Brown Recluse Spider Envenomation: A Prospective Trial of Hyperbaric Oxygen Therapy - RegenQuest
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184 ACADEMIC EMERGENCY MEDICINE MAR 1997 VOL 4/NO 3
Brown Recluse Spider Envenomation: A Prospective
Trial of Hyperbaric Oxygen Therapy
Michael L. Maynor; MD, Richard E. Moon, MD, Bruce Klitzman, PhD, Philip J. Fracica, MD,
Andrew Canada. PhD
I .ABSTRACT
...................................................................................................................................................
Objectives: Loxosceles reclusa (brown recluse) spider bites can produce severe skin lesions that may neces-
sitate extensive surgical repair. This study delineated the effects of hyperbaric oxygen (HBO) therapy on these
lesions by performing a prospective controlled animal study.
Methods: After approval by the Institutional Animal Care and Use Committee, 41 New Zealand white rabbits
received 64 intradermal injections of 73 p L of raw venom extract mixed with physiologic buffered saline
(Dulbecco’s solution). Control injections were made with buffer. The animals were divided into 5 groups: 1)
venom and no HBO; 2) venom and 1 immediate HBO treatment (100% 0,);3) venom and immediate HBO
with 10 treatments (100% 02); 4) venom and then delayed (48 hr) HBO therapy with 10 treatments (100%
0,); and 5 ) venom and immediate hyperbaric treatment with normal inspired PO, for 10 treatments (8.4%
0,). Three animals in group 2 also received a control sodium citrate buffer injection. HBO treatments were
at 2.5 atm absolute (ATA) for 90 minutes twice daily. Daily measurements were made of the lesion diameter,
and skin blood flow using a laser Doppler probe.
Results: There was no significant effect of HBO on blood flow at the wound center or 1-2 cm from the
wound center. Standard HBO significantly decreased wound diameter at 10 days (p < 0.0001; ANOVA),
whereas hyperbaric treatment with normoxic gas had no effect. Histologic preparations from 2 animals in
each group revealed that there were more polymorphonuclear leukocytes in the dermis of all the HBO-treated
animals when compared with the venom-alone and sodium-citrate controls.
Conclusion: HBO treatment within 48 hours of a simulated bite from L. reclusa reduces skin necrosis and
results in a significantly smaller wound in this model. The mechanism appears unrelated to augmented local
blood flow between treatments.
Key words: hyperbaric oxygen therapy; HBO; brown recluse; spider bite; envenomation; wound healing.
Acad. Emerg. Med. 1997; 4:184-192.
I The venom of Loxosceles reclusa causes a lesion that dose, the location of the bite, and immunity conferred by
is usually painless but can progress to become an enlarg- previous bites.2 A large number of bite victims do not seek
ing blue macule, then a zone of aseptic necrosis, leading medical attention, at least initially, because the initial bite
to gangrenous slough and a subsequent large stellate ul- is often painless with minor urticaria and erythema. More
ceration. Envenomation leads to a wide variety of clinical serious envenomations progress 8-24 hours later to de-
responses, ranging from no sensation of the initial bite to velop local induration, erythema, pruritus, and pain.3 5 p -
pruritus with a small (5-mm) erythematous papule, to se- ically, serious lesions develop a blanching ring that in-
vere dermatonecrosis, to systemic loxoscelism, and even creases in diameter, forming a central pustule and
death.’ This broad spectrum of bite manifestation has been becoming an enlarging blue macule that usually sinks be-
attributed to variable inconsistencies of injected venom low the level of the surrounding skin over 1-3 days.3 The
.................................................... surrounding erythema enlarges and becomes characteris-
tically violaceous, and is encircled by a ring of pallor that
From Duke University Medical Center; Durham, NC, Department of
is bordered by normal skin. This local reaction has been
Anesthesiology (MLh4, REM, AC), Department of Plastic Surgery
IBK), and Department of Pulmonary Medicine (PJF). described as a “halo effect.”’
The lesion and any edema spread by continuity, de-
Received: December I I , 1995; revision received: June 12, 1996; ac-
cepted: June 16, 1996; updated: August 8,1996. pending on gravity to involve a whole limb or a large part
of the trunk.’ The blue macule becomes a central zone of
Prior presentation: SAEM annual meeting, San Francisco, CA, May
1993, and the Undersea and Hyperbaric Medical Society annual meet- aseptic necrosis, turning black and irregular in 3-4 days.
ing, Nova Scoria. Canada, July 1993. In another 4-7 days later there may be gangrenous slough
Address for correspondence and reprints: Michael Maynor. MD, 1901
with eschar formation that results in induration. Around
Clifford Street, Unit 103 Sunset Vista, Ft. Myers. FL 33901. e-mail: 7- 14 days, the central necrotic area becomes mummified,
mmaynorl O3@aol.com allowing the eschar to separate from the edematous bor-HBO for Loxosceles Envenomation, Maynor er al. 185
dering viable skin. The eschar falls off, exposing fatty was dissected and homogenized in 20 mmol TRIS*-
necrosis 1-3 cm deep and eventually leaving a stellate buffered saline (pH 7.4). Centrifugation removed all in-
ulceration ranging 1-30 cm in diameter.4 Healing can re- soluble material. The venom protein extract was mixed
quire 1-6 months by means of epithelialization, granu- with 25 mmol sodium citrate, at pH 5, to be placed in 10
lation, and contracture. Secondary skin grafting or plastic different miniature vials to achieve individual concentra-
surgery may be r e q ~ i r e d . ~ tions of 0.21 pg/mL and a total shipment amount of 1.4
The therapy for these bites is based on few controlled mg. This was shipped to Duke University Medical Center
human or animal studies. Recent case series evidence in a Styrofoam container on dry ice. On arrival the venom
shows that hyperbaric oxygen (HBO) therapy may be ef- was stored at -70 “C until it was used.
