LABORATORY INVESTIGATION

Therapy of Brown Spider Envenomation: A Controlled Trial of Hyperbaric Oxygen, Dapsone, and Cyproheptadine

From Toxicology Associates, Ro&y Mountain Poison and Drug Center, Denver General Hospital, U)iversity of Colorado Health Sciences Center, Denver*; and Cotorado State University, School of Veterina U Medicine, Fort Collins, Colorado.*

Presented at the American Academy of Clinical Toxico!ogy Annual 5cientific Meeting in Tampa, Florida, October i992.

Copyright @ by the American College of gmeNency Physicians.

Scott Phillips, MD* Michael Kohn, MD* Dale Baker, DVM PhD* Rob Vander Leest, MB* Hernan Gomez, MD* Patrick McKinney, MD* John McGoldrick, MO* Jeffrey Breot, MD, PhD*

Study objective: To determine whether hyperbaric oxygen (HBO), dapsone, or cyproheptadine decreases the severity of skin lesions resulting from experimental Loxosceles envenomation.

Design: Randomized, blinded, controlled study.

Setting: Animal care facility.

Interventions: We used New Zealand white rabbits. All groups received 20 pg of pooled L deserta venom intradermally. Our control group received 4 ml of a 5% ethanol solution by oral gavage every 12 hours for 4 days. The HB0 group received hyperbaric oxygen at 2.5 ATA for 65 minutes every 12 hours for 2 days, plus 5% ethanol solution for 4 days. The dapsone group received dapsone 1.1 mg/kg in 5% ethanol by gavage every 12 hours for 4 days. The cyproheptadine group received cyprohepta- dine .125 mg/kg in 5% ethanol by gavage every 12 hours for 4 days.

Results: Total lesion size and ulcer size were followed for 10 days. The lesions were then excised, examined microscopically, and ranked by the severity of the histopathology. The groups did not differ significantly with respect to lesion size, ulcer size, or histopathologic ranking.

Conclusion: Given the negative result in this study with ade- quate power to detect meaningful treatment benefits, we cannot recommend hyperbaric oxygen, dapsone, or cyproheptadine in the treatment of Loxosceles envenomation.

[Phillips S, Kohn M, Baker 13, Vander Leest R, Gomez H, McKinney P, McGoldrick J, Brent J: Therapy of brown spider envenomation: A controlled trial of hyperbaric oxygen, dapsone, and cyproheptadine. Ann Emerg Med March 1995;25:363-368.]

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INTRODUCTION

The ulcerative skin lesions resulting from envenomation by the Loxoscdes species of spiders are known as necrotic arachnidism (also gangrenocutaneous arachnidism or lox- oscelism). Despite considerable literature concerning the therapy of koxosceles bites, there have been few well-con- trolled studies.

Hyperbaric oxygen (HBO) has been suggested as a poten- tial therapy, 1-3 both to decrease the potency of the major dermonecrotic factor in koxoscdes venom (sphingomyelinase D) 4 and to promote wound healing by increasing tissue oxy- gen tension, The leukocyte inhibitor dapsone has also been recommended 5,6 because neutrophil infiltration is necessary to lesion development. 7 The potent serotonin antagonist cyproheptadine could also be a potential therapy; Loxoscdes venom stimulates serotonin release from platelets, s This serotonin release may cause platelet aggregation resulting in small vessel plugging and tissue ischemia.

This report describes our randomized, controlled study evaluating HBO, dapsone, and cyproheptadine in the treatment of experimental Loxosceles envenomation in New Zealand white rabbits.

MATERIALS AND METHODS

Laboratory animals exhibit a wide variety of responses to Loxoscdes envenomation. Mice and rats will show systemic toxicity without significant cutaneous lesions. 9 The white

Figure 1. Average lesion area for each treatment group over time.

cm 2

7O

6O

50

40

30

2O I 10

0 -0.5

'\ _ "\

Dapsone

--~- Control

- k - HBO

q Cypmhaptadine

1.~- 2~ 4.'0 5~5 7.;- 8.'5 10.Q Days

Time 0 was 4 hours after envenomation. Repeated-measures ANOVA revealed no statisti- cally significant difference among the groups.

rabbit has been described as the ideal animal model ~° for cutaneous arachnidism because it displays local and sys- temic reactions similar to those of human beings.

