Electrolyzed Strong Acid Aqueous Solution IrrigationPromotes Wound Healing in a Burn Wound Model

Hajime Nakae and Hideo Inaba

Department of Emergency and Critical Care Medicine, Akita University School of Medicine, Akita, Japan

Abstract: The purpose of the study was to understand theeffectiveness of electrolyzed strong acid aqueous solution(ESAAS) for acceleration of epithelialization in a rat burnwound model. Eighteen anesthetized Sprague-Dawley ratsreceived 30% total body surface area third-degree burns,and 2 days after injury, the wound eschars were removed.Rats were divided into 3 groups: Group 1, no irrigation;Group 2, irrigation with physiological saline; and Group 3,irrigation with ESAAS. Wounds were observed macro-scopically until they were fully epithelialized, and epithe-

lialized wounds were examined microscopically. Epitheli-alization of the wounds occurred significantly early in therats treated with ESAAS (p < 0.05). Proliferation of asso-ciated lymphocytes and macrophages was more extensivein all rats that underwent irrigation than it was in controlrats. ESAAS irrigation may promote tissue growth in burnwounds. Key Words: Electrolyzed strong acid aqueoussolution—Burn—Excised wound—Eschar—Epitheliali-zation.

Major burn injury can easily lead to sepsis due towound infection, even when acute burn shock is un-der good control. The larger the wound, the greaterthe chance of burn wound infection. The importanceof preventing burn wound infection goes withoutsaying.

Electrolyzed strong acid aqueous solution(ESAAS), which has a high positive oxidation-reduction potential (ORP) and contains high con-centrations of dissolved chloride and oxygen, func-tions as a bactericide and is used clinically for thetreatment of various types of infection (1–6). How-ever, it is unclear whether ESAAS causes tissuedamage or promotes epithelialization. We studiedwhether ESAAS promotes tissue growth withoutcausing tissue damage in a rat burn wound model.

MATERIALS AND METHODS

The protocols for animal experimentation de-scribed in this article were approved by the AnimalResearch Committee of Akita University School of

Medicine, and all animal experiments adhered to theGuidelines for Animal Experimentation of the Uni-versity.

AnimalsMale Sprague-Dawley rats (8 weeks of age; 235 to

255 g; Nippon SLC Ltd., Sendai, Japan) were used inthe experiment. All rats were housed in individualcages in a temperature-controlled room with alter-nating 12 h light:dark cycles, and the animals wereacclimated for a minimum of 3 days before the study.Standard laboratory chow was available ad libitum.

Burn injuryExperimental burn injury was induced according

to the method described by Walker and Mason (7).In short, rats were anesthetized by i.p. injection ofpentobarbital (40 mg/kg). Hair on the back of eachrat was removed with an electric clipper, and dorsalskin surfaces were exposed to 95°C water for 10 sthrough a template designed to produce a third-degree burn that covered approximately 30% of thetotal body surface area. Directly after the burn in-jury, 5 ml physiological saline was administered i.p.to each rat for fluid resuscitation (8).

Electrolyzed strong acid aqueous solutionWe used an Oxilyzer (Miura Denshi Co., Ltd.,

Akita, Japan) to produce ESAAS (1). The ESAAS

Received October 1999; revised December 1999.Address correspondence and reprint requests to Dr. Hajime

Nakae, Department of Emergency and Critical Care Medicine,Akita University School of Medicine, 1–1–1 Hondo, Akita 010–8543, Japan.

Artificial Organs24(7):544–546, Blackwell Science, Inc.© 2000 International Society for Artificial Organs

544

generated is acidic (pH of ø2.7) and has an effectivechlorine concentration of 50 ppb to 40 ppm and adissolved oxygen (DO) concentration of 50 ppb to 20ppm, with a redox potential of at least +1,000 mV.

ExperimentTwo days after injury, the eschar of the burn

wound was surgically removed from each rat undertotal anesthesia (40 mg/kg i.p. injection of pentobar-bital). Rats without infection were assigned to thefollowing groups: Group 1, no irrigation (n 4 6);Group 2, irrigation with 20 ml physiological salineonce a day starting 8 h after removal of the eschar(n 4 6); and Group 3, irrigation with 20 ml ESAASonce a day starting 8 h after removal of the eschar(n 4 6). Wounds were observed macroscopically un-til they were fully epithelialized. With complete epi-thelialization, rats were sacrificed by overdose ofpentobarbital, and the epithelialized wound area wasexcised for microscopy after staining with hematoxy-lin-eosin to investigate inflammatory reactions. Weconsidered 80 postburn days as the cutoff for com-plete epithelialization, considering the age of therats.

Statistical analysisData are expressed as mean ± SD. The Kruskal-

Wallis rank test was used for group comparisons. Ap value of less than 0.05 was considered significant.

RESULTS

Full epithelialization was observed in 1 of 6 rats inGroup 1 (Epithelialization: 70 days), 1 of 6 in Group2 (48 days), and in 5 of 6 rats in Group 3 (58.4 ± 12.0days); the differences were significant (p 4 0.0292)(Table 1).

Although extensive regenerative epidermis wasseen in each group, lymphocytes, macrophages, andmast cells proliferated in Groups 2 and 3 more thanin Group 1. Moreover, these cells were larger, andcollagen tissues were denser. These findings wereparticularly evident in Group 3 (Fig. 1).

