CHAPTER IV

Regeneration and repair of wounds in Clarias batrachus

Introduction

If there is no regeneration, there could be no l f e ,

I f everything regenerated there could be no death,

Life exists between two extremes

- Goss (1969)

There is no doubt that wound healing and regeneration play a useful

part in the life and survival of animals. The skin in its normal state is

continually undergoing regeneration.The epidermal cells which are shed from

the surface are constantly replaced from below to maintain an equilibrium

(as described in previous chapters).

The active state of regeneration normally displayed by the epidermis

is probably of evolutionary significance because the skin is continually

subjected to trauma and without an effective mechanism of quick repair of

the outer surface, the life and survival of an individual would be precarious

(Mittal and Munshi, 1974).

The process of regeneration in the broadest sense is vegetative

reproduction and the capacity for regeneration varies in different groups of

animals. In fact, all organisms posses the power to produce new cells.

The term 'wound' refers to the break in continuity of a tissue. This

break may not be associated with a loss of tissue as in incised wounds or

there may be varying degree of loss of substance caused by either physical,

chemical, microbial or immunological insult to tissue (Johnson and Mc Minn,

1960). There does not seem to exist a precise definition of the term 'healing'

in the literature. Acco:rding to many authors (Mittal and Manshi, 1974;

Phromsuthirak, 1977; Nlittal et a/ . , 1978; Al-Hassan e t a / . , 1991; Ramesh et

ai., 1993; Martin et a/ . , 1994) a wound is said to be fully healed up when it

becomes fully epithelised. But it ignores the fact that many chanses still

continue to occur in the underlying connective tissue long after the surface

cells have been restored. Regeneration. as it is well established, is the renewal

of lost/removed part of the body. It is therefore resolved that, once a wound

is formed the first step undergone is repair of the wound which is then

followed by regeneration. The former is characteristic of all organisms, but

the capability of an organism for the latter varies, being restricted to some

organs in some animals. However, the skin of all animals is capable of

regeneration.

Localised destruction of the skin sets in long lasting sequence of

changes in the activities of the cells which surround the wound. It results in

the production of numerous substances by the injured cells. The course of

events taken by the constituent cells of the skin leading towards healing

seems to be largely determined by interaction between the cells at the site

of the wound.

Wound healing has aroused the investigative curiosity of man since

the beginning of his history. The literature on wound healing is vast and

deals with many different aspects of regeneration and repair in mammals

(Sequeira et al., 1989; Hunt, 1990; Ramesh et al., 1990; Martin and Lewis.,

1992; Martin et al., L992; Be~nent er a/. , 1993; Mc Cluskey et al., 1993;

Ramesh er al., 1993; Martin et al., 1994; Meyer et al., 1996; Oxland el al.,

1996; Walsh et al., 19526:). Wound repair in mammals involves various phases:

1 . Haemostasis and fibrin deposition : which blocks the wound and

thus prevents loss of body fluid.

2. Inflammation : by vascular dilation and cellular migration which

prevents infection and brings the substances for wound healing to

the injured site.

J . Wound debredi~nent : involves the removal of debris or tissue that is

heavily contaminated by dirt and bacteria.

4. Fibroplasia : collagen and other fibres are synthesized by fibroblasts

which are invariably seen in the wound.

5 . Neoangiogenesis : involves migration and proliferation of endothelial

cells followed by the formation of capillary buds which later canalize.

6. Wound contraction : is the process by which the size of the full

thickness open wound is reduced by the centripetal movement of the

whole thickness of surrounding skin. It is independent of size but

depends on shape of the wound. the rate being faster in rectangular

wound than circnlar wound.

7. Re-epithelisation : is most important as the wound remains as wound

until new epithelium is formed. It depends on several factors and

there are 4 stazes in the process of epithelisation as- cell mobilization.

cell migration, proliferation and differentiation.

8. Scar modulation : is the final and long continuing process, during

which the original size of the'scar is reduced.

Compared to many organisms, fishes have been studied very little in

terms of wound healing and regeration, the reasons being low tolerance or

viability of many fishes following injury to infections (Vorontsova and

Liosner, 1960).