ficacious in managing the dermatonecrosis associated with To determine the proper dosage of venom necessary
L. reclusa. Svendsen reported accelerated healing in 6 pa- to produce a 2-cm lesion, 1 test animal was injected in-
tients with at least class I11 or IV envenomations’ up to tradermally 3 times over a shaved area of the back using
2-6 days post bite with a twice daily regimen of HBO at 5, 10, and 20 p g of venom. A 20-pg injection resulted in
2.0 atm absolute (ATA) for 60-90 minutes for 3 days in a 2-cm diameter lesion within 3 days, whereas the lower
a Sechrist monoplace chamber. Kendall and Caniglia re- amounts caused insignificant lesions.
ported similar results in 47 cases with an average of 5.6 Thereafter prior to each study, the animal was shaved
treatments. Both reported no third-degree skin slough, over both flanks to 6 cm from the midline 24 hours in
scarring, or need for surgery, grafting, or hospitalization.6 advance to allow for any resulting changes in blood flow
Maynor et al. electively treated 14 adult patients out of to subside. No animal instrumentation or sedation was
40 who had suspicious lesions at 2-2.5 ATA for 90 used. On the day of injection, the venom protein extract
minutes twice a day for an average of 7 treatments. All was thawed and kept on ice until used. To prepare a single
the patients healed without scarring, disability, or the need injection, 73 p L of raw extract was mixed with 73 pL of
for skin grafting.’ Strain et al. performed the first con- double-strength (2X) Dulbecco’s physiologically buffered
trolled animal study and found significantly increased his- saline (PBS) and then with an additional 54 p L of normal-
tologic healing with twice-a-day HBO therapy (2 ATA for strength (1X) Dulbecco’s PBS to make a total amount of
2 hr) compared with control.* However, in another rabbit 200 pL. The PBS (1X) had the following molar concen-
study, Phillips et al. compared HBO, dapsone, cyprohep- trations: CaC1, 0.9 mmol; KC1 2.68 mmol; KH,PO, 1.47
tadine, and control; they found no lesion size or histo- mmol; MgSO, 0.491 mmol; NaCl 136.7 mmol; Na,HPO,
pathologic benefit with any of these therapies.’ 15.22 mmol. For 2X PBS, the concentrations are doubled.
We sought to further delineate the effect of HBO on This amount was injected intradermally within the shaved
wound healing after L. reclusa envenomation in a con- area with a I-mL tuberculin syringe using a 26-gauge nee-
trolled rabbit model primarily by assessment of wound dle.
size, in vivo blood flow, and wound histopathology. One untreated control animal did not receive an injec-
tion (i.e., no venom or sodium citrate injection) or HBO
I ...................................
METHODS treatment. This animal was sacrificed for the myeloperox-
idase portion of the study. The remaining animals received
Study Design: We used an evaluator-unblinded, con- intradermal injections; 25 received only 1 venom injection
trolled rabbit model of L. reclusa envenomation to eval- and 16 received 2 venom injections. Duplicate injections
uate the effect of immediate and delayed HBO therapy on were assigned randomly. All injections were made at least
experimental wound size, blood flow, histopathology, 7 cm apart. The rabbits were randomized to the following
venom sphingomyelinase activity, tissue polymorphonu- 5 treatment groups:
clear leukocyte (PML) count and tissue lipid peroxidation
up to 10 days after injury. 1. L. reclusa venom and n o HBO treatment [n = 15; 6
animals with 2 venom injections];
Animal Subjects: Adult New Zealand white rabbits 2. Venom and 1 immediate HBO treatment at 2.5 ATA
from the Duke vivarium were caged individually in an (100% 0,) [n = 6; 3 animals with 2 venom injections];
environmentally controlled room under a 12: 12 lighting
schedule with food and water as needed. The animals 3. Venom and immediate HBO for 10 treatments at 2.5
weighed on average 2.5 kg and were at least 12 months ATA (100% 0,) [n = 9; 2 animals with 2 venom in-
old. Prior approval for the study was obtained from the jections];
Duke University Animal Care and Use Committee.
4. Venom and then delayed HBO (48 hr) for 10 treatments
.............................................................................
Experimental Protocol: Captured spiders were frozen
at the University of Arkansas and the venom apparatus *TRIS = rris(hydroxymethy1 )aminomethane.186 ACADEMIC EMERGENCY MEDICINE MAR 1997 VOL 4/NO 3
-VenCont r
A D D e l H 8 3
L. 5-7
--lrnHBOlO
A l m H B O l
-*-NaCitCon
-8 %02Con
Time in Days
I FIGURE 1. Wound diameter vs treatment. Abbreviations are as follows: VenContr: venom control, no hyperbaric oxygen (HBO) treatment;
DelHBO: 10 HBO treatments beginning 48 hours after venom injection; ImHBO 10: 10 HBO treatments beginning immediately after injection;
ImHBOl: a single HBO treatment immediately after injection; NaCitCon: sodium citrate control injection; 8%O,Con: 10 hyperbaric treatments
while breathing 8.4% Oz to provide a normoxic inspired 0, tension. Wound diameter vaned significantly with time (p < 0.0001). Wounds treated
with ImHBOl, ImHBO10, or DelHBO were significantly smaller (p < 0.0001). Wound diameters in the 8%0,Con group were not significantly
different from those in the untreated animals.
