Topical use of Rectogesic® and Emla® to improve cutaneous blood perfusion following thermal injury. A comparative experimental study

Summary

Early post-burn ischemic necrosis of the skin is of particular interest in modern burn research. The purpose of this study was to test the hypothesis that blood perfusion could be improved by the topical application of vasoactive substances. A sample of 55 wistar rats was used to investigate the effect of 0,4% nitroglycerin ointment (Rectogesic^®) comparatively to no application and placebo. The beneficiary action of 5% prilocaine/lidocaine cream (EMLA^®) in burn blood perfusion was also tested comparatively to Rectogesic^®. Both preparations were tested respectively to non burned controls. Laser Doppler assessment of blood flow at 15, 30, 45, 60, 120 and 180 minutes after preparation application, demonstrated that the use of Rectogesic^® improved perfusion at all measurements compared to placebo and to no preparation application (p&0,05). There was no statistical significant difference in the effect of the two preparations.

Introduction

Jackson¹ described three concentric zones of a burn wound: the central zone of coagulation, the intermediate zone of stasis and the outer zone of hyperemia. He also proposed that the tissue between the irreversible necrosis of the zone of coagulation and the edematous red tissue of the zone of hyperemia (zone of stasis), involves a complete cessation of blood flow within 24h resulting in tissue necrosis, although it is not avascular.² Since then a challenging question has been raised: what factors contribute to or ameliorate the progression of tissue destruction in the zone of stasis?

Several experimental studies are focusing to establish adequate perfusion levels within the zone of stasis by reversing vasoconstriction, inflammation, edema-formation and thrombosis of microvessels in order to avoid tissue integrity harm due to hypoxia, lack of nutrients and accumulation of toxic metabolites.^3-4 There are no accepted nor standardized therapeutic approaches in humans today for salvaging the intercalated zone of stasis and preventing secondary burn wound progression.⁵

The hypothesis of this study was founded in previous similar studies attempting to give answers to this question, using topical application of vasoactive substances in order to improve deep partial thickness thermal burns blood perfusion.^6-32

Burn injury induces a pronounced inflammatory reaction, involving various vasoactive mediators such as histamine,^33-34 serotonin,^35-36 leukotrienes^37-38 and prostaglandins^39-40 with effects on vascular patency and edema formation.^6-42 Local anesthetics have been shown to interfere with inflammation induced, plasma extravasation^41-42 and the release and synthesis of several mediators such as prostaglandins,^43-44 leukotrienes,45 interleukins46 and histamine.⁴⁷ The mechanism which is responsible for the inhibition of burn edema by local anesthetics is related to the action of local anesthetics on vascular tone. This action could be secondary to inhibition of leukocyte adherence to the endothelium of injured vessels^46-47 and migration into injured tissues,^48-49 as well as the inhibition of the release of tissue toxic lysozymes,⁴⁹ oxygen free radicals^43-44, ⁵⁰ and histamine.⁵¹ All these factors are known to be involved in the control of vascular permeability in burns.⁵² The inhibition of burn edema by local anesthetics could, in conjunction with the antithrombotic⁵³ effects of these agents, account for the prevention of progressive burn ischemia reported following experimental burns.²¹

The vasoactive properties of 5% prilocaine/lidocaine cream (EMLA^®) and their effect in burn blood perfusion have already been described.^{13,17,20-23,37}

Organic nitrates, including nitroglycerin, are known for their vasodilatory and antithrombotic action. They are used systemically for the prevention and therapy of coronary disease and left heart failure.⁵⁴ By topical application, 0,4% nitroglycerine ointment (Rectogesic^®) is used for the chronic anal fissure⁵⁵ and hemorrhoids⁵⁶ treatment. Topical application of nitroglycerine has been used experimentally for the treatment of flap ischemia.^57-63 Very little is known about their effect in burn injury blood perfusion.²⁴

In the present study we used the Laser-Doppler, a non invasive method for repetitive measurements of blood flow,⁶⁴ to investigate the effects of topically applied 0,4% nitroglycerine ointment (Rectogesic^®) in burn blood perfusion. This was the first time that the vasoactive properties of this preparation were investigated in thermal injuries. In addition, the first attempt ever was made to compare its action to the beneficial action of the 5% prilocaine/ lidocaine cream (EMLA^®) on standardized deep partial thickness burns in rats.

Materials and methods

The study took place at the experimental laboratory of “G. Papageorgiou” General Hospital of Thessaloniki in Greece.

Materials

We used 55 male and female wistar rats because their skin has all the characteristics of mammals. Their weight ranged from 220 to 250 gr. For at least seven days prior to the experiments the animals were housed in special cages in a ventilated and temperature controlled room with food and water provided ad libidum. The day and night cycle was available naturally through windows.