Brief Preliminary studies on wound healing in taleost fishes were

done by Becker (1941), Kudokotsev and Silkina (1967) Ramachandran and

Thangavelu (1969) and Mawdesley-Thomas and Bucke (1973). Criddle et al.

(1986) A1 Harson et al. (1985) and A1 Hassan (1990) studied the role of fish

skin secretion in wound :healing.

Hase (1935) discussed the regeneration of gold fish fins. Karnrin and

Singer (1955) and Gors (1969) discussed barbel regeneration in the catfish

Alneilus nembulosus. Weis (1972), Sherwoods (1977) and Wiklund & Bylund

(1996) made detailed study of fin re~eneration.

Skin-wound healing in experimentally wounded fish was studied at

the ultra structural 1eve.l by Phromsuthirak (1971) and Iger and Abraham

(1990).

According to Bereiter-Hahn (1986) wound repair in fish involve two

steps: One quick event the elimination of injured tissue and re-epithelization

of the dermal connective tissue. Tlie inflammatory reaction after wounding

was studied by Finn and Nielson (1971a,b) Mittal and Munshi (1979)

Anderson and Roberts (1975) and Phromsuthirak (1977).

Iger and Abraham (1990) investigated the cellular activity including

mitosis during wound healing in wounded carp. Al-Hassan et al. (1991)

studied the accelerated wound healing by epidermal secreation of Arius

bil~neatus.

Avenant-Oldewage (1994) studied the integumental damage caused

by wounding inflicted by crustacean Dolops ranarzmz on Clarias gariepinus.

Most of the studies reveal that wound healing in fishes follows the

same pattern as in mammals.

Wound healing and regeneration seems to be influenced by various

intrinsic as well as extrinsic factors. Roubal aud Bullock (1987) studied the

effect of hydrocortisone on wound liealing in Atlantic Salmon Salmo salar.

Anderson and Roberts (1975) investigated the effect of temperature on wound

healing in teleost. Effect of ultraviolet rays in regeneration has been examined

by Johnson and Denton (:1977). Nail1 et al. (1982) studiedthe role of epideimal

factors in wound healing.,

The need for studies on repair and regeneration of wounds in fishes

is increasing, as the chance of i n j u ~ y to fishes by competition for various

causes is increasing. Anthoropogenic pollution of aquatic habitat results in

crowding of fishes in habitable space. Intensive and composite fish culture

results in competition fclr living space. Aquarium keeping of fishes lead to

fight: for various reasons In addition to the above reasons fishes fight during

breeding season for gettxng mate. All these cause severe injury.

Clarias batrachus, having great tolerance and survivability, is a good

experimental animal foi: wound healing studies in fishes. In the present

investigation, an attempt has been made to study the dynamic process of

growth and regeneration of superficial wounds on fish skin.

Materials and Methods

Fishes (Clarlas batrachus) approximately 15 cm to 18 cm in length

were collected from the ponds and rivers near Pathanamthitta, Kerala and

were acclimatized to laboratory conditions before experiments began. The

fishes were fed dally with chopped goat liver and earthworm.

The region between anterior end of the dorsal fin and lateral line

was selected for the study (Fig. 1) . Two types of wounds were applied to the

fishes.

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Experimental aniinal Clnrins bntrachus. Region marked by * is

the site where wound is made.

Diagrammatic :representation of excision wound - surface view.

Diagrammatic representation of excision wound - sectional view.

D~agramrnatic representation of contacted, healing excision

wound - surface view.

Fig. 5 Incision wound - diagrammatic representation, surface view.

Fig. 6 Incision wound - diagrammatic representation, sectional view.

Fig. 7 Diagrammatic representation of different stages in the healing

of incision wound (marked A - F). Sectional view.

(For fuller explanation see text)

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fig. 4

Fig. 6 4h. I,,

Fig. 7

1. Excision \Yound : For one group of fishes. wounds approximately 12 x

10 mm size and 2-3 nlm~ deep, parallel to the longitudinal axis of the body

were made with a sharp sterile scalpel blade (Fig. 2).

2. Incision wound : For the second group of fishes wounds approximately 6

mm in length and 2-3 mm in depth lying parallel to the longitudinal axis of

the body were made. (Fig. 5)

After the infliction of wound the fishes were returned to fresh water

aquaria until sacrificed.