at 2.5 ATA (100% 0,) [n = 8; 4 animals with 2 venom Gross Wound Size: Daily photographs were made
injections]; and documenting the gross appearance for 10 days. The di-
ameter of the wounds for all groups were measured daily
5. Venom and immediate hyperbaric treatment for 10 for 10 days using a centimeter ruler according to previ-
treatments at 2.5 ATA (8.4% 0,) [n = 3 ; all 3 animals ously standardized Auer-Hershey wound classifications."
with 2 separate venom injections]. We measured the black necrotic lesions or eschar in each
animal. As most of the wounds were of roughly circular
In addition, 3 animals in group 2 above also received a shape, diameter measurement was adequate to assess
harmless sodium citrate control injection. These injection wound size, following the precedent of a previous report."
sites were monitored as a control during the flow studies. If wounds were irregular in shape, the maximum diameter
All HBO treatments lasted 90 minutes and were ad- was used.
ministered twice daily. The treatments were given in an Wound Blood Flow: Laser Doppler Vasomedic
animal research chamber, with the rabbits enclosed in a BPM-2 (St. Paul, MN) with P403 right-angle probe flow
94 X 94-cm inflatable plastic bag (Instruments for Re- measurements were made to evaluate superficial skin
search and Industry, Cheltenham, PA) especially designed blood flow. These measurements were taken at the center
for animals, connected to a single pass-flow system of each lesion as well as 1 and 2 cm from the center in
through which gas was delivered at 100 L/min. An outside 4 quadrants. The 1- and 2-cm readings were 4 eachnesion
observer carefully watched the bag size on every com- and the mean was taken for each group.
pression to treatment pressure to prevent it from collaps- Histological Examination: After euthanizing 1 ani-
ing around the rabbits. All the groups received 100% 0, maVgroup for groups 1-4 at 3 days post venom injection,
in the flow system except those who received a premixed and 2 animaldgroup for groups 1-4 at 10 days post
gas containing 8.4% 0, in N, (group 5), to create an ef- venom injection, skin was obtained from the center, and
fective treatment Po, equal to that of room air at 1 ATA 1 and 2 cm from the center, yielding 36 gross tissue sam-
(i.e., 160 torr). Gas within the bag was continuously sam- ples. These 36 samples were divided to equally represent
pled via a through-hull penetrator to maintain the Po, treatment groups 1-4, at 3- and 10-days postinjection,
within 1% of the target concentration. Both 0, and CO, with half being placed in glutaraldehyde preservative and
were monitored using calibrated analyzers to ensure that the other half frozen at -70°C. Subsequently, Wright stain
the ambient O2and CO, concentrations were >99 torr and slides were then made 100 F m apart using the 3- and 10-
~ 0 . 5 %(Pco, 3-8 torr), respectively, for all groups except day gross tissue samples placed in glutaraldehyde. In ad-
1 and 5. dition, the animals euthanized at 3 days post injection
were not used in the daily wound size and blood flow
Measurements: The induced lesions were subsequently measurement arms of the study because they could not be
evaluated with the following methods without blinding of followed for the whole 10 days.
the investigator. Myeloperoxidase Assay: To assess the tissue PMLHBO for Loxosceles Envenomation, Maynor et al. 187
40 T
1 -venContr
R -DelHBO
-~IrnHBO10
A ImHBO1
-A- NaCitCon
-8 %02Con
I
Time in Days
I FIGURE 2. Blood flow at the center of the wound as measured by laser Doppler. Abbreviations as for Figure 1 . The buffer control group had
higher flow rates (p < 0.0001).
content, myeloperoxidase assays were performed on the margin) from the 10-day samples to yield 8 specimens
frozen skin (wound margin) of the 3-day (1 animavgroup; (i.e., 1 sample/animal; 2 animals/group; groups 1-4 sam-
2 specimensfanimal) and 10-day ( 2 animals/group; 2 spec- pled). These were homogenized with phosphate buffer
imendanimal) samples from groups 1-4 to yield 12 spec- mixed with NaOH. Then HCI, acetonitrile, and vortex
imen pairs."-13 The specimens were homogenized using were added to precipitate proteins. The centrifuged su-
a Kinematica Gmblt CH-6010 (Luzern, Switzerland) in pernatant was subsequently mixed with 2,4 dinitrophen-
phosphate buffer and then put in a Branson #250 op- ylhydrazine. Samples of total and free MDA were assayed
sonifier (Shelton, CT) for 5 times at 5 minutes each. The using high-performance liquid chr~matography.'~.''