Preliminary experimentation revealed that the intrader- mal injection of 20 lag of L deserts venom (Sigma Chemical Co; lyophifized venom reconstituted to 100 lag/ml) created a lesion characteristic of necrotic arachnidism, L deserts venom was used because L reclusa venom was unavailable. Pooled reconstituted venom was refrigerated between uses.

The total lesion area peaked approximately 1.5 to 2.0 days after envenomation. At the times of peak lesion area, we estimated the SD of the lesion size to be 30% of the mean lesion size. Using this estimate, and assuming that a clinically significant decrease in lesion size would be one third, or 33%, we chose a sample size of 12 in each group to give the experiment a power of 80%. (P=.05.)

From August 1991 through January 1992, four shipments of New Zealand white rabbits (Starry Pines Ranch, Inc) were obtained for the study. All rabbits were housed in an animal facility at 22 ° to 2~r°C under a 12-hour fight/dark cycle. They were allowed rabbit chow and water ad libitum. The study was approved by the Animal Use in Research Committee of the Denver General Hospital.

Animals were injected intradermally with 20 lag of venom and randomized into a control group and three treatment groups. Because of the death, before envenoma- tion or randomization, of a rabbit in the fourth shipment, one group (the cyproheptadine treatment group) ultimately comprised 11 rather than 12 rabbits. Animals were ran- domized by number drawing. We noted no significant dif- ferences in the minimum, maximum, and mean weights of the animals in each study group. Analysis of the ship- ment breakdown into treatment groups also revealed no significant differences.

Table 1. Average days to peak total lesion area and peak ulcer size, by group.

Days to Peak Total Days to Peak Group Lesion Area * t Ulcer Size * t

Control 1.5 (.7) 3.9 (2.1) Elapsone 1,5 (.6) 3.4 (3.1) HBO 2.0 (.8) 3.4 (2.6) Cyproheptadine 2.0 (.5) 3.6 (2.6)

*Time g was 4 hours after envenomation. Differences are net statistically significant (two-sample ttest of each treatment group versus the control group). tMean (SD).

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To simulate early recognition of the lesion and maximize the potential effects of the various therapies in all four study groups, we began treatment '~ hours after enveno- marion. The control group received ~ mL of 5% ethanol (control solution) orally (with a l'¢-gauge gavage needle) every 12 hours for eight doses. The HBO group received control solution on the same schedule, plus HBO treat- meats in a special animal chamber (Hyperbaric Oxygen Therapy Systems). The animals were dived to 2.5 ATA for 65 minutes, including diving and surfacing. This was repeated every 12 hours for a total of four treatments. The dapsone group received dapsone, 1.1 mg/kg orally, every 12 hours for eight doses. Finally, the cyproheptadine group received cyproheptadine, . 125 mg/kg orally, every 12 hours for eight doses. Both dapsone and cyprohepta- dine were suspended in 5% ethanol. Limited resources precluded the use of combined therapies.

Total lesion area (induration or erythema) and central ulcer areas were followed for 10 days. The outlines of the lesions were traced on acetate sheets by an investiga- tor who was not blinded to this portion of the study at 12-hour intervals for seven measurements, then every 24 hours for 6 days. The area in the tracing was measured with the computer design program ProDesign II.

After 10 days, the animals were killed with ketamine anesthesia and intracardiac pentobarbital. The lesions were excised and immersed in 10% formalin for preserva- tion. One to three samples of tissue were cut from the longest axis of each skin lesion and from the subcutaneous tract that developed in some specimens. The samples were embedded in paraffin, sectioned to a thickness of 6 t~m, and stained with hematoxylin and eosin. The sections from each animal were examined in random order with- out knowledge of treatment group. The depth of tissue necrosis, distance of adjacent epithelial migration under the eschar, and histologic composition of each lesion were recorded. The lesions were then ranked from least to most severe overall by a blinded investigator.

Table 2. Presence or absence of migration of neeros~s, by group.

Group Present Absent

Control 6 6 Dapsone 7 5 HBO 5 7 Cyproheptadine 8 3 Total 26 21

Fisher'sexacttest:~.54.

