Keio Journal of Medicines Vol. 14, No. 2, June, 1965

A COMPARISON OF AQUEOUS HUMOUR FORMATION AND CSF FORMATION ELECTROLYTES TRANSPORT ACROSS BARRIER

MICHIO SAKANOUE

Department of Ophthalmology, School of Medicine, Keio University, Tokyo

(Received for publication June 15, 1965)

Aqueous humour was once thought to be a dialysate of plasma, or to be secreted as such by the in a manner analogous to secretion of saliva. Many observations on aqueous humour, both experimental and clinical, could not be accounted for the basis of either of these suggested mechanisms of aqueous humour formation.

Kinsey recently reported bicameral nature of aqueous humour , and sug gested these two processes, secretion, diffusion, and dialysis as taking place simultaneously. The recent studies of the chemistry of the posterior chamber aqueous humour have provided additional evidence that aqueous humour is

formed by at least two processes, i.e. active transport and passive transport . Abundant evidence is available to suggest that a not insignificant proportion of all constituents normally present in the aqueous humour of the posterior chamber enters from the blood across the several barriers presented by the . Morphologically ciliary body is quite similar to those of plexus which is concerning with CSF formation. The author has recently taken into consideration the process within the "aqueous forming ciliary cells ," and suggested a comparison of ciliary body and choroid plexus. Beginning with the experiments of Dandy and Blackfan , substantial evidence has accumulated that the choroid plexus are important, perhaps the main, site for the bulk formation of CSF. Recent studies with tracers , reviewed by Sweet indicate that following the initial appearance of the CSF its various constituents continue to be in dynamic relationship with the blood in all major portion of the ventricular and subarachnoid spaces i.e. water and electrolytes move rapidly between the blood and CSF through the lining tissues of the subarachnoid spaces. If this be true, CSF as conventionally sampled would reflect in its composition a mixture of the characteristics

73 74 MICHIO SAKANOIJE imposed by the processes at the prexuses , plus those superimposed by the sub sequent exchanges.

The purpose of the studies to be presented in this paper is as follows . (1) To determine the electrolyte composition of fluids sampled directly from the ciliary body and choroid plexus . (2) To compare the both compositions each other, and with that of serum to see how they may differ from an ultrafiltrate . (3) To compare ciliary body fluid with those of posterior chamber and anterior chamber aqueous humour. (4) To compare the choroid plexus fluid with that of CSF obtained from sites progressively further along the course of its circulation.

METHOD

Samples of serum, ultrafiltrate, newly formed ciliary body fluid , choroid plexus fluid, anterior tap and posterior tap of aqueous humour, and cisternal

CSF were obtained from cats. Each specimen was analyzed for water , Na, K, Cl, Mg; and Ca. The cats were anesthetized with intraperitoneal Nembutal 30 mg/kg. Two samples of blood were obtained from the femoral artery, one before and the other after collecting the aqueous humour and the CSF specimens. The blood was centrifuged immediately, and a 2-6 ƒÊl samples of the serum was transferred with small pyrex glass capillary from the centrifuge tube to a tared vycor glass tube for weighing and analyses. The blood for ultrafiltration was collected by siliconized cold syringe, and was centrifuged immediately at 0•Ž. Siliconation of the syringe was not only for prevent clotting of blood but also prevent CO2 leak from the blood. The unclotted serum was maintained during ultrafiltration in equilibrium with a gas phase of 95%o 02 and 5% C02 at 37•Ž.(12) Sampling for newly formed ciliary body fluid is shown in Fig. 1. The was incised with the triangular knife (Keratome) at the lever of the limbus where it is covered by a thin layer of , penetration into the anterior chamber was done carefully to avoid injuring the and . Put silicone rubber tube coated with pantopaque outside through the corneal wound very carefully and gently, to avoid injury the both iris and lens and reach to posterior chamber. For this purpose the spatula is introduced through the corneal opening. Following taking the samples of anterior chamber and posterior chamber AQUEOUS HUMOUR FORMATION AND CSF FORMATION 75

Fig. 1

aqueous humour, chamber was filled with pantopaque and the fluid which collected on ciliary processes was sampled. Sampling for newly formed choroid plexus fluid is shown in Fig. 2.(8)(11) The corpus callosum was then carefully divided to unroof the lateral ventricle whose content of CSF immediately displaced with pantopaque (ethy liodophenylundecylate) according to Ames and Rougemont's technique . Panto paque is a contrast medium for myelography and a mixture of ethyl esters of

Fig. 2 76 MICHIO SAKANOUE isomeric iodophenylundecylic acids , is an absorbable heavy oil type contrast medium of low vescosity. The choroid plexus could be easily seen through the clear oil and the secretion which collected on its surface was sampled with a fine pyrex pipette lowered through the oil with a micromanipulator.

