The entry of glycerol into brain tissue

305

Journal of the neurological Sciences

Elsevier Publishing Company, Amsterdam - Printed in The Netherlands

The Entry of Glycerol into Brain Tissue J. M. W A T E R H O U S E AND R. V. C O X O N University Laboratory of Physiology, Oxford (Great Britain)

(Received 17 March, 1969)

INTRODUCTION Glycerol is generally regarded as a solute which penetrates the membranes of animal cells very readily. In fact in a well-known textbook of general physiology it is used as a typical example of a freely-penetrating solute in a discussion of the osmotic behaviour of the sea-urchin egg. In the case of another favoured object for osmotic studies, namely the human red cell, once again glycerol penetrates rather readily, although here there is some evidence that the process is not one of simple diffusion, since it can be interfered with by a number of inhibitory compounds; moreover, the red cells of other species behave in relation to glycerol somewhat differently from those of the human (HARRIS 1960). In whole animal studies in the cat it has been found that the volume of distribution of glycerol is between 50 and 65 o~, of body weight (HOLST 1944; LARSEN 1963), which points to a rather wide-spread entry into the cellular water. However, the fact that reduction of intracranial pressure can be brought about by the introduction of glycerol into the blood-stream suggests that free permeation into nervous tissue does not take place (BuCKELLAND WALSH 1964; CANTORE et al. 1964). Similarly, the use of glycerol as a means of reducing intraocular pressure again suggests that there are certain areas of the body where a barrier to the penetration of glycerol must exist (CANTOREet al. 1964; TREVOR-ROPER 1964). With these considerations in mind, a series of experiments was undertaken which will be described in the present paper.

MATERIAL AND METHODS Rabbits of both sexes were used and were anaesthetised by an intravenous dose of pentobarbitone at the beginning of the experiment, supplemented, as necessary, by further small doses. Paper presented at a symposium on The Blood-Brain Barrier, held 11 September, 1968, in New York, N.Y.(U.S.A.). J. M. WATERHOUSE:Medical Research Council Scholar. J. neurol. Sci., 1970, 10:305-311

306

J. M. WATERHOUSE, R. V. COXON

Intravenous infusion Where it was desired to maintain the blood concentration of glycerol at a high level this was achieved by administering a priming dose followed by a continuous infusion into the saphenous vein. The priming dose consisted of 1.15 g glycerol /kg of body weight dissolved in 10 ml of 6.6°,~, glucose solution. This was given over a period of 2 min and zero time was taken as the mid-point of this period. The maintenance infusions were given by means of a constant-speed injection apparatus (C.F, Palmer, London). During the first 21 h of a long experiment the infused solution contained 12.6 g glycerol and 6.6 g glucose/100 ml water, and after 2~ h this was substituted by 6.3 g glycerol plus 6.6 g glucose/100 ml water. Both solutions were given at a rate corresponding to 0.09 ml/min/kg body weight. Ventriculo-cisternal perfusions Cannulae were implanted into the lateral ventricles as described by POLLAY AND DAVSON (1963). The perfusion fluid was made up as described by PAPPENHEIMERet al. (1961) and was saturated with 5% CO2 in oxygen before use, the bicarbonate content being adjusted to give a final p H of 7.35-7.45. The artificial cerebrospinal fluid was introduced at a rate of 0.08 ml per min through each ventricular cannula and was drained through a third cannula inserted into the cisterna magna. Outflow was promoted by applying a negative pressure of up to 20 cm of water, the actual pressure being adjusted so that the fluid flowed out at the same rate as it was being injected, allowance being made for an endogenous production of cerebrospinal fluid (CSF) which was assumed to be about 1/20th of the rate of introduction of the perfusion fluid, in experiments where glycerol was included in the perfusate the strength was 30 mM, and Dextran 2000 (Pharmacia, Uppsala) combined with a blue dye was also added to facilitate post-mortem identification of the perfusion pathway. Sampling procedure When glycerol was administered by intravenous infusion, blood samples were taken at appropriate intervals from a catheter inserted into the femoral artery on the opposite side of the body. At the end of the experiment the animals were given a lethal dose of pentobarbitone. Aqueous humour was then aspirated from both eyes, muscle samples were obtained from the back of the neck and CSF by aspiration from the cisterna magna. For brain analyses portions of the frontal and parietal lobes were removed. The frontal lobes were used for glycerol estimation and the parietal lobes for determination of water content. When glycerol was infused into the cerebral ventricles muscle samples were obtained from the quadriceps femoris, and both frontal and parietal lobes were removed for glycerol estimation; the temporal lobe was removed for the determination of water content. Samples of CSF were obtained from the cannula in the cisterna magna. When glycerol was being introduced both intravenously and intraventricularly the quadriceps on the opposite side from the venous cannula was used; brain determinations were made on the same lobes as for intraventricular injection alone. Analytical methods Glycerol was measured by the enzymatic method of GARLAND AND RANDLE (1962) J. neurol. Sci., 1970, 10:305 -311

