MEDICINA (2003) 39 tomas, Nr. 10
955
Evaluation of peculiarities of the acetylcholinesterase-positive
nerve plexus and its length in the cornea
Vidmantas Lasys, Edvinas Stanevičius, Gintaras Zamokas1
Department of Anatomy and Histology,
1
Department of Internal Diseases, Lithuanian Veterinary Academy, Lithuania
Key words: acetylcholinesterase-positive nerve plexus, dog’s cornea.
Summary. The objective of this study was to evaluate age-related peculiarities of acetylcholinesterase-positive nerve plexus and its length in the dog cornea.
Material and methods. Nine mixed breed dogs (male and female) of 1.8–20 kg weight were
used. They were divided into three age groups: young (until 1 year), adult (1–7 years) and old (8
and more years). Acetylcholinesterase-positive nerve plexus was identified using acetylcholinesterase method of M. J. Karnowsky and L. A. Roots (1964) modified by D. H. Pauza et al.
(1996). The length of nerve bundles was measured by mm in 1 mm2 of cornea.
Results and conclusions. Branching thick, medium and thin nerve bundles form acetylcholinesterase-positive nerve plexus in dog cornea. The average of the length of nerve bundles
between the left and the right corneas was similar in different age groups (p>0.05). The length of
acetylcholinesterase-positive nerve bundles in 1 mm2 of the cornea in the group of adult dogs
(10.32±0.11 mm) was higher than in the groups of young (9.42±0.02 mm) and old dogs (7.75±0.14
mm) (p<0.001).
Introduction
The cornea is one of eyeball’s structures, which
carries out important optical functions. The cornea has
peculiar anatomical structure and innervation. The
mammalian cornea is one of the most innervated parts
of the body surface (1–3). The greatest number of sensory nerve fibers originating from trigeminal ganglion
was observed in the corneal nerve plexus (4, 5).
The enzyme acetylcholinesterase (AChE) performs
an important function in the metabolism of neurotransmitter acetylcholine (ACh). Because AChE hydrolyses ACh rapidly, there are no direct histochemical
methods for determining the localization of this neurotransmitter. The localization of AChE in nerve structures is determined by identification of acetylcholinesterase or cholinacetyltransferase, which assists in
synthesis of ACh (6).
Using the AChE-histochemical reaction for identification of nerve plexus, the fine granules of copper
thiocholine locate themselves along nerve bundles and
provide optimal uniformity to the smaller nerves and
terminals (7). N. Ishida et al. (8) maintains that the
product of this reaction is stable and such preparations
are suitable for the quantitative evaluation of nerve
plexus. This reaction is not specific because acetylcho-
linesterase and butyrylcholinesterase are distributed in
the other tissues (9). Therefore nerves determined by
AChE-histochemical reaction are named as AChEpositive nerves.
This reaction is better to perform in whole-mount
preparations than in sections because it is possible to
analyze nerve plexus in three dimensions (10).
By this method (8, 11, 12) it was determined that
trunks of AChE-positive nerve plexus were located
evenly in rat cornea (2–4 trunks in each quadrant).
Nerves that run from limbus to central part branched
and conjoined. They were varicosed in some places. T.
Tervo and A. Palkama (13) noticed AChE-positive
nerves in the stroma and epithelium of rabbit’s cornea.
According to these researches the thickest nerve bundles
are located in limbal part.
J. L. Jacot et al. (7) measured the length of AChEpositive nerve bundles in the rat cornea. But they did
not investigate age-related changes of the length in
nerve plexus.
Information about researches of length of dog corneal nerve plexus elements was not found in the literature.
The aim of this study was to evaluate age-related
peculiarities of the acetylcholinesterase-positive nerve
plexus and its length in the dog cornea.
Correspondence to V. Lasys, Department of Anatomy and Histology, Lithuanian Veterinary Academy, Tilžės 18,
3022 Kaunas. E-mail: [email protected]
956
Vidmantas Lasys, Edvinas Stanevičius, Gintaras Zamokas
Materials and methods
Nine mixed breed dogs (male and female) of 1.8–
20 kg weight were used. They were divided into three
age groups: young (until 1 year), adult (1–7 years)
and old (8 and more years).