samples received 1% Hetab, and were frozen and thawed Sphingomyelinase Assay: To investigate the possi-
with liquid N2 followed by sonication. The centrifuged bility that HBO might have an inactivating effect on the
supernatants were assayed using phosphate buffer, o-di- toxic component of L. reclusa venom, normal rabbit skin
anisidine, and hydrogen peroxide. The liquid samples was homogenized, then mixed with venom and exposed
were placed in a Beckman Du-64 spectrophotometer (DU to HBO. One New Zealand white rabbit was anesthetized
Beckman Instruments, Wilmington, DE). The resulting with ketamine/diazepam and then an area of skin (1.5 g)
data were expressed as a change in absorbanceiminlmg was excised. This was homogenized in a Kinematica-
protein, as 1 unit of myeloperoxidase activity equals that Gmblt CH-6010 homogenizer and put in a Branson op-
degrading 1 kmol of pero~ide/rnin."*'~.'~ sonifier and then mixed with 8 mL PBS to facilitate dis-
Malondialdehyde (MDA) Assay: To determine solution. This produced 8 mL homogenized skin + PBS,
whether HBO had an effect on the amount of lipid per- of which 6 mL subsequently was mixed with 900 pL of
oxidation induced by L. reclusa venom, MDA assays were raw venom. Two mL of this skin-PBS was kept on ice.
also performed on the frozen skin (1 cm from the wound The other 6 mL was divided to produce 300 pL venom/
30 T
-Del HBO
I I
Time in Days
I FIGURE 3. Blood Row 2 cm from the center of the wound as measured by laser Doppler. Abbreviations as for Figure 1. There was no
significant effect of treatment group on blood flow 2 cm from the center of the wound.~~
188 ACADEMIC EMERGENCY MEDICINE MAR 1997 VOL 4/NO 3
The remaining homogenized specimens from each
2-mL group were used in an assay for sphingomyelin-
ase-D. The assay was performed on the previously men-
tioned groups in addition to regular control venom and
venom treated with 2.5 ATA. Each sample was then prein-
cubated with HEPESt and CaCl,. Then 14C-sphingomye-
lin in choline (Sigma, St. Louis, MO) was mixed with
each preincubation sample. The reaction was terminated
with the addition of chloroform, KCl, and methyl chlo-
ride. The samples were then centrifuged and 500-pLali-
I FIGURE 4. Low-power section of the wound of an untreated animal
on day 3. The wound crater is evident with attached eschar. quots of each sample’s supernatants were used to obtain
counts in the gamma spectrometer. Sphingomyelinase-D
is cleaved by endogenous phospholipase-D to produce
2 mL for skin-PBS + venom and 600 pL v e n o d 4 mL free choline measured in counts per minute (cpm).’8-20
skin-PBS + venom + HBO. The 4 mL of skin-PBS +
venom + HBO was split evenly into 2-mL groups and
placed under ice in a Bethlehem experimental treatment Data Analysis: Statistical evaluation was performed on
chamber (SA-1) (0.1696 m3) and the first group was ex- wound diameters and blood flow measurements using re-
posed to 100% 0, at 4 ATA and the second group at 2.5 peated-measures ANOVA. Post-hoc comparisons were
ATA, both for 90 minutes. Two rabbits were shaved, and made using the Bonferroni -Dunn adjustment for multiple
each was given 3 injections in small increments out of the comparisons. In the evaluation of the myeloperoxidase,
2-mL groups: 1) homogenized skin, 2) homogenized skin MDA, and sphingomyelinase data, the sample sizes were
+ venom, 3) homogenized skin + venom + HBO at 4.0 too small for statistical comparisons; descriptive statistics
ATA. are reported.
I . .RESULTS
..........................................................................
Survival: Two animals (from groups 1 and 4) died 2
days post venom injection and were subsequently ex-
cluded from the study (i.e., not used for data collection).
It was presumed that they had succumbed to systemic
loxoscelism because they both received 2 injections each.
Gross Wound Appearance: The HBO-treated animals
had significantly smaller wounds than did the control an-
imals (p < 0.0001). The animals treated with 8.4% O2 at
2.5 ATA had wounds that were not different from those
in the untreated animals. However, both delayed and im-
mediate HBO treatment regimens, which produced supra-
normal 0, levels, dramatically decreased the size of L.
reclusa lesions (Fig. 1). All lesions had some evidence of
necrosis, ranging from control animals with complete
black eschars to HBO-treated animals with small lesions
with red granulation bases.$
Wound Blood Flow: However, there was no significant
effect of HBO on wound blood flow as measured by laser
Doppler at the center and 2 cm from the center (Figs. 2
and 3, respectively).
..................................................................................
7HEPE.S = N-2-hydroxyerhylpiperazine-N-2-erhanesulfonic
[acid].
I FIGURE 5. Same section as Fig. 4 at higher power. The junction
between the eschar and the wound crater can be seen. There is moderate $The authors will provide representative photographs of the wounds to
leukocyte infiltration in the subeschar tissue. interested readers on request.HBO for Loxosceles Envenomation, Maynor el al. 189
Histology: We also looked at necrosis in the 3-day post-
injection histologic sections showing increased numbers
of PMLs in those animals treated with HBO compared
with the untreated control animals (Figs. 4-7). For ex-
ample, the delayed HBO-treated wound centers showed
more consistent dense accumulations of PMLs (pannicu-
litis) in the dermis and fascia1 layers than those in any of
the control lesions. The control lesions had a larger, more
complete central eschar or necrosis, even to the exclusion
of all fatty layers in addition to necrotic myocytes. The
HBO-treated groups had more underlying deep granula-
I FIGURE 6. A wound 3 days after venom injection in an animal
tion tissue evidenced by new fibroblasts than did the con- administered 10 hyperbaric oxygen (HBO) treatments beginning im-
trol group. Sections of lung tissue showed no difference mediately after injection of venom. The wound crater is smaller than
in the overall number of PMLs between the controls and that in Fig. 4. A dark area can be seen adjacent to the eschar, which
all HBO-treated animals. consists of a layer of polymorphonuclear leukocytes (see Fig. 7).