Skin fragments with incised wounds were excised, at various intervals

of time (i.e., 1 3 :12h, 1811, 24h, 2 Days, 3d, 5d, 6d, 8d, 10d, 12d, 14d,

16d, lSd, 20d. 36d) and fixed in 10% neutral buffered formalin. The tissues

were dehydrated using giraded alcohol. Standard methods (Pearse, 1985) for

clearing and embedding were followed. Paraffin sections were cut at 5mm

and n-ere stained with Hermatoxylin in conjunction with eosin.

Macroscopic study of the healing process on excised wound was also

done. Fishes were brought close to the side of aquarium glass gently, using a

plastic trav and wound margin was traced on a transparent sheet which is

then plotted on a graph paper to count the wound area. The wound area were

measured at regular intervals and the rate of wound contraction calculated,

as percent of wound area, assuming initial wound area as cent per cent.

Observations

A. hlacroscopic Observations

Macroscopic observations are made on excision wound. After a lesion

is made in the skin, the free borders of the excoriated part retract, increasing

the area of the wound. For convenience we may divide the wounded area of

the, skin into three regions: region I- tlie wound area proper or the wound

eap: region II- the small area surrounding the wound and region I11 - the rest v

of the skin (Fig. 2,3).

In some wounds blood appears immediately after the cut is made.

But in others blood appe:ars late in the wound. Within 5-10 minutes after the

infliction of the wound the skin in region 11 start changing its colour, becoming

very dark b!. 1-2 brs. It i.s possible that the pigment cells in this region react

pliysiologically to cut made in tlie skin. However, the normal shade is

reestablished after 24h.

Initially, within 30 minutes there is an expansion of the wound

represented by an increase in the percentase of wound area. Within about

24h, the wound gap starts contracting gradually. However, the curved borders

of the square-shaped wound remain distinct even upto 6d, though it becomes

less conspicuous in later stages.

Within 6h, the wound gap' appears to be covered with a thin

transparent sheet through which muscles of the wound can be observed. This

gradually becomes translucent. By 2d. blood capillaries in the form of red

specks may be observed in the centre of the wound, which gradually becomes

more prominent in the later stages. After 6d the blood spots get concealed

behind a thick whitish mass that appears in the region.

The square-shaped wound develops semilunar margins by 6d

(Fig. 4). Gradually the wound gap narrows down as shown in graphi I and

Table I and finally by 48d the wound gap gets completely closed due to

healing and no scar is 1e:ft on the sur,face.

Table I

Wound contraction, in excised ~vound. in Clal-zas batrachzis

represented as percentage of wound area

% Wound area

0 h:r 100

1 day

4 days

6 days

10 days

24 days

36 days

48 days

Microscopic observations have been made only on incision wourids.

This type of wound can be divided into three zones, in addition to the three

regions described previiously; the zone B representing the middle central

part of the wound and zone A and C representing the two ends of the wound.

.At zones A and C, the depth and width of the wound gap is less than at zone

B (Fig. 5 )

Changes in t h e Epider.mis

As a vertical illcision is made in the skin the epitheliums get

discontinued and a V-shaped gap appears in the wound (Fig. 6). The size of

the breach depends on the tension of the skin in individual fish. This

unfortunately cannot be controlled and will make a difference in the time

scale of healing from fish to fish.

After a period of inactivity. of nearly 30 min., remarkable changes

may be noticed in the epithelial cells present at the margins of the wound.

Tlie epidermal cells increase in volume. The basal cells loose their firm

anchorage with derm:is and the epidermis is lifted from the dermis.The

epidermis is gradually dragged towards the wound gap (Fig. 7c). The cells

migrate - as a layer rather than individually. During this movement they bend

in the direction of movement (Fig. 10, l l ) and displays phagocytic activiry

of mainly cellular debris.

Due to the migration of the epidermal cells, the epidermis in the

rezion I1 of the wouud be~comes ver). thin. Mean while the epidermis in regicn

111 also starts migrating towards region I1 and reaches the wound margin and

ultimately by -lh the migrating epidsrnmal cells from all the sides move deep

into the wound. Mean while. blood cells and mucus appear in the wound

gap, the quantity of nrh~~ch gradually increases and results in the temporary

closure of the wound gap within 2h of illjury. The migrating epithelial cells

from all sides come close to each other meet and coalesce together resulting

in the complete epithe:lization of the wound forminy a continuous layer

(Fig. 13,14) by 411.