All dogs were pre-treated with chlorpromazine (50
mg/kg i. m.) and deeply anaesthetized with on overdose
of sodium pentobarbital (100 mg/kg i. v.).
Scientific research was carried out according to
“Law of Lithuanian Republic on animal care, keeping
and using” No. 8-500 and according to law statements
such as Lithuanian Republic orders of veterinary
service concerning veterinary requirement of breeding,
care and transportation of laboratory animals (December 31, 1998, No. 4-361) and concerning usage of
laboratory animals for scientific experiments (January
18, 1999, No. 4-16).
Removed eyes were washed in 0.1 M phosphate
buffer (pH 7.4). Later eyes were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer with 18%
sucrose for 30 min. Corneas were isolated by circumferential cut leaving 2–3 mm scleral ring surrounding
the cornea.
Every cornea was divided into upper, nasal, lower
and temporal quadrants (Fig. 1). Then the corneas were
rinsed with 0.1 M phosphate buffer for 18 hours at 4°C.
AChE-positive nerve plexus was identified using
acetylcholinesterase method of M. J. Karnowsky and
L. A. Roots (1964) (9) modified by D. H. Pauza et al.
(1996) (10). Histochemical demonstration of AChEpositive reactivity was performed on whole-mounts
preparations. Chemical composition of the incubation
medium (pH 5.6) was (in mM): sodium acetate buffer,
60; acetylthiocholine iodide, 2; sodium citrate, 15;
CuSO4, 3; K3Fe(CN)6, 0.5; Triton-X 100, up to 1%.
From time to time during incubation the staining of
nerve structures was controlled by a light microscope.
Superior
Temporal
Nasal
Inferior
Fig. 1. Scheme of quadrants of right cornea
1
2
3
Fig. 2. Parts of adult dog cornea
1 – central; 2 – pericentral; 3 – limbal.
Later corneas were dehydrated in graded alcohol steps
and then placed in xylene for 10 min. Corneas were
mounted epithelium upwards and cover slipped on glass
slides using balsam.
Each corneal quadrant was marked into central, pericentral and limbal parts for computerized morphometric evaluation of length of nerve bundles (Fig. 2).
Length of nerve bundles was measured by mm in 1
mm2 of cornea. Camera (SPOT RT Slider Digital by
Diagnostic Instruments Inc., St., Sterling Heights,
Michigan, USA), program Image Pro Plus (version
4.1 by Media Cybernetics, Silver Spring, Maryland,
USA), microscope (Leica DMRXA, by Leica
Microsystems AG, Ernst-Leitz-Strasse 17-37, 35578
Wetzlar, Germany), were used for computerized
quantitative estimation of the length of nerve bundles.
Results of length of AChE-positive nerve plexus
elements were processed using Student- Gaset’s criteria
(14, 15).
Results
Dog’s cornea is innervated by 12–15 thick of AChEpositive nerve bundles, which are distributed evenly
round limbus. Some thick nerve bundles, which come
to peripheral part of cornea branch into 2 nerve bundles
of equal thickness and running to the central part they
split into various nerve bundles. Other thick nerve
bundles run almost straight to the center and split into
medium and thin nerve bundles. Thick nerve bundles
run along intermediate stromal layer from limbus to
the center.
Having branched from of AChE-positive thick,
medium and thin nerve bundles are distributed into two
hardly separated interlocated layers. These nerve bundles split and repeatedly join. Medium and thin nerve
MEDICINA (2003) 39 tomas, Nr. 10
Evaluation of peculiarities of the acetylcholinesterase-positive nerve plexus
Right cornea
young
Young
adult
Adult
old
Old
7.96±0.06 c2
10.25±0.17 b2
7.53±0.24 c1
10.39±0.19 b1
11
10
9
8
7
6
5
4
3
2
1
0
9.41±0.19 a1
Lenght, mm/mm2
Discussion
Structures of AChE-positive nerve plexus were described in rat cornea by N. Ishida et al. (8) and T. Tervo
(11, 12), in rabbit cornea by T. Tervo and A. Palkama
(13), in guinea pig cornea by F. A. Moustafa et al.
