On the presence of subarachnoid fluid in the mudpuppy, Necturus maculosus

Comp.Biochem.Physiol., 1974, Vol. 48A, pp. 145 to 151. Pegamon Press. Printed in Great Britain

ON THE PRESENCE OF SUBARACHNOID FLUID THE MUDPUPPY, NECTURUS MACULOSUS* HELEN

F. CSERR+

and

IN

L. H. OSTRACH

Division of Biological and Medical Sciences, Brown University, Providence, Rhode Island 02912; and Mount Desert Island Biological Laboratory, Salsbury Cove, Maine 04672, U.S.A.

(Received 4 June 1973) Abstract-l.

Experiments examine the relationship between ventricular cerebrospinal fluid (CSF) and extrabrain fluid in lower vertebrates, with emphasis on the amphibian Necturus maculosus. 2. Analyses of Necturus extrabrain fluid identify this fluid as CSF, while experiments with methylene blue demonstrate communication between this fluid and ventricular CSF. 3. Extrabrain fluid or tissue collected from Myxine glutinosa, Raja erinacea, Hemitripterus americanus and Ginglymostoma cirratum is not CSF.

INTRODUCTION THE VENTRICLES

and subarachnoid spaces of the mammalian brain are interconnecting compartments filled with cerebrospinal fluid (CSF). In the fishes-cyclostomes, elasmobranchs and teleosts-there is no subarachnoid space and CSF is confined to the ventricular cavities of the brain (Ariens-Kappers, 1926). The point in evolution when CSF first surrounds the outer surface of the brain is unclear. Much of the earlier literature (reviewed by Davson, 1967) suggests that subarachnoid fluid first appears in birds, while other studies in amphibians (Palay, 1944; Brightman, 1953; Cohen et al., 1968) and reptiles (Heisey, 1970) indicate an earlier phylogenetic origin. Although lacking a subarachnoid space, the fish brain is surrounded by an external layer of fluid or tissue commonly referred to as extradural fluid (EDF) or perimeningeal tissue, respectively. This external layer may be distinguished from subarachnoid CSF on the basis of several physiological characteristics including: a protein concentration closer to plasma than CSF (Zubrod & Rall, 1959); lack of a blood-tissue barrier comparable to the blood-CSF barrier (Lundquist, 1942; Zubrod & Rall, 1959); and, for sharks at least, failure to communicate with ventricular CSF (Klatzo & Steinwall, 1965; Oppelt et al., 1966). We have investigated the possibility that there is subarachnoid fluid in the amphibian, Necturus maculosus. Internal and external brain fluids have been * Supported by National Science Foundation Grant GB-28472. t Recipient of Career Development Award NS-70500. 145

146

HELEN F. CSERR ANDL. H. OSTRACH

collected and analyzed. The question of continuity between the two fluid compartments has also been examined. For comparison, we have also examined a number of species which lack a subarachnoid space. MATERIALS

AND

METHODS

Animals Adult Necturus were purchased from Mogul-Ed, Oshkosh, Wisconsin, and maintained in the laboratory at either 4 or 15°C. Those maintained at 15” ate live goldfish added to their aquaria. For collection of fluid samples, a Necturus was anesthetized in Tricaine solution (0.6 g/l.) (Sigma), wrapped in wet paper towels and placed on its back in a dissecting pan. Additional Tricaine was applied to the gills as necessary. Hagfish (Myxine glutinosa), little skate (Raja erinacea) and red sculpin (Hemitripterus americanus) were caught off the coast of Maine and maintained at the Mount Desert Island Biological Laboratory in large aquaria provided with a continuous supply of fresh sea water. Nurse shark (Ginglymostoma cirratum) were caught off Bimini, Bahamas, and kept in live cars. Hagfish, skate, sculpin and nurse shark were anesthetized with a single i.p. or i.v. injection of sodium pentobarbital with doses of 100 mg/kg for hagfish and of 12.5 mg/kg for the remaining species. Anesthetized fish were ventilated by perfusing the gills with fresh salt water. It is essential that anesthetized fish be adequately ventilated during the sampling period (S-20 min) as anoxia leads to rapid changes in CSF electrolyte composition (Cserr & Rall, 1967). Sampling technique With the exception of nurse shark, most fluid and tissue samples were collected with the aid of a dissecting microscope. In Necturus ventricular CSF was sampled from the third ventricle using the ventral approach described by Cohen et al. (1968). The lower jaw was clamped and removed. A small hole was drilled in the hard palate slightly anterior and lateral to the pituitary. The hole was then extended using forceps to expose the dura overlying the ventral surface of the third ventricle. The dura was pierced with a fine-tipped dissecting needle or hook. A sharpened glass micropipette with a tip diameter of 25-50 p was lowered with the aid of a micromanipulator through the hole in the dura and into the ventricle, immediately anterior to the pituitary. The micropipette was connected to a 2-ml syringe via polyethylene tubing. A 10-20 ~1 sample of CSF was aspirated slowly into the micropipette. CSF samples were examined under the microscope and only those free of erythrocytes were analyzed. Fluid located outside the amphibian brain was collected from a cavity located anterior to the telencephalic hemispheres, between the origins of the olfactory nerves. Access to this fluid compartment was through a second hole in the hard palate located 6-10 mm posterior to the angle formed by the anterior row of teeth. This compartment generally yielded larger fluid samples (1 S-30 ~1) than the third ventricle. Hagfish blood was sampled through a ventral skin incision from the dorsal aorta. Perimeningeal tissue was taken from the dorsal surface of the brain or spinal cord. For skate and sculpin, sampling techniques were similar with the exception that skate blood was sampled from the heart, while sculpin blood was collected from a dorsal tail vessel by percutaneous puncture using a 22-gauge needle. The skull overlying the dorsal surface of the brain was removed using ronguers and a scalpel. The brain occupies only a small fraction of the cranial cavity in these species, the remaining volume being EDF. This fluid was aspirated into a 5 or 10 ml heparinized syringe. Skate CSF (S-30 ~1) was collected from either the third ventricle or the optic lobe using a glass micropipette; sculpin CSF (5-l 5 ~1) from the ventricle of the optic lobe.

