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A guinea-pig model of corneal jellyfish envenomations
0041-0101/93 56.00 + .00 ® 1993 Pagittwn Prer Ltd
Toxlca~, Vol . 31, No. 6, pp. d08-812, 1993 . Printed in Cmat Bduin .
A GUINEA-PIG MODEL OF CORNEAL JELLYFISH ENVENOMATIONS Dwvro B. Gr wssr~,' JosePx W. BuRNm-r,2* Sal>;evw S. KwTxuxiw' and M11tLYN M. RODRIGUES' Dcpartments of IOphthahnology, and Dermatology, University of Maryland School of Medicine, Baltimore, MD 21201, U.S.A. (Received 30 September 1992; accepted 8 December 1992)
D. B. Gr,wssm, J. W. BURNETT, S. S. KwTxuitiw and M. M. RODRIGUES. A guinea-pig model of corneal jellyfish envenomations. Toxicon 31, 808-812, 1993 .~orneal jellyfish stings are painful, self-limited injuries which usually produce conjonctival hyperemia and chemosis, corneal edema, and mild iridocyclitis with resolution in 48 hr . We have developed a guinea-pig model of corneal jellyfish stings in order to study the pathogenesis of human injury . Guinea-pig eyes were exposed to sea nettle (Chrysaora quinquecirrha) venom in vivo in the following ways : contact with live tentacles, intracameral injection of crude venom, and intracorneal injection of crude jellyfish venom. Slit lamp examination and histologic sections of the eye performed at intervals up to 8 days after exposure to the venom demonstrated conjonctival hyperemia and chemosis, corneal stromal inflammatory edema, anterior chamber inflammation, and opacities on the anterior capsule of the lens . Nematocysts adherent to the corneal epithelium were noted in eyes exposed to live tentacles. INTRODUCTION OPHTHALMOLOGICwL inj11r1eS to man caused by jellyfish envenomations have been reported from multiple species across the world (CLELAND and SoclTxcoz-r, 1965; Hsxcus, 1944 ; Mrrci~t.I,, 1970; Woxa and Mo~rosw, 1985 ; RA>'osw et al., 1986; BURNETT and BURNETT, 1980; PARADICE, 1924 ; Gt,wsssx et al., 1992). These injuries usually produce brief corneal inflammation and abrasions, although cases of iritis, chronic unilateral glaucoma, mydriasis, decreased accommodation, peripheral anterior synechia, iris depigmentation and visual blurring have been recorded. Injury to the external eye is caused by the projection of a toxin coated thread (nema) into the tissue . This thread is ejected from an intracytoplasmic organelle (nematocyst) located in the outer epithelium of the jellyfish tentacle (Cr.Fr.exn and Sou'rxcol-r, 1965). The toxin is composed of numerous polypeptides with enzymatic actions capable of destroying local tissue and inducing systemic sequelae (BURNETf and CALTON, 1987 ; BURNiI'f, 1992). Millions of nematocysts are discharged during a single sting, some or Author to whom correspondence should be addressed. 808
Short Communications
809
many of which may discharge into the cornea, conjunctiva, lids and tear film . Although these nematocysts are usually not apparent they have been detected on physical examination using tangential light and low magnification (GL~.~t et al., 1992). The purpose of this study was to design an animal model for ocular injuries from jellyfish envenomations and to define the time course and dosage necessary to detect these injuries . Nematocyst venom from adult sea nettle (Chrysaora quinquecirrha) medusas (Meredith Creek, St. Margaret's, MD, U.S.A., 1991) was prepared by a technique using autolysis, centrifugation and sonic treatment (Buitxm-r et al., 1992). A balanced salt solution, (Iosare, Iolab Pharmaceuticals, Claremont, CA, U.S.A.) was used as the diluent. Topical proparacaine hydrochloride 0.5% (Alcon, Inc., Forth Worth, TX, U.S.A.) anesthesia was used for all animals. Daily examinations for a week using an ophthalmoscope, a slit lamp and a portable electronic tonometer (Tonopen, Biorad, Santa Anna, CA, U.S.A .) were done. Histological specimens were prepared from animals sacrificed by i.p. barbiturate injection. Eyes from at least two animals were examined in duplicate experiments for each time point which included 2 hr, and 2, 4, and 8 days post-venom exposure. In direct sting experiments the mass of mixed tentacles was placed on the cornea for 90 sec. A microsyringe equipped with a 30 gauge needle was used for corneal intrastromal inoculations (5 pl). Control injections with salt solution of Chesapeake Hay water diluted with Iosare were performed. Serial threefold dilution of the venom was employed to detect the minimum dose capable of injuring the eye. Direct tentacle contact resulted in erythema in the surrounding conjunctival tissues 4 hr later. At this time the intraocular pressure was