By 12h. after wounding a basement membrane starts differentiating.

It then reestablishes its anchorage with the dermis. The mucous gland lining

the wound area appear to be very active as they secrete copious amounts of

mucus. At this stage b a d cells start differentiating which are almost flat or

low cuboidal in shape each having a flat nucleus.

At about 12h the basal cells acquire a cuboidal shape (Fig. 12) each

having a centrally placed spherical nucleus. The thickness of the epidermis

gradually increases. But there may be seen. sometimes, reduction in the

number of layers due to poor healing.

The basal cells are closely approximated to each other without having

intercellular spaces (Fig. 13). In between the polygonal cells of the middle

layer however. prominent intercellular spaces are discernible that give a

vacuolated appearance to the epidermis. Mucous cells show an increase in

the number and are arranged mainly in the outer layer of the epidermis.

At about 18h the epithelium that covers the wound gets its support

on the underlying tissue. The basal cells around the wound area (region

II).which get lifted up from the dermis now take up their firm anchorage on

the dermis. The central core of the epidermis of the newly closed up region

is characterised by vertically arranged irregular spindle-shaped epithelial

cells. The giant cells in and around the wound region show the maximum

degree - of vacuolization and attenuation. Few blood cells are seen in between

the epidermal cells on. the wound area.

At about id , the l~olygonal cells in the middle layer become compactly

arranged, giving the epidermis a comparatively thin appearance. The mucous

cells show vely high activity secreting copious amount of slime, forming a

thick layer on the surface of the epidermis.

The vacuolization of the giant cells reach its peak and then these

cells slowly disappear (by 2d) their place being taken by other epithelial

cells (Fig. 15). The basal cells take their usual columnar or high - cuboidal

shape (Fig. 15). The lymphatic spaces in between these cells likewise,

~~radual ly enlarge and get gorsed wifli small lymphocytes (Fig. 15). 3

After 2d, severa.1 rows of small cells may be differentiated just above

the basal layer. The cytoplasm of these cells show a high degree of

contraction. Few newly formed club cells may also appear at the sides of the

wound (Fig. 16). At A. and C regions of the wound the number of such cells

is more than at region B. The whole epidermis over the n-ound area, specially

neal the basal layer appear to be infested by a large number of red blood

corpuscles and phagocytes.

6d after wounding, the epidermis becomes thick and is mainly

composed of polygonal cells with distinct intercellular cytoplasmic bridges

and spaces. Mucous c'ells in different stage of their development are

discernible resulting simultaneously in an increase in the number of the

voluminous, sac-like cl.osely approximated mucous cells in very active

secretory stages (Fig. 17).

The basement n~embrane becomes comparatively very thick and

distinct. By this time, the repair of the derniis takes place and the epidermis

covering the wound area is pushed outwards. The deep V-shaped trough in

the wound region becomes shallow and finally by 6d the epitheliums over

the wound area comes to line. at the level of the normal epidermis.

The old club cells, by this time. are completely replaced by the newly

formed small epidermal cells. A good number of new club cells may appear

at the sides of the wound area. The basal layer of the epidermis in region I

and I1 of the wound show a high rate of activity. The clear rounded cells

situated in the basal layer enlarge in size and the nuclei divide and redivide

without the cytoplasmic division giving rise to nests of nuclei. Later on these

nuclei accumulate cytoplasm and give rise to new epidermal cells. Pigments

though comparatively s~na l l and less' in number may be seen.

After 8d, the epidermis appear wavy in out line, giving out several

papilla like projections (Fig. 30). Subsequently the epidermis becomes thinner

and acquires its normal appearance b!- about 20d. During this period the

epidermis over the wound area is mai111y occupied by newly formed giant

cells in different stages of their development. No other special activity was

noticed in the epidermis.

Changes in the Sub-epidermal tissue

In an artificial wound in the skin. the tough collagen layer of the

dermis along with the subcutis gets apart and the muscle bundles bordering

the mound gap get denuded.