Myeloperoxidase: Wound margin samples were ob-
tained from the animals in all groups except those receiv- These preliminary results do not suggest major differences
ing 8.4% O2 during HBO therapy (group 5). Duplicate in MDA content at 10 days.
assays from each animal were compared for assay consis-
tency. The duplicate assays were within 20% of each Sphingomyelinase: The results of the preliminary ho-
other. The mean results at 3 days post “bite” (units = mogenized skin studies initially show that the HBO
change in absorbance/min/mg protein = 1 U myeloperox-
idase activity) for these groups were as follows:
Normal skin: 1.24
Venom-no HBO: 1.31
Venom-HBO (1 immediate): 1.98
Venom- HBO (10 immediate): 2.45
Venom-HBO (10 delayed): 0.76
These results support the observation in this study and by
other authors of increased PMLs on histologic specimens
in animals that were immediately treated with HB0.10-’4
However, the small sample size prohibits definitive state-
ments.
The mean results at 10 days post “bite” were as fol-
lows:
Venom-no HBO: 0.189
Venom-HBO (1 immediate): 0.097
Venom-HBO (10 delayed): 0.176
MDA: Single wound margin samples were obtained
from the animals in all groups except those receiving
8.4% 0, during HBO therapy (group 5). The mean results
at 10 days post “bite” (units = nmoles/mg protein) for
these groups were as follows:
Buffer-HBO (1 immediate): 0.60
Venom-no HBO: 0.43
Venom-HBO (1 immediate): 0.33 I FIGURE 7. Same section as Fig. 6 at higher power. The layer of
Venom-HBO (10 immediate): 0.70 inflammatory cells is more evident. This area is thicker than in the control
Venom-HBO (10 delayed): 0.47 animal, suggesting that HBO enhances the inflammatory response.~~~~ ~
190 ACADEMIC EMERGENCY MEDICINE MAR 1997 VOL 4/NO 3
treated specimens had smaller lesions than did the venom misses the surge of PMLs 1 hour post bite, and it can
+ skin control specimens. At days 2 , 4 , and 6, the wound cause seizures and hemolytic anemia.' The most promis-
diameters in the animals injected with skin and venom ing therapy, intralesional antivenin, inhibits the activity of
were 6.5, 10, and 12 cm, respectively. In the animals in- sphingomyelinase-D.34The obvious disadvantage is that it
jected with skin and venom pretreated with 4 ATA (HBO) is effective only if given in the first 24-48 hours post
the diameters were smaller: 1, 1.5, and 2 cm, respectively. bite; however, because these bites can appear initially in-
Homogenized skin alone produced no lesion. There was nocuous, most patients are seen much later. Presently, an-
no demonstration of decreased sphingomyelinase-D levels tivenin is not commercially available.33For those patients
in our study samples. bitten by the brown recluse spider who go on to form
necrotic ulcers, HBO administration may offer a safe and
I DISCUSSION effective mode of therapy. The most common side effects
of HBO administration include ear, sinus, and pulmonary
Microdissection of venom sacs of L. reclusa has demon- barotrauma, as well as the benign 0, toxicity seizure.35
strated alkaline phosphatase, hyaluronidase, protease, es- Possible mechanisms for a beneficial effect of HBO in
terase, and c01lagenase.~'-~~ Most of these enzymes pro- the healing of the bites of L. reclusa include the seques-
mote the swift diffusion of venom through tissues; tration of PMLs away from the major wound the
however, the dennonecrotic component of L. reclusa inactivation of sphingomyelinase or venom,7 the direct
venom is sphingomyelinase-D, a complex phospholipase inhibition of PML adherence at the cellular-endothelial
of 32,000 da that causes a severe cytotoxic reaction in interface and subsequent decreased effects of any venom-
viwo.21,2425 induced ischemia-reperfusion injury,"-39 and the increas-
Sphingomyelinase-D attaches to, structurally alters, ing O2 delivery to ischemic ti~sue.~'.~'
and degrades sphingomyelin on human red blood and Our preliminary observations using homogenized skin
other cellular membranes in the presence of calcium, lead- + venom pretreated with HBO might suggest that the
ing to cell lysis, hemolysis, and death.'8.2' This leads to HBO mechanism in this injury could be some inactivation
the release of inflammatory mediators such as arachidonic of a venom component. However, we could show no dif-
acid and prostaglandins, followed by platelet aggregation ference in the levels of sphingomyelinase-D between the
and serotonin release, resulting in microvascular throm- HBO-treated and control groups ( N = 2). This could be
bosis and i s ~ h e m i a . ' . ' ' ~ ~Most
. ~ ~ important, there is a secondary to the type of assay chosen and to the presence
significant chemotactic infiltration of PMLs, leading to in- in the venom of sphingomyelinase-C, which can compete
travascular clotting, degeneration of vessel walls, hemor- with sphingomyelinase-D." We also cannot exclude the
rhage into the dermis, and liquefaction and abscess for- possibility that the mechanical action of the bubbles or
mation in 3-5 '' Finally, there is amplification of the environment of the chamber also might have had an
the initial inflammatory reaction by the intrinsic vascular inactivating effect on the venom.