unchanged but a hazy irregular corneal epithelial surface was visible. Multiple small white punctate opacities (nematocysts) could be seen in the cornea for the next day. The following day the lids were edematous and partially closed, the cornea was opaque and the conjunctival tissues were injected and chemotic . Some conjunctival hemorrhage was visible and crusting appeared on the lid margins. The examination of the pathologic specimens from three eyes tested by tentacle envenomation showed that nematocysts could be visualized within the cornea in specimens 2 hr after envenomation (Fig. 1 a). At that time there were only mild changes of edema within the corneal epithelium (Fig . l b). By two days, a considerable amount of corneal stromal edema and cellular infiltration had appeared (Fig.lc) . By 4 days, significant inflammatory infiltration was observed within the cornea and there was evidence of moderate iridocyclitis (Fig. ld). The inflammatory response had resolved by the seventh day post-evenomation (Table 1). Multiple guinea-pig eyes were injected intrastromally with 5 pl volume of various dilutions (1 : 3 to 1 : 1000) of crude nematocyst venom and then enucleated after 2, 4 and 7 days. At no time was an increase in intraocular pressure noted. Changes noted included : conjunctival hyperemia, superficial punctate keratopathy, corneal stromal edema and infiltrates, anterior chamber inflammation, dilated iris vessels and anterior lens capsule opacities. Most venom dilutions produced iridocyclitis and the 1 : 3 dilution of the venom resulted in a permanent corneal scar . Corneal inflammation could be detected by slit lamp examination on eyes injected with venom up to a dilution of 1 : 300. Resolution of these abnormalities began within 3 days and almost complete healing occurred by 1 week (Table 1). Injury to the cornea, conjunctiva and surrounding orbital tissues by venom coated jellyfish nematocyst threads can produce significant inflammatory responses. These lesions
81 0
Short Communications
Fta. l . (a) An empty nematocyst capsule impacted in the wrneal epithelium 2 hr after envenomation ( x 400) . (b) Mild basal edema of the corneal epithelium 2 hr after envenomation ( x 200) . (c) Two days after envenomation. Acute inflammatory cellular infiltrates and edema of the cornea . Polymorphonuclear cells are present in the anterior chamber (avow) and iris (curved arrow) ( x 40). (d) Four days after envenomation . Inflammatory cell infiltrates are present in the ciliary body ( x 100) .
Short Communications TARIE I. FFaTURF3 OF OPHTHALMIC JELLYFISH INJURY 4 hr
Visible nematocysts in the cornea Hazy corneal epithelial surface Slight lid and conjonctival erythema and edema
l-3 days
Nematocysts start to disappear from the cornea Lid edema, hemorrhage and marginal exudate Conjonctival hyperemia and edema Punctate keratopathy progressing to an opaque wrnea Corneal stromal edema and cellular infiltration advance Anterior chamber inflammation Dilated iris vessels Anterior lens capsule opacities Intraocular pressure ranged from 5-21 mm Hg
4 days
Moderate iridocyclitis Findings of 1-3 days begin to resolve
8 days
Resolution
resolved within a week in our experimental model. For several hours after the sting, empty nematocysts were visible on the corneal epithelial surface by careful direct examination or by examination of envenomated histologic specimens. The degree of edema in the eyelid tissue did not correspond to that present in the cornea and no case of elevated intraocular pressure was recorded even though there was considerable iridocyclitis. The discrepancy between the elevated intraocular pressure noted in a recent clinical report (GLASSER et al., 1992) and the normal pressure recorded in guinea-pig stinging may be due to anatomical variations or the unusually severe nature of the human envenomation . Many hours were required for significant cellular infiltrations to appear. This correlates with the delay for severely stung patients to present with significant ophthahnological injury, and it might explain the necessary interval for a delayed hypersensitivity or a slow toxic reaction to unfold (BtrxNETT, 1992; GLASSER et al., 1992). The observation that the venom could be diluted at least 300-fold and still produce significant pathological damage illustrates the potential severity of an inflammatory reaction to jellyfish envenomations and suggests that better case surveillance is needed . Aclrnowledgements-The authors would like to thank Ms KELLY Jogs and Ms HELExE RueIxsTEnv for the aid and assistance with these studies .
