183
Discussion should be directed towards decreasing the curvature of
The integral mean absolute curvature of the tubules
in infertile patients was abnormally high. The tubules in
these patients also had an abnormally small mean sec-
tional area and perimeter which, because the patients,
tubular circular sectional area was normal, supports the
finding that the patients’ tubules were more curved than
normal (sections of a highly curved or contorted cylinder
will tend on average to be much smaller than sections of
a straighter cylinder of equal radius).
The well-known spontaneous movement of semi-
niferous tubules exposed by capsular division involves
forces that are not well defined. There is evidence for
testicular contractile e1ements5-7 (especially in the cap-
sule, but myoid cells and actin filaments are also associ-
ated with the tubules); however, the effect of mechanical
or other insult on the overall positioning of each tubule
is not known. The grossly visible pouting of the testicu-
lar mass on section suggests lengthening (as opposed to
coiling up) of the tubules in response to insult, but this
may not be so because cutting the capsule could mar-
kedly decrease tubule packing efficiency while increasing
tubular curvature. For example, a loss of smoother but
higher amplitude coiling could produce overall tubule
displacement which might appear as lengthening in spite
of microscopic increase in curvature. As a result conclu-
sions about overall testis shape, gross or low magnifica-
tion appearance of individual tubules, and so on, cannot
be predicted simply from our microscopic findings.
Because of this and the (unlikely) possibility of proce-
dural artefact we used normal surgical as well as normal
necropsy material as controls and specimens from adult
rats with which the fixation procedure was varied. While
a functional defect leading to abnormal curvature or
contortion (luring excision is, of course, possible, the
data from both the human and rat material suggested
that the patients’ increased tubular integral mean abso-
lute curvature and surface area per unit volume were
not caused by procedural artefact.
It seems clear that there are at least as many, if not
more total cells per unit volume in the idiopathic infer-
tile group, and that the increased integral mean absolute
curvature and surface area per unit volume reflects a
contortion of the tubules, which are apparently other-
wise normal. Excurrent obstruction or slowing is an
accepted mechanism in some instances of azoospermia
and oligospermia (e.g., post-testicular blockage). Sperm
transport is considered to involve extrinsic factors-
seminiferous tubule fluid secretion, the contractile acti-
vity of cells within the testicular tunica and tubular
lamina propria, and possibly a small amount of ciliary
action in the efferent ducts.7 Excessive curvature (con-
tortion) of the tubules would be likely to further impede
these transport factors, which are probably severely de-
ranged (primarily and/or secondarily). The possibility
that undetected post-testicular blockage (e.g., in epidi-
dymis or vas deferens) contributed to the derangement
of transport factors has not been fully ruled out and
therefore deserves further investigation.
Hormonal therapy tends to predominate in male in-
fertility but an endocrine cause is proven in less than
10% of cases. Our series suggests that the testicular
hypercurvature syndrome may be the most common
form of human male infertility. Logically, treatment
the tubules or enhancing the compromised transport
process. Thickening of peritubular tissue has been
observed ultrastructurally in some cases of idiopathic
oligospermia,8 and localised obstructive epididymal
lesions ("brown patches")9 are a common necropsy find-
ing, both of which suggest a structural rather than
purely functional abnormality. If, on the other hand, the
defect is one of transport or contraction (manifest either
as hypercurvature or hypercurvature in response to exci-
sion), then pharmacological agents directed to the tubu-
lar smooth muscle would seem to hold more promise.
We thank A. Baddeley, Dr H. Langevin, and Dr B. M. Herbertson
for helpful criticisms, and Prof. 1. Mills for permission to study the
patients. Use of the quantimet was kindly provided by the department
of metallurgy, University of Cambridge (Dr B. Ralph and J. Ward).
This work was supported in part by the Medical Research Council of
Canada (P. A.)
Requests for reprints should be addressed to P. A.
REFERENCES
1.
2.
Kleinteich, V. B. & Schickedanz, H. Zschr. Urol. Bd. 70, 663-668 (1977).
