Dead Space Wound Model

8/2/2019 Dead Space Wound Model

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Protocol of Dead Space Wound Model

1. Name of the study: Dead space Wound Model

2. Animal species used : Male/Female- Albino rats

3. Criteria for choosing the animal:

Rat:

a. Age: Adult

b. Body weight: 200 to 250 g.

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:


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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


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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


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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


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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


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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 wasdiluted 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.


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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 groups-

Median 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)


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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


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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 c so as to obtain a constant dry weight. Their weights

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,


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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


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Plate No.3

Implantation of Grass Pith


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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

12. Total number of group : 05

13. Dosing frequency: Repeated (Approximately 10 days) topical

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.


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Turner R.A., Screening methods in pharmacology. New

York, Academic press. 1965; 61, 62 & 152-153pp.

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Dead Space Wound Model