Lesion and ulcer sizes over the 10 days after envenoma- tion were compared by means of ANOVA for repeated mea- sures. All groups were compared, and each treatment group was compared individually against the control group. Also, the mean times to peak lesion area and peak ulcer areas for each treatment group were compared with the mean times to peak lesion area and peak ulcer area for the control group by use of the two-sample Student t test. The histopathologic rankings were compared by use of the Kruskal-Wallis test (all four groups) and the Wilcoxon rank- sum test (each treatment group against the control group). Finally, Fisherg exact test was used to compare the groups with regard to the presence ol- absence of ulcer migration.

RESULTS

The experimental lesions showed the typical progression from. induration to erythema, with the formation of cen- tral ulcers. The total lesion area peaked an average of 1.9 days after envenomation, and the ulcer area peaked an average of 3.8 days after envenomation. Figure 1 depicts each group's average total lesion area over time. ANOVA for repeated measures, comparing all four groups, revealed no statistically significant difference. Similarl, repeated measures ANOVA for each group against the control group revealed no statistically significant difference. We noted no trends or tendencies toward decreased total lesion size in any of the three treatment groups relative to the control group.

Figure 2, Average uIcer area for each treatment group over time.

cm 2 4 I ~ Dapsone

Control

-0.5 1.0 2.5 4.0 5.5 7.0

Days

&5 10.0

Time 0 was 4 hours after envenomation. Repeated-measures ANOVA reveals no statistically significant difference among the groups.

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Figure 2 depicts each group~ average ulcer size over time. Again, repeated-measures ANOVA showed no statis- tically significant difference among the groups or between any single treatment group and the control group. We did note a statistically insignificant trend toward larger ulcers in the cyproheptadine group compared with the controls.

Table 1 shows the average time to peak total lesion area and peak ulcer size for each study group. (Time zero was 4 hours after envenomation.) The differences in time to peak lesion or ulcer area were not statistically significant (using the two-sample Student t test to compare each treatment group with the control group).

Microscopically, the epidermis in the central part of the lesions demonstrated coagulation necrosis. The superficial dermis also showed coagulation necrosis and, frequently, hyaline degeneration of collagen. The deeper dermis exhibited edema separating collagen bundles, and infiltra- tion of heterophils (the rabbit equivalent of neutrophils). Adjacent epithelium migrated underneath the areas of coagulation necrosis. Liquefaction necrosis and heterophil accumulation appeared in the subcutaneous tissue under- neath the areas of coagulation necrosis and in the adjacent, dependent subcutaneous tissues. The margins of these areas of liquefaction necrosis showed increased numbers of fibroblasts and macrophages containing eosinophilic, granular material. Platelet plugging of vessels was not a prominent feature. In summary, the lesions were charac- terized by epidermal coagulation necrosis and subcuta- neous liquefaction necrosis with the accumulation of heterophils and macrophages.

The blinded histopathologic specimen rankings revealed no significant differences among groups (Kruskal- Wallis test/% 2 approximation: P=.51). Table 2 shows the microscopic presence or absence of migration of necrosis by group. Differences among groups were minimal and not statistically significant (Fisher's exact test: P=.54). The SDs of the total lesion area of the control group over the study period ranged from 29% to 124% of the mean and peaked on day 1 (Figure 1).

DISCUSSION

This randomized, controlled study failed to show any bene- fit of HBO, dapsone, or cyproheptadine therapy in the treat- ment of animals with experimentally induced cutaneous arachnidism. At the time points where lesion size is greatest (1.0, 1.5, 2.0, 2.5, and 3.0), the SD averaged 35%. Therefore, assuming a clinically significant reduc- tion in lesion size would be one third, or 33%, the power

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of a simple, one-tailed t test comparison of means at these individual time points would be approximately 75%.

However, this applies only to a t test comparison of means at individual time points. This study failed to show signif- icant differences among the study groups on ANOVA for repeated measures, a more appropriate and powerful test in this setting. 11 Moreover, time to peak lesion size was not significantly different between the treatment groups and the control group. Finally, the negative result was consistent between the lesion-area analysis and the histo- pathologic analysis, which included blinded, systematic rankings of the lesions. Especially because of these sys- tematic histopathologic rankings, we feel that this study would have detected any clinically significant benefit to the therapies that were evaluated.