307

ENTRY OF GLYCEROL INTO BRAIN TISSUE

on protein-free filtrates. These filtrates were obtained from brain and muscle by homogenizing in water and precipitating the proteins with cadmium sulphate and sodium hydroxide. The same protein precipitants were also used for plasma, CSF and aqueous humour. Water contents were determined by drying to constant weight. Calculations All results are expressed as concentrations per kg of tissue water. In the experiments employing intravenous infusion the reference values for blood were based on linear interpolation using the measured values over the last hour of the experiment when the total duration was less than 3 h, and over the last 2 h in longer experiments. In the experiments employing ventriculo-cisternal perfusion the CSF concentration was taken to be the arithmetical mean of the inflow and outflow values at the same time as that for estimating the reference value for blood.

RESULTS

A plot of the blood glycerol concentration from a typical experiment is shown in Fig. 1 and illustrates that after the first 1½ h of the infusion the plasma concentration of glycerol fluctuates only narrowly.

6 tJ

235

!3o 20

d

15

x

~----~x ............

x ~

25

0

tO <

xjxJ

qO

5 0

i

0

I

~

-

2

-

J

i

J

3

4

5

TIME AFTER COMMENCEMEIJT OF PERFUSION (h ) Fig. 1. Graph showing the plasma concentration of glycerol in a typical experiment involving intravenous infusion.

Fig. 2 shows the concentration of glycerol in the brain, CSF and aqueous humour expressed as a fraction of that in the plasma at various times after raising the blood concentration of glycerol and maintaining it at about 30 mmoles/l. Some data on muscle are also included for comparison and it is evident that the concentration of glycerol attained in the brain and CSF remains very much lower than that in muscle water at all times up to 9 h. Concentrations in the aqueous humour are also lower than those found in muscle, although the difference is not so marked as in the case of CSF and brain water. J. neurol. Sci., 1970, 10:305-311

308

J.M. WATERHOUSE, R. V. COXON

1o[ AQUEOUS HU,~IOUR

0.8 ! o% [

/O f j -

BRAIN

.....

O'4!

CSF

i

O2 I L.._. ~

©

I

.......c

__~______~L

....

k

.

~

_

3 4 5 6 7 [DURATION OF INFUSION (H) 2

i

_

,~

9

Fig. 2. The ratio of concentration of glycerol in tissue water to that in plasma (T/P). The vertical bars represent the standard deviation of the means of 5 experiments.

80

o

o

qR

79

"~ i

P--O 0 2

P<) 02

P
77,

7S

,:~

74

73

P'~O

I

?

5

4

5

6

;'

8

'-~

DUf~ATION OF INFUSION (H) Fig. 3. Changes in the water content of muscle and brain following intravenous infusion of glycerol.

