CHARACTERIZATION OF THE NORMAL OCULAR OXYGEN TENSION IN THE EYE OF RAINBOW TROUT (SALMO GAIRDNERI) USING A MICRO OXYGEN POLAROGRAPHIC ELECTRODE Thesis for the Degree of M. S. MICHIGAN STATE UNIVERSITY MICHAEL B. FAIRBANKS 1968- A A ' "'3‘ 9"" ..' 4i’1\~'co'-¢.. War-4' THESIS If: I. u ' . ‘. ‘I- ‘ "ny :. ~. ~. RQILLLJJ ‘|~ “Igugcv ’9‘ University . J u ’9' ~ _, BINDING at " HUAB 8. SUNS' BUSY WWW INI‘ ABSTRACT CHARACTERIZATION OF THE NORMAL OCULAR OXYGEN TENSION IN THE EYE OF RAINBOW TROUT (Salmo gairdneri) USING A MICRO OXYGEN POLAROGRAPHIC ELECTRODE by Michael B. Fairbanks The apparent oxygen concentrating mechanism in the fish eye has been compared with the mechanism for oxygen concentra- tion in the swim bladder of most Teleosts. A brief review of this latter mechanism is given, along with a review of the use of the oxygen polarographic electrode for 1_n_ vivo measurements of the partial pressure of oxygen (ppOz). A micro oxygen polarographic electrode was constructed for use in in vivo and in vitro ocular, in vitro arterial blood, and environmental water ppO2 measurements, using the rainbow trout (Salmo gairdneri) as the experimental animal. The ppO2 values found behind the retina (445 :t 68. 5mm Hg) and in the vitreous body (103 j: 6. 7mm Hg) compared with arterial blood (21 i 2.2 mm Hg) and environmental water ppO2 (133 :t 3. 7) are evidence for an oxygen concentrating mechanism in the eye of the rainbow trout. Michael B. Fairbanks After administration of Diamox (0. 5mg/kg body wt) the ocular ppO values behind the retina and in the vitreous body were 2 reduced to values not significantly above arterial blood ppOz. After unilateral pseudobranchectomy (removal of the source of pseudobranch carbonic anhydrase) the ppO2 behind the retina was significantly lower than in the controls, but still significantly above the ppO of arterial blood. 2 Carbonic anhydrase, either extracellular from the pseudo- branch or intracellular from the RBC, retina and choroid, through catalyzation of the reaction between CO2 (produced by the retina) and H20 (from the blood plasma) to form H+ and HCO-, has been implicated as the agent responsible for the single concentrating effect. The single concentrating effect is an increase in the ppO2 of the blood in the choriocapillaris network of the choroid, as a result of a decrease in the pH of this blood. This increase in ppO2 is then multiplied, by counter current diffusion multiplication, in the choroid gland and lentiform body retes of the Teleost eye. CHARACTERIZATION OF THE NORMAL OCULAR OXYGEN TENSION IN THE EYE OF RAINBOW TROUT (Salmo gairdneri) USING A MICRO OXYGEN POLAROGRAPHIC ELECTRODE By Michael B‘: Fairbanks A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Physiology 1968 é .f/ L/ 5.5. ACKNOWLEDGMENTS The author wishes to express his thanks and appreciation to Dr. J. R. Hoffert for his guidance and support throughout this study and for his aid in the preparation of this dissertation. In addition, special thanks to my wife, Geri, for her patience over the last several months, and for the typing of the rough drafts. The author is also indebted to the National Institutes of Health for the support of this work through grant No. 04125 from the National Institute of Neurological Diseases and Blindness. ii TABLE OF CONTENTS Page INTRODUCTION . . . . . . . . . . . . . . . . . . . . . 1 ADDENDUM 5 LITERATURE REVIEW . . . . . . . . . . . . . . . . . . 7 Anatomy of the Choroidal Vessels . . . . . . . . . . . 7 Secretion of Oxygen in the Swim Bladder . . . . . . . . 12 The Polarographic Oxygen Electrode . . . . . . . . . . 19 The Clark Electrode . . . . . . . . . . . . . . . . . 23 MATERIALS AND METHODS . . . . . . . . . . . . . . . 26 Polarographic Electrode Circuitry . . . . . . . . . . . 26 Constant Temperature Chamber . . . . . . . . . . . 28 Construction of Polarographic Electrodes . . . . . . . . 29 Cathode Assembly . . . . . . . . . 29 Silver- Silver Chloride Anode Assembly . . . . . . 36 Ageing of Polarographic Electrode . . . . . . . . . . 38 Calibration of the Polarographic Electrodes . . . . . . 39 Animals . . . . . . . . . . . . . . . . . . . . . 41 Experimental Design . . . . . . . . . . . . . . . . 41 In Vi__v_o pp02 Determinations . . . . . . . . . . . 41 E Vitro ppO2 Determinations . . . . . . . . . . . 43 Blood pp02 . . . . . . . . . . . . . . . . . . . 44 Diamox Treatment . . . . . . . . . . . . . 44 Unilateral Pseudobranchectomy . . . . . . . . . . 45 RESULTS........................46 Characterization of the Oxygen Polarographic Electrode . . . . . . . . . . . . . . . . . . . . . . 46 Current-Voltage Polarograms . . . . . . . . . . . 46 CurrentvsSalinity................ 48 iii Current vs ppO2 Current vs pH . . Current vs Temperature Current vs %COZ . . Electrode Response Time . Electrode Stability . . . . . . Effect of Stirring on Electrode Signal . I_n Vivo Determinations of Ocular pp02 _In Vitro Determinations of Ocular ppOz _I_r_1 Vivo Ocular pp02 after Pseudobranchectomy Arterial Blood ppO2 Values DISCUSSION . .19. Vivo Ocular ppO2 SUMMARY AND CONCLUSIONS LITERATURE CITED . APPENDD( iv Page 49 51 51 53 53 55 55 57 59 64 65 69 71 80 82 86 Table LIST OF TABLES Changes in the electrode current at a constant ppOZ and 13C as a result of variations in the salinity of the solution Comparison of normal pp02 values found behind the retina, normal arterial ppOz, normal hematocrit, normal opercular rate, and ppOz of the environmental water with those values found in fish 24 hours after the injection of 0. 5mg/Kg of a 5g/100ml solution ofDiamox. Comparison of the ppOz values found behind the retina with those found in the vitreous body from the control and Diamox treated fish The effect of unilateral pseudobranchectomy on the ppOZ values found behind the retina during i_n vivo measurements Page 49 58 61 64 Figure LIST OF FIGURES Vascular patterns of the choroid gland and lentiform body of the teleostean fish 1a. Frontal View of the back of the eye (after Barnett, 1951) lb. Schematic diagram of blood flow through the back of the eye Current-voltage polarogram Construction of platinum cathode 3a. Platinum wire soldered to copper wire and electropolished to a tip diameter of 10- 20y, 3b. Platinum cathode seated in inner glass capillary and positioned in the platinum heating coil 3c. Platinum cathode being fused to the inner glass capillary 3d. Platinum cathode assembly showing tip coated with collodion Completed polarographic electrode . Current-voltage polarograms using cathodes of various types Current vs pp02 and OloCO2 Current vs temperature vi Page 10 21 31 32 32 33 33 37 47 50 52 Figure 10. 11. 12. Electrode response time Stability of electrode output for a given ppO2 Semilogarithmic graph of in vitro pp02 values recorded from the vitreous chamber . Illustration of in vitro experiment designed to show that the pp02 decay in the excised eye is not the result of oxygen consumption by the electrode Recording of the electrode output in mv from arterial blood samples vii Page 54 56 6O 63 67 INTRODUCTION At this time there are two different, but related, cases of the ”secretion" of 02 against an apparent concentration gradient. The first is the well-publicized instance of 02 "secretion" into the swim bladder of many species of fish. More recently, Wittenberg and Wittenberg (1962) have published data indicating that there is also an O concentrating mechanism in the eye of some marine 2 teleosts. In both instances of O2 "secretion” against an apparent concentration gradient there is the conspicuous presence of a rete mirabile, i. e. , the swim bladder rete and the choroid rete of the eye. The swim bladder rete is thought to act in conjunction with the glandular gas gland of the swim bladder to "secrete" oxygen. Wit- tenberg and Wittenberg (1962) have called attention to the analogy between the gas gland and the pigment cell layer of the retina. These structural resemblances, the swim bladder rete mirabile-gas gland complex and the choroid rete mirabile pigment cell layer complex were the