About 2-10 minutes were required to collect each of two duplicate 2-6

ƒÊl specimens. Pantopaque (specific gravity 1 .26) is heavier than the choroid plexus fluid so that the latter tends to rise up within the pool of oil. If a sample of the choroid plexus fluid and the ciliary body fluid contained even a trace of blood visible with the operating microscope , the experiment was considered a failure and terminated. The high specific gravity of the pantopaque tended to prevent contamina tion of the ciliary body and choroid plexus secretions by fluids from other sources, and the evaporation was eliminated since neither the secreting source nor the sample being collected were exposed to air. Chemical trauma ap peared, unlikely in view of the clinical evidence for nontoxity of pantopaque; and K concentrations in the samples collected were low, but the pantopaque was demonstrated not to certain appreciable quantities of Na, Cl, K, or water nor to interfere with the analyses of these substances. Cisterna pericallosa fluid was sampled as follows : The dura over the cisterna pericallosa was exposed through an interhemispheric approach and a sample of CSF from this site was simultaneously collected. Sampling of fluid was done before sampling of choroid plexus fluid. Cisterna magna fluid was sampled as follows : The dura over the cisterna magna was operatively exposed and penetration with a fine pyrex pipette into which a 2-6 µl sample of the CSF was allowed to flow by capillarity. Sampling of fluid was also done before sampling choroid plexus fluid to avoid contamination. Evaluation of sampling method : According to Dr. Ames' opinion the method described has been used with apparent success of cats, and the author reconfirmed the sampling technique of choroid plexus fluid. In addition the author adopted the almost same technique for ciliary body fluid sampling. An important effectiveness of a sampling method is its reproducibility. Duplicate samples showed the good agreement. The most difficult feature to evaluate is whether the fluid, formed by the ciliary body and the choroid plexus under these circumstances, are the same as that formed in the intact animal. The cats were maintained on artificial respiration throughout the proce- AQUEOUS HUMOUR FORMATION AND CSF FORMATION 77 dure, but the other feature of a sampling method is change in the CO2 tension of the fluid bathing the secreting cells. Since CO2 has been implicated in the formation of aqueous humour and CSF it may be important to maintain its tension at normal level by preequilibrating the pantopaque with 5% CO2. This was not done in the studies. Analysis: Wet weight was measured immediately after collection, dry weight was measured after 4 hours at 105•Ž in a mechanical convection oven, and sample water determined from the differences. The dried salts for Cl, Na, Ca, Mg analyses were then redisolved in a volume of extracting solution 1000 times greater than the volume of water in the original specimen, i.e. 1 ml of extracting fluid for each al of original sample water. The extracting solution contains AgNO3 0.220 mM/L (for Cl analysis), Li2SO4 5 mM/L (As internal standard for flame photometry), and HNO3 0.1 N. Cl was determined indirectly according to the method of Lowry by meas uring the free Ag remaining after precipitating the Cl in the sample with known amount of Ag added in the extracting solution. Na and K were measured by internal standard flame photometry with an instruments specially designed for microanalysis by Baird Atomic Inc., Cambridge, Mass., U.S.A. Ca was measured by spectrphotometry with murxide, and Mg with titan yellow. (1)(2)

The acuracy of analytical system was periodically checked by analyses of aliquots of a salt solution of known composition containing electrolytes in amounts similar to those expected in a 2-6 ƒÊl specimen of extracellular fluid.

RESULTS

Preliminary inspection of the data revealed the following four basic features, of importance for the further interpretation of the results. (1) Composition of the analyses of the two serum samples obtained from each animal before and after operation indicated no significant change in composition due to the operative procedure. Consequently the values on these two samples were averaged and these averages were used for comparison with aqueous humour and CSF samples . (2) Similary, since the two samples of choroid plexus fluid and ciliary body fluid obtained on each animal showed no significant differences between the first and second, the averages of the pairs were used for the subsequent treatment of the results. (3) There appeared to be no significant differences between animal, i.e., there were no cats that tended to have high K throughout or low Cl through out, etc. Therefore the animals were treated as a homogeneous group. 78 MICHIO SAKANOUE

PC: Posterior chamber fluid of aque ous humor CB: Ciliary body fluid as newly form ed aqueous humour CP: Choroid plexus fluid as newly formed CSF CM: Cisterna magna fluid CC: Cisterna pericallosa fluid UF: Ultrafiltrate AqH: Aqueous humour CSF: Cerebrospinal fluid Fig. 3 AQUEOUS HUMOUR FORMATION AND CSF FORMATION 79