cascade that is dependent on the mediators C-reactive pro- The pathophysiologies of ischemic perfusion injury
tein and amyloid P component, as well as complement and L. reclusa bites share the increased adherence of
inhibition in producing the local and systemic reactions PMLs to microvascular endothelium and subsequent base-
of necrotic arachnidi~rn.~~.~' In contrast, snake and bee ment membrane penetration and interstitial tissue penetra-
venoms produce a lesser necrotic amplification reaction in tion. These PMLs produce reactive 0, metabolites, i.e.,
spite of much higher levels of hyaluronidase and phos- hydroxyl, superoxide, and peroxidase, as well as myelo-
pholipase A Z 4 peroxidase, an enzyme that increases the production of
There continues to be controversy regarding the de- hypochlorous acid and N-chloroamines, known cytotoxic
finitive therapy for cutaneous Ioxoscelism. Few con- oxidants. 14*15.42
trolled human studies have demonstrated that any one Zamboni et al. recently found that HBO provided a
therapeutic modality decreases the chance of dermatone- protective rather than an exacerbation effect for reperfu-
c r o ~ i s .Conservative
~ measures include local cleansing, sion injury, by decreasing venular PML adherence, mye-
sterile dressings, cryotherapy, elevation, and administra- loperoxidase, and arteriolar vasoc~nstriction.~~ Thom and
tion of antipruritic, analgesic, and sedative medi~ation.~'Elbuken recently observed in animal models of carbon
htralesional, oral, and systemic steroids do not augment monoxide (CO) poisoning that if HBO was given post C O
wound healing because of vasoconstriction and increased exposure, there was a dramatic decrease in B2 integrins
intralesional p r e s s u ~ e . Early
~ ~ . ~ wide
~ excision is expen- (CD11- 18) and therefore PML endothelial binding, reduc-
sive, disabling, and disfiguring; furthermore, the extent of ing the effects of ischemia-reperfusion i n j ~ r y . ~The ' . ~ ~ac-
venom distribution margins is difficult to Dap- cumulation of PMLs within the intravascular and interstitial
sone, traditionally used for leprosy therapy, has been used tissue of L. reclusa bites may be vitally dependent on vas-
with some success in treating these dermatonecrotic le- cular endothelial adherence and could explain the ischemic
s i o n ~ However,
.~~ the administration of dapsone usually injury pattern responsible for the ultimate necrosis.HBO for Loxosceles Envenomation, Maynor et al. 191
We observed more preliminary PML activity in the (diameter) were based on previously recognized L. reclusa
HBO-treated animals when comparing histologic and wound protocols set by Auer and Hershey.” As our le-
myeloperoxidase assays of our wound tissue sections. sions were uniformly circular, no difference in results
This was noted by the more consistent dense accurnula- would have occurred comparing total wound surface ar-
tions of PMLs (panniculitis) in the dermal and fascia1 lay- eas. The measurement method for blood flow using laser
ers. This seems paradoxical, in light of inhibition of PML Doppler readings proved to be limited as it produced day-
binding in other models by HB037-39and that sphingo- to-day biologic variability secondary to motion artifact.
myelinase-D causes a massive influx of PMLs into the The study was also limited because the observer was not
wound area in venom control animals. This could be ex- blinded to solution used during lesion induction at the
plained by our limited number of study samples for each time of wound measurement.
treatment group ( N = 2 ) , and warrants future prospective This study provides important prospective data related
large-sample animal studies. to the effect of HBO alone in L. reclusa bites. This study
Hyperbaric oxygen could minimize wound size in the opens many intriguing possibilities for future studies.
bites of L. reclusa by 3 possible mechanisms. First, the Does HBO affect the inactivation on the toxic venom
increased PO? in wound extracellular fluid may prevent component sphingomyelinase? What role does HBO play
ischemic necrosis that might otherwise occur in the pen- in the manipulation of PML function, and how does that
umbra of the envenomated area by extending the usual 0, affect the healing of L. reclusa bites? Perhaps a future
gradient from 30-80 p m away from the vessel walls.4o study using much larger sample sizes would compare
This increased O2 tension also could enhance capillary HBO and a PML monoclonal antibody or other PML in-
endothelial cell (angiogenesis) into new wounds. Wound hibitor to assess whether any difference in lesion sizes
margin fibroblasts require a tissue Po2 of 40-55 torr to occurs.
replicate and produce a collagen matrix strong enough to
support angiogenesis and extension into the w o ~ n d . ~ ~ I - ~ .’.CONCLUSIONS
.......................................
Fibroblasts are also attracted to new wound centers by
mitogen-releasing macrophages. This supports the use of In this study, there was a positive effect of HBO on re-
intermittent HBO with normobaric intervals. It also has ducing the size of lesions caused by L. reclusa venom.
been shown that PMLs have bursts of 0, consumption HBO was effective not only when given immediately in
after phagocytizing bacteria. The provision of extra 0, via varying doses, but also when it was delayed by 2 days.