influencing factors which prompted Wittenberg and Wittenberg (1962) to measure the 02 tension in the eye of the marine teleosts . In some Species they found 0 tensions in the eye much 2 greater than those in the surrounding environment. They were also able to find a positive correlation between the measured 02 tensions and the degree to which the choroid rete mirabile was developed. The lowest 0 tensions were found in those species which lacked a 2 choroid gland (the eel, Anguilla rostrata and conger, Conger oceanica). The development of the choroid gland is also related to the degree of development of the pseudobranch (Walls, 1942), the pseudobranch being absent in the eel and conger. The pseudobranch and choroid are anatomically related in that the choroid receives its afferent blood supply via the ophthalmic artery which is the major efferent vessel from the pseudobranch. Maetz (1953) working with Egg and Serranus found high carbonic anhydrase activity in the pseudobranch, retina, choroid and swim bladder; the highest activity being found in the pseudobranch. Hoffert (1966) confirmed these results in the lake trout (Salvelinus namaycush) except in the case of the swim bladder where low enzyme activity was found. The apparent reason for the discrepancy was that Maetz was working with fish that had well developed gas glands while Hoffert was working with a species which has no specialized secretory tissue (gas gland) in the swim bladder. Fange (1953) has shown that intramuscular injections of Diamox (acetazolamide), which inhibits the catalytic action of car- bonic anhydrase, is also capable of abolishing the secretion of gas into the swim bladder. Carbonic anhydrase catalyzes the reaction: CO (1) 4—— co +H20 ___,H2 3 2 The hydration or dehydration of CO is thus under enzymatic con- 2 trol. HZCO3 can also undergo the following reaction: H co :H+ + HCO 2 3 3 (2) This reaction is virtually instantaneous and not subject to enzymatic acceleration. These factors: 1. the anatomical similarity between the choroid gland and swim bladder rete; 2. the direct vascular connection between the pseudobranch and the choroid gland; 3. the high concentration of carbonic anhydrase in the pseudobranch, retina, ‘choroid gland and swim bladder; 4. and the ability of Diamox to inhibit gas "secretion" into the swim bladder; all suggest that the apparent oxygen concentrating ability of the choroid rete mirabile is dependent on the presence of carbonic anhydrase and the source of this carbonic anhydrase may be the pseudobranch. A polarographic oxygen electrode was constructed for use in ppO measurements in order that this hypothesis could be 2 explored further. After characterization of the polarographic electrode,experiments were designed to: 1. Determine the normal oxygen tensions existing in the vitreous body and behind the retina of the fish eye (rainbow trout, Salmo gairdneri). 2. Determine the effect which Diamox (acetazolamide) has on the maintenance of normal oxygen tension in the eye. 3. Determine the effect of unilateral pseudobranchectomy on the normal oxygen tension in the eye. 4. Compare the above results with ppO levels of arterial 2 blood and environmental water. ADDENDUM Beginning with the promising work in the control of sea lamprey in the Great Lakes in 1957, a movement has been under way to restock these waters with hatchery-raised lake trout. At all hatcheries it became apparent that hatchery-raised lake trout were prone to develop several types of eye abnormalities (Hoffert and Fromm, 1965). Coincident with the eye lesions is the development of pseudobranch pathology characterized by initial hyperplasia of the gland, subsequent dissociation of the cells and finally atrophy (Hoffert, unpublished). Vacuoles appear within the pseudobranch and have been described as resembling the pathology noted in the brains of pilots who have undergone explosive decompression, with the exception that in pilots there would be cerebral hemorrhage, whereas, there is no hemorrhage associated with the pseudobranch pathology. Occurring also in the trout eye diseases are changes in the retina and choroid, involving disorganization of the cells and hyalinization, without any signs of inflammation. The work on the characterization of O tensions in the fish 2 eye is in conjunction with other experiments which are aimed at describing the normal physiology of the fish eye in comparison with the mammalian eye, and the pathological physiology of the lake trout eye lesions. The relationship between pseudobranch pathology and lake trout eye lesions, and the anatomical and chemical relationship between the pseudobranch and the fish eye, suggests that the pseudo- branch is necessary for the normal physiological functioning of the eye. The experiments were designed to characterize the 02 tensions in the fish eye and also to investigate the role of the pseudobranch in metabolic gas exchange of the retina. LITERATURE REVIEW Anatomy of the Choroidal Vessels Albers in 1806 (Barnett, 1951) was the first to show that the horseshoe-shaped body (the choroid gland) found in the choroidal layer of the eye in most bony fish is neither a muscle nor a secretory gland, as was currently supposed, but a collection of small blood vessels, a rete. Barnett (1951) has given a detailed description of the vascular anatomy of the gland. Blood from the first efferent gill artery supplies the pseudobranch, the remnant of the first gill arch in teleosts. Within the pseudobranch this gill artery is divided into a system of capillaries which reunite to form the ophthalmic artery (Figure 1). This efferent vessel of the pseudobranch supplies the choroid gland of the eye. It enters the eye dorsal to the optic nerve . and passes into the lumen of the ophthalmic venous sinus which lies along the inner border of the choroid gland. It then divides into two branches, a branch going to each limb of the horseshoe-shaped choroid gland. Within the gland the branches give off parallel col- umns of capillaries which pass through the gland to the choriocapil- laris network of the choroid proper. Blood is returned from the AC CH CG EBA FB IC LB OA OM ON ONA OV OV S RV RVS SP VC VCV SC L FIGURE 1. -- Vascular patterns of the choroid gland and lentiform body of the teleostean fish 1a. Frontal View of the back of the eye (after Bar- nett, 1951) lb. Schematic diagram of blood flow through the back of the eye Arterial capillaries Choroid (choriocapillaris) Choroidal gland Efferent branchial artery (lst gill arch) Falciform body Internal carotid artery Lentiform body Ophthalmic artery Extra-ocular muscle artery Optic nerve Optic nerve artery Ophthalmic vein Ophthalmic venous sinus Retinal artery Retinal vein Retinal venous sinus Spiracular pseudobranch Venous capillaries Ventral choroidal vein Sclera .30 0.? U< .mH WMDUHEH m>O ./ 1 w k ‘ \ ,. 4QQ§III , I ( Md \\ \’MflV\‘H\ 2/1.--- .5. a . \A/ 54% §$vevx L 2.0 <0 10 a: HMDOHEH "I l I I I l | I I l ., ! __l 9:; >o> . Flillllullulllllllllll L , \ N. _ 5 1. 4/ f _ . _ _ mo 1.. ._ . _ 1 _ TI... II... _ _. - . _ . _ _ _ _I .L n , . u _ g 26 _ m>m _ _ CU Em. VIII. ...!.IL L. 20 /\ <20 I ,_ 0 A .35 _. . <0 11 choriocapillaris by way of the venous capillaries which pass through the choroid gland in juxtaposition with the arterial capillaries, the two blood streams being separated by only two layers of endothelial cells. The venous capillaries open into the ophthalmic venous sinus which drains into the large ophthalmic vein. Barnett (1951) noted that the eye of the fish is also supplied with blood from the retinal artery, a branch of the internal carotid. While the main branch of the retinal artery supplies the lentiform body, small branches from the artery go to the optic nerve and extraocular muscles. Within the lentiform body the artery passes through the lumen of a small venous sinus before dividing into a capillary system. After emerg- ing from the lentiform body the capillaries go on to form the central part of the choriocapillaris of the choroid. In the choriocapillaris they communicate freely with capillaries from the ophthalmic artery. Two striking features of the vessels supplying the chorio- capillaris plexus (Figure 1) are: (1) the regular juxtaposition of arterial and venous capillaries in the choroid gland and lentiform body and (2) in both the choroid gland and the lentiform body the artery passes through a venous sinus before dividing into a capillary network. The parallel arrangement between the arterial and venous capillaries is also a feature of the rete mirabile of the swim bladder. 