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"33 .33 ‘ 33b..- .21; 33;... ‘1'“ “1"" '1' - 33"” ’. -‘ "if 3" «.31.... .33. 7' “1 ‘1tv 31.136133 ‘ , 11311113? “flag. a \ $53; .1 I. r. ' “,2th 3 . I. ' 3:1: w;""" 3~ - _ ,1..." l 1: 3:: J‘ v . 'Wkflfm; -_. _ -4" - l 1"? 4‘11; 51111211111, 1 1m ‘1 ,3! $3111} 1.9234 ‘1: x," V..- x ". ' ‘I U ' E Z ' '14"??? ;v. w. i 1 . 1! $11 , 11" .LJ‘R. 3‘ ‘...,‘1'31{‘ 93,"! .V 3 "111213 . . 1.711111%;th {if 11:3:25‘3313'1'11 1:313 1:111?“ ,. -' '13; ‘ '1'?" 4‘ " " " “W! 513913“. 3‘3"“‘9333 31'3131‘1 :3 r- .i . .3 .nu.‘1\jq ' 311'”. .w. “511111.71“! 1‘13" b. .3: “‘1 g "311313134 3.331 3'. 33:233.333.33.3331. w... . .. . 13“ 1“ ”F’- ' ’w .9; 311... 2.13.33... $3.3}...- . cH cocoeouom mmo cu oocmpmfimom m> mmHommm :oHuQLomnm mmo on oocmpmwmom ucm Am¢v oumc coHpmsLom UHSHm HmcHamocnoLoo :H :Owumwcm> moflooqm .F anmh 12 reactions leading to the secretion of CSF by the choroid plexus. However, Maori gg gl. (1966) postulated that part of the effect of Diamox on CSF formation was mediated via the vascular system, since they observed choroidal arteriolar constriction with intravenous Diamox administration that was comparable to that produced by intravenous norepinephrine, both of which led to a reduction in CSF formation rate. The effects of acidosis and alkalosis on CSF formation rates were studied by Oppelt gg gl. (1963) using a ventriculocisternal perfusion technique in adult dogs. They reported that acidosis induced by either 5—10% CO2 inhalation, or by the intravenous administration of 0.1—0.3N HCl produced no significant effect on the CSF production rate. Metabolic alkalosis produced by intravenous infusion of 0.5M NaHCO3 reduced CSF production 23% below control levels, whereas respiratory alkalosis (hyperventilation) resulted in a A6% reduction in CSF production rate. Ames g2 31. (1965) observed that inhalation of a 10% CO2 gas caused marked dilation of the choroid plexus blood vessels and an increase in the rate of choroid plexus fluid formation which averaged 66% above control levels after 30 minutes and 90% after 1 hour of CO2 exposure. They also observed that with hyperventilation there was a 68% drop in PaCO2 and constriction of the choroid plexus blood vessels which produced difficulty in obtaining fluid from the choroid on CSF plexus. They suggested that the effects of PaCO2 formation were secondary to its vascular effect and that the 13 rate—limiting factor in the formation of CSF is the blood supply to the choroid plexus. Pappenheimer also suggested that the rate of CSF formation may be limited by the blood supply to the choroid plexus (cited by Ames _g _l., 1963). Welch (1963) demonstrated that 25% of the plasma entering the rabbit's choroid plexus is converted into CSF. There is further evidence that there is a relationship between cerebral blood flow and the formation rate of CSF in mammals. Snodgrass and Lorenzo (1972) found that CSF formation rates in cats were altered by 11% when rectal temperature increased or decreased by 1°C within the range 31—A10C. They postulated that this effect was similar to that of temperature on cerebral blood flow. Rosomoff (195A) demonstrated that cerebral blood flow varies linearly with body temperature between the range 30—U50C, and that a 1°C change produced a 6.7% change in cerebral blood flow. A more direct relationship was found by Bering (1959) in which he observed that changes in CSF formation rate correlated with both cerebral blood flow and cerebral oxygen consumption. Carey and Vela (197”) found that the reduction of systemic blood pressure which was sufficient to reduce cerebral blood flow also reduced CSF formation rates in dogs. These workers demonstrated that when systemic arterial blood pressure was reduced to 62 i 1 mm Hg from control levels (119 1 9 mm Hg) by hemorrhage, there was a significant reduction (”0%) in CSF formation rate as measured using the inulin dilution technique. With the restoration of mean arterial 1“ blood pressure by total replacement of blood volume, CSF production rate rose to approximately pre-hemorrhagic control levels. Weiss and Wertman (1978) reported that when systemic blood pressure was lowered to an extent at which cerebral perfusion pressure (CPP; mean arterial blood pressure minus intracranial pressure) fell below 55 mm Hg, CSF formation rate decreased. They found there was no difference in the CSF production rate due to variations in intracranial pressure or to systemic blood pressure for those animals in which CPP was maintained at levels greater than or equal to 70 mm Hg. They suggested that cerebral autoregulatory mechanisms could not maintain adequate choroidal blood perfusion when cerebral perfusion pressure was below 55 mm Hg. 2.2. Cerebrospinal fluid absorptigg Early studies indicated that CSF moving from the cerebral ventricles to the subarachnoid spaces was being drained into the blood vascular system. Dandy and Blackfan (191A) and Frazier and Feet (191”) showed that when the aqueduct of Sylvius was blocked, internal hydrocephalus developed and there was an increase in intraventricular pressure. This suggested that the main CSF drainage route is distal to the aqueduct. Weed (191”) showed that fluid in the subarachnoid space was drained into the large endocranial venous sinuses via the arachnoid villi. He injected dyes into the subarachnoid space of dogs and found them in the 15 arachnoid villi and sagittal venous sinuses. Welch and Friedman (1960) showed histologically that the arachnoid villus in monkeys is a labyrinth of small interconnecting tubules which project into the lumen of the sinus. These investigators also excised pieces of dural membrane containing arachnoid villi and mounted the membrane so that it separated two fluid filled chambers. A pressure of 10 mm H20 was observed to be the critical opening pressure which allowed fluid flow from the CSF side of the membrane to its perfused side. When sagittal pressure exceeded CSF pressure, fluid flow stopped. These authors concluded that the arachnoid villi act as unidirectional valves which permit the flow of CSF into the venous system while preventing the flow of blood in the opposite direction. 2.2.1. Quantitative measurement of cerebrospinal fluid absorptigg Two lg gigg techniques have been used to measure the rate at which molecules are removed from CSF: 1) intracisternal injection of test substances, and 2) perfusion of the ventriculocisternal system with an artificial CSF containing test molecules. Davson gg gl. (1962) injected a measured volume of fluid (0.1 ml) which contained known concentrations of zuNa, inulin and sucrose into the cisterna magna of anesthetized rabbits to determine the relative rates at which substances were removed from the subarachnoid space. One hour after injection, the total cranial volume of CSF (1.0-2.0 ml) was 16 withdrawn from the cisterna magna and analyzed for 2“Na, inulin and sucrose. The removal rate for different substances from the CSF system was determined by the difference in the amount of test substance injected into the CSF and the amount remaining in the withdrawn fluid at the end of 1 hour, expressed as a ratio of the amount injected. 23Na was used as the reference substance and the removal of injected molecules from CSF were expressed as a percentage of 21‘Na loss in which the relative rates of loss for 2uNa, sucrose and inulin were: 100%. 64% and 43%, respectively. Davson concluded that the slow loss of inulin from the CSF was due to its being removed only by bulk absorption from the subarachnoid space. The larger rate of loss of sucrose was due to its diffusion in the extracellular spaces of the brain in addition to its bulk absorption. Pappenheimer gg gl. (1962) perfused the ventriculocisternal system of unanesthetized goats and estimated quantitatively a value for CSF absorption by calculating an inulin clearance from the CSF system. Inulin was assumed to be removed mainly by bulk absorption, since at a cerebroventricular pressure of -15 cm H2O all inulin entering the lateral ventricles was recovered in the cisternal effluent. This indicates that inulin does not diffuse across the ventricular ependyma. They also observed that when intraventricular pressure was changed, inulin clearance varied linearly with pressure. This suggested that there was a direct relationship between the rate of bulk 17 fluid absorption and CSF hydrostatic pressure. Bulk absorption of CSF varied from zero at an intraventricular pressure of -15 cm H20 to approximately 0.4 ml/min at an intraventricular pressure of +30 cm H20. The technique of ventriculocisternal perfusion has been employed in the determination of CSF absorption in a variety of species and with other high, molecular weight molecules. Anderson and Heisey (1972) use the clearance of radio—iodinated human serum albumin (RIHSA; M.W. = 65,000) as a measure of bulk absorption rate in chickens, and showed that it was removed from CSF by a pressure-dependent process which was comparable to results found in monkeys, using blue dextran (M.W. = 2 X 106) as the non—diffusible indicator (Martins g3 31., 1976). 