HBO in a normally hypoxic wound could facilitate phag- When given immediately after induced L. reclusa bites, a
ocytic killing of microorganism^.^^ single HBO treatment was as effective as multiple HBO
treatments. These beneficial effects were not simply due
to the effect of hyperbaric treatment alone. Since using an
I . .LIMITATIONS AND FUTURE QUESTIONS
................................................................. inspired PO, of 160 torr in a hyperbaric environment (2.5
ATA) did not result in any effect on wound size, the mech-
The rabbit model that we used was an excellent one in anism appears to be due to the effects of hyperoxia at
which to study the effects of HBO on the lesions of L. increased pressure, rather than any nonspecific effect of
reclusa. To our knowledge, no controlled human study has hyperbaric normoxic exposure.
addressed HBO therapy for this injury; however, several There was no indication that blood flow in the vicinity
case reports have supported the use of HBO? Our ani- of the wound was altered as a result of HBO therapy.
mal lesions were similar to those of humans as supported Also, PML accumulation in these induced lesions seems
by our pathophysiology review. Furthermore, previous au- to be enhanced by immediate HBO administration. Our
thors have found rabbit skin, though not identical, to be animal model results support the use of HBO in the ther-
a close approximation to human skin regarding dermal apy of the bites of L. reclusa. For those individuals who
thickness, underlying layers, and t e ~ t u r e . ~ ~ ~ ~ ~either ~ ~ ~ see the L. reclusa spider during the bite or develop
The effect on ultimate healing (>lo days) was not de- a typical 2-2-cm diameter lesion 2 days after being in a
termined in this study because all rabbits were killed after place consistent with the spider’s habitat, we recommend
10 days according to our animal committee protocol. the initiation of HBO therapy with at least 1 treatment.
However, our observations of the successful outcome of Whether a single HBO treatment is as effective as mul-
the lesions in the first 3 - 10 days support the use of HBO tiple treatments for L. reclusa bites in humans remains to
in the therapy of the bites of L. reclusa. The sample sizes be proven.
of the myeloperoxidase, MDA, and sphingomyelinase as-
says were inadequate to draw definitive conclusions.
The authors express appreciation to the following individuals: Collis
However, these preliminary findings should help guide fu- Geren, PhD, University of Arkansas, Little Rock, for his generous do-
ture studies with larger sample sizes. nation of brown recluse venom; the diving technicians, nurses, and other
The measurement methods used to assess lesion size personnel of the F. G. Hall Hyperbaric Laboratory at Duke University192 ACADEMIC EMERGENCY MEDICINE MAR 1997 VOL 41NO 3
Medical Center for their tireless contributions in the completion of the D. Arch Biochem Biophys. 1978; 187:355-65.
study; the Department of Dermatology at Duke University Medical 22. Geren CR, Chan TK, Ward BC, Howell DE, Pinkston K, Odell GV.
Center for their help with histology interpretation; and Mrs. Carmen Composition and properties of extract of fiddleback spider venom ap-
Hurst for the preparation of the manuscript. paratus (L. reclusa). Toxicon. 1973; 11:471-9.
23. Heitz JR, Norment BR. Characteristics of an alkaline phosphatase
activity in brown recluse venom. Toxicon. 1974; 12:181-7.
HREFERENCES 24. Geren CR, Chan TK, Howell DF. Odell GV. Partial characterization
of the low molecular weight fractions of the extract of the venom ap-
1. Wong RC, Hughes SE, Voorhees JJ. Spider bites: review in depth. paratus of the brown recluse spider and of its hemolymph. Toxicon.
Arch Dermatol. 1987; 123:98- 103. 1975; 13:233-8.
2. Edwards JJ, Anderson RL. Loxoscelism of the eyelid. Arch Ophthal- 25. Kurpiewski G, Forrester W,Barrett JT, Campbell BJ. Platelet ag-
mol. 1980; 98:1997-2000. gregation and sphingomyelinase-D activity of a purified toxin from the
3. Binder LS. Acute arthropod envenomation: incidence, clinical fea- venom of Loxoscetes reclusa. Biochem Biophys Acta. 1981; 678:467 -
tures and management. Med Toxicol Adverse Drug Exp. 1989; 4:163- 76.
73. 26. Rees RD, Gates C, Timmons S, Des Prez RM, King LE. Plasma
4. Hobbs GD, Jarrell RE. Brown recluse spider bites: a common cause components are required for platelet activation by the toxin of Loxos-
of necrotic arachnidism. Am J Emerg Med. 1989; 7:309-12. celes reclusa. Toxicon. 1988; 11:1035-45.
5. Svendsen FJ. Treatment of clinically diagnosed brown recluse spider 27. Smith CW, Micks DW. The rate of polymorphonuclear leukocytes
bites with hyperbaric oxygen; a clinical observation. J Ark Med SOC. in the lesion caused by the venom of the brown spider, Loxosceles
1986; 83~199-204. reclusa. Lab Invest. 1970; 22~90-3.
6. Kendall TE, Caniglia RJ. Hyperbaric oxygen in the treatment of 28. Geren CR, Rekow MA, Beasley JN, Jones JL. The mechanisms of
clinically diagnosed brown recluse spider bites: a review of 48 cases action of the mammalian toxin isolated from Laxosceles reclusa venom
[abstract]. Undersea Biomed Res. 1989; 16:21. [abstract]. Toxicon. 1988; 23:567.
7. Maynor ML, Abt JL, Osbome P. Brown recluse spider bites: bene- 29. Rees RS, Gates C. Platelet activation stimulated by the toxin of the
ficial effects of hyperbaric oxygen. J Hyperbar Med. 1992; 7:89- 102. brown recluse spider requires serum amyloid P component, not C-re-
8. Strain GM, Snider TG, Tedford BL, Cohn GH. Hyperbaric oxygen active protein. Toxicon. 1989; 27:953-4.
effects on brown recluse spider envenomation in rabbits. Toxicon. 1991; 30. Hufford DC, Morgan PN. C-reactive protein as a mediator in the
29: 989-96. lysis of human erythrocytes sensitized by brown recluse spider venom.