12 Because of this anatomical similarity between the rete mirabile of the choroid and that of the swim bladder, the possibility exists that the mechanism of 0 concentration in the eye is similar to that in 2 the swim bladder. Secretion of Oxygen in the Swim Bladder Haldane (1922) was the first to suggest that the mechanism for concentration of O2 in the swim bladder is a counter current multiplication, in the rete, of a primary O2 gradient caused by the addition of acid on the venous side. The walls of the rete capillaries are single layered endothelial cells and the distance between the arterial and venous capillaries is on the average 1. 5 FL (Scholander, 1954) or approximately the same as the distance between air and blood in the human lung alveoli. The structure of the rete thus seems well-suited for gas exchange by diffusion. A rete, whose main property is diffusion of gases between arterial and venous cap- illaries, will constitute a barrier for gases leaving the swim bladder. However, the same type of barrier exists for the entry of gases into the swim bladder unless the solubility of the gas is decreased in some manner in the venous blood. If a solubility decrease in the venous blood leaving the swim bladder does occur, then gas will dif- fuse from the venous side to the arterial side in the rete. The 13 enrichment of the arterial blood will in turn result in a further in- crease of gas in the venous blood returning from the bladder to the rete and this will again result in an increase in diffusion of gas to the arterial blood. This process has been termed the single concentrat- ing effect (Kuhn $31. , 1963). This type of counter current diffusion results in high gas tensions at the bladder pole of the rete, allowing gas to enter the swim bladder by diffusion. A rapidly rising tension gradient on the arterial side of the rete will also guard against gas loss through circulation. Hemoglobin, from all fish known to have a swim bladder capable of concentrating 02, shows a decreased 02 affinity on addi- tion of acid (Bohr effect) and also (in some Species) a reduced oxy— gen capacity (Root effect). Scholander (1954) developed equations for expressing rete function in the concentration of 02' In theory he found that if acid were added to the venous blood stream in a quantity sufficient to decrease the amount of O bound to hemoglobin 2 by 10% and the physical solubility by 1%, a rete such as found in deep sea fish would be capable of creating pressures in excess of those actually recorded. Scholander and van Dam (1954) have found ppO2 values in these fish greater than 50 atmospheres. However, they also found that the blood of several deep sea fishes acidified with lactic acid to pH 6 was fully saturated at O2 tensions much 14 lower than those existing in the swim bladder. Scholander (1954) argued that the results suggested a mechanism other than dissocia- tion of 02 from oxyhemoglobin. He felt that the effects of an acid added to the venous rete blood could produce 02 by counter current multiplication only at pressures at which the lowered pH would dis- sociate oxyhemoglobin. The anatomical arrangement of the gas gland and the rete caused Scholander (1954) to suggest that there was "glandular secretion" of 02’ in addition to a mechanism that dis- sociates oxygen from the hemoglobin. Wittenberg (1961) and Wittenberg and Wittenberg (1961) have provided evidence that this latter theory of ”glandular secretion" may have some significance. Their arguments were based on the analysis of swim bladder gases of toadfish (Opsanus tau) maintained in sea water equilibrated with gas mixtures of 50% 02 and various concentrations of CO. They found that the proportions of carboxy- and oxyhemoglobin present in the blood were grossly different from the proportions of CO and 02 found in the swim bladder. Since the gas proportions found in the swim bladder did not reflect those in the blood, they argued that the gases were not generated by acidifi- cation of the blood. Ruling out blood hemoglobin as the primary source of the secreted gases, they substituted an active mechanism by which an intracellular carrier facilitated the transport of gases into the swim bladder. 15 Kuhn et al. (1963) criticized this interpretation and pro- posed an experimental model including detailed mathematical treat- ment to show that swim bladder 02 could indeed be derived directly from the plasma O and blood oxyhemoglobin. Their analysis is 2 based on hairpin counter current diffusion multiplication in which an initial single concentrating effect such as a change in solubility on the venous side of the rete will be cascaded into a concentrating mechanism sufficient to create the high 02 tensions found in the swim bladder of deep sea fish. Three mechanisms may be involved in the single concen- trating effect. They are: 1. Bohr effect (a shift in the oxygen dissociation curve) 2. Root effect (reduced hemoglobin binding capacity) 3. The salting out effect (decrease in the solubility coef- ficient upon the addition of solutes) Kuhn et_a_l. (1963) used the salting out effect to show that Scholander' s (1954) data on the inhibition of the Root effect at high partial pressures did not warrant the postulation of cellular secre- tion of 02. An example would be a fish in which a lowered pH will dissociate oxyhemoglobin at pressures of up to 50 atm in the first part of the rete from the heart pole, while the rest of the rete will continue concentrating 02 above this level due to the salting out effect. l6 Acidification of the venous blood in the rete has been shown to be due at least in part to the production of lactic acid by the gas gland, even in the presence of a high concentration of 02 (Ball, Strittmatter and Cooper, 1955). The influence of the lactic acid on the venous blood will not only be a Bohr or Root shift causing the dissociation of O2 from hemoglobin, but the acid will also have a general salting out effect. Steen (1963) and Berg and Steen (1968) have in turn raised some objections to Kuhn' 8 model of the O concentrating mechanism 2 in the swim bladder. The ability of the system to multiply a small initial ppO increment depends, according to Kuhn' 8 model, on the 2 permeability properties of the arterial—venous partition. The model requires that the partition allows rapid diffusion of 02 but not an equilibration of pH. This seems to be a rather difficult requirement to fulfill since CO2 is known to pass through biological membranes more rapidly than 0 Steen (1963) made direct measurements of 2. blood pH in actively secreting swim bladders of eels (Anguilla vulgaris) and found a high degree of equilibration of lactic acid across the rete. In the same study, he also found a lower venous than arte- rial pH at the bladder pole of the rete, as required by the model, but this situation was reversed at the heart pole, which is contradictory 17 to the model. Rather than discarding the model, Steen suggested that the decrease in ppO2 that would occur as a result of an increase in pH (as CO and H+ diffused to the arterial side of the rete) may 2 be slow compared with passage of blood through the rete. Experi- mental verification of this hypothesis came as a result of the mea- surements of the Root shift. Forster and Steen (quoted by Berg and Steen, 1968) introduced the terminology of the Root-Hoff shift (acidi- fication of blood and subsequent unloading of 02) and the Root-on shift (an increase in blood pH and subsequent loading of O2). They found