An experimental model to investigate the dynamics of wound contraction

British hournnl of Plastic Surgery (1995). 48, 189-197 0 1995 The British Association of Plastic Surgeons

An experimental model to investigate the dynamics of wound contraction S. E. Cross, I. L. Naylor, R. A. Colemant

and T. C. Teo*

Postgraduate School of Studies in Pharmacology, *Plastic Surgery and Burns Research Unit, Department Biomedical Sciences, University of Bradford, Bradford, and t Peripheral Pharmacology, Glaxo Group Research, Ware, Hertfordshire, UK

of

SUMMARY. An excisional wound model in the Hooded Lister rat is described. The methods used to inflict, measure and characterise the process of wound contraction are comprehensively discussed. The model is shown to be reliable, reproducible and capable of detecting the effect of systemically administered prednisolone.

Experimental models to investigate the process of wound contraction have been described in both marP and laboratory animals.The aims of such studies were twofold. They attempted to gain an understanding of the dynamics of wound contraction and, to develop models in which dressing’s6 topical treatments7 or systemically administered drugs’ could be evaluated for their potential effect on the process of contraction. Experimental wound healing studies in man are, by necessity and ethics, very limited. Consequently, animal experimentation has been used even though it is recognised that the extrapolations of results from animals to man should be guarded. The usual approach has been to study the processes of repair in full thickness, excisional skin wounds under a variety of treatments. To analyse quantitatively the progress of wound repair under such treatments, a combination of macroscopic and histological (both optical and electron microscopic) observations, biochemical and physical measurements of the surface area of the residual wound defect over a period of time have been used. A further advantage in using animals is that the wound healing process may be conveniently studied over days rather than weeks, as in man. In 1910 Alexis Carrel introduced the concept of using the measurement of surface area change as an index of wound contraction rate.’ His initial observation prompted Lecomte Du Nouy to produce the relevant mathematics to characterise the overall process.lO Although designed originally for wounds in humans, this procedure also proved to be applicable to animal studies. In contrast to the studies in man, those performed using animals have some methodological problems associated with them. These include the choice of species, the site of the wound (which has to be ‘out of reach’ of the animal) and the depth, size and shape of the wound. This diversity in experimental conditions is clearly seen in the many wound healing studies which have used rabbits. Excisional wounds have been made on the dorsum,*~” flanks,12 thorax13s1* and ear.“-” The depth of the wound has varied in thickness from down to the level of the deep fascia overlying the muscles of the back to above the panniculus carnosus. Sizes and

shapes have ranged from squares8s12’20-25and rectangle?* to circles. 26-28A similar diverse range of sites, depths and sizes have been used in the guinea pig29-33 and rat.34-3g Although experiments are reported for other species such as the hamster,40-42 mouse,43-45 dog9 and pig,46-48 most experiments now use rats. A critical part of all these experiments is the technique of measuring the area of the residual wound. In loose skinned animals such as rats and rabbits, it is essential to keep the animal in a “ standard position” during the measurement. Yet no report clearly states what this position is and how it is achieved unless by the use of anaesthesia. However, the repeated exposure of an animal to anaesthetic procedures may be deleterious to its health, be time consuming and could cause wound damage during the induction of anaesthesia. Various methods have been used to record the wound areas. Tracings of the wound edges using cellophane sheets’, 12,‘*, *‘, 5o and transparent plastic sheets8j35 have all been used, as has still photograpb30z 43 and, more recently, a video camera.51 All the tracing procedures suffer from the problem of an error due to parallax, as perhaps do the photographic techniques, unless the wound is compressed under a flat glass plate.52s53 Usually only two or three tracings are made of each wound area and, for photography, one exposure. The combination of photography incorporating a grid 54,55is unusual but does confer its own internal standard. Once a tracing is obtained the next decision is how to measure its surface area. Traces onto a constant thickness paper or cellophane and weighing,43z44.56,57 planimetryl2,21,49.58 or, more recently, computer digitisation*s ‘3js have been used. Once the measurements have been made, the next step is to find the most appropriate way of expressing the area changes versus time so that statistical tests can be used to compare various treatments. The simplest way is to plot graphs of abso1ute37,42.61 or percentage of original area versus time in days.8.3’,60 However, this produces exponential curves which are difficult to compare. To facilitate analysis, the area may be transformed into logarithms (log). A plot of the log area versus time should produce a straight line; 189