Weibel, E. R., Fisher, C., Gahm, J., Schaefer, A. J. Microscopy, 1972, 95,
367.
3. DeHoff, R. T. in Geometrical Probability and Biological Structures (edited
by R. E. Miles and J. Serra,); p. 99 Vienna, 1978.
4. DeHoff, R. T., Gehl, S. M. in Proceedings of Fourth International Congress
of Stereology (edited by E. E. Underwood); p. 29, Washington, 1976.
5. Ross, M. H., Long, I. R. Science, 1966, 153, 1271.
6. Furuya, S., Kumamoto, Y., Suzuki, T., Takanji, M., Nagai, T. Andrologia,
1977, 9, 349.
7. Hargrove, J. L., MacIndoe, J. H., Ellis, L. C. Fertil. Steril. 1977, 28, 1146.
8. DeKretser, D. M., Kerr, J. B., Paulsen, C. A. Biol. Reprod. 1975, 12, 317.
9. Mitchinson, M. J., Green, S. F., Sherman, K. P. J. Path. 1973, 109, 9.
RATIONALISATION OF FIRST-AID
MEASURES FOR ELAPID SNAKEBITE
S. K. SUTHERLAND A. R. COULTER
R. D. HARRIS
Department of Immunology Research, Commonwealth Serum
Laboratories, Melbourne 3052, Australia
Summary The plasma of monkeys envenomated
with tiger snake (Notechis scutatus)
venom was monitored by radioimmunoassay for both
crude venom and a neurotoxin. When the injected limb
was immobilised and a pressure of 55 mm Hg applied to
the injection site, only very low levels of circulating
venom or neurotoxin were detectable. In practical terms,
venom movement can be effectively delayed for long
periods by the application of a firm crepe bandage to the
length of the bitten limb combined with immobilisation
by a splint. Pressure alone or immobilisation alone did
not delay venom movement.
Introduction
FEWexperimental studies on the movement of snake
venom published. Fairley1 found that arterial
have been
tourniquets sheep and goats did not significantly pro-
in
long life when a lethal dose of tiger snake (Notechis scu-
tatus) venom had been injected subcutaneously. Lym-
phatics have a role in the central movement of venom,
and their ligation delays absorption of N. scutatus
venom and Vipera russelli venom, but not that of Naja
naja venom.2
184
The survival of rabbits injected with the non-elapid
venom Crotalus adamanteus improved in animals that
were immobilised and less antivenom was required to
prevent death when venom movement was obstructed.4
Attempts were made to make first-aid for snakebite safe
and practical,5-7 but the lack of a suitable experimental
model allowed the emergence and temporary popularity
of extreme measures such as cryotherapy8 and wide
incision and/or excision.9
Our experience10 suggested that an arterial tourni-
quet was impracticable as a first-aid procedure because
it caused extreme distress in the conscious patient and
could be used only for a short period. We decided to in-
vestigate the treatment for snakebite in monkeys (the
anatomical similarities of their limbs to those of man
allows the application of relevant first-aid measures to
the envenomated limb) by radioimmunoassay monitor-
ing of the distribution of whole-venom components and
a neurotoxin in envenomated animals.
Materials and Methods
Male or female adult monkeys (Macaca fascicularis) of
weight range 2.3±0.3 kg were used in the experiments. Well-
padded frames were used to make the immobilisation of the
monkeys comfortable since in most experiments neither anaes-
thetics nor tranquillisers could be used. The monkey’s arms
were restrained by the frame, and the left leg was splinted to
protect the venous cannula through which blood-samples were
collected from the inferior vena cava. The right leg, into which
venom was injected, was either free to move or was subjected
to the various restrictions, pressures, &c. under study.
Plasma-samples were assayed in triplicate for crude venom
(tiger-snake venom, T.s.v. and the major neurotoxin in N. scu-
tatus venom, the presynaptic toxin, notexin," by a solid-phase
Fig. 1-Chamber designed to apply uniform pressure to limb
with minimum restriction of movement.