REFERENCES BuRr>Err, J. W. (1992) Human injuries following jellyfish stings. Maryland med. J. 41, 509-513. BuRxerr, H. W. and BURNErf, J. W. (1990) Prolonged blurred vision following coelenterate envenomations. Toxicon 28, 731-733. Buxr>E-rr, J. W. and CALTON, G. J. (1987) Venomous pelagic coelenterates: chemistry, toxicology, immunology and treatment of their stings. Toxicon 2S, 58102. BURNETr, J. W., LONG, K. O. and RUBRVSIEIN, H. (1992) Beachside preparation of jellyfish nematocyst tentacles . Toxicon 30, 794-796. CiELAND, J. B. and SOUTHCOTT, R. V. (1965) Injuries to Man from Marine Invertebrates in the Australian Region . Commonwealth of Australia, Canberra . GLASSrat, D. B., NoELL, M. J., BuRx~rr, J. W., KATHURIA, S. S. and RonRIGUes, M. M. (1992) Ocular jellyfish stings . Ophthalmology 99, 1414-1418 . HERCUS, J. (1944) An unusual eye condition. Med. J. Aunt . 1, 98-99.
81 2
Short Communications
Mtrc~.t,, J. H. (1970) Ocular injuries due to jellyfish. 7Yons. Aunt. Coll. Ophthabnol 2, 96-99. PARADICS, W. E. J. (1924) Injuries and lesions caused by the bites of animals and in .9ects . Med. J. Aunt. 2, 650-652 . Renosn, P. A., WEST, S. K., Nswurtn, H. S. and TeYIAn, H. R. (1986) Ocular jellyfish stings in Chesapeake Bay watenaan. Am. J. Ophthabnol. 102, 536-537. WoNG, S. K. and Morose, A. (1985) Jellyfish sting of the cornea . Am. J. Ophthal»wl. 100, 739-740.
Toxlca~, Vol . 31, No. 6, pp. d08-812, 1993 . Printed in Cmat Bduin .
A GUINEA-PIG MODEL OF CORNEAL JELLYFISH ENVENOMATIONS Dwvro B. Gr wssr~,' JosePx W. BuRNm-r,2* Sal>;evw S. KwTxuxiw' and M11tLYN M. RODRIGUES' Dcpartments of IOphthahnology, and Dermatology, University of Maryland School of Medicine, Baltimore, MD 21201, U.S.A. (Received 30 September 1992; accepted 8 December 1992)
D. B. Gr,wssm, J. W. BURNETT, S. S. KwTxuitiw and M. M. RODRIGUES. A guinea-pig model of corneal jellyfish envenomations. Toxicon 31, 808-812, 1993 .~orneal jellyfish stings are painful, self-limited injuries which usually produce conjonctival hyperemia and chemosis, corneal edema, and mild iridocyclitis with resolution in 48 hr . We have developed a guinea-pig model of corneal jellyfish stings in order to study the pathogenesis of human injury . Guinea-pig eyes were exposed to sea nettle (Chrysaora quinquecirrha) venom in vivo in the following ways : contact with live tentacles, intracameral injection of crude venom, and intracorneal injection of crude jellyfish venom. Slit lamp examination and histologic sections of the eye performed at intervals up to 8 days after exposure to the venom demonstrated conjonctival hyperemia and chemosis, corneal stromal inflammatory edema, anterior chamber inflammation, and opacities on the anterior capsule of the lens . Nematocysts adherent to the corneal epithelium were noted in eyes exposed to live tentacles. INTRODUCTION OPHTHALMOLOGICwL inj11r1eS to man caused by jellyfish envenomations have been reported from multiple species across the world (CLELAND and SoclTxcoz-r, 1965; Hsxcus, 1944 ; Mrrci~t.I,, 1970; Woxa and Mo~rosw, 1985 ; RA>'osw et al., 1986; BURNETT and BURNETT, 1980; PARADICE, 1924 ; Gt,wsssx et al., 1992). These injuries usually produce brief corneal inflammation and abrasions, although cases of iritis, chronic unilateral glaucoma, mydriasis, decreased accommodation, peripheral anterior synechia, iris depigmentation and visual blurring have been recorded. Injury to the external eye is caused by the projection of a toxin coated thread (nema) into the tissue . This thread is ejected from an intracytoplasmic organelle (nematocyst) located in the outer epithelium of the jellyfish tentacle (Cr.Fr.exn and Sou'rxcol-r, 1965). The toxin is composed of numerous polypeptides with enzymatic actions capable of destroying local tissue and inducing systemic sequelae (BURNETf and CALTON, 1987 ; BURNiI'f, 1992). Millions of nematocysts are discharged during a single sting, some or Author to whom correspondence should be addressed. 808
Short Communications
809
