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Effect of spinal fluid pressure on cerebrospinal fluid formation
EXPERIMENTAL
32, 30-40
XEUROLOGY
Effect
of
(1971)
Spinal
Cerebrospinal GERALD Departments Medical Rcrci~d
M.
HOCHWALD
Fluid
Pressure
Fluid
Formation
AND
ABRAHAM
of Neurology and Neurosurgery, Center, 550 First dvcrm-, New March
1. 19il;
Kczisiorl
York,
Kcccizd
on
SAHAR
I
New York University Nezw York 10016 April
R, 1Vl
The effect of spinal fluid pressure on cerebrospinal fluid (CSF) formation was measured in rabbits and cats. A decreased rate of CSF production with increased perfusion pressure was found, which was best illustrated by paired comparisons in individual animals during ventriculocisternal perfusion at both low (-5 to -10 cm H,O) and high (2025 cm H,,O) perfusion pressure. Each animal was perfused at either pressure for 2 hr (rabbits) or 2.5 hr (cats) for steady-state measurements. After a gradual change in hydrostatic pressure, perfusion continued for an additional 1.5 hr (rabbits) or 2 hr (cats) when steady-state measurements were repeated. The mean rate of CSF production decreased in rabbits from 0.0081 to 0.0045 ml/min after the perfusion pressure was elevated. This effect was independent of whether the animals were perfused initially under high or low pressure. In cats, after a similar increase in perfusion pressure, the mean rate of CSF formation decreased from 0.0212 to 0.0102 ml/min. Introduction
Various studies using the technique of verticulocisternal perfusion have shown that most of the cerebrospinal fluid (CSF) is formed within the cerebral ventricles (7, 10). Much evidence favors the choroid plexus as the site of formation of the fluid (1, 6, 8, ZOj, although the possibility for the flow of fluid across the ventricular walls must be considered (4, 11, 20). There are several factors which influence spinal fluid production. It has been postulated that the rate of production of fluid probably depends upon mechanisms such as the transport of solutes from the blood with water following passively, or the hydration of CO? (4, 7, 13). The secre1 This study was supported by Grants NS-05024 and NS-06.599 from the USPHS. Dr. Hochwald was a recipient of Special Research Fellowship Award 2 Fll NB01431 from USPHS. Dr. Sahar’s present address is Dept. of Neurosurgery, Hebrew University-Hadassah Medical School, Jerusalem, Israel. The authors thank Miss J. Brown, B.Sc., and S. Sullivan for their technical assistance. Reprint requests to Dr. Hochwald. 30
CEREBROSPINAL
FLL-ID
31
tion of CSF is dependent on metabolic energy since its rate of formation decreases under the influence of enzyme inhibitors such as acetazolamide, dinitrophenol, and ouabain (1, 7, 14, 20). Other factors influencing spinal fluid formation may be the blood supply to the spinal fluid-forming organs (, 1). The reduction of CSF formation with a decrease in plasma PCO, resulting from hyperventilation has been attributed in part to the narrowing of choroid plexus vessels (1 j. hlacri it al have suggested that the effect of acetazolamide may be from its capacity to constrict the choroid artery (12). Attempts to relate CSF formation with intraventricular pressure have been made by several investigators (2, 5, 10, 11, 16). From data obtained during ventriculocisternal or ventricle-to-lumbar perfusion, they concluded that alterations of intraventricular pressure had little or no significant effect on CSF production. However, other studies (3, 17) have demonstrated that the rate of CSF formation decreased with increased perfusion pressure. Calhoun ct nl. (3) were able to show this in calves during ventriculocisternal perfusion. Sahar, Hochwald, and Ransohoff ( 17) reached this conclusion from data obtained during steady-state lateral ventricle-tolateral ventricle perfusion in kaolin-induced chronic, hydrocephalic cats. In these experiments, changes in CSF formation were measured during a single perfusion under both high and low pressures. The present experiments were designed to detect the effect, if any, of intraventricular pressure on CSF production in normal rabbits and cats. Since the individual rates of CSF formation were varied, the results from each animal were analyzed separately in order to emphasize the effects of pressure changes. Materials
and
Methods
Perfusion of the ventricular system of rabbits was carried out by a slight modification of the technique described by Pollay and Davson (15). The animals were anesthetized with intraventus sodium Thiamylal (20 mg/kg) and were maintained on it throughout the experiment. During the course of the experiment rabbits were also given intermittent injections (6 mg ‘kg) of Flasedil (gallamine triethiodide) and artificial respiration. The lateral ventricle was penetrated with a 19gauge needle inserted with the aid of a stereotasic apparatus through a burr hole 10 mm behind the coronal suture and 8 mm lateral from the sagittal suture. The correct placement of this inflow needle was recognized by a sudden drop in pressure in the inflow tubing. The outflow system consisted of a 19-gauge needle which was attached to plastic tubing. The height of the outflow tubing determined the perfusion pressure. The cisterna magna was penetrated with this needle between the occiput and Cl. The technique for the perfusion of the ventricular system of cats has
32
HOCHWALD
AND
SAHAR
been described ( 11) . However, in these experiments, Flaxedil was omitted and the cats were allowed to breathe spontaneously. The perfusion fluid for all animals used was an artificial CSF which has been described previously (11). It also contained inulin and 1311-labeled rabbit or cat serum albumin as tracers for measuring CSF formation and absorption ( 11) . Inulin concentrations were determined in triplicate by the resorcinol method (9) ; radioactivity was measured in a well-type scintillation detector (Nuclear-Chicago). Both determinations were made to a probable error of 2%. The perfusion technique in general was similar to that previously described; the perfusion pressure was measured with a Statham transducer and was continuously recorded (11). The outflow volume was measured gravimetrically at 15min ilitervals. A successful experiment was one in which perfusion was maintained for approsimately 5 hr, and in which at the termination of the experiment, addition of dye to the perfusate verified the pathway taken by the fluid. Evans blue dye (1%) was perfused through the system for 20-30 min ‘prior to the