Twdcoa, 1975, Vol. 13, pp . 16169. Pin Pray . Printed in Great Britain.
HISTOPATHOLOGICAL AND HISTOCHEMICAL CHANGES IN SKELETAL MUSCLES AFTER BITIS GABONICA ENVENOMATION A. H. MOIiAI~, A. M.
$ALEH,
S. Ai-tn~t» and M.
EL-MACiHRABY
Faculty of Medicine, Ain Shams University, Cairo, Egypt (Accepted for publication 20 October 1974)
A. H. Moxw~n, A. M. S~r etl, S. AI~o and M. E~.-M~oHIUSV. Histo~athological and histochemical changes in skeletal muscles after Bibs gabonica envenomauon. Toxicon 13, 165-169, 1975 .-The histochemical activities of succinic dehydrogenase and cholinesterase in skeletal muscles of the mouse were studied after i.m. administration of B. gabonica venom. There was generalized depletion of succinic dehydrogenase activity 48 hr after injection of an i.n~, (18 ug per 25 g body wt). The subsarcolemmal density which typi~ type C fibres was reduced . In the chronically treated animals which received 0~5 rn~o at ~~ mtervals for S weeks, the normal granular pattern of the reaction became diffuse thus diminishing the differences between the enzyme activities in different fibres. Generalized reduction in fibre size similar to denervation atrophy was observed . The cholinesterase activity was not affected in either in vitro or in vlvo experiments. In acutely treated animals receiving one i.Dso haemorrhages and extravasation of blood cells was observed . Animals receiving 0~5 rn showed haematoma at the site of injection after 24 days . Histopathological changes included extensive vacuolization along the course of dilated intramuscular blood vessels. Interruption of their endothelial lining was detected in certain areas. Muscle tissue was either absent or showed vacuolization with non-specific cellular infiltration . Fasciculation, infra-fibral vawolization and loss of transverse striations was observed . Nerve trunks were partially degenerated and wntained variably sized empty vesicles . Degenerated motor end plates were present. Away from the site of injection normal muscle fibres, intramuscular nerve trunks and motor end plates were observed. INTRODUCTION
Tl->E >~CT of Bitis gabonica venom on skeletal muscles of mice has been recently investigated (Tu et al., 1969) . They examined formalin fixed leg muscles of mice 6 hr after i .m. injection of venom and reported that B. gabonica venom, although highly hemorrhagic, did not cause any myonecrosis. The aim of the present work is to re-investigate the effect of B. gabonica venom on some histological and enzymological properties of skeletal muscle . MATERIALS AND METHOD$
Bids gabonica venom was obtained from the serpentarium of the Faculty of Medicine, Ain-Shams University. The crude venom was collected from adult B. gabonica, dried in a desiccator and stored at 3°C
in the dark. The crystals were reconstituted in physiological saline on the day of the experiment . tn,° was calculated according to the method of Raen and Mu>?rrcx (1938) . Albino mice of both sexes, weighing 25 g were divided into five groups of five mice per group. Grou I served as control animals which were injected with 03 ml physiological saline i.p . or i.m. Two anima of group II received an i.p . dose of one tn,o (18 ltg per ZS g body wt) of B. gabonlca venom. The other throe animals received the same dose i.m . in the back of the left thigh. Grou III received an i.m . sublethal dose (9 Itg per 25 g body wt). Two animals were sacrificed after 24 and 48 ~ and the rest after 21-24 days . Group TV received suocesaive i.m . sublethal doses (9 itg per 25 g mouse) at weekly intervals for 5 weeks, tho total dose boing 2~5 times the 165 TOX7CON 1973 Vot. )3
166
A. H. MOHAr~ , A. M. SALEH, S. AHMED and M. EL-MAGHRABY