Open symbols: data from experiments in which the vehicle only without glycerol was introduced; full symbols: results of expernments in which glycerol was included in the fluid introduced. Circles apply to brain, and squares to muscle. Vertical bars: the standard deviation of the means of 5 experiments.

Fig. 3 shows the effect of a high blood-glycerol level on the water content of brain and muscle. There is clearly a selective dehydration of the brain during the course of the glycerol infusion up to some 5 h from the start of the experiment. Between 5 and 7 h there is some dehydration also of muscle but this is thought to be due to an overall loss of fluid from the animal as a result of osmotic diuresis. However the difference between brain and muscle during the first 5 h of the infusion is very striking, Fig. 4 shows the results of perfusion of the ventriculo-cisternal system with artificial CSF containing some 30 mmoles glycerol/l. As in the case of intravenous infusion there is a marked difference of concentration o f glycerol in the brain from that in the perfusing fluid. These results demonstrate the existence of a CSF-to-brain barrier for glycerol in addition to a blood-brain barrier. J. neurol. Sci., 1970, t0:305.-311

309

ENTRY OF GLYCEROL INTO BRAIN TISSUE

T/F 06

/c~ (F)

05[

BL~.

~o (P)

04 03 02 f OI © 0

"°-~

I

2

CS F

3

4

6

5

7

DURATION OF INFUSION (H)

Fig. 4. The results of experiments in which glycerol was introduced intravenously (the line labelled blood) and intraventricularly (the 2 lower lines labelled CSF). (F): signifies frontal lobe; (P): parietal lobe. T/F is the ratio of glycerol in tissue water to that in plasma or artifical CSF.

[/po~ 0.5 04 05 0.2 0-1 O

I

0'6

I

I

I

I

I

I

0"8 I-O I '2 CSFconcn/Plasrnaconcn.

I

I

1"4

Fig. 5. The ratio of the concentration of glycerol in brain to that in plasma (T/P) plotted against the ratio of the concentrations in artificial CSF and in plasma when glycerol was introduced both intravenously and intraventricularly. The duration of the experiments was 3 h in all cases.

Since it is possible that the low concentration found in brain during both intravenous and ventriculo-cisternal perfusion might be due to a loss of glycerol via the CSF in the case of intravenous infusions and via the blood in the case of ventriculo-cisternal perfusions a final series of experiments was done in which the glycerol concentration was maintained high in both. The results of these experiments are shown in Fig. 5, from which it is evident that even ifa high glycerol level is maintained in both CSF and blood the brain concentration still remains low.

DISCUSSION

We interpret the low concentration of glycerol in the brain as being due to a failure to penetrate but two other possible explanations deserve some consideration. One alternative has already been mentioned, namely that there might be a process leading J. neurol. Sci.,