2.3. The quantitative measurement of molecular movement from the CSF Material is removed from CSF by several mechanisms: bulk flow of fluid via the arachnoid villi into the blood, diffusion of substances into nervous tissue or blood, and active transport of substances into nervous tissue or blood. Bulk absorption refers to the pressure-dependent removal of CSF and its contents through the arachnoid villi. For example, large molecules such as inulin (M.W. = 5200) and dextran (M.W. = 70,000) leave the CSF system by a process comparable to glomerular filtration in the kidneys (Heisey gg gl., 1962). Since there is no filtration across the arachnoid villi (Welch and Pollay, 1961; Davson, 1967). clearance by bulk absorption is one mechanism for a substance to leave the CSF system. The passive or active (non—bulk) movement of substances into the blood can also account for the loss of some materials from the CSF system. Heisey g3 g1. (1962) measured molecular movement of tritiated water (TOH). urea, creatinine and fructose from the CSF system using the ventriculocisternal perfusion technique. They perfused various test substances into the brain's ventricular system in goats and calculated the rate of molecular loss from the cerebral ventricles as a function of the difference between the total clearance of test substance and the amount lost through bulk absorption. They found that diffusional loss of test molecules from CSF was inversely related to the size of the molecule, in which TOH was greater than urea, creatinine and fructose, in terms of decreasing permeability. The transependymal efflux rates for these test substances were independent of CSF intraventricular pressure over the range —10 to +30 cm H O. 2 2.3.1. Molecular exchange between CSF, brain and blood during hypercapnia Early studies using intravenously injected vital dyes demonstrated that although many body tissues stain, the brain is relatively free of staining. which provided the concept of blood-brain and blood-CSF barriers. Breathing C02 (hypercapnia) appears to increase the permeability of the blood—CSF and blood-brain barriers to a variety of substances. Clemedson gg a1. (1958) found that 19 inhalation of 7-30% CO2 caused staining of the brain parenchyma by intravascularly injected trypan blue, an acidic dye which does not normally penetrate the central nervous system (CNS). They further observed that hypercapnia in rabbits and guinea pigs produced more staining of the brain parenchyma than it did in cats. A longer exposure to CO2 was necessary to produce trypan blue staining in the CNS in all animals using 7-10% CO concentrations than when 11—30% C02 2 concentrations were used. These investigators demonstrated that there was no dye penetration if trypan blue was administered after the period of 7-30% C02 inhalation. This indicated that the abnormal permeability of the blood—brain barrier was rapidly reversible. Cutler and Barlow (1966) studied the uptake of labelled 125—1 albumin in male guinea pigs breathing 10% or 25% C0 for periods of time varying 2 from 5 minutes to 8 hours. They suggested that albumin entry into the brain was related both to the length of CO exposure 2 and to the elevation of PaC02. They observed no labelled albumin in the brain parenchyma during metabolic acidosis produced by intravenous infusion of 0.1N HCL. Lending t al. (1961) found that inhalation of 7% CO2 increased the rate of entry of intravenously injected 131-I albumin twenty times over its normocapnic levels into the cisterna magna fluid of puppies while only a four-fold increase was observed in the adult dogs over its normocapnic level. They attributed the differences in results between puppies and adult dogs to be due to the arterial pH and CO contents produced in puppies 2 20 but which were not produced in the adult dogs. Hochwald gg El' (1973) reported that during steady-state perfusion of the ventriculocisternal system, 10% C02 inhalation in anesthetized cats caused more than an 11-fold increase in the amount of intravenously injected 125—1 albumin into the brain ventricles. With the removal of 10% CO as the breathing gas 2 mixture, the amount of 125—1 albumin in the CSF came back approximately to normocapnic steady—state levels, indicating that the effect of hypercapnia on the blood-CSF barrier was reversible . These permeability changes of the blood—brain and blood-CSF barriers observed during hypercapnia were observed to be related to the magnitude of elevated PaCO2 (Clemedson _g _l., 1958; Lending gg gl., 1961) but not following reduction of blood pH by acid infusion (Cutler and Barlow, 1966). Elevation of blood PCO2 causes vasodilation of the cerebral vasculature and an increase in cerebral blood flow (Wolff and Lennox, 1930: Kety and Schmidt, 1948; Reivich, 1964). Cerebral blood flow has been shown to increase linearly with PaCO2 over the range 15 to 76 mm Hg (Smith gg gl.. 1971: Grubb gg _l., 1974), but is not correlated with blood pH (Reivich, 1964). Changes in cerebral vasculature hemodynamics during hypercapnia may be factors leading to the increased entry rate of substances into the brain and CSF since the effects of vascular permeability of the CNS were observed to be readily reversible when CO2 inhalation was discontinued as the breathing gas mixture (Goldberg gg gl., 21 1963; Hochwald gg 1., 1973). 2.4. Cerebrospinal fluid volume Pappenheimer gg g}. (1962) introduced a method for estimating the volume of CSF during perfusion of the CSF system of goats with an artificial solution containing test molecules. They intégrated the transient rise in molecular concentration as a function of time. Their method of estimating the distribution volume of a test molecule accounts for the total amount of material added to the system by perfusion inflow and subtracts from the amount recovered from perfusion effluent and the amount lost by clearance from the ventricles. It is assumed that the calculated steady state clearance rate occurred throughout the period of rising effluent concentration preceeding the steady state concentration. These investigators perfused the lateral ventricles to the frontal subarachnoid space and calculated the distribution volumes of inulin (22.7 ml), creatinine (26.0 ml), and Diodrast-I131 (25.3 ml) to be approximately equal to the total volume of fluid that can be drained from the goat's cranium. They also estimated the distribution volumes of these test substances in the perfused ventriculocisternal system. They found them to be slightly less than half of the total CSF volume; inulin (11 ml). creatinine (8.5 ml), and Diodrast-I131 (8.5 ml). Other investigators have reported distribution volumes using Pappenheimer's method in other species and with 22 other test substances. Bering and Sato (1963) found for the anesthetized dog, that the distribution volume in the ventriculocisternal system was 3.0 ml for inulin and 5.2 ml for RISA (131-I serum albumin). They attributed the distribution volume to include the lateral, third. and the fourth ventricles, the subarachnoid space of the posterior fossa, and the cisterna magna. The combined volumes of the two lateral ventricles and the third ventricle calculated from ventriculo—aqueductal perfusions were 1.6 ml for inulin and 2.2 ml for RISA. Cutlerb t 1. (1968) perfused the ventriculocisternal system of kittens and adult cats with 125-1 albumin. They recovered less than 2% of the total 125-1 albumin from the brain, and suggested that the distribution volume of 125-1 albumin represents the perfused compartment volume. They used a negative outflow pressure so that bulk fluid absorption was zero. They found distribution volumes of 0.84 ml and 0.75 ml in the kitten and adult cats, respectively. Sahar gg g}. (1970) measured the distribution volume of 131—I cat serum albumin in normal and kaolin-induced hydrocephalic cats and found correspondence between the calculated distribution volumes and the direct volume measurements of silicone rubber casts of the perfused space. These investigators found that the volume of the silicone cast made during ventriculocisternal perfusion at zero cm H20 intraventricular pressure was 0.74 ml. a cast volume which included not only cerebral ventricles but some portion of the cranial subarachnoid space in which residual 23 casting material was found. They suggested that during ventriculocisternal perfusion in normal cats, the distribution volume (0.50 ml) measured at —8 cm H20 represented only the cerebral ventricular volume and not that of the subarachnoid spaces. At higher perfusion pressure, they found an increase in measured volume which they attributed to perfusion fluid entering the subarachnoid spaces. This conclusion was supported by perfusions with silicone casting material, which also entered the subarachnoid space when perfusion pressure was high. III. STATEMENT OF THE PROBLEM The purpose of this study was to determine in anesthetized cats the effect of hypercapnia on cerebrospinal fluid (CSF) dynamics. The ventriculocisternal perfusion technique was used to establish steady—state concentrations of inulin and sucrose in the cerebral CSF compartments. Analysis of steady—state conditions, during both normocapnia and hypercapnia allowed deductions about the effect of hypercapnia on CSF absorption and formation rates as well as the molecular exchange between CSF and blood, and CSF and brain. Mathematical analysis of the transient approach to steady-state concentration of test molecules and the transient decrease in concentrations using a perfusion inflow free of test molecules provided information concerning the effects of hypercapnia on the volume of the cranial CSF compartment. 24 IV. MATERIALS AND METHODS 4.1. General operative procedures Mongrel, male or female cats, Fglig domesticus, weighing 3.0-4.5 kg were anesthetized with an intraperitoneal injection of Dial—urethane (0.6 ml per kg; Appendix E). A catheter (PE—60 tubing) was placed in the femoral artery for monitoring arterial blood pressure and for obtaining arterial blood samples for pH and PCO2 measurements (Appendix A). A femoral venous catheter (PE—60 tubing) allowed for administration of anesthesia supplements and of a 0.9% sodium chloride drip to maintain hydration of the animal. The trachea was cannulated with a 1 cm diameter vinyl tube which was attached to a respiratory valve assembly for administering gas mixtures and connected to a pneumotachograph (Model 7324 Dynasciences, Bluebell, PA) for monitoring respiration. A heating pad was used to keep body temperature at 38° 1 0.50C which was continuously monitored using a rectal thermistor probe (Model 400 Tele-Thermometer, Yellow Springs Instrument 00., Yellow Springs, Ohio) placed 10 cm beyond the anal sphincter. 