Envenomation of Trimeresurus flavoviridis
Venom and its Components, Phospholipase A2 Isozymes
Motoo KITANO,1* Masato HIRANO,1 Makoto YAHATA,1
Eri UMEMURA,1 Aichi YOSHIDA,2 Shosaku HATTORI ,3
Naoko UEDA,4 Daisuke TSURU,5 Motonori OHNO4

Abstract
The lesions caused by sublethal doses of Trimeresurus flavoviridis (Tf) venom injected into the anterior thighmuscle (quadriceps femoris muscle; QFM) of rats were studied with paraffin sections. A dose of 50 µg of Tf
venom produced a large area of necrosis in QFM together with neighboring muscles. Phagocytosis of necrotic
remnants was followed by marked regeneration of the muscle fibers. Myonecrosis was microscopically evident 30
minutes after the injection, and over the following 72 hours the necrotic muscles remained acellular and devoid of
inflammatory reaction except at the very edge where liquefaction necrosis associated with inflammatory infiltrate
was marked from the beginning. Blood vessels within and outside the necrotic foci were often involved ininflammatory changes and thrombosis, to some degree. However, marked hemorrhage was never noted in andaround the foci. Phagocytosis of debris proceeded from the periphery, and after 24 hours the periphery of thenecrotic foci was replaced by granulaion tissue containing many regenerating myoblastic cells. Abscesses
developed in the vicinity of the injection site in several rats receiving the crude venom, but never after injectionof its phospholipase A2 isozymes, BPI or BPII. Muscle necrosis after envenomation of Tf venom seems due
primarily to direct action of the venom, though vascular thrombosis and ischemia may contribute. Interestingly, inthe rats, the myonecrotic Tf venom itself may cause marked and extensive activation of muscle fiber regeneration,and the venom seems to be a great myotoxin but not a hemorrhagenic toxin.
Key words: Trimeresurus flavoviridis (Tf), Envenomation, Snake venom, Myotoxin, Skeletal muscle, Quadriceps
femoris muscle (QFM), Myonecrosis, Regeneration, Myoblast, Phospholipases A2 isozymes, Hemorrhage

Introduction
Envenomation of Trimeresurus flavoviridis (Tf) snake venom causes two main symptoms,
one local and the other systemic in humans. The local symptoms are severe pain, swelling,
hemorrhage and necrosis in compliance with the bitten extremity, result in permanent

rhage (HOMMA and TU, 1971).
We have examined the short term effects of the various components of Tf venom on
rat skeletal muscle, quadriceps femoris muscle (QFM), with closer attention to impairment,
necrosis and regeneration of muscle fibers, the latter occurring even at the earlier stages.
Moreover, the rats have shown a strong resistance to hemorrhage by envenomation of Tf
venom. Renewed interest in this venom has stemmed from our recent studies of its action
on muscle and vascular wall of the rat and from its fractionation of the venom, which may
shed light on the pathogenesis of local lesions caused by the snake bites.

Materials and Methods
Venom and its phospholipase A2 isozymes
Tf venom was collected in Amami-Oshima island and lyophilized. Its phospholipase A2(PLA2) isozymes, [Asp49] PLA2, BPI and BPII, the latter two being [Lys49] PLA2s, wereseparated by conventional chromatographies at Sojo University (LIU et al, 1990; K IHARA etal., 1992). The crude venom and PLA2 isozymes were weighed and dissolved in sterile
physiological saline immediately prior to use.
Rats
Young adult female and male rats (80-100g) of F344 (Charles River Japan, Inc.,), DarkAgouti (Shizuoka Laboratory Animal Center), Wistar/Furth (maintained in our laboratory
by sister-brother mating) strains were divided into two to five groups, each group consistingof 6 rats (total; 90) (Table 1).
Experimental procedures
The rats of the first four groups were anesthetized with pentobarbital sodium (Nembutal:
Abbot Lab, U.S.A.). The crude venom and three PLA2 isozymes (50 µg each) in 100 µl
physiological saline were injected into each rat of four groups at the upper two-thirds of theright QFM. The fifth group, the control rats, were similarly injected with 100 µl sterile
physiological saline.
The animals were allowed to survive for the periods ranging from 30 minutes to 72
hours and were sacrificed under ethyl ether anesthesia. Both the right and left hindlimbs andvisceral organs including the brain were immersed for several days in 10% bufferedformalin (pH 7.4), and processed for paraffinic embedding. The sections of 5-6 mm

Of
snake
(Tf)

Crude
Venom

[Asp49]
PLA２

BPI

＊

Dura- (F344 ,InflamMusc- Inflam- Changes
tion DA, WF Necro- matory Hemoular re- matory in the Hemo♯＊＊
Edema
Edema Others: f+m ) sis
infiltr- rrhage
generat- infiltr- vascular rrhage
＊＊ ＊
ation
ion
ation walls
30m