British Journal of Plastic Surgery

190

the gradient of which-generally called the coefficient of wound contractionl’+an then be calculated. Another option is to use the log of the percentage of the original wound area versus time, which again results in a straight line graph for which the slope can be calculated.‘j’ More recently, Snowden claimed that the transposition of the area data to its square root before it is plotted against time would produce a more representative constant for wound contraction.24’63 A further modification has been suggested64 specifically for those wounds whose initial size prevents them from producing a final scar with an insignificant residual area. Clearly a consensus on how to calculate this fundamental property of wound contraction is lacking. Just as there are differences in graphical presentation, there are differences for the number of days over which the slope has been calculated, e.g. days &7 or O-12.62 This reflects the fact that in some studies the wounds are only traced after 5 or 7 days following injury and then only every 34 days. With such diversity of approaches to the study of wound contraction in mind, this paper describes a series of studies we have conducted in order to develop a simple rat excisional wound model whose experimental variables can be rigorously controlled. Standard wounds have been made in a specific area and accurately measured under precisely controlled conditions. The area measurements have been compared using a range of graphical techniques to establish the most useful representation to adopt for studying the dynamics of wound contraction. Methods

The studies were divided into two phases: first, to determine the best animal model to adopt and, second, to validate the usefulness of the chosen model by studying its response to a drug known to delay wound contraction. Animals

(i) Rats. Two strains of rats were studied: Hooded Lister (HL, Bradford strain)-adult males and females (200-350 g, 334 months old), young males and females (11 O-160 g, 6 weeks old)---and Sprague Dawley (CD)“aged” males (550-750 g, 7 months old), and adult females (200-300 g, l&12 weeks old). Before surgery the animals were housed in wiretopped cages with sawdust bedding (maximum 5 per cage) and allowed standard food pellets (CRM, Labsure) and tap water ad libitum. During the experimental period the animals were housed individually and given the same diet and water. Holding rooms were illuminated between 07.00 and 20.00 hours with the ambient temperature maintained at 21 f 1°C and the humidity at 45-50 %. (ii) Guinea pigs. Male (42&540 g) and female (330400 g) Dunkin Hartley guinea pigs (Bradford strain) were also studied. Both before and after surgery, the animals were housed in wire-topped cages with hay and sawdust bedding, with a maximum of 3 per cage throughout the study, and allowed standard food

crouching position of the rat ,wound

cotton duster Fig. 1 Figure l-Animal studies.

held in standard crouching position used in all the

pellets (SGl + ascorbic acid) and tap water ad libitum. Holding rooms were illuminated between 07.00 hours and 20.00 hours with the temperature controlled at 21 f 1°C and the humidity at 45-50 %. Excisional wounding procedure

Rats and guinea pigs were anaesthetised with halothane (0,NO carrier) and their lower back and left flank clipped with electric clippers and wet shaved with a scalpel blade. After cleaning with alcoholic chlorhexidine and drying of the area, the mobile skin high on the left flank was gently held taut, by simply stretching with the fingers, and a template measuring 20x20mm,15x15mmor10x10mm,dependingon the study (Table 1) was placed on the skin and the outline traced using a fine felt-tipped pen. The medial border of the template was orientated parallel with the sagittal axis of the animal. Full-thickness wounds were made by excising the skin within the confines of the square down to the level of the loose subcutaneous tissues. All dissections were performed using only a size 15 scalpel blade and forceps, with particular care being taken to ensure that the wound edges were sharply defined and not contused. The wounds were immediately dressed with a Tegaderm@ self-adhesive dressing secured at a distance over 10 mm from the wound edges with spots of Perrnabondm cyanoacrylate adhesive. The animals were then held in a standard “crouching’ position (Fig. 1) while the wound areas were traced onto acetate strips, after which each animal was placed in a separate cage for full recovery from the anaesthesia before being returned to the holding rooms. Wound measurement and area determination

For the first 48 h postoperatively the dressings were left undisturbed and during this time 2 wound measurements were performed at 24 and 48 h, tracing through the dressing with the animals fully conscious in the standard “crouching” position. Then, the dressings directly overlying the wound and approximately 5 mm beyond were cut away, leaving the parts that remained firmly adherent to the skin to fall off by themselves. This generally happened over the next 5-7 days and did not appear to interfere with the process of wound contraction. The outline of each wound was recorded every 24 h for the next 12 days. For this, the

