Copaiba oil effect under different pathways in mice subjected to sepsis¹

Introduction

Sepsis is the leading cause of death in intensive care units (ICU) with a mortality rateranging from 20 to 80%, being diagnosed 18 million new cases of severe sepsis per year worldwide^{1 - 4}.

The incidence of severe sepsis increased 91.3% over the past 10 years occurring in 1 to 3 per 1000 individuals in different regions, with annual growth of 1% per year^{1 , 5}, representing still a severe public health problem in the world, both from the point of view of social and economic terms⁶. Brazil is appointed, along with the United Kingdon, as one of the countries with the highest rate of occurrence of sepsis in ICU, responsible for occupying about one quarter of the ICU beds³.

The treatment of sepsis represents a challenge for medicine⁷, because it still remains an entity of difficult clinical management. Possible interventions in the inflammatory response and coagulation with the goal of reducing morbidity and mortality, and improve the prognosis of sepsis, has been extensively investigated⁸.

The use of plants for medicinal purposes, for treatment, prevention and cure of diseases, is one of the oldest forms of medical practice of mankind. In the early 1990s, the World Health Organization reported that 65-80% of the population in developing countries depended on medicinal plants as the only form of access to basic health care⁹, and in relation to world population, about 60% makes use of medicinal plants¹⁰.

In Brazil, many species are still used empirically, without scientific support for efficacy and safety, which shows that, in a country with enormous biodiversity, there is a huge gap between the supply of plants and little research¹¹.

One of the main medicinal plants used in the Amazon region, and the subject of many scientific studies is the copaiba oils, whose effects were tested and found effective in various activities, such as anti-inflammatory and antimicrobial¹², especially against gram-positive bacteria¹³.

It is, therefore, a potential source of new agents against major infectious diseases¹³ having already reports in the literature about its benefits in sepsis induced in mice¹⁴. Still, the oil released by copaiba tree resin is low cost and easy access¹⁵ present in pharmacies throughout Brazilin various presentations¹⁶. Thus, the aim of this study is to evaluate the effects of copaiba oil administered by different routes on survival of mices subjected to cecal ligation and puncture.

Methods

Research approved by the Ethics Committee in the Use of Animals of the State University of Para (UEPA), protocol 17/13.

Thirty two adults males mice (Mus musculus) were used, weighing between25 - 30grams, provided from the Animal Colony of the Experimental Surgery Laboratory of UEPA, kept in a controlled environment, with food and water ad libitum. The animals were randomized distributed into four groups, with eight animals each:

-Sham Group (SG): The animals were used as normal standard for survival and histological analysis;

-Control Group (CG): Animals were only realized the cecal ligation and puncture (CLP);

-Gavage Group (GG): Animals were realized the cecal ligation and puncture and treat with copaiba oil by gavage;

-Subcutaneous Group (SG): Animals were realized the cecal ligation and puncture and treat with copaiba oil by subcutaneous injection.

The animals were anesthetized with ketamine hydrochloride (100 mg/Kg) and xylazine hydrochloride (10 mg/Kg), intraperitoneally. After was performed the epilation and antisepsis of the abdominal region. Subsequently, was performed a laparotomy of one centimeter.

After opening the abdominal cavity, the cecum was located, exposed and isolated, leaving the rest of the small and large intestine into the peritoneal cavity, taking care not to violate or damage the mesenteriais' vessels. The cecal ligation and puncture as realized by a 75% cecum ligated with silk 4-0 immediately after the ileocecal valve, to induce a high-grade sepis. After this, the cecal stump was transfixed by a single through-and-through puncture with a 21 G needle. After the surgical procedure, was administered pre-heated saline (5 ml per100 g) by subcutaneously.

The gavage and subcutaneous group was tread by copaiba oil (Copaifera officinalis) 0,63ml/Kg once, for five days before the cecal ligation and puncture, differentiating according to the administration route.

Confirmed the death of the animal was collect the lung of the animal, that were stored in 10% buffered formaldehyde and used for histopathological analysis by means of hematoxylin and eosin. It was analyzed in the lung the presence of necrosis, vascular congestion, and alveolar hemorrhage. It were analyzed by a semi-quantitative scale: 0 - absent, 1 - mild, 2 - moderate, 3 - severe.

Kruskall-Wallis and Fisher's exact test was used to compare the histopathological results. Survival curves of groups were plotted using the Kaplan-Meier method and then compared by the log-rank test. Was adopted a significance level of 5% to reject the null hypothesis.