4.2. Brain ventricular and cisternal puncture 25 26 The animal's head was secured in a stereotaxic frame (Model 1430, David Kopf Instruments, Tujunga, CA), by infra-orbital and serrated palate clamps and ear bars inserted into the external auditory meatus. A 4 cm long incision was made through the skin of the head from a point 4 cm from the supraorbital ridge to extend posteriorly to a point 1 cm behind a line posterior to the near dege of the ear margin. Skin, subcutaneous fascia, and muscle were retracted to expose a 3 cm2 area of skull surface. Using a 2 mm diameter dental burr, two 4x4 mm holes were drilled through the skull 13.5 mm anterior to the ear bars and 2.5 mm lateral from the animal's cerebral midline. Two ventricular cannulae (Figure 1) which were connected to a motor driven syringe pump (Model 940, Harvard Apparatus Co., Dover, MA) and to a pressure transducer (Model P230. Grass Instrument Co., Quincy, MA) for recording CSF pressure were lowered separately through the holes drilled in the skull and through brain matter until the probe tips penetrated the lateral cerebral ventricle. Communication with the cerebral ventricles was indicated by a drop in the CSF pressure as the probes penetrated the walls of the lateral ventricles. Figure 2 shows the arrangement of the apparatus used for the ventriculocisternal perfusion experiments. A single cisternal needle (21 ga.: metal hubless needle) held in a micromanipulator (Model MM-3; Eric Sobotka Co., Inc., Farmingdale, NY) was positioned with its point on the atlanto—occipital membrane at the midline. midway between Figure 1. 27 The drawing is a cross-sectional view of the lateral ventricular cannula assembly which is attached to a stereotaxic electrode carrier (Model 1460, David Kopf Instrument, Tujunga, CA) by the carrier bar (A). A brass block (E) is drilled to accept two pieces of 23 ga. needle tubing (B+C) and 21 ga. needle tubing (D) which are press fitted and soldered to the block. Polyethylene tubing (PE-50) from a syringe-drive pump is attached at (B) and to a pressure transducer for measurement of intraventricular pressure (C). A 1 mm diameter glass capillary used to make the probe (F) is sharpened and heat sealed using a vertical micropipette puller (Model 7008, David Kopf Instrument). Holes in the side of the glass probes are made under magnification by grinding with a fine grit rotary wheel. The glass cannula is cemented (Devcon, 5-min epoxy) onto the 21 ga. needle tubing (D). 28 FROM ' " . T0 PRESSURE PUMP _ TRANSDUCER F————GLASS FIGURE 1 29 .Aczogm uocv Looznmcmcu oesmmoea w on nouooccoo mfizccmo Hmfleopcm HmLOEom o no memos an uoLOpHcoe mmz oczmmoeq vocab HmHLouL< .Looscmcmcp oczmmoea m mafia: voLOpwcoe mm: zufi>wpom accumcfiamoc scan: sock .masccmo awesome» mpH on cocomuum zaneommm o>~w> m cmSOLSp oczuxfie an soon CH moo wowlw so go cam Econ Eocene nocwamcfi Hweficm one .mofisoonoe Looms» mo mwmzamcm com new mewzmwoz com wouooHHoo cum umo ho moHaEmm new A.oo unmascpmcH mmmcm .Pham Honozv Lopczoo noun m wean: nousmmoe ma Ao>v one; onmpzo .mcmme mccopmwo on» oacfl noncomcw masccmo m spas .mocwso ..oo pcoechmcH mmmgo .ummm Hovozv Loozvmcmep ocsmmoea m nmsocnp notepficoe zfimsoscfipcoo we oczmmoca cmasoficuco> mmo .Aoa ..oo pcmsscumcH vLm>me .ozo Hocozv assn o>figu1omaL>m m >n coHHocucoo an AH>V mum; zofiucfi mmo .oaofieuco> Hmcnocoo Hmcmpma on» nmsognu vomsucoq m“ Nmmov ofisam HmchmocnoLoo HwHonHme cm ofifinz osmgm oflxmuoogoum m 2H coczoom mfi umo venwuosumocm an no omen one .mcofimsucoq Emflsowcuco> Hmcnogoo Lou coHmewqoLa amazoeficoaxo on» no OHmeosom .m oeswfim N MMDUHW m_< mmoaomz nM maesqm fl. muonomzqmp mmnmmmme 31 the atlas and the base of the skull and in a plane parallel to the stereotaxic frame. A 50 cm length of polyethylene tubing (PE-90) was attached to the needle as the outflow cannula. The needle was advanced approximately 8 mm to puncture the atlanto-occipital and dural membranes and then withdrawn slowly until CSF could be withdrawn freely through the outflow cannula Ventricular perfusion was begun using an artificial cat cerebrospinal fluid (CSF), the electrolyte composition of which is reported in Appendix D. The outflow cannula was positioned in a photocell drop counter (Model PTT1, Grass Instrument Co., Quincy, MA) and the ventricular hydrostatic pressure was adjusted by the level of the cisternal outflow tubing with repect to the level of the stereotaxic ear bars which were set at zero pressure. Arterial blood pressure, respiration, perfusion outflow rate and ventricular pressure were monitored throughout the experiment using a polygraph (Model 5?, Grass Instrument Co., Quincy, MA). 4.3. Experimental perfusion with test molecules 4.3.1. Protocol A. The animal inspired either room air (normocapnia) or an 8-10% CO in air gas mixture (hypercapnia) while the CSF 2 perfusion fluid was substituted with another, containing known concentrations of inulin and tritiated sucrose. After approximately 30 minutes of perfusion during either 32 normocapnia or hypercapnia, a steady-state concentration of test molecules in the cisternal effluent was reached. At 60 minutes, the inspired gas was changed to either room air or 8-10% C02; opposite of the mixture used initially. Perfusion of the ventricular system continued for an additional 30 minutes at which time the CSF perfusion fluid was changed to one containing inulin and 1"C labelled sucrose, and perfused for an additional 60 minutes. Data from a representative experiment are shown in Figure 4 (Section 5; Results). 4.3.2. Protocol B. Perfusion experiments were similar to those described in Protocol A, time prior to 60 minutes, using inulin, radiolabelled sucrose and dextran (New England Nuclear, Boston, MA) as test molecules. After steady state concentrations of test molecules in the cisternal effluent were achieved the inflow perfusion fluid was changed to one containing no test molecules and perfusion continued for an additional 60 minutes under the same breathing condition (normocapnia or hypercapnia). In some animals, the entire experiment was repeated but under the alternate respiratory condition (room air or 8-10% CO2). These experiments lasted approximately 120 minutes for each respiratory condition or a total time of 240 minutes if both breathing conditions were achieved in the same animal. Representative data from one such experiment in which the animal breathed room air only is shown in Figure 5 (Section 5: Results). 33 4.4. Experimental criteria Following every ventriculocisternal perfusion experiment, a methylene blue solution was perfused for ten minutes through the ventricular needles to confirm cannula placements. The animal was killed with an intravenous injection of sodium pentobarbital, the skull was removed, and the ventricles were examined for methylene blue stain. The appearance of methylene blue staining on the cortex would suggest perfusion had occurred other than in the ventriculocisternal spaces. This precluded using data from that experiment. When an excessive amount of blood was found in the cisternal effluent samples during the perfusion experiment, data from that experiment were not used. Steady state measured and calculated data were selected from perfusion experiments on the basis of two experimental criteria: 1) the fluid mass balance equation (Vi + Vf = V0 + Va) was satisfied and 2) variances of test molecule concentrations in the perfusion effluent were less than or equal to 0.0010. Data which did not meet these two criteria were not used in this study. 4.5. Measurement of perfusion inflow and outflow rates and concentrationg The inflow and outflow rates of ventriculocisternal perfusion were determined gravimetrically for each experiment by collecting CSF effluent over timed periods in tared vials. The inflow concentraion of test molecules was determined by 34 analysis of an aliquot from the inflow syringe and outflow concentrations were determined similarly from measured drops of the cisternal effluent. Concentrations of radio-labelled sucrose and dextran were analyzed using a liquid scintillation counter (Model 3150?, Beckman Instruments, Fullerton, CA). (Appendix B). Inulin concentrations were determined colorimetrically by the resorcinol method without alkali treatment (Appendix C). 4.6. Mathematical analysis and calculations Principles and equations used in the calculation of bulk absorption rate, CSF formation rate, and the transependymal efflux rate have been previously described (Pappenheimer g3 g1., 1961; Heisey t al., 1962) and are ———_ presented below. 4.6.1. Definition of symbols: V = volume flow rate (ul/min) i,o = subscripts denote inflow and outflow volumes or concentrations f,a : subscripts refer to bulk formation and absorption of CSF, respectively. 0 = concentration (quantity/ul) Ev = exponential mean concentration in the ventricular system. = ci-co/ln(c./co) (quant1ty/pl) 1 when c1 = 0, the function above is undefined 30 __.,,~- «o.- 35 and Ev was calculated as: co + 0'37(°i'co) as described by Pappenheimer gg _l. (1961). steady-state transport of any substance, x, from or into the CSF perfusion system = V.c.-V (quantity/min). clearance of x = nx/c. c may be chosen as Ev or 00 depending upon whether one evaluates clearance from the ventricular system or from fluid reabsorbed distal to the fourth ventricle (pl/min). efflux coefficient of any substance, x, from the perfusion system by means other than bulk absorption (ul/min) Rate of bulk absorption, Va Heisey gg g1. (1962) determined that the ependymal linings of the ventricular system are virtually impermeable to inulin. The inulin lost from the CSF system can be accounted for by bulk fluid absorption distal to the fourth ventricle since the rate of inulin lost varies linearly with the hydrostatic pressure in the CSF system (Heisey g3 g1., 1962). .c - V c )/c = clearance of inulin, F. (1) o o o in where c's are inulin concentrations. 