exsanguination of each animal. A careful examination was made in the region of the inflow and outflow needles for loss of dye to tissue around these needles. Although no leaks were observed under these conditions, it is conceivable that a very small loss of fluid may have occurred during perfusion at high pressures. However, the measurement of CSF formation at all pressures during ventriculocisternal perfusion was dependent upon the dilution of a nondiffusible indicator and a leak after complete mixing would not affect this dilution. The effect of intraventricular pressure on the rate of CSF formation was studied in 18 rabbits and in 7 cats. Each rabbit was initially perfused at either high (20-25 cm H?O) or low ( -5 to - 10 cm H,O) perfusion pressure with respect to the interaural line. Subsequent to steady-state measurements, the pressure was gradually (30 min) reduced or elevated. Perfusion continued until a new steady state was reached and measurements were repeated. In these experiments the rate of inflow was 0.077 ml/min and approximately 1.5 hr was needed for equilibrium to be reached. Twelve of these rabbits were initially perfused at a high pressure and then subsequently at a low pressure. All of the cats were perfused at a high (20-25 cm H30) pressure for 2.5 hr and then at a low ( -5 to - 10 cm H?O) pressure for 2 hr. The rate of inflow of these animals was also 0.077 ml/min. The perfusion pressure represents the range within which all the perfusion took place, and for purposes of some calculations, each range was also considered as a group. The choice of perfusion at these two pressures was made only in order to determine whether the rate of CSF formation was sensitive to the hydrostatic pressure of the system, and therefore, does not indicate whether this relationship was linear. The perfusion
CEREHROSPINAL
FLUID
33
of one animal at two pressures during one experiment partially eliminates wide ranges of CSF formation rate values measured in a group of animals at the same pressure or in individual animals perfused on different occasions. In addition, perfusion at two different pressure intervals reduced the importance of the definition of the zero pressure at the time of perfusion. Cnlclrln/io~zs. The rates of formation and absorption of CSF were calculated according to Heisey rt nl. (10). At steady state, for substances removed by hlk flow, the rate of absorption of CSF is :
where C, clearance of test substance ( innlin or albun~in-l”‘I by bulk absorption; 1’. rate of flow; i, o, f, a, inflow, outflow, formation, and absorption of CSF, respectively : C, exponential mean ventricular concentration ; f, concentration of test substance. The rate of formation of CSF is therefore:
Results
The 13*1-labeled albun~in in the perfusate was monitored at 1.5~min intervals. With an inflow rate of 0.077 ml/min steady state was reached in appro-ximately 1 hr for the rabbit and 2 hr for the cat. This inflow rate was found to be satisfactory since it provided a sufficient volume of outflow fluid at elevated perfusion pressures, and it also allowed significant changes in concentrations of albumin-‘“‘1 and inulin to be detected.? Calculations were based on samples taken approximately 2.5 hr after perfusion began and 2 hr after the perfusion pressure was changed. The results of a representative experiment in which the effect of hydrostatic pressure on these physiological parameters in one rabbit is demonstrated can be clearly seen in Fig. 1. A decrease of approximately 30 cm H,O pressure resulted in a fourfold increase in CSF production. Under similar conditions the increase in absorption of fluid was variable because at the lower pressure the absorption was very small. The mean rates of formation and absorption from 18 rab2 Results calculated from inulin or albuminagreement. On occasion, however, dilutions of The reason for this was subsequently learned experiments with very small amounts of labeled fluid, and in the absence of a carrier protein, the apparatus. These animals were eliminated ratio of unbound/bound 1311 in the preparations
Is11 dilutions xvere generally in good these indicator substances did differ. to be due to the fact that in some protein ( < 1 pg/ml) in the perfusion some of the radioactivity adhered to from CSF turnover calculations. The used was less than 0.01%.
34
HOCHWALD
AND
SAHAR
p~*----------~~~ 100
20T025cmH20
-5 TO-IOcmHZO
90 80 70
FIG. 1. Effect of hydrostatic pressure on rates of CSF formation during ventriculocisternal perfusion of an individual rabbit. Co/C; X 100, outflow concentration as percentage of inflow concentration (albumin-1311). Vi, V,, V,, rates of inflow, absorption, and formation, respectively. These rates were calculated from measurements made during steady-state perfusion at 20-25 and -5 to -10 cm H,O pressures.
bits perfused at both high and low pressures can be seen in Table 1. The percentage decrease in formation at the higher perfusion pressure was approximately 45%. Absorption of CSF increased fivefold over this same pressure range. The individual and mean rates of CSF formation and absorption from seven cats perfused at both high and low pressures are summarized in Table 2. In addition, a comparison of the rates of formation determined from inulin and albumin-1311dilutions can be seen in Table 3. These rates (V,) were calculated according to the formula, Y, = Vi (Ci - C,)/C,, where Vi is the rate of inflow, Ci and C, are the concentrations of tracer substances in the inflow and outflow fluids, respectively. In this series of .animals, the percentage reduction in CSF production was about 50% dP
Perfusion pressure (cm H@)
20to25 -5 (1 Mean
to -10 f
SE.
I
OF HYDROSTATIC PKESSURE ON CSF AND ABSORPTION IN RABBITS CSF
FORMATION
formation (ml/min)
CSF
absorption (ml/min)
0.0045 f 0.0006= 0.0082 f. 0.0009
0.0298 0.0049
f f
0.0045 0.0020
CEREBROSPIKAL
EFFECT
OF HVDKO~TATIC FROM
FORMATION
rix INDIVIDUAL
CATS
pressure
(20-25
cat
ON CSF
PKESSLXE
AND ABSOK~~TION Perfusion
35
FLUID
cm
(-
HsO)
Perfusion pressure 5 to - 10 cm H,O)
Vf h (ml/mill)
V& c (ml/min)
v’r h (ml/min)
1; c (ml/min)
0.077 0.077 0.077
0.0100 0.0202 0.0060
0.0614
0.0250 0.0273
0.0010 0.0030
0.077 0.077 0.077
0.0195 0.0046
0.0217 0 ,055.~ 0.0650
0.0207 0 .0236
0.0002 0.0060
0.0118 0.0204 0.0196
0.0101 0.0091
no.
0.0386 0.0291
0.0027 0.0091 0.0102 f0.0026
0.077
” Vi, rate b Vf, rate
of inflow. of CSF formation.
c I’%, rate
of CSF
0.0356 0.0438 ~0.0063
0.0212 f0.0018
0.0071 0.0051 *o .OOl‘l
absorption.
