J. Phy8iol. (1969), 202, pp. 83-95 83With 4 text-ftgurewPrinted in Great Britain

CHICKEN CEREBROSPINAL FLUID:NORMAL COMPOSITION AND RESPONSE TO INSULIN

ADMINISTRATION

BY D. K. ANDERSON AND R. L. HAZELWOODFrom the Department of Biology, University of Houston,

Houston, Texas 77004, U.S.A.

(Received 12 September 1968)

SUMMARY

1. With the exception of Na, K and C1 clear cerebrospinal fluid (c.s.f.)obtained from chickens varying in age from 6 weeks to 2 years did notreveal significant alterations in composition as could be related to ageper se.

2. Considerably higher levels of total protein and glucose are found inchicken c.s.f. than are found in mammalian fluid; slightly more chloride isfound in chicken than most mammalian c.s.f.'s.

3. Intravenous beef insulin depressed chicken cerebrospinal fluid glucoselevels; insulin placed intracisternally had no effect on the avian glycogenbody glycogen content indicating that c.s.f. glucose constancy, with orwithout glycogen body assistance, is not responsible for the resistance ofthe chicken to pharmacological doses of insulin.

4. Bovine insulin injected into the cisterna magna of chickens depressesplasma glucose partially by acting over vagal pathways to release endo-genous insulin and partially by diffusion across the c.s.f.-blood barrier toexert a peripheral effect.

INTRODUCTION

Evidence has been presented by several investigators that, with thepossible exception of geese, birds are very resistant to large doses of insulinas well as to the diabetic sequelae attending surgical ablation of thepancreas (Nelson, Elgart & Mirsky, 1942; Chen, Anderson & Maze, 1945;Hazelwood, 1958; Hazelwood & Lorenz, 1959). In an attempt to explainthe tolerance of chickens to upwards of 5000 u. beef insulin per kilogrambody weight (Chen et al. 1945), it has been suggested that exogenousinsulin may not decrease avian cerebrospinal fluid (c.s.f.) glucose levelsto any great extent. Alternatively, the avian glycogen body may assist incontrolling avian central nervous system (C.N.S.) metabolism by releasing

84 D. K. ANDERSON AND R. L. HAZELWOOD

the hydrolysed polysaccharide to the c.s.f. compartment and therebyprotecting birds from hypoglycaemic convulsions (Hazelwood, Hazelwood& McNary, 1962; Hazelwood, 1965).

Before investigating the above possibilities further it was found that adearth of literature existed germane to the composition of avian c.s.f.Therefore, it was necessary to develop a technique whereby adequateamounts of chicken c.s.f. could be obtained from birds of various ages forthe purpose of biochemical characterization. Only following this couldsubsequent experimental studies be carried out to determine the role, ifany, which avian c.s.f. and/or the glycogen body plays in protecting theC.N.S. from insulin-induced hypoglycaemic reactions. Results of the studiespresented herein describe a method for obtaining chicken c.s.f., its normalcomposition in birds up to two years of age, and the influence of intra-venously administered insulin on c.s.f. glucose levels. Additionally, someinformation was obtained as to the passage of insulin across the c.s.f.-blood barrier and the role which the vagus nerve plays in regulating endo-genous insulin release from the chicken pancreas.

METHODS

Animals. All chickens employed were single comb white Leghorns varying in age from6 weeks to 2 years. Birds were grouped arbitrarily into three age groups, 6-10 weeks, 11-18weeks and 1-2 years of age. All birds were fed Purina (Growina) chicken mash and waterad libitum and were kept at a temperature of 75+ 10 F. When employed, bilateral vagotomywas performed between levels C 7 and T 2 and segments of the nerve removed.

Cerebrospinal fluid sampling. Anatomy of the avian sacro-lumbar vertebral region aswell as pilot studies employing fluoroscopy and perfusion of a radio-opaque dye indicatedthe infeasibility of approaching the c.s.f. compartment by conventional (mammalian)means. Therefore chickens were narcotized with sodium pentobarbital and a 2-3 inchincision was made on the dorsal neck region exposing the occipital region of the skull. A25-gauge hypodermic needle containing a stylet was inserted through the muscle layersbetween the occipital protuberance and atlas vertebra directly into the cisterna magna(Fig. 1). The needle was inserted on a line running from the point of insertion to the eye andto a depth of approximately one quarter of an inch. The stylet was then removed and thecavity of the needle examined for appearance of colourless c.s.f. Sometimes rotation of theneedle facilitated c.s.f. appearance; in most cases the closer the insertion was made to theoccipital protuberance the less likely that medullary trauma occurred. When c.s.f. wasvisible in the needle a 1 ml. tuberculin syringe was attached and gentle aspiration applied.Frequently it was found that injection of a small amount of air into the cisterna magnafacilitated subsequent c.s.f. aspiration. By this procedure samples ranging in size from 0-1to 0-4 ml. were obtained. All c.s.f. samples were obtained from well fed chickens in goodphysiological condition and usually between 8.00 a.m. and 12 noon. C.s.f. samples werefrozen immediately to await subsequent analysis; samples were not pooled. Only dataobtained on completely clear-colourless c.s.f. samples are reported here.

