In vivo Studies for wound healing activity

IN VIVO STUDIES FOR WOUND HEALING: 

IN VIVO STUDIES FOR WOUND HEALING Presented By, D.Kumarasamyraja Research scholar, Department of pharmacy, Annamalai University, Chidambaram.

INTRODUCTION : 

INTRODUCTION The small mammals have emerged as the model of choice for such studies , Like wound healing activity, which are beneficial for multiple reasons. They are inexpensive, easily obtainable, require less space, food, and water, easy to maintain, and can be genetically modified. Additionally , they often have multiple offspring, which develop quickly allowing experiments to proceed through multiple generations. Small animals usually have accelerated modes of healing in comparison to humans, thus experiment duration lasts for days, in contrast to weeks or months in human experiments. Some small mammals can easily be altered genetically and provide a wound model capable of approximating defective human conditions such as diabetes, immunological deficiencies, and obesity . Another advantage of small mammal models is their ability to serve in experiments where death is an endpoint.

ANIMAL HANDLING : 

ANIMAL HANDLING Animal of either sex, same age group, and approximately of similar weight are employed following an acclimatization period of 2–14 d. they are maintained at a well ventilated animal house under standard controlled conditions at temperature 22 } 1 ◦ C to 30 } 1 ◦ C, relative humidity 35 } 5 to 65 } 5%, and kept under either 10/14 or 12/12 h light/dark cycles with free access to food and water ad libitum . The animals are housed individually in clean, sterile polyvinyl/propylene/metal cages containing autoclaved paddy husk or paper cuttings as bedding. The animals are fasted/starved for 12–14 h before tests to achieve better drug absorption through the gastrointestinal tract but given unrestricted access to clean drinking water [25].

WOUND CREATION : 

WOUND CREATION All the surgical interventions are carried out under sterile conditions under general anaesthesia . The predetermined area for wound infliction at the back of the animal is prepared for surgery by removing hairs with depilatory cream/shaving machine/razor. The animal is anaesthetized with anaesthetic ether/chloroform by open mask method or intraperitoneally with anaesthetic drug (35 mg pentobarbitone sodium/25mg thiopentone sodium/10mg ketamine/40mg thiopental/60 mg pentobarbital sodium/0.3mg chloralhydrate solution per kg body weight of an animal).

WOUND CREATION: 

WOUND CREATION The animals are allowed to recover, housed individually in their cages, and monitored for respiration, color, and temperature. They are maintained under standard husbandry conditions and on a uniform diet and managed throughout the experimental period. Animals are closely observed for any infection; those who show signs of infection are separated and excluded from the study. They are periodically weighed before and after the experiments.

EXCISION WOUND MODEL : 

EXCISION WOUND MODEL Excision wounds are inflicted on the dorsal thoracic region 1–1.5 cm away from the vertebral column on either side and 5 cm away from the ear. After wound area preparation with 70% alcohol, using a sterile round seal of 2.5 cm diameter or a surgical blade or 5–8mm biopsy punch, the circular skin from the predetermined area on the depilated back of the animal is excised to its full thickness to obtain a wound area of about 200–500mm2 diameter and 2mm depth. Haemostasis is achieved by blotting the wound with a cotton swab soaked in normal saline. The respective therapeutic treatment is administered either orally or topically to the animals of respective groups until complete epithelialization starting from the day of operation. Collagen estimation, percentage wound contraction, and period of epithelialization parameters are studied

INCISION WOUND MODEL : 

INCISION WOUND MODEL After wound area preparation with 70% alcohol, two longitudinal Para vertebral incisions are made through the skin and cutaneous muscles at a distance of about 1.5 cm from the midline on either depilated side of the vertebral column with a sterile sharp surgical blade. Each incision made is 4–6 cm in length, and after complete haemostasis, the parted skin is stitched with interrupted sutures, 0.5–1.0 cm apart using black braided silk surgical thread (no. 000) and a curved needle (no. 11). The continuous threads on both wound edges are tightened for good closure of the wound. The wounds were left undressed and mopped with a cotton swab. The respective therapeutic treatment is administered either orally or topically to the animals of respective groups until 7th– 9th day starting from the day of operation. The sutures were removed on 7th day, and the skin breaking strength of the healed wound is measured on 8th–10th day.

