AIN

ing centres of man's brain.the shrouded veil is not yettransform the world and to

h the sea, is probably notit is difficult to imagine howays and means of destroyingbrain most of the hard pro-face to face with indeter-

icians ultimately succeed in) complete with 10 billion

ing. What that "something"y. Some will call it a soul.'Passion. They will suggestssess,not to his soul or his

and description from his envi-er excluded and inspiration

180, 193;Univ. Mich. Med.

and J. G. Mil1~r, p. 86-105.,

Frontiers, editor. N. S. Kline,

anism and Learning, Blackwell

rand j. G. Miller, p .• 1-85,

cad sa., 66 (3),784.. Clin. Neurophysiol., 5,91.

homas.n, Neurophysiol., 1, 455.

.J. Pharm., 18,433.

tional Anatomy of the Human Brain,

slol, Psycol., 50, 74.

Psycol., 51, 130.rurophysiol., 12, 1.

CAPILLARY PERMEABILITY-I CREASING PROPERTY OF

HYALURONIDASE IN RATBy

M. N. GHOSH!, R. M. BANERJIE AND S. K. MUKHERJI

Defence Research Laboratory, Kanpur, India(Received March 28, 1962)

Toe swelling produced by the subcutaneous injection of a small volume of fluidin rat's foot was measured plethysmometrically every 5 min over a period of 30 min.Following the injection of normal saline (control), the swelling decreased in volumeslowly. The swelling produced by the injection of hyaluronidase, on the otherhand, increased in size at first, reaching maximum in 10 to 15 min after which itgradually decreased but not to its original size. The duration-action response wasgraded depending on the concentration of hyaluronidase. There was markedinhibition of the response when the enzyme was inactivated by heating at

100·C for 15min at pHIO.

It is concluded that the oedema produced by hyaluronidase is due to increasein the capillary permeability. Furthermore, the effect is specific and is not due to

contamina!lts like histamine or other permeability factors.

Whether hyaluronidase increases the permeability of capillaries is stilla controversial point. A group of workers have shown that the extravasationof Evans blue from the blood was accelerated by addition of a crudeextract from testes (Duran-Reynals, 1939; Aylward, 1942)); while othershave failed to confirm this (Rocha e Silva and Dragstedt, 1941; Zweifachand Chambers, 19:>0). Benditt et al. (1951) found a decrease in the permea-bility-inducing action with increase in the purity of the preparations ofhyaluronidase. This latter finding suggests that the capillary permeability-increasing action of crude hyaluronidase demonstrated by some workersmay be due to the presence of impurities such as histamine or other permea-

bility factors .

The present work, which was taken up with the idea of developing anin vivo bioassay method for hyaluronidase in rat, however, lent some evidencetowards the capillary permeability-increasing action of hyaluronidase. Thishas been presented here.

METHODS

Rats, 100-180 g were lightly anaesthetised by intraperitoneal injectionof pentobarbitone sodium 5 mgJlOO g. The degree of swelling produced

1 Present address: Department of Pharmacology, Medical College, Pondicherry, S. India.

18 HYALURONIDASE ON CAPILLARY PERMEABILITY

by a constant volume of fluid injected subcutaneously in rat's foot wasmeasured plethysmometrically by the method described by Buttle et al.(1957), with the modifications that sodium pentobarbital was used inplace of sodium amy tal; only the upper mark A was graduated on the widemouth of the burette and a mark put below the knee of the rat with somewater-proof ink and this was made to coincide with the mark A. The rat.was allowed to lie on a wooden platform just over the burette and througha hole in it the leg was introduced and dipped in the water in theburette.

The volume of the foot was first measured before the injection.Normal saline 0.4 ml with or without hyaluronidase, was then injectedsubcutaneously in the dorsum of the foot and the volume of the foot measuredagain and then every 5 min for a period of 30 min. Difference in thevolume at any given time from the initial normal volume gave the volumeof the injected fluid still remaining unabsorbed in the foot.

The concentrations of hyaluronidase used were 400, 500, 600, 700 and800 i.u.jrnl. The different concentrations of hyaluronidase as well as thecontrol saline were randomly distributed amongst a total of 14 rats so thateach rat had either control and hyaluronidase, or two different concentra-tions, or same concentrations of hyaluronidase on the two feet. For eachconcentration as well as for the control 3-4 replications were obtained.

Later, in a separate series of experiments, the effect of heating theenzyme preliminary to injection was observed. In one foot of each of 3 rats700 i.u.jrnl of hyalurohidase was injected, while in the other foot same wasinjected after preliminary heating on a water bath at 1000e for 15 min ata pH adjusted to 10.

The hyaluronidase used in these experiments was highly purifiedpreparation Rondase (Evans Medical Supplies, Ltd.) available In ampoulesof 1,500 i.u, and Kinaden (Schering A. G. Berlin) available in ampoulesof 350 i.u.

RESULTS

The average time courses of action following varying concentrations ofhyaluronidase have been presented in Fig. 1. With hyaluronidase, between400-800 i.u.jml., the swelling of the foot increased as evidenced by theupward rise of the graphs from the initial level, reaching maximum in about10.15 min. Within 30 min period of observation, none of the swellingsexcepting that caused by the lowest concentration (i.e., 400 i.u.) returned tothe initial normal volume. Only in this lowest concentration group, there

EABJLITY

neously in rat's foot wasescribed by Buttle et al.

ntobarbital was used inas graduated on the wideknee of the rat with someith the mark A. The rat

r the burette and throughin the water in the

ed before the injection.idase, was then injectedlume of the foot measured

min. Difference in thevolume gave the volumee foot.