The animals were sacrificed on the 5th and the 6th weeks. Animals of the Sth group were used for in vitro studies. The injected muscle was carefully dissected after the onset of deep surgical ether anaesthesia, except in group II animals, where the muscles were taken immediately after death (1-b hr after injection of one Lne o) . Longitudinal and transverse fresh frozen section 35 Eun thickness were processed for histochemical localization of cholinesterase and succinic dehydrogenase (SDH) activity according to GOMORI (1952) and Nachlas technique (Peaase, 1960), respectively. For ordinary histological studies the specimens were fixed in 10 per cent neutral formalin. Dehydration, clearing and embedding were carried out in the usual manner and the specimens were cut serially into longitudinal and transverse section 8-101.ßm in thickness. Different stains were used, e.g . haematoxylin and eosin, Van Geison's, Holme's silver, Glees and Marsland, Bielschowsky, phosphotungstic acid haematoxylin and periodic acid Schiff (PAS) techniques (DRURY and Wnu.u~rcTOx, 196 . Distal muscles from the leg of the injected limb (fibialis anterior) and the same muscle from the contralateral limb were processed in the same way as the injected muscles. In vitro studies Fresh frozen sections from the limb muscles as well as whole preparations of respiratory muscles (diaphragm and intercostals) were incubated in venom (1 mg per ml normal saline) at 37°C for varying periods which ranged from 20-60 min. Control sections and whole preparations were incubated for similar periods in normal saline. This was followed by localization of cholinesterase and succinic dehydrogenase activity . ~so"
Acute in
RESULTS
vivo experiments Group II animals given a dose of one LD fio i .p. showed haemorrhages in the peritoneal cavity . Animals given the same dose i .m . developed subcutaneous haemorrhages as well as extensive haemorrhages in the injected muscle . Localization of cholinesterase activity in longitudinal sections from the injected muscle showed a normal histochemical pattern of reaction, compared to the control. Various forms of motor end plates showed a well-defined cholinesterase active border of the subneural apparatus (Fig. 1). Localization of intramitochondrial succinic dehydrogenase (SDH) in transverse serial sections of the injected muscle showed a normal histochemica.l pattern of reaction in all the three fibre types of extrafusal muscle fibres compared to the control. According to S~rEIN and PADYKULA (1962), type A fibres in a heterogeneous muscle have a low enzymatic activity and represent the classical white fibres . The red fibres are divided into two subcategories, B and C. Type A has the largest diameter while B is intermediate and C is the smallest. Type B shows concentric lamination of SDH activity while type C has a high subsarcolemmal activity . This confirmed the inverse relationship between the size of the striated muscle fibre and its mitochondrial content (DUßOWITZ and PnaxsE, 1960a, b, 1961). Ordinary histological techniques revealed haemorrhage and extravasation of blood cells from dilated vessels. Animals given an i .m . sublethal dose (0~5 t,ns~ suffered from paralysis of the left injected limb. No changes were observed in the cholinesterase activity of the injected muscle after 24-48 hr. A generalized depletion of SDH was observed in all fibre types 48 hr after injection. This was manifest in both intermediate and small fibres (types B and C) . The subsarcolemmal activity which typifies type C fibres was reduced while the diformazan granules in its centre diminished (Fig. 2). Animals given i.m . sublethal dose (0~5 Lnbo) developed paralysis of the injected limb. Gradual recovery started after 7 days, so that after 24 days the injected limb was normal as far as mobility is concerned. This shows that Bitis gabonica venom has a reversible or a pseudo-paralytic effect. In animals sacrificed after 24 days, gross inspection of the injected muscle revealed the TOXICON 1975 Vol. 73
FIG . 1 . MOTOR END PLATE OF THE INJECTED MUSCLE (ONE LDao) "
Note the well-defined ChE active border of the subneural apparatus. 50 um scale. FIG . i. INJECTED MUSCLE (O'S LDso) AFTER 48 hr, SHOWING SDH ACTIVITY. Note a depleted B fibre. Type C fibres are partially depleted with reduced subsarcolemmal activity . 50 Nm scale. FIG . 3 . INJECTED MUSCLE (O'S LDso ) AFTER 24 DAYS.
Extensive vacuolization along the course of intramuscular blood vessels. 50 Nm scale. Haematoxylin and eosin (H 8c E). FiG . 4 . INJECTED MUSCLE (O'S LD6o) AFTER ZA DAY3.