Fig.2-Venom and neurotoxin levels in conscious monkeys (nos.
13, 18, and 20);
no first-aid measures applied to envenomated
limb.
sandwich radioimmunoassay12 which has been useful in the in-
vestigation of human snakebite victims. 12
The venom used had been pooled from mainland tiger
snakes and its subcutaneous LDso was 2.35µ g (95% confidence
limits 1.90-2.92) for mice (weight range 18-21 g). Stan-
dardised venom solutions were kept frozen until use. Monkeys
were injected with 300 µg of venom (estimated to be six certain
lethal doses for monkeys in this weight range14) in 150 µl of
saline via a 25 guage needle to a depth of 2-5mm over the
lower third of the lateral gastrocnemius. The depth of penet-
ration, which was limited by use of a special guard, represents
the average length of a tiger-snake’s fang. 14 The venom was
deposited on or in the aponeurosis of the muscle. A slow infu-
sion of Hartmann’s solution was given between blood-sam-
plings.
Venom movement was delayed (1) by a standard pædiatric
sphygmomanometer cuff 10 x 5 cm, placed round the thigh of
the monkey and inflated to 140 mm Hg to achieve complete
arterial occlusion; (2) since preliminary studies had suggested
that direct pressure applied to the site of the bite should be
further investigated, by inflatable air splints (Alsafe Industries,
Melbourne); or (3) by crepe bandages and a splint.
When anxsthesia was required, open ether was used because
it allowed rapid recovery from profound anaesthesia.
With the exception of the arterial tourniquet (30 min) all
methods of venom restriction were employed for 60 min.
Crepe Bandages and Special Pressure Chamber
The requirement for the additional apparatus arose after
the initial experiments in which the application of a firm crepe
bandage to the whole of the envenomated limb and the immo-
Fig. 3-Venom and neurotoxin levels in monkey no. 22.
Fig.4-Venom and neurotoxin levels in monkey no. 24: pro-
found anaesthesia until 50 min after venom injection; no first-
aid measures.

Fig. 5-Venom and neurotoxin levels in monkey no. 21 (cons-
cious) with leg in pressure chamber at 55 mm Hg pressure for
60 min.
Fig.6-Venom and neurotoxin levels in monkey no. 23 after
combination of profound anaesthesia and use of pressure
chamber at 55 mm Hg for 60 min.
bilisation of the limb by a splint had given encouraging results.
It then became desirable to determine subjectively the approxi-
mate pressure exerted by a firm crepe bandage and to con-
struct a chamber which could exert such a pressure on a mon-
key’s limb with minimum restriction of movement.
Ten volunteers had a firm crepe bandage applied to one
forearm and their other forearm was inserted into an air splint
that was then slowly and repeatedly inflated and deflated. One
of us (S.K.S.) judged that a pressure of 555 mm±5 mm Hg was
the likely pressure exerted by a firm crepe bandage.
The chamber (fig. 1) had a rigid outer casing. The limb was
placed in a rubber bag which was then pushed into the
chamber, the upper part of the bag being used to make a seal.
In this way pressure can be applied uniformly through the rub-
ber bag to the whole limb without immobilisation of the limb.
This method of applying pressure to the limb mimics that pro-
duced by a firm crepe bandage.
Results
25 monkeys were studied and the significant findings
in 11of these are described below (figs 2-8). The other
14 monkeys, which include non-envenomated controls
and those that took part in failed first-aid experiments,
will be reported on later. Venom injections were given at
ume zero. Except in monkey 24 (fig. 4), which was
anæsthesised before venom was injected, the first-aid
measures were applied within 60 to 90 s of envenom-
ation.
185
Fig. 7-Venom and neurotoxin levels in monkeys which had
crepebandage firmly applied for 60 min to the whole limb
(monkeys no. 11 and 16) or below the knee only (monkey no.
17).
Fig. 8-Venom level in monkey no. 12 whose envenomated limb
wasplaced in a faulty inflatable air splint at 55 mm Hg.
