Cell Tissue Res (2005) 321: 401–410
DOI 10.1007/s00441-005-1139-z
REGULAR ARTICLE
José Roberto Machado Cunha da Silva . Edwin Lowell Cooper .
Idércio Luiz Sinhorini . João Carlos Shimada Borges .
Bernard Ernesto Jensch-Junior . Laércio Ribeiro Porto-Neto .
Francisco Javier Hernandez-Blazquez . Bruno Cossermelli Vellutini .
Leandro Nogueira Pressinotti . Frederico Azevedo Costa-Pinto
Microscopical study of experimental wound healing in Notothenia
coriiceps (Cabeçuda) at 0°C
Received: 29 October 2004 / Accepted: 5 April 2005 / Published online: 13 July 2005
# Springer-Verlag 2005
Abstract Notothenia coriiceps (Cabeçuda) is an Antarctic
benthic fish frequently found with lesions in the tegument
caused by seal predation. We have investigated epidermal
repair in these animals by means of a microscopic study of
experimental wound healing at 0°C. At 24–48 h after
wound induction, mucous exudate and necrotic lining cells
covered the wound. At 7–14 days, an epidermal “tongue”
could be discerned, folded at the tip, with intercellular
oedema between the tip and the wound border. After 23–30
days, the wound was completely closed and the migrating
epidermis, with intercellular oedema, was reduced. By 45–
90 days, melanocytes progressively increased in the epi-
dermis but no scales were formed. The inflammatory in-
filtrate was mainly composed of neutrophils after 7 days,
at which time they were mostly replaced by macrophages;
lymphocytes and plasma cells were also present. The
border epidermis slid towards the centre, folding at the tip
and finally fusing to form a diaphragm. The cells of the
epidermis began to multiply only after complete closure of
the wound. The lack of scale formation on induced and
naturally found wounds, even after 90 days, suggests that
different mechanisms in wound repair occur at 0°C from
those in fish from temperate and tropical environment. This
is the first report of successful wound repair at polar
temperatures, indicating the adaptation of N. coriiceps to
the Antarctic environment.
Keywords Wound healing . Inflammation . Skin .
Antarctic fish . Notothenia coriiceps (Teleostei)
Introduction
Metchnikoff (1891) demonstrated that phagocytosis and
the inflammatory process occurs in almost all animals, with
a few remarkable exceptions (Metchnikoff 1891; Tauber
and Chernyak 1991; Silva et al. 1995, 1998b). The in-
flammatory process, as an essential physiological mecha-
nism, is a component of wound repair. It is essential for
the maintenance of homeostasis, being present from the
beginning of ontogeny (Silva 2000, 2001).
The intensity and velocity of wound resolution in ecto-
thermic vertebrates varies according to local temperature
(Reddan and Rothstein 1965; Finn and Nielsen 1971;
Grout and Morris 1987; Hardie et al. 1994). When the
temperature is reduced, cellular responses tend to decrease
(Finn and Nielsen 1971). Other studies have shown that
Antarctic low temperatures do not inhibit phagocytosis in
invertebrates (Silva and Peck 2000; Silva et al. 2001;
Borges et al. 2002) or in Antarctic fish (Silva et al. 2002).
The inflammatory process is not inhibited either in the
Antarctic fish Pleuragramma antarcticum (O’Neill et al.
1987, 1988) or Notothenia coriiceps (= N. neglecta; Silva
The financial support from CNPq (68.0047/00-0 and 48.0262/00-4
grants), PROANTAR, SECIRM and FAPESP is greatly appreciated.
Professor E.L. Cooper is partially supported by funds from the
Hewlett Foundation to the Latin American Center, UCLA.
J. R. M. Cunha da Silva (*) . J. C. S. Borges .
B. E. Jensch-Junior . L. R. Porto-Neto . B. C. Vellutini .
L. N. Pressinotti
Department of Cellular and Developmental Biology,
Biomedical Sciences Institute,
University of São Paulo,
Lineu Prestes 1524, Sala 409,
São Paulo, 04601-000, Brazil
e-mail: jrmcs@usp.br
E. L. Cooper
Laboratory of Comparative Neuroimmunology,
Department of Neurobiology,
David Geffen Scholl of Medicine at UCLA,
University of California,
Los Angeles, CA 90095-1763, USA
I. L. Sinhorini . F. A. Costa-Pinto
Department of Pathology,
Faculty of Veterinary Medicine,
University of São Paulo,
São Paulo, Brazil
F. J. Hernandez-Blazquez
Department of Surgery,
Faculty of Veterinary Medicine,
University of São Paulo,
São Paulo, Brazil

et al. 1998a, 1999). These inflammatory studies have de-
tected the same pattern as that observed in temperate fishes
but with a reduced speed of healing. Macrophages and
neutrophils predominate during acute phases but the former
do not present the activation signs that characterize their
ultrastructure (Silva et al. 1998a).
