ACUTE INFLAMMATION INDUCED BY AGAR: A MODEL FOR IN

VIVO PHARMACOLOGICAL ANALYSIS

TROVÃO, J. E.1, ÁVILA, P. E. S.2, BASTOS, G. N.T3, DO NASCIMENTO, J. L.M4.

1,2,3,4Laboratório de Neuroquímica Celular e Molecular, Universidade Federal do Pará, Belém (PA), BrasilE-mail: josetrovao@gmail.com

Abstract. Inflammation is a reaction of tissue to injury, expressed by five cardinal points: pain, swelling, redness, heat and loss of function. There are standard models for analysis of the pathophysiological process. One pharmacological model is experimental air-pouch used to study the mechanisms of anti-inflammatory drugs. The hydrocolloid agar is a polycationic, consisting agarose and agaropectin used in food, pharmaceutical and cosmetics, and together with the carrageenans, It’s a polysaccharide from red algae. In the present study has adapted the model of the air-pouch, replacing the phlogistic agent carrageenan  by the agar. The objective of this work is to standardize the air pouch model using the agar in rats Wistar. The pouch were produced by a subcutaneous injection of sterile air in the intraescapular, forming a cavity. In the first and second day were injected 10 ml of air in the third day the agar was administered and after 16 hours the samples were collected. Five concentrations of agar 1%,  2%, 3%, 4% were tested. Getting the 2% concentration, as more satisfactory, further tests were performed with groups carrageenan, 2% agar, 2% agar + celebra, 2% agar + Aspirina,  for to infer the mechanism of action. All agar concentrations induced inflammation, however, the group 2% was most evident vasodilation, increased production of nitric oxide, lack of necrosis, greater cell migration and production of exudate similar to carrageenan. The groups treated with agar and anti-inflammatory drugs celebra and aspirina reduced exudation and cell migration. The model produced significant differences in the induction of inflammation, compared to the carrageenan, as observed with this signs of inflammation. Thus, the model of acute inflammation induced by agar demonstrated applicability for use in experimental studies of drugs with potential anti-inflammatory activity that are in any presentation: pure substances, extracts or nanoparticle..

Keywords: Inflammation, Air-pouch, Ágar.

INTRODUCTION.

For new substances with anti-inflammatory effects is tested, it is necessary to produce the inflammation in the tissue in order to prove that their use is effective.

For this, there are several experimental techniques that induce the process of acute inflammation such as paw edema model [described by Levy (1969)], air-pouch, pleurisy model [descrito por HENRIQUES et al, (1990)], among others, but it requires the use of a pro-inflammatory agent (phlogistic agent) as carrageenan, LPS, bradykinin, dextran and zymosan.

However, the phlogistic agent have a high value, which increases the implementation costs of the study. Thus the process of identifying a pharmacological agent with properties that result in clinical benefits ends being slow and expensive. The experimental model of inflammation air-pouch, previously described by Selye (1953) and modified by Ghosh et al (2000) is used to study the mechanisms of inflammatory responses, besides serving as a model for various treatments with anti-inflammatory drugs (Ellis et al., 2000). This model

consists of a subcutaneous injection of sterile air in the region intraescapular being subsequently injected into an inflammatory agent (carrageenan).

The air cavity formed subcutaneously in the non-inflamed pocket is lined with a thin layer of fibroblasts and macrophages, similar to the synovial layer. Injection of carrageenan into the cavity produces an inflammatory reaction characterized by infiltration of cells, and increased exudate production of inflammatory mediators such as prostaglandins, leukotrienes and cytokines (EDWARDS et al., 1981).

The polycationic hydrocolloid agar is widely used in food, pharmaceutical and cosmetic industries. The hydrocolloid is carrageenans together with one red seaweed polysaccharide and consists of a mixture of agarose and agaropectin (ARMISEN; GALATAS, 2009)..

MATERIALS AND METHODS.

Animals.

For this study, we used 50 male Wistar rats weighing between 150 and 220 grams, supplied by the vivarium of Universidade Federal do Pará The animals were maintained with water and balanced ration ad libitum.

Air-Pouch.

The air-pouchs were produced by a subcutaneous injection of 10 ml of sterile air in the intraescapular forming a cavity (bubble).

On the first day was injected 10 ml of sterile air, the second day was added additional 10 ml of sterile air for maintaining the bag, on the third day the animals was administered into the agar and, after 16 hours, there sacrifice.

Experimental Groups.