Results

No animals died in the Sham group, presenting good surgical recovery, and euthanized 30 days after the death of the animals in the other groups.

All animals that received copaiba, regardless of the route used, survived longer when compared to the control group (p<0.0001), whereas the survival time (Figure 1) ranged from 20 hours for the control group up to 52 hours for the animals of group Subcutaneous, corresponding to a survival time of 2.6 times greater (p=0.0003). The animals that received gavage copaiba lived about 12 hours more than the control (p=0.0017) and about 20 hours unless the subcutaneous group (p=0.0042).

Figura 1
Graph showing the survival curve of animals after performing cecal ligation and puncture, according to the group.

As regards the histopathological (Table 1) there was no statistical difference when compared the intensity of inflammatory response (p>0.05), having all groups displayed similar pattern of lung inflammatory response, with the presence of diffuse polymorphonuclear interstitial infiltration, vascular congestion, emphysema and focal areas of interstitial hemorrhage. However, it was observed the presence of tissue necrosis in 62.5% of the control group animals and none of the animals of the other groups (p=0.02).

Tabela 1
Main histopathological features according the group.

Group	Interstitial infiltrate	Emphysema	Vascular congestion	Alveolar hemorrhage	Necrosis
Control	2.00 ±0.93	1.75 ±0.71	2.50 ±1.07	0.62 ±0.52	0.75 ±0.71
Subcutaneous	1.71 ±0.49	1.86 ±0.69	3.00 ±0.00	0.71 ±0.76	0.00 ±0.00
Gavage	2.00 ±0.58	2.00 ±0.58	3.00 ±0.00	0.43 ±0.53	0.00 ±0.00

• Source: Protocol search p>0.05 (Kruskall-Wallis)

Discussion

The present study demonstrated that there is significant difference in the organic response to severe sepsis when using different routes for the administration of copaiba oil. This maybe related to the longer time required for drug absorption according to the specific route, thus delaying the on set of it, which is a critical factor to the prognosis and survival of animals with sepsis.

Furthermore, the bioavailability of active molecules with antimicrobial and anti-inflammatory present in copaiba oil may be changed due to the mechanism of the first passage through the liver when administered by gavage. Thus, using the same dose for both routes (subcutaneous and gavage) probably resulted in increased action of the copaiba in subcutaneous group, culminating in the increased animal survival on this group.

Copaiba oil has been used by several authors to test its anti-inflammatory and antimicrobial effects^{17 - 19}, and the results have shown that the main active components responsible for these properties are the diterpenes and sesquiterpenes, as bisabolol and beta betacariofileno⁹. Knowing that the copaiba used in this study does not show effects against gram-negative bacteria¹³ and that the model of CLP primarily deflagrates gram-negative septicemia, it is believed that the determining factor for increasing animal survival was the anti-inflammatory action of this plant, by modulating the inflammatory response and subsequent issue damage mediated by free radicals and pro-inflammatory citocinais against sepsis.

When analyzed histologically in the lungs of mice, was observed that the use of copaiba oil was crucial to the progression of pulmonary status, been observed pattern of focal pulmonary necrosis only in animals that don't received the oil. This finding suggests appropriate systemic anti-inflammatory action of copaiba, in agreement with that described by Veiga Junior et al.²⁰, which showed a decrease in the number of lung neutrophils and leukocytes, besides inhibiting the production of nitric oxide.

Regardless of theroute used, the prophylactic use of copaiba oil for five days was effective to delay and mitigate the evolution of sepsis, and, judging by its wide use in Brazilian culture, especially Amazon, shows itself as an important component to be further studied for evidence of its role in popular usage.

Conclusion

Prophylactic subcutaneous administration of copaiba (copaifera reticulada) in mice subjected to severe sepsis by cecal ligation and puncture, resulted in a survival time of 2.6 and 1.5 times higher compared to non-use or use of this oil by gavage, respectively.

References

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• Financial sources: Institutional Program for Scientific Initiation (PIBIC), National Council for Technological and Scientific Development (CNPq), Ministry of Science, Brazil
• 1
  Research performed at Experimental Surgery Laboratory. School of Medicine, State University of Para (UEPA), Belem-PA, Brazil. Tutor: Nara Macedo Botelho.

Publication Dates

• Publication in this collection
  Aug 2014

History

• Received
  10 Mar 2014
• Reviewed
  12 Mar 2014
• Accepted
  11 June 2014