DEAD SPACE WOUND MODEL : 

DEAD SPACE WOUND MODEL In this model, the physical and mechanical changes in the granuloma tissue are studied. The subcutaneous dead space wounds are inflicted one on either side of axilla and groin on the ventral surface of each animal, by making a pouch through a small nick in the skin. The cylindrical grass piths (2 . 5 × 0 . 3 cm) or sterile cotton pellets (5–10mg each) are introduced into the pouch. Each animal received 2 grass piths/cotton pellets in different locations. The dead space wound is created by subcutaneous implantation of a sterilized, shallow, metallic ring (2 . 5 × 0 . 3 cm) known as the cylindrical pith or polypropylene tube (2 . 5 × 0 . 5 cm) on each side beneath the dorsal paravertebral lumbar skin surface, and wounds are sutured. The respective therapeutic treatment is administered either orally or topically to the animals of respective groups for 10 consecutive days. The physical changes in the granuloma tissue are studied in this model .

BURN WOUND MODEL : 

BURN WOUND MODEL PARTIAL THICKNESS BURN WOUND A special metal plate 2 × 2 cm with holder is heated to 60 ◦ C and applied to the dorsal area of the animals for 30 s to induce partial thickness burn wound. SECOND-DEGREE BURNS WOUND It can be made by placing the 90 ◦ C hot plate on the selected dorsal area of the animal for 10 s. FULL THICKNESS BURN WOUND The metal plate is heated to 100 ◦ C and applied to the dorsal area for 30m.

BURN WOUND MODEL : 

BURN WOUND MODEL RECTANGULAR BURN WOUNDS The animal can also be subjected to rectangular burn wounds (20 × 25mm2) using hot (180 ◦ C) brass brick weighing 300 g, which is pressed against the shaved skin for 10 s in the treatment group. A cylindrical metal rod (10mm diameter) is heated over the open flame for 30 s and pressed to the shaved and disinfected surface for 20 s on selected dorsal area of animal under light anesthesia. Animals are placed in individual cages after recovery from anesthesia. The respective therapeutic treatment is administered either orally or topically to the animals of respective groups until the day of scab falling starting from the day of operation. The parameters studied are percentage wound contraction, hydroxyproline content and epithelialization time

PowerPoint Presentation: 

Wound contraction contributes to wound closure, is expressed as a reduction in percentage of the original wound size is studied starting from the day of operation until the day of complete epithelialization and evaluated to calculate the degree of wound healing . EVALUATION OF WOUND HEALING Percentage Wound Contraction. Collagen Estimation. Hexosamine Estimation. Skin Breaking Strength. Granuloma Studies.

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The progressive reduction in the wound area is monitored planimetrically by tracing the raw wound boundaries initially on a sterilized transparency paper sheet in mm2 without causing any damage to the wound area, and then, the wound area recorded is measured using a graph paper on every 2–4 d interval. The period of epithelialization is expressed as the number of days required for falling of the eschar (dead-tissue remnants) without any residual raw wound is considered as the end point of complete epithelialization. Percentage wound contraction is calculated as: Percentage wound contraction on PERCENTAGE WOUND CONTRACTION

COLLAGEN ESTIMATION: 

COLLAGEN ESTIMATION On the 11th post wounding day, the animals from each group are euthanized and the wound tissue is excised, weighed, and dried in an oven at 60 ◦ C– 70 ◦ C for 12–18 h, and the dry weight is noted. The tissues are hydrolyzed in 6NHCl for 24 h at 110 ◦ C in sealed glass tubes. The hydrolysate is neutralized to pH 7.0. The sample (200 μ L) is mixed with 1mL of 0.01MCuSO4 followed by the addition of 1mL of 2.5N NaOH and then 1mL of 6% H 2 O 2 . The solution is mixed and shaken occasionally for 5min. All the tubes are incubated at 80 ◦ C for 5min with frequent vigorous shaking. Upon cooling, 4mL of 3NH2SO4 is added with agitation. Finally, 2mL of 5% Para dimethyl amino benzaldehyde is added to develop a pink color.