Fasciculation and loss of transverse striations with intrafibral vacuolization. Note that the peripheral rim of the fibre still has transverse striations . Phosphotungstic acid haematoxylin . 50 kirl scale. FIG . S . INJECTED MUSCLE (O'S LD SO) AFTER
is DAYS
Partially degenerated nerve trunks. Note that some axons are interrupted and faintly stained Glees and Marsland stain. 50 Nm scale. FIG . 6 . INJECTED MUSCLE (O'S LDyo AT WEEKLY INTERVALS FOR S WEEKS) SHOWING REDUCITON IN THE FIBRE DIAMETER AND DIFFUSE SDH AGTIVfTY IN CONTRAST TO THE GRANULAR APPEARANCE OF THE NORMAL MUSCLE FIBRE . 50 um scale. TOXICON 1975 Vol. 13
f.p. 166
Histopathology of Muscle after B . gobonica Envenomation
]67
presence of a localized haematoma at the site of injection. Histological examination showed areas of extensive vacuolization along the course of intramuscular blood vessels (Fig. 3). There was an associated fibroblastic infiltration . Histiocytes, plasma cells and occasional lymphocytes were present. The vacuoles were oval and well circumscribed of small, intermediate or large size. In formalin-fixed preparations they were empty. Muscle fibres were either completely absent in the areas containing this cellular infiltrate or showed an early beginning of vacuolization . The blood vessels were dilated and the endothelial lining was interrupted at certain areas. Extravasated blood cells were observed between the muscle fibres . One of the large vacuoles had a fine communication to the nearby vessel . Its lumen was lined by endothelium and contained few red blood corpuscles, so that it appeared as an outpouch of the parent trunk. The other end of the blood vessel opened into a wide interstitial space containing blood cells. In other areas discontinuous spaces appeared between the muscle fibres which lost their normal nucleation and transverse striations. Such spaces either contained fragments of muscle or fibrous connective tissue . A striking feature was the presence of superimposed intact bundles of intramuscular nerves on top of degenerated muscle fibres . Fasciculation and complete loss of transverse striations in the centre of an individual fibre gave it the appearance of a shell containing variably sized vacuoles and bounded by a peripheral rim which still acquired its transverse striations (Fig. 4). Remnants of the muscle tissue and its nuclei could still be identified in the centre of the fibre. Fresh sections from the injected muscle of another animal sacrificed after 24 days were stained with Sudan III to investigate the nature of such vacuoles . No fat cells were detected inside individual muscle fibres denoting that intrafibral vacuolizations are not due to fatty degeneration . Nerve trunks in this injected muscle were divided into two categories after silver impregnation . The first category was formed ofunaffected normal intramuscular nerve trunks. Normal preterminal fibres, spirals and vesicular axonic swellings were observed. The majority of the motor end plates showed normal axons ending in fine ramifications on the soleplate nuclei . Perivascular nerves were also unaffected . Nerve trunks in the immediate vicinity of the site of injection were partially degenerated. Interruptions in the continuity of the axons and loss of affinity to the stain was quite evident (Fig. 5). When stained with Periodic acid schiff (PAS) small empty vesicles were apparent inside the trunk, while peripheral large vesicles interrupted nerve continuity. A minority of the motor end plates were partially degenerated. The degenerated axon lost its normal affinity to the stain. Distal muscles from the injected limb ofanimals sacrificed after 24 days
The tibialis anterior muscle contained occasional small and less extensive vacuoles . The intramuscular nerve trunks, the preterminals and the motor end plates with their fine terminal axonal sprays were normal . The muscles of the contralateral limb were unaffected . Animals given an i.m . sublethal dose (0~5 L.n 6o) at weekly intervals and sacrificed after 5 weeks had similar hispopathological changes in the injected muscle . However, a striking reduction of the fibre diameter similar to denervation atrophy was observed . Sections incubated for SDH were diffusely stained in contrast with the granular appearance of the normal muscle fibres. The characteristic subsarcolemmal density oftype Cfibres disappeared, thus diminishing the differences between the activities of this enzyme in the different muscle ToxrcoN ~grs v~r. s3
168
A. H. MOHAMED, A. M. SALER, S. ARMED and M. EL-MAGHRABY
fibres (Fig. 6). Fibres maintaining normal morphology were observed in addition to those in various stages of degeneration . In vitro studies When whole preparations of diaphragm and intercostal muscles were incubated in venom, the majority of the motor end plates were normal . The cholinesterase active border of the subneural apparatus was well defined. Transverse sections from the limb muscles incubated for SDH activity after venom application revealed no changes. DISCUSSION