The dynamics of wound contraction

191

Table 1 The rates of excisional wound healing for various rodent groups Study

categories

(a) Control (i) (ii) (iii) (iv)

(v) (vi) (vii) (viii)

animals

Wound

Adult female Hooded Lister (HL) rats (200-300 g) Adult Female Sprague Dawley (CD) (200-300 g) Adult male HL rats (25G350 g) Young female HL rats (lo&160 g) Young male HL rats (110-160 g) Aged male CD rats (SS(r750 g) Adult female guinea pigs (3OtK400 g) Adult male guinea pigs (42&550 g)

(b) Drug treated animals (female HL rats, 15 x 15 mm initial Drug Prednisolone (Deltastab@)

20 x 20 mm 10xlOmm 10xlOmm 2Ox20mm 15xl5mm 15xl5mm

5 6 6 11 6 6

size)

Route

Regimen

Vehicle

(4 1 w/kg

subcut.

daily (days daily (days

saline

6

saline

4

saline

10

control

n = number

8 8 5

Dose

(b) 10 mg/kg Vehicle

wound

size : n

(a) 15 x 15 mm (b)20x20mm 15xl5mm

1 ml/kg

subcut. subcut.

daily

2-7) 2-5)

of animals

animals were held, fully conscious, in the standard “crouching” position on a wooden board with their head covered by a clean soft cloth (Fig. 1). To trace the wound outline, the investigator always stood in the same position facing the left flank of the animal in order to minimise, or at least keep constant, any error due to parallax. Tracings of each wound were made, usually three times, but more commonly 10 times, onto acetate strips (length 26 cm, width 2.5 cm) with a finetipped permanent marker pen (Staedtler Lumocolor 3 13). Scabs overlying the wounds were never physically removed as this would almost certainly traumatise the wound beds; instead they were allowed to fall off by themselves. Furthermore, leaving the scabs did not seem to impede the satisfactory recording of the wound edges. The acetate strips were photocopied normal size, and the areas of the wounds determined in mm’, using a digitiser (Summagraphics Bit Pad Two) attached to a BBC micro-computer using DIGIT0 software (Department of Ophthalmology, University of London). The accuracy of the measurements made using this equipment, as determined by digitising regular shapes with known surface area, was found to produce an error of < 0.5 %. The combination of digitiser tracing error, as determined by calculating the area of ten wound tracings taken from the same animal at the same time was < 1.5 %. Treatment groups

The animals were grouped as shown in Table la for studying the influence of species, strain, sex and size of initial wounds on the rate of excisional wound healing. From the experience gained from these studies, the adult female Hooded Lister rats with an initial 15 x 15 mm were selected for assessing the effect of systemic prednisolone on the rate of wound contraction. The treatment protocol is shown in Table 1 @I.

The effects of wounding and drug treatment on the animals were monitored through daily weight measurements. In addition, for those animals treated with steroid, the wounds were observed carefully for any signs of infection which could contribute to any delay in wound healing and their exclusion from the study. Histology

At day 15 the animals were killed by an overdose of pentobarbitone. The wounds of all animals were excised, leaving a 5 mm margin of normal skin around the edges of the wound, and placed on thin card and fixed in 10% formal saline for histological examination for a minimum of 7 days. After the tissues were processed, mid-wound vertical sections of each specimen were cut and stained with haematoxylin and eosin as well as with Goldner’s modification65 of Masson’s trichrome stain. The specimens were assessed under light microscopy for the progression of new epithelium, inflammation, vascular responses and the formation of collagen in the wound defect. Statistics

The data are expressed as mean (standard error of the mean (s.e.m.)). The coefficient of wound contraction between the different groups of animals was compared using analysis of variance (ANOVA) for multi-group comparison. Where ANOVA was significant (p < 0.05) between two groups, then further comparisons using Student’s non-paired t-test were carried out. All tests for significance were performed using StatsView 5 12 + @ on a Macintosh@ microcomputer.

