Interaction of anaesthetic drugs and UV-B irradiation in the anterior segment of the rat eye.

Abstract

To determine the impact of anaesthesia on acute transient cataractogenesis and ultraviolet radiation (UVR)-induced cataractogenesis.
Sprague-Dawley rats were anaesthetized with pentobarbital, which caused almost full eyelid closure, or xylazine/ketamine, which caused eyelid retraction and proptosis. The eyelids of one eye were kept open with either a suture or adhesive tape, or both. The other eye was kept closed with either a suture or tape. Cataract was graded clinically and quantified in vitro as intensity of forward light scattering. In two UVR experiments, anaesthetized rats were irradiated unilaterally with 5 kJ/m2 UVR-B 300 nm for 15 mins. The difference between the two UVR experiments was the degree of proptosis in the pentobarbital group. Corneal drying was judged clinically with a grading scale.
Within 60 mins of anaesthesia induction in the first experiment, almost all lenses in open eyes developed cataract, whereas all lenses in closed eyes remained clear. In the first UVR experiment the lens light scattering was significantly higher in the xylazine/ketamine group. In the second UVR experiment the pentobarbital group was treated to achieve proptosis similar to that in the xylazine/ketamine group, which led to a smaller difference in lens light scattering between the two anaesthesia groups. Lens light scattering in the pentobarbital groups was significantly higher with forced proptosis than without prominent proptosis.
Xylazine/ketamine anaesthesia facilitates the development of UVR-induced cataract, whereas pentobarbital anaesthesia does not. Xylazine/ketamine anaesthesia induces more proptosis and therefore leads to increased exposure of the cornea and, secondarily, the lens.