venom produced haemorrhagic effects and tissue destruction at the site of injection. The endothelial lining of the dilated blood vessels was interrupted at certain areas and this was associated with extravasation of blood cells. These changes may be due to the proteolytic enzymes of this venom. Our findings coincide with the previous work of Tu et al. (1969), who found a correlation between haemorrhagic effects and proteolytic activities of viper venoms . GITTEIt et al. (1960) reported transitions of vessel wall deterioration. They attributed the `per diapedesis' haemorrhage to the effect of proteolytic enzymes on the intercellular spaces of the vascular endothelium which coincides with our results. Inhibition of the intramitochondrial enzyme succinic dehydrogenase was produced by B. gabonica venom 48 hr after injection. V1DAL et al. (1966) reported that succinic dehydrogenase was inhibited by treatment with proteolytic enzymes. Our in vitro studies with high venom concentrations did not indicate a direct immediate succinic dehydrogenase inhibition. The inhibiting factor may need a longer time to produce its effect . In our chronically treated animals, given i.m . sublethal doses at weekly intervals for 5 weeks, the inhibition of succinic dehydrogenase was so profound that the granular pattern of the reaction was transformed to a diffuse type. This was accompanied by a reduction of fibre size and the subsarcolemmal density, which typifies type C fibres, was reduced, thus diminishing the differences between the activities of the enzyme in different fibre types. Similar changes have been reported in denervation atrophy by ToMnNEx and LuND (1973) . The reduced subsarcolemmal density also coincides with the previous electron microscopic studies of NnssAIZ (Ph.D . Thesis, University of London, 1967). She reported that the mitochondria move from under the sarcolemma 1 week following denervation . Since the muscle is known to have a neural dependence, such changes observed in the present study may be attributed to the effect of the venom on the nerve supply of the muscle producing a type of toxic denervation. This is further supported by the presence of degenerated and vacuolated intramuscular nerve trunks and degenerated motor end-plates . Furthermore, the direct myotoxic action of the venom was observed at the site of injection in the form of absence of muscle tissue, vacuolization, fasciculation and loss of transverse striations . All these histopathological and histochemical changes were irreversible for up to 6 weeks. Examination of the peripheral part of the injected muscle as well as distal muscles from the injected limb proved that they maintained their normal morphology . Localization of cholinesterase activity proved that B. gabonxca venom contains no cholinesterase inhibitory factor. The haemorrhagic effects, the local tissue damage and the inhibition of succinic dehydrogenase, which is linked to cell respiration (GREEN et al., 1960) may be contributing factors to the toxicity of this venom. B, gabonica
work was supported by Grant 03-006-1 from The National Institute of Health, Hethesda, Maryland, U.S .A .
Ackirowledgement-This
TOXlCON 1975 Vol. 13
Histapathology of Musck after B. gabonka Bnvemomation
169
REFERENCES Dxvxv, R. A. B. and Weunaa~rox, E. A. (1967) Carleton's Histologieal Teclutique, 4th ed. Oxford : Oxfea+d University Press. Duaownz, V. and PR~R~ A. G. E. (1960x) A comparative histochemical study of oxidative enzyme and phospharylase activity in skeletal muscle. Histochemie 2,105. DvHOwrrt, V. and PBexsB, A. G. E. (1960b) Reciprocal relationship of phosphoryhue andoxidativeenzymes in skeletal muscle. Nestare, Load. 185, 701. Dueowrrz, V. and Pa~utss, A. G. E. (1961) Enzymatic acclivity of normal and dystrophic human muscles, a histochemical study. J. path. Bact . 81, 365. Grrr~, S., Lsvr, G., Kocxwe, S., na Van~v, A., Rec~n~rrc, J. and C~~t, J. (1960) Studies on the varom of Echis coloratus. Am. J. trop. Med. Hyg. 9, 391. Go~oar, G. (1952) Microscopic Histochemistry : Principles and Practice. Chicago : Chicago University Press. G~rr, D. E. and F~rscm x, S. (1960) The mitochondrial system of enzymes. In : Metabolic Pathways, p. 41, (GxES~eea, D. M., Ed.). New Yack : Academic Press. Pew, A. G. E. (1960) Histochemistry, 7lreonetlcal and Applied, 2nd ed. London : J. & A. Churchill . Rs®, L. J. and Muexcx, H. (1938) Asimple method of estimating füty per cent ead pointa Am. J. Hyg. 27, 493. Ste, J. M. and P~wvgur.~, H. A. (1962) Histochemical classification of individual skeletal muscle ßbres of the rat. Am . J. Anat. 110, 103. Torves;, R J. and Lune, D. D. (1973) Degeneration of diffament types of skeletal muscle fibres--I . Denecti vation . J. Anat. 116, 395. Tu, A. T., Horn, M. and Hoxa, B. S. (1969) Haemaorrhagic, myorsrcrotic, thrombotic aad proteolytk activities of viper venoms . Toxicon 6, 175. Vro~r., J. C., B~n~uvo, B. N., Szorrexi, A. O. M. and Hovesn, A. (1966; published 1968) Inhibition of electron transport chain by purified phospholipase A from Botlrrops neawledivenom. Menu Inrt. Brrtantmr 33, 913.