CSF formation and absorption were greater in the cats than in the rabbits. The results from the larger group of animals, i.e., the IS rabbits, were analyzed more completely. Data from individual rabbits can be seen in Fig. 2. The range of values for CSF production in normal rabbits perfused at
EFITCT
01; HYDROSTATIC Fwohl D1Ltwo~ Perfusion (20-25
Cat
no. 1 2 3 -I 5 6 7
Mean fSE * Vf, rate b Vf, rate
cm
0~
pressure HsO)
vp1-CS.4) (ml/min)
V~(inulin) (ml/mh
0.0143 0.0230 0.0054
0.0131 0.0238 0.0065
0.0190 0.0024 0.0026 0.0094 0.0108 f0.0020
0.0158 0.0042 0.0041 0.0109 0.0112 fO.0026
of CSF of CSF
formation, formation,
ON CSF NONDIITIMBIX
PKESS~IW
calculated calculated
FORMATION CALCULATED I~XDKXTOKS (-5
b 1
Perfusion pressure to - 10 cm H?OI
vpr-CSA) (ml/min)
Vf(inulin) (ml/mini
0.0290
0.0214
0.0268 0.0161 0.0241
0.0270 0.0154 0.0183 0.0108 0.0225 0.0212
0.0111 0.0199 0.0191 0.0208 f0.0022 from from
dilution dilution
of cat serum of inulin.
0.0195 I!z0.0017 albumin-‘3’1.
36
HOCHWALD
AND
SAHAR
both pressures was about 15-fold. The mean difference in formation rate between both groups was 0.0036 ml/min (P
Prior to the development of the method of ventricular perfusion, studies of CSF formation could not readily delineate the effect of elevation of intraventricular pressure on absorption or formation of fluid. Recently, this pressure effect has been the subject of several studies with various species.
160 t-
.
1
I.
140
80I 60-
l .
-15 -10 -5 0 5 10 15 20 PRESSURE, cm Hz0
25
FIG. 2. Rates of CSF formation measured in 18 rabbits, each perfused at both low (-5 to -10 cm H,O) and high (20-Z cm H20) pressures. Formation rates of individual rabbits grouped according to perfusion pressure. Bars represent mean rates of formation, 0.0081 and 0.0045 ml/min * SE ( [ ). Note that in this nonpaired comparison, the ranges for both groups are similar and there is a good deal of overlapping.
CEREBROSPIKAL
FLUID
37
The most detailed studies reported were those of Heisey et al. on the goat ( 10) and Calhoun et al. on the calf (3). With the aid of the technique of ventriculocisternal perfusion, both groups of authors measured CSF turnover at various hydrostatic pressures. The former concluded that CSF production was independent of hydrostatic pressure, while the latter found that fluid formation decreased with increased perfusion pressure. The opposing conclusions of these authors may be the results of such factors as differences in species employed or the use of the anesthetized versus the unanesthetized animals. La more careful consideration of experimental data from both of those laboratories, however, may resolve this apparent discrepancy. In the experiments of Calhoun st al., CSF formation as a function of pressure was measured only at a limited number of pressure intervals between - 10 and 20 cm H,O in anesthetized calves. Heisey et al., in an attempt to establish a relationship between CSF formation and absorption with hydrostatic pressure, made measurements on unanesthetized goats at smaller pressure intervals. It is conceivable that, with the free movement of the head, frequent but transient changes in the hydrostatic pressure of these unanesthetizecl animals occurred. These transient pressure changes could have influenced fluid formation, and thereby, have resulted in a scatter of data which made their measured decrease in the mean rate of CSF formation not statistically significant.
PRESSURE,
cm Hz0
FIG. 3. Effect of hydrostatic pressure on rates of CSF formation demonstrated by paired comparisons. The data are the same as those seen in Fig. 2 except that corresponding rates of formation measured in individual rabbits under both high and low pressures are identified. The effect of pressure is shown to be independent of whether the initial perfusion pressure was high (solid lines) or low (broken lines). Since the effect of pressure was measured at only two intervals, these lines do not imply a linear relationship.
38
HOCHR’ALD
AND
SAHAR
Similarly, the effect of hydrostatic pressure on CSF production in hydrocephalic animals was studied by Bering and Sato (2 j and Sahar et al. ( 17). The latter investigators, by perfusion of individual, kaolin-induced hydrocephalic cats at both - 15 and 20 cm H,O pressures, were able to demonstrate a 70% decrease in bulk formation of fluid. The rate of CSF synthesis decreased from 0.0157 to 0.0049 ml/min with increased intraventricular pressure. The pressure gradient from the ventricular-to-pial surfaces was thought to be greater in these animals because of lack of communication between the ventricles and the subarachnoid spaces. This may explain the larger effect of hydrostatic pressure on CSF formation in these cats as compared to the normal ones reported here. The fact that hydrocephalic cats also show the inhibitory effect of increased pressure on CSF formation diminishes the possibility that inadequate mixing of ventricular fluid with newly formed CSF in the craniai subarachnoid space is the major cause of the observed phenomenon. Bering and Sato (2) suggested that there is CSF formation in the subarachnoid space of normal dogs, because they thought that the decreased formation of CSF in kaolin hydrocephalic clogs was due to the exclusion of fluid formed in the subarachnoid space. There is little direct quantitative data available concerning CSF formation in the cranial subarachnoid space. During cranial subarachnoid space-to-cisterna magna perfusion of cats, Lorenzo has been unable to detect any newly formed CSF in this spinal fluid compartment (personal communications). However, Sato and Bering (18) detected 0.014 ml/min in the cranial suharachnoid space of the clog. Although in normal animals mixing of perfusion fluid with CSF from the cranial subarachnoid space cannot be excluded, it is not the most likely explanation for the observed phenomenon. Changes in bulk absorption of CSF would not cause the observed changes in the concentration of the indicator substances used for measuring CSF production. Therefore, the results of the experiments reported here suggest that the rate of CSF formation in normal rabbits and cats is affected by intraventricular pressure. That this effect may be linear within the hydrostatic pressure range employed in these studies cantlot be concluded from the data. The percentage decrease in CSF formation within the pressure limits examined (-5 to - 10 and 20-25 cm H20) in these experiments was approximately 45% in the rabhit and 50% in the cat. Similar results can be approximated from studies on the goat [Heisey et al., Fig. 1 (10) 1 or calf [Calhoun et al., Fig. 4 (3)]. Comparable effects were described in man by Rubin et al. (16) ; however, these authors attrihuted this apparent reduction of CSF formation, under increased outflow pressure, to a loss of fluid distal to the fourth ventricle. The mechanisms by which cerebrospinal fluid production is influenced