400, 500, 600, 700 andluronidase as well as thea total of 14 rats so that

two different concentra-the two feet. For each

ions were obtained.

the effect of heating thene foot of each of 3 ratsthe other foot same wasat 100°C for 15 min at

ts was highly purified) available in ampoules

available in ampoules

rying concentrations ofhyaluronidase, between

as evidenced by theing maximum in aboutnone of the swellings

e., 400 i.u.) returned toncentration group, there

M.N. GHOSH, R.M. BANERJIE AND S.K. ,MUKHERJI 19

---- -- - - -- -- ...•... - - ..

o 10 305 10 IS lS

Fig. I.TIME IN MIN. AFTER INJECTION

Mean duration-action curve of oedema produced by different concentrations hyaluro-nidase injected subcutaneously in rat's paw. .---. control, normal saline (4) ;9i---!i Hyaluronidase 400 i. u./m!. (3); ._--. 500 i. u./m!. (4);~---. 600 i. u./rnl. (4); 0---0 700 i. u.jml. (3), l::. l::. 800

i. u./mJ. (4). Thefigures in parenthises indicate the number of obseroa ons,

ril(4);> OS~;:J

QZ........o~••• ..l~<QC)<Cooz>-o~-<E-<~<CEo<~(il;:J~Cl<----••••0 0'1<CIol~<

400 ..t:;()(') 600 700 80CHYALURONIDASE i. u./ml.

Fig. 2. Concentration-action curve of hyaluronidase. Abscissa: concentraticns of hyaluro-nidase plotted on a Jogscale. Ordinate: area of the duration-ac tion curve measur-ed with a planimeter. Vertical lines represent standard errors ()f the mear.s,Figures in parentheses indicate the number of observations•

.~

20 HYALURONIDASE ON CAPILLARY PERMEABILITY

was some sign of decrease in the swelling beyond the initial size after 20min. There was no sign of increase in the volume of the control foot whileincrease in the volume of the hyaluronidase foot had been produced in a gra-ded fashion depending on the concentration. This has been clearly shown inFig. 2, where area of each duration-action curve above the respective baselines was measured with a planimeter and the mean values plotted againstthe corresponding log-concentrations.

Since hyaluronidase is known to lose its activity at a temperature above570C and at a pH beyond the range of 4-7.5, it was of interest to investi-gate whether inactivation of the enzyme produced any change in theresponse. Fig. 3 shows the effect of 700 i.u. of hyaluronidase corn pared tothat of the same heated at IOOvC for 15 min at a pH 10. There hadbeen marked diminution in the volume of the swelling produced by theheated hyaluronidase as compared to the contro 1.

06

0'5

- _ ..•.•... ---- ~--------0·4

0·3

o2.5 30$ 10 15 10

TIME IN MIN. AFTER INJECTION

Fig. 3. A comparison of the mean duration-action curves of oedema produced by hyaluro-nidase 700 i. u./ml 0--0 (3) and hyaluronidase 700 i. u.jrnl heated at 100'Cfor 15min. at pH 10 A---A (3) Figures in parentheses indicate the number of obser-

vations.

DISCUSSION

The results support the capillary permeability-increasing action ofhyaluronidase. The mechanism of oedema production by hyaluronidase inthe present series can be explained in the following lines. Hyaluronidasedepolymerises the hyaluronic acid not only of the intercellular substances of thesubcutaneous tissues but of the capillary endothelium as well, producing an in-crease in the capillary permeability. As the total volume of fluid injected wasvery small (i.e., 0.4 rnl), the hydrostatic pressure exerted by the intracapillary-blood was much higher than the surrounding fluid. As a conseq uence, the

RMEABILITY

yond the initial size after 20e of the control foot while

ad been produced in a gra-is has been clearly shown in'e above the respective basemean values plotted against

ivity at a temperature aboveit was of interest to investi-duced any change in thehyaluronidase corn pared toin at a pH 10. There hade swelling produced by the

...•..----+----

10ION

f oedema produced by hyaluro-c 700 i. u.jrnl heated at 100'Ctheses indicate the number of obser-

15 lO

ility-increasing action ofction by hyaluronidase inving lines. Hyaluronidaseercellular substances of the

as well, producing an in-olume of fluid injected waserted by the intracapillary. As a conseq uence, the

M.N. GHOSH, R.M. BANERJIE AND S.K. MUKHERJ[ 21

fluid came out of the capillaries into the surrounding tissues giving rise tooedema. The work of Elster, Freeman and Dorfman (1949) producingoedema with subcutaneous injection of hyaluronidase also points to a capil-lary permeability-increasing action. The argument that the said action isprobably due to impurities like histamine, etc., does not apply at least tothe present series where a comparatively pure substance was used. Thedemonstration that inactivation of the enzyme markedly decreased theamount of swelling, confirms that the increase in the capillary permeabilityis due to the enzyme itself and not due to any impurities.

Thanks arc due to our director Dr. J. N. Nanda, for his kind permission to publishthiswork.

REFERENCES

Aylward,F. X. (194-2). Proc, Soc. Exp. Bioi. (N. T.), 49, 342.Benditt, E. P., Schiller, S., Mathews, M. B. and Dorfman, A. (1951). Proc, Soc. Exp. Bioi.

(N. T.), 77,643.Buttle,G. A. H., D'Arcy, P. F., Howard, E. M. and Kellett, D. K. (1957). Nature, 179,629.

Duran-Reynals, F. (1939). Yale]. Bioi. u«, 11,601.Elster,S., Freeman, M. and Dorfman, A. (1949). Amer. J. Physiol., 156,429.Rocha e Silva, M. and Dragstedt, C. A. (1941). J.Pharmacol. 73,405.Zeifach. B. and Chambers, R. (1950). AI/n. N. Y. Acad. Sei., 52, 1047.