SUBARACHNOID

FLUID IN THE MUDPUPPY

147

Nurse shark EDF was sampled from the cavity located anterior to the telencephalic hemispheres following removal of the overlying skull. Dye experiments The question of possible communication between internal and external brain fluids in Necturus was examined using methylene blue. A sharpened glass micropipette containing 24 ~1 of 2.5% methylene blue was inserted into the third ventricle as described above. To confirm proper pipette placement, gentle suction was applied until clear fluid appeared in the pipette. The dye was then injected slowly into the fluid cavity. The pipette was removed carefully and any leakage of dye noted. The injection procedure lasted about 5 min. The injected animal was kept at room temperature for periods ranging from 30 min to 4 hr to allow intracranial distribution of the dye. At the end of this period, the brain was fixed by vascular perfusion as suggested by Bodenheimer & Brightman (1968). The external gills were ligated and isotonic saline followed by 100 ml of buffered formalin was perfused via the dorsal aorta. The fixed brain was photographed in situ, removed from the cranial cavity and sliced on a freezing microtome. 14C-Inulin penetration

into internal and external brain fluids

Inulin-carboxylJ4C (crystalline solid; New England Nuclear) was prepared fresh daily in isotonic saline and given as a single i.p. injection. The amount administered varied from 2 to 5 &i/100 g body wt. Twenty hours following isotope administration samples of plasma and of brain fluids were collected and analyzed for radioactivity. Results are given as the distribution ratio: counts/mm per ml CSF, EDF or perimeningeal tissue +-counts/ min per ml plasma. Radioassay of serial plasma samples revealed that plasma isotope concentration remained relatively constant during the 20-hr experimental period in Necturus (N = 2), little skate (N = 3), red sculpin (N = 4) and hagfish (N = 2). Analyses Protein concentrations were estimated by the method of Lowry et al. (1951), potassium and sodium concentrations by flame photometry (Instrumentation Laboratory, Inc., Model 143). Radioactivity was assayed in a liquid scintillation counter. Radioactive samples were digested in a scintillation vial in O-5 ml Protosol (New England Nuclear) at 40°C. After freezing (to prevent chemiluminescence), 15 ml of counting medium (New England Nuclear’s Liquifluor) was added to each vial for subsequent radioassay. Automatic external standardization was used and corrections were made for sample quenching.

RESULTS

Table 1 compares protein concentrations of plasma and cerebral extracellular fluids (CSF or EDF) in mudpuppy (Necturus), man (Hunter & Smith, 1960) and nurse shark (Cserr et al., 1972). CSF is characterized by a low protein concentration, relative to plasma, whether the fluid is sampled from the ventricles or subarachnoid space. EDF in nurse shark, as well as in other species of shark (Zubrod & Rail, 1959; Rasmussen & Rasmussen, 1967), differs from CSF in having a much higher protein concentration. According to this criterion, the low protein concentration of fluid collected from outside the Necturus brain identifies this fluid as subarachnoid CSF. CSF may also be distinguished from EDF or perimeningeal tissue by determining the extent of inulin penetration into the tissue. Due to the presence of the

148

HELEN

TABLE

I-PROTEIN

F. CSERR AND L. H.

CONCENTRATIONS

OSTRACH

op PLASMA AND BRAIN FLUIDS IN MAN, MUDPUPPY

AND

NURSE SHARK

Total

protein

(mg/lOO ml) -__

Species Man Mudpuppy Nurse

Plasma

Ventricular CSF

Subarachnoid CSF

N 6.500 2920 f 140

15 24+5

36 25-t2

(3) 176

(7)