many of which may discharge into the cornea, conjunctiva, lids and tear film . Although these nematocysts are usually not apparent they have been detected on physical examination using tangential light and low magnification (GL~.~t et al., 1992). The purpose of this study was to design an animal model for ocular injuries from jellyfish envenomations and to define the time course and dosage necessary to detect these injuries . Nematocyst venom from adult sea nettle (Chrysaora quinquecirrha) medusas (Meredith Creek, St. Margaret's, MD, U.S.A., 1991) was prepared by a technique using autolysis, centrifugation and sonic treatment (Buitxm-r et al., 1992). A balanced salt solution, (Iosare, Iolab Pharmaceuticals, Claremont, CA, U.S.A.) was used as the diluent. Topical proparacaine hydrochloride 0.5% (Alcon, Inc., Forth Worth, TX, U.S.A.) anesthesia was used for all animals. Daily examinations for a week using an ophthalmoscope, a slit lamp and a portable electronic tonometer (Tonopen, Biorad, Santa Anna, CA, U.S.A .) were done. Histological specimens were prepared from animals sacrificed by i.p. barbiturate injection. Eyes from at least two animals were examined in duplicate experiments for each time point which included 2 hr, and 2, 4, and 8 days post-venom exposure. In direct sting experiments the mass of mixed tentacles was placed on the cornea for 90 sec. A microsyringe equipped with a 30 gauge needle was used for corneal intrastromal inoculations (5 pl). Control injections with salt solution of Chesapeake Hay water diluted with Iosare were performed. Serial threefold dilution of the venom was employed to detect the minimum dose capable of injuring the eye. Direct tentacle contact resulted in erythema in the surrounding conjunctival tissues 4 hr later. At this time the intraocular pressure was unchanged but a hazy irregular corneal epithelial surface was visible. Multiple small white punctate opacities (nematocysts) could be seen in the cornea for the next day. The following day the lids were edematous and partially closed, the cornea was opaque and the conjunctival tissues were injected and chemotic . Some conjunctival hemorrhage was visible and crusting appeared on the lid margins. The examination of the pathologic specimens from three eyes tested by tentacle envenomation showed that nematocysts could be visualized within the cornea in specimens 2 hr after envenomation (Fig. 1 a). At that time there were only mild changes of edema within the corneal epithelium (Fig . l b). By two days, a considerable amount of corneal stromal edema and cellular infiltration had appeared (Fig.lc) . By 4 days, significant inflammatory infiltration was observed within the cornea and there was evidence of moderate iridocyclitis (Fig. ld). The inflammatory response had resolved by the seventh day post-evenomation (Table 1). Multiple guinea-pig eyes were injected intrastromally with 5 pl volume of various dilutions (1 : 3 to 1 : 1000) of crude nematocyst venom and then enucleated after 2, 4 and 7 days. At no time was an increase in intraocular pressure noted. Changes noted included : conjunctival hyperemia, superficial punctate keratopathy, corneal stromal edema and infiltrates, anterior chamber inflammation, dilated iris vessels and anterior lens capsule opacities. Most venom dilutions produced iridocyclitis and the 1 : 3 dilution of the venom resulted in a permanent corneal scar . Corneal inflammation could be detected by slit lamp examination on eyes injected with venom up to a dilution of 1 : 300. Resolution of these abnormalities began within 3 days and almost complete healing occurred by 1 week (Table 1). Injury to the cornea, conjunctiva and surrounding orbital tissues by venom coated jellyfish nematocyst threads can produce significant inflammatory responses. These lesions
81 0
Short Communications
Fta. l . (a) An empty nematocyst capsule impacted in the wrneal epithelium 2 hr after envenomation ( x 400) . (b) Mild basal edema of the corneal epithelium 2 hr after envenomation ( x 200) . (c) Two days after envenomation. Acute inflammatory cellular infiltrates and edema of the cornea . Polymorphonuclear cells are present in the anterior chamber (avow) and iris (curved arrow) ( x 40). (d) Four days after envenomation . Inflammatory cell infiltrates are present in the ciliary body ( x 100) .