Full text

Page 1

Introduction
The current study aimed to study the
effect of anaesthesia on crystalline lens
clarity.
Cataract is the leading cause of
blindness in the world (World Health
Organization2004).
multifactorial disease characterized by
loss of vision due to the scattering of
Cataractis a
light in the lens. Ageing, diabetes,
exposure to solar ultraviolet radiation
(UVR), steroid treatment and smo-
king are all factors that have been
epidemiologically
cataract.
In order to increase understanding
of the mechanism of UVR-induced
cataract, it is necessary to expose
experimental animals to UVR. In
experiments on the immediate devel-
opment of cataract after experimental
exposure to UVR, anaesthetics are
used to immobilize animals during
UVRexposure
So ¨ derberg 1990a). Two common types
of anaesthetic have been used by our
group andothers:
(So ¨ derberg 1990b) and a combination
of xylazine and ketamine (Michael
2000; Lo ¨ fgren2001;
Dong 2005). Traditionally, barbitu-
rates, such as pentobarbital, have been
the anaesthetics of choice for laborat-
ory rodents. However, pentobarbital
has a narrow therapeutic dose range
and increased mortality in the dose
rangeneeded to
anaesthesia (Brown & Ferner 1985).
The combination
ketamine began to be used as an alter-
native to barbiturate anaesthesia in
veterinary and laboratory work in the
late 1970s. Xylazine itself is an analge-
sic, but it is mainly used for its seda-
tive and muscle relaxant effects when
applied in combination with the dis-
sociate anaesthetic ketamine.
Acute reversible
described by Fraunfelder & Burns
(1970) as cataract of rapid onset,
associatedwith
(So ¨ derberg1988;
pentobarbital
Ayala2005;
achieveefficient
ofxylazine and
cataract was
Interaction of anaesthetic drugs
and UV-B irradiation in the
anterior segment of the rat eye
Fengju Zhang,1,2Stefan Lo ¨ fgren1and Per G. So ¨ derberg1,2
1St Erik’s Eye Hospital, Karolinska Institute, Stockholm, Sweden
2First Hospital attached to Dalian Medical University, Dalian, China
ABSTRACT.
Purpose: To determine the impact of anaesthesia on acute transient cataracto-
genesis and ultraviolet radiation (UVR)-induced cataractogenesis.
Methods: Sprague-Dawley rats were anaesthetized with pentobarbital, which
caused almost full eyelid closure, or xylazine⁄ketamine, which caused eyelid
retraction and proptosis. The eyelids of one eye were kept open with either a
suture or adhesive tape, or both. The other eye was kept closed with either a
suture or tape. Cataract was graded clinically and quantified in vitro as intensity
of forward light scattering. In two UVR experiments, anaesthetized rats were
irradiated unilaterally with 5 kJ⁄m2UVR-B 300 nm for 15 mins. The difference
between the two UVR experiments was the degree of proptosis in the pentobar-
bital group. Corneal drying was judged clinically with a grading scale.
Results: Within 60 mins of anaesthesia induction in the first experiment,
almost all lenses in open eyes developed cataract, whereas all lenses in closed
eyes remained clear. In the first UVR experiment the lens light scattering was
significantly higher in the xylazine⁄ketamine group. In the second UVR
experiment the pentobarbital group was treated to achieve proptosis similar to
that in the xylazine⁄ketamine group, which led to a smaller difference in lens
light scattering between the two anaesthesia groups. Lens light scattering in
the pentobarbital groups was significantly higher with forced proptosis than
without prominent proptosis.
Conclusions: Xylazine⁄ketamine anaesthesia facilitates the development of
UVR-induced cataract, whereas pentobarbital anaesthesia does not. Xyla-
zine⁄ketamine anaesthesia induces more proptosis and therefore leads to
increased exposure of the cornea and, secondarily, the lens.
Key words: xylazine – ketamine – pentobarbital – ultraviolet radiation – lens – reversible – cata-
ract – light scattering – rat
Acta Ophthalmol. Scand. 2007: 85: 745–752
ª 2007 The Authors
Journal compilation ª 2007 Acta Ophthalmol Scand
doi: 10.1111/j.1600-0420.2006.00856.x
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usually appearing within 1 hour, and
clearing after a couple of hours, whe-
ther or not the initiating stimulus was
continued. A series of experiments
showed that cataract could be induced
if the eyelids were held open, regard-
less of the technique for holding them
open and whether or not the animal
was anaesthetized
Burns 1962, 1966, 1970; Hanna &
Fraunfelder 1971). In all cases, closure
of the eyelids prevented the develop-
ment of lens opacity. The use of
xylazine⁄ketamine
research was cautioned by Calderone
et al. (1986), who had observed the
development of acute reversible cata-
ract in rats and mice anaesthetized
with xylazine⁄ketamine or xylazine
alone, even after topical administra-
tion of xylazine. They concluded that
the evaporation of water from the
cornea and the subsequent hyper-
osmolarity of the aqueous humour
caused the lens opacification. Pento-
barbital in combination with ketamine
was shown not to induce cataract and
was thus suggested as a safe anaes-
thetic for ophthalmic research (Kufoy
et al. 1989).
In the present study, we wanted to
complement earlier qualitative experi-
ments with quantitative measurements
ofthedegreeof
xylazine⁄ketamine and pentobarbital
anaesthesia, combined with varying
eyelid positions. Further, we wanted
to quantify the effect of pentobarbital
andxylazine⁄ketamine,
on cataract development after in vivo
exposure of the eye to UVR.
(Fraunfelder&
inophthalmic
cataractafter
respectively,
Materials and Methods
Animals and anaesthetic drugs
The experiments were approved by
the Stockholm Animal Experiments
EthicalCommittee
with the ARVO statement on the use
of animals in vision and ophthalmic