(14) 3670

shark

EDF

2880+

140

(5) Values for man from Hunter & Smith (1960); those for shark plasma and CSF from Cserr et al. (1972). Mudpuppy and shark EDF data given as means + S.E. with number of observations in parentheses.

blood-CSF barrier, steady-state inulin concentrations of ventricular CSF (summarized in Table 2) are only 2-3 per cent of those of plasma in Necturus, sculpin and skate. In contrast to CSF, there is no barrier to drug penetration into EDF or perimeningeal tissue, although the rate of penetration may be very slow (Zubrod & Rall, 1959). Twenty hours after isotope administration, inulin concentrations of TABLE

2-INULIN

PENETRATION

INTO CSF,

EDF

AND PERIMENINGEAL

SYSTEMIC ADMINISTRATION

Inulin

Species

distribution

ratio

(counts/mm

Ventricular CSF

TISSUE 20 hr AFTER

OF W-INULIN

per ml fluid +cpm/ml

Subarachnoid CSF

plasma)

EDF or perimeningeal tissue

Mudpuppy

0.02 * 0.002

0.02 +_0.001 (5)

Red sculpin

(7) 0.03 f 0*005

Little

(4) 0.03 + 0.006

(6) 0.50 * 0.03

(6)

(9) 0.54 + 0.02

skate

0.77 + 0.04

Hagfish

(6) Data

given as mean + S.E.

with number

of observations

EDF or perimeningeal tissue were between 50 and tion in sculpin, skate and hagfish. Presumably, The low inulin revealed complete equilibration. from outside Nectwus brain substantiates further subarachnoid CSF.

in parentheses.

80 per cent of plasma concentralonger time periods would have concentration of fluid collected the conclusion that this fluid is

SUBARACHNOID

FLUID IN THE MLJDPUPPY

149

Necturus fluids were also analyzed for potassium and sodium. Mean + SE. concentrations (m-equiv/l.) for plasma, ventricular CSF and subarachnoid CSF, respectively, were 2.5 + 0.2 (N = S), 2.1 (2.3, 1.9) and 1*9+0*1 (N = 6) for potassium and 95 + 2 (N = 8), 94 (88, 101) and 97 f 3 (N = 6) for sodium. Values for plasma and ventricular CSF are similar to those given by Cohen et al. (1968). Since the concentrations of sodium and of potassium were similar for all three fluids, these determinations do not contribute further to the identification of subarachnoid CSF as distinct from EDF. In mammals, there is free communication between ventricular and subarachnoid CSF, so that substances injected into one compartment rapidly distribute throughout the CSF spaces. Methylene blue injected into the third ventricle of Necturus distributed throughout the ventricular system and much of the subarachnoid over the ventral brain surface is illustrated in space. The extent of dye distribution Fig. 1. The external surface of the brain stained heavily from the posterior portion of the telencephalon to the caudal medulla, including cranial nerve roots. The surface of the pituitary appears unstained. This agrees with Palay’s (1944) finding that the amphibian pituitary lies outside the subarachnoid space. Following visual inspection, the fixed brain was removed from the cranium and sliced at 40 p on a freezing microtome. Examination of the cut surface of the frozen brain confirmed that dye had stained both the inner (ventricular) and outer surfaces of the brain. Anterior portions of the telencephalic hemispheres and the olfactory nerves were not stained by the dye. There is no reason to suspect that this result indicates that the cephalic telencephalon lacks a subarachnoid space, since Palay (1944) and Flexner (1929) both found that the subarachnoid space completely surrounds the amphibian brain. DISCUSSION

The results of these experiments establish the presence of a subarachnoid space, continuous with the ventricles, in the amphibian N. maculosus. An amphibian subarachnoid space is also indicated by anatomical descriptions of a well-defined arachnoid membrane and underlying subarachnoid space in Ambystoma (Flexner, 1929), several species of toads (Palay, 1944) and Necturus (Brightman, 1953). Experiments with methylene blue demonstrate mixing between ventricular and subarachnoid CSF, but do not identify the route of this exchange. Brightman (1953) also demonstrated mixing between internal and external fluids in Necturus brain. Following intracisternal injection of HgS, he found sulfide particles both within the ventricles and subarachnoid space. Based on his anatomical findings he suggested that the route of fluid exchange may be “across the thin wisp-like meninx that connects the choroid plexus to each end of the 4th ventricle”. Whether there is communication between ventricular CSF and EDF in fish is not known. Klatzo & Steinwall (1965) and Oppelt et al. (1966) found no exchange in sharks and according to Lundquist (1942) there is also no communication in teleost fish. The large difference in inulin concentration maintained between CSF and