Short Communications TARIE I. FFaTURF3 OF OPHTHALMIC JELLYFISH INJURY 4 hr
Visible nematocysts in the cornea Hazy corneal epithelial surface Slight lid and conjonctival erythema and edema
l-3 days
Nematocysts start to disappear from the cornea Lid edema, hemorrhage and marginal exudate Conjonctival hyperemia and edema Punctate keratopathy progressing to an opaque wrnea Corneal stromal edema and cellular infiltration advance Anterior chamber inflammation Dilated iris vessels Anterior lens capsule opacities Intraocular pressure ranged from 5-21 mm Hg
4 days
Moderate iridocyclitis Findings of 1-3 days begin to resolve
8 days
Resolution
resolved within a week in our experimental model. For several hours after the sting, empty nematocysts were visible on the corneal epithelial surface by careful direct examination or by examination of envenomated histologic specimens. The degree of edema in the eyelid tissue did not correspond to that present in the cornea and no case of elevated intraocular pressure was recorded even though there was considerable iridocyclitis. The discrepancy between the elevated intraocular pressure noted in a recent clinical report (GLASSER et al., 1992) and the normal pressure recorded in guinea-pig stinging may be due to anatomical variations or the unusually severe nature of the human envenomation . Many hours were required for significant cellular infiltrations to appear. This correlates with the delay for severely stung patients to present with significant ophthahnological injury, and it might explain the necessary interval for a delayed hypersensitivity or a slow toxic reaction to unfold (BtrxNETT, 1992; GLASSER et al., 1992). The observation that the venom could be diluted at least 300-fold and still produce significant pathological damage illustrates the potential severity of an inflammatory reaction to jellyfish envenomations and suggests that better case surveillance is needed . Aclrnowledgements-The authors would like to thank Ms KELLY Jogs and Ms HELExE RueIxsTEnv for the aid and assistance with these studies .
REFERENCES BuRr>Err, J. W. (1992) Human injuries following jellyfish stings. Maryland med. J. 41, 509-513. BuRxerr, H. W. and BURNErf, J. W. (1990) Prolonged blurred vision following coelenterate envenomations. Toxicon 28, 731-733. Buxr>E-rr, J. W. and CALTON, G. J. (1987) Venomous pelagic coelenterates: chemistry, toxicology, immunology and treatment of their stings. Toxicon 2S, 58102. BURNETr, J. W., LONG, K. O. and RUBRVSIEIN, H. (1992) Beachside preparation of jellyfish nematocyst tentacles . Toxicon 30, 794-796. CiELAND, J. B. and SOUTHCOTT, R. V. (1965) Injuries to Man from Marine Invertebrates in the Australian Region . Commonwealth of Australia, Canberra . GLASSrat, D. B., NoELL, M. J., BuRx~rr, J. W., KATHURIA, S. S. and RonRIGUes, M. M. (1992) Ocular jellyfish stings . Ophthalmology 99, 1414-1418 . HERCUS, J. (1944) An unusual eye condition. Med. J. Aunt . 1, 98-99.
81 2
Short Communications
Mtrc~.t,, J. H. (1970) Ocular injuries due to jellyfish. 7Yons. Aunt. Coll. Ophthabnol 2, 96-99. PARADICS, W. E. J. (1924) Injuries and lesions caused by the bites of animals and in .9ects . Med. J. Aunt. 2, 650-652 . Renosn, P. A., WEST, S. K., Nswurtn, H. S. and TeYIAn, H. R. (1986) Ocular jellyfish stings in Chesapeake Bay watenaan. Am. J. Ophthabnol. 102, 536-537. WoNG, S. K. and Morose, A. (1985) Jellyfish sting of the cornea . Am. J. Ophthal»wl. 100, 739-740.