that the Root-off shift has a t% (biological half time) of ,05sec at 23C while the Root-on shift has a t% of 10 - 20sec. The fact that the ppO2 of venous blood leaving the rete decreases with an apparent half-time of 10-205ec and that the t% of the Root-on shift is also 10- 208ec influenced Berg and Steen (1968) in their modification of the Kuhn model. Blood enters the rete with arterial pH and ppOz. Dur— ing the circulation of blood through the bladder epithelium, 02 dif- fuses into the bladder. At the same time, lactic acid is being added to the blood, which results in a further increase in blood ppOz, either through the Root-off shift, the Bohr effect, or the salting out effect. As the blood returns to the venous side of the rete it has a lower pH and 02 content but a higher ppO2 than when it entered the bladder epithelium on the arterial side. As this blood flows through 18 the rete, 0 will diffuse from the venous to arterial side of the rete, 2 as will CO2 and H+. The increase in venous pH will result in the concomitant Root-on shift, but this is a slow process when compared to the Root-off shift, so that the arterial venous ppO2 gradient will be maintained in favor of movement of O2 to the arterial blood. Ac- cording to this model, 0 is still being concentrated by counter cur— 2 rent multiplication of a ppOZ gradient caused by acid, as suggested by Kuhn, but this gradient is not based on impermeability of the rete to acid, but rather on the slow response of the 0 capacity of blood 2 to an increase in pH (Root-on shift). Féinge (1953) showed that Diamox (acetazolamode) when injected intramuscularly abolished gas deposition in the swim blad— der. Forster and Steen (quoted by Berg and Steen, 1968) have found a likely explanation for this observation in terms of the Root—off shift. They found that Diamox lengthens the t% of the Root-off shift from a normal of .05sec to at least 3OSec . According to the model of Berg and Steen (1968) acid secreted by the bladder epithelium would act too slowly to be manifested as an increased ppO2 while the blood still circulates through the bladder wall and the venous side of the rete. The similarities between the rete of the choroid and that of the swim bladder suggest that the mechanism for the high O:2 tensions 19 found in the eye of the fish are similar to those involved in 02 con- centration in the swim bladder. The Polarographic Oxygen Electrode Polarography is essentially a method of chemical analysis which was discovered and developed by Heyrovsky and his colleagues in the 1920' s. The method depends on the observation that when an electrolyte solution is polarized between a dropping mercury and a nonpolarizable electrode, the shape of the current-voltage curve obtained depends on the presence, composition and concentration of electro-oxidizable or electro-reducible substances (Silver, 1967). Each substance, whether it is a simple metalic ion or a complex protein, gives a unique current-voltage curve. Oxygen is one such substance which is reduced on the electrode surface according to the following equations (Longmuir, 1963). O2 + e—->O2 (3) 02 + e--9O2 (4) = + 02 + 2H —->H202 (5) Most of the hydrogen peroxide formed in equation (5) then undergoes the following reactions: 20 02 + H202—9- OH + 02 + OH (6) OH + e —=-0H' (7) The terms polarograph and polarogram were first used by Heyrovsky. The polarograph is the instrument used for the mea- surement of current-voltage curves while the polarogram refers to the record obtained from the instrument (Kolthoff and Lingane, 1952). Characteristically the polarogram consists of a sigmoid curve as depicted in Figure 2. Each electro-oxidizable (or electro-reducible) substance has a characteristic decomposition potential and also a specific half-wave potential. Neither of these two characteristics have been exploited in the construction of polarographic oxygen electrodes but instead the limiting current plateau has been of major interest, since it is in this region of the polarogram that electrode current is directly proportional to the concentration of electro-reducible O The ap- 2. pearance of the limiting current plateau for O can be explained as 2 follows: when an emf greater than the decomposition potential for 02 is applied to the electrode, 02 is reduced and removed from the surface of the electrode. This in turn establishes a concentration gradient for 02 between the solution and the electrode surface. Further increase in the emf will result in a faster rate of reduction Current —9 PI 21 FIGURE 2. -- Current-voltage polarogram -- Decomposition Potential (Substance A) -- Half Wave Potential (Substance A) Decomposition Potential (Substance B) -- Residue Current -- Limiting Current Plateau MUOIIIKD l I P----——- 1 I I I | l | I II I II I gl L AB C Polarization Voltage a 22 at the electrode and the resulting lower concentration of 02 at the electrode surface causes an increased rate of diffusion from the surrounding solution to the electrode. When the applied emf is high enough (between 0. 6 and 1. 0V in the case of 02) the average concen- tration of O on the electrode surface will be zero. Under these 2 conditions the diffusion of O2 from solution to electrode surface will be maximum, and dependent only on the concentration of 02 in the solution. Further increase in the applied emf will not result in an increase in current output, since the increase will not result in any increase in the concentration gradient between solution and electrode surface. The reduction current in the plateau region is directly pro- portional to the amount of O reaching the electrode surface, which 2 is a function of the driving force for the diffusion of 02, i. e. , the partial pressure of oxygen (ppOz). According to Silver (1967) cer- tain conditions must be maintained to insure that the electrode is actually measuring ppO the most important of which are that the 2’ electrode area should be small and that the partial pressure of 02 should not be too great (less than 1000 mm Hg for electrodes with cathode diameters of 20M. or less). The dropping mercury electrode has been used extensively in the analysis of biological fluids, but its use as anin vivo 02 mea- suring device is limited because of the toxicity of mercury and its 23 inaccuracy when only small amounts of O are present. Today, 2 polarographic O electrodes are made from solid metals (platinum, 2 gold, etc.) in conjunction with nonpolarizable reference electrodes. These solid electrodes are of three basic types. (1) The open or bare metal electrodes (Davis and Brink, 1942; Montgomery and Howitz, 1950) which consist of a noble metal wire insulated except near the tip. (2) The recessed electrodes of Davis and Brink (1942) in which platinum is fused in a glass capillary which projects a short distance beyond the platinum tip. (3) The Clark electrode (Clark _eia_l. , 1953) which incorporates both the cathode and a non- polarizable anode behind an insulating membrane which is freely permeable to 02' The Clark electrode is the electrode of choice for £1 vivo measurements although modifications of the other two types have been used in recent years (Longmuir, 1963; Silver, 1966). The Clark Electrode In the Clark electrode both the cathode and anode are situ- ated in a standard electrolyte behind an electrically insulating but 02 permeable membrane. The original Clark electrode had a large active surface area. It gave marked differences in current output depending on whether or not it was exposed to unstirred or stirred 24 gases and fluids of the same ppOz. Silver (1965) and Fatt (1964) showed that these undesirable effects could be eliminated by reduc- ing the cathode size to less than 20/.L. Fatt (1964) also found that if the thickness of the insulating membrane was six times the diameter of the exposed cathode tip, then there would be no difference in cur- rent output in stirred or unstirred fluids. The theoretical reason for this observation as described by Fatt is as follows: If the membrane thickness is about six times the electrode diameter and if the product of the oxygen diffusion coefficient multiplied by oxygen solubility is the same in the membrane as in the solution outside, then the electrode sees only the oxygen in the membrane. This oxygen content is maintained by the solution that sweeps over the membrane and is in equilibrium with the solution at all solution velocities. For membranes of this kind which are thinner than six electrode diameters the electrode sees into the solution and the oxygen concentration seen by the electrode is influenced by flow. The problem with using a thick membrane is that the time necessary to reach equilibrium after a change in oxygen tension increases in proportion to the membrane thickness. Recently de- veloped electrodes have smaller cathodes and a membrane thickness that combines minimum sensitivity to fluid movement with minimum response time to changes in ppOZ. The response time of 1p, elec- trodes covered with a membrane was found to be 99% of the reading in 12sec (Fatt, 1964) or 99% of the reading in 0. 