36 4.6.3. Rate of cerebrospinal fluid formation, Vf The diffusion of inulin from the ventricular system into brain tissue is negligible (Heisey g3 g1., 1962; Rall gg gl., 1962), therefore any dilution of inulin during passage through the ventricles results from newly-formed inulin-free fluid by the animal. Vf = Vi(ci—co)/co (2) where c's are inulin concentrations 4.6.4. Efflux coefficient, K (non-bulk clearance) The total clearance of molecules from the CSF system which are smaller than inulin or dextran and are not present in significant concentrations in blood has two components: (1) the clearance due to diffusion and/or active transport, and (2) clearance due to bulk absorption. The clearance of the small molecule by bulk absorption is given by: c F = F B in _° (3) c x where: co = ratio of the cisternal outflow and exponential mean 5 x ventricular of the small molecule, x. F = »inulin clearance 37 FB = clearance of x by bulk absorption Subtracting the bulk absorption clearance of x from its total clearance yields clearance due to diffusion or active transport. K = FX — FB (4) where: K = efflux coefficient of x (pl/min) 4.6.5. Volume of distribution, VD The distribution volume is determined by numerically integrating the transient rise of molecular outflow concentration to the steady-state with respect to time. n ;E% [Vici — oojcoj - anj:]Atj (5) VDx = ‘ vds c where: VDx = distribution volume of x (ul) Vi = perfusion inflow rate 01 : perfusion inflow concentration V = perfusion outflow rate associated with the jth OJ 38 sample. coj : perfusion outflow concentration of the jth sample. Fx = steady state clearance of the test molecule AtJ : outflow collection time between the j-1st and jth sample in minutes Ej = mean concentration of x in the CSF during the jth sample. c = E, steady-state mean ventricular concentration for molecules smaller than inulin. = co, steady state outflow concentration for inulin. n = total number of samples VdS : volume of the perfusion needles and tubing (dead space volume). j = first outflow sample beginning at time zero = or first outflow sample after time zero was adjusted (Appendix F) in which dead space volume was omitted. 4.7. Statistical analysis All data were analyzed statistically with a 0.05 probability criteria, using the Student's "t" (paired and group comparisons) or the analysis of variance tests on group comparisons (Sokal and Rohlf, 1969). Statistical comparisons were made using a programmable calculator (Model 67, Hewlett-Packard, Corvallis, Oregon) and a mini-computer 39 (PDP-11, Digital Equipment Corp., Maynard, MA). 4.7.1. Optimization routine The mean steady-state concentration for each test molecule was determined statistically by a direct search method called the Pattern Search routine (Gottfried and Weisman, 1973). This method evaluates the linear path of steady-state concentrations by calculating a summated variance and mean for every molecular concentration in that path, starting with the concentration at the longest perfusion time and sequentially working backward in time. The criterion for determining an optimum mean steady—state concentration for any molecule is to find the series of concentrations with the smallest calculated variance until the largest constant step increase in variance is encountered. 4.7.2. Non-linear curve analysis Transient data obtained during the "washout" phase of the perfusion experiments (Protocol B) were fitted by a multiple exponential equation by the method of non—linear least squares (Appendix F). The criterion for a "good fit" of experimentally observed points to exponential equations, was evaluated by a chi—square test (Equation F-2: Appendix F). V. RESULTS 5.1. Effects of hypercapnia on arterialng and PCO2 These experiments were designed to study the effects of increased brain blood flow on cerebrospinal fluid formation (Vf), absorption (Va) and on brain ventricular permeability (K). Carbon dioxide inhalation was used to increase PaCO because it is known to cause cerebral 2 vasodilation and an increase in brain blood flow. Data reported in Table 2 show that in 18 animals spontaneously breathing room air, PaCO2 was 28 mm Hg but it increased to 54 mm Hg when the inspired gas was an 8-10% COZ-in-air mixture. In addition, arterial pH decreased significantly (P<0.05) from 7.33 during room air breathing to 7.11 when the animal breathed the CO2 mixture, indicating a significant respiratory acidosis. 5.2. Effects of hypercapnia on steady state CSF dynamics Successful ventriculocisternal perfusions as judged by the criteria outlined in Section 4.4 were obtained in 18 cats breathing room air and 11 cats breathing 8—10% C02. Measured steady state data are reported in Table G-1 (Appendix G) for animals used in Protocol A and Protocol B 240 41 experiments (Methods; Sections 4.3.1. and 4.3.2.). In 9 of the 11 cats where comparisons can be made between room air and CO2 breathing, the most consistent finding was an increase Vo during the period of CO2 breathing. This agrees with data reported in Figure 3. Hypercapnia caused a decrease in the steady state outflow concentration (Co/Ci) of inulin in 4 animals and of sucrose in 6 animals. This is consistent with data for dextran and sucrose reported in Figure 5. There was no consistent pattern of change in the intraventricular pressure (CSFP) between air and C02 breathing and no change in this parameter is seen in the representative data reported in Figure 3. Arterial pH and PCO2 values in these animals were similar to those reported in Table 2. Calculated parameters derived from the measured quantities reported in Table G-1 are shown in Table G—2 (Appendix G). The mean values for Vf, Va and Ksuc determined in animals breathing air were not significantly different (P>0.05) from those breathing 8-10% C02. In addition, the resistance to bulk absorption, calculated as the ratio of intraventricular pressure (CSFP) to the bulk absorption rate (Va), was not significantly different (P>0.05) under the two respiratory conditions. The increase in VG (Table G-1; Figure 3) is not apparently caused by an increased resistance to bulk absorption. In some cases, the level of the outflow cannula (which determines cerebroventricular pressure in the perfused system) was such that bulk absorption was zero which 42 causes absorption resistance to be undefined. The mean values for Vf and K reported in Table G-2 suc (Appendix 6) did not show significant differences between air breathing and C02 breathing animals whether analysis was for means for all animals or for those exposed to both respiratory conditions. However, some animals showed marked increases in Vf during hypercapnia compared to the air breathing period. Weiss and Wertman (1978) showed that rate of CSF formation was affected by cerebral perfusion pressure (CPP) which they defined as the difference between mean arterial blood pressure (MABP) and cerebroventricular pressure (CSFP). We found that in 5 of the animals reported in Table G-2 which were exposed to both air and C02 breathing, CPP was maintained throughout the experiment at 68 mm Hg or higher. Data from these animals are shown in Table 3. Rate of CSF formation (Vf) increased significantly (P<0.05) from 17.9 ul/min during air breathing to 35.4 ul/min during 002 breathing. The Ksuc’ which is a measure of non-bulk clearance of sucrose from the cerebral ventricles, also increased signficantly from 7.7 ul/min to 23.4 ul/min in response to hypercapnia. In 3 of the animals (Cat# 8, 12 and 13) the air breathing period preceeded the CO2 breathing period while in the others (Cat# 17 and 22) the hypercapnic period was first. This indicates that the changes in V and f Ksuc were independent of experimental time and not due to "preparation deterioration". In addition. hypercapnia produced a significant increase (P<0.05) in V0 in these 5 43 animals, but Va and the absorption resistance were not changed (P>0.05) significantly. 5.3. Effects of hypercapnia on intracranial fluid volumes Data reported in Figure 3 are representative of responses of V0 and CSFP to hypercapnic induction and withdrawal. Steady state CSF pressure was not different under the two breathing conditions, but V0 was elevated during C02 breathing when compared to that during air breathing (see also 70's in Table G—1 and Table 3). Data in Figure 3 show, in addition, transient changes in V0 and CSFP to hypercapnia induction and withdrawal. Both V0 and CSFP rose transiently during hypercapnia induction (time:60 min). the rise in pressure lasting a shorter time (3 minutes) than the rise in 70, the latter returning to steady state levels in approximately 6-7 minutes. The change in response to C02 withdrawal (Figure 3; time:156 min) was a transient fall in CSFP accompanied by a very dramatic slowing in V0. These data are interpreted to indicate a shift in intracranial fluid volumes and/or a transient change in CSF secretion during hypercapnia induction and removal. In addition to causing an increased brain blood flow, hypercapnia may also increase brain blood volume. Since the skull is a rigid container the increase in brain blood volume causes a competitive displacement of CSF volume through the relatively low resistance cisternal needle. Data reported in Figure 4 show the steady state and 44 transient changes in cisternal effluent concentration observed during air and C02 breathing. The steady state sucrose concentration in cisternal effluent (Co/Ci) was 85% and 74% of perfusion inflow concentration during the air and C0 breathing periods, respectively. Similar changes in 2 effluent sucrose concentration were observed in all animals reported in Table 3. Data reported in Figure 4 show that hypercapnia induction caused a transient fall in the cisternal effluent concentration of sucrose. A transient reduction in perfusion effluent concentration of the test molecules was observed in every animal in which perfusion of the ventriculocisternal system with CSF containing inulin and/or sucrose was continued during the period of hypercapnic induction. These data, as well as those reported in Figure 3. indicate that a transient increase in Vf occurred during hypercapnia induction which caused a dilution of the test molecules. An alternative explanation is that a CSF volume free of test molecules is mixed with the perfused CSF compartment as a result of the expansion of brain blood volume and consequent reduction of cranial CSF volume. This results in a dilution of perfusion effluent concentrations. 