CEREBROSPINAL
39
FLUID
by intraventricular pressure are not clear. It does not seem feasible that 30 cm H,O pressure would directly affect the active or passive transport of anions or cations, although no systematic esamination of newly formed CSF under various pressures has made. Curl and Pollay (4) estimated that rate of formation of CSF across rabbit ventricular ependyma (0.37 pliter min -* cm -“) would require a driving force of about 1.5 X 1&G dynes cn?. In the experiments reported here, the effect (an opposite force) of approximately 30 cm H,O or 3.1 X IO4 dynes cm-? would not seem great enough to affect CSF formation of 1.8 pliters mill-l cm-? in the cat. The driving force resulting from a hydrostatic pressure of 30 cm HZ0 would not be expected to reduce CSF secretion by the choroid plexus because of the relatively small effect on the filtration coefficient (20). In addition, no evidence is available to suggest that such processes as the catalytic hydration of CO, or other enzyme-mediated reactions involved in CSF secretion would he suppressed 1,~ a 30 cm H,O increase in intraventricular pressure. It seems more likely that the effect of pressure of CSF formation is mediated directly or indirectly by the blood supply. Ames. Higashi, and Nesbett (1) demonstrated that the production of CSF and intracranial blood s~lpply is enhanced 1)~ increasing the Pco?. :\lthough no effort was made in our experiments to measure Pcoy levels at various intraventricular pressures, the effect of increased perfusion pressure on CSF formation was observed equally well in rabbits respired artificially as in cats allowed to breathe spontaneously. In addition, an attempt was made here in rabbits to minimize the effects of a possible gradual increase of Pco2 in time, hy reversing the order of high-low perfusion pressures. Blood flow to the spinal fluid-forming organs may have a limiting influence on CSF formation, ancE may to some degree, regulate it. Experimental data, however, shows that CSF pressure in dogs must be very close to arterial pressure before cortical 1~100~1 flow is affected (9). In the experiments reported here the relatively low pressure under which these animals were perfused would not have been expected to influence total cerebral blood flow. This does not rule out the possibility that local blood flow changes could occur in such an organ as the choroid plexus which is l&xc1 in spinal fluid. References 1.
;\., III, K. HIGASHI. AKD F. R. NESBETT. 1965. Effects of PCO, acetazolamide and ouahain on volume and composition of choroid-plexus fluid. J. Pltysiol.
Alhrts,
Lomforr
181 : 516-524.
2. BERING, E. A.. and 0. SATO. absorption of cerebrospinal 20 : 1050-1063. 3.
CALHOUN,
M.
C., H.
D.
1963. Hydrocephalus: Changes in formation and fluid Gthin the cerebral ventricles. J. Nc~vosz~rg.
HURT,
H.
D.
EATOS.
J. E.
ROLTSSEAU, JR., AND R. C.
40
4. 5. 6. 7. 8. 9.
10. 11.
12.
13. 14. 15. 16.
17.
18. 19. 20.
HOCHWALD
AND
SAHAR
HALL, JR. 1967. Rates of formation and absorption of cerebrospinal fluid in Holstein male calves. Bzt11. Unizq. Corm., Co/l. Agri. Exp. Sta. 401 : 22-26. CURL, F. D., and M. POLLAY. 1968. Transport of water and electrolytes between brain and ventricular fluid in the rabbit. Exp. Nmrol. 20 : 558-574. CCTLER, R. W. P., L. PAGE, I. GALICICH, AND G. V. WATTERS. 1968. Formation and absorption of cerebrospinal fluid in man. Brain 91: 707-720. DANDY, W. E., and K. D. BLACKFAX. 1914. Internal Hydrocephalus, an experimental clinical and pathological study. Amer. J. Dis. Child. 8 : 406412. DAVSON, H. 1967. “Physiology of Cerebrospinal fluid,” p. 132. Churchill, London. DE ROUGEMONT, J., A. AF~IES III, F. B. NESBETT, AND H. F. HOFMANN. 1960. Fluid formed by choroid plexus. J. Nrzlrophysiol. 23 : 485-495. HXGGENDAL, E., J. LBFGREN, N. J. NILSSON, AND N. ZWETNOW, 1969. Influence of induced changes in the cerebrospinal fluid pressure on the cerebral blood flow of dogs, pp. 275-285. In “Research on the Cerebral Circulation.” Third International Salzberg Conference. J. S. Meyer, H. Lechner, and 0. Eichhorn [eds.]. Thomas, Springfield, Ill. HF.ISEY, S. R., D. HELD, AND J. R. PAPPENHEIMER, 1962. Bulk flow and diffusion in the cerebrospinal fluid system of the goat. Amer. J. Physiol. 203: 775-781. HOCH~ALD, G. M., AND M. WALLENSTEIK. 1967. Exchange of albumin between blood, cerebrospinal fluid and brain in the cat. Amer. J. Physiol. 212: 11% 1204. MACRI, F. J., A. POLITOFF, R. R~BIK, R. DIXON, AND D. RALL. 1966. Preferential vasoconstriction properties of acetazolamide on the arteries of the choroid plexus. Int. J. Nenropltnrmacol. 5 : 109-115. MAREN, T. H., AND L. E. BRODER. 1970. The role of carbonic anhydrase in anion secretion into cerebrospinal fluid. 1. Plzarmucol. Exp. Ther. 172: 197-202. OPPELT, W. W., C. S. PATLAK, AND D. P. RALL. 1964. Effect of certain drugs on cerebrospinal fluid production in the dog. AIIZEY. J. Physiol. 206: 247-250. POLLAY, M., AND H. DAVSON. 1963. The passage of certain substances out of the cerebrospinal fluid. BraiN. 86 : 137-150. RUBIN, R. C., E. S. HENDERSON, A. EC. OMMAYA, M. D. WALKER, AND D. P. Rw. 1%6. The production of cerebrospinal fluid in man and its modification by acetazolamide. J. Nerkroszkrg. 25 : 430-436. SAHAR, A., G. M. HOCHWALD, AND J. RAXSOHOFF. 1970. Experimental Hydrocephalus: Cerebrospinal fluid formation and ventricular size as a function of intraventricular pressure. J. Nezwol. Sri. 11 : 81-91. SATO, O., AND E. A. BERING, JR. 1%7. Extra-ventricular formation of cerebrospinal fluid. Brain Nerzw 19 : 883-885. SCHREINER, G. E. 1950. Determination of inulin by means of resorcinol. Proc. Sot. E.Q. Biol. Med. 74 : 117-120. WELCH, K. 1963. Secretion of cerebrospinal fluid by choroid plexus of the rabbit. Amer. J. Physiol. 205 : 617624.
