) in Environmental Atmosphere
114
154
304
455
609
680
745
Oxygen Tension (mm. Hg) in Vitreous Humor*
30 26 29 29 31 27 28 27
53 53 53 52 47 53 51 53 53 72 47 57 54 53 53 53 53 61 53 62
115 122 118 99 121 115 118 112 115 118 136 115 115 101 115 110 118 118
100 133 121 144 129 144 121 133 136 141 133 130
162 160 190 162 175 182 174 181 160 170
175 177 176 176 173 174 178 176 177 174
178 180 175 179 172 180 178 177 178 176
* Temperature corrections have been made for the tabulated data.
768
ARLINGTON C. KRAUSE AND SEYMOUR B. GOREN TABLE 2 RELATIONSHIPS BETWEEN OXYGEN TENSION AND TIME IN OXYGEN
0
2
4
6
8
10
12
14
100 133 121 144 129 144 121 133 136 141 133 130
88 90 86 87 89 88 85 90 91 87
70 72 63 74 70 71 69 70 71 73
62 58 59 61 60 60 63 61 60 62
55 53 52 51 58 57 56 55 57 55
51 55 53 53 50 54 53 53 54 52
53 53 53 58 49 54 53 53 54 53
52 54 54 50 53 52 53 53 52 53
Minutes out of Incubator Oxygen Tension (mm. Hg) in Vitreous Humor*
Temperature corrections have been made for the tabulated data. incubator and 60-percent oxygen adminis tered to them for 45 minutes. T h e animals were then removed from the incubator, placed in 20.9-percent oxygen, and the oxy gen tension in the vitreous humor deter mined at various intervals. T h e results are tabulated in Table 2 and graphed in Figure 4. The ideal equation of this curve was also found to have an exponential form. The oxygen tension in the vitreous humor sharply decreased initially, and then levelled off at 53 mm. H g after the animal had been
0
4 MINUTES
8
/
2
OUT OF INCUBATOR
Fig. 4 (Krause and Goren). The exponential curve expressing the relationship between the oxy gen tension in the vitreous humor and the length of time which the animal had been transferred from 60-percent oxygen to 20.9-percent oxygen.
removed from the incubator for eight to 10 minutes. DISCUSSION
W h e n the animal was under conditions of hypoxia, the oxygen tension in the vitreous humor was subnormal. This is to be ex pected since the blood hemoglobin holds less than the normal amount of oxygen when the oxygen tension in the environmental atmos phere is low. Therefore, less oxygen is available to the body tissues, thereby causing a decreased oxygen tension in the vitreous humor. W h e n the animal was placed under con ditions of increasing degrees of hyperoxia, a corresponding increase in the oxygen ten sion in the vitreous humor resulted until a maximum of approximately 175 mm. H g was reached. This value was attained when the oxygen tension in the inspired air was 609 mm. H g . It is of interest to note that the blood hemoglobin is fully saturated with oxygen at values far below 609 mm. Hg. The increased oxygen which goes into simple solution in the blood plasma under severe hyperoxia is not sufficient to explain the difference in oxygen tensions in the vitre ous humor found under moderate and se vere hyperoxia. F o r some unknown reasons, a greater quantity of oxygen diffuses into the vitreous humor when the animal is under severe hyperoxia than is the case un-
OXYGEN TENSION IN VITREOUS der moderate hyperoxia, although basically the same amount of oxygen is being carried through the blood vessels of the eye in both conditions. F u r t h e r research is necessary be fore this fact may be adequately explained. W h e n an animal was removed from an environment in which the oxygen tension was 455 mm. H g , and placed in air having one of 154 mm H g , the oxygen tension in the vitreous humor was found to level off at the normal value of 53 mm. H g after eight to 10 minutes (fig. 4 ) . These results are in agreement with the sharp initial decrease and the subsequent leveling off of the blood hemoglobin saturation with oxygen seen when an animal is transferred from an at mosphere having a high oxygen tension to one having a normal oxygen tension. It is well known that no blood vessels penetrate the vitreous humor. W e may there fore assume that its nutritional requirements are satisfied by the diffusion of substances from surrounding blood vessels. Anatomic evidence indicates that the anterior portion of the vitreous humor derives its oxygen primarily from the blood vessels of the cili
769
ary body, while the posterior portion re ceives its oxygen principally from the retinal vessels. F u r t h e r investigation in both kittens and adult cats is necessary to ascertain the relative importance of the vessels of the inner layers of the eye in the maintenance of normal oxygen tensions in the vitreous humor. SUMMARY
1. U n d e r normal physiologic conditions, the oxygen tension in the vitreous humor of the adult cat was 53 mm. H g . 2. Hypoxic conditions caused an exponen tial decrease in the oxygen tension in the vitreous humor. 3. T h e maximum oxygen tension in the vitreous humor was approximately 175 mm. H g , this value being reached when the at mospheric oxygen tension was 610 mm. H g . 4. T h e oxygen tension in the vitreous humor of a cat, when it was removed from hyperoxic conditions and placed under normal physiologic conditions, was found to decrease exponentially. Medical Teaching Group Hospital (15).
