Shrimp diet and skin healing strength in rats

Dieta com camarão e resistência cicatricial da pele, em ratos

ORIGINAL ORIGINAL

Shrimp diet and skin healing strength in rats

Dieta com camarão e resistência cicatricial da pele, em ratos

Elizabeth Lage Borges^I,*; Fernanda Kelley Silva Pereira^I; Jacqueline Isaura Alvarez-Leite^II; Luiz Ronaldo Alberti^III; Mônica Alves Neves Diniz Ferreira^IV; Andy Petroianu^III

^IUniversidade Federal de Minas Gerais, Instituto de Ciências Biológicas, Departamento de Fisiologia e Biofísica. Av. Antônio Carlos, 6627, Bloco A4, Sala 249, Pampulha, 31270-010, Belo Horizonte, MG, Brasil

^IIUniversidade Federal de Minas Gerais, Instituto de Ciências Biológicas, Departamento de Bioquímica e Imunologia. Belo Horizonte, Minas Gerais, Brasil

^IIIUniversidade Federal de Minas Gerais, Faculdade de Medicina, Departamento de Cirurgia. Belo Horizonte, Minas Gerais, Brasil

^IVUniversidade Federal de Minas Gerais, Instituto de Ciências Biológicas, Departamento de Patologia. Belo Horizonte, Minas Gerais, Brasil

ABSTRACT

OBJECTIVE: Surgical scar tensile strength may be influenced by several factors such as drugs, hormones and diet. The purpose of the present study was to determine the influence of a shrimp-enriched diet on the tensile strength of rat scars.

METHODS: Forty male Wistar rats were submitted to a 4 cm dorsal skin incision and the wounds were sutured with 5-0 nylon interrupted suture. The animals were divided into two groups: Group 1 (control) received a regular diet, and Group 2 (experimental) received a shrimp-enriched diet. The two diets contained the same amounts of proteins, lipids and carbohydrates. The rats in each group were divided into two subgroups according to the time of assessment of the scar tensile strength: subgroup A, studied on the 5^th postoperative day, and subgroup B, studied on the 21^st postoperative day.

RESULTS: The tensile strength of the scar on the 5th postoperative day was lower in the animals that received the shrimp-enriched-diet (303.0, standard error of mean= 34.1) than in the control group (460.1, SEM = 56.7) (p<0.05).

CONCLUSION: A shrimp diet reduces the tensile strength of the scar. The next step of this study will be to clarify the mechanism in which shrimp affects tensile strength.

Indexing terms: diet; wound healing; eating; rats.

RESUMO

OBJETIVO: A resistência cicatricial da pele pode ser influenciada por diversos fatores como medicamentos, hormônios e dieta. Este trabalho foi delineado para determinar a influência da dieta com camarão na resistência cicatricial na pele.

MÉTODOS: Quarenta ratos machos Wistar foram submetidos a incisão (4cm) e suturas interrompidas da pele dorsal, com fio de nylon 5-0, e foram divididos em dois grupos: o Grupo 1 (controle) recebeu uma dieta convencional e Grupo 2 (experimental), recebeu dieta com adição de com camarão. As duas dietas continham quantidades semelhantes de proteína, lipídeos, e carboidratos. Os ratos de cada grupo foram divididos em dois subgrupos de acordo com os distintos períodos pós-operatórios de avaliação da resistência tecidual: subgrupo A, estudado no 5° dia pós-operatório, e subgrupo B, estudado no 21° dia pós-operatório.

RESULTADOS: A resistência cicatricial da pele no 5° dia pós-operatório foi menor nos animais que receberam dieta suplementada com camarão (303,0, erro padrão da média=34,1), quando comparada ao grupo controle (460,1, erro padrão=56,7) (p<0,05).

CONCLUSÃO: A dieta suplementada com camarão reduziu a resistência cicatricial da pele de ratos. Dando continuidade ao estudo, será averiguado o mecanismo pelo qual ocorre essa redução.

Termos de indexação: dieta; cicatrização de feridas; ingestão de alimentos; ratos.