Blood samples. Immediately after successful c.s.f. aspiration, blood samples were obtainedfrom the same bird by cardiac puncture. Blood samples were centrifuged at 3000 rev/min inan International clinical (table) centrifuge, the plasma decanted and frozen immediately toawait subsequent analysis. At no time were plasma samples pooled.

CHICKEN C.S.F. AND INSULIN RESPONSE 85Analyses. Determinations on c.s.f., plasma and tissue samples included total reducing

substances (Somogyi, 1952), tissue glycogen (Seifter, Dayton, Novic & Muntwyler, 1950),total protein (Lowry, Rosebrough, Farr & Randall, 1951), amino-nitrogen (Frame, Russell& Wilhelmi, 1943): sodium and potassium were determined by flame photometry (Eppen-dorf model), chlorides by a Cotlove chloridometer (Buchler) and specific gravity and pH byconventional means (the latter two analyses were made only on fresh c.s.f. samples).

Cerebrum Calvarium Cerebellum

'Occipital

magna

Optic Lateral Medulla Spinalchiasma ventricle oblongata cord

Fig. 1. Sagittal section through chicken brain (modified from van Tienhoven &Juhasz, 1962). Section indicates position of needle and syringe slightly beneathoccipital protuberance to gain access to the cisterna magna. Note location ofmedulla with respect to needle angle and position.

Insulin and tolbutamide. Insulin (Iletin) was a gift of W. R. Kirtley, Eli Lilly Co., andsodium tolbutamide (1-butyl-3-p-tolysulphonylurea) was a gift of C. J. O'Donovan, UpjohnCo. The doses of each employed were chosen because previous work (Hazelwood, 1958) hadshown that they were equal in hypoglycaemic potential in birds when administered intra-venously. Additionally, in the intracisternal injection series (Figs. 3 and 4) the concentra-tions were adjusted so that equal volumes could be injected maintaining the same dose perunit body weight. Rarely was the volume injected intracisternally greater than 0-15 ml.and in all cases it was followed by an equal volume of 0-85 % saline (w/v) as a flush into thec.s.f. compartment. The needle and saline syringe were left in place after injection as adeterrent to back leakage from the cisterna magna. Control chickens which received twovolumes of saline were employed in both studies, i.e. experiments with intact or sectionedvagi.

RESULTS

One hundred and fourteen chickens were grouped by age as indicated inTable 1 and the chemical characteristics of the samples of c.s.f. obtainedare presented in this table and in Table 2. Only one c.s.f. sample wasobtained from each bird; plasma samples obtained simultaneously

86 D. K. ANrDERSON AND R. L. HAZELWOOD

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CHICKEN C.S.F. AND INSULIN RESPONSEallowed for comparisons to be made between plasma and c.s.f. concen-trations of each variable (Table 2). While the levels of glucose, protein andamino nitrogen in c.s.f. did not fluctuate with age, the levels of bothprotein and glucose are far in excess of those found in the literature formammals (Millen & Woollam, 1962; Cornelius, 1963). Avian plasma levelsof these two components are excessive relative to mammalian levels onlyin the case of glucose, thus indicating possible permeability differencesbetween chicken and mammalian c.s.f.-blood barriers. Large moleculesplaced intravenously, as insulin, could possibly gain access to the c.s.f.compartment in the chicken.