After 10 minutes the pressure had fallen to 30 mm Hg. It was im-
mediately reinflated to 55 mm Hg and maintained at this pressure for
a further 50 minutes.
Discussion
The application of firm crepe bandages combined
with splinting of the injected ’limb prevents rapid rise of
the plasma-levels of both whole tiger-snake venom and
the major neurotoxin. The plasma-levels after the re-
moval of the crepe bandages are, in this small series, sig-
nificantly lower than those when venom movement has
not been restricted.
Venom immobilisation appears to be the result of a
combination of compression of lymphatics at the site of
injection and reduction of lymph-flow because of immo-
bilisation ; it may also partly be due to compression of
the superficial veins.
The nature of venom absorption and distribution was
well demonstrated in monkey 12 (fig. 8) in which a delay
in the achievement of effective pressure and immobilis--
ation slowed absorption of residual venom and reduced
plasma-levels of whole venom components.
Our findings suggest that the application of a firm
crepe bandage and splint (or suitable air splint if avail-
able) is an excellent method of retarding the absorption
of elapid venom. This measure seems almost as effective
 186
as an tourniquet in the short term, but it is far
arterial
more practical because of the comfort, unlikelihood of
ischxmia, and most important of all, the period over
which such first-aid measures can be employed.
Since delayed application of the measures retards
movement of the remaining venom, a crepe bandage
should be applied even in severely envenomated patients
who are seen late, and it should be re-applied if a patient
becomes critically ill on removal of the bandage. The ap-
plication of the bandage allows time for neutralisation of
the venom by antivenom. It may also reduce antivenom
requirements.
None of the 16 or more distinct, antigenic proteins in
tiger-snake venom were detected in significant amounts
in the plasma of the envenomated animals during the 60
min of restriction of venom movement. The lower mole-
cular weight (M.w.) components (M.w. 6000-13 574)
are the neurotoxins;" notexin has a M.w. of 13 574. It
is noteworthy that these neurotoxins were immobilised
by this procedure since all polypeptide snake neuro-
toxins isolated to date,6 except crotamine (M.w. 5450)
from Crotalus d. terrificus, 15 have a M.w. greater than
6000. The other major components of snake venoms
which cause severe tissue damage, coagulation distur-
bances, and haemolysis, have M.w.s far greater than
10 000. Thus on a basis of molecular size, bandaging
and splinting may well immobilise a wide range of snake
venoms and even non-reptilean (e.g., scorpion) venoms.
Requests for reprints should be addressed to S. K. S., Department
of Immunology Research, Commonwealth Serum Laboratories, Mel-
bourne, 3052, Australia.
REFERENCES
1. N. H. Med. J. Aust. 1929, i, 377.
Fairley,
2. Barnes, J. M., Trueta, J. Lancet, 1941, i, 623.
3. Leopold, R. S., Huber, G. S., Kathan, R. H. Milit. Med. 1957, 120, 414.
4. Christensen, P. A. S. Afr. med. J. 1969, 43, 1253.
5. Reid, H. A. Br. med. J. 1968, iii, 359.
6. Minton, S. A. Venom Diseases; p. 169. Springfield, Illinois, 1974.
7. Sutherland, S. K. Aust. fam. Physns, 1976, 5, 272.
8. Russell, F. E. Toxicon, 1969, 7, 33.
9. Glass, T. G. Surg. Gynec. Obstet. 1973,136, 774.
10. Sutherland, S. K. Med. J. Aust. 1975, i, 30.
11. Karlsson, E., Eaker, D., Ryden, L. Toxicon, 1972, 10, 405.
12. Coulter, A. R., Cox, J. C., Sutherland, S. K., Waddel, C. J. Immun. Meth.
1978, 23, 341.
13. Sutherland, S. K., Coulter, A. R. Med. J. Aust. 1977, ii, 177.
14. Kellaway, C. H. ibid. 1929 i, 348.
15. Gongalaves, J. M., Giglio, J. R. Proc. 6th Int. Biochem. New York. 1964,
2, 170.