REFERENCES
1. Friedenwald, J. C, and Pierce, H. F.: Circulation of the aqueous: VI. Intraocular gas exchange. Arch. Ophth., 17 :477, 1937. 2. Patz, A.: Experimental studies. Tr. Am. Acad. Ophth., 59:2S, 1955. 3. Davies, P. W., and Brink, F.: Microelectrodes for measuring oxygen tension in animal tissues. Rev. Scient. Instruments, 13:524, 1942. 4. Montgomery, H., and Horwitz, O.: Oxygen tension of tissues by the polarograph method: I. Intro duction : Oxygen tension and blood flow of the skin of human extremities. J. Clin. Investigation, 29: 1120, 1950.
OPHTHALMIC
MINIATURE
Sclerosis of the Cribiform Ligament is in itself the predisposing factor in Glaucoma, the effect of which is to obstruct the free passage of Aque ous to Schlemm's Canal. Thomas Henderson, Glaucoma, London, E. Arnold, 1910, p. 136.
AUSTIN I. FINK AND IRVING BARAS
2. Ashton, N., and Cook, C.: Effect of cortisone on healing of corneal wounds. Brit. J. Ophth., 35 :708 (Nov.) 1951. 3. Newell, F. W., and Dixon, J. M.: Effect of subconjunctival cortisone upon the immediate union of experimental corneal grafts. Am. J. Ophth., 34:977 (July) 1951. 4. Ragan, G., Howes, E. L., Plotz, C. M., et al.: Effect of ACTH and cortisone on connective tissue. Bull. New York Acad. Med., 26 :25l (Apr.) 1950. 5. Plotz, C. M., Blunt, J. W., Ragan, C.: Effect of pituitary adrenocorticotropic hormone (ACTH) on disseminated lupus erythematosus. Arch. Dermat. & Syph., 61:913 (June) 1950. 6. Bourne, G. H.: Effect of cortisone and vitamin C on wound healing. Internat. Rev. Vitamin Re search, 24:318, 1952. 7. Palmerton, E. S.: The effect of local cortisone on wound healing in rabbit corneas. Am. J. Ophth., 40:344-53 (Sept.) 1955. 8. Bangham, A. D.: Effect of cortisone on wound healing. Brit. J. Exper. Path., 32:77 (Apr.) 1951. 9. Maumenee, A. E.: The influence of donor recipient sensitization on corneal grafts. Am. J. Ophth., 34:142 (May) 1951. 10. Ragan, C, Howes, E. L., Plotz, C. M., Meyer, K., and Blunt, J. W.: Effect of cortisone on granu lation tissue in rabbits. Proc. Soc. Exper. Biol. & Med., 72:718, 1949. 11. Howes, E. L., Plotz, C. M., Blunt, J. W., Ragan, C.: Retardation of wound healing by cortisone, Surgery, 28:177 (Aug.) 1950. 12. Findlay, C. W., and Howes, E. L.: The combined effect of cortisone and partial protein depletion on wound healing. New Eng. J. Med., 246 :597 (Apr.) 1952. 13. Greene, R. W., Faloon, W. W., and Lozner, E. L.: Use of ACTH in preparing patients with idiopathic thrombocytopenic purpura for splenectomy. Am. J. Med. Sc, 226:203 (Aug.) 1953. 14. Grant, G.: Effect of cortisone on healing of tendons in rabbits. J. Bone & Joint Surg., 35-A:525 (Apr.) 1953. 15. Gonzalez, R. I.: Effect of cortisone of healing of tendons in rabbits. J. Bone & Joint Surg., 35-A: 525 (Apr.) 1953. 16. Ashton, N.: Cortisone in ocular conditions. Lancet, 2:919 (Nov.) 1951. 17. Cole, J. W., Orbison, J. L. Holden, W. D., et al.: A histological study of the effect of cortisone on wound healing per primum. Surg Gynec. & Obst., 93:321 (Sept.) 1951. 18. Kay, J. A., Odell, R. T., and Taylor, L. W.: Failure of cortisone to delay or prevent the healing of fractures in rats. J. Bone & Joint Surg., 34-A :665 (July) 1952. 19. Daily, L., Jr., and Daily, R. K.: Present status of corneal transplantation. Texas State M. J., 49: 706 (Sept.) 1953. 20. McDonald, P. R., Leopold, I. H., Vogel, A. W., and Mulberger, R. D.: Hydrocortisone (Com pound F) in ophthalmology. Arch. Ophth, 49 :400 (Apr.) 1953.