The preparations used were the 0,4% (4mg/gr) nitroglycerine ointment (Rectogesic^®, ProStrakan Limited, UK) and the 5% (25mg+25mg/ml) prilocaine/lidocaine cream (EMLA^®, ?stra Zeneca, Recip Karlskoga AB, Sweden).

Method

The sample was based on previous studies. Animals in the study were distributed randomly⁶⁵ into 4 groups with burns and 3 control groups without burns. Group A was given Rectogesic^®, group B EMLA^®, group C was given nothing and group D were given the respective placebo without nitroglycerin. The control group A^c had Rectogesic^®, group B^c had EMLA^® and group D^c was given the respective placebo without nitroglycerin.

Preparation of the animals

The animals were anesthetized using intramuscular injection of 20-30mg/kg ketamine hydrochloride (Ketalar amp, 50mg/5ml, Warner Lambert, Hellas), in combination with 1-2mg/kg midazolam (Dormicum amp, 15mg/3ml Roche, Hellas). This method gives quick induction and maintenance for at least one hour of anesthesia, as well as analgesia and, if necessary, can be repeated.⁶⁶ This was followed by the non traumatic depilation of the dorsal skin.

Experimental burn

A scald was created to the back of the animals.¹² The standardized process was as follows: The boiling water was placed in a 5cm diameter heat resistant container. Following that, the temperature was monitored with an infrared thermometer descending down to 75?C. At this temperature the back of the anesthetized animal was kept immersed for 10 sec?nds in the hot water, creating a standard deep partial thickness burn. This process was followed by the measurements.

Blood flow evaluation

Measurement of the blood flow in ml/min/100gr was performed using Laser–Doppler equipment (PERIFLUX^®, PF 4001, PERIMED AB, Sweden). The Laser-Doppler technique allowed non invasive, repetitive measurements as opposed to other described methods requiring removal of experimental tissue.⁶⁴ The accuracy of this method as a quantitative tool was evaluated and it was suggested that multiple measurement points be used to increase validity and reliability of measurements.⁶⁷ In this study five standard points of the burned area were used for blood flow evaluation and the mean value of these measurements was recorded as the variable for analysis. Six different measurements were performed on a standardized timescale during the early post-burn period, to assess the blood perfusion changes. The preparations were wiped from the experimental area before the measurement process using 95° alcohol wipes and were replaced during the intervals between measurements.

Experimental protocol

The standardized experimental process was performed by the same investigator.

The experimental steps were common for all groups. No burn was performed in controls. The first measurement took place before the burn formation or preparation placement and was used as a reference measurement of the normal skin flow. This was followed by the experimental burn. Five minutes after the burn 0,5gr of the respective preparations were applied in groups A, B and D. In group B the preparation was covered with occlusive dressing (Tegaderm^®, 3M, USA). The timescale of blood perfusion measurements was at 15, 30, 45, 60, 120 and 180 minutes after the first placement of preparations. The measurement at 180 minutes was not followed by replacement of the preparation. In group C all measurements were performed without placement of preparation. The experimentation protocol is presented concisely in Table I.

Table I. Experimental protocol.

Histolopathology

The depth of the experimental burn was confirmed preliminary by the histological examination of burn tissue specimens performed in 3 anesthetized animals.

Ethical considerations

All animals received human care. Experimental protocols were approved by the Thessaloniki District Veterinary Services Direction Ethics committee.

Statistics

All data were stored using ?S Excel 2007 for PC. Mean comparison analysis was performed using t-tests. Statistical significance was set at <0.05.

Results

Descriptive statistics for group A (Rectogesic®)

Mean values of blood flow during measurements, presented in Table II and Fig. 1, ranged from 25,3750 (S.D.=14,05502) to 31,9020 (S.D.=24,92093) ml/min/100gr.

Table II. Descriptive statistics for group A (Rectogesic®).

Fig. 1. Comparative diagram of the measurements for groups A, B, C and D.

Compared to the intact skin controls, the flow was higher to the injured skin at the 15 minute measurement (t=3,427, p=0.008). It should be mentioned that there was also a trend at the 180 minute measurement (t=1,927, p=0,086).

Descriptive statistics for group B (EMLA®)

Mean values of blood flow during measurements are presented in Table III and Fig. 1 and ranged from 20,2500 (S.D.=8,39248) to 24,4917 (S.D.=14,25861) ml/min/100gr. Compared to the intact skin controls, the flow was higher to the injured skin at the measurements taken at 120 minutes (t=2,823; p=0,02) and at 180 minutes (t=3,166; p=0,011). There was also a trend at the 60 minute measurement (t=1,849; p=0,097).

Table III. Descriptive statistics for group B (EMLA®).

Descriptive statistics for group C (no preparation)

Mean values of blood flow during measurements are presented in Table IV and Fig. 1 and ranged from 11,6000 (S.D.=4,84271) to 14,5833 (S.D.=7,62478) ml/min/100gr.