The animals were divided into eight groups that were used in different concentrations of 1% agar, 2%, 3%, 4% and the control group. Subsequently, after checking the optimal concentration to be used, an experiment was performed in which animals received 2% agar and these were treated with anti-inflammatory celecoxib, a selective COX-2, or acetylsalicylic acid, COX-1 and COX-2, to analyze the mechanism of action. Furthermore, analyzes were performed with animals treated with the substance carrageenan.

Analysis of the exsudato.

After therapeutic procedures in the treated groups, animals were sacrificed in a chamber of carbon dioxide (CO2). The wells were washed subcutaneously with 1 ml of saline (0.9%) and EDTA (1 mM). Then, the exudates were collected and analyzed for volume and the total number of leukocytes.

The leukocyte count was performed on smears stained with Giemsa staining.

Evaluation of Vasodilatation of experimental groups.

After removal of the exudate, evaluation was made in vasodilatation this dorsal area, which was recorded by photography.

Evaluation of nitrergic activity in Inflammatory Exudate.

The nitric oxide production in the exsudato supernatant after treatment with agar was assessed by measurement of metabolite nitrite, using the method of Griess reagent (1% sulfanilamide and nafitiletileno 0.1%) (Grenn et al, 1982), after 10 minutes of reaction, the samples were read in a microplate reader with wavelength of 570 nm.

So that interference does not happen accumulation of exudate proteins during the analysis in the microplate reader, the samples were diluted 1:1 with a solution of 3% zinc sulfate and centrifuged for 5 minutes at 10,000 rpm, prior to the procedure pattern.

Evaluation of Activity Through the painful test Lick Paw.

To evaluate the nociceptive activity caused by the agar solution was performed to test the time of paw licking the adapted model and Hunskaar hole (1987). Was injected in 1% formalin into the plantar region of the right paw of the animals. The reactions of licking the affected paw were recorded in the first phase, which corresponds to the initial 5 minutes and the second phase that corresponds to the final 15 minutes, corresponding to the nociceptive and inflammatory pain, respectively. Each animal was examined for 30 minutes.

Statistical Analysis

The data collected was used variance analysis (ANOVA), followed by the Bonferroni test. Being used levels of significance of p <0.05 and p <0.001.

RESULTS

Evaluation Process-Induced Vasodilation in Agar Air-Pouch Model.

In Figure 1 can be observed in varying degrees of vasodilation different experimental groups.

In the control group (saline), vasodilation was not observed (Figure 1A). In the group treated with a solution of 1% agar was not observed any change in the control group (Figure 1B). While in the group treated with a solution of 2% agar is observed a strong vasodilation (Figure 1C). In the group administered with a solution of 3% agar, an increase in vasodilation, with respect to group 2%, and the onset of ischemic points, which suggests evidence of necrosis (Figure 1D).

In the group treated with a solution of 4% agar were perceived increase in the number of points ischemic been observed also foul odor to make the opening of the air pocket (Fig. 1E).

It should be noted an important fact observed in Figures 1D and 1E, which was the formation of agar gel, caused by the gelation of this polysaccharide.

Figure 1: (A) only with animals given saline, (B) animals administered with a solution of 1% agar, in (C) animals administered with a solution of 2% agar, in (D) animals administered with solution 3% agar, in (E) animals administered with a solution of 4% agar. The arrows show the yellow vasodilation, increased according to the treatment group in question, while arrows indicate green indication of the process of necrosis.  The area in question is the back of the animals, which were injected doses of sterile air, and these solutions during the experiment. Source: Data from author

Evaluation of nitrergic activity in inflammatory exudate into Agar-Induced Model of Air-pouch.

Graphic1: Production of nitrite in the experimental groups treated with a solution of 1% agar, 2%, 3%, 4% and a control group (saline). All groups treated with the substance agar showed significant differences when compared to the control group (** p <0.001).Source: Data from author.

In this analysis, we compared the concentrations of nitrite, a metabolite of NO, between the control and treated groups with a solution of agar (1%, 2%, 3% and 4%).

In graphic 1, there was a dose-dependent increase in the nitrite concentration of metabolite, but the group treated with 4% agar, decreased by 14% compared to group 3%. All groups treated with a solution of agar showed highly significant differences (p <0.001) when compared to the control group (saline).

Evaluation of Cell Migration into Inflammatory Exudate Agar-Induced Model of Air-Pouch.

Graphic 2: Total cell count experimental group treated with solution of 1% agar, 2%, 3%, 4% and a control group (saline). * p <0.05 and ** p <0.001 compared to control group.Source: Data from author.