INTRODUCTION

Skin wound healing involves a cascade of cellular and molecular events in which biological processes such as proliferation, differentiation and cell migration play pivotal roles^1-5. The process may be influenced by factors such as vitamin C, hormones, diet, and systemic or local diseases^6-9. Some hormones and other mediators, mainly angiotensin II and angiotensin-(1-7), accelerate skin repair by means of keratinocyte proliferation¹⁰. In contrast, obstructive jaundice and glucocorticoids inhibit the healing of jejunal anastomoses and skin wounds¹¹.

Crustaceans are a common source of coastland diet and folk culture mentions that food based on crustaceans interferes with wound healing. However, no information is available regarding this topic in the scientific literature. Therefore, a study of the influence of a shrimp-rich diet on skin healing should be relevant. The results of the present investigation showed that the addition of shrimp to the diet (33% of the diet) reduces the tensile strength of healing wounds.

METHODS

Forty male Wistar rats weighing 210-236 g were housed individually in acrylic metabolic cages (Nalgene, Rochester, NY, USA) with free access to food and water available ad libitum. The animals were maintained under standard laboratory conditions of a 12/12-h light-dark cycle and temperature of 25°C.

The composition of the experimental and control diets is shown in Table 1.The diet was based on Association of Official Analytical Chemists (AOAC)¹² with modifications, in order to maintain the composition of both, experimental and control diets, and to avoid interferences on the results. The protein concentration of dried shrimp flour (made from shell as well as the flesh of the shrimp) was 33.5mg/100mg as determined by the method of Lowry et al.¹³. Both diets contained the same amounts of proteins, lipids and carbohydrates.

To determine if the salty taste of the shrimp diet induced an increase in food intake, in another experimental stage, we added salt to the regular diet so that it would contain the same amount of salt as the shrimp-enriched diet.

After a four-day adaptation period, the animals were randomly divided into two groups: Group1 (control) received a regular rat chow, and Group 2 (experimental) received a diet enriched 33% with shrimp. The rats of each group were divided into two subgroups according to the time that the tensile strength of the scar was studied: rats of subgroup A were investigated on the 5^th postoperative day and rats of subgroup B on the 21^st postoperative day (times that are well acknowledged in literature^6-7). Food and water intake were assessed daily during the entire experimental period.

Under general anesthesia with intraperitoneal thionembutal (40mg/kg), all rats were submitted to a 4cm incision in the dorsal thoracic skin. The wound was closed with four interrupted sutures using 5-0 nylon suture.

After 5 or 21 days, the rats were anesthetized (thionembutal 40mg/kg), the skin fragment containing the scar was cross-sectionally removed and the tensile strength of the scar was determined. Each skin segment was 3cm long and 1cm wide and included the scar in its middle part. The nylon suture was carefully removed to avoid damage to the skin and the two ends of the skin sample were lifted with two Duval clamps. One clamp was suspended and fixed on a support and the other was connected to a plastic container, which was filled with distilled water at the rate of 1.4 liter/minute. The tensile strength of the wound was estimated by the total weight of the plastic container, of one clamp and of the amount of water at the time of scar rupture¹¹.

Another wound sample was removed, fixed in Bouin solution, dehydrated and embedded in paraffin. Sections of 5 µm were prepared and stained with hematoxylineosin (HE) for light microscopy analysis. Other sections were stained with Picrosirius solution and examined by polarization microscopy in order to identify collagen fibers^14-15.

Serum sodium and potassium ion concentrations were assessed by flame photometry (FC-180, CELM, Brasil).

The present investigation was in agreement with the Ethical Principles in Animal Experimentation, adopted by the Declaration of Helsinki (2000) and by the Ethics Committee in Animal Experimentation (CETEA/UFMG).

The results (mean and standard error of mean-SEM) obtained for the two groups were compared by the Student t-test and Mann-Whitney rank sum test, with the level of significance set at p<0.05.

RESULTS

The animals did not present any abnormality during the experimental period. No sign of toxicity was verified.

Table 2 shows total food intake, water intake, and weight of the animals on the 5^th and 21^st postoperative days. Food and water intake were higher in the group that received the shrimp-enriched diet (p<0.001). No difference in body weight was observed between the groups (Table 2).