DISCUSSION

The environment required by mitochondria in allmicroorganisms is within a range of 2 to 12 pH and−400 to 850 mV ORP. However, ESAAS requires apH below 2.7, DO at 20 ppm, hypochlorite ion above30 ppm, and ORP above 1,100 mV (1). The fact thatsome ESAAS requirements fall outside the rangerequired by mitochondria in microorganisms is onereason for its germicidal effects. Moreover, the so-lution contains a high concentration of hypochlorous

acid, a well-known and potent germicidal agent ofwhich bactericidal action increases markedly in acidsolution. The hydroxy radical also has a germicidaleffect, and the electrical energy of the solution itselfdestroys microorganisms including viruses, fungi,mycobacteria, spirochetes, and other bacteria. It isknown that almost all germs will perish within 10 s insuch a solution. It is important to note that the so-lution loses both its electrolytic potential and its ger-micidal action when it comes into contact with mul-ticellular organic matter and reverts to ordinarywater with no added function (1–6).

Because the dermis necroses in full-thickness in-jury, epithelialization can be expected to occur onlyfrom the burn edges (9). This healing process alonerequires a long time, and the chance of infectionincreases during that time. However, when the full-thickness burn area is small in patients or skin graft-ing is rejected for personal or medical reasons, thereis nothing to rely on but the natural healing process,which is why we selected the excised burn woundmodel. In this study, the epithelialization of burnwounds occurred significantly early in the grouptreated with ESAAS. Compared with that in thenonirrigation group, the proliferation of lympho-cytes and macrophages associated with dense colla-gen deposition was noted more frequently in thegroup treated with physiological saline and in theESAAS group. Wound irrigation may cause inflam-matory reactions, with tissue proliferation resultingin the epithelialization of burn wounds. However, nosignificant differences were noted in the completionof epithelialization between the control group andthe saline group. It may be that wound irrigationalone is somewhat effective but not sufficient forcomplete epithelialization. Therefore, chemical me-diators such as cytokines that accelerate tissue repairmay be produced by the stimulation of EOW irriga-tion, thus promoting tissue growth and acceleratingepithelialization.

In our study, ESAAS irrigation did not cause tis-sue damage resulting in delayed healing of burn in-juries. The prevention of sepsis is critical to a goodprognosis in patients with severe burn injury. It isimportant to control infection at the wound site. Ir-

TABLE 1. Epithelialization per group

Group Number of rats

Group 1 1/6 (16.7%)Group 2 1/6 (16.7%)Group 3 5/6* (83.3%)

Group 1: control, Group 2: physiological saline, Group 3: elec-trolyzed oxidized water.

* p < 0.05 (Kruskal-Wallis rank test).

ELECTROLYZED STRONG ACID AQUEOUS SOLUTION 545

Artif Organs, Vol. 24, No. 7, 2000

rigation and disinfection with ESAAS may preventburn wound infection without inhibiting healing ofburn injuries. Further clinical studies are needed toclarify the usefulness of ESAAS irrigation in burnwounds.

CONCLUSIONS

The epithelialization of burn excised wounds oc-curred significantly early in the group treated withESAAS in our rat model. Compared with that in thenonirrigation group, the proliferation of lympho-cytes and macrophages associated with dense col-lagen deposition was noted more frequently inthe group treated with physiological saline and theESAAS group. ESAAS irrigation promoted tissuegrowth in burn excised wounds.

Acknowledgments: We thank Professors Junichi Chi-hara (Department of Clinical and Laboratory Medicine)and Hiroki Masuda (Second Department of Pathology) fortheir guidance. This study was supported in part by Grant10470311 from the Ministry of Education, Science, andCulture of Japan.

REFERENCES

1. Kumon K. What is functional water? Artif Organs 1997;21:2–4.

2. Inoue Y, Endo S, Kondo K, Ito H, Omori H, Saito K. Trial ofelectrolyzed strong acid aqueous solution lavage in the treat-ment of peritonitis and intraperitoneal abscess. Artif Organs1997;21:28–31.

3. Sekiya S, Ohmori K, Harii K. Treatment of infectious skindefects or ulcers with electrolyzed strong acid aqueous solu-tion. Artif Organs 1997;21:32–8.

4. Hayashi H, Kumon K, Yahagi N, Haruna M, Watanabe Y,Matsui J, Hattori R. Successful treatment of mediastinitis af-ter cardiovascular surgery using electrolyzed strong acidaqueous solution. Artif Organs 1997;21:39–42.

5. Shirahata S, Kabayama S, Nakano M, Miura T, Kusumoto K,Gotoh M, Hayashi H, Otsubo K, Morisawa S, Katakura Y.Electrolyzed-reduced water scavenges active oxygen speciesand protects DNA from oxidative damage. Biochem BiophysRes Com 1997;234:269–74.

6. Tanaka N, Fujisawa T, Daimon T, Fujiwara K, Yamamoto M,Abe T. The cleaning and disinfecting of hemodialysis equip-ment using electrolyzed strong acid aqueous solution. ArtifOrgans 1999;23:303–9.

7. Walker H, Mason A Jr. A standard animal burn. J Trauma1968;8:1049–51.

8. Nakae H, Inaba H, Endo S. Usefulness of procalcitonin inpseudomonas burn wound sepsis model. Tohoku J Exp Med1999;188:271–3.

9. Matsumura H, Yoshizawa N, Kimura T, Watanabe K, GibranNS, Engrav LH. A burn wound healing model in the hairlessdescendant of the Mexican hairless dog. J Burn Care Rehabil1997;18:306–12.

FIG. 1. Shown are epithelialized tissue samples representing the 3 groups (original magnification ×20). Compared with that of the controlrats (A), the proliferation of lymphocytes and macrophages associated with dense collagen deposition was more extensive in the ratstreated with physiological saline (B) and electrolyzed strong acid aqueous solution (C).

H. NAKAE AND H. INABA546

Artif Organs, Vol. 24, No. 7, 2000