Zentralblatt für Chirurgie 1967 Heft 13 (Central Journal for Surgery 1967, Volume 3)

[pdf] German original (with graphs)

From the Institute of Pharmacology and Toxicology  (Director: Prof. Dr. F. Hauschild) and the Institute for Medical Microbiology and Epidemiology (Director: Prof. Dr. Dr. h. c. G. Wildführ) of the Karl-Marx University, Leipzig

Concerning the effect of Hydrogen Peroxide on Tetanus toxin

in skin lesions

By von Ludewig

With 1 Illustration

In previous papers we have been able to demonstrate that hydrogen peroxide in the form of high percentile preparations penetrates the katalase-free epidermis in almost undisintegrated form, without the unjustifiably feared burn-like effect and that it can bring about the destruction in large measure of the sub-cutanely present tetanus culture material (Hauschild et al., Ludewig; Francke and Ludewig). The question of whether apart from the inhibition of resorption and damage to the katalase-poor, oxygen-sensitive clostridium the chemical detoxification of the toxin in the tissue also plays a role, remained unresolved. In vitro, the toxin can indeed be destroyed by H₂O₂, but because no conclusions can be reached from the results of the incubation tests concerning the conditions in hyperoxidase-active tissues, we carried out relevant tests on skin lesions of comparable size.¹   

¹ We thank Prof. Dr. Wildführ for the valuable support

Methodology

We used a total of 245 male, unqualified mice weighing between 20 to 25 g.

The animals were sedated with Urethane (0,2 ml of a 10% solution per mouse i.p.) (I don’t know what i.p. means, so have left it untranslated; Erwin) and slightly narcotised with a mild application of ether for the duration of wound-making and toxin-application. On the skin of their backs, which had been shaved the previous day, a stencil pattern was used to cut wounds of a diameter of 6 mm and reaching down to the muscle face, to each of which 75 µg of tetanus toxin in 0,05 ml of physiological Na Cl-solution was applied after one hour. Twenty control animals which are not listed in table 1 received ceteris paribus (my Latin is no longer up to scratch so left this term untranslated; Erwin. It could possibly mean ‘for the sake of comparison/in order to compare) saline solution without toxin added. After 2 resp. 10 minutes the bowl-shaped wound was filled to the brim with the “antidote”. Hydrogen peroxide of 12% was added to an undifferentiated powder base¹, which ensured good adhesion to the wound and made a comparison with conventional wound powders possible. Apart from an

H₂O₂-free basis (placebo), we used the used Becarmal (10% salt of amino-benzol-sulfo-thiocarbamide of the amino-methyil-benzol-sulfonoamid + 40% amino-benzol-sulfonamid) which is commonly used in the care of infected wounds, in order to have a substance for comparison. The occurrence of death in typical stretch position was registered on a daily basis. The results were compiled in a “2 x 2 table” and evaluated in accordance with the corrected x² test by Yates.      

¹ We thank Pharm. Councillor Dr. Börngen for the preparation of the stabilized H₂O₂ powder, which in the meantime is now also being manufactured by the VEB Leipziger Arzneimittelwerk (a German pharmaceutical company; Erwin).   

Results

The test results which have been listed in table I and graphically depicted in the column diagramme (illustration 1) allow the following observations: The untreated tetanus toxin-infected mice died in this test set-up largely within 2 days. No further mice died in typical stretch-position later than after 5 days. Solely as a result of the dry powder-treatment starting 2 minutes after the inoculation, it was possible to delay the onset of death (mortality rate within 2 days significantly lowered); here it initially seems rather unimportant whether an inactive powder base, H₂O₂^-powder or Becarmal is used. By the 5^th test day however, the mortality rate of animals treated with powder base or Becarmal becomes so similar, that a definite certainty in regard to their differentiation is no longer possible. The mortality of the animals treated with H₂O₂ lies however highly significantly below that of untreated (compare table 1) or that of with Becarmal X² = 21,03; p < 0,001) treated mice. The results of the complementary series show that the effect of H₂O₂ as opposed to the application of Becarmal  lead to a statistically markedly ascertained success even when there is a delay of 10 minutes between the inoculation of tetanus and the treatment start. All 20 control animals, whose wounds were filled with only Na Cl-solution instead of tetanus toxin, survived the post-observation period of 14 days.