In this test was to assess the total amount of exudates present in cells induced agar. In grapic 2, it can be seen that the agar has promoted an increase in cell migration with increasing concentration of agar solution administered up to a concentration of 3%. As seen in the graph the concentration of 4% promoted a decrease in cell migration compared to the concentration of 3%, but this result was still superior to the group that deal only with saline.  Significant differences between control group and groups treated with a solution of agar (p <0.05). The groups which are outstanding 3% and 4% (p <0.001) and group 2% (p = 0.0032), which showed significant differences compared to the control group. 

Evaluation of the Volume-Induced Inflammatory Exudate Agar Model for Air-

pouch.

Graphic 3: Volume of exudate (ml) of the experimental groups treated with a solution of 1% agar, 2%, 3% and 4% and group control * p <0.05.Source: Data from author.

To evaluate the relationship between dosage and agar effect on the inflammatory exudate, there was an analysis of the volume of exudate formed.

In Graphic 3, there is a dose-dependent increase in the production of exudate plasma when compared to control.

Statistical analysis showed a significant difference only in group 4% compared to the control group. The groups treated with a solution of agar, when compared, significant differences compared to the group only 4%.

Count the cells differentiated Induced Inflammatory Exudate Agar 2% in Air

Pouch Model.

Leukocytes Eosinophils Lymphocytes Monocytes Neutrophils

Average ± DP 6,00 ± 5,0 6,63 ± 10,4 27,33 ± 2,5 60,33 ± 8,3Table 1: Differential leukocyte counting.Source: Data from author.

Figure 2: Neutrophils and monocytes present in the inflammatory exudate group of 2% agar.Source: Data from author.

The differential count was performed in order to identify cell types which are induced by 2% agar solution.

As the control group (saline) does not exude cell was not possible to compare group with 2% agar.

The analysis of the data in Table 1 and Figure 2 show a large population of neutrophils followed by monocytes and small amount of lymphocytes and eosinophils.

Evaluation of painful activity, induced a 2% agar in Model Lick Paw.

Grafiphic 4: Average duration of paw licking.Source: Data from author.

Graphic 5: Average time licking the paw. The column red expresses the time of the animals under the effect of the substance 2% agar (with pH adjusted to 2.5).Source: Data from author.

To check the activity of nociceptive solution of 2% agar was performed measuring the time of paw lick.

Analyzing the data, as shown in graphic 4, it is perceived that there was no significant difference between the substance and saline solution agar (neutral pH) in the induction of pain in both phases of testing.

As shown in grapic 5, the change of pH of 2% agar, licking time in the animals treated with the solution of 2% agar, pH modified with (2:5) showed no significant difference in nociceptive phase (0 -5 '). During the inflammatory phase (15-30 '), the substance agar also showed significant difference compared to the control group, treated with saline, and no difference with the group treated with formalin, a substance which is standard for this test.

Evaluation of Anti-Inflammatory Activity Patterns in Process-Induced Vasodilation Agar 2% in Air Pouch Model.

Figure 3: In (A) animals administered with carrageenan, (B) administered with a solution of 2% agar, in (C) animals administered with a solution of 2% agar / Celecoxib, in (D) animals administered with a solution of 2% agar / ASS. The area in question is the back of the animals, which were injected doses of sterile air and the solutions mentioned above.Source: Data from author.

Figure 3 shows the process of vasodilation induced by carrageenan phlogistic agent (A), 2% agar (B) treated with 2% agar Celecoxib (C) and 2% agar treated with acetylsalicylic acid (D).

It is observed in Figure 3B vasodilation caused by 2% agar solution, being greater than the vasodilation induced by carrageenan substance.

Furthermore, it is evident intense redness caused by the increase in vascular tone location higher in the group treated with solution of 2% agar in comparison with the animal treated with carrageenan.

Figure 3C, celecoxib blocked the activity of 2% agar which promoted the considerable decrease in vasodilation compared with groups 2% carrageenan and agar. In Figure 3D, aspirin blocked the activity of 2% agar which promoted the reduction of vasodilation and flushing process, compared to groups carrageenan and agar 2%, yet this group had a greater vasodilation compared to the group treated with celecoxib.

Evaluation of Anti-Inflammatory Activity in Activity Patterns Induced Nitérgica Agar 2% in Air Pouch Model.

Graphic 6: Concentration of nitrite experimental group treated with solution of 2% agar, carrageenan, agar 2% / 2% agar celecoxib and / acetylsalicylic acid (AAS).Source: Data from author.

