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Lipoperoxide level of the retina of chick embryo exposed to high concentration of oxygen
355
Clinica Chimica Acta, 0 Elsevier/North-Holland
80 (1977)
355-360
Biomedical
Press
CCA 8937
LIPOPEROXIDE LEVEL OF THE RETINA OF CHICK EMBRYO EXPOSED TO HIGH CONCENTRATION OF OXYGEN
KUNIO YAGI a,*, SHIGERU MATSUOKA a, HIROSHI YOSHIKO K. TAKEUCHI b and HISASHI SAKAI b a Institute of Nagoya,
of ~ioc~ern~t~ and b Department Nagoya 466 {~apan~
(Received
May l&h,
of Anatomy.
OHKAWA Faculty
a, NOBUKO
OHISHI
a,
of Medicine,
U~lil~er~ity
1977)
Summary To approach the mechanism of degeneration of the retina in retinopathy of prematurity, a model experiment using chick embryo was carried out. Upon exposing chick embryo at various stages to a high concentration of oxygen at 2 atm pressure, lipoperoxide levels of both the blood and the retina were elevated. The change in lipoperoxide level of the liver was not significant, except for a slight increase at the 9th day of incubation. Upon exposing chick embryo of the 14th day to high concentration of oxygen at ambient pressure, lipoperoxide levels of both the blood and the retina were elevated at 6 h and 12 h of exposure, but significant change was not observed in the lipoperoxide level of the liver.
Introduction In a previous paper [ 11, it was reported that lipoperoxide increased in the retina of a rabbit exposed to high concentration of oxygen, followed by a decrease in the ~plitude of the electroretino~am and degeneration in the visual cell layers. Lipid peroxidation in living cells must be associated with serious damage to essentially structural components such as proteins and enzymes [Z--4]. Therefore, the increase in lipoperoxide of the retina explains, at least partly, the mechanism of retinal degeneration caused by oxygen poisoning. In the field of clinical medicine, the etiology of retinopathy of prematurity is one of the most urgent problems to be elucidated. The retinopathy of prematurity is considered to be caused by the administration of high concentrations of oxygen to immature infants [ 51. Accordingly, the increase in lipoperoxide in Abbreviations: TBA. thiobarbituric acid; TMP. 1,1,3,3-tetramethoxypropane. * To whom corwspondence should be addressed.
356
the retina of animals exposed to high concentration of oxygen is reasonably related to the etiology of the retinopathy of prematurity. To confirm the validity of the above view, the present study deals with a detailed investigation on the etiology of the retinopathy of prematurity by using chick embryo as a model for an immature infant. Chick embryo seemed to be advantageous in the present study, in that the embryo at a definite developmental stage is easily obtained and that the retina is relatively large and easily separable. Materials and methods Fertilized eggs of the White Leghorn breed were obtained from a local poultry farm. They were kept in an incubator at 38°C with high humidity until the experiments. Thiobarbituric acid (TBA) was obtained from BDH Chemicals Ltd., Poole, England. 1,1,3,3-Tetramethoxypropane (TMP) was purchased from Tokyo Kasei Kogyo, Ltd., Tokyo. The eggs were placed in a hyperbaric chamber or in an ordinary incubator. The oxygen concentration was maintained at nearly 95% at 2 atm pressure or at ambient pressure. Temperature was maintained at 38°C. The levels of lipoperoxide of the retina and liver were determined by a TBA method using TMP as an external standard and expressed in terms of nmol of malondialdehyde [6]. The retina and liver were homogenized in cold 0.9% NaCl solution and their homogenates were subjected to TBA reaction. The amount of lipoperoxide in the blood was measured by a micromethod using fluorometry [ 71. To check histological change in the retinal tissue, light microscopic observations were made on the specimens which were embedded in paraffin, sectioned at 10 pm thickness and stained with Masson-Goldner’s trichrome staining method after fixing in the mixture of saturated aqueous solution of picric acid, formalin and glacial acetic acid. Results and discussion Table I shows the data of the changes in lipoperoxide levels of the blood, retina and liver upon exposing chick embryos of various stages to a high concentration of oxygen under 2 atm pressure for 6 h. As shown in Table I, the lipoperoxide levels of the blood and retina increased at each stage. In the liver, the increase in lipoperoxide was not significant except for a slight increase at the 9th day of incubation. To check the effect of high concentrations of oxygen under conditions similar to those of immature infants, the changes in lipoperoxide level were followed under milder conditions. A chick embryo of the 14th day was placed in an incubator at ambient pressure with 95% oxygen gas flowing. The data are summarized in Table II. The lipoperoxide level of the blood did not change after exposure for 3 h, but it was elevated after exposure for 6 h. The elevation was also found at 12 h of exposure, but it was not observed at 24 h and 48 h of exposure. It should be noted that elevation in the retina coincided with elevation in the blood. No significant elevation in the liver was observed at any time of exposure.
