Protocol of Dead Space Wound Model

1.

Name of the study: Dead space Wound Model Animal species used : Male/Female- Albino rats

2.

3. Criteria for choosing the animal: Rat:

a.

Age: Adult Body weight: 200 to 250 g.

b.

4.

Housing procedure: Animals should be caged individually. They were housed in polypropylene cage in aircondition area at 22 oC (+ 3o) for rodents and the relative humidity 30 to 70 % with 12 hr light & dark cycle. All the animals had free access to standard pellet diet clean water ad libitum. Prior to the day of experimental procedure they were starved overnight.

5.

Duration of study: Animals were treated with test samples for approximately 10 days (On 10th post wounding day tensile strength was measured).

6.

Route of administration : Topical

7. Evaluation parameters:

 Granuloma weight Granuloma breaking strength Granuloma histology Hydroxyproline estimation Breaking strength: Anesthetized animal was secured to operation table, in its natural position and lines were dawn on either side of the incision wound, 3mm away from the wound margin on adjacent normal skin leaving about 5mm wound towards both the ends. Two Allis forceps were firmly applied on the lines, facing each other, the forceps on one side is hooked to a metal rod, fixed firmly to the operation table, while the other to a light polythene container through a string run over a pulley (Plate 1). Water was allowed to flow at a constant rate into the polythene container so as to build a gradual pulling force necessary to disrupt the wound. The flow of water was regulated by means of an occlusion clamp on rubber tubing connected to water reservoir, kept at a suitable height. As soon as the gaping of the wound was observed, the water flow was cut off. Further opening of the wound was avoided by releasing the pulling force on the wound immediately by lifting up the polythene container. The

volume of water in the polythene container was measured and converted to the corresponding weight. The breaking strength is expressed as the minimum weight of water necessary to bring about the gaping of the wound. Three such reading were recorded for a given incision wound and the procedure was repeated on the other, thus obtaining six readings for each animal. The mean breaking strength in each animal (average of six readings) was used to calculate the group mean.

Figure No.1

Assembly to Estimate the Breaking Strength

Estimation of Hydroxyproline (OH-P) Hydroxyproline was estimated on 10th day from the granulation tissue grown around the pith. The granulomas were dissected out from the animal treated with drug for a period of 10 days. Procedure: Estimation of hydroxyproline is an amino acid present in the collagen fibres of granulation tissue helps clinically to understand progress rate at which the healing process is going on in the connective tissue on the wound. Chemicals Required: Hydroxyproline (Loba chemicals, Bombay), Methyl ed, Conc. HCl, Sodium Hydroxide Pellets (LR, Pune chemicals), Chloramine T(LR), P dimethyl amino benzaldehyde(LR, Loba chemicals, Bombay), Citric acid monohydrate (LR), Glacial acetic acid, Sodium acetate trihydrate (LR), Toluene (LR), Methyl cellusolve (Thomas Baker Chemical Co. Bombay), Perchloric acid (Thomas Baker Chemical Co. Bombay). Procedure: Preparation of chemical required:
1.

Hydroxyproline Standard: A stock solution was prepared by dissolving 25mg of vacuum dried l Hydroxyproline in 250ml of 0.001N HCl. Standards were prepared daily by diluting the stock with water to obtain concentrations of 1 10g /2ml.

2.

Buffer: 50gms of citric acid monohydrate, 12ml of glacial acetic acid, 120mg of sodium acetate trihydrate and 34gms of sodium hydroxide were

dissolved in distilled water and made to final volume of 1ltr. The pH was carefully adjusted to 6 and it was stored in refrigerator under toluene.
3.

Chloramine T: 0.05N solution of Chloramine T was prepared freshly before use by dissolving 1.41gm of Chloramine T in 2oml of water to which 30ml of methyl cellusolve (ethylene glycol mono methyl ethanol) and 50ml of the buffer were added.

4.

Perchloric acid: A 3.15M solution was prepared by diluting 27ml of 70% Perchloric acid AR to 100ml with water.

5.

P dimethyl aminobenzaldehyde: A 20% solution of p dimethyl amino benzaldehyde was prepared shortly before use by adding methyl cellusolve to 20gm P dimethyl amino benzaldehyde to give a volume of 100ml.

HYDROXYPROLINE ESTIMATION In order to obtain the standard curve the following procedure was followed. Six test tubes were taken each containing 2ml of distilled water (blank) and 2g, 4g, 6g, 8g and 10g of hydroxyproline obtained from freshly prepared stock solution dissolved in 2ml of distilled water. 2drops of 0.02% methyl red were added to each of the above test tubes and shaken thoroughly. This was followed by addition of 2.5N NaOH drop by drop till color changes pink to yellow and by addition of 0.01N HCl, pH was adjusted to values of 6 7. To each was added 1ml of Chloramine T solution in a sequential order. The contents were mixed and allowed to stand for 20min. at room