(I don’t think I would fancy being a lab mouse or rat or any experimental animal for that matter; Erwin) 

 Table I. The effect of a 12% H₂O₂^-containing powder on tetanus-toxin in wounds (compare text)

Treatment                        2^nd test day                      5^th test day

Start after  with       dead        alive                           dead    alive 

                          Animals %  Animals % p    Animals %      Animals %  X² p

 2 minutes    untreated 

                   H₂O₂ powder

                   placebo

          Becarmal

10 minutes    untreated

                    H₂O₂ powder         

Illustration 1. Comparison of the mortality of mice after inoculation with tetanus toxoid. Treatment start after 2 respectively 10 minutes

by the 2^nd day         by the 5^th day         by the 2^nd day         by the 5^th day                              2 min interval                            10 min interval

(I have only reproduced the translated text of Table 1 and Illustration 1 here; please refer to German original for figures; Erwin)

Discussion

The remarkable aspect of the results obtained is first of all the fact that even the powder base (placebo) by itself delays death following inoculation with tetanus toxin significantly. Its temporary adhesion to the powder, apparently influenced by the size of the powder granules and their solubility, can be regarded as the cause of this. This results in a delayed resorbtion and the gain of a time period for an oxidative and immunobiolgical effect of diminishing the toxin’s effect. The situation on the 5^th day however allows the observation that in the end effect neither a powder base nor the commonly used Becarmal can secure a statistical lowering of the mortality. Decisively effective however is the hydrogen peroxide, as the infiltration of minute oxygen bubbles into the tissue and its capillaries (Ludewig) achieves an even stronger inhibition of resorbtion while the high oxygen-potential effects a detoxification, before lethal amounts of toxin are resorbed or the H₂O₂ is decomposed by hydroperoxidasis by the tissue or wound secretions. Even the oxygen resulting from catalytic H₂O-splitting may still contribute toward the inactivation of the toxin.

The results achieved by our model are of course only transferable by maintaining corresponding clinical conditions: favourable conditions such as those we created in our test animals in order to compare the application of various substances will only rarely be found in skin lesions. It should however be noted that H₂O₂  possesses an unusually deep effect and is able to penetrate even into jagged wounds (I have chosen jagged for lack of a better word for unübersichtlich; übersichtlich literally means overlookable, denoting something which can be easily looked over or assessed because it is smooth or tidy; unübersichtlich by contrast is something which can not be looked at or assessed easily because it has hidden aspects or contains an element of disorder or chaos; untidy or messy would convey a meaning similar to unübersichtlich; Erwin)  

In the event of a wound subjected to tetanus-infected material there is also not such a massive intoxication as in our case, as under favourable conditions the toxin is only produced gradually and is therefore easier to influence. The fact that H₂O₂ also cleans wounds mechanically and develops bactericidal and anti-mycotic (= fungicidal? Erwin)  effects, without having the disadvantage of resorbtion or allergy development, makes high-percentile hydrogen-peroxide in contrast to other wound medications appear a valuable complement to the active and passive tetanus vaccination.

Summary

In addition to earlier investigations it is demonstrated in mice that hydrogen peroxide in the form of a 12% powder obstructs the resorption of tetanus toxin applied to skin lesions and detoxifies it. The advantages of high-percentile hydrogen-peroxide preparations compared to conventional wound remedies are discussed.

We are indebted to Miss Ch. Holfeld and Miss E. Rehwald for their valuable technical assistance.

Bibliography

(Please refer to original German article; Erwin)