5sec (silver, 1965). According to Charlton (1961) electrodes with small cathode diameters are also relatively insensitive to hydrostatic pressure. All 02 25 electrodes regardless of size are temperature sensitive, the sensi- tivity being anywhere from 2-4% increase in current per degree centigrade increase in temperature. Other characteristics of O2 polarographic electrodes are an insensitivity to changes in the pH of the medium between pH 1 and 13 (Naylor and Evans, 1960) and an insensitivity to changes in the per cent carbon dioxide of the medium (Clark and Sachs, 1968). Charlton (1961) has shown that an initial period of aging (4-6 hours) with a polarizing voltage of 0. 7V is needed before the current output of the electrode becomes stable (1 0. 5%). Subsequent use of the electrode requires only a 30 minute warming up period. Stability of the electrode has been shown to coincide with the deposi- tion of an alkaline layer on the surface of the cathode; any disturbance of this layer leads to erratic results. Cater (1966) calibrated electrodes to be used for i_n vivo measurements in an artificial extracellular fluid equilibrated with a gas mixture of 5% C02 plus 95% room air. He found that he could obtain identical calibration currents when using physiological saline equilibrated with room air, justifying the use of this much simpler method for calibration of the electrodes. MATERIALS AND METHODS Polarographic Electrode Circuitry Oxygen tension measurements were made using a polaro- graphic electrode constructed as described below. A polarizing voltage of 0. 7V was applied between the platinum cathode and the silver-silver chloride anode (see Appendix 1). The polarizing voltage source consisted of two 1. 5V 4FM Burgess dry cells con- nected in parallel with the voltage monitored using a Model 410B Hewlett Packard vacuum tube voltmeter (Hewlett Packard, Palo Alto, California). The electrode signal was amplified by a Grass 5P1 Low Level DC Preamplifier (Grass Instruments, Quincy, Mass.) with a Grass Model 5E Driver Amplifier used as the power supply. The signal was displayed as voltage fluctuations on a Beckman Ten Inch Potentiometric Recorder, Model 1005 (Beckman Instruments, Inc. , Fullerton, California). Linear current measurements can be made using the 5P1 Preamplifier if the input impedance of the preamplifier is a small fraction (less than 0. 1) of the source impedance of the polarographic electrode. Using electrodes with tip diameters 26 27 between 10 and 20,1” the requirement of a high source impedance with respect to the input impedance was met. The rationale for using the polarographic electrodes for O2 tension measurements is that the current from the electrode is di- rectly proportional to the partial pressure of oxygen (ppOz). The electrode current was calculated from Ohms law: (8) II FUI Q :< a N0% assess a .s v a o AmOdd @003 333.3 H.993: m> «33.3 05 wagon. NOdd 38.373 :5 .o v do 23w? “macoafiohgao do Odd m> math.“ on» @5509 Axofiflm m> 33:08 3 .o v dn A8835 E 8588 :5 .o v as Amnofimtmmno mo pong—b .m— .m H 532.. saves .e a m .fi. 85 c. A we 3:. .m A 2: 83 .m A «N as: .m s 2.. 08:35 858 .o a «.3 8H: .m A 2: e85 .m a $3 sacs.” A R $3,.“ .3 a new 8,380 poem? 0 a 38 .83 Seaseasosnrsm 8 S 323$ «53.8 as”. 33% . A E fihooamgom «coapmonH 3mg .33“;me . Amm 88V mode onflQ mo nofigom HSooHEm .m mo mvimfim .0 do :ofiomnfi 05 non—mm mason em and.“ 5 p238 moans. omofi 8:3 .8ng dflcoficongco on» do «Odd 98 Jump Agnonodo 38.8: #300380: Hugo: .NOdd Riots 38.8: .953.“ on» Eugen Ugo.“ mead; mOdd Ransom mo comEdeoO II .N mqmaefi. 59 ppO2 values found behind the retina in the control fish were 95. 3% higher than the oxygen tensions of the arterial blood samples and 71. 3% higher than the ppO of the environmental water. These 2 values were significant at p < 0. 001 and p < 0. 01 respectively. This table also compares the results from Diamox-treated fish with the controls. Diamox treatment resulted in a 94. 4% decrease in the ppO2 found behind the retina, from a mean of 445mm Hg in the con- trols to a mean of 25mm Hg for the Diamox-treated fish. There was also a significant increase in the hematocrit and decrease in the opercular rate of the Diamox—treated fish at p < 0. 01 and p < 0. 001 respectively. In Vitro Determinations of Ocular ppOg Measurements were made on excised eyes with the electrode tip in the vitreous body. Characteristically the recorded values decayed in an exponential manner (Figure 10). Extrapolation of the semi-logarithmic plot of the i_n vitro ppO values to zero time gave 2 a value (103mm Hg) which correSponded to the ppO2 in the vitreous body at the time the Spinal cord was cut. The results are presented in Table 3 as normal ppO values in the vitreous body, but because 2 of the depth of penetration they are probably more representative of the vitreous ppO2 near the retina than that near the lens. 60 FIGURE 10. -- Semilogarithmic graph of E vitro ppOZ values recorded from the vitreous chamber. The dotted line represents extrapolation of graph to zero time, which corresponds to the ppOZ in the vitreous at the time the spinal cord was cut. Measurements were made at 13C. 100 \ 50 I- 20 '- 60 m E g 10 P- * N 0 CL Q. 5 n- 2 I- 1 I I I I 0 10 20 30 40 Time (Min. ) =I< = mean 3: SE. (11 observations per point) 61 08:85 as 8588 :3 .o v as .m.m “:82 Am:ofiw>:0mno do none—Ev Z used :04 Hp m wag.“ Ham w Nofiwfim mm 4. N. .m H m3 2 m .ww H 33. m: Hoax—:00 anon. 9:00.53 05 5 BE 8:: .300. 0:00.53 Z «:30: 05 Z 30:50:39 mOddHEE: 05 338 Am: 88C Nos: 2: 5 Am: 880 N0%.. senses Am: 880 ~03 and.“ 03.8.5 onSO 0:: 33:00 05. 50:.“ moon m:0 I033 05 5 6:30.: among. 5:» MEN—0: 05 0:30p 0:33 :95? NOdd 05 .0 :03:de00 I .m midmafl. 62 That decay in the vitreous ppO2 was not due to O2 consump- tion by the electrode was determined by i_n v_it£ studies, in which the electrode was placed in the contralateral eye after sufficient time had elapsed so that the mv recording in the first eye had reached a value near zero. In these instances the mv recording from the con- tralateral eye came to within :t 1. 0% of the extrapolated value for the first eye (Figure 11). Table 3 is also a comparison of the Control and Diamox in vivo ppO values (ppO found behind the retina) and _i_n_ vitro ppO2 2 2 values (ppO2 found in the vitreous body). As in the case of the _i_n_ vivo values, Diamox treatment resulted in a significant decrease (p < 0.