Results General observations on wound healing in non-treated animals

Immediately after the operation, the measured wound area increased by about 15 % over the area of the original template used because the wound edges generally bowed outwards due to the elastic tension from the surrounding skin and its loose attachment to the deep fascia. During the first 2 days, relatively minor changes were seen in the wound area, this phase of wound healing being generally known as the “ lag” phase (Fig. 2). Between the 2nd and 3rd day, a sharp decrease in the area of the wounds was seen. This diminution was associated with a visible shrinkage and desiccation of the wound. Over the wound bed a fine scab formed and this appeared to thicken during the next few days before spontaneously detaching from the wound during the 2nd week following wounding. After the 3rd day, the wound area appeared to decrease at a constant rate, which progressively declined towards the 10th to 12th day (Fig. 2). After this time, the wound area approached zero and was very difficult to trace accurately since it was in the final stages of its closure.

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British

Journal

of Plastic Surgery

Table 2a Summary of the mean coefficient of wound contraction, k, for each of the wound contraction with measurements taken from the day of wounding 0) Study group Control (9

(ii) (iii) 0

LO

5

(iv)

Day

(v)

Fig. 2 Figure 2-The area of a wound plotted against time showing the division of the repair process in the rat into the: (a) lag phase, (b) active contraction phase and (c) consolidation phase.

pgression

(vi)

animals

studies, (i.e. day

Area excised (mm)

k (s.e.m.)

R

15x 15

-0.128 (0.008)

-0.99

- 0.129 (0.007) - 0.076 (O.OOS)**

-0.99 -0.98

-0.095 (0.008)

-0.99

-0.087 (0.009)**

-0.99

- 0.072 (0.007)*

- 0.98

-0.062 (0.002)***

-0.98

15x 15

-0.099 (0.008)

-0.98

15x 15

-0.073 (0.007)*

-0.89

:

Adult female HL rats

20x20 Adult female 15 x 15 CD rats Adult male 20x20 HL rats Young female 10 x 10 HL rats Young male 10 x 10 HL rats Aged male 20x20

Fvii) E?uEmale guinea pigs (viii) Adult male guinea pigs

line (days Z-12)

Table 2b Summary of the mean coefficient of wound

Lo~AQ..-

LogA= kt+LogAo

4

Wound Area

contraction,

k, for each of the wound

contraction

studies,

with measurements taken from days 2-12 of wounding Study grow

Area excised (mm)

k (s.e.m.)

R

15 x 15

-0.131 (0.007)

- 0.97

-0.123 (0.008) -0.078 (O.OOS)**

-0.98 -0.95

-0.103 (0.008)

-0.97

-0.092 (O.OlO)**

- 0.97

-0.066 (0.012)*

-0.90

-0.061 (0.002)***

-0.96

-0.106 (0.010)

- 0.96

-0.078 (0.007)*

-0.89

Regression line (days 012) I

Time

(days) Fig. 3

Figure SCalculation of the regression coefficient. Log A = log of the area of the wound at any particular time point. Log Ao = log of the original area of the wound at time 0. k = specific rate of contracture, the amount by which a unit area of wound decreases in one day. t = time, in days since the day of wounding.

Guide to expression of results

In order to determine the best method of treating the data from these studies, they were initially analysed by graphically plotting: (a) the wound area, (b) logarithm (log) to base “e” of wound area, (c) percentage original area, (d) log percentage original area, (e) square root, as well as (f) log square root of the wound area, all versus time in days after wounding. We found that plotting the log wound area versus time was simplest and conveniently allowed the regression line to be calculated for both the whole study period (days G12) and the phase of rapid wound contraction days (2-12). The gradient of each regression line is the log coefficient of wound contraction, k, for each group of animals (Fig. 3). This constant specifies the amount by which a unit area of the wound diminishes in one day. Even more significant than the simplicity of the approach was that the correlation coefficient, R, was found to be near to unity for most of the groups studied, suggesting a high reproducibility of the result of these studies. Consequently, all the results were subsequently expressed as the mean k (s.e.m.) for purposes of comparison.

Control (9

animals

:

Adult female HL rats

20x20 Adult female 15 x 15 CD rats (iii) Adult male 20x20 HL rats (iv) Young female 10 x 10 HL rats (v) Young male 10x 10 HL rats (vi) Aged male 20x20 CD rats (vii) Adult female 15x 15 guinea pigs (viii) Adult male 15x 15 guinea pigs (ii)

= k R = s.e.m. = * = ** ***

mean coefficient of wound contraction. correlation coefficient. standard error of mean. p < 0.05; young male HL rats versus adult male HL rats; male guinea pigs versus female guinea pigs; adult male guinea pigs versus adult male HL rats. = p < 0.01; adult female CD rats versus adult female HL rats; young female HL rats versus adult female HL rats. = p < 0.001; aged male CD rats versus adult male HL rats.