357
TABLE
1
CHANGES
OF
VARIOUS Chick
embryo
control The
was
chick
level
values
....__I___
LEVEL
HIGH
OF
was placed
of
was
in the
RETINA
with
OF
chamber same
determined
malondialdehyde
obtained
THE
CONCENTRATION
placed in a hyperbaric
lipoperoxide
as nmol the
TO
embryo
of
expressed between
LIPOPEROXIDE
STAGES
L6.71.
the main
stages
at 2 atm
hyperbaric by
and
UPON
method
Means
rt S.E.
control
Lipoperoxide
of 95%
pressure
chamber
TBA the
EXPOSING
CHICK
EMBRYO
at 2 atm using
are
TMP
given.
oxygen
at 38°C
pressure as
n = 6.
an
for
day
13th
16th
of incubation
external
Student’s
t-test
day
day
of incubation
285.0
+ 33.6
Control
111.4
i
02
259.2
+ 54.9
Control
of incubation
wet
weight) t I .l
23.8
+ 1.5
16.0
+ 0.9
**
24.3
+ 2.3
15.0
c 0.5
19.1
+ 1.3
3.4 i
159.5
17.2 *
8.9
72.9?
02
8.3
and made
Liver
(nmo1/100
02
was
__--
Retina
+ 21.4
6 h.
level
(nmol/ml)
181.7
for
standard
experiment.
Blood
Controi
6 h. The
or air at 38°C
(nm01/100
mg
wet
9th
AT
OXYGEN
***
43.0
+ 2.8
***
54.1
It 3.4
31.8
? 2.5
**
39.0
?- 2.7
**
mg
weight) _I_--
9.1
? 1.0
9.1
? 1.1
*
~11* P <
0.05,
* * P < 0.02.
* **
p <
0.01.
Histological examination of oxygen under milder conditions show the microphotographs of concentration of oxygen for 6 TABLE
II
CHANGES
IIIGH
the retinal tissue of chick embryo exposed to was made by light microscopy. Figs. lb and Id the retinas of chick embryo exposed to a high h and 48 h, respectively. By comparing Fig. Id
OF
LIPOPEROXIDE
CONCENTRATION
LEVEL
OF OXYGEN
UPON
EXPOSING
FOR DIFFERENT
CHICK
EMBRYO
OF
THE
14TH
DAY
TO
INTERVALS
Chick embryo of the 14th day was placed in an incubator flowed with 95% oxygen at ambient pressure at 38°C for definite times. The control chick embryo was placed in an incubator with air at ambient pressure at 38°C. The Ieve of lipoprroxide was determined by TBA method using TMP as an external standard and expressrd as nmoi of malondialdrhvde I6.71. Means f. S.E. ax given. n = 6. Student’s f-test was made between the valurs obtained with the main and the control experiment. -__I_ -. -._Exposuw
Lipoperoxide --
(h)
level
Biood (nmoI/mlf
Retina (mno1/100 mg wet weight)
Liver (nmo1/100 mg wet weight)
109.3 ? 15.6 109.4 + 15.6
17.8 It 0.6 17.8 + 0.4
23.7 + 1.5 21.8 -f 2.0
110.7 ? 16.2 253.4 t 24.6 **
17.3 + 1.3 18.8 t 1.1
21.7 t 1.5 23.8 It 1.7
109.0 ? 24.9 254.3 + 42.5 **
17.5 t 1.6 24.1 t 1.9 *
25.3 t 2.5 26.4 t 2.3
109.2 + 20.9 129.7 + 19.9
17.5 f 1.5 22.0 + 2.3
24.4 If 1.7 27.5 t 2.6
107.3 i 11.7 148.7 It 21.1 --..___
17.2 t 2.8 16.0 + 2.5
17.7 + 1.9 23.9 t 3.3
___-. 3
Control
6
Control
02 02 12
Control 02
24
Control 02
48
Control 02
* p < 0.05, ** p < 0.02.