temperature. Then 1ml of Perchloric acid was added to each of the test tubes in same order. Contents were mixed and allowed to stand for 5min. Finally 1ml of P-dimethyl amino benzaldehyde solution was added to each tube and shaken until no play of color (schlieren) could be seen. Then tubes were placed in 60C water bath and heated for 20 min. cooled under running tap water for 5min. the absorbency of the solution was determined colourimetrically at 570nm and standard curve was plotted. A sample of granulation tissue weighing around 300mg was homogenized in glass homogenizer, 10ml of 6N HCl was added to the homogenizer in 25ml glass ampoules. The ampoules were sealed and hydrolyzed for 3hrs. at 130C. They were then opened and contents were decanted to graduated glass cylinders, the tubes were washed thoroughly with water and the washings were combined with the hydrolysate. Further they were processed in manner similar to that described for obtaining the standard curve. After adjusting pH ( 6 to 7) the sample was diluted to a volume of 50ml water such that 2ml of these diluted sample contain approximately 1 to 10 g of hydroxyproline. To each test tube 2ml of sample 1ml of Chloramine T solution was added. The contents were mixed and allowed to stand for 20min. at room temperature. Then 1ml of perchloric acid was added to each of the test tubes in same order. Contents were mixed and allowed to stand for 5min. Finally 1ml of P dimethyl amino benzaldehyde solution was added to each tube and shaken until no play of color (Schlieren) could be seen. Then tubes were placed in 60C water bath and heated for 20min. cooled under running tap water or 5min. The absorbency of the solution was determined colourimetrically at 570nm (Plate 4). The hydroxyproline content of the granulation tissue were calculated from standard curve.

All the results of various experiments carried out in the present study were analyzed by Students t test. The value (P < 0.05) was considered to be significant.

8.

Instrument used :

9. Methodology: Standard drug: Take standard drug (ointment) as per requirement. Preparation of test drug: These should be sufficient in number, at least three, to produce test groups with a range of toxic effects and mortality rates. Experiments: Animals are divided into 5 groupsMedian test dose level: 6 (male/female) Higher test dose level (Satellite group): 6 (male/female) Lower test dose level: 6 (male/female) Control: 6 (male/female) Vehicle control (If necessary): 6 (male/female)

Note: If application of the test substance produces severe skin irritation, the concentration may be reduced, although this may result in a reduction in, or absence of, other toxic effects at the high dose level. However, if the skin has been badly damaged early in the study it may be necessary to terminate the study and undertake a new study at lower concentrations.

Performance of the test: Physical, mechanical and histological changes in the granuloma tissue were studied in this model. Under light ether anesthesia, subcutaneous dead space wounds were inflicted in the region of the axillae and groin, by making a pouch through a small nick in the skin for implanting either sterile cotton pellets or grass piths to induce granuloma. a) Two sterile cotton pellets weighing 10mg (sterilized by

autoclaving) were used to grow granulation tissue by the technique of DArcy et al as described by Turner, but the granulomas were removed on the 10th day instead of 4th day (Plate 2). b) Similarly two cylindrical grass piths measuring 25mm in length

and 3mm diameter were also introduced into the subcutaneous pouch in each animal in different locations at random. Thus one animal had two cotton pellets and two grass piths. The sutured were mopped with an

alcoholic swab and animals were placed into their individual cages for recovery from anesthesia (Plate 3). Excision of the granuloma from the surrounding tissue was performed on the 10th post-wounding day under light ether anesthesia. Cotton pellet granulomas excised from dead space wounds were dried overnight at 60 so as to obtain a constant dry weight. Their weights c were noted and expressed as mg/100gm body weight as suggested by Dipasquale and Meli. 98 Granuloma surrounding the grass piths were excised and slit open by a longitudinal incision in one plane so as to obtain rectangular strips. The breaking strength of a strip of granuloma measuring about 15cm in length and 8mm in width (obtaining by trimming the rectangular strip of granuloma tissue) was measured employing the method described under incision wounds. The pieces obtained at the end of these measurements were weighed to obtain 300mg of granulation tissue, which was kept in 6N HCl for estimation of hydroxyproline. The other granulation tissue was put in 10% formalin for further histological studies. Paraffin sections of the preserved granuloma tissue were stained with Van Gieson so as to enable the assessment of fibroblasts population,

infiltrating cells, collagen content and thickness of the tissue under light microscope. The above parameters were rated by subjective comparison.

Plate No.2

Implantation of Cotton Pellet

Plate No.3

Implantation of Grass Pith

10.Dose selected a. Median test dose level b. Higher test dose level c. Lower test dose level
11.

Total number of animal used for the study : 30 Total number of group : 05 Dosing frequency: Repeated (Approximately 10 days) topical

12.

13.

dose in all 05 groups

14.

Euthanasia (If applicable) : Higher dose of ether

15.Reference: Ehrlich H.P., and Hunt T.K., Effect of cortisone and anabolic steroids on the tensile strength of healing wounds. Ann. Surg., 1969; 170: 103-206pp. Weossner J.F. T.Jr., The determination of Hydroxyprolline in tissue and protein samples containing small proportions of this amino acid. Arch. Biochem 1961; 93: 440pp.

 Turner R.A., Screening methods in pharmacology. New York, Academic press. 1965; 61, 62 & 152-153pp.