Wound healing patterns in individual study groups

(i) Adult female Hooded Lister (HL) rats. The mean coefficients of wound contraction over the whole study period (days &12) were calculated as -0.129 (20 x 20 mm wounds) and - 0.128 (15 x 15 mm wounds), and during the phase of rapid contraction (days 2-12) they were - 0.123 and - 0.13 1 respectively (Tables 2a and b). Statistically, there was no significant difference (p > 0.05), using either of the time periods calculated, in the

193

The dynamics of wound contraction coefficient of wound contraction between the two wound sizes. This demonstrates that the initial area of the wound, at least for the two sizes studied, appeared to have no effect on the overall coefficient of wound contraction. (ii) Adult female Sprague Dawley (CD) rats. Female Sprague Dawley (CD) rats were found to be more averse to being handled than Hooded Lister females. They tended to struggle more during the wound measuring sessions and it was felt that precise area measurements could not be obtained as accurately as those from the HL rats. This behaviour was more marked during the first week, after which time the CD rats became slightly accustomed to being handled and restrained in the “crouching’ position for wound tracing. However, they were never as “tame’ as the HL rats. Occasionally, the necessary restraint led to the edge of the wound being disrupted and these then had to be excluded from the study. The mean coefficient of wound contraction of the two groups when taken from day 0 were -0.076 for the CDs and -0.128 for the HLs (Table 2a) and -0.078 and -0.131 (Table 2b) respectively when taken during the phase of rapid contraction. Statistically, there was a highly significant difference between the coefficient of wound contraction for the two strains (p < 0.005) for both of the time periods studied, apparently indicating that CD rats healed more slowly. (iii) Adult male Hooded Lister rats. To establish whether the sex of the animal played an important role in the rate of excisional wound contraction,the healing of 20 x 20 mm wounds in male Hooded Lister rats was studied. The mean coefficient of wound contraction was calculated to be - 0.095 for days &12 (Table 2a), and -0.103 for days 2-12 (Table 2b), indicating a slightly slower rate of wound healing than the female animals of the same strain. Statistically, however, this difference was not significant. Although both sexes gave results with little individual variation within a group, suggesting that either would have been suitable for the subsequent pharmacological studies, it was decided to use female animals. The thinner female skin allowed a faster rate of wound healing with a higher wound contraction rate with less of a tendency for the defect to heal by epithelialisation as occurs in male animals. (iv) Young female and young male Hooded Lister rats. The mean coefficients of wound contraction for 6week-old female and male Hooded Lister rats were found to be -0.087 and -0.072 respectively for measurements taken from day 0 (Table 2a), with values of -0.092 and -0.066 respectively when these were calculated for days 2-12 (Table 2b). There was no statistically significant difference between the coefficient of wound contraction of the two sexes at this age (p > 0.05), for either time period studied. However, there was a significant difference in the rates of healing between young and adult female rats, with p = 0.0027 (days O-12) and p = 0.006 (days 2-l 2). This difference was reflected between young and

adult male rats only during the phase of rapid contraction, days 2-12 where p = 0.033, days O-12 being not significant. (v) Aged male rats. The effect of ageing on the process of wound contraction was studied in a group of “aged” male Sprague Dawley (CD) rats weighing 550-750 g. In contrast to the behavioural problems encountered with female CD rats, the “aged” male animals were much more docile and throughout the study none of the wound margins were disrupted while the animals were being restrained for wound tracing. The mean coefficients of wound contraction were calculated to be - 0.062 (days Crl2) and - 0.061 (days 2-12) as shown in Tables 2a and b; these were much lower rates than those observed for the adult male HL rats, which were - 0.095 and -0.103 respectively. The difference between the coefficient of wound contraction of these two age groups was found to be highly significant, p = 0.0001 for both time periods studied, indicating that aged animals had a slower rate of wound contraction.