No significant change or acti\-in. in the organisation of the dermal

tissue and muscle budle is evident upta 211 after the injury

After 3h remarkable changes occur in the denuded muscle bundles.

Some of the loose bundles may be observed lying freely in the wound gap

and by 1611 these muscular elernents came to lie just below the epidermal

In between the disintegrating muscle bundles, connective tissue cells,

blood capillaries and blood sinuses may be observed (Fig. 22). After 2d a

few blood capillaries may even be observed to penetrate through the basal

layer of the epidermis. There is marked increase in the number of phagocytic

cells. The subcutis layer near the margin of the wound area gets a rich supply

of blood capillaries. The cut end of the collagen layer still remain unchanged.

The disintegrating muscle bundles, and the acellular amorphous

substance disappear conipletely from the wound gap which is now by 3d

occupied by a large number of ~rregnlarly and loosely arranged fibroblasts,

fine blood capillaries. wandering blood cells and connective tissue cells

resulting in the formation of the granulation tissue (Fig. 20). The blood

capillaries are comparatively more numerous in the outer layers of the

granulation tlssue.

At about 5d, the granulation tissue become comparatively more

compact having the fibroblasts in the outer layers,Oriented more or less

parallel to the body surface and show the besinning of the process of fibre

formation. At the lateral and inner margin of the granulation tissue, small

myoblasts start appearing at the level of the old muscle bundles (Fig. 22). A

large number of vertically oriented blood capillaries and small pigmente cells

are also obsel -~sd in the granulation tissue (Fig. 22). The cut ends of the

compact collagnous layer of the dermis still remain distinct at 6d.

By 7d to 8d the fibroblasts become more compact and get arranged

in a regular fashion parallel to the body surface and later on undergo

fibroplasia (Fig. 25). The lying down of the fibre starts first at the margins

from outwards to the inner side due to the formation of collageous fibres at

the margins of the wound just below the epidermal cells, the wound area

now looks like an inverted 'V' .

Along with the differentiation of new collagen fibres the pigment

cells start appearing scattered irnmehiately below the epidermis at region I.

.At the margin of the wound area these cells are however well developed and

Illore distinct at 9d. The melanophores gradually increase in number and

enlarge to form a thick and distinct layer below the epidermis. Well developed

fat cells may be observed at the anterior and posterior margins of the wound.

At the margins of the wound near the old muscle bundles a large

number of new muscle cells (myoblasts) appear, which differentiates

nradually into muscle bundles (Fig 16). In this area, the connective tissue - cells are usually arranged in a circular fashion mingled with fine blood

capillaries.

After IOd new muscle bundles of appreciable size are distinctly seen

111 the wound area. At this stage it is \.cry difficult to establish the original

margins of the wound in the dermal area, because of the formation of new

collagen fibres that cannot be distiniuished from the rest. On the outerside.

the central area of the wound just below the epidermis is occupied b ~ .

compactly arranged collagen fibres intermingled with few fibroblasts (Fin - 7 5 ) , oriented parallel to the body surface. The rest of the area still remain

infiltrated with fibroblasts and blood capillaries. Pigment cells may be

observed forming a complete layer below the epidermis of the newly

regenerated wound area (Fig. 18).

After 12d the granulation tissue may be differentiated into the stratum

lasum and the stratum compactuxn and also the muscular layer. The subcutis

layer reappears only by 20d. The deeper part of the central core of the wound

are still infiltrated with fibroblasts, which take blue colour in haematoxylini

eosin preparations (Fig. 23) slowly this colour reaction changes to red as the

young collagen fibres get matured.

After 20d the wound area is hardly distinguishable from the rest of

the skin except that the stratum compactum is comparatively thinner than

that of the unwounded areas and that the dermis is heavily infiltrated with

blood cells. h o other special activity was noticed in the subepidermal tissue.

Abnormal Observations in \Vound healing

Certain abnormalities are noticed in the wound healing process.

Sometimes the quality of healing is poor. where re-epithelisation and collagen

reorganization is delayed, basement membrane poorly formed, epidermis thin,

and collagen fibres not strongly bound (Fig. 26). Over healing of the skin may

sometimes occur resulting in abnormal structure in the regenerated skin.