‘ 001) in the ppO in the vitreous body, from 103mm Hg in the 2 Controls to 7mm Hg in the Diamox-treated fish. The vitreous body pp02 values from the Diamox-treated fish were obtained during in vivo measurements. After recording the ppO behind the retina, the electrode was raised so that the tip 2 was in the vitreous body, and values for vitreous ppO2 recorded. The comparison of values obtained during i_n_ vivo studies with those from _1r_1 vitro studies (Table 3) was justified by the fact that the ratio of ppO behind the retina (i_n vivo measurements) to the ppO in the 2 2 vitreous body (in vitro measurements), for the controls, is similar to the ratio found in the Diamox-treated fish. In the case of the 63 FIGURE 11. -- Illustration of in vitro experiment designed to show Electrode Output (mv) 0.3 0.2 that the ppOZ decay in the excised eye is not the result of oxygen consumption by the electrode. After recording for 44 min. from the left eye, the electrode was placed in the right eye. Dotted line represents extrapolation of the plot for the left eye. Left Eye )- Right Eye ] 1 I J I J 0 10 20 30 40 50 60 Time (Min. ) 64 Diamox-treated fish both of the measurements were made during in vivo experiments . In Vivo Ocular ppOz after Pseudobranchectomy Table 4 is a comparison of ppO values found in the eyes 2 of fish which were subjected to unilateral pseudobranchectomy. TABLE 4. -— The effect of unilateral pseudobranchectomy on the ppO2 values found behind the retina during _12 vivo measurements ppOg (mm Hg) . Treatment N behind the Arterial Opercular. rate retina PPOZ (mm Hg) (per mm) Control 5 239i 24.7 20: 1.2 98¢ 3,8 Pseudobranch- 57 i 9. 63 20 d: 1. 2b 98 i 3.8 ectomy 5 N (number of observations) Mean i S. E. ap < 0. 031 (Control vs Pseudobranchectomy. Walsh test) bp < 0. 01 (When comparing the ppOz at the retina with arterial blood pp02 after pseudobranchectomy. Mann Whitney U test) _I_1_1_ vivo measurements were first made on the side of the pseudo- branchectomy, then the fish was turned over and i_n vivo 65 measurements made in the other eye, these latter values represent— ing the controls. There was a 76. 3% decrease in the ppO2 behind the retina (from 239mm Hg in the controls to 57mm Hg after pseudo- branchectomy) as a result of the pseudobranchectomy. However, the ppO values behind the retina on the side of the pseudobranch- 2 ectomy were still significantly higher (p < 0. 01) than the ppO values 2 found in the arterial blood. The ppO2 behind the retina in the control eyes was lower than in the previously reported controls (239mm Hg vs 445mm Hg). A possible reason for this observation may be the added stress to these animals from the operation, plus the stress from measuring of O2 tensions in both eyes. Arterial Blood pp02 Values Tables 2 and 4 report the ppO2 values that were found in the arterial blood samples of both the control and experimental fish. The mean for these values was close to a ppO of 20mm Hg, with no 2 significant difference existing between the controls and treated fish. This blood was taken from the dorsal aorta and is representative of blood which had just moved through the gills. The samples were dark red, similar in color to mammalian venous blood and indicative of a low oxygen content since the blood became bright red if room air was bubbled through it. 66 The ppO of the arterial blood was determined by extrapo- 2 lating the linear plot of electrode output in mv back to zero time (Figure 12). Zero time in this case corresponded to the time at which the blood samples were drawn. The mv value at zero time was converted to current, and the ppO2 read from a calibration graph of current vs ppOZ. Because of the slow rate at which the blood ppO2 decayed, this was a more accurate method than conver- sion of each mv recording to ppO and extrapolation of the resulting 2 plot. Consumption of O by the electrode probably did not con- 2 tribute significantly to the decay rate. Experiments were designed to determine quantitatively the amount of O consumed by the elec- 2 trode. A 1. Oml sample of air saturated water at 13C was placed in a small bottle, beneath a 6cm column of mineral oil. The mv out- put from the electrode dropped at the rate of O. 0011mv/hour (cor- responding to a decrease in ppO of 0. 15mm Hg/hour) over a 6 hour 2 interval. However, subsequent work indicated that O diffuses quite 2 rapidly through mineral oil. In this latter experiment 1ml of N2- saturated H20 was covered with a 6cm column of mineral oil which had been degassed under vacuum for 15 minutes. The mv recording in this case rose from 0. 100 to 0. 312mv in 4 hours, or at the rate of 0. 053mv/hour, which corresponds to a ppO2 increase of 27. 5mm Hg/hour. This evidence for the rapid diffusion of 02 through mineral 67 :55 68:. m N @UOHHO 0am 1;, mm 8862: 11’ - l 1 j, m: 3882: mm 000.5003: I.( N .5220 was» 035.3 0:3. 0:» «a 0:: 0003 02:32.” m0 :03m58:300 0:» mkofiw 0::3 Ohms op :03 005 N06 «56 mod mod (ALU) indino apono 913 -325:me .moaawm 0003 30:3,; 80:.“ >:: 5 35:0 000.3030 05 m0 m50uooom In .NH HmDUHm 68 oil indicates that the ppO2 in the distilled water sample of the first experiment was probably in equilibrium with the ppO2 of the mineral oil. Therefore, no quantitative determination of the O2 consumption by the electrode could be made from this data. Servinghaus (1968) has found that the 0 consumption of electrodes with cathode diam- 2 eters between 12 and 2011, is insignificant when compared with the O2 consumption of red and white blood cells. The oxygen consump- tion by the nucleated red blood cells of fish blood would probably be even greater than that of non-nucleated red blood cells of mammals. DISCU SSION Characterization of the oxygen polarographic electrode indi- cated that it would not be necessary to calibrate the electrode in a simulated extracellular or ocular fluid, nor for that matter, even in a saline solution. Temperature and the effect of stirring were found to cause the greatest variation in electrode output for a given ppOz, but these two variables were relatively constant during the experi- mental measurements. Temperature was controlled within i 0. SC in the constant temperature chamber, while all ppO measurements 2 were made 'in static solutions (distilled H 0, blood and vitreous 2 body) or at the retina so that there was no stirring effect on the electrode current. The reSponse time of the electrode would make it unsuitable for measurements in which the response of the animal to a rapid change in environmental ppO2 was being monitored. However, for the i_n v_iv_o steady state experiments and the _12 _vi_tr_‘c_) experiments the electrode response was not a critical factor to be dealt with in explaining the results. The long response time was a result of the relatively thick collodion and outer silicone membrane and perhaps 69 70 the O2 diffusion coefficient of the membranes. Even though the membranes resulted in a long re3ponse time for the electrodes, they did protect the cathode from poisoning by proteins, as occurs in electrodes where the cathode is not protected. That poisoning did not occur was shown by the fact that the calibration graphs were the same before and after blood ppO2 determinations. The question remains whether or not the electrode mea- sures the actual oxygen tension (ppOz) in tissues. Interfering fac- tors such as the viscosity, concentration of solids and solubility coefficient for O2 in biological fluids will all influence the availabil- ity of O to tissues. These same factors govern the availability of 2 O2 to the electrode. Charlton (1961) drew the analogy between an oxygen electrode with a small cathodediameter and a tissue cell; both have a small 0 consumption and both derive their 02 from the 2 available 02 in the region in which they are situated. Therefore, positioning of the electrode will have some influence on the mea- sured O2 tension of a biological fluid or tissue. There was some indication during the i_n_ _v_iv_o_ measurements that even though the vitreous is a relatively uniform body, a ppO2 gradient exists between the retina and the lens. This gradient was small and no attempts were made to characterize it. The fact that it exists is mentioned to reiterate the idea that positioning of the 71 electrode tip will influence‘the availability of O2 to the electrode and that a ppO value for a specific area in a tissue cannot be assumed 2 to be the average 02 tension of that tissue. Another factor which will bias the actual versus the mea— sured ppOZ of a tissue is the diameter of the cathode. The larger the cathode, as a general rule, the higher the