9. Phillips S, Kohn M, Baker D, et al. Therapy of brown spider en- Proc SOCExp Biol Med. 1981; 167:493-7.
venomation: a controlled trial of hyperbaric oxygen, dapsone, and cy- 31. Rees RS, King LE. Treatment of brown recluse spider bites. J Am
proheptadine. Ann Emerg Med. 1995; 25:363-8. Acad Dermatol. 1986; 14:691-2.
10. Auer AL, Hershey FB. Surgery for necrotic bites of the brown 32. Fardon DW. The treatment of brown spider bites. Plast Reconstr
spider. Arch Surg. 1974; 108:612-8. Surg. 1967; 40:482-8.
11. Kensler T, Egner P, Moore G, Taffe BG, Twerdok LE, Trush MA. 33. Rees RS, Shack RB, Withers H, Madden JJ, Franklin JD, Lynch
Role of inflammatory cells in the metabolic activation of polycyclic JB. Management of the brown recluse spider bite. P l a t Reconsb Surg.
aromatic hydrocarbons in mouse skin [abstract]. Toxicol Appl Phar- 1981; 68:768-73.
macol. 1987; 90:337. 34. King LE, Rees RS. Dapsone treatment of a brown recluse spider
12. Suzuki K, Ota H, Sasagawa S, Sakatani T, Fujikura T. Assay bite [abstract]. JAMA. 1983; 250:648.
method for myeloperoxidase in human polymorphonuclear leukocytes. 35. Clark JM, Fisher AB. Oxygen toxicity and extension of tolerance
Anal Biochem. 1983; 132345-52. I
in oxygen therapy. In: David JC, Hunt TK (eds). Hyperbaric Oxygen
13. Bradley PB, Priebat DA, Christensen RD, Rothstein G. Measure-
Therapy, 4th ed. Bethesda, MD: Undersea Medical Society, 1988, pp
ment of cutaneous inflammation: estimation of neutrophil content with
61 -77.
an enzyme marker. J Invest Dermatol. 1982; 78:206-9.
36. Zamboni WA, Roth AC, Russell RC, Nemiroff PM, Casas L, Smoot
14. Smith JK, Grisham MB, Granger DN, Korthuis J. Free radical de-
EC. The effect of acute HBO therapy on axial pattern skin flap survival
fense mechanisms and neutrophil infiltration in postischemic skeletal
when administered during and after total ischemia. J Reconstr Micro-
muscle. Circ Res. 1990; 66:789-93.
surg. 1989; 5:343-50.
15. Carden DL, Smith JK, Korthuis RJ. Neutrophil-mediated micro-
37. Zamboni WA, Roth AC. Russell RC, Graham B, Suchy H, Kucan
vascular dysfunction in postischemic canine skeletal muscle. Circ Res.
J. Morphologic analysis of the microcirculation during reperfusion of
1990; 66~1436-43.
16. Ekstrom T, Garberg P, Egestad B, Hogberg J. Recovery of malon-
ischemic skeletal muscle and the effect of hyperbaric oxygen. Plast
dialdehyde in urine as a 2.4 dinitrophenylhydrazine derivative analyzed Reconstr Surg. 1993; 91:lllO-23.
with high-performance liquid chromatography. Chem Biol Interact. 38. Thom SR, Elbuken ME. Oxygen-dependant antagonism of lipid
1988; 66: 177-87. peroxidation. Free Radic Biol Med. 1991; 10:413-26.
17. Lee HS, Csallany AS. Measurement of free and bound malondi- 39. Thom SR. Functional inhibition of leukocyte B2 integrins by hy-
aldehyde in vitamin E deficient and supplemented rat liver tissues. perbaric oxygen in carbon monoxide-mediated brain injury in rats. Tox-
Lipids. 1987; 22:104-7. icol Appl Pharmacol. 1993; 123:248-56.
18. Rees RS, Nanney LB, Yates RA, King LE. Interaction of brown 40. Hunt TK, Zederfeldt B. Oxygen and healing. Am J Surg. 1969;
recluse spider venom on cell membranes: the inciting mechanism? J 118:523-5.
Invest Dermatol. 1984; 83:270-5. 41. Hunt TK, Pai MP. The effect of varying ambient oxygen tensions
19. Babcock JL, Geren CR, Civello DJ. Purification and characteriza- on wound metabolism and collagen synthesis. Surg Gynecol Obstet.
tion of a toxin from brown recluse spider venom gland extracts. Toxi- 1972; 135:561-7.
con. 1981; 5:677-89. 42. Weiss SJ. Tissue destruction of neutrophils. N Engl J Med. 1989;
20. Dressler KA, Kolesnick RN. Ceramide 1-phosphate, a novel phos- 3 2 0 10- 20.
pholipid in human leukemia cells. J Biol Chem. 1 9 9 0 265:14917-21. 43. Mader JT, Brown GL. A mechanism for the amelioration by hy-
21. Forrester LJ, Barrett JT, Campbell BJ. Red blood cell lysis induced perbaric oxygen of experimental staphylococcal osteomyelitis in rabbits.
by the venom of the brown recluse spider, the role of sphingomyelinase- I Infect Dis. 1980; 142:918-21.You can also read