Insulin and tolbutamide injected intravenously caused the expectedhypoglyeaemia over the observed time course; however, the plasma glucosecurves in response to the two substances, while qualitatively similar, werenot in accord quantitatively (Fig. 2). Of particular interest are the c.s.f.data presented in Fig. 2 which indicate that, indeed, the c.s.f. glucoselevels are markedly affected by intravenous hypoglycaemic agents and thatthese alterations appear to follow closely the changes observed in plasmaglucose throughout the observation period. In both cases changes inglucose levels of c.s.f. lagged behind similar changes in plasma glucose,indicating possibly that c.s.f. glucose levels reflect plasma levels and theformer are altered according to the concentration differences expressedacross the avian c.s.f.-blood barrier.The possibility that intravenous insulin and/or tolbutamide gained

access to c.s.f. compartments is not precluded by the results presentedabove. Thus, insulin (exogenous or endogenously released) may havecrossed the blood-c.s.f. barrier and encouraged glucose uptake by thechicken C.N.S. in a manner similar to its action on the periphery. To testthis possibility, saline, tolbutamide or insulin was injected intracisternally(cisterna magna) and peripheral blood samples taken for 100 min. Fortechnical reasons c.s.f. could not be obtained from these birds; repre-sentative data from a successful series of experiments are presentedherein. Pilot studies employing Evans Blue (T-1824; MW 961) placedintracisternally yielded information pertinent to circulation of c.s.f. overthe intended period of observation. After 20 min of equilibration dye wasobserved on the lateral and dorsal aspects of the cerebellum and on thedorsal cerebral surface; after 60, 80 and 100 min post-injection equilibra-tion periods, dye concentration decreased in the cisterna magna and in-creased over the complete surface of the cerebrum and on dorsal and ventralaspects of the spinal cord extending at least to the C10 level.The hypoglyeaemia observed in response to beef insulin injected intra-

cisternally (Fig. 3) closely paralleled that response seen in Fig. 2 for intra-venous administration of the same hormone. The glycaemic response to

87

D. K. ANDERSON AND R. L. HAZELWOODtolbutamide placed intracisternally was considerably less than that seenwhen the sulfonylurea was administered intravenously (Fig. 2). Since theaction of tolbutamide in the chicken is probably mediated through theperiphery (Mirsky & Gitelson, 1957; Hazelwood, 1958) and is not known tohave any effect on the c.N.s., it may be assumed that the hypoglycaemiarecorded in Fig. 3 is the direct result of transport of the sulfonylurea acrossthe c.s.f.-blood barrier. Hypoglycaemia induced by insulin could be the

/a

Difference in response (%) between plasmaand c.s.f. levels of glucose

464 22 9 21 0 17 8 2 6 8-3 Beef insulin422 200 72 -202 -81 80 Tolbutamide

20 40 60 80 100 120

Minutes following injection (i.v.)

Fig. 2. Effect of intravenous insulin and tolbutamide on chicken plasma andc.s.f. glucose levels. Representative data shown from twenty-four birds; each pointrepresented by one bird. Cardiac blood samples taken immediately after c.s.f.sample obtained. Initial plasma glucose levels of the insulin and tolbutamide groupswere 261+9-2 and 287+3-3mg/100ml. plasma, respectively. Plasma glucose(*) and c.s.f. glucose (A) in response to 1 u. beef insulin/kg body weight; plasmaglucose (0) and c.s.f. glucose (A) in response to 10 mg tolbutamide/kg body weight.

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CHICKEN C.S.F. AND INSULIN RESPONSE

result of transport of the hormone across the barrier or due to stimulationof neural centres which activate vagal endings in the pancreas. Strongstimulation of the vagus in dogs increases endogenous release of pre-formedinsulin (Chowers, Lavy & Halpern, 1966; Frohman, Ezdinli & Javid, 1967).

It was of interest to note that at no time did the avian glycogen bodyglycogen levels change significantly in response either to insulin or totolbutamide injection (Fig. 3).

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Fig. 3. Effect of insulin or tolbutamide injected into cisterna magna on plasmaglucose (upper) and glycogen body glycogen levels (lower) in normal chickens.Shaded area on glycogen body data indicates s.E. of mean of nine birds; all otherdata are typical/representative data obtained from a total of fourteen birds.Initial plasma glucose levels ranged from 234 + 6-5 to 266 + 4-4 mg/100 ml. plasmafor the three groups shown. Dosage of beef insulin ( 0) and tolbutamide (0) wassame as in Fig. 2; saline (A) injections were made in control birds. Total volumeinjected in each case was 0-1 ml.

89

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90 D. K. ANDERSON AND R. L. HAZELWOODTo ascertain what role, if any, the vagi played in the response of the

chicken to insulin injected intracisternally such injections were made intochickens which had been doubly vagotomized 5-15 min previously and thedata obtained relative to plasma glucose alterations are presented in Fig. 4.Immediately after severing the vagi respirations decreased from 33 + 2 to9 + 1 per minute. It is obvious that severing the vagi had little or no effect

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Fig. 4. Effect of insulin injected intracisternally on plasma glucose levels ofbilateral vagotomized chickens. All data are means; vertical bars are standarderrors of means. Initial plasma glucose levels ranged from 264 + 7-2 to 278 + 12 8mg/100 ml. plasma for the four groups shown. Dosage and volume identical withFig. 3. Five birds were bilaterally vagotomized controls (A), three birds weresimilarly prepared and injected with saline intracisternally (Fl), and seven birdswere doubly vagotomized and injected with beef insulin intracisternally (0).Filled circle data (@) are from Fig. 3 and obtained on five intact chickens forcomparison.