The evaluation of the nitrite concentration was performed to characterize the effects of anti-inflammatory celecoxib and acetylsalicylic acid in the animals administered with solutions of 2% agar, and identify the mechanism of action of the substance agar.

The analysis of Graphic 6 shows a reduction in the concentration of metabolite in the groups treated with nitrite celecoxib (85%) and ASA (81%) as compared to group that received treatment with a solution of 2% agar. This fact confirms the reduction in the vasodilation of those groups, as shown in Figure 10C and 12D.

The group treated with 2% agar showed higher concentration of nitrite, compared to the carrageenan group (p <0.05).

Evaluation of Anti-Inflammatory Activity Patterns in Cell Migration Induced Agar 2% in Air Pouch Model

Graphic 7: Total cell count experimental group treated with solution of 2% agar, carrageenan, agar 2% / 2% agar celecoxib and / acetylsalicylic acid (AAS).Source: Data from author.

The analysis of total cell count was performed in order to evaluate the effects of anti-inflammatory celecoxib and acetylsalicylic acid on cell migration and to compare the effect of agar carrageenan.

Cell migration in the groups treated with celecoxib and acetylsalicylic acid showed a significant reduction when compared to group treated with 2% agar (p <0.05).

The groups treated with anti-inflammatory celecoxib and acetylsalicylic acid reduced 55% and 40% the number of cells, respectively, compared to group 2% agar. The acetylsalicylic acid and celecoxib groups showed no significant differences when compared. Groups 2% agar and carrageenan did not show significant differences when compared.

Evaluation of Anti-Inflammatory Activity Patterns in Volume-Induced Exudate Agar 2% in Air Pouch Model

Graphic 8: Volume of exudate experimental group treated with solution of 2% agar, carrageenan, agar 2% / 2% agar Celecoxib and / Acetylsalicylic Acid (AA).Source: Data from author.

The anti-inflammatory effects of celecoxib and acetylsalicylic acid were analyzed by the production of exudate formed, and comparisons made with the group 2% agar and carrageenan.

The production of exudate from the group agar was 86% higher when compared to the carrageenan. Celecoxib groups and ASA were reduced by 72% and 63% respectively, compared to group treated only with 2% agar.

The volume of exudate collected from the agar 2% group showed a significant difference compared to the carrageenan group (p <0.05). The groups treated with celecoxib and aspirin had a significant decrease in the volume of the exudate, as compared to those administered only with a solution of 2% agar.

The groups celecoxib, aspirin and carrageenan group showed no significant differences when compared.

DISCUSSION

The purpose of this study was to assess the activity of the phlogistic agar hydrocolloid in an experimental model of air pouch in Wistar rats. As well as its ability to observe its mechanism nociceptive and flogistic The results demonstrate that the substance agar, 2% concentration, phlogistic activity produced by activating the prostaglandin pathway, it has not been able to produce a nociceptive response in a short time. In the present study we used the experimental model of air bag to prove the efficacy of hydrocolloid agar as phlogistic agent, using the pharmacological model of air pouch, which can demonstrate the four cardinal signs of inflammation: swelling, redness, heat and loss function. Inflammation may be considered as classical biological response of an organism, in injury. The expression of the inflammatory process is characterized by five cardinal signs: pain, heat, swelling, erythema and loss of function (RUSSEL, 2005). To assess the amount of agar suitable for use solutions were tested with different concentrations of agar and were assessed for cell migration, nitrite concentration and volume of exudate to the cardinal signs of inflammation were shown.

The ON increases vascular permeability and an intermediate in the production of prostaglandins, for lipid oxidation, and is a potent vasodilator (DAVIS et al., 2001). The results show that injection of agar in the air pouch cavity, causes an increase in the concentration of nitrite, as the concentration of the solution administered (Graphic 1) induced inflammation in agar. These results are confirmed by Szabo, 1996, 1998, which showed that during the first hours after an insult capable of initiating an inflammatory process, production of NO by iNOS-mediated begins to be upregulated, resulting in a burst release of NO, which exceeds the basal levels of free radicals. This production, increased NO, leads a cellular damage. First, NO may directly promote an exacerbation of peripheral vasodilation, resulting in a vascular decompensation, NO may also positively regulate NF-κB by initiating an inflammatory signaling pathway that culminates in the production of proinflammatory cytokines (SZABO, 1996, 1998 , cited, Horton, 2003).