The ability of fish and other animals to maintain integ-
ument integrity is essential for their survival. Tissue repair
always aims at closing the wound with new tissue, in order
to recover the original function or to recreate a physical
barrier (Majno and Joris 1996). The inflammatory process
is the initial phase of tissue regeneration or wound repair
and is attributable to the activity of inflammatory cells that
act as scavengers, removing necrotic tissue and debris,
killing potentially pathogenic microorganisms and stimu-
lating fibroblasts and epidermal proliferation (Rowley and
Ratcliffe 1988).
Studies have been carried out concerning the influence
of acclimatization of fish from tropical and temperate
waters on the regenerating process (Mittal and Munshi
1974; Anderson and Roberts 1975; Bullock et al. 1978;
Phromsuthirak 1977; Whitear et al. 1980; Majno and Joris
1996; Quilhac and Sire 1998, 1999). However, only one
study has reported wound repair under polar temperatures
(Silva et al. 2004).
Wound repair is a vital mechanism for animal survival in
any environment and, as there is only one description of
fish wound healing at Antarctic temperatures (Silva et al.
2004), we have studied the histological processes occurring
at 0°C to verify their similarity to those occurring at higher
temperatures.
Materials and methods
Fish maintenance and wound induction
Specimens of N. coriiceps (according to Gon and Heemstra
1990; common name: Cabeçudas; n=21), with a mean
weight of 792.45±220.16 g (range: 438.00–1,835.00 g),
standard size of 34.61±4.73 cm (range: 25.50–47.00 cm)
and total size of 38.44±5.23 cm (range: 29.00–53.00 cm)
were collected in January–February in 2000 and 2001 in
Admiralty Bay, King George Island, South Shetland
Islands (S 62°10.168′, W 058°26.959′). The fishes were
acclimatized for 1 week. Experiments were carried out in
fibreglass tanks (1,000 l) filled with running sea water
(1±1.0°C) in a temperature-controlled room at 0±1.0°C at
the Biology Laboratories of the Brazilian Antarctic Sta-
tion, “Comandante Ferraz”, in which the recording of bio-
metric data and identification of the fish took place (Silva
et al. 1998a, 1999). Following anaesthesia with benzocaine
(50 ppm; Silva et al. 2002), two square (2.0×2.0 cm) full-
thickness excision wounds, removing scales, epidermis,
dermis, hypoderm and most of the perimysium, were in-
flicted on both sides of the dorsal–lateral anterior region
in each of the 21 fishes. Three fish were used as normal
skin controls. After time spans of 0 (n=1), 1 (n=2), 2 (n=2),
7 (n=3), 15 (n=4), 23 (n=4), 30 (n=2), 45 (n=1), 60 (n=1)
and 90 (n=1) days, the fish were killed by immersion in
70 ppm benzocaine. Tissue samples from the border to the
centre of the wound and the area covered with the new
epidermis were collected and processed for light and trans-
mission electron microscopy (Fig. 1). One N. coriiceps was
found with a wound scar on the posterior ventral lateral
region. This fish was photographed and tissue samples from
the wound centre and periphery was processed for light
microscopy.
This work was carried out in accordance with the Ethical
Commission of Animal Experimentation of the Biomedical
Sciences Institute of the University of São Paulo (protocol
number 067/03).
Light microscopy
All samples were fixed in cold McDowell’s fixative so-
lution (McDowell and Trump 1976) for 48 h and decal-
cified in 10.0% Na3C6H5O72H2O (sodium citrate) and
21.25% HCOOH (formic acid) for 7 days. Afterwards, the
tissues were dehydrated in 95% ethanol and embedded in
Historesin (Leica). Sections (2 μm) were stained by the
fuchsin–toluidine blue, Romanowsky, periodic-Schiff (PAS)
and Picrosirius techniques and the peroxidase (H2O2) test
for melanin characterization (Bancroft and Stevens 1982;
Junqueira et al. 1979; Silva et al. 1999). Observation, doc-
umentation and section measurement were carried out on a
Zeiss (Axiomat) microscope equipped with a scale to mea-
sure the “leap-frog” diameter (see below) in tissue samples
and on an Olympus (BX-60) photomicroscope.
Fig. 1 Wound area. Region A Wound periphery composed of
normal skin with scales. Region B Regenerating epidermis. The re-
gion next to the normal skin is the border. The area in contact with
the wound centre is the tip. Region C Wound centre in which the
muscle with the remaining perimysium is in contact with the ex-
ternal environment. Scale (bottom) is in centimetres
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