T H E EFFECTS OF HYPOXIA AND
HYPEROXIA*
U P O N T H E OXYGEN T E N S I O N I N T H E VITREOUS H U M O R OF T H E CAT A R L I N G T O N C.
K R A U S E , M.D.,
AND S E Y M O U R B.
GOREN,
B.A.
Memphis, Tennessee INTRODUCTION
It is obvious that oxygen is necessary for the maintenance of retinal life. The mecha nisms by which oxygen is transported to and metabolized by the retina, however, are * From the Division of Ophthalmology, Depart ment of Surgery, of The University of Chicago. This investigation was supported by the Douglas Smith Foundation for Medical Research and the Chicago Community Trust of Chicago. Presented at the Midwest Section of the Association for Research in Ophthalmology, March 17, 1956, at Chicago.
not so well known. The diffusion of oxygen through the vitreous humor is one factor in
retinal metabolism. It has been found that, under normal conditions, the oxygen tension in the anterior chamber of the eye is 40 to 50 mm. Hg, while that in the posterior chamber is 100 to 120 mm. Hg. 1 The pur pose of the present study was to determine the relationships between the oxygen ten sion in the vitreous humor of the adult cat and the environmental conditions of hypoxia and hyperoxia.
OXYGEN TENSION IN VITREOUS T h e determination of oxygen tensions in the inner cavities of the eye is pertinent to the better understanding of the relationships between oxygen tensions in the environ mental atmosphere and disease processes of the eye. Perhaps the most dramatic ocular disease caused by excess oxygen administra tion is retrolental fibroplasia. Patz 2 concluded that the eye is susceptible to this damage only when the retina is in completely vascularized. This, of course, ex plains the high incidence of retrolental fibroplasia in premature infants who are ad ministered large quantities of oxygen for prolonged periods of time. Since the vitreous humor derives much of its oxygen by diffusion from the retinal vessels, the oxygen tension in the vitreous humor is of interest in the study of this dis ease process. Although retrolental fibro plasia is not incurred by the adult, it is of equal interest to know how much oxygen is present in the vitreous humor of the fully vascularized eye under conditions of hypoxia and hyperoxia. METHOD
The polarographic method, similar to that used by Davies and Brink 3 and Montgomery and Horwitz, 4 was utilized for measuring the oxygen tension in the vitreous humor of the cat under conditions of hypoxia and hyperoxia. Platinum electrodes were used because, in this type of circuit, platinum is capable of reducing the oxygen in the tissue being studied, thereby creating a current which can be measured with a galvanometer. T h e galvanometer serves as an ammeter whose deflections are directly proportional to the oxygen tension in the tissue or solution be ing studied. The electrodes were prepared of soda-lime glass tubing having a 3.0-mm. O.D. and a 1.5-mm. I.D., a 2.0-cm. strip of platinum wire having a diameter of 0.02 to 0.03 mm., and mercury. A recessed electrode was not used in this experiment because of the in
765
creased recovery time necessary when using such an electrode; an open type of electrode was not used because only relative values could be obtained with it. This is in agree ment with the results obtained by Davies and Brink. 3 Consequently, 1.0 mm. of platinum wire was allowed to project beyond the edge of the glass tubing. Such an electrode was found to be the most favorable for this experiment. A 5.5 to 6.5 cm. column of mercury was placed in the open end of the glass tubing. No. 18 insulated copper wire was inserted into the mercury, and the completed elec trode was stored in a solution of 0.9-percent sodium chloride. T h e electrodes were re moved from this solution only when in use. T h e saline was renewed every 48 hours. T h e electrical circuit is illustrated in F i g u r e 1. T h e main switch ( E ) was kept closed throughout all determinations and the zero adjustment of the string galvanometer was controlled by the 500-ohm variable re sistance ( H ) . T h e sensitivity of the galva nometer was 2.5 X 10~8 amperes. Through out all experiments the voltage was kept at