32, 30-40
XEUROLOGY
Effect
of
(1971)
Spinal
Cerebrospinal GERALD Departments Medical Rcrci~d
M.
HOCHWALD
Fluid
Pressure
Fluid
Formation
AND
ABRAHAM
of Neurology and Neurosurgery, Center, 550 First dvcrm-, New March
1. 19il;
Kczisiorl
York,
Kcccizd
on
SAHAR
I
New York University Nezw York 10016 April
R, 1Vl
The effect of spinal fluid pressure on cerebrospinal fluid (CSF) formation was measured in rabbits and cats. A decreased rate of CSF production with increased perfusion pressure was found, which was best illustrated by paired comparisons in individual animals during ventriculocisternal perfusion at both low (-5 to -10 cm H,O) and high (2025 cm H,,O) perfusion pressure. Each animal was perfused at either pressure for 2 hr (rabbits) or 2.5 hr (cats) for steady-state measurements. After a gradual change in hydrostatic pressure, perfusion continued for an additional 1.5 hr (rabbits) or 2 hr (cats) when steady-state measurements were repeated. The mean rate of CSF production decreased in rabbits from 0.0081 to 0.0045 ml/min after the perfusion pressure was elevated. This effect was independent of whether the animals were perfused initially under high or low pressure. In cats, after a similar increase in perfusion pressure, the mean rate of CSF formation decreased from 0.0212 to 0.0102 ml/min. Introduction
Various studies using the technique of verticulocisternal perfusion have shown that most of the cerebrospinal fluid (CSF) is formed within the cerebral ventricles (7, 10). Much evidence favors the choroid plexus as the site of formation of the fluid (1, 6, 8, ZOj, although the possibility for the flow of fluid across the ventricular walls must be considered (4, 11, 20). There are several factors which influence spinal fluid production. It has been postulated that the rate of production of fluid probably depends upon mechanisms such as the transport of solutes from the blood with water following passively, or the hydration of CO? (4, 7, 13). The secre1 This study was supported by Grants NS-05024 and NS-06.599 from the USPHS. Dr. Hochwald was a recipient of Special Research Fellowship Award 2 Fll NB01431 from USPHS. Dr. Sahar’s present address is Dept. of Neurosurgery, Hebrew University-Hadassah Medical School, Jerusalem, Israel. The authors thank Miss J. Brown, B.Sc., and S. Sullivan for their technical assistance. Reprint requests to Dr. Hochwald. 30
CEREBROSPINAL
FLL-ID
31
tion of CSF is dependent on metabolic energy since its rate of formation decreases under the influence of enzyme inhibitors such as acetazolamide, dinitrophenol, and ouabain (1, 7, 14, 20). Other factors influencing spinal fluid formation may be the blood supply to the spinal fluid-forming organs (, 1). The reduction of CSF formation with a decrease in plasma PCO, resulting from hyperventilation has been attributed in part to the narrowing of choroid plexus vessels (1 j. hlacri it al have suggested that the effect of acetazolamide may be from its capacity to constrict the choroid artery (12). Attempts to relate CSF formation with intraventricular pressure have been made by several investigators (2, 5, 10, 11, 16). From data obtained during ventriculocisternal or ventricle-to-lumbar perfusion, they concluded that alterations of intraventricular pressure had little or no significant effect on CSF production. However, other studies (3, 17) have demonstrated that the rate of CSF formation decreased with increased perfusion pressure. Calhoun ct nl. (3) were able to show this in calves during ventriculocisternal perfusion. Sahar, Hochwald, and Ransohoff ( 17) reached this conclusion from data obtained during steady-state lateral ventricle-tolateral ventricle perfusion in kaolin-induced chronic, hydrocephalic cats. In these experiments, changes in CSF formation were measured during a single perfusion under both high and low pressures. The present experiments were designed to detect the effect, if any, of intraventricular pressure on CSF production in normal rabbits and cats. Since the individual rates of CSF formation were varied, the results from each animal were analyzed separately in order to emphasize the effects of pressure changes. Materials
and
Methods
Perfusion of the ventricular system of rabbits was carried out by a slight modification of the technique described by Pollay and Davson (15). The animals were anesthetized with intraventus sodium Thiamylal (20 mg/kg) and were maintained on it throughout the experiment. During the course of the experiment rabbits were also given intermittent injections (6 mg ‘kg) of Flasedil (gallamine triethiodide) and artificial respiration. The lateral ventricle was penetrated with a 19gauge needle inserted with the aid of a stereotasic apparatus through a burr hole 10 mm behind the coronal suture and 8 mm lateral from the sagittal suture. The correct placement of this inflow needle was recognized by a sudden drop in pressure in the inflow tubing. The outflow system consisted of a 19-gauge needle which was attached to plastic tubing. The height of the outflow tubing determined the perfusion pressure. The cisterna magna was penetrated with this needle between the occiput and Cl. The technique for the perfusion of the ventricular system of cats has
32
HOCHWALD
AND
SAHAR
been described ( 11) . However, in these experiments, Flaxedil was omitted and the cats were allowed to breathe spontaneously. The perfusion fluid for all animals used was an artificial CSF which has been described previously (11). It also contained inulin and 1311-labeled rabbit or cat serum albumin as tracers for measuring CSF formation and absorption ( 11) . Inulin concentrations were determined in triplicate by the resorcinol method (9) ; radioactivity was measured in a well-type scintillation detector (Nuclear-Chicago). Both determinations were made to a probable error of 2%. The perfusion technique in general was similar to that previously described; the perfusion pressure was measured with a Statham transducer and was continuously recorded (11). The outflow volume was measured gravimetrically at 15min ilitervals. A successful experiment was one in which perfusion was maintained for approsimately 5 hr, and in which at the termination of the experiment, addition of dye to the perfusate verified the pathway taken by the fluid. Evans blue dye (1%) was perfused through the system for 20-30 min ‘prior to the exsanguination of each animal. A careful examination was made in the region of the