Hyperplasia, irregular surface of the regenerated epithelium and increased

number of epidermal and dermal papillae (Fig. 27) are some observations of

abnormal healing. These alterations may be caused by some external factors

which need further investigations. Irregular surface of the regenerated

epithelium niay also be noticed in bruiced skin, where the epidermis is

damaged and the dermis is irregularly cut at many planes (Fig. 29,30)

Abnormally high concentration of melanin may observed during some

stages in the process of wound healing. (Fig. 28).

Discussion

The process of healing of superficial wounds in fishes varies

considerably from that of mammals and other higher terrestrial vertebrates.

The epitlielization in mammals starts after a latent period of 24 hrs, while in

fishes the mo\.ernent of the epidermis bezins within 1-2 hrs after the infliction

of tlie wound. In fishes (Mitral & Munshi, 1974; tlie present investigation),

the granulation tissue is formed long after the complete epithelization of the

wounds. Thus the quicker rate of ebithelization in fish may also be correlated

with the late formation of the granulation tissue as also suggested by XIitral

and Munshi (1974).

The present investigation reveals that the epithelization of the wound

in C. batrachzis takes place by mass movement of the epidermal cells towards

the wound gap resulting in the bridging of the wound gap. A relatively very

thin epidermis in region I1 during the process of epithelization supports this

\.iew. Kudokotsev and silkima (1967), h,littal and Munshi (1974) also reported

epithelization by movement of the epidermis from the areas surrounding the

wounds in fishes.

According to van Oosten ( 1957). the epithelization of wounds in fishes

takes place by proliferation of cells at the edges of the wound. No significant

illcrease in the mitotic activity in the epidermis is observed at the wound

edge in Clarias batrachzis. The mass movement of epidermal cells is initiated

by the detachment of the epidermis near the wound edge from the basement

membrane as observed by Lash (1955j and Weiss (1956) in amphibians. The

migrating epithelial cells retain their general morphological charactenstics

for some time. The orientation of cells, however, changes as they move

towards the wound gap.

The present investigation reveals that, in Clarias batrachus the

epithelization of the incised wounds takes place by 4 hrs. Ramachandran

and Thangavelu (1969) reported that it takes place by 3-4 days in Channa

sp. hlittal and Munshi (1974) reported complete epithelisation of an incised

wound in Rira rita within 4-6 11. The rate of epithelialization is different in

excised wound in Clarias batrach~ls which takes place by 6h.

Mittal and hlunshi (1974) correlated the rate of epithelisation with

the thickness of the epidermis in different fishes and speculated that in fishes

having thick epidermis the epithelisation is very quick than those having

thinner epidermis.

The quicker rate of epithelization in fishes may be correlated with

the late formation of granulation tissue. In Clarias batrach1rs the formation

of granulation tissue starts only after three days, long after complete

epithelization.

The process of epithelization takes place much more rapidly in fishes

than in marnnlals and other land vertebrates. In mammals the epithelization

starts after a latent period of approximately 2411 and is completed in 2d

(Bullough and Lawrance, 1957) or 6-7 days (winter, 1964) depending on the

slze of the wound. Apart from the reason that the average thickness of the

epidzrn~is in fishes is comparatively greater than that of mammals (according

to Bloom and Fawcett (1968) in mammals the epidermis varies from 70 mm

to 120 mm in thickness over most of the body), the quicker rate of

epithelization in fish may also be due to the fact that the fish remains in

water and secretes mucous so that the wound iilval.iably remains moist. This

is supported by the observations made by winter (1964) who studied the

speed of epithelization on dry as well as moist wound surfaces in pig skin.

Rovee et al. (1972) also reported that in mammals the maintenance of wound

tissue hydration overtly alters the speed of migration of epidermal cells, a

single and most important factor in re-establishing a continuum of epidermis.

During wound repair there occurs detachment of the stratum

germinativum layer of the epidermis from the underlying basement membrane,

as observed by Lash (1955) and Weiss (1956) in amphibians. This makes the

cells to move freely towards the wound gap resulting in quicker epithelization

of the wound in fislles and amphibians. According to Weiss (1956) the

detachment is due to the swelling and hydration of the cement substance

present between the epidermal cells and the basement lamella. It may also

be due to some enzymatic activity going on in the basal layer of epidermis

during injury. This needs more experimental work to confirm the reason.

