INTRODUCTION
The eye is a complex and highly developed photosensitive organ that permits
an accurate analysis of the form light intensity and color reflected from objects.
Each eye is composed of 3 concentric layers: an external layer (sclera and cornea),
middle layer (choroid, ciliary body and iris) and an inner layer of nervous
tissue (retina). The optical retina consists of an outer layer of photosensitive
cells, the rods and cones. They are sensory retinal receptors and play an important
role in visual system.
The effects of chronic retinal ischemia in an animal model involving permanent
carotid occlusion investigated by Stevens et al.
(2002). They indicated that ischemic damage to the optic nerve caused loss
of pupillary reflex and death of retinal ganglion cells in a subset of rats.
Block et al. (1997) suggested that the intraluminal
suture method leads to retinal ischemia and immunohistochemistry of the retinas
revealed that the vascular occlusion induced the expression of glial fibrillary
acidic protein in retinal Müller cells. Barnett and
Osborne (1995) showed that reduction of the retinal blood flow by occlusion
of both common carotid arteries suppressed the b-wave of the rat's electroretinogram.
Optic nerve atrophy in rats has been shown following bilateral ligation of common
carotid artery (Takamatsu, 1988). The permeability of
retinal blood vessels to intravenously injected horseradish peroxidase was examined
two to seven days after bilateral common carotid ligation in rats by Korte
and Henkind (1986).
Various histological studies were undertaken on photoreceptor structures of
the retina in different animals using light and electron microscopy (Braeckevelt,
1983, 1987, 1990, 1992,
1993, 1998; Braeckevelt
et al., 1996, 1998; Garcia
and De Juan, 1999; Haacke et al., 2001).
No ultrastructural evaluation of the effect of unilateral ligation of common
carotid artery on the photoreceptor layer has yet been conducted. The objective
of the present study, therefore, was to determine effects of unilateral ligation
of common carotid artery on photoreceptor cells of retina.
MATERIALS AND METHODS
For this study the eyes of 20 adult healthy male dogs were investigated by
light and electron microscopy. The animals were obtained from the animal house
of Islamic Azad University Kazerun Branch. This study was conducted from Dec.
2008 to Nov. 2009. The animals had free access to food and water. The same environmental
conditions (temperature, light history and diet) were used before and after
ligation of common carotid artery.
The dogs were randomly divided into 4 groups (n = 5 in each group): control
group (CON), experimental group I (EXP-I), experimental group II (EXP-II) and
experimental group III (EXP-III). The dogs in the CON group were kept in normal
environmental conditions. Unilateral ligation of common carotid artery was done
in the animals of EXP-I, EXP-II and EXP-III for 12 h, 24 h and 1week, respectively.
The histological effects of ligation of common carotid artery were evaluated
using a transmission electron microscope.
The dogs were anesthetized with xylazine-ketamine and their eyeballs quickly
removed. The cornea, lens and vitreous body were removed and opened at the equator
and thereafter fixed for 4 h in 4% glutaraldehyde buffer (pH 7.3) with sodium
cacodylate at 4°C. The posterior half of the eyeball was removed and the
retina was then separated near the optic nerve of the superior hemisphere and
fixed for an additional hour. Thereafter, the retinal tissue was washed in sodium
cacodylate and cut into less than 1 mm2 pieces. The tissue was postfixed
for 1.5 h in 1% osmium tetroxide, washed briefly in distilled water, dehydrated
through a graded ethanol series and then cleared in propylene oxide and embedded
in agar resin.
Semithin sections (0.5 μm) were obtained using an ultramicrotome (Omu
3; Reichert, Vienna, Austria), stained with toluidine blue and examined first
under a light microscope. Ultrathin sections (60 nm) were also obtained and
collected on copper grids. These sections were stained in aqueous uranyl acetate
and lead citrate and were examined under a Philips CM-10 transmission electron
microscope (Philips, Eindhoven, The Netherlands).
The laboratory care, anesthesia and euthanasia of animals used in this study
were performed in accordance with the Guide for the Care and Use of Laboratory
Animals (Institute of Laboratory Animal Resources, 1996).
RESULTS AND DISCUSSION
The rod outer segments were slender, whilst the cone outer segments were slightly
less slender. The rod and cone outer segments were composed of bimembraneous
discs (Fig. 1, 2). The inner segments of
the photoreceptor layer had few mitochondria (Fig. 1).
|
| Fig. 1: |
Electromicrograph of photoreceptor layer from the control
group. A: Outer segment; B: Inner segment; C: Outer nuclear layer; Arrow:
Outer limiting membrane; Arrowhead: Cone nucleus; Thin arrow: Rod nucleus
(x1650) |
|
| Fig. 2: |
Electromicrograph of outer segment of photoreceptor cells
from the control group. r: Outer segment of rod cell; c: Outer segment of
cone cell; Asterisk: Disc membrane of outer segment (x8900) |
The outer limiting membrane was composed of a series of zonulae adherents between
the rods and cones (Fig. 1). The cone nuclei are located nearest
the outer limiting membrane. They are larger than rod nuclei (Fig.