Fig. 1 (Krause and Goren). Diagram of electric circuit. (A) Calomel half-cell containing 0.9-per cent NaCl (anode). (B) Tissue electrode (cathode). (C) Variable resistance of 10,000 ohms. (D) Fixed resistance of 40,000 ohms. (E) Main switch. (F) 1.5-volt dry cell. (G) Galvanometer (Rubicon No. 3418). (H) Variable resistance of 500 ohms. (J) Fixed resistance of 2,000,000 ohms. (K) Aryton shunt (Rubicon No. 1243). (L) Electrode switch. (V) Voltmeter.
766
ARLINGTON C. KRAUSE AND SEYMOUR B. GOREN
0.7 V. Higher voltages did not give an ap preciable deflection of the galvanometer, while lower voltages decreased the accuracy and the sensitivity of the galvanometer be cause of the increased current flow. The electrodes were calibrated by measur ing the current created by the reduction of oxygen in standard saline solutions of known oxygen tension and temperature. They were calibrated with 0.9-percent solution of so dium chloride equilibrated with 20.9-percent oxygen and then with the same solution equilibrated with 100-percent oxygen at con stant temperatures. In order to equilibrate 0.9-percent solution of sodium chloride with 100-percent oxygen, pure oxygen was bub bled into the solution for 45 minutes before readings were taken and discontinued there after for only 20 seconds of each minute (that is, 10 seconds before the switch was closed and 10 seconds necessary for the de termination of a reading). Experimental data revealed that the saline solution was equilibrated with the desired oxygen tension after 45 minutes (not tabled). The bubbling was stopped 10 seconds prior to closing the switch so as to allow the solution to stop swirling. This precaution was taken because agitation of the solution produced increased and inconstant deflections of the galvanome ter as a result of convection currents in creasing the rate of oxygen supply to the electrode. In accordance with the work of Montgomery and Horwitz,4 it was found that errors caused by excessive reduction of oxygen by prolonged current flow and from the period swing of the galvanometer were avoided by reading the galvanometer 10 seconds after closing the electrode switch. For calibration, the electrodes were dipped into a 150-ml. beaker which contained 100 ml. of 0.9-percent sodium chloride solution. The calomel half-cell was placed in the same beaker as the electrode, thereby com pleting the circuit. Care was taken so that no air bubbles were trapped in the calomel halfcell. Determinations were made at 110-second intervals to allow sufficient time for the
electrode to recover from its previous use. All readings were recorded 10 seconds after the electrode switch was closed. Multiple determinations were made with 0.9-percent solution of sodium chloride at 14°C, 24°C, 34°C, and 37°C. equilibrated with 20.9-percent and 100-percent oxygen respectively as shown in Figure 2. Increased temperatures up to 60° C. resulted in in creased galvanometric deflections because of the greater diffusibility of oxygen at higher temperatures. From 60°C. to 85°C, how ever, the readings decreased as a result of the increased temperature agitating the mole cules of oxygen to a point where they were forced out of solution (not tabled). Cats weighing approximately four kg. were anesthetized with an intraperitoneal in oculation of Nembutal-Sodium.® They were then placed in an Isolette incubator and the desired oxygen tension was maintained for 45 minutes. After 45 minutes, the oxygen tension in the vitreous humor had reached a plateau. It was therefore assumed that the oxygen tension in the tissue being studied was at equilibrium with the oxygen tension in the environmental atmosphere. A Beckman oxygen analyzer, Model D, was used to measure the oxygen tension in the circulating air in the incubator. Blood on the tip of the electrode caused inaccurate
o
to
-to
60
80
PERCENT OWSEN IN ENVIRONMENTAL ATMOSPHERE
Fig. 2 (Krause and Goren). Temperature cor rection curves. (A) 37°C. (B) 34°C. (C) 24°C. (D) 14°C.