6

-〜+

-

-〜+

+

-

-

-

-

+

-

6h

6

+

+

-〜+

+

-

-〜+

-

-

+

-

24h

6

++〜+++

+++

-〜+

++

+

++

-〜+(Th)

-〜+

++

-

48h

6

++〜+++

+++

-〜+

++

++

++

-〜+(Th)

-〜+

++

+

72h

6

++〜+++

++

-〜+

++

++

++

-〜+(Th)

-〜+

++

+

Total

30

24h

6

++

++

-〜+

++

+

++

-〜+(Th)

-

++

-

48h

6

++

++

-〜+

++

+

++

-

-

++

-

Total

12

24h

6

++

+〜++

-〜+

++

+

+

-〜+(Th)

-

++

-

48h

6

++

+〜++

-〜+

+

++

+

-〜+

-

+

-

Total

12

24h

6

++

+〜++

-〜+

+

++

+

-〜+

-

++

-

48h

6

++

+〜++

-〜+

+

+++

+

+

-

++

-

72h

6

+

+

-〜+

+

+++

+

-

-

++

-

Total

18

30m

6

-

-

-

-

-

-

-

-

-

-

24h

6

-

-

-

-

-

-

-

-

-

-

48h

6

-

-

-

-

-

-

-

-

-

-

Total

18

BPII

Control(physiologicalsaline)kinds of toxins, among three strains of rats and among female and male rats, so we preferred
to describe here all together.
Macroscopically, there was a more or less marked necrotic change with a mild
swelling of the muscle mass in the venom-injected right hind leg. A close examination of
injected QFM revealed an absence of marked hemorrhage (Fig 1).
Histologically, the muscle tissue injected with physiologic saline solution had a typical
histology of skeletal muscle with no abnormality in muscle fibers, nerves or blood vesseles.

Fig. 1: A large necrotic lesion in the QFM of a F344 male rat 48 hours after the
inoculation of the crude venom of Tf. Note the severe and extensive atrophy ofthe skeletal muscles of the anterior thigh due to necrotic changes without amarked hemorrhage.

There were groups of affected cells accompanied by the decrease of fine cytoplasmic
structures to some degree, which were located in the periphery of the muscle as early as 30
minutes after inoculation. At this time edema without inflammatory cells were observed,
and many of the skeletal muscle cells presented edematous swelling (Fig.2).
Necrosis of muscle fibers in QFM was indicated by nuclear pyknosis and fragmentation of the myofibrils into homogenous eosinophilic masses separated by empty-looking
segments 6 hours after injection. There were many areas where necrotic cells predominated.
Some of them appeared to be in the initial stages of cell degeneration, i.e. some muscle cells
had only edematous swelling, whereas other cells were in a more advanced stage of damage
with the formation of dense clumps of myofibril alternating with cellular spaces apparently
devoid of myofibrillar material. The findings support the view that the earliest alterations
seem to take place in the periphery of the necrotic focus. A very mild inflammatory
infiltrate was observed at this time.
After the elapse of 24 hours there was widespread myonecrosis; the myofibrillar

granulation tissue with neutrophils, macrophages and fibroblasts surrounded the central
mass of necrotic fibers. Myofibrillar material in affected cells was pale and amorphous.
Very little fibrosis was observed.
Simultaneously, regenerative proliferation of myoblasts, which were characterized by
the presence of myofibrils and rows of centrally-located nuclei, were observed in the
periphery of necrotic cells. The activity of proliferation of small regenerating muscle fibers
were seen most markedly in BPII group, and in the BPI, crude venom, and [Asp49] PLA2
groups in descending order of degree. Twenty-four hours after toxin inoculation, theregenerating cells were spindle in shape with scanty basophilic cytoplasm and a central
nucleus, some revealing mitotic activities at 24 hours (Fig.3). Muscle regeneration wasobserved in the peripheral fibers of necrotic muscles, but the inner parts of those muscles
appeared still necrotic. The regenerating cells gradually became hypertrophic with an
acidophilic light cytoplasm at 72 hours (Fig.4). The muscle tissue examined histologically
8 weeks after the onset of muscle necrosis was characterized by the presence of abundantFig. 2: Massive necrosis of the QFM of a F344 male rat 48 hours after the inoculationof the crude venom of Tf. Marked inflammatory infiltration is focally seen in
the necrotic layer, most of which is composed of liquefaction necrosis andpartly coagulative necrosis. Hemorrhage is negligible.

normal, but in some animals inflammatory infiltrate was noted in the vascular walls andplatelet thrombi were found in some medium-sized veins and arteries, occasionally forming
occlusive masses (Fig.5). There were several peripheral nerves showing marked edema of
the endoneurium, but no apparent structural disintegration was found.

