INTRODUCTION:

Inflammation is reaction of living tissues towards injury, and comprises systemic and local responses^[1]. Inflammation is part of the complex biological response of vascular tissues to harmful stimuli, such as pathogens, damaged cells, or irritants ^[2]. Inflammation is a protective attempt by the organism to remove the injurious stimuli as well as initiate the healing process for the tissue ^[3]. The nature of the inflammatory response following tissue injury depends upon both the time elapsed since injury as well as the extent of tissue damage. ^[5]. Inflammation can be classified as either acute or chronic ^[4].Acute inflammation is usually of short duration and sudden onset. The typical sequence of events is an initial transient arteriolar constriction followed by vasodilation, increased vascular endothelial permeability, exudation of fluid and plasma proteins, and transmigration of leukocytes from vessels into the injured tissues.

The vasodilation gives rise to calor (heat) and rubor (redness), while the exudation and leukocyte infiltration give rise to tumor ^[5] Dolor (pain) and functio laesa (loss of function) are associated with increased pressure on nerves as a result of tissue edema. There are several causes of increased vascular permeability. The shortest-lived and most reversible follows the release of chemical mediators, such as histamine from mast cells and bradykinin, prostaglandins and serotonin from injured cells. A more long-lived endothelial cell retraction is induced by inflammatory mediators, such as tumor necrosis factor alpha (TNFa) and interleukin-1 (IL-1), following their release from injured cells. ^[6].

Chronic inflammation can begin 2–4 days after the onset of the acute response and can last for weeks to months or years due to the persistence of the initiating stimulus, interference of the normal healing process, repeated bouts of acute inflammation, or low-grade soldering due to continued production of immune response mediators^.[7]. Chronic inflammation on the contrary is granuloma formation ^{[8, 9, and 10]}. Acute and chronic inflammatory disorders account for most of the painful medical problems encountered all over the world ^[11]. The most important mechanism of anti-inflammatory action of NSAIDs ^[12] is the inhibition of cyclooxygenase enzymes which are responsible for the conversion of Arachidonic acid to prostaglandins ^[13] It is believed that current drugs available such as Opoids and NSAIDs drugs are not useful in all cases of inflammatory disorders, because of their side effects, economy and potency ^{[14, 15]}. Inflammation research involves a number of experimental models to study the anti-inflammatory activity ^[16], these models are two types: acute inflammation model and chronic inflammation models ^[17]. The anti-inflammatory agents exert their effect through a spectrum of different modes of action ^[18]. Carrageenan induced edema in the hind paw (acute inflammation) and Cotton pellet granuloma (chronic inflammation) are widely employed in screening the new anti-inflammatory compound ^[19]. Adjuvant induced arthritis is also an often used model of inflammation ^[20].

IN VIVO SCREENING MODELS:

Methods for testing acute inflammation are:

Ø Paw edema in rats (various modifications and various irritants)

Ø UV-erythematic in guinea pigs

Ø Vascular permeability

Ø Oxazolone-induced ear edema in mice

Ø Croton-oil ear edema in rats and mice

Ø Pleurisy tests

Ø Granuloma pouch technique (various modifications and various irritants)

The proliferative phase is measured by methods for testing granuloma formation, such as:

Ø Cotton wool granuloma

Ø Glass rod granuloma

Ø PVC sponge granuloma.^[21]

CARRAGEENAN INDUCED PAW EDEMA MODEL:

Carrageenan – induced paw edema is the most commonly used method in experimental pharmacology. Carrageenan is a sulphated polysaccharide obtained from seaweed (Rhodophyceae), and by causing the release of histamine, 5-HT, bradykinin and prostaglandins it produces inflammation and edema.^[22]

PROCEDURE:

Albino Wistar rats weighing between 150-200gms were divided into 5 groups of 6 rats each; three animals being housed in a labelled cage each. Animals were given a period of time to adjust to the new environment provided with food & water ad libitum ^[21,
23]. The test compounds and standard drugs are administered by oral or intra-peritoneal route. Thirty minutes later, the rats are challenged by a subcutaneous injection of 0.05 ml of 1% solution of carrageenan into the plantar side of the left hind paw. The paw is marked with ink at the level of the lateral malleolus and immersed in mercury column of plethysmometer for measuring the paw volume. The paw is measured immediately after the carrageenan injection and then at 2, 3, 4 and 6 hours. The peak effect of carrageenan usually occurs at 3 hours after the injection ^[24].