-
placed at 38’C.
’ was i!mre
a: control
for
in an incubator
359
with the control (Fig. lc), it is clear that the blood vessels became narrowed. A loose arrangement and broadening of the bipolar cell layer are also observed. These changes can also be seen in the retina of chick embryo after 6 h of exposure (compare Fig. lb with la), though the changes are less pronounced. Fig. 2 shows the enlarged photomicrograph of the specimen of 48 h exposure. These features are less pronounced, in comparison with those obtained previously in the rabbit [ 11, but suggest the initial stage of the degeneration of the visual cell layer. From these results it can be supposed that the lipoperoxide, increased in the blood upon incubating animals under high concentrations of oxygen, would cause an increase in the lipoperoxide level of the retina and provoke the degeneration of the retinal tissue. Though the primary site of lipid peroxidation in animals exposed to high concentration of oxygen remains to be elucidated, the results obtained in the present study indicate that the retinopathy of prematurity seems to be caused by tipoperoxide incorporated into the retina via the blood. Considering this, it could reasonably be recommended to physicians that the concentration of oxygen might be controlled by monitoring the level of lipoperoxide in the blood. For this purpose, the microdetermination of lipoperoxide in the blood seems to be applicable [ 71.
itration [posed
of for
360
References 1 IIiramitsu, T., Hasegawa, Y., Hirata, K.. Nishigaki. I. and Yagi. K. (1976) Experientia 32, 622423 2 Desai, I.D. and Tappel, A.L. (1963) J. Lipid Res. 4, 204-207 3 IIochstein, P. and Ems&x, L. (1964) in Microsomal Peroxidation of Lipids and its Possible Role in CeIIular Injury, J. & A. Churchill Ltd., London 4 Tappel, A.L. (1973) Federation Proc. 32.1870-1874 5 Patz, A., Hoeck. L.E. and Cruz, E.D.L. (1952) Am. J. Ophthal. 35, 1248-1253 6 Ohkawa. H.. Ohishi, N. and Yagi, K. (1977) Arch. Riochem. Biophys.. in Press 7 Yagi. K. (1976) Biochem. Med. 15,212-216
Clinica Chimica Acta, 0 Elsevier/North-Holland
80 (1977)
355-360
Biomedical
Press
CCA 8937
LIPOPEROXIDE LEVEL OF THE RETINA OF CHICK EMBRYO EXPOSED TO HIGH CONCENTRATION OF OXYGEN
KUNIO YAGI a,*, SHIGERU MATSUOKA a, HIROSHI YOSHIKO K. TAKEUCHI b and HISASHI SAKAI b a Institute of Nagoya,
of ~ioc~ern~t~ and b Department Nagoya 466 {~apan~
(Received
May l&h,
of Anatomy.
OHKAWA Faculty
a, NOBUKO
OHISHI
a,
of Medicine,
U~lil~er~ity
1977)
Summary To approach the mechanism of degeneration of the retina in retinopathy of prematurity, a model experiment using chick embryo was carried out. Upon exposing chick embryo at various stages to a high concentration of oxygen at 2 atm pressure, lipoperoxide levels of both the blood and the retina were elevated. The change in lipoperoxide level of the liver was not significant, except for a slight increase at the 9th day of incubation. Upon exposing chick embryo of the 14th day to high concentration of oxygen at ambient pressure, lipoperoxide levels of both the blood and the retina were elevated at 6 h and 12 h of exposure, but significant change was not observed in the lipoperoxide level of the liver.
Introduction In a previous paper [ 11, it was reported that lipoperoxide increased in the retina of a rabbit exposed to high concentration of oxygen, followed by a decrease in the ~plitude of the electroretino~am and degeneration in the visual cell layers. Lipid peroxidation in living cells must be associated with serious damage to essentially structural components such as proteins and enzymes [Z--4]. Therefore, the increase in lipoperoxide of the retina explains, at least partly, the mechanism of retinal degeneration caused by oxygen poisoning. In the field of clinical medicine, the etiology of retinopathy of prematurity is one of the most urgent problems to be elucidated. The retinopathy of prematurity is considered to be caused by the administration of high concentrations of oxygen to immature infants [ 51. Accordingly, the increase in lipoperoxide in Abbreviations: TBA. thiobarbituric acid; TMP. 1,1,3,3-tetramethoxypropane. * To whom corwspondence should be addressed.