5.3.1. Effect of hypercapnia on intracranial CSF volume The volume of the perfused ventriculocisternal system was calculated from the molecular distribution volumes of 3H 14 and C-labelled sucrose in animals breathing both room air and 8-10% 002. Distribution volumes were calculated by 45 integrating cisternal effluent concentrations as a function of time (Figure 4). Two different methods were used to calculate distribution volume. In Protocol A (Methods) the numerical integration was performed after which the volume of the perfusion cannulae (dead space) was subtracted from the calculated volume (Methods; Section 4.6.5.). In the other, using Protocol B (Figure 5). a curve fitting routine (Appendix F) was used to analyze the transient decrease of molecular concentrations (time 63-120 min; Figure 5) to fit a general washout equation. The intersection of the generated washout curve with the steady state concentration resulted in the determination of a time lag due to the dead space of the perfusion cannulae (Figure F-2; Appendix F). This time was subtracted from the initial perfusion time zero. From this, a distribution volume was calculated from the transient approach of effluent molecular concentations to its steady state (time 3-15 min; Figure 5). Data presented in Table 4 show the comparisons of sucrose distribution volumes in animals breathing both air and C02. Distribution volumes for cats D34 and D86 were calculated using the "time lag" method (Appendix F) while the other cats were calculated using Equation 5 (Methods; Section 4.6.5.). There was no apparent difference in volume, determined by the two methods and there was no significant difference (P>0.05) in sucrose distribution volume between air and CO2 breathing. The distribution volume for inulin (not shown) showed no difference (P>0.05) between animals us breathing room air (593 1 32 ul in 8 cats) to those breathing 8-10% 002 (726 1 79 ul in 4 cats). These data indicate that the distribution volume (approximately 600 Hi), which represents largely the volume of the cerebral ventricles, was not changed significantly by 002 breathing. Also there was no significant difference (P>0.05) between sucrose and inulin distribution volumes indicating that the method for estimating a perfused volume is independent of test molecule's weight and size. 5.4. Effects of hypercapnia on molecular distribution volume using compartmental analysis Data reported in Figure 5 show the steady state and transient changes in sucrose and dextran concentrations in cisternal effluent. The steady state concentrations in cisternal effluent were 85% and 78% of perfusion inflow concentration for dextran and sucrose, reapectively. These are similar to molecular concentrations observed for inulin and sucrose reported in Table G-1 (Appendix C). After steady state concentrations were attained in the cisternal effluent, an artificial CSF free of the test molecules was infused into the ventriculocisternal system (time=60 min). This resulted in a subsequent washout of the test molecules from CSF-containing spaces (time 60-120 min). The transient washout data for sucrose and dextran were mathematically analyzed using a curve fitting routine (Appendix F) and are described by a double exponential equation (Equation F-3: Appendix F) indicating that the CSF perfusion system is a two 47 compartment system. The evaluation of the best exponential fit between the predicted curve (Y(t) = A + Be-k2t) and the experimental data was by the chi-square minimization test (Equation F-2; Appendix F). For the exponential curve to be judged as a good fit to the experimental data points in the present study, the chi-square value must be less than or equal to 0.0005. Data presented in Table 5 show the coefficients and rate constants of a double exponential washout equation obtained by non—linear curve stripping technique in animals breathing room air (Table 5A) and 8-10% CO2 (Table SB). These data which indicate the washout of test molecules from a two compartment system is presumed to be from the cerebral ventricles and the subarachnoid space (based on the positions of the perfusion cannulae). Analysis of the rate at which test molecules are being removed from each compartment can indicate the relative volume of each CSF compartments during air and CO2 breathing. From this, it was found that there were no significant differences (P>0.05) detected in mean values of coefficients and rate constants between animals breathing room air and those breathing CO Also, there were 2. no significant differences (P>0.05) in washout rates between the two different size molecules indicating that the removal rates of sucrose and dextran from CSF-containing spaces were independent of molecular weight, size and breathing stimulus. These data suggest that the volumes of the two compartments are not altered significantly by hypercapnia. This does not 48 support the hypothesis of a reduced intracranial CSF volume induced by hypercapnia as suggested by data in Figure 3. However, the double exponential equations derived from the washout data may only be describing apparent volume changes and not functional, anatomical changes of the CSF compartments between air and CO2 breathing, since our data do not show a well mixed perfused system as indicated by the transient decrease in outflow concentration in all test molecules when the animal initially breathed C0 (Figure 4). 2 This may cause misinterpretation of the washout data in reference to volume changes of either the cerebral ventricles or the subarachnoid space under air and CO breathing if an 2 unmixed compartment exist. 49 Table 2. Effect of hypercapnia on arterial pH and P002 No. cats Inspired gas pH PaCO2 mixture (mm Hg) 7.33 28.3 18 » room air 1302 :1.6 a a 7.11 53.8 17 8-10% CO2 1’02 :3-9 Mean :_SEM * (P<0.05) room air 1 8-10% CO2 50 cofipqeomnm mmo on mocmumwmoe n m confluencs u a N 00 «ovum m Lam EOOL Amo.ovmve m. 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Coco-000000 o .0 Loo . . _... C C C C C C . _ .0 ex; o _ . o .oo. ...DOIWSS 2.0mm 558 Table 5. Curve fitting analyses of the washout of sucrose and dextran during room air (A) and 002 (B) breathing. Table 5A 3H-sucrose 1uC-dextran 2 2 - - - -k X Cat A B k1 k2 X A B k1 2 ' (x 10'3) (x 10‘3) 28 .838 .162 .483 .113 .21 - - - - - 031 .926 .074 .374 .005 .13 .885 .115 .413 .083 .12 034 .884 .116 .427 .063 .03 - - - - - 035 .770 .230 .789 .084 .44 .823 .177 .611 .069 .39 036 .926 .074 .572 .050 .10 .956 .044 .537 .050 .13 087 .785 .215 .468 .078 .39 .839 .161 .386 ..073 .44 Mean .856 .145 .519 .074 .876 .124 .487 .069 ISBN .028 .028 .060 .009 .030 .030 .053 .007 Table SB 3H—sucrose 1uC-dextran 2 2 Cat A 3 'k1 -k2 x A B -k1 -k2 x 9 (x 10'3) (x ,0-3) 30 .736 .264 .614 .059 .20 .832 .168 .305 .038 .48 032 .903 .097 .391 .040 .09 — _ - - - D34 .931 .069 .326 .038 .08 - _ - _ _ 037 .839 .161 .496 .045 .11 .850 .150 .500 .055 .32 Mean .852 .148 .457 .046 :SEM .043 .043 .063 .005 The fit of the experimental washout data points to the general exponential equation: Y(t) = Ae’k1t +Be -k t is by a chi-square (x2) minimization criterion (the least sum of squares between the predicted data point and the experimental data points) which were less than or equal to 0.0005. VI. DISCUSSION 6.1. Effect of hypercapnia on arterial pH and FCC 2 Normal arterial blood pH values reported for awake, unrestrained cats vary in a range 7.37 to 7.43 and for P002, 26.6 to 32.5 mm Hg (Fink gg gl., 1963; Herbert _g gl., 1971). These values are similar to those reported in the present study (Table 2 and Table G-1, Appendix G) for barbiturate anesthetized cats. The relatively low arterial PC02 (compared to man) is considered to be normal for the domestic cat (Fink 33 31.. 