Ramachandran and Thangavelu (1969) reported that the newly formed

epidermis in Channa species is devoid of mucous glands.

The present investigation, however, reveals the presence of PAS

positive mucous galnds secreting copious amounts of mucous that forms a

thick layer on the surface. Mittal and Munshi (1974) also made similar

observations and correlated the presence of thick mucous layer on the surface

playing an important role in protection. This hypothesis may be supported

by the observatio~ls that in Clarias batrachzrs, the number of mucous glands

In the wounded area increases enorn~ously during the early hours of repair.

It is interesting to note that the epidermis covering the wound gap is thicker

than the adjacent regions during the,wound healing process.

The problem, whether the basement membrane is formed by the

epithelium or by connective tissue has been a subject of controversy. Some

illvestigators (Nadol et a[., 1969) considers the basement membrane to be

formed from collagen while Pierce et al. (1964) considers it to be formed by

epithelial cells. Phromsuthirak (1977) considers the formation of basement

nnembrane by the combined action of the two tissues. Iger and Abraham

( 1990) observed the regeneration of the basal lamina in carp corresponding

to the renewed organisation of the basal fi la~nent cells from the epidermis

and the fibroblasts in the dermis. In the present investigation the basement

membrane starts appearing by 1111 aftel. incisio~i but the granulation tissue

appears only by 3d. This supports the view that the basement membrane is

formed by epithelial cells. It may also be possible that the basement membrane

is formed from the amorphous non cellular substance formed from the

degenerating tissue.

The presence of well defined lymphatic spaces in the stratum

eerminativum layer of the skin in teleostean fishes is very significant (Mittal L

and Munshi, 1971, 1974). In Clarras batrachzls these lymph spaces in the

epidermis adjoining the wound enlarge on the 2nd day after formation of the

wound and become gorged with lymphocytes. This condition continues for

several days and may be an i~nmunological reaction of the body playing an

important role in the local defense mechanism.

The repair of the wound involves considerable reorganization of

fibrous and cellular systems to the dermis. blittal and Munshi (1974) observed

the formation of a granulation tissue in the wound gap composed of large

number of f ibroblasts and fine blood capi l lar ies , in Rita rita. Rich

vascularization in granulation tissue facilitate the active migration, of

leucocytes in these areas that may act as an efficient protective barrier

preventing the absorption of toxins and the penetration of bacteria into the

underlying tissues. It niay further be correlated with supplying the various

nutrients efficiently, in view of the increased need of the actively proliferating

cells in wound regions.

The question of origin of the iibroblasts in the granulation tissue has

been a matter of considerable debate. -4ccor-ding to one school o f thought

these cells are derived by metaplasia of mononuclear cells of the blood stream.

(Fichtelius and Diderholum, 196 1; Petrakis et al., 196 1). Another view is

that fibroblasts are derived from the local mesenchymal cells. (Dunphv. . . 1963;

Hadfield, 1963; Grollo, 1963; kIch,I~nn_ 1967), their source being the loose

areolar perivascular tissue.

In the late stages of healing in C'lnrins batrachrrs, there is a decrease

in the amount of granula t ion t issues present in wound gap by the

disappearance of the many fibroblasts and other cell types. According to

McMinn (1967) some of the fibrobalsts having done their job of synthesis

remain as resting fibrocytes, while the fate of the remaining fibroblasts and

other cells is not precisely known because there are no very obvious sign of

~ ~ e c r o s i s or degeneration of these cells.

In the present study, the phenomenon of wound contraction is clzarly

evident in excised wound. Mittal and l l u ~ l s h i (1974) denied such phenomenon

in Rira rira, \vhere the scar remained after healing. But in Clarias batrachus

wound contraction occurs which is fast in the beginning and very slow after

10 days. The repair of the dermis takes long time compared to re-epithelisntion

of the wound gap. In incised wound, where the wound gap is narrow repair

rnay occur at a rapid rate than the excised wound, as observed in the present

study.

There has been extensive work on the phenomenon o f wound

contraction in mammals and a number of conflicting hypotheses concerning

the location and site of this mechanism were put forth from time to time.