The tensile strength of the skin segment from the two groups is presented in Figure 1 A and ^B. The skin of the rats that received the shrimp-enriched diet demonstrated a lower tensile strength on the 5^th postoperative day (p<0.05) (Figure 1A).

Table 3 shows the total food and water intake, and weight of the animals that received a salt-enriched diet on the 5^th postoperative day. The salt-enriched diet did not affect food intake.

Serum sodium and potassium levels (mM) did not differ significantly between the two groups on the 5^th (n=7) and 21^st (n=4) days: serum sodium on the 5^th postoperative day (125.9, SEM=1.7 versus 127.6, SEM=2.7) and on the 21^st day (121.8, SEM=0.8 versus 119.8, SEM=0.4); serum potassium on the 5^th postoperative day (6.0, SEM=0.1 versus 6.2, SEM=0.1) and on the 21^st day (6.0, SEM=0.3 versus 6.5 and 0.2). Data are reported as means and SEM. The two groups were homogeneous after being submitted to the specific diets (control vs. experimental shrimp-enriched diet).

No histological difference was found between the groups (control and experimental) on the 5^th and 21^st postoperative days. On the 5^th postoperative day, the dermis showed loose connective tissue fibers, neovascularization and granulation tissue (Figure 2A). On the 21^st day, the healing tissue was mature and fibrous with a less intense inflammatory reaction (Figure 2C). Both groups presented type III collagen on the 5^th postoperative day, but type I fibers were predominant after 21 days (Figures 2B and D).

DISCUSSION

In the healing process, fine and disorganized collagen fibers appear first (type III)^16-17, being then replaced with thicker fibers, with the progressive occurrence of organization of type I collagen. Collagen type, more than the amount of collagen fibers, is important in maintaining the strength of healed tissue¹⁷. The present findings agree with literature data showing the occurrence of type III collagen in granulation tissue and its later replacement with type I collagen in the fibrous tissue of the mature scar. This replacement provides more resistance to mechanical tension.

The hypothesis of increased food intake induced by the salty taste of the shrimp¹⁸ was not verified in this investigation. The consequent enhancement of water consumption observed in tables 2 and 3 for the shrimp-enriched diet and salt-enriched diet respectively, suggests an osmotic pressure regulation. The rats of the salt-enriched diet group did not increase the food intake, and no ionic alteration occurred in either group with the shrimp-enriched diet and its control.

Shrimp is also rich in chitin fiber, increasing the total amount of feces in the group that ate shrimp; unfortunately this was only observed, not measured. Chitin is called an animal fiber because of its low digestibility in the animal gastrointestinal tract. However, the percentage of cellulose (10%) was reduced in the shrimp diet to compensate the percentage of chitin present in it. Although the total energy of the two diets differs in about 9%, the result suggests that the increased intake of the shrimp-enriched diet was also due to the reduced energy density of the diet caused by the animal fiber. The animals tried to compensate the lower energy value of this diet by consuming more food.

In conclusion, these data suggest that, even though the groups receiving the two diets ingested the same amount of energy, the shrimp-enriched diet reduced the tensile strength of the scar of the animals, in agreement with folk belief. The next step of this study will be to clarify the mechanism in which shrimp affects tensile strength.

ACKNOWLEDGMENTS

Fernanda K.S. Pereira was sponsored by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

CONTRIBUTORS

E.L.BORGES is the coordinator of this group, designed the study, did biochemical assays, statistical analysis, preparation of the manuscript. F.K.S.PEREIRA daily care of rats in metabolic cages. J.I. ALVAREZ_LEITE preparation of diets. L.R. ALBERTI surgical procedures. M.A.N.D. FERREIRA histology. A. PETROIANU designed the study and preparation of manuscript.

Received on: 30/11/2005

Final version resubmitted on: 10/10/2006

Approved on: 9/1/2007

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Publication Dates

• Publication in this collection
  27 July 2007
• Date of issue
  June 2007

History

• Accepted
  09 Jan 2007
• Received
  30 Nov 2005