1963) due to the smaller (40%) buffering capacity of the blood found in this species compared to that in man. The elevation of PaCO induced by C0 inhalation 2 2 (hypercapnia) has been well documented to cause vasodilation of the cerebral vasculature and an increase in brain blood flow (Reivich, 1964; Smith gg gl., 1971: Grubb gg gl., 1974). Brain blood flow (BBF) has been shown in mammals to vary 2 over the range 15 to 70 mm Hg (Smith gg gl., 1971; Grubb gg gl., 1974), but it is not correlated with linearly with Paco changes in arterial pH, other than those produced by changes in PaCO2 (Reivich, 1964). In the present study, it appears that BBF may be increased above air breathing levels since the PaCO2 values of hypercapnic cats (54 mm Hg) are at levels 59 60 which have been shown to induce increases in BBF. 6.2. Effect of hypercapnia on CSF formation The technique of ventriculocisternal perfusion was used in this study to investigate the influence of an hypercapnic-induced increase in BBF on CSF phenomena since it permits the quantitative studies of CSF formation and absorption, the transport of substances among blood, brain and CSF, and an estimation of a CSF volume, simultaneously. Inulin, a non-metabolizable molecule (M.W. = 5200) was used to estimate CSF formation since it has been shown that transependymal flux of inulin during perfusion of the brain ventricular system was negligible (Heisey g3 g1., 1962; Rall g2 gl., 1962). In the present study, mean CSF formation rate was 22.8 ul/min, a value similar to that previously reported for barbiturate anesthetized, air breathing cats (Snodgrass and Lorenzo, 1972; Weiss and Wertman, 1978), and it is similar to CSF formation values when inulin or albumin were used as the non-diffusible indicator (Table 1). The formation of CSF has been shown to be altered by changes in body temperature (Snodgrass and Lorenzo, 1972). intracranial pressure (Hochwald and Sahar, 1971), systemic arterial blood pressure (Carey and Vela, 1974) and when PaCO2 is decreased below normocapnic levels by hyperventilation (Oppelt g3 g1., 1963; Ames gg gl., 1965). However, the effect of raising PaCO by C02 inhalation on CSF formation in 2 mammals has been the subject of several experimental studies 61 which have yielded inconsistent results. In this study, hypercapnia caused increases in CSF formation rates which are in agreement with data reported by Ames gg gl. (1965) who demonstrated an increased rate of CSF formation with an lg 3332 preparation of the lateral ventricular choroid plexus in cats during 10% 002 inhalation. They indicated that this was due to the dilation of the choroidal blood vessels observed during hypercapnia. Our results are contrary to data from investigators who used the ventriculocisternal perfusion technique as in this study to study the influence of hypercapnia on CSF production. They found that the rates of CSF formation were not altered by C02 breathing in various mammals (Oppelt gg gl., 1963 in dogs; Hochwald gg gl., 1973 in cats; Martins gg 31., 1976 in monkeys). The discrepancy between the results is explained on the basis of a cerebral perfusion pressure (CPP) criterion (Section 4.4., Methods) used in our study to account for the variations of both intracranial and systemic arterial pressures among experimental animals. Weiss and Wertman (1978) indicated that CSF formation rates were unaffected by alterations in either blood pressure or intracranial pressure as long as CPP was maintained above 70 mm Hg. They also found that CSF formation was dependent on blood pressure and intracranial pressure when CPP was lowered to 55 mm Hg or less. Using this as a criterion, we found animals in the present study which maintained a CPP greater than or equal to 68 mm Hg showed an increase in CSF formation 62 rates during hypercapnia (Table 3). This would indicate that the relative status of both intracranial and systemic arterial pressures must be considered in the estimation of CSF formation especially under conditions of hypercapnia where these parameters are more variable (Figure 3; Small gg gl., 1960). Based upon the observation of Welch (1963) that about 25% of choroidal blood flow is secreted as CSF, the hypercapnia-induced increases in CSF formation are proposed to be due to an increase in BBF which influences the secretory activity of CSF producing sites (choroid plexus and/or extrachoroidal sites). However, a simple correlation does not exist between BBF and CSF formation since it has been shown that CSF formation decreased during hypoxia (Michael and Heisey, 1973), a period of increased BBF (Kety and Schmidt, 1948). These data suggest that CSF formation is performed by an oxidative metabolic system and that blood flow plays a secondary role in the formation of CSF. 6.3. Effect of hypercapnia on ventricular permeability The permeability of the cerebral ventricles to lipid insoluble molecules varies inversely with molecular size (Heisey g3 g1., 1962). It is presumed to be affected by changes in BBF (Heisey gg gl., 1970; Anderson and Heisey, 1975) and deterioration of the experimental preparation (Cserr, 1965; Anderson and Heisey, 1972). In the present study, the increase in Ksuc (Table 3) may be explained by an 63 hypercapnia-induced increase in BBF since changes in Ksuc were considered to be independent of time as indicated by reversing the animal's breathing sequences but still finding an increased Ksuc (Table 3). Brain blood flow is hypothesized to cause a "sink" effect for some materials moving from CSF or brain into blood and increasing this sink function by hypercapnia may steepen the diffusion gradient and/or lessen the diffusion distance for material moving from CSF to brain tissue and to blood; In mammals, if the test materials are elevated significantly in the blood or infused simultaneously into CSF and blood, the sink function provided by BBF would be compromised. This would affect the concentration gradients of test molecules within the brain and indirectly affect the calculation of the molecular transfer coefficient (Section 4.6.4.; Equation 3, Methods) leading to more variability in CSF clearances. 6.4. Effect of hypercapnia on intracranial CSF volume The initial response to inspiration of 8—10% CO2 was a transient increase in the intraventricular pressure and a translocation of CSF perfusate (Vo) through the low resistance cisternal needle (Figure 3). These data, which are similar to results reported by Hochwald _g _l. (1973) and Martins g3 g1. (1976), indicate a hydraulic shift in intracranial fluid spaces during hypercapnia which reduced CSF volume. These competitive changes observed during hypercapnic induction and the subsequent displacement of CSF 64 volume through the cisternal needle have been related to an increase in brain vascular volume as a consequence of cerebral vasodilation within the confinement of a non-compliant dura mater and skull (Small gg gl., 1960: Smith _5 _l., 1971). Wolff and Lennox (1930) observed an increase in pial vessel diameter with the induction of 5% C02 inhalation. Also, they measured a transient increase in intracranial pressure by a needle inserted in the cisterna magna which they interpreted was the result of an increased BVV as a consequence of pial vessel dilation induced by hypercapnia. From these data and the results of the present study, it is hypothesized that the reduction in CSF volume appears to occur predominantly in the subarachnoid space, the lining of which contains compliant veins and venous sinuses. When 002 is removed as the inspired gas, a rapid decrease in BVV is speculated to occur. This would result in an accumulation of perfusion effluent within the brain case until a new perfusion outflow is attained as suggested by the evidence following hypercapnia withdrawal (Figure 3). In the present study, the perfused CSF volumes were estimated in animals exposed to both air and CO2 breathing (Section 4.6.5., Methods) which was to lend support to the hypothesis of a changing CSF volume following hypercapnia induction and withdrawal. Data reported in Table 4 show that the perfused volumes, from the lateral ventricle to cisterna magna of cats, were unaltered by hypercapnia, compared to those under air breathing conditions. These data are 65 interpreted to represent an unchanging ventricular CSF volume. Our perfusion volume for air breathing cats (approximately 600 pl) was found to be less than values previously reported for this species (750 ul, Cutlerb t al., 1968; 740 pl, Sahar gg gl., 1970) indicating that our volume may be an estimate of the cerebral ventricles since it has been implicated that the estimation of distribution volumes included some portion of the cranial subarachnoid space (Cutlerb t al.. 1968: Sahar gg al., 1971). These data suggest that with an unchanging ventricular volume, the displacement of CSF volume during hypercapnic induction is occurring distal to the cisterna magna, in the subarachnoid space. Washout of test molecules from the perfused ventriculocisternal system were performed to demonstrate further that hypercapnia resulted in an unchanging ventricular CSF volume and a reduced subarachnoid space volume. A two compartment washout was indicated by the double exponential equations reported in Table 5. It is suggested that the initial exponential washout of test molecules is representing the clearance from the perfused volume, the cerebral ventricles. Based on the similarity of the initial rate constants of the washout equations under air and CO breathing, it would suggest that ventricular CSF 2 volume is unaffected by hypercapnia. This would support our distribution volume data (Table 4) of an unchanging cerebral ventricular volume during CO2 breathing. The second 66 exponential which is slower than that representing the ventricular washout is speculated to represent the molecular clearance from a larger CSF volume, the subarachnoid space. It is hypothesized that if the subarachnoid space were reduced during hypercapnia, its exponential washout rate would be slower than that during air breathing but our data did not show this response. The lack of significance in these data may be due to the subarachnoid space being an unperfused compartment which behaved as a reservoir for fluid that is not mixed with the inflow perfusate, thereby causing a variable washout from this compartment. Data presented in Figure 4 is interpreted to support the hypothesis that there is an unmixed subarachnoid space compartment. This is indicated by the transient reduction in outflow concentration of test molecules when the animals initially inspired C02. These reductions in effluent concentrations occurred concurrently with the transient increase in perfusion effluent (V0; Figure 3) and long after steady state molecular concentrations were established in the cisternal effluent. These data suggest that if the subarachnoid space were reduced during hypercapnia, it would contribute unmixed fluid to our effluent and reduce molecular concentrations. It is not clear whether a transient increase in CSF formation occurred upon hypercapnia induction which caused dilution of perfusion effluent concentrations (Figure 4) and raised perfusion effluent (Vo) transiently (Figure 