OXYGEN TENSION IN VITREOUS
and variable results because the small sur face to volume ratio of a droplet of blood prohibited it from giving off oxygen as easily as the tissue cells could. Therefore, bloodless surgery was performed. The cornea was removed and the aqueous humor allowed to drain. Forceps were used to retract the lens, and the electrode was in serted into the vitreous humor. Absorbent cotton soaked in 0.9-percent saline was used to make contact between the eye and the calomel half-cell, thereby completing the elec trical circuit. Temperature corrections were extrapolated from the experimental data shown in Figure 2. RESULTS
The oxygen tension in the vitreous humor of the cat under conditions of hypoxia and hyperoxia are tabulated in Table 1 and graphed in Figure 3. The ideal equation of the curve was found to have an exponential form. Under normal physiologic conditions, the average oxygen tension in the vitreous humor was 53 mm. Hg. When the animal was submitted to moderate hypoxia, such as 15-percent oxygen in the inspired air, the average oxygen tension in the vitreous hu
767
rt
o
UJ
g 180 > ? 135
£ o
I
30
45
-+-
0
200
■+■
WO
600
H 600
OXYGEN TENSION (MM. H&) IN ENVIRONMENTAL ATM05PHERE
Fig. 3 (Krause and Goren). The exponential curve expressing the relationship between the oxy gen tension in the vitreous humor and that in the environmental atmosphere.
mor was 28 mm. Hg. The maximum oxygen tension attained in the tissue being studied was 172 to 177 mm. Hg. Oxygen tensions in the environmental atmosphere above 610 mm. Hg failed to cause any significant in crease in the oxygen tension in the vitreous humor. Another series of experiments were per formed in which cats were first placed in an
TABLE 1 OXYGEN TENSION IN VITREOUS OF CAT Oxygen Tension (mm. Hg) in Environmental Atmosphere
114
154
304
455
609
680
745
Oxygen Tension (mm. Hg) in Vitreous Humor*
30 26 29 29 31 27 28 27
53 53 53 52 47 53 51 53 53 72 47 57 54 53 53 53 53 61 53 62
115 122 118 99 121 115 118 112 115 118 136 115 115 101 115 110 118 118
100 133 121 144 129 144 121 133 136 141 133 130
162 160 190 162 175 182 174 181 160 170
175 177 176 176 173 174 178 176 177 174
178 180 175 179 172 180 178 177 178 176
* Temperature corrections have been made for the tabulated data.
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ARLINGTON C. KRAUSE AND SEYMOUR B. GOREN TABLE 2 RELATIONSHIPS BETWEEN OXYGEN TENSION AND TIME IN OXYGEN
0
2
4
6
8
10
12
14
100 133 121 144 129 144 121 133 136 141 133 130
88 90 86 87 89 88 85 90 91 87
70 72 63 74 70 71 69 70 71 73
62 58 59 61 60 60 63 61 60 62
55 53 52 51 58 57 56 55 57 55
51 55 53 53 50 54 53 53 54 52
53 53 53 58 49 54 53 53 54 53
52 54 54 50 53 52 53 53 52 53
Minutes out of Incubator Oxygen Tension (mm. Hg) in Vitreous Humor*
Temperature corrections have been made for the tabulated data. incubator and 60-percent oxygen adminis tered to them for 45 minutes. T h e animals were then removed from the incubator, placed in 20.9-percent oxygen, and the oxy gen tension in the vitreous humor deter mined at various intervals. T h e results are tabulated in Table 2 and graphed in Figure 4. The ideal equation of this curve was also found to have an exponential form. The oxygen tension in the vitreous humor sharply decreased initially, and then levelled off at 53 mm. H g after the animal had been
0
4 MINUTES
8
/
2
OUT OF INCUBATOR
Fig. 4 (Krause and Goren). The exponential curve expressing the relationship between the oxy gen tension in the vitreous humor and the length of time which the animal had been transferred from 60-percent oxygen to 20.9-percent oxygen.