150

HELEN F. CSERRANDL. H. OSTRACH

EDF in red sculpin (Table 2) supports this conclusion. On the other hand, both Van Rijssel(l946) and Bakay (1947) claim that there is mixing between internal and external brain fluids in teleosts. In understanding the conflicting results for teleosts, it may be important to consider certain differences in technique employed in these studies. Lundquist injected a few drops of dye solution into the EDF of teleost fish by inserting a fine cannula through a small drill hole in the skull. No dye penetration into the underlying brain tissue was observed except in those fish in which damage to the delicate meninges was suspected. Our samples of fish CSF and EDF were also obtained using microsurgical technique. Van Rijssel injected a relatively large volume of dye solution (0.1-0.2 ml for a fish 10 cm in length) by percutaneous, intracranial puncture. Either the large volume or the percutaneous puncture might be expected to result in brain damage and, in fact, many fish did not survive the procedure. The brains of amphibians, as well as those of lungfishes and primitive teleosts, are characterized by hollow, thin-walled hemispheres and large choroid plexuses. According to Herrick (1921), these morphological features may have been of adaptive value in the transition from water to land which occurred during the During periods of drought and consequent late Silurian or early Devonian. hypoxia, such as accompanied this prehistoric period, primitive fishes with these characteristics were ensured of a dual oxygen supply to the brain-( 1) from parenchymal cerebral capillaries and (2) via CSF, from large, well-vascularized choroid plexuses. In evaluating Herrick’s theory it is important to consider whether these fish had a subarachnoid space, since this affects the estimated path length for diffusional exchange of oxygen between CSF and brain. Herrick assumed that oxygen-rich CSF bathed both the inner and outer surfaces of the brain. In that our results indicate an earlier phylogenetic origin for the subarachnoid space than is frequently assumed, they are compatible with Herrick’s assumption of a subarachnoid space in the transitional fishes.

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FIG. 1. Ventral view of Necturt~ brain 4 hr after injection of methylene blue into the third ventricle. Results demonstrate communication between ventricular CSF Dye has entered the subarachnoid and fluid surrounding the amphibian brain. space and, with the exception of the pituitary, stained the ventral surface of the brain from the mid-portion of the telencephalic hemisphere to the caudal medulla. The extent of dye distribution on the dorsal surface was similar. The brain was fixed by vascular perfusion with formalin and exposed by removing part of the hard palate.

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FLEXNER L. B. (1929) The development of the meninges in amphibia: a study of normal and experimental animals. Con&. to Embryol. No. 110: Publ. 394, Carnegie Inst., Washington 20, 31-48. HEISEY S. R. (1970) Cerebrospinal and extracellular fluid spaces in turtle brain. Am. J. Physiol. 219, 1564-I 567. HERRICK C. J. (1921) A sketch of the origin of the cerebral hemispheres. r. Cotnp. Neurol. 32,429-454. HUNTER G. & SMITH H. V. (1960) Calcium and magnesium in human cerebrospinal fluid. Nature, Lond. 186, 161-162. KLATZO I. & STEINWALL 0. (1965) Observation on cerebrospinal fluid pathways and behaviour of the blood-brain barrier in sharks. Acta neuropath. 5, 161-175. LOWRY 0. H., ROSEBROUGHN. J., FARR A. L. & RANDALLR. J. (1951) Protein measurement with the Folin phenol reagent. r. biol. Chem. 193, 265-275. LUNDQUIST F. (1942) The blood-brain barrier in some freshwater teleosts. Acta phys. scandinav. 4, 201-206. OPPELT W. W., ADAMSONR. H., ZUBROD C. G. & RALL D. P. (1966) Further observations on the physiology and pharmacology of elasmobranch vgntricular fluid. Comp. Biochem. Physiol. 17, 857-866. PALAY S. (1944) The histology of the meninges of the toad. Anat. Rec. 88, 257-270. RASMUSSENL. E. & RASMUSSENR. A. (1967) Comparative protein and enzyme profiles of the cerebrospinal fluid, extradural fluid, nervous tissue, and sera of Elasmobranchs. In Sharks, Skates and Rays (Edited by GILBERT P. W., MATHEWSONR. F. & RALL D. P.), pp. 361-379. Johns Hopkins Press, Baltimore. VAN RIJSSEL T. G. (1946) Circulation of cerebrospinal fluid in Carassius gibelio. Arch. Neural. Psychiat. 56, 522-543. ZUBRODC. G. & RALL D. P. (1959) Distribution of drugs between blood and cerebrospinal fluid in the various vertebrate classes. J. Pharmacol. 125, 194-197. Key Word Index-Necturus maculosus; cerebrospinal fluid; subarachnoid space; extradural fluid; blood-brain barrier; choroid plexus; protein; potassium; sodium; inulin; fish.