CHTICKEN C.S.F. AND INSULIN RESPONSE

on plasma glucose levels; neither did the injection of 0 15-0-30 ml. salineinfluence plasma glucose in vagotomized control chickens (Fig. 4). How-ever, the injection of insulin intracisternally into the operated birds(bilateral vagal section) produced a marked hypoglyeaemia, the nadir ofwhich was 20, 22 and 22 % less than those values obtained in chickens withintact vagi at the 40, 60 and 80 min times, respectively. Thus, in absenceof the vagal nerves the degree of hypoglycaemia was reduced greatly butwas not abolished.

DISCUSSION

Primary among the objectives of this study was the development of atechnique for obtaining chicken c.s.f. samples for the purpose of submittingthese samples to chemical analysis. Analysis of chicken c.s.f. indicates thatit contains considerably higher glucose and protein levels relative tomammals and slightly higher levels of chloride (Table 3). Hyperglycor-rhachia is a common observation in diabetes mellitus and apparently is areflexion of the existing hyperglycaemic condition (Cornelius, 1963). Theconcentration of constituents other than glucose and protein in avianc.s.f. are in good accord with mammalian values selected from the litera-ture. While the high glucose levels of avian c.s.f. probably reflect the highplasma glucose levels normally found in birds the very high c.s.f. proteinlevels cannot be attributed to elevated plasma protein levels. Thus, c.s.f.protein levels of chickens are six times greater than that found in mam-malian c.s.f., yet the plasma protein levels of the two classes rarely differby more than 6-9 % from each other. Such observations on chickens mayindicate that the blood-c.s.f. barrier is not as effective and/or selective asnormal mammalian barriers in regulating protein levels of c.s.f. Thepossibility therefore exists that the protein hormone, insulin, crosses theblood-c.s.f. barrier and thereby comes into intimate contact with avianneural tissue via c.s.f. circulation.A second objective of this investigation was to ascertain whether or not

the resistance which chickens demonstrate to pharmacological doses ofinsulin could be due to unwavering levels of c.s.f. glucose with or withoutauxiliary aid from the polysaccharide-rich glycogen body of birds. Therelatively rapid, marked and possibly plasma-linked drop in c.s.f.-glucosein response to intravenous beef insulin (Fig. 2) argues against the suggestionthat constancy of this moiety protects the avian C.N.s. against convulsionsinduced by insulin. Furthermore, the data presented in Fig. 3 indicate thateven when insulin is injected intracisternally, glycogen body glycogenlevels remain unaltered for at least 100 min, casting considerable doubt onany role this structure might play in protecting the chicken from immediateconvulsive effects of the hormone. These observations expand the growing

91

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CHICKEN C.S.F. AND INSULIN RESPONSE

list of evidence (Hazelwood et al. 1962; Hazelwood, Hazelwood & Olsson,1963) confirming the physiological inactivity of this avian structure.Additional information was gained from these studies, however, con-

cerning transport of insulin across the blood-c.s.f. barrier as well as con-cerning possible vagal control over release of endogenous insulin from thechicken pancreas. Decrease in c.s.f. glucose as a result of intravenous beefinsulin may not have been the result of shifting c.s.f. glucose to the plasmasince at no time did the induced hypoglycaemic nadir reach the highest levelof c.s.f. glucose. Thus, no downhill concentration gradient was establishedfavouring a shift of glucose from c.s.f. to plasma compartments. Alter-natively, insulin could have crossed the blood-c.s.f. barrier and encouragedgreater C.N.s. uptake of glucose from the c.s.f. compartment or the hor-mone could have acted on blood-brain facilitative or active transportcarriers of glucose. Further work is needed to clarify these possibilities.Intravenous use of tolbutamide did not clarify to any extent the mechanismby which glucose was reduced in c.s.f. However, previous work obtainedin mammals has led to equivocal interpretations, some workers favouringthe increased c.N.s.-glucose uptake postulate, other investigators proposethe plasma versus c.s.f. glucose concentration difference hypothesis (cf.Kasahara & Uetani, 1924; Day, Niver & Greenberg, 1938). More recently,Mahon, Steinke, McKhann & Mitchell (1962), Margolis & Altszuler (1967,1968), re-approached this problem employing radioactive insulin and againreached opposite conclusions as to the mobility of [131I]insulin across theblood-c.s.f. barrier. While the former workers could not demonstrateinsulin in the c.s.f. ofhumans after a single injection, the latter investigatorsfound a tenfold increase in c.s.f. insulin following intravenous infusion ofphysiological levels of beef insulin in dogs. The infusion technique mayparallel the normal endogenous situation more closely than the singleinjection method and thereby yield results which can be interpreted as ofgreater physiological significance. Similar work is yet to be done inchickens.