EVALUATION:

The increase of paw volume after 3 or 6 h is calculated as percentage compared with the volume measured immediately after injection of the irritant for each animal. Effectively treated animals show much less edema. The difference of average values between treated animals and control groups is calculated for each time interval and statistically evaluated. The difference at the various time intervals gives some hints for the duration of the anti-inflammatory effect. A dose- response curve is run for active drugs and ED50 values can be determined. ^[25][26][21].

ULTRAVIOLET ERYTHEMA IN GUINEA PIGS:

UV augments blood flow and infiltration by blood leukocytes, such as macrophages and neutrophils into the skin, observed clinically as inflammation. Increased production of no and prostaglandins contribute to these events ^{[27, 28]}. UV radiation-induced lipid peroxidation increases production of prostaglandins (pg), including pge2, which in turn cause inflammation in the skin ^[29].

PROCEDURE:

Albino guinea pigs of both sexes with an average weight of 350g are used. Four animals are used each for treatment and control group. Eighteen hr prior testing, the animals are shaved on both the flanks and on the back. Then they are chemically depilated by a commercial depilation product or by a suspension of barium sulphide. Twenty min later, the depilation paste and the fur are rinsed off in running warm water. On the next day the test compound is dissolved in the vehicle and half of the test compound is administered by gavage (at 10 ml/kg) 30 min before UV exposure. Control animals are treated with the vehicle alone. The guinea pigs are placed in a leather cuff with a hole of 1.5×2.5 cm size punched in it, allowing the UV radiation to reach only this area. Then animals are exposed to UV radiation. After 2 min of expose the remaining half of the test compound is administered. The erythema is scored 2 and 4 hr after exposure.

EVALUATION:

The degree of erythema is evaluated visually by 2 different investigators in a double blinded manner. The followings scores are given:

0 = no erythema,

1 = weak erythema,

2 = strong erythema,

4 = very strong erythema.

Animals with a score of 0 or 1 are considered to be protected. The scoring after 2 and after 4 h gives some indication of the duration of the effect. ED50 values can be calculated ^{[30, 21]}.

VASCULAR PERMEABILITY:

During inflammation, vascular permeability increases to allow plasma constituents such as antibodies and complement to access injured or infected tissues. The test is used to evaluate the inhibitory activity of drugs against increased vascular permeability which is induced by phlogistic substances. Mediators of inflammation, such as histamine, prostaglandins and leucotrienes are released following stimulation e.g. of mast cells. This leads to a dilation of arterioles and venules and to an increased vascular permeability. As a consequence, fluid and plasma proteins are extravasated and edemas are formed. The increase of permeability can be recognized by the infiltration of the injected sites of the skin with the vital dye Evan’s blue.

PROCEDURE:

Albino Wistar are used each group containing 4 rats. Control group will receive distilled water 1%w/v 1ml/100g by oral route and other group will receive test compound by oral route and standard group will receive diclofenac 10ml/kg by intra-peritoneal route. After 1h of these administration rats are injected with 0.25ml of 0.6% v/v solution of acetic acid intraperitoneally. Immediately, 10 ml/kg of 10%w/v Evans blue is injected intravenously via tail vain. After 30 min, the animals are anesthetized with ether anaesthesia and sacrificed. The abdomen is cut open and exposed viscera. The animals are held by a flap of abdominal wall over a Petri dish. The peritoneal fluid (exudates) collected, filtered and made up the volume to 10 ml using normal saline solution and centrifuged at 3000 rpm for 15 min. The absorbance (A) of the supernatant is measured at 590 nm using spectrophotometer.

EVALUATION:

Decreased concentration of dye with respected to absorbance indicates reduction in permeability. The result of test is compared with that of standard. ED50 values can also be calculated.^[31][21].

OXAZOLONE-INDUCED EAR EDEMA IN MICE:

The oxazolone-induced ear edema in mice is a model of delayed contact hypersensitivity that permits the quantitative evaluation of the topical and systemic anti-inflammatory activity of a compound following topical administration. The oxazolone-repeated challenge increased the level of Th2 cytokines and decreased that of a Th1 cytokine in the lesioned skin. The Th2 cytokines, especially IL-4, play major roles in the development of dermatitis in the present mouse model.