1970, 10:305-311

310

J . M . WATERHOUSE, R. V. COXON

to the rapid removal of glycerol either into the blood or into the CSF. We believe that the results of the experiments in which both CSF and blood contained raised and approximately equal concentrations of glycerol tend to exclude this possibility. A further alternative is that glycerol might be rapidly utilised by nervous tissue. This again we consider unlikely for several reasons. The first derives from results obtained by CRONE (1965) who measured the rate of entry of glycerol into the brain by determinations of arteriovenous gradients and found that glycerol disappeared no more readily than did Evans blue from the cerebral circulation. Secondly, we ourselves obtained some indirect evidence pointing in the same direction by studying the concentration of glycerol in the water of the liver, which is the organ in the body where most active utilisation of glycerol appears to take place (LARSEN 1963). We found that the concentration in liver differed very little from that in muscle water. Finally~ some unpublished experiments carried out in our laboratory (LARKINS AND COXON 1968) have failed to reveal any substantial degradation of radioactive glycerol by slices of rabbit brain in vitro. It therefore seems that some barrier must exist which prevents the free movement of glycerol into the brain either from the blood or from the subarachnoid space and ventricular cavities. In the case of the blood-brain barrier this seems to be of such a nature that osmotic movement of water can take place across it. This barrier could be an intracerebral one separating one population of cells which are permeable to glycerol from another population which is not. The glycerol space is appreciably greater than the inulin or sucrose space (OLDENDOREAND DAVSON 1967) SO that some intracellular penetration is likely in the case of glycerol. A part of the same restricted intracellular space may be accessible to glycerol introduced into the CSF or a different space may be involved in this ease. The results obtained by separate perfusion of the cerebral blood vessels on the one hand and of the ventricuto-eisternal system on the other indicate clearly that the two "spaces" are not co-extensive. Nevertheless since together they amount to a value very close to the total volume of the brain water these results do not indicate whether or not there is overlap between them. However, the results obtained by maintaining a high level of glycerol in both blood and CSF do exclude the possibility of the spaces accessible from the two routes being independent and additive, since the space found by simultaneous perfusion of both the vascular and the ventriculo-cisternal systems does not appreciably exceed that found by perfusion of the vascular system alone.

ACKNOWLEDGEMENT

Our thanks are due to Mrs. Sandra Holmberg ibr skilled technical assistance.

SUMMARY

Experiments are described in which the concentration of glycerol in brain water, cerebrospinal fluid and aqueous humour has been measured following the maintenance of the plasma concentration at a level of about 30 m M by means of a continuo us intravenous infusion. The results indicate an incomplete penetration of glycerol into J. neurol. Sci.~ 1970, 10:305 -311

ENTRY OF GLYCEROL INTO BRAIN TISSUE

3 11

brain water. A second series of experiments is described in which glycerol was perfused t h r o u g h the ventriculo-cisternal system, a n d the b r a i n c o n c e n t r a t i o n measured after varying intervals of time. Here too p e n e t r a t i o n was incomplete. A third series of experiments involving the simultaneous i n t r o d u c t i o n of glycerol both into the blood stream a n d into the ventriculo-cisternal system is reported a n d in these circumstances also penetration into brain water was incomplete. The possible interpretation of these data is discussed.

REFERENCES

BUCKELL,M. AND L. WALSI-I(1964) Effect of glycerol by mouth on raised intracranial pressure in man, Lancet, ii: 1151-1152.

CANTORE, G., B. GUIDETTIAND M. VIRNO(1964) Oral glycerol for the reduction of intracranial pressure, J. Neurosurg., 21 : 278-283. CRONE, C. (1965) The permeability of brain capillaries to non-electrolytes, Acta physiol, scand., 64: 407-417. GARLAND,P. B. AND P. J. RANDLE(1962) A rapid enzymatic assay for glycerol, Nature (Lond.), 196: 987-988. HARRIS, E. J. (1960) Transport and Accumulation in Biological Systems, 2nd edition, Butterworths, London. HOLST,E. J. (1944) Glycerol oxidation in the animal organism, Actaphysiol. scand., 7: 69-79. LARSEN,J. A. (1963) Elimination of glycerol as a measure of the hepatic blood flow in the cat, Acta physiol, scand., 57: 224-234. OLDENDORF,W. H. AND H. DAVSON(1967) Brain extracellular space and the sink action of cerebrospinal fluid, Arch. Neurol. (Chic.), 17: 196-205. PAPPENHEIMER, J. R., S. R. HEISEYAND E. F. JORDAN (1961) Active transport of Diodrast and phenolsulfonphthaleinfrom cerebrospinal fluid to brood, Amer. J. Physiol., 200: 1-10. POLLAY, M. AND H. DAVSON(1963) The passage of certain substances out of the cerebrospinal fluid, Brain, 86: 137-150. TREVOR-ROPER.P. D. (1964) The use of oral glycerol in glaucoma, Proc. roy. Soc. Med., 57: 37-38.

J. neurol. Sci., 1970, 10:305-311