removed from the incubator for eight to 10 minutes. DISCUSSION
W h e n the animal was under conditions of hypoxia, the oxygen tension in the vitreous humor was subnormal. This is to be ex pected since the blood hemoglobin holds less than the normal amount of oxygen when the oxygen tension in the environmental atmos phere is low. Therefore, less oxygen is available to the body tissues, thereby causing a decreased oxygen tension in the vitreous humor. W h e n the animal was placed under con ditions of increasing degrees of hyperoxia, a corresponding increase in the oxygen ten sion in the vitreous humor resulted until a maximum of approximately 175 mm. H g was reached. This value was attained when the oxygen tension in the inspired air was 609 mm. H g . It is of interest to note that the blood hemoglobin is fully saturated with oxygen at values far below 609 mm. Hg. The increased oxygen which goes into simple solution in the blood plasma under severe hyperoxia is not sufficient to explain the difference in oxygen tensions in the vitre ous humor found under moderate and se vere hyperoxia. F o r some unknown reasons, a greater quantity of oxygen diffuses into the vitreous humor when the animal is under severe hyperoxia than is the case un-
OXYGEN TENSION IN VITREOUS der moderate hyperoxia, although basically the same amount of oxygen is being carried through the blood vessels of the eye in both conditions. F u r t h e r research is necessary be fore this fact may be adequately explained. W h e n an animal was removed from an environment in which the oxygen tension was 455 mm. H g , and placed in air having one of 154 mm H g , the oxygen tension in the vitreous humor was found to level off at the normal value of 53 mm. H g after eight to 10 minutes (fig. 4 ) . These results are in agreement with the sharp initial decrease and the subsequent leveling off of the blood hemoglobin saturation with oxygen seen when an animal is transferred from an at mosphere having a high oxygen tension to one having a normal oxygen tension. It is well known that no blood vessels penetrate the vitreous humor. W e may there fore assume that its nutritional requirements are satisfied by the diffusion of substances from surrounding blood vessels. Anatomic evidence indicates that the anterior portion of the vitreous humor derives its oxygen primarily from the blood vessels of the cili
769
ary body, while the posterior portion re ceives its oxygen principally from the retinal vessels. F u r t h e r investigation in both kittens and adult cats is necessary to ascertain the relative importance of the vessels of the inner layers of the eye in the maintenance of normal oxygen tensions in the vitreous humor. SUMMARY
1. U n d e r normal physiologic conditions, the oxygen tension in the vitreous humor of the adult cat was 53 mm. H g . 2. Hypoxic conditions caused an exponen tial decrease in the oxygen tension in the vitreous humor. 3. T h e maximum oxygen tension in the vitreous humor was approximately 175 mm. H g , this value being reached when the at mospheric oxygen tension was 610 mm. H g . 4. T h e oxygen tension in the vitreous humor of a cat, when it was removed from hyperoxic conditions and placed under normal physiologic conditions, was found to decrease exponentially. Medical Teaching Group Hospital (15).
REFERENCES
1. Friedenwald, J. C, and Pierce, H. F.: Circulation of the aqueous: VI. Intraocular gas exchange. Arch. Ophth., 17 :477, 1937. 2. Patz, A.: Experimental studies. Tr. Am. Acad. Ophth., 59:2S, 1955. 3. Davies, P. W., and Brink, F.: Microelectrodes for measuring oxygen tension in animal tissues. Rev. Scient. Instruments, 13:524, 1942. 4. Montgomery, H., and Horwitz, O.: Oxygen tension of tissues by the polarograph method: I. Intro duction : Oxygen tension and blood flow of the skin of human extremities. J. Clin. Investigation, 29: 1120, 1950.
OPHTHALMIC
MINIATURE
Sclerosis of the Cribiform Ligament is in itself the predisposing factor in Glaucoma, the effect of which is to obstruct the free passage of Aque ous to Schlemm's Canal. Thomas Henderson, Glaucoma, London, E. Arnold, 1910, p. 136.