Table IV. Descriptive statistics for group c (no prepatation).

Descriptive statistics for group D (placebo)

Mean values of blood flow during measurements are presented in Table V and Fig. 1. These ranged from 11,2877 (S.D.=5,36349) to 13,1924 (S.D.=5,98779) ml/min/100gr. Compared to the intact skin controls, the flow remained higher to the intact skin in all measurements (p<0.001).

Table V. Descriptive statistics for group D (placebo).

Comparison of blood flow after burn between groups A and C

The results from Restogesic^® use compared to no preparation use are presented in Table VI. It appears that nitroglycerine improves blood perfusion in all measurements (p<0,05) (at the 180 minute measurement p<0.001). These results are presented schematically in Fig. 1.

Table VI. Comparative results between groups A and C.

Comparison of blood flow between groups A and D

The results from Rectogesic® use compared to placebo use are presented in Table VII. It appears that nitroglycerine improves blood perfusion in all measurements (p<0,05) (at the 180 minute measurement p=0.001). These results are presented schematically in Fig. 1.

Table VII. Comparative results between groups A and D.

Comparison of blood flow between groups B and C

The results from EMLA^® use compared to no preparation use are presented in Table VIII. The anesthetic cream improves blood perfusion in most of the measurements (p<0,05) and there was a trend to the rest of them [at 30 minutes (p=0,075) and at 45 minutes (p=0,054)]. These results are presented schematically in Fig. 1.

Table VIII. Comparative results between groups B and C.

Comparison of blood flow between groups A and B

The comparative results between Rectogesic^® and EMLA^® use are presented in Table IX. The analysis did not demonstrate any significant difference from their use, however there was a trend in favor of nitroglycerine at the measurement taken at 180 minutes (p=0.80). These results are presented schematically in Fig. 1.

Table IX. Comparative results between groups A and B.

Comparison of blood flow between groups D and C

The comparative results between these groups are presented in Table X. There was no significant difference from the use of placebo compared to no use of preparation. These results are presented schematically in Fig. 1.

Table X. Comparative results between groups D and C.

Discussion

The main outcomes of the present study confirm the initial hypothesis suggesting that post-burn topical application of Rectogesic^® improves blood perfusion after a thermal injury. These results are in agreement with the results of Gorman et al.²⁴ However it should be mentioned that they used a 2% galenic nitroglycerine ointment. The experimental burn model was also different. They used a heated metallic probe creating a full thickness “comb shape” injury to the back of 4 animals and they used histological examination, assessing at 24 hours post infusion, the degree of Evans Blue pigmented albumin extravasation.

The primary outcomes of this study also confirm that the use of EMLA^® may improve blood perfusion after thermal injury. This is in agreement with the findings of Cassuto et al.¹³ Although their sample was similar, the experimental full thickness burn model used was different and they used histological examination, assessing the degree of Evans Blue pigmented albumin extravasation at 24 hours post infusion. The results are also in agreement with those of Jonsson et al.^20-21 They used image digital color analysis of Evans Blue pigmented albumin extravasation in 20 rats and Laser Doppler flow measurements in 29 rats respectively, testing the same hypothesis. The experimental full thickness burn model used was different.

Overall, the primary outcomes did not demonstrate any difference in use of the two preparations.

As compared to the intact skin controls, at the 15 minute measurement, the action of Rectogesic^® was demonstrated higher in injured skin. The same appeared to happen with EMLA^® but at a different time measurement. Initially the blood flow was higher to the intact skin controls (p<0.001) but this was later inverted starting with a trend at the 60 minute measurement (p=0,097), which became significant at the measurements taken at 120 minutes (p=0,020) and at 180 minutes (p=0,011). These fluctuations of blood flow by the use of EMLA^® have been previously documented.³¹

It is suggested that the transcutaneous absorption of the preparations in injured skin is increased.

At this point it should be useful to briefly mention the limitations of the study, creating a framework that should be considered for future studies.

Because of the complexity of burn injury, it shouldn’t be feasible to hypothesize that only one variable, such as blood perfusion, could represent burn wound progression. The zone of stasis pathophysiological events leading to cell death either through apoptosis or necrosis are extremely complex. Several experimental animal studies have already attempted to address neutrophiles aggregation, inflammation mediators production and cell infiltration, infection, alterations to blood flow due to edema formation, vasoconstriction, thrombosis and free radicals formation in a selective way to prevent cell death with variable results.⁶⁸ Application of all these selective results to the clinical environment should be made with caution, recognizing the need for integration with the outcomes of future studies, testing the systemic, side and synergy effects, as well as different timings, doses and durations of treatment.

Conclusion

In conclusion, from the assessment of blood flow with the Laser Doppler it appears that Rectogesic^® and EMLA^® are equally beneficial to the perfusion of deep partial thickness burns.