inflow and outflow needles for loss of dye to tissue around these needles. Although no leaks were observed under these conditions, it is conceivable that a very small loss of fluid may have occurred during perfusion at high pressures. However, the measurement of CSF formation at all pressures during ventriculocisternal perfusion was dependent upon the dilution of a nondiffusible indicator and a leak after complete mixing would not affect this dilution. The effect of intraventricular pressure on the rate of CSF formation was studied in 18 rabbits and in 7 cats. Each rabbit was initially perfused at either high (20-25 cm H?O) or low ( -5 to - 10 cm H,O) perfusion pressure with respect to the interaural line. Subsequent to steady-state measurements, the pressure was gradually (30 min) reduced or elevated. Perfusion continued until a new steady state was reached and measurements were repeated. In these experiments the rate of inflow was 0.077 ml/min and approximately 1.5 hr was needed for equilibrium to be reached. Twelve of these rabbits were initially perfused at a high pressure and then subsequently at a low pressure. All of the cats were perfused at a high (20-25 cm H30) pressure for 2.5 hr and then at a low ( -5 to - 10 cm H?O) pressure for 2 hr. The rate of inflow of these animals was also 0.077 ml/min. The perfusion pressure represents the range within which all the perfusion took place, and for purposes of some calculations, each range was also considered as a group. The choice of perfusion at these two pressures was made only in order to determine whether the rate of CSF formation was sensitive to the hydrostatic pressure of the system, and therefore, does not indicate whether this relationship was linear. The perfusion
CEREHROSPINAL
FLUID
33
of one animal at two pressures during one experiment partially eliminates wide ranges of CSF formation rate values measured in a group of animals at the same pressure or in individual animals perfused on different occasions. In addition, perfusion at two different pressure intervals reduced the importance of the definition of the zero pressure at the time of perfusion. Cnlclrln/io~zs. The rates of formation and absorption of CSF were calculated according to Heisey rt nl. (10). At steady state, for substances removed by hlk flow, the rate of absorption of CSF is :
where C, clearance of test substance ( innlin or albun~in-l”‘I by bulk absorption; 1’. rate of flow; i, o, f, a, inflow, outflow, formation, and absorption of CSF, respectively : C, exponential mean ventricular concentration ; f, concentration of test substance. The rate of formation of CSF is therefore:
Results
The 13*1-labeled albun~in in the perfusate was monitored at 1.5~min intervals. With an inflow rate of 0.077 ml/min steady state was reached in appro-ximately 1 hr for the rabbit and 2 hr for the cat. This inflow rate was found to be satisfactory since it provided a sufficient volume of outflow fluid at elevated perfusion pressures, and it also allowed significant changes in concentrations of albumin-‘“‘1 and inulin to be detected.? Calculations were based on samples taken approximately 2.5 hr after perfusion began and 2 hr after the perfusion pressure was changed. The results of a representative experiment in which the effect of hydrostatic pressure on these physiological parameters in one rabbit is demonstrated can be clearly seen in Fig. 1. A decrease of approximately 30 cm H,O pressure resulted in a fourfold increase in CSF production. Under similar conditions the increase in absorption of fluid was variable because at the lower pressure the absorption was very small. The mean rates of formation and absorption from 18 rab2 Results calculated from inulin or albuminagreement. On occasion, however, dilutions of The reason for this was subsequently learned experiments with very small amounts of labeled fluid, and in the absence of a carrier protein, the apparatus. These animals were eliminated ratio of unbound/bound 1311 in the preparations
Is11 dilutions xvere generally in good these indicator substances did differ. to be due to the fact that in some protein ( < 1 pg/ml) in the perfusion some of the radioactivity adhered to from CSF turnover calculations. The used was less than 0.01%.
34
HOCHWALD
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p~*----------~~~ 100
20T025cmH20
-5 TO-IOcmHZO
90 80 70
FIG. 1. Effect of hydrostatic pressure on rates of CSF formation during ventriculocisternal perfusion of an individual rabbit. Co/C; X 100, outflow concentration as percentage of inflow concentration (albumin-1311). Vi, V,, V,, rates of inflow, absorption, and formation, respectively. These rates were calculated from measurements made during steady-state perfusion at 20-25 and -5 to -10 cm H,O pressures.
bits perfused at both high and low pressures can be seen in Table 1. The percentage decrease in formation at the higher perfusion pressure was approximately 45%. Absorption of CSF increased fivefold over this same pressure range. The individual and mean rates of CSF formation and absorption from seven cats perfused at both high and low pressures are summarized in Table 2. In addition, a comparison of the rates of formation determined from inulin and albumin-1311dilutions can be seen in Table 3. These rates (V,) were calculated according to the formula, Y, = Vi (Ci - C,)/C,, where Vi is the rate of inflow, Ci and C, are the concentrations of tracer substances in the inflow and outflow fluids, respectively. In this series of .animals, the percentage reduction in CSF production was about 50% dP
Perfusion pressure (cm H@)
20to25 -5 (1 Mean
to -10 f
SE.
I
OF HYDROSTATIC PKESSURE ON CSF AND ABSORPTION IN RABBITS CSF
FORMATION
formation (ml/min)
CSF
absorption (ml/min)
0.0045 f 0.0006= 0.0082 f. 0.0009
0.0298 0.0049
f f
0.0045 0.0020
CEREBROSPIKAL
EFFECT
OF HVDKO~TATIC FROM
FORMATION
rix INDIVIDUAL
CATS
pressure
(20-25
cat
ON CSF
PKESSLXE
AND ABSOK~~TION Perfusion
35
FLUID
cm
(-
HsO)
Perfusion pressure 5 to - 10 cm H,O)
Vf h (ml/mill)
V& c (ml/min)
v’r h (ml/min)
1; c (ml/min)
0.077 0.077 0.077
0.0100 0.0202 0.0060
0.0614
0.0250 0.0273
0.0010 0.0030
0.077 0.077 0.077
0.0195 0.0046
0.0217 0 ,055.~ 0.0650
0.0207 0 .0236
0.0002 0.0060
0.0118 0.0204 0.0196
0.0101 0.0091
no.
0.0386 0.0291
0.0027 0.0091 0.0102 f0.0026
0.077
” Vi, rate b Vf, rate
of inflow. of CSF formation.
c I’%, rate
of CSF
0.0356 0.0438 ~0.0063
0.0212 f0.0018
0.0071 0.0051 *o .OOl‘l
absorption.