356
the retina of animals exposed to high concentration of oxygen is reasonably related to the etiology of the retinopathy of prematurity. To confirm the validity of the above view, the present study deals with a detailed investigation on the etiology of the retinopathy of prematurity by using chick embryo as a model for an immature infant. Chick embryo seemed to be advantageous in the present study, in that the embryo at a definite developmental stage is easily obtained and that the retina is relatively large and easily separable. Materials and methods Fertilized eggs of the White Leghorn breed were obtained from a local poultry farm. They were kept in an incubator at 38°C with high humidity until the experiments. Thiobarbituric acid (TBA) was obtained from BDH Chemicals Ltd., Poole, England. 1,1,3,3-Tetramethoxypropane (TMP) was purchased from Tokyo Kasei Kogyo, Ltd., Tokyo. The eggs were placed in a hyperbaric chamber or in an ordinary incubator. The oxygen concentration was maintained at nearly 95% at 2 atm pressure or at ambient pressure. Temperature was maintained at 38°C. The levels of lipoperoxide of the retina and liver were determined by a TBA method using TMP as an external standard and expressed in terms of nmol of malondialdehyde [6]. The retina and liver were homogenized in cold 0.9% NaCl solution and their homogenates were subjected to TBA reaction. The amount of lipoperoxide in the blood was measured by a micromethod using fluorometry [ 71. To check histological change in the retinal tissue, light microscopic observations were made on the specimens which were embedded in paraffin, sectioned at 10 pm thickness and stained with Masson-Goldner’s trichrome staining method after fixing in the mixture of saturated aqueous solution of picric acid, formalin and glacial acetic acid. Results and discussion Table I shows the data of the changes in lipoperoxide levels of the blood, retina and liver upon exposing chick embryos of various stages to a high concentration of oxygen under 2 atm pressure for 6 h. As shown in Table I, the lipoperoxide levels of the blood and retina increased at each stage. In the liver, the increase in lipoperoxide was not significant except for a slight increase at the 9th day of incubation. To check the effect of high concentrations of oxygen under conditions similar to those of immature infants, the changes in lipoperoxide level were followed under milder conditions. A chick embryo of the 14th day was placed in an incubator at ambient pressure with 95% oxygen gas flowing. The data are summarized in Table II. The lipoperoxide level of the blood did not change after exposure for 3 h, but it was elevated after exposure for 6 h. The elevation was also found at 12 h of exposure, but it was not observed at 24 h and 48 h of exposure. It should be noted that elevation in the retina coincided with elevation in the blood. No significant elevation in the liver was observed at any time of exposure.
357
TABLE
1
CHANGES
OF
VARIOUS Chick
embryo
control The
was
chick
level
values
....__I___
LEVEL
HIGH
OF
was placed
of
was
in the
RETINA
with
OF
chamber same
determined
malondialdehyde
obtained
THE
CONCENTRATION
placed in a hyperbaric
lipoperoxide
as nmol the
TO
embryo
of
expressed between
LIPOPEROXIDE
STAGES
L6.71.
the main
stages
at 2 atm
hyperbaric by
and
UPON
method
Means
rt S.E.
control
Lipoperoxide
of 95%
pressure
chamber
TBA the
EXPOSING
CHICK
EMBRYO
at 2 atm using
are
TMP
given.
oxygen
at 38°C
pressure as
n = 6.
an
for
day
13th
16th
of incubation
external
Student’s
t-test
day
day
of incubation
285.0
+ 33.6
Control
111.4
i
02
259.2
+ 54.9
Control
of incubation
wet
weight) t I .l
23.8
+ 1.5
16.0
+ 0.9
**
24.3
+ 2.3
15.0
c 0.5
19.1
+ 1.3
3.4 i
159.5
17.2 *
8.9
72.9?
02
8.3
and made
Liver
(nmo1/100
02
was
__--
Retina
+ 21.4
6 h.
level
(nmol/ml)
181.7
for
standard
experiment.
Blood
Controi
6 h. The
or air at 38°C
(nm01/100
mg
wet
9th
AT
OXYGEN
***
43.0
+ 2.8
***
54.1
It 3.4
31.8
? 2.5
**
39.0
?- 2.7
**
mg
weight) _I_--
9.1
? 1.0
9.1
? 1.1
*
~11* P <
0.05,
* * P < 0.02.