(vi) Female and male guinea pigs. Wound contraction rates for 15 x 15 mm excisional wounds in Dunkin Hartley guinea pigs were determined in both adult female and male animals. The coefficients of wound contraction were calculated to be - 0.099 and - 0.073 for females and males respectively over the whole study period, with rate constants of -0.106 and -0.078 over the phase of rapid contraction (days 2-12). Statistically, the female guinea pigs gave significantly higher rate constants than the males between day 2-12 (p = 0.034), indicating a faster rate of wound contraction than the males over this time period. However, the difference between the rate constants taken over the whole study period just failed to reach statistical significance (p = 0.052).

The coefficient of wound contraction for male guinea pigs was significantly lower than those for male rats between day 2-12 (p = 0.035) but, when measurements were taken from day 0, there was no significant difference between the two species. The coefficient of wound contraction for the female animals proved to be similar, and only just failed to reach statistical significance between days 2-12 (p = 0.056). Studies using prednisolone

Prednisolone treatment was started on the second day following wounding and was continued until day 5 for the group given 10 mg/kg, and until day 7 for the group treated with 1 mg/kg. As shown in Table 3, the mean coefficients of wound contraction for 1 and 10 mg/kg prednisolone were - 0.058 and - 0.032 respectively over the whole study period and -0.053 and -0.029 respectively between days 2-12. Statistically, there was a significant decrease in the coefficient of wound contraction in animals treated with 10 mg/kg prednisolone as compared with vehicle treated controls during both time periods, with p = 0.0075 (days O-12) and p = 0.004 (days 2-12). There was no difference between the animals dosed

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British Journal of Plastic Surgery

Table 3 Effect of prednisolone on the mean coefficient of wound contraction ascompared with vehicle treated controls Days

regimen (s.c.daily) Dose

?I

(a) 1 mg/kg pred.

6 1 ml/kg saline# 6 (b) lOmg/kgpred. 4 1 ml/kg saline# 4

O-12

Days

k

k

R

-0.058 -0.073 -0.032** -0.121

2-12

-0.98 -0.98 -0.90 -0.97

-0.053 -0.076 -0.029** -0.116

R

-0.92 -0.95 -0.88 -0.96

= number of animals. n S.C. = subcutaneous injection. med. = nrednisolone. = coefficient of wound contraction. = correlation coefficient. = p < 0.01 versus saline treated control animals. = both 1 mg/kg and 10 mg/kg prednisolone were given in a dose volume of 1 ml/kg-hence each control is the same volume.

matory response, (c) the vascularity and (d) the collagen fibres within the granulation tissue. Epithelialisation. At 7 days, none of the wounds was fully epithelialised and even at 14 days only 35 % showed full epithelialisation. The thickening of the epidermis in a zone near the edge of the defect was characteristic of rat wounds. Inflammatory response. In all the wounds examined at 7 days, polymorphonuclear leucocytes were present in limited numbers. At 14 days they were absent. Vascularity. At 12 days the vertical orientation of the blood vessels in the granulation tissue was a characteristic finding. Notably, the upper areas of the granulation tissue were much more vascular than the lower areas. Collagen jibres. Goldner’s modification of Masson’s trichrome staining for collagen clearly demonstrated a greater density of collagen fibres in the lower areas of the wound than the upper areas of the granulation tissue at 7 days. At 14 days the staining pattern was still variable, with the upper areas still less intensely stained. The edge of the original defect could always be clearly seen, since the collagen of the dermis stained an intense green quite unlike that of granulation tissue. Prednisolone treated animals

0

I

2

3

4

5

6

7

8

9

10 11 12

13 14

DAY Fig. 4 Figure &The effect of prednisolone therapy (1 mg/kg and 10 mg/ kg) on the body weight of rats compared to vehicle treated controls [Mean (s.e.m.)], n = 6 and 4 for the two dose groups respectively.

with 1 mg/kg prednisolone and their vehicle treated controls (p > 0.05) during both time periods. During the study, the animals treated with prednisolone did not suffer from any wound infection. However, they lost a significant amount of body weight in a dose dependent fashion-a 30% weight loss at day 12 when treated with 10 mg/kg (Fig. 4). Macroscopically, the wounds appeared to be contracting during the first 2 days of steroid administration but by days 4-5 it was evident that the wound areas were decreasing at a much slower rate than controls. With 10 mg/kg of prednisolone, the wound measurements were static on day 9-and subsequent contraction was slight-ven though drug treatment had by then been withheld for 4 days. Histological observations