apparent oxygen ten- sion (Jamieson and Van den Brenk, 1965). This appears to be due to damage of the tissues, caused by insertion of the electrode, where bleeding into the electrode track would result in recordings of higher 02 tensions than actually exist in the tissue. There was probably some tissue damage during the i_n_ H9. studies, but this would not account for the very high ppO values found in the control fish, since 2 the ppO value found for arterial blood was only slightly over 20mm 2 Hg. In Vivo Ocular ppOz The normal 02 tensions found behind the retina in the fish eye were significantly higher than the pp02 values for either the en- vironmental water or the arterial blood, while the ppO2 values for the arterial blood were significantly lower than those of the environ- mental water. This apparent concentration of O behind the retina 2 is particularly significant when one realizes that the retina of most 72 teleosts is a relatively avascular tissue. Thus, the high 02 tensions behind the retina would serve as a pressure head for diffusion of O2 to meet the high 0 demands of the retina. The question then arises 2 as to how such a high diffusion gradient is achieved when such a low arterial blood ppO exists. 2 Hoffert and Fromm (1966) found that at 20C arterial blood hemoglobin of lake trout was less than 30% saturated, while Irving (1941) found that brook trout (Salvelinus fontinalis) hemoglobin at 20C would be about 30% saturated at a ppO of 20mm Hg. At the lower 2 temperature of 13C the partial pressure of 0 required to cause a 2 30% saturation of hemoglobin would be lowered (Bohr shift to the left), so that the recorded ppO value of 20mm Hg may represent hemo- 2 globin saturation of as high as 50%. Irving (1941) also found that the 0 capacity of rainbow trout 2 blood at 15C is 13. 8vol%. If one assumes that the ppO2 value of 20mm Hg found in the arterial blood represents a hemoglobin satu- ration of 30% (which may actually be a low estimate), then the blood would contain 4. 14vol% 02' If a closed container was filled with this blood and all the O2 released from oxyhemoglobin through addition of an acid, then the O2 tension which would develop would be the ratio of the 0 content of the hemoglobin and the solubility coefficient 2 for 02. The solubility coefficient for O2 in water at 15C and one 73 atmosphere is 0. 0344ml 02 /ml H O or 3. 44vol% (Umbreit et a1. , 2 1964). Thus the O 4. 14V01% 3. 44V01% 2 tensions which could develop in this example would be = 1. 3, and 1. 3 times 1 atmosphere is equal to 1. 3 atmospheres or a ppO of 988mm Hg, a higher value than found 2 during the in vivo measurements. However, the choriocapillaris network of the choroid is not a closed container nor has anyone (Scholander and van Dam, 1954) been able to liberate all the 02 from HbO through the addition of an acid with a pH as low as 6. On the 2 other hand, the vascular anatomy of the choroid gland rete suggests the possibility of a mechanism of 02 concentration based on the counter current multiplication of an initial ppO2 increase in the venous blood of the rete as a result of acidification, as is postulated for the concentration of O2 in the swim bladder. In this way the choroid gland rete would be acting as a semiclosed system in protecting against gas loss through circulation. The fact that the 1_n_ vitro ppO recordings decayed with a t% 2 of 9 minutes, plus the apparent existence of a high ppO2 gradient from the back of the retina to the vitreous chamber suggests that the retina is a very metabolically active tissue in fish as well as in mammals. According to de Vincentiis (quoted by Davson, 1962) aerobic glycolysis does not occur in the fish retina although it does in the mammalian retina and apparently in the gas gland of fish 74 capable of producing lactic acid even in the presence of high 02 ten- sions (Ball 9131. , 1955). Whereas the "single concentrating effect" in the swim bladder rete may be a result of the addition of lactic acid on the venous side, some other mechanism for acidification of blood in the choroid gland rete should be explored. After Diamox treatment the ppO2 behind the retina was reduced to a value which was not significantly different from the ppO2 of the arterial blood, a mean of 24. 8mm Hg behind the retina and 22. 4mm Hg for the arterial blood. However, there was a sig- nificant increase in the hematocrit for the Diamox-treated fish, which indicates a greater 0 capacity. Earlier reports (Hoffert and 2 Fromm, 1966) have shown that Diamox treatment results in a de- creased pH of the blood. The decreased pH would lead to a decreased HbO binding capacity, so that the 0 content of the arterial blood 2 2 in the Diamox-treated fish may have been lower than in the controls. Fange (1953) has shown that inhibition of carbonic anhydrase with Diamox abolishes gas "secretion" into the swim bladder while Copeland (1951) has shown that removal of the pseudobranch causes a 50% reduction in the ability of the fish to regenerate the gas needed to refill the swim bladder. Both of these observations suggest a direct role for carbonic anhydrase in the O2 concentrating mechanism of the swim bladder. The fact that Diamox treatment eliminates the 75 high 02 tensions found in the eye, suggests that carbonic anhydrase may also be directly involved in the O concentrating mechanism of 2 the fish eye. Four sources of carbonic anhydrase in the eye are: from the pseudobranch, red blood cells, retina, and choroid. Unilateral pseudobranchectomy eliminates one of these sources, unless car- bonic anhydrase from the contralateral pseudobranch reaches the eye through the retinal artery. In any case the results from the unilateral pseudobranchectomies clearly showed a significant de- crease from the controls in the ppO values found in the eye. Un- 2 fortunately they do not show to what extent the disruption of a major portion of the blood supply to the eye has contributed to the decrease in the recorded ppO2 values. In the case of gas secretion into the swim bladder, the effects of pseudobranchectomy are not the result of any decrease in the blood supply to the swim bladder, since there is no direct vascular connection between the pseudobranch and the swim bladder. Swim bladderand RBC carbonic anhydrase are still present in these fish and yet they are unable to completely refill their swim bladder after pseudobranchectomy. It may be that con- centration of O2 in the eye and the swim bladder is dependent on an extracellular carbonic anhydrase carried in the plasma from the pseudobranch, or that there is a slight difference in the chemical nature of this enzyme, depending on in which cells it is produced. 76 Even after pseudobranchectomy the ppO levels in the eye 2 are significantly above those in the arterial blood. Pseudobranch- ectomy eliminates the blood supply to the choroid gland, so that there is no longer‘any possibility that O2 is being concentrated by the choroid gland rete. Previously only the rete of the choroid gland has been emphasized, but it appears from this data that the rete of the lentiform body is also capable of concentrating 02' The mechanism of concentration is no doubt the same in both retes, but its true nature can only be speculated on at this time. Forster and Steen (quoted by Berg and Steen, 1968) have shown that the Root-off shift (dissociation of 02 from HbO2 upon the addition of acid) is increased from a normal t% (biological half-time) of .053ec to 303ec after the addition of acid to the blood sample. If the Root-off shift were the only mechanism by which 02 could be dissociated from HbOz, then this data might suffice to explain the effects of Diamox. However, the Bohr shift and the salting out effect may also play a role in the establishment of the high 02 tensions behind the retina. The fact that Diamox does completely inhibit the concentrating mechanism has prompted the following hypothesis: Blood enters the choroid gland rete and lentiform body rete with arterial pH and ppO2 (Figure 1). The plasma of this