COLLAGEN ESTIMATION: 

COLLAGEN ESTIMATION The samples are incubated at 70 ◦ C for 16 min, cooled by placing the tubes in water at 20 ◦ C, and the absorbance is measured at 540nm using a colorimeter/spectrophotometer. The amount of hydroxyproline in the samples is calculated using a standard curve prepared with pure L-hydroxyproline at the same time . The concentration of the sample is Calculated as:

HEXOSAMINE ESTIMATION : 

HEXOSAMINE ESTIMATION The granulation tissue is obtained from wound area on 11th post wounding day is dried in an oven at 60 ◦ C and hydrolyzed with 2NHCl at 100 ◦ C for 2 h. The hydrolyzed solution is filtered and pH of the filtrate is adjusted to 6-7. This acid hydrolysate solution is subjected to deamination and non deamination of hexosamine to determine the amount of hexosamine . For deamination, 0.5mL of a 5% solution of sodium nitrite and 0.5mL of a 33% solution of acetic acid is added to 0.5mL of acid hydrolysate solution. The tubes are shaken and left for 10 min for complete deamination. The excess nitrous acid is removed by adding 0.5mL of a 12.5% solution of ammonium sulfamate and by shaking the mixture for 30 min. For indole reaction, 2mL of 5% HCl and 0.2mL of a 1% solution of indole in alcohol is added to 2mL of the deaminated hexosamines.

HEXOSAMINE ESTIMATION : 

HEXOSAMINE ESTIMATION The tubes are immersed for 5min in a boiling water bath. An intense orange colour and a slight turbidity is seen. To remove turbidity, 2mL of alcohol is added, and tubes are shaken. For nondeamination of hexosamine , 1.5mL of a mixture of equal volumes of solutions of 5% sodium nitrite, 33% acetic acid and 12.5% ammonium sulfamate are added to 0.5mL of acid hydrolysate solution. This serves as the control without deamination. The indole reaction is carried out on this mixture as described above. The absorbances of the solutions are determined spectrophotometrically at 492 and 520 nm. The absorbance value for the nondeaminated solutions is subtracted from the corresponding absorbance values for the deaminated unknown and standard solutions of glucosamine hydrochlorides. The increase in the difference in absorbance after the deamination procedure is considered as the measure of the amount of hexosamine . The hexosamine value in μ g is computed from the standard curve.

SKIN BREAKING STRENGTH : 

SKIN BREAKING STRENGTH The anesthetized animal is secured to the table, and a line is drawn on either side of the wound 3mm away from the suture line. Two Allice forceps are firmly applied on to the line facing each other. One of the forceps is supported firmly, whereas the other is connected to a freely suspended light weight metal pan/measuring graduated container through a string run over to a pulley. Weight is added to the pan/water is allowed to flow from the reservoir slowly and continuously into the container. A gradual increase in weight is transmitted to the wound site pulling apart the wound edges. As soon as wound gaping appeared, the addition of weight/water flow is stopped, and the weights added to pan/volume of water collected in the container (approximately equal to its weight) is determined and noted as a measure of breaking strength in grams.

SKIN BREAKING STRENGTH : 

SKIN BREAKING STRENGTH In another approach, the animal is anaesthetized, and healing tissue along with normal skin at the two ends is excised. Strips of 8mm width and 20mm length are cut out from the excised tissue, which is loaded between the upper and lower holder of the tensile testing machine in such a way that the effective load bearing size is 8 × 8mm with the wound remaining in the centre . The total breaking load is measured in Newtons (N), and the tensile strength is calculated as mass in kg by the following equation: Total breaking load Tensile strength = ---------------------------- Cross-sectional area

GRANULOMA STUDIES: 

GRANULOMA STUDIES The day of the wound creation is considered as day zero. Granulation tissue forms on the dead space wound surrounding the implanted pellets/piths is harvested by careful dissection on the 10th post wounding day under light ether anesthesia. After noting the wet weight of the piece of granuloma excised, it is dried in an oven at 60 ◦ C for 12–24 h to obtain a constant dry weight expressed as mg/100 g body weight. The granuloma tissues are trimmed to obtain the rectangular strip measuring about 15mm in length and 8mm width to determine its breaking/tensile strength by continuous water flow technique. The dry granulation tissue is used for the estimation of hexuronic acid, hexosamines, hydroxyproline content, which can be assayed calorimetrically/ spectrophotometrically , and a piece of wet granuloma is preserved in 10% formaldehyde for histological studies to evaluate the effect of the herbal extract on collagen formation .

REFERENCE: 

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