PROCEDURE:

Mice of either sex with a weight of 25 g are used. Before each use a fresh 2% solution of oxazolone (4- ethoxymethylene-2-phenyl-2-oxazolin-5-one) in acetone is prepared. The mice are first sensitized by application of 0.1 ml on the shaved abdominal skin or 0.01 ml on the inside of both ears under halothane anaesthesia. The mice are challenged 8 days later again under anaesthesia by applying 0.01 ml 2% oxazolone solution to the inside of the right ear (control) or 0.01 ml of oxazolone solution, in which the test compound or the standard is solved. Special pipettes of 0.1 ml or 0.01 ml are used. Groups of 10 to 15 animals are treated with the irritant alone or with the solution of the test compound. The left ear remains untreated. The maximum of inflammation occurs 24 h later. At this time the animals are sacrificed under anaesthesia and a disc of 8 mm diameter is punched from both sides. The discs are immediately weighed on a balance. The weight difference is an indicator of the inflammatory edema. The method can be used for testing both the steroid and non-steroid compounds and for the evaluation of various topical formulations.

EVALUATION:

Average values of the increase of weight are calculated for each treated group and compared statistically with the control group ^{[32, 21, 24]}.

CROTON-OIL EAR EDEMA IN RATS AND MICE:

Croton oil contains 12-o-tetracanoilphorbol-13-acetate (TPA) and other phorbol esters as main irritant agents. TPA is able to activate protein kinase C (PKC), which activates other enzymatic cascades in turn, such as mitogen activated protein kinases (MAPK), and phospholipase A2 (PLA2), leading to release of platelet activation factor (PAF) and AA. This cascade of events stimulates vascular permeability, vasodilation, polymorphonuclear leukocytes migration, release of histamine and serotonin and moderate synthesis of inflammatory eicosanoids by cyclooxygenase (COX) and 5- lipoxygenase (5-LOX) enzymes. COX and 5-LOX inhibitors, leukotriene B4 (LTB4) antagonists and corticosteroids show topical anti-inflammatory action in animal models of Croton oil or TPA induced skin inflammation. ^[33].

PROCEDURE:

For tests in mice the irritant is composed as follows (v/v): 1 part Croton oil, 10 parts ethanol, 20 parts pyridine, 69 parts ethyl ether. For tests in rats the following mixture is prepared (v/v): 4 parts Croton oil, 10 parts ethanol, 20 parts pyridine, 66 parts ethyl ether. The standards and the test compounds are dissolved in this solution. For tests in mice male NMRI-mice with an weight of 22 g, for tests in rats male Sprague-Dawley rats with a weight of 70 g are used. Ten animals are used for controls and each test group. The test compounds are dissolved in a concentration of 0.03 mg/ml to 1 mg/ml for mice and in a 3 to 10 times higher concentration for rats in the irritant solution. On both sides of the right ear 0.01 ml in mice or 0.02 ml in rats are applied. Controls receive only the irritant solvent. The left ear remains untreated. The irritant is applied under ether aesthesia. Four hours after application the animals are sacrificed under anaesthesia. Both ears are removed and discs of 8 mm diameter are punched. The discs are weighed immediately and the weight difference between the treated and untreated ear is recorded indicating the degree of inflammatory edema.

EVALUATION:

The antiphlogistic effect can be determined by expressing the increase in weight of the treated ear as percentage of the weight of the contralateral control ear. The difference between both ears and excised discs is calculated as the average values for treated and control groups and the effect is evaluated by statistical methods ^[21].

PLEURISY TESTS:

In experimental animals pleurisy can be induced by several irritants, such as histamine, bradykinin, prostaglandins, mast cell degranulators, dextran, enzymes, antigens, microbes, and nonspecific irritants, like turpentine and carrageenan. Carrageenan-induced pleurisy in rats is considered to be an excellent acute inflammatory model in which fluid extravasation, leukocyte migration and the various biochemical parameters involved in the inflammatory response can be measured easily in the exudates ^[34].