(16).
We have not found information about dog corneal
AChE-positive nerve plexus elements in literature. We
determined that 12–15 thick AChE-positive nerve
bundles, which distributed evenly around limbus, formed nerve plexus of dog cornea. Some thick nerve
bundles, which came to peripheral part of cornea,
branched into 2 nerve bundles of equal thickness and
run to the central part and split into nerve bundles of
various thicknesses. Other thick nerve bundles run
almost straight to the center of cornea and split into
medium and thin nerve bundles.
These data coincide with N. Ishida’s et al. (8), T.
Tervo’s (11, 12), T. Tervo’s and A. Palkama’s (13)
results. N. Ishida et al. (8), T. Tervo (11, 12) wrote
that AChE-positive nerve trunks were distributed
evenly in all quadrants of rat cornea (2–4 nerves in
each). Nerves run from limbus to central part; they
branch and rejoin. T. Tervo and A. Palkama (13) noticed that the thickest AChE-positive nerve bundles
were in the limbal part of rabbit cornea.
In this study we noticed that having branched from
of AChE-positive thick, medium and thin nerve bundles
were distributed into two hardly separated interlocated
layers. These nerve bundles split and joined repeatedly.
Medium and thin nerve bundles dominated in superficial stromal layer of cornea in central and pericentral
parts. Part of medium nerve bundles split from thick
9.42±0.03 a2
bundles are dominating in superficial stromal layer of
cornea both in central and pericentral parts of cornea.
Part of medium nerve bundles split from thick nerve
bundles and branch coming back to the limbal part of
cornea. Medium and thin nerve bundles run from limbus to peripheral part but they don’t reach the central
part of cornea. These nerve bundles are distributed
among thick nerve bundles. Thin and medium nerve
bundles run along superficial stromal layer and innervated peripheral part of cornea.
Thin nerve bundles run to epithelium and are abundantly anastomosed with one another. Some thin nerve
bundles end in conic enlargements.
Average of the length of nerve bundles between left
and right corneas was similar in different age groups
(p>0.05). Average of the length of nerve bundles of
young dogs was in right (9.4±0.19 mm) and left
(9.42±0.03 mm) corneas; of adult dog respectively
(10.39±0.19 mm) and (10.25±0.17 mm) (p>0.05); old
(7.53±0.24 mm) and (7.96±0.06 mm) (p>0.05). We
added average of the length of nerve bundles in right
and left corneas. The length of acetylcholinesterasepositive nerve bundles in the 1 mm2 of the cornea in
the group of adult dogs (10.32±0.11 mm) was higher
than in the groups of young (9.42±0.02 mm) and old
dogs (7.75±0.14 mm) (p<0.001).
Left cornea
Fig. 3. The lenght of AChE-positive nerve bundles (mm) in 1 mm2 of the right and left corneas
in different age dog groups (X±Sx)
Difference between a1:a2, b1:b2, c1:c2 is statistically non-significant (p>0.05).
MEDICINA (2003) 39 tomas, Nr. 10
957
Vidmantas Lasys, Edvinas Stanevičius, Gintaras Zamokas
7.75±0.14 c
young
Young
adult
Adult
old
Old
10.32±0.11 b
11
10
9
8
7
6
5
4
3
2
1
0
9.42±0.02 a
Lenght, mm/mm2
958
Age groups
Fig. 4. The total lenght of AChE-positive nerve bundles (mm) in 1 mm2 of cornea in different age
dog groups (X±Sx)
Difference between a:b, b:c, a:c is statistically significant (p<0.001).
nerve bundles branching well came back to limbal part
of cornea. Medium and thin nerve bundles run from
limbus to peripheral part but they don’t reach central
part of cornea. These nerve bundles distributed among
thick nerve bundles. Thin and medium nerve bundles
run along superficial stromal layer and innervated
peripheral part of cornea.
Our results were coincident with N. Ishida’s et al.