The hypothesis of Grillo et al. (1958) proposes that a specialised zone of

cells the'picture frame', developed beneath the wound margins, migrate

centripetally carrying the overlying skin with it. Again Ambercrombis et al.

(1960) suggested that the contraction mechanism actually lies in the

granulation tissue mass itself and that the other hypothesis explaining this

phenomenon may apparently be excluded.

The degeneration of the denuded muscle bundles during the early

hours of healing is interesting Kudoltotsev and silkima (1967), Roberts et a1

(1973), Mawdesly-Thomas (1973) Mittal and Munshi (1974) also made

similar observations. Pomerat (1956) correlated this lysis with the relsase of

nutrients and in loosening of the strorna in the wounded area. the appearance

of an accellular amorphous slightly eosinopl~ilic substance in the woundgap

in C:larias brrrrach~is with the degeneration of the muscle bundles is thus

significant. The amount of this substance is maximum in the wound gap

when the vacuolizatiorl of the ~nuscle bundles reaches its peak, then it

gradually decreases with the formation of granulation tissue. Yamada (1964)

and Roberts et al. (1973b) also reported the presence of such substance in

the n-oundgap and called hyaline plasma and fibrinous exudate respectively.

In Clarias batrachzrs the colour of the skin close to the w-ound

chanses, becoming dark, and remains so for about id . It is possible that

when the cut is made, the nerves alons with the pigment concentrating fibres

supplying the pigment cells in tlie area are damaged, resulting in the passive

dispersal of the pigments. This observation corroborates the findings of sage

(1970) that the dispersal of the pis~nents is under neural control. Other

workers in the field (Becker, 1942; Kudokotsev and Silkima 1967; Conant.

1970) made no reference to such a change in colour when a cut is made in

the skin of fish. Melamin granules may have bactericidal properties (Ellis.

1977). Iger et a1 (1988a) and l ~ e r and .Abraliam (1990) presumed that melamin

from the demal melanocytes may form a barrier against invading bacteria in

fish living in polluted water and fish inflicted by a wound respectively. The

abnormal increase in melanin observed at some instances during the present

i~lvestigation may also be due to the same reason.

Fig. 8-18

Fig. 8

Fig. 9

Fig. 10

Fig. 11

Fig. 12

Fig. 1.3

Fig. 14

Fig. 15

Fig. 16

Fig. 17

Fig. 18

Different stages in the wound healing process in Clarias batrachus.

Wound gap showing blood cells and amorphous, eosinophilic fluid (marked by arrow).

A thin layer of epithelial cells have migrated towards the wound gap. The wound gap still contains blood cells and amorphous fluid (marked by arrow).

Epithelisation in the zone A of the wound, where the dermis is not fully cut.

Epithelisation in the zone B of the wound, were the incision is made upto the muscle.

Migrating epidermal cells from both sides meet and undergoes hyperplasia because the wound gap was little.

Migrating epithelial cells meet and move towards the interior of the wound gap.

Epidermal cells from both sides of the wound meet and form a continuous layer above the wound gap which still remains in the dermis.

Wound is fully re-epithelised. Fibroblasts originate from the granulation tissue.

Granulation tissue undergoes maturation where the haphzardly arranged collagenous fibres start arranging parallelly.

Increased number of mucous cells in the healed tissue (PAS reaction).

The wound is completely healed

Fig. 20-30

Fig. 20

Fig. 21

Fig. 22

Various histological observations during wound healing in

Clnrias batrachus.

Granulation tissue showing blood cells, and young fibroblasts

(marked by arrows).

Dermo-epidermal junction in the wound region. Basement

membrane begins to get invested. The granulation tissue contains

more fibroblasts.

Granulation tissue in the dermo-muscular junction, blood

capillaries (marked by arrows) are discernible.

Fig. 23 Collagenous fibres begin to form from fibroblasts

Fig. 24 Collagenous fibres arranged haphazardly (arrow)

Fig. 25 Collagenous fibres get arranged parallelly

Fig. 26 Poor healing. See the epidermis is thin and the dermal collagen

loose.

Fig. 2'7. Overhealing showing hyperplasia of the epidermis. Note an

island of club cells in the dermis, surrounded by pigment cells.

Fig. 28 Hypermelanism in the wound region

Fig. 29 & 30 Irregularity of the, regenerated skin