Four features of the wounds were qualitatively assessed by histology: (a) the degree of epithelialisation of the wound defect, (b) the degree of inflam-

10 mg/kg of prednisolone markedly decreased the quantity of granulation tissue in the wound defect. In addition, the epithelialisation process had not started, as there was no epithelialisation at the wound edges, the characteristic vascularity was absent and the collagen fibres were greatly reduced throughout the whole wound. Discussion

The aim of the present studies was to establish a simple, reproducible, predictable and quantifiable animal model of excisional wound healing. The results suggest that the adult female Hooded Lister (HL) rat is the most suitable strain of animal to use. The technical aspects of wound creation in the left flank of the rat and the application of the dressings are simple procedures to both perform and standardise. All the animals recovered rapidly and throughout the studies there was no fatality. It should be stressed that no further anaesthesia was used for the handling and measurement of wound area. This contrasts with other reported studies38s66where the repeated exposure of an animal to anaesthesia must increase the stress upon it as well as increase the risk of injury, especially during the induction phase, to the delicate granulation tissue of the wound. These factors are difficult to control and yet they are important in that they would almost certainly contribute to a delay in wound contraction.67 In these studies the lack of stress of the animals during the tracing procedures was shown by the observation that the wooden board on which the

The dynamics of wound contraction animals were placed for all these procedures remained dry even after the measurement of 24 consecutive animals, because none of the rats had stress micturition. Wood is to be used in preference to metal since the latter does not provide a surface on which the animals can grip and so they become stressed. The lack of stress of the entire procedure was also shown by the progressive regain in body weight of the animals throughout the experimental period. Our concern to minimize the stress imposed upon the animals also led to the choice of a 15 x 15 mm rather than a 20 x 20 mm wound for the HL rat model. Some studies have used wounds which seem unnecessarily large-for example, 40 x 60 mm in rats of unspecified size3gand 40 mm diameter circular wounds in 300 g rats. 38 The physical and metabolic stress that a wound of such size places upon a small animal must surely be a source of concern and independently influence the results of the studies. With these additional variables, any effect of a particular treatment could be quite difficult to detect. The results of the wound contraction coefficients show that the choice of strain, sex and age of animal are extremely important variables to standardise in the search for reproducibility. We found that the “docility” of Hooded Lister rats as compared with Sprague Dawley rats was a crucial factor which enabled multiple precise wound area measurements to be obtained daily for each animal. Although this ability to trace accurately the wound area is absolutely fundamental to the subsequent mathematical treatment of the data, few studies have even commented upon it. However, a model which is merely reproducible is insufficient to investigate modifications to the wound healing process as it must also be capable of detecting the effect of a potential treatment or procedure; hence we used a steroid which is well known from animal studies and clinical observations to inhibit the wound healing process. This was an important consideration in our present studies since the Hooded Lister rats, chosen as the experimental model, have only been used on one previous occasion for wound healing studies.34 Since these rats clearly showed a significant dose dependent inhibition of wound healing by prednisolone (Table 3) this makes them comparable to other strains of rats.” During the daily weighing, tracing and handling of the animals it was noted that the animals which received 10 mg/kg prednisolone, although showing a marked inhibition of wound contraction, were not directly comparable in terms of body weight with the control animals. Surprisingly, this decrease in body weight was not commented upon when this drug was used in a recent wound healing study,64 since it has important implications. The significant loss of body weight by steroid treatment animals over control animals suggests that the mechanism of steroid action on wound healing may be non-specific. Our observations suggest a number of important considerations that any wound healing study must contain. Firstly, the body weight of every animal should be measured frequently and shown in the results. This is to ensure that all the animals, both test