Margolis & Altszuler (1968) reported that [1311]insulin crosses the dogc.s.f.-blood barrier following injections directly into the cisterna magna.The data obtained in chickens presented here (Figs. 3 and 4) are in partialaccord with their work in the dog. Insulin and tolbutamide placed intra-cisternally both were effective in depressing plasma glucose in chickens.The small molecular size (mol. wt. 278) and the lack of known directneural effect of the sulphonylurea would indicate in all probability that itcrossed the c.s.f.-blood barrier to exert its hypoglycaemic effect. (Insulininjected intravenously in chickens which were bilaterally vagotomizedresulted in an hypoglycaemic response which is indistinguishable from thatobserved when the hormone is placed inthe c.s.f.of similarly prepared birds.)

93

D. K. ANDERSON AND R. L. HAZELWOODBeef insulin placed intracisternally depressed chicken plasma glucose

markedly (Fig. 3), an effect which could be due to c.s.f.-blood barriercrossing of insulin (Sloviter & Sakata, 1963) or due to stimulation of neuralcentres which activate endogenous release of insulin over vagal-pancreaticpathways (Chowers, Lavy & Halpern, 1961). In a more recent paper Chowerset al. (1966) reported that insulin injected intracisternally promoted hypo-glycaemia in dogs by stimulating the motor nuclei of the vagus since dogswhich are bilaterally vagotomized failed to demonstrate an hypoglycaemiceffect in response to adding insulin to the c.s.f. Similarly, electrical stimula-tion of the peripheral end of the cut vagus nerve in dogs produces animmediate elevation of immunoreactive insulin in pancreatic vein plasma(Kaneto, Kosaka & Nakao, 1967). The possibility is raised, therefore, that indogs basal insulin release may be partially under the control of vagal acti-vity. Such may also be the case in chickens since the saline control chickens(injected intracisternally) as well as the control chickens which had doublevagal section demonstrated a modest yet continuous blood glucose eleva-tion shortly after section of the vagi was carried out (Fig. 4). The role ofthe vagi in mediating insulin-induced hypoglycaemia in chickens receivinginjections intracisternally is punctuated further in Fig. 4. If the insulininjected into the cisterna magna of chickens, whose vagal pathways wereintact, induced hypoglycaemia via stimulation of some neural parasym-pathetic pathway (Fig. 3) the results in the series ofbilaterally vagotomizedbirds of Fig. 4 would have been expected to fall close to those observed inthe birds injected with saline and/or control chickens which were bilaterallyvagotomized. If, on the other hand, the induced hypoglycaemia in theintact bird was due primarily to passage of the hormone from the c.s.f.compartment to the periphery, one would expect the hypoglycaemia ofthechickens which were vagotomized bilaterally to be in accord with thatobserved in the intact chicken. The observation that the degree andcharacter ofthe hypoglycaemia differed markedly and significantly from theintact chicken receiving intracisternal injections (Fig. 4, filled circles) aswell as from the vagotomized birds injected with saline (Fig. 4, open squares)indicates that the hypoglyeaemia induced by insulin which was observedin vagotomized chickens was due in part to insulin stimulation of C.N.s.-vagal centres as well as due in part to significant amounts of the hormonepassing across the c.s.f.-blood barrier to exert a peripheral effect. Thedifference in hypoglycaemia of the vagotomized birds injected with insulinfrom that observed in the intact chickens indicates the role which vagal' centres' may play in mediating the observed hypoglycaemia. Similarly,it is concluded that the difference in hypoglyeaemia observed in the vago-tomized chickens injected with insulin from those birds injected withsaline (also vagotomized) reflects the extent to which intracisternal insulin

94

CHICKEN C.S.F. AND INSULIN RESPONSE 95left the c.s.f. compartment and passed into the vascular system to en-courage peripheral glucose uptake.

This work was supported by NSF: GB-6012 and GB-8457.

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