TOYICON I97S Vd. 19
HISTOPATHOLOGICAL AND HISTOCHEMICAL CHANGES IN SKELETAL MUSCLES AFTER BITIS GABONICA ENVENOMATION A. H. MOIiAI~, A. M.
$ALEH,
S. Ai-tn~t» and M.
EL-MACiHRABY
Faculty of Medicine, Ain Shams University, Cairo, Egypt (Accepted for publication 20 October 1974)
A. H. Moxw~n, A. M. S~r etl, S. AI~o and M. E~.-M~oHIUSV. Histo~athological and histochemical changes in skeletal muscles after Bibs gabonica envenomauon. Toxicon 13, 165-169, 1975 .-The histochemical activities of succinic dehydrogenase and cholinesterase in skeletal muscles of the mouse were studied after i.m. administration of B. gabonica venom. There was generalized depletion of succinic dehydrogenase activity 48 hr after injection of an i.n~, (18 ug per 25 g body wt). The subsarcolemmal density which typi~ type C fibres was reduced . In the chronically treated animals which received 0~5 rn~o at ~~ mtervals for S weeks, the normal granular pattern of the reaction became diffuse thus diminishing the differences between the enzyme activities in different fibres. Generalized reduction in fibre size similar to denervation atrophy was observed . The cholinesterase activity was not affected in either in vitro or in vlvo experiments. In acutely treated animals receiving one i.Dso haemorrhages and extravasation of blood cells was observed . Animals receiving 0~5 rn showed haematoma at the site of injection after 24 days . Histopathological changes included extensive vacuolization along the course of dilated intramuscular blood vessels. Interruption of their endothelial lining was detected in certain areas. Muscle tissue was either absent or showed vacuolization with non-specific cellular infiltration . Fasciculation, infra-fibral vawolization and loss of transverse striations was observed . Nerve trunks were partially degenerated and wntained variably sized empty vesicles . Degenerated motor end plates were present. Away from the site of injection normal muscle fibres, intramuscular nerve trunks and motor end plates were observed. INTRODUCTION
Tl->E >~CT of Bitis gabonica venom on skeletal muscles of mice has been recently investigated (Tu et al., 1969) . They examined formalin fixed leg muscles of mice 6 hr after i .m. injection of venom and reported that B. gabonica venom, although highly hemorrhagic, did not cause any myonecrosis. The aim of the present work is to re-investigate the effect of B. gabonica venom on some histological and enzymological properties of skeletal muscle . MATERIALS AND METHOD$
Bids gabonica venom was obtained from the serpentarium of the Faculty of Medicine, Ain-Shams University. The crude venom was collected from adult B. gabonica, dried in a desiccator and stored at 3°C
in the dark. The crystals were reconstituted in physiological saline on the day of the experiment . tn,° was calculated according to the method of Raen and Mu>?rrcx (1938) . Albino mice of both sexes, weighing 25 g were divided into five groups of five mice per group. Grou I served as control animals which were injected with 03 ml physiological saline i.p . or i.m. Two anima of group II received an i.p . dose of one tn,o (18 ltg per ZS g body wt) of B. gabonlca venom. The other throe animals received the same dose i.m . in the back of the left thigh. Grou III received an i.m . sublethal dose (9 Itg per 25 g body wt). Two animals were sacrificed after 24 and 48 ~ and the rest after 21-24 days . Group TV received suocesaive i.m . sublethal doses (9 itg per 25 g mouse) at weekly intervals for 5 weeks, tho total dose boing 2~5 times the 165 TOX7CON 1973 Vot. )3
166
A. H. MOHAr~ , A. M. SALEH, S. AHMED and M. EL-MAGHRABY