Previous histopathologic studies of cutaneous arachni- dism 12,~3 described early eosinophilic infiltration, platelet thrombi, intravascular thrombosis, and heterophil (neutrophil) infiltration. We did not note infiltration of eosinophils, probably because this is an early phenomenon, and our specimens were taken l0 days after envenoma- lion. Also, we did not consistently see vascular thrombo- sis, but we did note thrombosis in some tissues, and early thrombosis probably caused the superficial coagulation necrosis that we did see consistently.

The major dermonecrotic factor in Loxosceles venom appears to be a sphmgomyelinase D. 4 Whereas phos- pholipases of the A group are present in reptilian ven- oms, phospholipases of the D group are usually found in plants and bacteria.14 The sphingomyelinase D in Loxosceles venom stimulates platelet aggregation and release of serotonin s, but only in the presence of calcium and a plasma glycoprotein called serum amyloid protein. 15,16 Along with platelet aggregation, complement-dependent neutrophil infiltration is essential to the development of necrotic arachnidism, r However, the effect of the sphin- gomyelinase on neutrophils has not been investigated.

In vitro studies 13 suggest that HBO will inactivate sulfhydryl-containing enzymes such as sphingomyelinases by favoring the formation of disulfide bridges between adjacent sulfhydryl groups. Also, HBO increases tissue oxygen tension that supports the production of collagen by fibroblasts, thereby speeding angiogenesis and wound healing. Finally, HBO causes the pulmonary sequestration of neutrophils, thereby reducing the number of circulating neutrophils and the degree of neutrophil infiltration at the lesion site. For these reasons, HBO is a potential therapy for necrotic arachnidism.

Strain et al 2 used the rabbit model of cutaneous arach- nidism to evaluate HBO therapy and reported results sim-

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ilar to ours wiLh regard to lesion size. However, they did note qualitative histopathologic benefit in those rabbits that received HBO twice a day. In contrast, our systematic, bfinded histopathologic rankings showed no statistically significant difference between the HBO group and controls.

Maynor et a113 treated 1'~ consecutive patients with clinically diagnosed brown spider envenomations and reported beneficial effects of HBO. However, the patients received a variety of adjunctive treatments; they were treated at different times after envenomation, and there was no control group. Although this study had the advantage of involving human beings rather than animals, the lack of randomization and controls makes its conclusion in favor of HBO premature.

Because neutrophil infiltration is essential to the devel- opment of necrotic arachnidism, the leukocyte inhibitor dapsone has been suggested as a potential therapy. King and Rees 5 showed that pretreatment of guinea pigs with oral dapsone (1.73 mg/kg) before intradermal injection of partially purified brown recluse spider venom resulted in marked reduction in lesion size compared with that in untreated controls. Our study used the more clinically rel- evant protocol of administering the dapsone after enveno- marion. This model shows that dapsone does not appear to be beneficial.

In an uncontrolled, nonrandomized study involving human patients with clinically diagnosed cutaneous arachnidism, Rees et al ~ reported improved outcome with dapsone and delayed surgical excision as compared with early surgical excision. As with the HBO study by Maynor 13 this study had the advantage of involving human subjects rather than animals. But, again, the lack of randomization and control makes its conclusion in favor of dapsone pre- mature.

We chose cyproheptadine as a potential therapy because of its potent antiserotonergic activity. Serotonin release has been associated with the platelet aggregation and plugging characteristic of cutaneous arachnidism. 8 Our cyprohep- tadine group showed a trend (not statistically significant) toward increased ulcer size relative to the ulcer size of controls. With regard to the other measures of wound severity (lesion size, time to peak lesion size, time to peak ulcer size, histopathologic ranking, and presence of migration of necrosis), the cyproheptadine group did not differ front controls.