195 and control, were at least comparable in respect of body weight change. This simple yet fundamental precaution used to be taken in some of the earlier wound healing studies.6,6g Regrettably, the practice has been discontinued in more recent investigations. Secondly, daily tracing ensures that the progress of the wound is closely monitored both visually and by the area measurement so that any changes, as well as problems like injury and infection, can be rapidly detected. In addition, any aberrant results-such as may be produced by an unduly adherent scab-are minimised and the coefficient of wound contraction maintains its accuracy. This clearly would not be the case if readings were carried out at 3-5 days intervals, since such anomalous results could then have a significant effect on the value of this constant.3g Finally, conclusions about the specificity of the inhibition or otherwise of a procedure on wound contraction should always take into account the contribution of the above factors and, in the absence of any indication that these measurements/observations have been made, any such claims should be treated with caution. Once the model was found to be simple, reliable, reproducible and sensitive, consideration was given as to the most suitable way to express the results since there are a number of methods reported in the literature. This inevitably complicates any attempt at a direct comparison of the results of one study with another. Many studies35, 53,6o,70*71have used graphs of wound area versus time but this produces a curve which does not facilitate quantitative comparison. An alternative has been to use a graph of the percentage or fraction of the original wound area versus time. 32,38,58164 This procedure may be inappropriate when wounds of different sizes and shapes are being compared since the figures produced become relative rather than absolute values. The transposition of the wound area to its square root and then expressing this against time as suggested by Snowden28*63 is a further complication yielding no additional useful information on the overall process of wound contraction and has not found widespread use. In these present studies we found that the most suitable graphical presentation of the results for comparison was a simple plot of log. wound area versus time since this results in a straight line graph, the slope of which is a constant known as the log. coefficient of wound contraction. Modern microcomputers with appropriate statistical packages are readily able to calculate this constant and its associated correlation coefficient accurately and without bias. In our studies the correlation coefficients obtained for these slopes (Tables 2a and 2b) suggest that a highly reliable result could be obtained with this model. In the calculations of the coefficient of wound contraction, a decision has to be made about which time period is thought appropriate. Taking measurements over the whole study period will include the “lag” phase where little wound surface activity is evident when the wound is dressed, and it may be argued that measurements confined to the phase of rapid wound contraction (Fig. 2) would be more relevant when studying the effect of a treatment.“”

196

Likewise, the results after 12 days could be ignored since by then active contraction in the rat wound has ceased, being maximal at day 7.62As long as the choice of study period is clearly stated in each experimental protocol, comparison of the result of different studies may be possible. We conclude that the adult female Hooded Lister rat excisional wound model described here is a simple, reproducible, sensitive and quantifiable model for the study of wound contraction. Furthermore, from these studies it appears that the coefficient of wound contraction is a simple yet reliable measure of wound contraction rate and its use may facilitate the direct comparison between the results from groups of animals differing by species, age, sex and treatments. What are the significant clinical applications of such experimental studies? Even though we recognise that the extrapolation of data from animal studies to man should be very guarded, nevertheless the results of pharmacological and other manipulations carried out on such models may yield potentially useful information on the mechanisms underlying certain clinical conditions characterised by progressive pathological contraction on one hand and those characterised by the failure of normal contraction on the other hand. Examples of the former conditions include Dupuytren’s contracture, capsules around silicone breast implants and hypertrophic scars, while chronic venous ulcers” are an example of the latter. It has been suggested that the common factor in many of these conditions is the activity of contractile fibroblasts or “myofibroblasts” which are also the cells found in the granulation tissue of healing wounds.73 Thus the identification of pharmacological agents or biological growth factors which could significantly alter the rate of wound contraction may prove to have a wider clinical application. Acknowledgments The authors would like to thank Glaxo Group Research for providing financial assistance for this study, and Mr James Kirkpatrick, Research Fellow at the Plastic Surgery and Burns Research Unit, Bradford, for his help with the completion of this paper.

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The Authors Sheree E. Cross, PhD, Postgraduate Student, Postgraduate School of Studies in Pharmacology, School of Pharmacy, University of Bradford. Current address: Department of Medicine, Queensland University, Brisbane, Australia. Ian L. Naylor, BPharm, MSc, WD, Lecturer, Postgraduate School of Studies in Pharmacology, School of Pharmacy, University of Bradford. Robert Coleman, PhD, Peripheral Pharmacology, Glaxo Group Research, Ware, Hertfordshire, SG12 ODP. Tiew Chong Teo, MD(Hons), FRCSEd, formerly Research Fellow, Plastic Surgery and Burns Research Unit, Department of Biomedical Sciences, University of Bradford. Current address: Plastic Surgery Department, Queen Victoria Hospital, Holtye Road, East Grinstead. Correspondence Pharmacology, Bradford, West Paper Paper

received accepted

to: I. L. Naylor, Postgraduate School of Studies in School of Pharmacy, University of Bradford, Yorkshire BD7 IDP. 23 June 1994. 5 January 1995, after

revision.