blood may also contain pseudobranch carbonic anhydrase. After leaving the 77 retes the blood enters the choriocapillaris network of the choroid proper and receives CO from the rapidly metabolizing retina. The 2 C02 from the retina reacts rapidly with plasma H20 in the presence of carbonic anhydrase to form HC0; and H+. The HCO; anions thus formed may diffuse into the vitreous chamber‘to account in part for the presence of this anion in the vitreous humor, or they may be buffered by some cation in the blood. The H+ formed would result in a decrease in the pH of the blood which would lead to dissociation of 02 from HbO2 either through the Bohr shift or the Root-off shift, and consequently an increase in the ppO2 of the blood. A further ppO2 increase may result from the salting out effect due to the addi- tion of retinal metabolites to the blood. The net effect would be an increase in the ppOz and a decrease in the pH of the blood leaving the choriocapillaris network (single concentrating effect). The "single concentrating effect" would not be due to lactic acid, as it may well be in the swim bladder) since the fish retina does not exhibit aerobic glycolysis. When the blood returns to the choroid gland rete and the lentiform body it has a lower pH, higher ppO2 and may also have a lower physical gas solubility because of salting out. As this blood flows through the rete 02 will diffuse according to the gradient in pp02 from venous to arterial blood. There may also be an 78 equilibration of pH, with H+ diffusing into the arterial blood, leading towards the concomitant Root-on shift (increased binding capacity of the Hb as a result of an increased pH) in the venous blood and the Root-off shift in the arterial blood. The net result in the arterial is lost through blood is an increase in the ppO As long as little O 2' 2 circulation, this mechanism could be repeated through counter cur- rent multiplication in the retes until ppO2 such as those recorded were established in the choriocapillaris network behind the retina. The capillaries of the swim bladder rete appear to be the longest in the animal kingdom, measuring 4mm in some species (Hoar, 1966). There is a positive correlation between the degree of development and the length of the rete capillaries and the maximum ppO2 values found in the swim bladder (Steen, 1963). Thus it would seem that capillary length is a protective mechanism to guard against the loss of gas through circulation, i. e. , the longer the capillaries, the greater the chance that gas diffusion from the venous to the arterial side of the rete is complete by the time the blood leaves the rete. No data are available for the length of capillaries in the cho- roid gland rete or the lentiform body rete, but the author doubts that they are as long as those of the swim bladder. Based on the length of the rete capillaries, it would seem more probable that there would be a loss of O2 through circulation in the retes of the eye than in the 79 swim bladder rete. In addition to the rete of the choroid and the lentiform body there is another peculiarity of the vascular anatomy in the eye of bony fish which may serve as a protective mechanism against the loss of O through circulation. This is the fact that both 2 the ophthalmic and retinal artery pass through a venous sinus (which drains the venous side of the retes) before dividing into a capillary system. One can hypothesize that this region guards against gas loss through circulation. If the blood entering the venous sinus still has a high ppOz, then there would be a large surface available for diffusion of oxygen to the artery as it passes through the venous sinus. This might be the case if the Root-on shift, of rete blood reaching the venous sinus, is not yet fully established. SUMMARY AND CONCLUSIONS The normal ocular ppO2 values behind the retina and in the vitreous body have been measured using micro oxygen polaro- graphic electrodes. The mean values, 445 d: 68. 5mm Hg ppO2 behind the retina (i_n vivo measurements) and 103 j: 6.7mm Hg ppO2 in the vitreous body (i_n Xitfi measurements), are an indication, by virtue of the large gradient, of a high metabolic rate for the retina. Intraperitoneal administration of Diamox (0. 5mg/ Kg body wt) resulted in a significant decrease (p < 0. 001) from 445 i 68.5 to 25 j: 5. 1mm Hg in the ppO values behind the retina and from 2 103 i 6. 7 to 7 i 1. 0mm Hg ppO2 for the values in the vitreous chamber. These findings implicate carbonic anhydrase in the oxygen concentrating mechanism of the eye. Unilateral pseudobranchectomy resulted in a 76.3% decrease from the control ppO values recorded behind the retina, from 2 239 :t 24. 7 to 57 j: 9. 6mm Hg ppOZ. It is difficult to determine if these results are due to removal of pseudobranch carbonic anhydrase or the disruption of the major blood supply to the eye. 80 81 After unilateral pseudobranchectomy the ocular ppO2 values were still significantly higher than arterial ppO2 (p < 0. 01). These results suggest that the small rete of the lentiform body in the fish eye is capable of concentrating oxygen. values in the eye with the ppO of Comparison of the ppO 2 2 arterial blood (mean of 21 2t 2. 2mm Hg) and with the environ- mental water (mean of 133 d: 3. 7mm Hg) show that there is a significant difference between ocular and arterial ppO2 (p < 0. 001) and a significant difference between the ppO2 behind the retina and the ppO2 of the environmental water (p < 0.01). A hypothesis is offered for the mechanism of oxygen concentra- tion at the retina of the fish eye. This mechanism involves acidification of blood in the choriocapillaris network of the cho- roid, as a result of carbonic anhydrase catalization of the reac- tion between CO from the retina and H20 from the plasma to 2 form H+ and HCOg. The decreased pH would result in an in- creased ppO2 (single concentrating effect) which would be mul- tiplied by a counter current mechanism in the choroid gland rete and the lentiform body rete. LITERATURE CITED Ball, E., C. F. Strittmatter, and 0. Cooper. 1955. Metabolic Studies on the Gas Gland of the Swim Bladder. Biol. Bull. , 108: 1-17. Barnett, C. H. 1951. The Structure and Function of the Choroidal Gland of Teleostean Fish. J. of Anat. , 85: 113-119. Berg, T. , and J. B. Steen. 1968. The Mechanism of Oxygen Con- centration in the Swim Bladder of the Eel. J. Physiol. , 195: 631-638. Cater, D. B. 1966. The Significance of Oxygen Tension Measure- ments in Tissues. In Payne, J. P. , and D. W. Hill (ed.). Oxygen Measurements in Blood and Tissues. J. 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Wittenberg, J. B., and B. A. Wittenberg. 1962. Active Secretion of Oxygen into the Eye of Fish. Nature, 194: 106-107. Umbreit, W. W., R. H. Burris, and J. F. Stauffer. 1964. Mano- metric Techniques, (4th ed. ). Burgess Publishing Co. , 426 South Sixth St. , Minneapolis 15, Minnesota, 305 p. APPENDIX 1 Diagram of Polarographic Electrode Circuitry 87 Symbol Function S1 Switch 1 When in up position circuit between polarographic electrode and preampli- fier is completed. When in down position input resistance of the pre- amplifier can be determined. 82 Switch 2 When in up position resistance across 50K potentiometer is measured. When in down position input impedance of preamplifier can be determined. S3 Switch 3 Electrode on-off switch. S4 Switch 4 Polarizing voltage on-off switch. T , T , T Output terminals to Grass 5P1 preamplifier and 1 2 3 . recorder. T1 to terminal post 3, T2 to post 2, and T3 to post 6 of Grass 5P1 preamplifier input cable. T4, T5 Output terminals from 50K potentiometer to Heathkit Impedance Bridge. T6’ T7 Input terminals from polarizing voltage source. T8’ T9 Input terminals from oxygen polarographic elec- trode. T3, T10 Ground. T , T Output terminals to Hewlett Packard Vacuum 7 8 Tube Voltmeter. 50KJL Potentiometer used to measure input impedance 40KIL, 500A, 2megf\_‘ IOKIL or preamplifier. Auxillary zero adjust. Polarizing voltage adjust. thufisofiv 000.500me oEQd:wo:wH0m mo :Hmnmmfifl