Addendum
Since the preparation of this paper the movement of
venom of the Eastern brown snake (Pseudonaja taxtilis),
the taipan (Oxyuranus scutellatus), and the death adder
(Acanthophis antarcticus) has been retarded to the same
degree as tiger-snake venom by application of crepe ban-
dages and a splint.
The first-aid measures have been effective when much
greater amounts of venom were injected. When a mon-
key of 2-6 kg was injected with 3 mg of tiger-snake
venom (i.e., 60 certain lethal doses) the maximum
plasma-level of tiger-snake venom components after 55
min of bandaging and splinting was 80 ng/ml. When the
crepe bandages were removed at 60 min a plasma-level
of 1700 ng/ml of venom components was reached 25
_
min later. At this stage neurotoxic signs appeared, anti-
venom was infused, and the monkey survived.
ÆTIOLOGICAL RELATION BETWEEN
KOREAN HÆMORRHAGIC FEVER AND
NEPHROPATHIA EPIDEMICA
HO WANG LEE PYUND WOO LEE
Institute for Viral Diseases, Korea University Medical College,
Seoul, Korea
JUHANI LÄHDEVIRTA
Third Department of Medicine, University of Helsinki,
Helsinki, Finland
MARKUS BRUMMER-KORVENKONTIO
Depatment of Virology, University of Helsinki, Finland
Summary A close ætiological relation between Kor-
ean hæmorrhagic fever and nephro-
pathia epidemica of Scandinavia has often been sug-
gested because of similarities in their clinical picture and
epidemiology. This relation has now been confirmed
serologically; raised titres of antibody to Korean
hæmorrhagic fever agent and seroconversions have been
found by the indirect fluorescent-antibody technique in
20 patients with nephropathia epidemica.
Introduction
KOREAN hæmorrhagic fever (K.H.F.) did not attract
great attention until the Korean War,1although dis-
eases similar to K.H.F. had been known in Russia as
hxmorrhagic nephroso-nephritis or haemorrhagic fever
with renal syndrome,34 and by the Japanese in Man-
churia as epidemic hæmorrhagic fever5-7 in the 1940s.
Very recently, Lee et awl. 89 reported the first isolation of
the aetiological agent Of K.H.F.
Nephropathia epidemica (N.E.) has been known in
North European countries since 1934,10 11 but the
causative agent has not yet been isolated. The sugges-
tion that there is a close aetiological relation between
K.H.F. and N.E.12 13 was strongly supported by the
detailed comparison of the two conditions by Lahde-
virta.14
Serological evidence has so far been lacking. We
report here on the positive antigenic relation between
K.H.F. and N.E. found by the method of Lee et al.9
Material and Methods
38 acute and convalescent sera from 20 typical N.E. patients,
seen in Finland between 1965-1977, and sera from 5 controls
(1 hxmaturia and proteinuria, 1 chronic pyelonephritis, 2
acute glomerulonephritis, and 1 secondary ansemia) were used.
The sera were stored for 0-12 years at -20°C until they were
tested in 1977. The first batch of coded sera was mailed from
Finland to Korea as lyophilised sera and the second batch in
dry ice. Details of patients and sera are presented in the
accompanying table. The diagnosis of N.E. was made according
to the clinical picture14 and assessment" of the severity of dis-
ease was based mainly on the peak value of serum-creatinine:
<200 &micro;mol/1-mild; 200-800 &micro;mol/l-moderate case; and
> 800 &micro;mol/l-severe case. Some other findings and symptoms
were also considered in the assessment of severity.
Sera from 7 cases of acute glomerulonephritis, from 6 cases
of upper respiratory illness, and from 4 cases of acute tonsillitis
in Korea were used as additional controls.
Antibody determinations were done blind by the immuno-
fluorescence (LF.) method of Lee et a1.9 The K.H.F. agent,
strain 76/118, 8th Apodemus agrarius coreae passage,8 105.3