This production of nitric oxide induced by agar may be influencing the process of vasodilation and cell migration (Graphic 2 and 3). According to Szabo and Bechara, 2006, during the acute inflammatory process is no change in vessel diameter, blood flow and vascular permeability which triggers cell migration. Salvemini and colleagues (1995) when used for iNOS inhibitors in inflammatory model of air bag and found that there was an anti-inflammatory action, not only by blocking iNOS, but also by decreased cellular infiltration and prostaglandins in the area where he was going the inflammatory process.

One of the first signs of inflammation in the microcirculation is the elevation of endothelial permeability. The endothelium becomes fragile for practically all the components contained in the plasma, water and ions, and a large number of protein species. The mechanisms by which permeability occurs involving transmembrane signaling and responses

in endothelial cells, with separation of proteins in the VE-cadherin junctions (Schonbein, 2006).

To prove this cardinal sign was measured the volume of exudate formed in the bag (Figure 3) which showed that the agar promoted an increase in vascular permeability, as the increase in concentration of the solution administered.

During exudation, activated enzymes in plasma or interstitial cells exsudated enzymes act on the ground substance of proteoglycans and break molecules, increasing the hydrophilicity site. Due to the abundance of hydroxyl groups, carboxyl and sulphate chain carbohydrate in most glisosaminoglicanas, proteoglycans are strongly hydrophilic and act as polyanions (BOGLIOLO, 2006; JUNQUEIRA, CARNEIRO, 2004).

The agar hydrocolloid possesses the property of retaining water, and colloidal substances forming water activity controlling a system, being formed by agarose with a small amount of sulfate and agaropectin, which is sulfated and has acid residues. (Hanus et al., 1967; Raven, 2005). With this we can infer that the increase in the volume of exudate formed, induced by agar, due to the action of NO in relaxation of blood vessels associated with the hydrophilic property of the agar in the interstitial space.

For confirmation of this event cell counts were performed in cells present in the exudate collected. All experimental groups (Graphic 2) treated with a solution of agar significant differences in cell migration, confirming that the agar substance induces the production of chemokines, with consequent cell migration to the site of inflammation.

Moreover, the leukocyte differential count agar 2% group showed a prevalence of neutrophils followed by monocytes and small amounts of eosinophils and lymphocytes (Table 2). Studies by Ramalho-Garcia et al., 2002 showed the role of inflammation resident cells which when stimulated release TNF-α, responsible for the synthesis of chemokines and, consequently, the migration of leukocytes. Other cytokines such as IL-1 and IL-6 also participate in this process. During the development of inflammation, cell migration of leukocytes is initiated by a significant number of polymorphonuclear neutrophils, followed by migration of monocytes. One of the main points on which we can evaluate the activity of the agar is phlogistic cell migration of polymorphonuclear leukocytes (PMN) and monocytic cells to air pocket region (Table 1). This increase in leukocyte migration could be due to induction of expression of adhesion molecules that are responsible for the "rolling" of leukocytes over endothelial. This process can be explained by the activation of nuclear transcription factors such as, NF-κB part which is responsible for transcription of genes iNOS, COX-2 and vascular cell adhesion molecule 1 (VCAM-1) (MARTIN et al., 2000; KIM et al., 2010). The binding of inflammatory mediators to receptors present on macrophages results in the phosphorylation and degradation of IκB and translocation of NF-κB to the nucleus and binds the promoter regions of DNA which will initiate the transcription of other inflammatory mediators (LASKIN et al. 2001).

As pain is involved in the inflammatory process in the experimental pharmacology paw licking test was introduced in 1967, which is performed in the subcutaneous injection of formalin produced a biphasic response (SHIBATA, 1989). The formalin test has two distinct phases of nociception, the first phase takes place immediately after intraplantar application of formaldehyde solution (the first five minutes) and the second stage corresponds to twenty minutes after injection. Thus, to evaluate the action of painful solution of agar used model paw lick. After tissue injury, inflammatory mediators are released promoting a synergistic way, a change in the transduction mechanism of the peripheral nociceptive stimulation, which increases the sensitivity of the transduction of high threshold nociceptors, exaggerated response to suprathreshold noxious stimuli and spontaneous pain (CARVALHO ; LEMÔNICA, 1998). Although the solution phlogistic activity agar 2%, not shown nociceptive activity, probably due to the time frame. Thus, the test was repeated with pH

adjustment the solution of 2% agar, to 2.5. According to Burke, 1933 formalin solution has a pH due to the biphasic response of the formalin solution which promotes an injury site injected. This observation confirms our results demonstrate that activation of nociceptors after the change of pH of the solution of agar used.