The animals were sacrificed on the 5th and the 6th weeks. Animals of the Sth group were used for in vitro studies. The injected muscle was carefully dissected after the onset of deep surgical ether anaesthesia, except in group II animals, where the muscles were taken immediately after death (1-b hr after injection of one Lne o) . Longitudinal and transverse fresh frozen section 35 Eun thickness were processed for histochemical localization of cholinesterase and succinic dehydrogenase (SDH) activity according to GOMORI (1952) and Nachlas technique (Peaase, 1960), respectively. For ordinary histological studies the specimens were fixed in 10 per cent neutral formalin. Dehydration, clearing and embedding were carried out in the usual manner and the specimens were cut serially into longitudinal and transverse section 8-101.ßm in thickness. Different stains were used, e.g . haematoxylin and eosin, Van Geison's, Holme's silver, Glees and Marsland, Bielschowsky, phosphotungstic acid haematoxylin and periodic acid Schiff (PAS) techniques (DRURY and Wnu.u~rcTOx, 196 . Distal muscles from the leg of the injected limb (fibialis anterior) and the same muscle from the contralateral limb were processed in the same way as the injected muscles. In vitro studies Fresh frozen sections from the limb muscles as well as whole preparations of respiratory muscles (diaphragm and intercostals) were incubated in venom (1 mg per ml normal saline) at 37°C for varying periods which ranged from 20-60 min. Control sections and whole preparations were incubated for similar periods in normal saline. This was followed by localization of cholinesterase and succinic dehydrogenase activity . ~so"
Acute in
RESULTS
vivo experiments Group II animals given a dose of one LD fio i .p. showed haemorrhages in the peritoneal cavity . Animals given the same dose i .m . developed subcutaneous haemorrhages as well as extensive haemorrhages in the injected muscle . Localization of cholinesterase activity in longitudinal sections from the injected muscle showed a normal histochemical pattern of reaction, compared to the control. Various forms of motor end plates showed a well-defined cholinesterase active border of the subneural apparatus (Fig. 1). Localization of intramitochondrial succinic dehydrogenase (SDH) in transverse serial sections of the injected muscle showed a normal histochemica.l pattern of reaction in all the three fibre types of extrafusal muscle fibres compared to the control. According to S~rEIN and PADYKULA (1962), type A fibres in a heterogeneous muscle have a low enzymatic activity and represent the classical white fibres . The red fibres are divided into two subcategories, B and C. Type A has the largest diameter while B is intermediate and C is the smallest. Type B shows concentric lamination of SDH activity while type C has a high subsarcolemmal activity . This confirmed the inverse relationship between the size of the striated muscle fibre and its mitochondrial content (DUßOWITZ and PnaxsE, 1960a, b, 1961). Ordinary histological techniques revealed haemorrhage and extravasation of blood cells from dilated vessels. Animals given an i .m . sublethal dose (0~5 t,ns~ suffered from paralysis of the left injected limb. No changes were observed in the cholinesterase activity of the injected muscle after 24-48 hr. A generalized depletion of SDH was observed in all fibre types 48 hr after injection. This was manifest in both intermediate and small fibres (types B and C) . The subsarcolemmal activity which typifies type C fibres was reduced while the diformazan granules in its centre diminished (Fig. 2). Animals given i.m . sublethal dose (0~5 Lnbo) developed paralysis of the injected limb. Gradual recovery started after 7 days, so that after 24 days the injected limb was normal as far as mobility is concerned. This shows that Bitis gabonica venom has a reversible or a pseudo-paralytic effect. In animals sacrificed after 24 days, gross inspection of the injected muscle revealed the TOXICON 1975 Vol. 73
FIG . 1 . MOTOR END PLATE OF THE INJECTED MUSCLE (ONE LDao) "
Note the well-defined ChE active border of the subneural apparatus. 50 um scale. FIG . i. INJECTED MUSCLE (O'S LDso) AFTER 48 hr, SHOWING SDH ACTIVITY. Note a depleted B fibre. Type C fibres are partially depleted with reduced subsarcolemmal activity . 50 Nm scale. FIG . 3 . INJECTED MUSCLE (O'S LDso ) AFTER 24 DAYS.