1).
|
| Fig. 3: |
Electromicrograph of photoreceptor layer from the EXP-1 group.
A: Outer segment; B: Inner segment; C: Outer nuclear layer; Arrow: Outer
limiting membrane; Arrowhead: Loss of mitochondrial cristae (x1650) |
The unilateral ligation of common carotid artery produced damage in the dog
retina. The ultrastructure of the dog retina was examined after unilateral ligation
of common carotid artery of different times. The photoreceptor layers in EXP-I
group showed the least damage, such as only some loss of mitochondrial cristae
(Fig. 3). In addition, in the EXP-I group, the outer segment,
mitochondria, outer limiting membrane and outer nuclear layer appeared normal
(Fig. 3).
The dogs in EXP-II group showed that a few vacuolization and loss of mitochondrial
cristae in inner segments (Fig. 4). But outer segment, outer
limiting membrane and outer nuclear layer had normal. The retina in EXP-III
group had major signs of damage (Fig. 5, 6).
Distention and disorganization of the outer segment disc membrane appeared in
this group (Fig. 5, 6). Extensive vacuolization,
more loss of mitochondrial cristae and cell swelling of the inner segment were
obvious (Fig. 5). Pyknotic and karyolysis nuclei were apparent
in the outer nuclear layer (Fig. 5).
The retina is the eye tissue layer that converts light into visual signals
transmitted to the brain. This process is carried out by two major types of
photoreceptors, rods and cones that are distinguished by their shape, type of
photopigment, retinal distribution and pattern of synaptic connections. These
properties reflect the fact that rod and cone systems are specialized for different
aspects of vision. The photoreceptor layer of the dog retina is very similar
to that of other animals (Braeckevelt, 1983, 1987,
1990, 1992, 1993,
1998; Braeckevelt et al.,
1996, 1998; Garcia and De Juan,
1999; Haacke et al., 2001). In the present
study, the photoreceptor damage produced by EXP-I group, EXP-II group and EXP-III
group was evaluated. At the electron microscopic level, the dogs in EXP-II and
EXP-III showed disorganization and distention of the outer segment disc membrane,
similar to the findings of Esfandiari et al. (2008).
By comparison, the outer segment of dogs in EXP-I appeared normal. However,
it is equally plausible that diurnal changes in RPE function unrelated to outer
segment phagocytosis and renewal underlie the effect of unilateral ligation
of common carotid artery for different times.
In the histopathology results presented in our study, ligation of common carotid
artery for 1 week appeared to cause more damage to the inner segment, outer
limiting membrane and outer nuclear layer of the dog retina than did ligation
of common carotid artery for the 12 and 24 h.
|
| Fig. 4: |
Electromicrograph of photoreceptor layer from the EXP-2 group.
A: Outer segment; B: Inner segment; C: Outer nuclear layer; Arrow: Outer
limiting membrane; Arrowhead: Loss of mitochondrial cristae; Thin arrows:
Vacoules (x1650) |
|
| Fig. 5: |
Electromicrograph of photoreceptor layer from the EXP-3 group.
A: Outer segment; B: Inner segment; C: Outer nuclear layer; Thin arrow:
Disorganization of outer segment; Thick arrow: Vacoule; S: Cell swelling;
Arrow: Outer limiting membrane; Arrowhead: Pyknotic nucleus; Asterisk: Karyolysis
(x1650) |
Present results suggested that the mitochondrial cristae in the inner segment
of dog retinas were more loss in EXP-III than EXP-II. Cell swelling and vacuoles
observed in the inner segment of retina in the EXP-III group. Vacuoles are a
common response to injury in sublethally damaged cells.
|
| Fig. 6: |
Electromicrograph of outer segment of photoreceptor cells
from the EXP-3 group. Arrows indicates disorganization and distension of
outer segment (x2950) |
In the EXP-3 group, mitochondrial cristae were lost, aerobic oxidative phosphorylation
stopped and ATP levels fell. Ultimately, this deficiency of ATP lead to a failure
of Na+-K+ pumps and loss of cell volume control (Na+ and
H20 moved into cells), maybe also causing cell swelling. In addition,
lethal injury to photoreceptor nuclei resulted in pyknotic and karyolysis nuclei.
We concluded that unilateral ligation of common carotid artery for 1 week caused
more photoreceptor cells damage than did short time ligation. Furthermore, ligation
of common carotid artery for 12 h did not cause photoreceptor cells damage.
ACKNOWLEDGMENT
This study was conducted under the sponsorship of the Islamic Azad University-Kazerun
Branch.
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