CSF formation and absorption were greater in the cats than in the rabbits. The results from the larger group of animals, i.e., the IS rabbits, were analyzed more completely. Data from individual rabbits can be seen in Fig. 2. The range of values for CSF production in normal rabbits perfused at
EFITCT
01; HYDROSTATIC Fwohl D1Ltwo~ Perfusion (20-25
Cat
no. 1 2 3 -I 5 6 7
Mean fSE * Vf, rate b Vf, rate
cm
0~
pressure HsO)
vp1-CS.4) (ml/min)
V~(inulin) (ml/mh
0.0143 0.0230 0.0054
0.0131 0.0238 0.0065
0.0190 0.0024 0.0026 0.0094 0.0108 f0.0020
0.0158 0.0042 0.0041 0.0109 0.0112 fO.0026
of CSF of CSF
formation, formation,
ON CSF NONDIITIMBIX
PKESS~IW
calculated calculated
FORMATION CALCULATED I~XDKXTOKS (-5
b 1
Perfusion pressure to - 10 cm H?OI
vpr-CSA) (ml/min)
Vf(inulin) (ml/mini
0.0290
0.0214
0.0268 0.0161 0.0241
0.0270 0.0154 0.0183 0.0108 0.0225 0.0212
0.0111 0.0199 0.0191 0.0208 f0.0022 from from
dilution dilution
of cat serum of inulin.
0.0195 I!z0.0017 albumin-‘3’1.
36
HOCHWALD
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both pressures was about 15-fold. The mean difference in formation rate between both groups was 0.0036 ml/min (P
Prior to the development of the method of ventricular perfusion, studies of CSF formation could not readily delineate the effect of elevation of intraventricular pressure on absorption or formation of fluid. Recently, this pressure effect has been the subject of several studies with various species.
160 t-
.
1
I.
140
80I 60-
l .
-15 -10 -5 0 5 10 15 20 PRESSURE, cm Hz0
25
FIG. 2. Rates of CSF formation measured in 18 rabbits, each perfused at both low (-5 to -10 cm H,O) and high (20-Z cm H20) pressures. Formation rates of individual rabbits grouped according to perfusion pressure. Bars represent mean rates of formation, 0.0081 and 0.0045 ml/min * SE ( [ ). Note that in this nonpaired comparison, the ranges for both groups are similar and there is a good deal of overlapping.
CEREBROSPIKAL
FLUID
37
The most detailed studies reported were those of Heisey et al. on the goat ( 10) and Calhoun et al. on the calf (3). With the aid of the technique of ventriculocisternal perfusion, both groups of authors measured CSF turnover at various hydrostatic pressures. The former concluded that CSF production was independent of hydrostatic pressure, while the latter found that fluid formation decreased with increased perfusion pressure. The opposing conclusions of these authors may be the results of such factors as differences in species employed or the use of the anesthetized versus the unanesthetized animals. La more careful consideration of experimental data from both of those laboratories, however, may resolve this apparent discrepancy. In the experiments of Calhoun st al., CSF formation as a function of pressure was measured only at a limited number of pressure intervals between - 10 and 20 cm H,O in anesthetized calves. Heisey et al., in an attempt to establish a relationship between CSF formation and absorption with hydrostatic pressure, made measurements on unanesthetized goats at smaller pressure intervals. It is conceivable that, with the free movement of the head, frequent but transient changes in the hydrostatic pressure of these unanesthetizecl animals occurred. These transient pressure changes could have influenced fluid formation, and thereby, have resulted in a scatter of data which made their measured decrease in the mean rate of CSF formation not statistically significant.
PRESSURE,
cm Hz0
FIG. 3. Effect of hydrostatic pressure on rates of CSF formation demonstrated by paired comparisons. The data are the same as those seen in Fig. 2 except that corresponding rates of formation measured in individual rabbits under both high and low pressures are identified. The effect of pressure is shown to be independent of whether the initial perfusion pressure was high (solid lines) or low (broken lines). Since the effect of pressure was measured at only two intervals, these lines do not imply a linear relationship.
38
HOCHR’ALD
AND
SAHAR
Similarly, the effect of hydrostatic pressure on CSF production in hydrocephalic animals was studied by Bering and Sato (2 j and Sahar et al. ( 17). The latter investigators, by perfusion of individual, kaolin-induced hydrocephalic cats at both - 15 and 20 cm H,O pressures, were able to demonstrate a 70% decrease in bulk formation of fluid. The rate of CSF synthesis decreased from 0.0157 to 0.0049 ml/min with increased intraventricular pressure. The pressure gradient from the ventricular-to-pial surfaces was thought to be greater in these animals because of lack of communication between the ventricles and the subarachnoid spaces. This may explain the larger effect of hydrostatic pressure on CSF formation in these cats as compared to the normal ones reported here. The fact that hydrocephalic cats also show the inhibitory effect of increased pressure on CSF formation diminishes the possibility that inadequate mixing of ventricular fluid with newly formed CSF in the craniai subarachnoid space is the major cause of the observed phenomenon. Bering and Sato (2) suggested that there is CSF formation in the subarachnoid space of normal dogs, because they thought that the decreased formation of CSF in kaolin hydrocephalic clogs was due to the exclusion of fluid formed in the subarachnoid space. There is little direct quantitative data available concerning CSF formation in the cranial subarachnoid space. During cranial subarachnoid space-to-cisterna magna perfusion of cats, Lorenzo has been unable to detect any newly formed CSF in this spinal fluid compartment (personal communications). However, Sato and Bering (18) detected 0.014 ml/min in the cranial suharachnoid space of the clog. Although in normal animals mixing of perfusion fluid with CSF from the cranial subarachnoid space cannot be excluded, it is not the most likely explanation for the observed phenomenon. Changes in bulk absorption of CSF would not cause the observed changes in the concentration of the indicator substances used for measuring CSF production. Therefore, the results of the experiments reported here suggest that the rate of CSF formation in normal rabbits and cats is affected by intraventricular pressure. That this effect may be linear within the hydrostatic pressure range employed in these studies cantlot be concluded from the data. The percentage decrease in CSF formation within the pressure limits examined (-5 to - 10 and 20-25 cm H20) in these experiments was approximately 45% in the rabhit and 50% in the cat. Similar results can be approximated from studies on the goat [Heisey et al., Fig. 1 (10) 1 or calf [Calhoun et al., Fig. 4 (3)]. Comparable effects were described in man by Rubin et al. (16) ; however, these authors attrihuted this apparent reduction of CSF formation, under increased outflow pressure, to a loss of fluid distal to the fourth ventricle. The mechanisms by which cerebrospinal fluid production is influenced
CEREBROSPINAL
39
FLUID
by intraventricular pressure are not clear. It does not seem feasible that 30 cm H,O pressure would directly affect the active or passive transport of anions or cations, although no systematic esamination of newly formed CSF under various pressures has made. Curl and Pollay (4) estimated that rate of formation of CSF across rabbit ventricular ependyma (0.37 pliter min -* cm -“) would require a driving force of about 1.5 X 1&G dynes cn?. In the experiments reported here, the effect (an opposite force) of approximately 30 cm H,O or 3.1 X IO4 dynes cm-? would not seem great enough to affect CSF formation of 1.8 pliters mill-l cm-? in the cat. The driving force resulting from a hydrostatic pressure of 30 cm HZ0 would not be expected to reduce CSF secretion by the choroid plexus because of the relatively small effect on the filtration coefficient (20). In addition, no evidence is available to suggest that such processes as the catalytic hydration of CO, or other enzyme-mediated reactions involved in CSF secretion would he suppressed 1,~ a 30 cm H,O increase in intraventricular pressure. It seems more likely that the effect of pressure of CSF formation is mediated directly or indirectly by the blood supply. Ames. Higashi, and Nesbett (1) demonstrated that the production of CSF and intracranial blood s~lpply is enhanced 1)~ increasing the Pco?. :\lthough no effort was made in our experiments to measure Pcoy levels at various intraventricular pressures, the effect of increased perfusion pressure on CSF formation was observed equally well in rabbits respired artificially as in cats allowed to breathe spontaneously. In addition, an attempt was made here in rabbits to minimize the effects of a possible gradual increase of Pco2 in time, hy reversing the order of high-low perfusion pressures. Blood flow to the spinal fluid-forming organs may have a limiting influence on CSF formation, ancE may to some degree, regulate it. Experimental data, however, shows that CSF pressure in dogs must be very close to arterial pressure before cortical 1~100~1 flow is affected (9). In the experiments reported here the relatively low pressure under which these animals were perfused would not have been expected to influence total cerebral blood flow. This does not rule out the possibility that local blood flow changes could occur in such an organ as the choroid plexus which is l&xc1 in spinal fluid. References 1.