PROCEDURE:

Male Sprague-Dawley rats weighing 220–260 g are used. The animal is lightly anaesthetized with ether, placed on its back and the hair from skin over the ribs of the right side is removed using animal clippers. The region is swabbed with alcohol. A small incision is made into the skin under the right arm between the seventh and eighth rib. The wound is opened and a further shallow incision is made into the exposed intercostals muscle. 0.1 ml of 2% carrageenan in solution is injected into the pleural cavity through this incision. The injection needs to be made swiftly to avoid the risk of injuring the lung. The wound is closed with a Michel clip. One hour before carrageenan injection and 24 and 48 h thereafter, groups of 10 rats are treated with the standard or the test compound subcutaneously or orally. A control group receives only the vehicle of medication. The animals are sacrificed 72 h after carrageenan injection by ether inhalation. The animal is pinned on a dissection board with the forelimbs fully extended. An incision in the skin over the xiphosternal cartilage is made to free the cartilage from overlying connective tissue. The cartilage is lifted with a forceps and a small cut is made with scissors in the body wall below to gain access into the pleural cavity. One ml of heparinised Hank’s solution is injected into the pleural cavity through this cut. The cavity is gently massaged to mix its contents. The fluid is aspirated out of the cavity using a pipette. This is made easier if the dissection board is raised to an angle of 45–60°; the contents then pool in the corners of the cavity. The aspirated exudate is collected in a graduated plastic tube.

EVALUATION:

One ml (the added Hank’s solution) is subtracted from the measured volume. The values of each experimental group are averaged and compared with the control group. ED50 values can be calculated using various doses ^[21]

GRANULOMA POUCH TECHNIQUE:

It is one of the classical methods for measuring subacute inflammation in rats. Earlier croton oil, freund adjuvant and lipo-polysaccharide were used in rats which led to granuloma formation. This replaced by carrageenan as a phlogistic agent for the induction of granuloma. ^[24] With the introduction of an irritant substance into an s.c. air pocket, granulation tissue begins to proliferate and soon covers the whole inside of the pouch. This tissue consists of fibroblasts, endothelial cells and an infiltrate of macrophages and polymorphonuclear leukocytes ^{[35, 36]}.

PROCEDURE:

Male or female Sprague-Dawley rats with a body weight between 150 and 200 g are used. Ten animals are taken for controls and for test groups. The back of the animals is shaved and disinfected. With a very thin needle a pneumoderma is made in the middle of the dorsal skin by injection of 20 ml of air under ether anaesthesia. Into the resulting oval airpouch 0.5 ml of a 1% solution of Croton oil in sesame oil is injected avoiding any leakage of air. Forty-eight hours later the air is withdrawn from the pouch and 72 h later any resulting adhesions are broken. Instead of croton oil 1 ml of a 20% suspension of carrageenan in sesame oil can be used as irritant. Starting with the formation of the pouch, the animals are treated every day either orally or subcutaneously with the test compound or the standard. For testing local activity, the test compound is injected directly into the air sac at the same time as the irritant. On the 4th or the 5th day the animals are sacrificed under anaesthesia. The pouch is opened and the exudate is collected in glass cylinders. Controls have an exudates volume between 6 and 12 ml, which is reduced dose dependent in the treated animals.

EVALUATION:

The average value of the exudates of the controls and the test groups is calculated. Comparison is made by statistical means. A clear dose response curve could be found by S.C. injection of 0.5, 1.0 and 2.0 mg hydrocortisone acetate/rat. Also doses of 1.5 mg/kg indomethacin were found to be active ^[21].

METHODS TO EVALUATE PROLIFERATIVE PHASE OF INFLAMMATION:

COTTON WOOL GRANULOMA

The foreign body granulomas were provoked in rats by subcutaneous implantation of pellets of compressed cotton. After several days, histologically giant cells and undifferentiated connective tissue can be observed besides the fluid infiltration. The amount of newly formed connective tissue can be measured by weighing the dried pellets after removal. More intensive granuloma formation has been observed if the cotton pellets have been impregnated with carrageenin.

PROCEDURE:

Male Wistar rats with an average weight of 200 g are anaesthetized with ether. The back skin is shaved and disinfected with 70% ethanol. An incision is made in the lumbar region. By a blunted forceps subcutaneous tunnels are formed and a sterilized cotton pellet is placed on both sides in the scapular region. The pellets are either standardized for use in dentistry weighing 20 mg or pellets formed from raw cotton which produce a more pronounced inflammation than bleached cotton. The animals are treated for 7 days subcutaneously or orally. Then, the animals are sacrificed, the pellets prepared and dried until the weight remains constant. The net dry weight, i.e. after subtracting the weight of the cotton pellet is determined.