(4) The measurement of water was the least accurate of several deter minations, errors apparently arising due to evaporation prior to the measure ment of wet weight or due to uptake of water by the dry salts prior to the measurement of dry weight. The concentration of Cl, Na, K, Ca, and Mg, found in each of the fluids are shown graphically in Fig. 3. (The magnitude of the standard error of the mean is indicated by the vertical line at each point.) Fluids obtained directly from the ciliary body had a higher concentration of Cl than serum and ultrafiltrate, and i.e. about the same as choroid plexus fluid. However, the differences are not significant statistically. The Cl ratios of ciliary body fluid to serum and choroid plexus fluid to serum are somewhat higher than that expected in an ultrafiltrate.

Cl in the CSF are gradually increased along its circulation to cisternal fluids. Cl in the aqueous humour are also increased along its circulation, but not remarkable than the CSF circulation. Posterior chamber aqueous humour Cl seems to be lower than cisternal fluid Cl. The concentration of Na in the ciliary body fluid and choroid plexus fluid are about the same concentration, and both of them are significantly higher than that expected in an ultrafiltrate of serum. The cisternal fluids also showed higher Na and posterior chamber aqueous showed more higher Na. All of the differences were statistically significant. 191(10)

On the contrary the concentration of K in the ciliary body fluid and choroid plexus fluid are lower than serum and ultrafiltrate. The K ratios ciliary body fluid to serum and choroid plexus fluid to serum are both lower than that expected in an ultrafiltrate. K in the CSF are gradually decreased along the course of its circulation but aqueous humour are not remarkably decreased along the course of its circulation. There was a progressive fall in K from the choroid plexus to cisterna magna to the cisterna pericallosa which was significant statistically. (3) The concentration of Ca in the ciliary body fluid and choroid plexus fluid are lower than serum and ultrafiltrate. Actual value of ciliary body fluid Ca is little lower than the choroid plexus fluid. Ca both in the CSF Circulation and aqueous humour circulation are the almost same progressive fall, but not re markably.

A more meaningful and interesting comparison would be the contrast quite opposite difference between the ciliary body fluid Mg and choroid plexus fluid Mg, i.e. Mg in the choroid plexus fluid is strikingly higher than serum and ultrafiltrate level contrary to the ciliary body fluid which is lower than serum 80 MICHIO SAKANOUE and ultrafiltrate level. Both of them are gradually decreased along their circulation in the same direction but the cisternal fluids in the cisterna magna and cisterna pericallosa are still higher than serum and ultrafiltrate level .(5) Fig. 4 shows the direct difference between the ciliary body fluid and the choroid plexus fluid of each electrolyte .(6)(7)

Fig. 4

Both of them are described in the ratios to ultrafiltrate. The distribution ratios, between ciliary body fluid and ultrafiltrate are shown by a dotted bars. The distribution ratios, between choroid plexus fluid and ultrafiltrate are shown by a solid bars. Na and Cl ratios of ciliary body fluid and choroid plexus fluid to ultrafiltrate are the same value in the same direction. K and Ca ratios of ciliary body fluid and choroid plexus fluid to ultrafiltrate are different value each other but in the same direction. Mg ratio in the choroid plexus fluid to ultrafiltrate is strikingly higher but ciliary body fluid to ultrafiltrate is lower. Both of them are in quite opposite direction.

DISCUSSION

Most striking feature is the finding that less Mg in the aqueous humour and more Mg in the CSF than expected in an ultrafiltrate of serum. On the AQUEOUS HUMOUR FORMATION AND CSF FORMATION 81 basis of such deviations from the composition expected of an ultrafiltrate, it is quite generally believed that aqueous humour and CSF are formed either entirely as a secretion by some combination of filtration plus superimposed elements of active transport.(4) The results of the present studies indicate that at least some of the present active transport phenomena take place at the site of the bulk formation of the aqueous humour at the ciliary body and the CSF at the choroid plexus. It is easier to demonstrate the presence of active transport than to identify just which of the ions are actively handled. For the latter purpose it would be helpful to know the electrical potential across the ciliary body and choroid plexus membrane, whether or not filtration is ocurring and, if so, whether or not the membrane contains a significant amount of fixed charge. Hogben has found the ventricular fluid of the dogfish to have a negative potential, of about 17 mV, with reference to systemic extracellular fluid.(9) Tschirgi has measured smaller potentials of the same sign between the blood and brain of mammals.(10) Cole has found the smaller potentials of the same sign in aqueous humour with reference to systemic blood. Lack of knowledge of the difference in electrical potential across the blood aqueous barrier and blood-CSF barrier makes speculation concerning which ions are being actively transported much more difficult. For these reasons detailed speculation about the transfer mechanisms would not appear useful. However, the above observations, taken in conjunction with the high concen tration of Cl in aqueous humour and CSF, all suggest that the transport of Cl may be the major somewhat rather active events in the formation of aqueous humour and CSF formation.