Our study has certain limitations. Foremost, it is an animal study The results should not be immediately generalized to human beings. Our treatment doses and regimens were chosen to be similar to those that would be used in clinical practice. However, it is possible that dif-

ferent results would have been obtained at different doses or durations of treatment. The tracing of the lesions by the lead investigator may have introduced bias into the mea- surement of the ulcers. However, the margins of the ulcers were sufficiently sharp that it is unlikely that significant error was introduced into the data collection at this step. The lesions were further evaluated by a blinded patholo- gist and ranked accordingly, thus limiting bias. Our study casts doubt on the efficacy of the tested treatments of brown spider envenomation.

CONCLUSION

On the basis of this animal study, we cannot recommend HBO, dapsone, or cyproheptadine as therapy for necrotic arachnidism. This study is not absolutely definitive; one could argue for another study using different dosing regi- mens, different HBO dive profiles and more than one sys- tem for blinded histopathologic lesion ranking, However, we believe that research efforts would be better spent on evaluating new potential therapies for cutaneous arachni- dism such as antagonism of sphingomyelinase D.

REFERENCES 1. Svendsen F J: Treatment of clinically diagnosed brown recluse spider bites with hyperbaric oxygen: A d inical observation. J Ark Mefl Soc 1986;83:199-204.

2. Strain GM, Snider TG, Tedford BL, et al: Hyperbaric oxygen effects on brown recluse spider (Lexosceles reclusa) envenomation in rabbits. Toxicon 1991;2£:989-998.

3. Maynor ML, Moon RE, Klitzman B, et ah HBO and the effect of brown spider venom in rabbits (abstract}. Undersea Hyperbaric Med I993;20(suppl):45.

4. Rekow MA, Civeilo D J, Geren CR: Enzymatic and hemolytic properties of brown recluse spider (Loxosceles reclusa)texin and extracts of venom apparatus, cephalothorax and abdomen, Toxicon 1983;21:443446.

5. King LE, Rees RS: Dapsone treatment of a brown recluse bite. JAMA 1983;250:648.

6. Bees RS, AItanbern DP, Lynch JB, et al: Brown recluse spider bites: A comparison of early sur- gical excision and dapsene and delayed surgical excision. Ann Surg I985;28:2126-2130.

7. Smith CW, Micks DW: Tbe rote of polymorphonudear leukocytes in the lesion caused by the venom of the brown spider, Lexosceles recluea. Lab Invest 1970;22:90-93.

8. Kurpiewski G, Forrester L J, Barrett JT, et al: Platelet aggregation and sphingomyelinase D activity of a purified toxin from the venom of Loxosce/es recluse. Bieehim Biophys Acta 1981 ;678:467476.

& Morgan PN: Preliminary studies on venom from the brown recluse spider Loxoseeles reclusa. Texicen 1 £69;6:161-165.

10. Jansen GT, Morgan PN, McQueen JN, et ah The brown recluse spider bite: Controlled evalu- ation of treatment using the white rabbit as an animal model. South MedJ 1971 ;64:1194-1202.

11. Rochon J: Sample size calculations for two-group repeated-measures experiments. Biometrics 1991;47:1383-1398.

12. Butz WC: Envenomation by the brown recluse spider and related species: A public health problem in the United States. C/in Toxicof1971;4:515-524.

13. Maynor ML, Abt JL, Osborne PD: Brown recluse spider bites: beneficial effects of hyperbaric oxygen. J Hyperbaric Med 1992;7:89-102.

14. Bernheimer AW, Campbell B J, Forrester L J: Comparative toxinology of Loxosceles recluse and Corynebacterium pseudotuberculosis. Science 1985;228:290.-291

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15. Rees RS, Gates C, Timmons S, et al: Plasma components are required for platelet activation by the toxin of Loxosceles recluse. Toxicon 1988;26:1035-1045.

16. Gates CA, Rees RS: Serum amyloid P component: Its role in platelet activation stimulated by sphingomyelinase D purified from the venom of the brown recluse spider (Loxosceles reclusa). Toxicon 1990;28:1303-1315.

The authors thank Caroline Vosti for assisting with the statistical analysis.

Reprint no. 47/1/62958 Address for reprints:

Scott Phillips, MD, FACP

Toxicology Associates

85467 East Arapahoe Road, J268

Greenwood Village, Colorado 80112-1430

303-762-2960

Fax 303-843-6053

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