Given the results above, was chosen solution of 2% agar as the optimal concentration for our model of inflammation. However, OKOLI et al., 2005, agar used in the model of induced paw edema. However, the described solution used was the concentration of 3% (v / v). In the experiments of this work were used solutions 1-4%, and it was observed that the highest concentration hydrocolloid has the ability to form a gel solution which needs to be constantly at a temperature of 37 ° C as the solution viscosity hampers administration of the substance, leading to easy clogging of the syringe.

In the second stage of labor, with the agar solution chosen, we tested the activity of anti-inflammatory patterns, celecoxib, and acetylsalicylic acid on the type of air bag. The results demonstrated that aspirin an anti-inflammatory estereoidal and celecoxib, a selective inhibitor of COX-2 inhibited the activity of the phlogistic agar (Graphics 6, 7 and 8).

The anti-inflammatory action of acetylsalicylic acid is due to the inhibition of prostaglandins and thromboxane by acetylation and blocking the catalytic activity of COX-1. Aspirin and other derivatives of the anti-inflammatory drugs (NSAIDs) lipooxigenaxe not inhibit, and thereby do not suppress the formation of leukotrienes (CARVALHO, LEMÔNICA, 1998; SERHAN et al., 2008).

Celecoxib exhibits anti-inflammatory, antipyretic and analgesic properties attributed to selective inhibition of COX-2. At therapeutic concentrations, celecoxib does not inhibit COX-1 does not alter platelet function (GILMAN, 2004).

The vasodilation is effected by mediators, including histamine, ON, prostaglandin (PGE2) and prostacyclin (PGI2). Furthermore, PGE2 PGI2 and cause redness and heat the tissue due stimulating increase local blood flow (BOGLIOLO, 2006; COTRAN 2001; RANG et al., 2006).

In the qualitative test of vasodilation was greater vasodilation and flushing the group agar 2% compared to the carrageenan group, suggesting an increased release of inflammatory mediators by substance agar. In groups treated with celecoxib and acetylsalicylic acid vasodilation and redness were removed by the action of these drugs (Figure 3). The activity of celecoxib suggests that the agar can be induced COX-2 in inflammation (HILARIO et al., 2006).

The results demonstrate that the activity induced by nitrergic agar is blocked by anti-inflammatory patterns used (Graphic 6). Jung et al (2010) analyzed the anti-inflammatory action of n-Propyl Gallato through the negative regulation of NF-κB in cultures of macrophage line RAW 264.7, coming to the conclusion that there is a decrease in concentration of nitrite in the treated cells with substances that have anti-inflammatory activity, this decrease is probably a result of inhibition of nuclear transcription factors (NF-κB). In experimental models of inflammation induced by carrageenan in air bag, there is an increased activation of NF-κB (Crippen, 2006), and when animals are treated with dexamethasone (anti-inflammatory steroid) for a reduction in the markup for NF -κB has been made when an immunohistochemistry assay for tissue removed from the stock area (Ellis et al., 2000). Analyzing the results of concentration of nitrite, it was observed that the agar could be inducing the activation of NF-κB.

Several studies have suggested that NSAIDs may act directly on the surface of mononuclear cells by preventing the migration of these cells to sites of inflammation. Interestingly, immunosuppressive agents may exert an anti-inflammatory activity by reducing the infiltration of PMNs (DUKE et al., 1973, apud, MEACOCK & KITCHEN, 1976). MEACOCK & KITCHEN (1976) testing the performance of various NSAIDs such as

indomethacin, phenylbutazone, ketoprofen, ibuprofen, acetylsalicylic acid a, fenoprofen and naproxen in the process of cell migration, they concluded that none of the tested drugs prevented the migration of PMNs in a carrageenan-induced inflammation, however, certain NSAIDs tested suppressed the migration of mononuclear cells (monocytes).

The decrease in leukocyte migration observed in the results (Graphic 7) may be due to blocking the expression of adhesion molecules that are responsible for "rolling" of leukocytes along the endothelium. This blocking the expression of these molecules can be demonstrated by inhibition of nuclear transcription factors such as, NF-κB part which is responsible for transcription of genes for iNOS, COX-2 and vascular cell adhesion molecule 1 (VCAM-1) (Martin et al ., 2000, Kim et al., 2010).

CONCLUSION

The present study demonstrated that the hydrocolloid agar can be used as template in phlogistic agent air bag (air-pouch), since this promotes the plasma and leukocyte exudation into the cavity of the bag, induction of vasodilation and redness.

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