;\., III, K. HIGASHI. AKD F. R. NESBETT. 1965. Effects of PCO, acetazolamide and ouahain on volume and composition of choroid-plexus fluid. J. Pltysiol.
Alhrts,
Lomforr
181 : 516-524.
2. BERING, E. A.. and 0. SATO. absorption of cerebrospinal 20 : 1050-1063. 3.
CALHOUN,
M.
C., H.
D.
1963. Hydrocephalus: Changes in formation and fluid Gthin the cerebral ventricles. J. Nc~vosz~rg.
HURT,
H.
D.
EATOS.
J. E.
ROLTSSEAU, JR., AND R. C.
40
4. 5. 6. 7. 8. 9.
10. 11.
12.
13. 14. 15. 16.
17.
18. 19. 20.
HOCHWALD
AND
SAHAR
HALL, JR. 1967. Rates of formation and absorption of cerebrospinal fluid in Holstein male calves. Bzt11. Unizq. Corm., Co/l. Agri. Exp. Sta. 401 : 22-26. CURL, F. D., and M. POLLAY. 1968. Transport of water and electrolytes between brain and ventricular fluid in the rabbit. Exp. Nmrol. 20 : 558-574. CCTLER, R. W. P., L. PAGE, I. GALICICH, AND G. V. WATTERS. 1968. Formation and absorption of cerebrospinal fluid in man. Brain 91: 707-720. DANDY, W. E., and K. D. BLACKFAX. 1914. Internal Hydrocephalus, an experimental clinical and pathological study. Amer. J. Dis. Child. 8 : 406412. DAVSON, H. 1967. “Physiology of Cerebrospinal fluid,” p. 132. Churchill, London. DE ROUGEMONT, J., A. AF~IES III, F. B. NESBETT, AND H. F. HOFMANN. 1960. Fluid formed by choroid plexus. J. Nrzlrophysiol. 23 : 485-495. HXGGENDAL, E., J. LBFGREN, N. J. NILSSON, AND N. ZWETNOW, 1969. Influence of induced changes in the cerebrospinal fluid pressure on the cerebral blood flow of dogs, pp. 275-285. In “Research on the Cerebral Circulation.” Third International Salzberg Conference. J. S. Meyer, H. Lechner, and 0. Eichhorn [eds.]. Thomas, Springfield, Ill. HF.ISEY, S. R., D. HELD, AND J. R. PAPPENHEIMER, 1962. Bulk flow and diffusion in the cerebrospinal fluid system of the goat. Amer. J. Physiol. 203: 775-781. HOCH~ALD, G. M., AND M. WALLENSTEIK. 1967. Exchange of albumin between blood, cerebrospinal fluid and brain in the cat. Amer. J. Physiol. 212: 11% 1204. MACRI, F. J., A. POLITOFF, R. R~BIK, R. DIXON, AND D. RALL. 1966. Preferential vasoconstriction properties of acetazolamide on the arteries of the choroid plexus. Int. J. Nenropltnrmacol. 5 : 109-115. MAREN, T. H., AND L. E. BRODER. 1970. The role of carbonic anhydrase in anion secretion into cerebrospinal fluid. 1. Plzarmucol. Exp. Ther. 172: 197-202. OPPELT, W. W., C. S. PATLAK, AND D. P. RALL. 1964. Effect of certain drugs on cerebrospinal fluid production in the dog. AIIZEY. J. Physiol. 206: 247-250. POLLAY, M., AND H. DAVSON. 1963. The passage of certain substances out of the cerebrospinal fluid. BraiN. 86 : 137-150. RUBIN, R. C., E. S. HENDERSON, A. EC. OMMAYA, M. D. WALKER, AND D. P. Rw. 1%6. The production of cerebrospinal fluid in man and its modification by acetazolamide. J. Nerkroszkrg. 25 : 430-436. SAHAR, A., G. M. HOCHWALD, AND J. RAXSOHOFF. 1970. Experimental Hydrocephalus: Cerebrospinal fluid formation and ventricular size as a function of intraventricular pressure. J. Nezwol. Sri. 11 : 81-91. SATO, O., AND E. A. BERING, JR. 1%7. Extra-ventricular formation of cerebrospinal fluid. Brain Nerzw 19 : 883-885. SCHREINER, G. E. 1950. Determination of inulin by means of resorcinol. Proc. Sot. E.Q. Biol. Med. 74 : 117-120. WELCH, K. 1963. Secretion of cerebrospinal fluid by choroid plexus of the rabbit. Amer. J. Physiol. 205 : 617624.