Extensive vacuolization along the course of intramuscular blood vessels. 50 Nm scale. Haematoxylin and eosin (H 8c E). FiG . 4 . INJECTED MUSCLE (O'S LD6o) AFTER ZA DAY3.
Fasciculation and loss of transverse striations with intrafibral vacuolization. Note that the peripheral rim of the fibre still has transverse striations . Phosphotungstic acid haematoxylin . 50 kirl scale. FIG . S . INJECTED MUSCLE (O'S LD SO) AFTER
is DAYS
Partially degenerated nerve trunks. Note that some axons are interrupted and faintly stained Glees and Marsland stain. 50 Nm scale. FIG . 6 . INJECTED MUSCLE (O'S LDyo AT WEEKLY INTERVALS FOR S WEEKS) SHOWING REDUCITON IN THE FIBRE DIAMETER AND DIFFUSE SDH AGTIVfTY IN CONTRAST TO THE GRANULAR APPEARANCE OF THE NORMAL MUSCLE FIBRE . 50 um scale. TOXICON 1975 Vol. 13
f.p. 166
Histopathology of Muscle after B . gobonica Envenomation
]67
presence of a localized haematoma at the site of injection. Histological examination showed areas of extensive vacuolization along the course of intramuscular blood vessels (Fig. 3). There was an associated fibroblastic infiltration . Histiocytes, plasma cells and occasional lymphocytes were present. The vacuoles were oval and well circumscribed of small, intermediate or large size. In formalin-fixed preparations they were empty. Muscle fibres were either completely absent in the areas containing this cellular infiltrate or showed an early beginning of vacuolization . The blood vessels were dilated and the endothelial lining was interrupted at certain areas. Extravasated blood cells were observed between the muscle fibres . One of the large vacuoles had a fine communication to the nearby vessel . Its lumen was lined by endothelium and contained few red blood corpuscles, so that it appeared as an outpouch of the parent trunk. The other end of the blood vessel opened into a wide interstitial space containing blood cells. In other areas discontinuous spaces appeared between the muscle fibres which lost their normal nucleation and transverse striations. Such spaces either contained fragments of muscle or fibrous connective tissue . A striking feature was the presence of superimposed intact bundles of intramuscular nerves on top of degenerated muscle fibres . Fasciculation and complete loss of transverse striations in the centre of an individual fibre gave it the appearance of a shell containing variably sized vacuoles and bounded by a peripheral rim which still acquired its transverse striations (Fig. 4). Remnants of the muscle tissue and its nuclei could still be identified in the centre of the fibre. Fresh sections from the injected muscle of another animal sacrificed after 24 days were stained with Sudan III to investigate the nature of such vacuoles . No fat cells were detected inside individual muscle fibres denoting that intrafibral vacuolizations are not due to fatty degeneration . Nerve trunks in this injected muscle were divided into two categories after silver impregnation . The first category was formed ofunaffected normal intramuscular nerve trunks. Normal preterminal fibres, spirals and vesicular axonic swellings were observed. The majority of the motor end plates showed normal axons ending in fine ramifications on the soleplate nuclei . Perivascular nerves were also unaffected . Nerve trunks in the immediate vicinity of the site of injection were partially degenerated. Interruptions in the continuity of the axons and loss of affinity to the stain was quite evident (Fig. 5). When stained with Periodic acid schiff (PAS) small empty vesicles were apparent inside the trunk, while peripheral large vesicles interrupted nerve continuity. A minority of the motor end plates were partially degenerated. The degenerated axon lost its normal affinity to the stain. Distal muscles from the injected limb ofanimals sacrificed after 24 days
The tibialis anterior muscle contained occasional small and less extensive vacuoles . The intramuscular nerve trunks, the preterminals and the motor end plates with their fine terminal axonal sprays were normal . The muscles of the contralateral limb were unaffected . Animals given an i.m . sublethal dose (0~5 L.n 6o) at weekly intervals and sacrificed after 5 weeks had similar hispopathological changes in the injected muscle . However, a striking reduction of the fibre diameter similar to denervation atrophy was observed . Sections incubated for SDH were diffusely stained in contrast with the granular appearance of the normal muscle fibres. The characteristic subsarcolemmal density oftype Cfibres disappeared, thus diminishing the differences between the activities of this enzyme in the different muscle ToxrcoN ~grs v~r. s3