3). This, 67 however, cannot be clearly differentiated from a reduction in CSF volume since an hypercapnia-induced increase in CSF formation occurred in our present study (Table 3). but it is a problem that warrants further investigations. VII. SUMMARY Brain ventricles of cats were perfused with an artificial CSF which yielded data on rates of CSF formation and absorption, molecular movement from the CSF, and the size of the cerebral ventricles. Elevation of arterial carbon dioxide pressure (PaC02) by 8-10% C02 inhalation resulted in increases in the rates of CSF formation (7f) and molecular clearances from the brain ventricles (Ksuc) in animals with cerebral perfusion pressure greater than or equal to 68 mm Hg. Hypercapnic-induced increases in Vf and Ksuc were independent of perfusion time suggesting that these were effects of an hypercapnia—induced increase in brain blood flow. Hypercapnia induction produced transient increases in perfusion effluent rate (70) and intraventricular pressure as well as a transient decrease in molecular outflow concentrations. These were interpreted to indicate a competitive reduction in CSF volume within the rigid brain case as a result of an increase in brain 68 69 vascular volume consequent to cerebral vasodilation. The brain ventricular volume of cats was approximately 600 ul and was not altered significantly by hypercapnia. It is suggested that the competitive shift of CSF volume was distal to the cisterna magna and from a volume that that was free of or which had a lower concentration of test molecules. Washout of test molecules from the ventriculocisternal system indicated a two compartment system which is speculated to be cerebral ventricles and subarachnoid space. The volumes of these compartments, as indicated by the rates of molecular removal during washout were not altered significantly by C02 breathing. The lack of significance in washout data to the hypothesis of a reduced CSF volume may be due to a nonmixing phenomenon in the subarachnoid space. APPENDICES APPENDIX A pH and PaCO2 measurements Reference: Radiometer instruction manual, Model PHA 927b, Radiometer A/S, Copenhagen, Denmark Principle: The pH electrode is designed to measure H+ ion activity. The sample is drawn into a H+—sensitive glass capillary which is surrounded by a buffer whose pH is approximately 6.40. The H+ ion difference between the fluid sample and the sealed buffer generates a voltage potential which is measured by a calomel reference electrode and a Ag-AgCl glass electrode amplified and displayed on a scale calibrated in pH units (Model PHM27, The London Co., Cleveland, Ohio). The CO2 electrode (Model E5036, The London Co.) is a pH electrode in direct contact with a bicarbonate solution. The bicarbonate solution and electrode are separated from the sample being analyzed by a 002-permeable silicon rubber membrane which allows only gas molecules to pass through and not charged particles or ions. The CO2 gas rapidly diffuses into the bicarbonate solution thus altering its pH. This altered pH generates a potential which is sensed by a galvanometer and the deflection is recorded on a scale calibrated in mm Hg PCO (Model PHM27, The London Co.). 2 7O Calibration: The CO2 electrode is calibrated using two gases with known CO2 composition (5% and 12% CO2 with balanced N2, 21% 02, The London Co.). The pH electrode is standardized with commercial buffers at 38 1 0.2°C (pH = 6.840 and pH = 7.383, Scientific Products Inc., Allen Park, MI). Specimen: Arterial blood is collected anaerobically via the femoral arterial cannula in a glass syringe, the dead space of which is filled with heparin (10,000 units/ml). The tip of the syringe is sealed with a metal cap filled with mercury. Mercury is introduced into the syringe to facilitate mixing of the blood specimen after collection and before analysis. Analyses of arterial pH and PCO2 were performed in triplicate or until duplicate pH readings were 1 0.010 pH units and duplicate PCO2 readings were 1 0.3 mm Hg. The thermostatted electrodes were adjusted approximately to rectal temperature (38 1 0.50C). APPENDIX B LIQUID SCINTILLATION COUNTING Reference: Beckman Liquid Scintillation Counter manual, Model 3150P, Beckman Instruments Inc., Fullerton, CA Principle: Liquid scintillation counting is a method for the detection of energy from an ionizing particle emitted by a decaying nucleus. This energy is converted from kinetic energy of an ionizing particle into light photons by a solution of organic fluors and is detected by a photomultiplier tube connected to amplifiers and a scalar circuit. The decaying nucleus and the scintillation detector (fluor) are in intimate contact, making the detection efficiency quite high for use with low energy beta emitters such as tritium and carbon-14. Counting channel selection: The energy spectra of tritium and carbon-14 overlap and when both beta emitters are present in the same sample, they are counted simultaneously on 2 separate analyzer channels. Channel 1 amplifiers are adjusted to give high efficiency of counting tritium with minimal interference of carbon-14; amplifiers of Channel 2 are set so that counting efficiency of tritium is insignificant while efficiency for carbon—14 is maximal. A separate amplifier is set to 72 73 maximize energy pulses from an external standard of known disintegration rate (137-cesium). Quench correction curve: Quenching is defined as any process which results in a decreased number or intensity of photons produced; this reduces the efficiency of counting the radioactivity. The amount of quench will be related to the probability of measuring the emissions from a sample: i.e., counting efficiency. To monitor the amount of quench in a sample and allow for corrections to give true sample activity, a method called external standard channel ratio (ESCR) is used. This measures the change in the ratio of pulses produced by an external gamma-ray source (137-cesium) between two selected counting channels as a function of the counting efficiency. T2‘32 ESCR : —~_———— (B-1) T1 — S1 where: T1 or T2 = counts per minute produced by the sample and external gamma source in channel 1 or 2. S1 or $2 : counts per minute produced by the sample alone in channel 1 or 2. 74 The properly scaled number of events obtained in each channel during the sample count is subtracted from the events obtained during the total count (sample plus 137-Cs source) to give the net contribution from the gamma source observed in each channel. The ratio of the two net counts (2/1) is computed, and the resulting ESCR number is printed. The quench correction curve (Fiqure B-1) is a plot of the efficiency of tritium and carbon-14 quench standards (Amersham/Searle, Boston, MA) as a function of the external standard channel ratio (ESCR). Counting efficiencies of each isotope in an unknown sample can be read directly from the quench correction curves (Figure 8-1) corresponding to the unknown sample's external standard channel ratio (ESCR). Tritium and carbon—14 detection In these studies, dual-labelled counting procedures 14 were used. 3H—sucrose and C—sucrose; or 3H—sucrose and 1uC—dextran (New England Nuclear, Boston, MA) activities were measured both in the cisternal effluent and in the inflow perfusion fluid. The disintegration rates of unknown samples 3H and 1”C and were calculated from the counts per minute of the selected efficiencies of counting each isotope in channels A or B. Figure 8—1. 75 Quench standards correction curves for 3 differential counting of H and 1“C samples. 3H and 14C quench Percent efficiencies of standards (ordinate) were calculated as net 3 H photopeak) or channel B (set for 1“C above 3H) counts per minute in channel A (set for divided by disintegrations per minute of tritium and carbon-14 quench standards. The abscissa is the external standard channel ratio (ESCR) as described and calculated by Equation B-1. Quench correction curve for 1“C efficiency in channel B (circles): quench correction curve for 3 H efficiency in channel A (squares); quench u correction curve for 1 C efficiency in channel A (triangles). Settings used on LS-3150P: Channel A Channel B Windows tritium iso—set carbon-14 iso-set Gain 283 283 AQC/ESCR .720 .720 Preset error 0.5% 0.5% Preset time 20 min 20 min 0 '4C in Channel B Ioor % EFFICIENCY I 3H in Channel A A ”C in Channel A 80 ' 60 ' 4o - 20 " ESCR O - I; 4 4 4 I #1 O 02 O 4 0.6 08 IO FIGURE B—l 77 Counting procedures and calculation of disintegration rates of 3H and 13 The disintegration rates for 3H and C 14 C were calculated using a Hewlett-Packard programmable calculator (Model 67, Corvallis, solving the following equations: 3H disintegration disintegration net count rate net count rate rate rate in Channel A (cpm—bkgd cpm) in Channel B (cpm-bkgd cpm) Oregon) which was programmed for (dpm) of tritium (dpm) of carbon-14 counting efficiency for tritium in Channel A (%) counting efficiency for carbon-14 in Channel A (%) counting efficiency for carbon-14 in Channel B (%) (B-2) (B-3) APPENDIX C INULIN ASSAY Direct Resorcinol Method without Alkali Treatment Modified from H.W. Smith, 1956 Principle: A method in which inulin is hydrolyzed to fructose by heating in acid. Fructose molecules combine with resorcinol to yield a colored complex: the intensity of the color is proportional to the amount of fructose present. Reagents: 1. Resorcinol (Fisher Sci. Co., Fairlawn, New Jersey) 2. Ethanol (95%) 3. Hydrochloric acid (conc.) Solutions: A. Resorcinol (1.0 mg/ml) Dissolve 200 mg resorcinol q.s. 200 ml with 95% ethanol. Prepare fresh daily. B. HCl (appoximately 8N) Add 224 ml of deionized water to 1000 ml conc. HCl. 78 79 Inulin standard solutions: Dissolve 200 mg inulin (Pfanstiehl Laboratories, Inc., Waukegan, IL) in deionized water q.s. 100 ml (2.0 mg/ml). Dilute 7.5, 5.0, 4.0, 3.0, 2.0, and 1.0 ml of 2.0 mg/ml inulin solution to 10 ml with deionized water yielding 1.5, 1.0, 0.8, 0.6, 0.4, and 0.2 mg/ml inulin standards. Standards are stored at 4°C. Procedure: To duplicate 0.05 ml unknown samples, inulin standards and a CSF—reagent blank, 1.0 ml solution A and 2.5 ml solution B are added and mixed (vortex) in pyrex test tubes. A glass marble is placed on top of the tubes to prevent evaporation and tubes are incubated for 25 minutes at 800 C. Tubes are cooled in tap water for 3 minutes and optical density is determined within 1 hour at 410 nm (peak of absorbency; Figure C-1) in plastic cuvettes (Scientific Products, Allen Park, MI) against a CSF—reagent blank in a Beckman DB spectrophotometer (Beckman Instruments, Inc., Fullerton, CA). 