* **
p <
0.01.
Histological examination of oxygen under milder conditions show the microphotographs of concentration of oxygen for 6 TABLE
II
CHANGES
IIIGH
the retinal tissue of chick embryo exposed to was made by light microscopy. Figs. lb and Id the retinas of chick embryo exposed to a high h and 48 h, respectively. By comparing Fig. Id
OF
LIPOPEROXIDE
CONCENTRATION
LEVEL
OF OXYGEN
UPON
EXPOSING
FOR DIFFERENT
CHICK
EMBRYO
OF
THE
14TH
DAY
TO
INTERVALS
Chick embryo of the 14th day was placed in an incubator flowed with 95% oxygen at ambient pressure at 38°C for definite times. The control chick embryo was placed in an incubator with air at ambient pressure at 38°C. The Ieve of lipoprroxide was determined by TBA method using TMP as an external standard and expressrd as nmoi of malondialdrhvde I6.71. Means f. S.E. ax given. n = 6. Student’s f-test was made between the valurs obtained with the main and the control experiment. -__I_ -. -._Exposuw
Lipoperoxide --
(h)
level
Biood (nmoI/mlf
Retina (mno1/100 mg wet weight)
Liver (nmo1/100 mg wet weight)
109.3 ? 15.6 109.4 + 15.6
17.8 It 0.6 17.8 + 0.4
23.7 + 1.5 21.8 -f 2.0
110.7 ? 16.2 253.4 t 24.6 **
17.3 + 1.3 18.8 t 1.1
21.7 t 1.5 23.8 It 1.7
109.0 ? 24.9 254.3 + 42.5 **
17.5 t 1.6 24.1 t 1.9 *
25.3 t 2.5 26.4 t 2.3
109.2 + 20.9 129.7 + 19.9
17.5 f 1.5 22.0 + 2.3
24.4 If 1.7 27.5 t 2.6
107.3 i 11.7 148.7 It 21.1 --..___
17.2 t 2.8 16.0 + 2.5
17.7 + 1.9 23.9 t 3.3
___-. 3
Control
6
Control
02 02 12
Control 02
24
Control 02
48
Control 02
* p < 0.05, ** p < 0.02.
-
placed at 38’C.
’ was i!mre
a: control
for
in an incubator
359
with the control (Fig. lc), it is clear that the blood vessels became narrowed. A loose arrangement and broadening of the bipolar cell layer are also observed. These changes can also be seen in the retina of chick embryo after 6 h of exposure (compare Fig. lb with la), though the changes are less pronounced. Fig. 2 shows the enlarged photomicrograph of the specimen of 48 h exposure. These features are less pronounced, in comparison with those obtained previously in the rabbit [ 11, but suggest the initial stage of the degeneration of the visual cell layer. From these results it can be supposed that the lipoperoxide, increased in the blood upon incubating animals under high concentrations of oxygen, would cause an increase in the lipoperoxide level of the retina and provoke the degeneration of the retinal tissue. Though the primary site of lipid peroxidation in animals exposed to high concentration of oxygen remains to be elucidated, the results obtained in the present study indicate that the retinopathy of prematurity seems to be caused by tipoperoxide incorporated into the retina via the blood. Considering this, it could reasonably be recommended to physicians that the concentration of oxygen might be controlled by monitoring the level of lipoperoxide in the blood. For this purpose, the microdetermination of lipoperoxide in the blood seems to be applicable [ 71.
itration [posed
of for
360
References 1 IIiramitsu, T., Hasegawa, Y., Hirata, K.. Nishigaki. I. and Yagi. K. (1976) Experientia 32, 622423 2 Desai, I.D. and Tappel, A.L. (1963) J. Lipid Res. 4, 204-207 3 IIochstein, P. and Ems&x, L. (1964) in Microsomal Peroxidation of Lipids and its Possible Role in CeIIular Injury, J. & A. Churchill Ltd., London 4 Tappel, A.L. (1973) Federation Proc. 32.1870-1874 5 Patz, A., Hoeck. L.E. and Cruz, E.D.L. (1952) Am. J. Ophthal. 35, 1248-1253 6 Ohkawa. H.. Ohishi, N. and Yagi, K. (1977) Arch. Riochem. Biophys.. in Press 7 Yagi. K. (1976) Biochem. Med. 15,212-216