Fig. 4: Numerous myoblastic cells around the necrotic focus in a F344 male rat 72
hours after inoculation of the BPII. Myoblastic cells are hypertophied and havea round nucleus in their central cytoplasms. Some of the regenerating cells have
two nucleoli.

Fig. 5: Thrombosis of a medium-sized vessels in the peripheral area of a necrotic focus
in a F344 female rat 48 hours after inoculation of the crude venom. In the wallof the vessels inflammatory infiltrate is observed.

Discussion
Thirty minutes after the injection of Tf venom, initial changes of muscle fiber necrosis was
widespread in QFM, where most blood vessels looked normal and contained no thrombi.

delay in phagocytosis was also observed after the inoculation of venom of other snakes, andvasculitis and thrombosis were also considered important in its pathogenesis. Similar
vascular changes were reported after the local administration of various other snake venoms(HOMMA and TU, 1971).
On the other hand, muscle necrosis by the local inoculation of various kinds of snake
venoms is usually followed by the rapid phagocytosis of debris at the periphery of thenecrotic area. The clearing of necrotic material by phagocytes was followed by a rapid
regeneration process. Myoblasts were observed 24 hours after the onset of myonecrosis, andby 8 weeks regeneration was complete (data, not shown). There were many regenerative
cells of size similar to that of adult muscle fibers, but with the centrally-located nuclei, atypical finding in regenerating muscle cells. The success of regeneration after myonecrosis
induced by Tf myotoxin might be due to the fact that neither nerves nor blood vessels aremarkedly affected by this toxin, since adequate blood supply and innervaton are essentiallyrequirements for muscle regeneration (Q UEIROZ et al., 1984; GUITIERREZ et al., 1989).
The most striking effects produced by the parenteral injection into animals of many snake
venoms, including venom of Tf, are local, consisting of marked hemorrhage, myonecrosis,
and edema (OHSAKA et al., 1960; OKONOGI et al, 1960; LOMONTE et al., 1994). It is wellknown that the pathological signs characteristic of Tf bite in humans, rabbits and mice aremarked by extensive hemorrhage and necrosis (OKONOGI et al., 1960). Our experimented
rats, however, failed to demonstrate that hemorrhage was a predominant sign of snake
venom injection. We used three strains of rats and four kinds of preparations of Tf venom
for our experiments. In spite of the difference of strains of rats and kinds of venompreparations, we could not note marked hemorrhage in and around the injection area.
It is of great interest whether or not the same principle is responsible for necrosis andhemorrhage. However, little is known concerning this point. Many researchers stated that
hemorrhage is due, at least in part, to the action of myonecrotic enzymes, such as
phospholipase A2 (LOMONTE et al., 1994). Concerning local hemorrhage, our results did
not agree with the general view that snake venoms produce bleeding at the site of injection.
Pathological observations indicated that the easily discernible local changes produced in thehumans, rabbits and mice by Tf bite injury were hemorrhage. However, in the present
experiment, Tf venom did not give rise to any prominent bleeding locally, but produced
myolysis with edema, in the rats.
Some investigators have suggested that proteolytic enzymes are factors in hemorrhage
and necrosis (OHSAKA et al., 1960; O KONOGI et al., 1960). Our observations concerning thedegree of hemorrhage and necrosis were not always in parallel with proteolytic activities.

devoid of hemorrhagic activity, elapidae venoms, fail to cause significant changes in thearterial walls. The important changes responsible for developing arterial lesions appear tobe the injury of the endothelium and the disintegration of the media. It has been reported
that Tf venom attacks the endothelium and smooth muscles of the media when the venom
is injected close to the arteries. This suggests that both hemorrhagic and myonecrogenetic
factors play an important role in the involvement of local blood vessels (M ATSUI et al.,2000). Thrombi were observed in local arteries at the sites injected with several kinds ofvenoms. In our experiments the frequency of thrombus formation was not always in parallelwith the severity of arterial lesions and of local hemorrhage and swellings. Important factors
favoring thrombosis are slowing or eddying of the blood stream, injury to the lining of ablood vessel and alterations of the blood. If a longer period of time allowed to elapse
between injection and autopsy, there would probably have been more thrombus formation,
as viperinae and crotalinae venoms produce conditions favorable to this kind of lesions(HOMMA and TU, 1971).

Acknowledgements
This work was supported, in parts, by a Fund for Promotion and Development of Amami
Islands sponsored by Ministry of Land, Infrastructure and Transport and Kagoshima
Prefecture and a Kagoshima University Fund for Improvement of Education and Research
Activities. Prof. Toru Aoyama kindly reviewed the manuscript.

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