80 .mneweoao>w3 En 0e: new E: OF: ew no>eomno oLw mxwoe ooewnLOmnw oze .Awwweownw "Eev neweoHo>w3 no eOHeoeee w mw noeeoHa Aoewewneov eOHeeHow eefleee He\ws o.e w no zeemeon Honeeo one .euo weewee 00m Omw own only HID mmDon Owe 00¢ 0mm 00m -1 RES Ieozmnw><>> q >._._mzmo 4<0_._.n_0 n 82 Calculations: Optical density at 410 nm plotted as a function of inulin concentration yields a straight line over the range 0-1.0 mg/ml inulin. Inulin concentration in unknown samples is calculated by multiplying the optical density of the unknown by the reciprocal of the slope of the standard curve (Figure C—2). cs C : X 0D x x OD s a where: C : concentration 0D : optical density 3 = standard a = average x = unknown sample 83 Figure C-2. The optical density of inulin standards (ordinate) plotted as a function of known inulin concentrations (mg/ml: abscissa). 0.8 0.6 0.4 0.2 OPTICAL DENSITY 84 CON CENTRATION (mg/ml) l l l I 0.4 0.6 0.8 1.0 FIGURE C-2 APPENDIX D Composition and Preparation of Artificial Cat Cerebrospinal Fluid (CSF) Cat CSF contains: Na, 130 mEq/l; K, 2.5 mEq/l; Cl, 132 mEq/l; HC03, 20 mEq/l: Ca, 2.5 mEq/l; Mg, 1.0 mEq/l (Vogh and Maren, 1975). Reagents 1. NaCl, Analytic reagent (A.R.) 2. KCl, A.R. 3. NaH2P0u°H2 u. Na2HPOu-7H2O, A.R. O, A.R. 5. NaHCO A.R. 3v 6. MgSOu°7H2O, A.R. 7. CaCl A.R. 2. Solutions A. 7.6 g of reagent 1; 186.4 mg of reagent 2; 169.0 mg of reagent 3; 134.0 mg of reagent 4; 1.68 g of reagent 5 are dissolved in deionized water; q.s. to one liter. B. 24.65 g of reagent 6 is dissolved in deionized water; q.s. to 100 m1. C. 27.75 g of reagent 7 is dissolved in deionized water; q.s. to 100 ml. 85 86 Procedure The pH of solutions A is adjusted to approximately 7.40 by bubbling with 5% CO2 at room temperature for 30 minutes. Then 0.1 m1 of solutions B and C are added to 100 m1 of solution A to give the normal ionic concentrations of cat CSF. APPENDIX E Dial-Urethane solution Reagents 1. Diallyl barbituric acid (crystalline; K&K Laboratories, Inc., Plainview, NY). 2. Monoethyl urea (Pfaltz and Bauer, Inc., Flushing, NY). 3. Urethane (Aldrich Chemical Co., Milwalkee. WI). 4. Disodium calcium ethylene diamine tetra acetate trihydrate (Pfaltz and Bauer. Inc., Flushing. NY) Procedure Dissolve 10.0 g urethane. 40.0 g monoethyl urea. 50.0 mg disodium calcium ethylene diamine tetra acetate trihydrate, and 40.0 g diallyl barbituric acid in 10 m1 of deionized water. Heat in a water bath to dissolve chemicals. Cool to room temperature and store in stoppered dark glass bottle at room temperature. 87 APPENDIX F Method of Non—Linear Least Squares Reference: Bevington. P.R. (1969) Principle The method of non-linear least squares is a technique for fitting experimental data points (xi.yi) with a function y(x) which is not linear with respect to its parameters such as: Y(x) = Aex (F-1) where: A : coefficient (parameter) ex = exponential rise for any given value of x (parameter) The application is to fit straight lines to a set of experimentally observed points for any dependent variable, y, and for any independent variable. x. For a straight line which comes close to fitting most of the observed data points. some of the deviations will be positive and some will be negative (random scatter of data points along the straight line). By squaring the deviations and requiring that the sum of squares of the deviations be minimized, the expression 88 89 counts 3 positive deviation and a negative deviation equally. The curve fitted to these experimentally observed points is considered to be of best fit when it produces the smallest sum of squares of the deviations or a "least-squares fit". The criterion for minimizing the parameters of a given equation is such that the discrepancy between the values of the measured yi and the corresponding predicted values y : f(xi) are relatively small and can be found utilizing a chi—square test. n 2 2 x = _ _ 2: [Ytj Ydj] (F 2) J=1 where: X2 : chi-square, a statistical measure of the dispersion of the observed (actual) distribution versus the theoretical (predicted) distribution. Ytj = theoretical jth data point (predicted) Ydj = experimental jth data point (actual) n = total number of data points The minimized value of the chi—square function is one which yields a value near zero (less than or equal to 0.0005) when the predicted fit of the curve is a close approximate of the experimentally observed curve. 90 Procedure The washout of CSF-containing spaces (Figure 5: 60—120 minutes: Results) from perfusion of the ventriculocisternal system were interactively and systematically analyzed by a mini-computer (PDP-11, Digital Equipment Corp., Maynard, MA) using the method of non-linear least squares (Figure F-1). The chi-square function (Equation F-2) is the best fit criterion for fitting non-linear equations to experimentally determined points. A double exponential equation which best fit the transient washout data obtained from the perfusion experiments is: Y(t) : Ae— (F-3) where: Y(t) = molecular concentration in the CSF ventricular system at any time (t) t = time (minutes) A.B = are coefficients (parameters) of an exponential function. When t:0: Y(O) = A + B = mean steady—state concentration. When t=m; Y(m) = 0. the washout of CSF brain fluid spaces is complete. k1,k2 = are rate constants (parameters) of the exponential function. Figure F-1. 91 Flow chart of algorithm (Bevington, 1969) used to find the best non-linear numerical equation to fit the experimental washout data to a double e‘k1t + Be’kzt (Equation F-3). Best fit is determined by finding iterative values of A, B, R1, k2 by a exponential equation: Y(t) = A chi-square minimization test computed from a non-linear least squares method. 92 Specify data set. devices and output location: output location terminal or printer (terminal or printer) I Read (n)th title of (n) data set Arrange final curve fit and Save estimated parameters truncated for stora e results K (Y or N)? again ulth Select specific curve fit TERMINATE (T) same data set ro tin f d t t u e or (n) a a se Y,N, or T)? Manually change default criteria of 0.00l value which is a relative change of each parameter with respect to each parameter's partial derivative for (n) data set use default criteria on (n) data set (Y or N)? Enter initial estimates of each parameter for curve fit routine for (n) data set Plot predicted Iterative results of best (curve fit) curve fit for experimental and experimental data points, measured as data points chi-square (minimization) (Y or N)” for (n) data set output location: terminal or printer FIGURE F—l 93 The application of the exponential washout equation can be extended towards predicting the relative time when perfusion of the ventriculocisternal system began (time zero) without having to include cannulae dead space volume. The dead space volume is represented by a time delay occuring between the time when CSF containing test molecules is begun and the time when a measurable concentration is detected in the cisternal effluent (Figure 5; 0—2 minutes) The curve fitting program generates a time zero (predicted) from the experimentally determined washout data. The predicted time zero is then subtracted from the actual time when CSF free of test molecules was first introduced through the ventricular needles (Figure F-2). This difference is the time lag that is presumably due to cannulae dead space volume. The value for the time lag can then be subtracted from the initial time when CSF containing test molecules was first introduced through the ventricular needles. This will result in an adjusted perfusion time zero from which to calculate a distribution volume of the ventriculocisternal system (Section 4.6.5.. 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P d d a a a la < 1 (1 1 <1 ‘i‘ 1‘1 a}! it! {Eta ) 00 3:5 ms: ON 0.? 00 4 on w}: a g _ we 00 0 APPENDIX G MEASURED AND CALCULATED STEADY STATE DATA Data in Table G—1 are measured steady state quantities from 9 cats breathing room air and 8-10% CO2 in room air mixture during ventriculocisternal perfusion which allowed calculation of CSF perfusion phenomena (Table G-2). Definition of symbols: 9.. 1 <- <- u n pH PaCO2 = CSFP : MABP : perfusion inflow rate perfusion outflow rate CSF formation rate CSF bulk absorption rate molecular outflow concentration expressed as a ratio of perfusion inflow concentration. variances of steady state molecular outflow concentration (x 10‘3). inulin and sucrose (logarithmic) hydrogen ion concentration arterial partial pressure of carbon dioxide intraventricular pressure mean arterial blood pressure outflux rate of sucrose from cerebral ventricles 96 97 Table G-1 and Table G—2 show data from experiments which satisfied two experimental criteria: (1) in the steady state the total fluid volume input (Oi + 9f) was equal to the total fluid volume output (90 + Ya) during ventriculocisternal perfusion and (2) the variances ($2) of inulin and sucrose concentrations were equal to or less than 0.0010. 98 Amuo— xv - 8 m4. om 3.0 m. P. 2. i... 2: a: 8 3. N 8; F. I 2.. I 9: 3: own on a cm 3.0 H. N. No. 3. a: mm. 8 m N 0N4. 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