168
A. H. MOHAMED, A. M. SALER, S. ARMED and M. EL-MAGHRABY
fibres (Fig. 6). Fibres maintaining normal morphology were observed in addition to those in various stages of degeneration . In vitro studies When whole preparations of diaphragm and intercostal muscles were incubated in venom, the majority of the motor end plates were normal . The cholinesterase active border of the subneural apparatus was well defined. Transverse sections from the limb muscles incubated for SDH activity after venom application revealed no changes. DISCUSSION
venom produced haemorrhagic effects and tissue destruction at the site of injection. The endothelial lining of the dilated blood vessels was interrupted at certain areas and this was associated with extravasation of blood cells. These changes may be due to the proteolytic enzymes of this venom. Our findings coincide with the previous work of Tu et al. (1969), who found a correlation between haemorrhagic effects and proteolytic activities of viper venoms . GITTEIt et al. (1960) reported transitions of vessel wall deterioration. They attributed the `per diapedesis' haemorrhage to the effect of proteolytic enzymes on the intercellular spaces of the vascular endothelium which coincides with our results. Inhibition of the intramitochondrial enzyme succinic dehydrogenase was produced by B. gabonica venom 48 hr after injection. V1DAL et al. (1966) reported that succinic dehydrogenase was inhibited by treatment with proteolytic enzymes. Our in vitro studies with high venom concentrations did not indicate a direct immediate succinic dehydrogenase inhibition. The inhibiting factor may need a longer time to produce its effect . In our chronically treated animals, given i.m . sublethal doses at weekly intervals for 5 weeks, the inhibition of succinic dehydrogenase was so profound that the granular pattern of the reaction was transformed to a diffuse type. This was accompanied by a reduction of fibre size and the subsarcolemmal density, which typifies type C fibres, was reduced, thus diminishing the differences between the activities of the enzyme in different fibre types. Similar changes have been reported in denervation atrophy by ToMnNEx and LuND (1973) . The reduced subsarcolemmal density also coincides with the previous electron microscopic studies of NnssAIZ (Ph.D . Thesis, University of London, 1967). She reported that the mitochondria move from under the sarcolemma 1 week following denervation . Since the muscle is known to have a neural dependence, such changes observed in the present study may be attributed to the effect of the venom on the nerve supply of the muscle producing a type of toxic denervation. This is further supported by the presence of degenerated and vacuolated intramuscular nerve trunks and degenerated motor end-plates . Furthermore, the direct myotoxic action of the venom was observed at the site of injection in the form of absence of muscle tissue, vacuolization, fasciculation and loss of transverse striations . All these histopathological and histochemical changes were irreversible for up to 6 weeks. Examination of the peripheral part of the injected muscle as well as distal muscles from the injected limb proved that they maintained their normal morphology . Localization of cholinesterase activity proved that B. gabonxca venom contains no cholinesterase inhibitory factor. The haemorrhagic effects, the local tissue damage and the inhibition of succinic dehydrogenase, which is linked to cell respiration (GREEN et al., 1960) may be contributing factors to the toxicity of this venom. B, gabonica
work was supported by Grant 03-006-1 from The National Institute of Health, Hethesda, Maryland, U.S .A .
Ackirowledgement-This
TOXlCON 1975 Vol. 13
Histapathology of Musck after B. gabonka Bnvemomation
169
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