EVALUATION:

The weight of the transudate and the granuloma as well as the percent granuloma inhibition of the test drugs was calculated. The body weight gain was also recorded ^{[37, 38,
39, 21, 24]}.

RHEUMATOID ARTHRITIS:

Rheumatoid arthritis is characterized by chronic swelling and inflammation of the synovial membrane that lines joints. As rheumatoid arthritis progresses, the linings of joints degenerate, leading to severe pain and decreased joint mobility and significantly impacting on the quality of life ^[40]. RA is genetically associated with the major histocompatibility complex (MHC) class II antigen HLA-DR4. The different HLA-DR4 alleles associated with RA susceptibility share a common amino acid motif in the peptide-binding groove ^[41].

RAT ADJUVANT-INDUCED ARTHRITIS MODEL:

The original use of the rat adjuvant-induced arthritis model was focused on the eicosanoid pathway and in particular on inhibitors of cyclooxygenases (COX 1 and COX2)—the NSAIDs (non-steroidal anti-inflammatory drugs) ^[42].

PROCEDURE:

Arthritis was induced by a single intra-dermal injection (0.1 ml) of Freund’s Complete adjuvant (FCA) containing 1.0 mg dry heat-killed Mycobacterium tuberculosis per milliliter sterile paraffin oil into a foot pad of the left hind paw of male rats.^[43] A glass syringe (1 ml) with the locking hubs and a 26G needle was used for injection. The rats were anesthetized with ether inhalation prior to and during adjuvant Injection, as the very viscous nature of the adjuvant exerts difficulty while injecting. The swelling paws were periodically examined (up to 21 days) in each paw from the ankle using Digital Plethysmometer ^[44].

PAPAYA LATEX INDUCED ARTHRITIS:

PROCEDURE:

Papaya latex induced model of experimental rheumatoid arthritis has been developed to test the anti-inflammatory activity of slow reaction antirheumatic drugs (SARDs).0.1 ml of 0.25 % solution of papaya latex (prepared in 0.05 M sodium acetate buffer, pH 4.5 containing 0.01% thymol) is injected into the rat hind paw. The peak effect occurs at 3 hours and lasts for more than 5 hours. The method is sensitive for evaluating NSAIDs like aspirin, ibuprofen and steroidal anti-inflammatory drugs (SAIDs). Papaya latex induced inflammation is known to be mediated through prostaglandins ^[26].

COLLAGEN-INDUCED ARTHRITIS:

CIA is induced by intradermal injections of CII together with an adjuvant. Most commonly, heterologous CII of bovine, chick or rat origin is used to induce the disease. CIA is major histocompatibility Complex (MHC) dependent and is characterized by erosive joint inflammation mediated by both T and B cells. The pathogenesis and disease course vary considerably, however, depending on the genetic background of the mice and on the origin of the CII used for disease induction. A bottleneck in the development of disease is the T cell recognition of an immunedominant CII-derived peptide bound to MHC class II with a certain affinity ^[45].

PROCEDURE:

All mice were 8-10 wk old when immunized. Native type II collagen was dissolved in 0.1 N acetic acid at a concentration of 2 mg/ml by stirring overnight at 4°C. Heat-killed Mycobacterium tuberculosis was ground with a mortar and pestle and added to incomplete Freund's adjuvant (IFA). Equal volumes of adjuvant and collagen solution were emulsified for 2 min at high speed with a homogenizer. Each mouse received an intradermal injection of 0.1 ml of the resulting emulsion in the tail. Other groups of mice were immunized in the same manner with 100 µg of denatured CII, or CB-peptides 8, 9, 11. A booster injection of 100 ~g of antigen emulsified in IFA was given by intradermal injection in the dorsal skin on day 28. A control group received an equal volume of 0.1 N acetic acid emulsified with IFA and containing 250 µg of Mycobacterium tuberculosis as a primary injection, and acetic acid in IFA alone as a booster on day 28.

EVALUATION:

An arthritic index was assigned to each mouse by using the following criteria: 0, no signs of arthritis; 1, swelling and redness in a single joint; 2, inflammation in multiple joints; and 3, severe swelling, joint erosion, and/or ankylosis. Each paw was scored from 0–3, and the arthritic paws then were multiplied by their score with the index being the sum of all of the paws ^[46].

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Received on 25.10.2013 Accepted on 01.12.2013

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