Becker also suggested that the relationship of Cl excess in aqueous humour of human to the rate of flow provides suggestive evidence for a primary role of the secretion of this anion in primate .(13)(14)(15)(17) The presence in aqueous humour of less K, and in CSF of more less K, but more Na than expected in an ultrafiltration suggest that at least one of these two ions is also actively handled. The fluid obtained from the posterior chamber and from the cisternae should have Cl at even higher and K at even lower concentrations than the fluid from the ciliary body and the choroid plexus seemed of considerable interest.(16)(17) It is possible that same derangement of the physiologic state associated with the process of sampling, led to the temporary formation by the choroid plexus and the ciliary body fluids intermediate between a filtrate and its normal product. This then might explain the difference between specimens 82 MICHIO SAKANOUE from the ciliary body and choroid plexus , and those from the posterior chamber and the cisternae, since the latter had been formed sometime prior to the sampling procedure. If this could be demonstrated , it would be interesting since it would be suggested that filtration and active transport were both operative in the formation of the choroid plexus fluid and ciliary body fluid. Some general conclusions, however, may be drawn. The fact that the distribution ratio between aqueous humour and ultrafiltrate, and the ratio between CSF and ultrafiltrate of monovalent and divalent cations is quite selective across each barrier. It does not prove beyond doubt that active transport is involved, but this is probably the most likely explanation. As other have pointed out, the fact that K, Mg, and (to a lesser degree) Ca in the CSF are maintained at quite constant levels in the CSF, in spite of marked and prolonged alteration in their serum levels, indicates some con trolling step in the transfer of each of these ions probably an ion specific carrier. This may serve a physiological role by providing additional home ostasis for the milieu of the neurons or by permitting controlled modification of this milieu in response to as yet undefined stimuli. Similar mechanisms operating at the blood-brain barrier may extend this control to portions of the brain beyond diffusion reach of the CSF. Even after it has lost contact with the ciliary body, posterior chamber aqueous humour to be exposed to the action of energy requiring transport process further modification in the same direction as those initiated during its formation by ciliary body. Especially progressive fall of Mg on the contrary to the CSF and pro gressive fall of K similar direction to the CSF would suggest that the me chanisms involved in cation transfer are quite selective at blood-aqueous barrier, and the mechanisms are also active but may be an active removal of cations. It is tempting to postulate that these further modification are the result of the same transport mechanisms as the initial forming function, residing in cells of similar origin, as those of ciliary body and choroid plexus. How ever at the initial forming sites, the ciliary body and choroid plexus are somewhat different and each of them has own speciality and selectivity at the barrier.

SUMMARY

Techniques are described for collecting newly formed aqueous humour from the ciliary body of the , and for collecting newly formed CSF AQUEOUS HUMOUR FORMATION AND CSF FORMATION 83 directly from the choroid plexus of the anaethetized cat. Both these two fluids were found to have a little lower concentration of K than plasma ultrafiltrate but did not so much differ from plasma ultrafiltrate in Cl. Na concentrations of these fluids were higher than expected in an ultrafiltrate. Most striking feature is the finding that less Mg in the newly formed aqueous humour and more Mg in the newly CSF than plasma ultrafiltrate. Ca in these two fluids were lower than plasma ultrafiltrate. The change of each electrolyte concentration along the course of aqueous circulation and CSF circulation was also reported. The fact that the destribution ratio across barrier was taken as evidence that the mechanisms involved in cation transfer are quite selective. Though not proven, active transport was considered the most likely explanation of these findings. It is postulated that the further modification of aqueous humour and CSF electrolyte pattern from the initial forming site to their circulation course may the result of the same mechanisms residing in cells of similar origin as those of ciliary body and choroid plexus, but the both of them are somewhat dif ferent each other at the blood-aqueous barrier and blood-CSF barrier.

ACKNOWLEDGEMENT

The author is much indebted to Professor Dr. Yasuharu Kuwahara, and to Dr. Adelbert Ames, ‡V, Department of Biological chemistry, Harvard Medical School, U.S.A.

REFERENCES

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