(8) data. They noticed that thin nerve bundles came
out of plexus and formed short terminal branches,
which went up vertically and diagonally to the epithelial
layer in the rat cornea. Nerves run from limbus to central part, they split and repeatedly join one with another.
T. Tervo with co-workers (17) investigated that
AChE-positive nerve bundles of human cornea split
into thinner branches and formed basal epithelial nerve
plexus. Branches of this plexus formed terminal
branches among epithelial cells.
T. Tervo and A. Palkama (13) determined that
AChE-positive nerves of rabbit cornea are located in
stroma but not in the epithelial layer. It differs from
Ishida’s et al. (8), T. Tervo’s et al. (17) and our results,
which showed that thin AChE-positive nerve bundles
innervate human, dog and rat corneal epithelial layer.
J. L. Jacot et al. (7) measured average of the length
of nerve bundles (5.52±1.31 mm/mm2) in the rat cornea. But these authors did not compare the average of
the length of nerve plexus between left and right corneas
in age groups. Results of our study showed that the
average of the length of nerve bundles between left
and right corneas was similar in different age groups
(p>0.05). When dogs grow up the length of AChEpositive nerve bundles increases, when dogs get older
it decreases (p<0.001).
We had no possibility to compare our results with
the results of other investigations because we have not
found any data in literature.
Conclusions
1. Branching thick, medium and thin nerve bundles
form acetylcholinesterase-positive nerve plexus in dog
cornea.
2. Average length of nerve bundles between left and
right corneas was similar in different age groups
(p>0.05).
3. The length of acetylcholinesterase-positive nerve
bundles in the 1 mm2 of the cornea in the group of
adult dogs (10.32±0.11 mm) was higher than in the
groups of young (9.42±0.02 mm) and old dogs
(7.75±0.14 mm) (p<0.001).
MEDICINA (2003) 39 tomas, Nr. 10
Evaluation of peculiarities of the acetylcholinesterase-positive nerve plexus
959
Ragenos acetilcholinesterazei pozityvaus nervinio rezginio ypatybės ir jo ilgio įvertinimas
Vidmantas Lasys, Edvinas Stanevičius, Gintaras Zamokas1
Lietuvos veterinarijos akademijos Anatomijos ir histologijos katedra, 1 Vidaus ligų katedra
Raktažodžiai: acetilcholinesterazei pozityvus nervinis rezginys, šuns ragena.
Santrauka. Darbo tikslas. Įvertinti įvairaus amžiaus šunų ragenos acetilcholinesterazei pozityvaus nervinio
rezginio ypatybes ir jo nervinių pluoštų ilgį.
Tyrimo medžiaga ir metodai. Naudoti devyni mišrūnai, abiejų lyčių, 1,8–20 kg masės šunys, kurie suskirstyti
į tris amžiaus grupes: jauni (iki 1 m.), suaugę (1–7 m.) ir seni (8 metų ir vyresni). Acetilcholinesterazei
pozityvus nervinis rezginys išryškintas naudojant M. J. Karnovsky ir L. A. Roots acetilcholinesterazės metodą
(1964), modifikuotą D. Pauzos ir kt. (1996). Išmatuotas ragenos nervinių pluoštų ilgis mm/mm2.
Rezultatai ir išvados. Šuns ragenos acetilcholinesterazei pozityvų nervinį rezginį formuoja besišakojantys
stori, vidutinio storio ir ploni nerviniai pluoštai. Skirtingų amžiaus grupių šunų nervinių pluoštų ilgio vidurkis
tarp kairės ir dešinės ragenų buvo panašus (p>0,05). Suaugusių šunų ragenos 1 mm2 acetilcholinesterazei pozityvių
nervinių pluoštų ilgis (10,32±0,11 mm) buvo didesnis negu jaunų (9,42±0,02 mm) ir senų šunų (7,75±0,14
mm) (p<0,001).
Adresas susirašinėjimui: V. Lasys, LVA Anatomijos ir histologijos katedra, Tilžės 18, 3022 Kaunas
El. paštas: [email protected]
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Received 16 June 2003, accepted 15 September 2003
Straipsnis gautas 2003 06 16, priimtas 2003 09 15
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