research.A total
oldfemalealbino
(B & KUniversal
Sweden)ratswere
experiments. Two rats died during
pentobarbital anaesthesia and were
replaced. The rats were anaesthetized
with an intraperitoneal injection of
either 40 mg⁄kg pentobarbital (Pento-
barbital; Apoteksbolaget, Stockholm,
Sweden), or a mixture of 80 mg⁄kg
andconformed
of1426-week-
Sprague-Dawley
AB,Stockholm,
usedinthree
ketamine
Stockholm, Sweden) and 10 mg⁄kg
xylazine (Rompun; Bayer, Stockholm,
Sweden).
(Ketalar;ParkeDavis,
Experiment 1: anaesthetic and eyelid
position versus anterior segment reaction,
eyelids sutured
In this experiment, we determined
qualitatively and quantitatively the
cataractogenic effect of anaesthetics in
closed versus open eyes. The animals
were randomized into two anaesthet-
ized groups and one non-anaesthetized
control group of 20 rats each (Fig. 1).
To facilitate slit-lamp inspection of
the lenses, the mydriatic tropicamide
Mydriacyl (5 mg⁄ml) (Alcon, Stock-
holm, Sweden) was instilled in both
eyes a few minutes after the drug
injection. Xylazine⁄ketamine anaesthe-
sia induces eyelid retraction and prop-
tosis (eye protrusion) in rats and mice,
whereas pentobarbital induces almost
completeeyelidclosure.
suture (5–0 Ethilon; Ethicon, Stock-
holm, Sweden) was used to close one
eye in all rats in the two drug groups
to achieve similar eyelid closure. In
the xylazine⁄ketamine group, the non-
sutured eye was naturally open. In the
pentobarbital group, the non-sutured
eye was held open by a suture in the
upper eyelid and adhesive tape on the
Anylon
lower. The tape pulled the lower eye-
lid down without causing added pres-
sure on the eye.
The eyes were inspected with a slit-
lamp microscope at 30 mins (open
eyes only) and 60 mins (both eyes)
after the induction of anaesthesia. The
stage of lens opacity was determined
in vivo according to the qualitative
method ofKufoy
(Table 1).
After 60 mins, all rats were killed
and the lenses extracted. The intensity
of forward light scattering in the lens
was then quantified (So ¨ derberg et al.
1990).
et al.(1989)
Experiment 2A: anaesthetic, eyelid
position and UV-B irradiation versus
anterior segment reaction, eyelids sutured
This experiment aimed to compare
cataract development after exposure
to 300-nm UVR in xylazine⁄ketamine
anaesthetized animals and pentobar-
bital anaesthetized animals.
Twogroupsof
were treated similarly to those in
Experiment 1, with the additional
treatment of 5 kJ⁄m2UV-B irradi-
ation at around 300 nm for 15 mins
(Fig. 2).
At 30 mins and 60 mins after induc-
tion of anaesthesia, the irradiated
eyes were inspected with a slit-lamp
20ratseach
Fig. 1. Experiment 1: design. Anaesthetic and eyelid position versus anterior segment reaction;
n ¼ 20 in each drug or control group.
Table 1. Cataract stages (Kufoy et al. 1989).
Stage Description
0
1
2
3
No cataract
Anterior sutural opacity
Superficially spread anterior opacity
Dense anterior cortical opacity seen without biomicroscope
if the eye is laterally illuminated with a pen-light
Dense opacity visible even without pen-light4
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microscope. The surgical sutures in
the irradiated and non-irradiated eyes
were released before the rats recovered
fromanaesthesia.
UV-B irradiation, corneal damage was
graded according to Kufoy et al.
(1989) (Table 2).
Thereafter, the rats were killed. The
degree of cataract in the lenses 1 week
after UV-B irradiation was quantified
in vitro as described in Experiment 1.
The 1-week period between UV-B
irradiation and cataract assessment
was based on our previous finding
that cataract development levels out
close to an upper asymptote at 1 week
aftera close-to-threshold
UVR dose in albino rats (So ¨ derberg
1990a; Michael et al. 1996). The 5-
kJ⁄m2
dose isslightly
threshold dose for albino rats anaes-
thetizedwith
(Michael et al. 1996; So ¨ derberg et al.
2002). It is also the lowest dose to
induce permanent cataract in rabbits
(Pitts et al. 1977).
One weekafter
300-nm
above the
xylazine⁄ketamine
Experiment 2B: anaesthetic, eyelid
position and UV-B irradiation versus
anterior segment reaction, eyelids taped
As in Experiment 2A, this experiment
aimed to compare cataract develop-
ment after exposure to 300-nm UVR
inxylazine⁄ketamine
animals and pentobarbital anaesthet-
ized animals, with the addition of
anaesthetized
enforced proptosis in the pentobarbit-
al group.
Theforcedlid
proptosis were achieved in the pento-
barbitalgroupby
application of adhesive tape. As in
Experiment 2A, the xylazine⁄ketamine
group was naturally proptotic. The
other eye was kept closed by adhesive
tape in both groups (Fig. 3).
retraction and
circumferential
Statistics
The sample sizes for experimental
groupswere based
acquired lens light scattering data,
accepting an a error of 5% and a b
error of 20% in the detection of a
20% difference (So ¨ derberg
1990). All tests were two-sided. Ortho-
gonal t-tests were used to test for
contrasts. Approximate t-tests were
used when variances for test groups
onpreviously
etal.
were found statistically significantly
unequal.
Results
Anaesthesia and eye responses
xylazine⁄ketamine
was deep, with a total absence of cor-
neal reflex. The pentobarbital group
exhibited a weak corneal reflex. The
rats anaesthetized with pentobarbital
started to recover after about 90 mins,
whereas the xylazine⁄ketamine group
remained under anaesthesia for at
least 120 mins.
The spontaneously open eyes in the
xylazine⁄ketamine
ments 1, 2A, 2B) exhibited prominent
proptosis. The eyes sutured open in the
pentobarbital groups in Experiments 1
and 2A had smaller eyelid openings
than eyes in the xylazine⁄ketamine
groups, but the
exposed to the surrounding air. The
eyes taped open in the pentobarbital
group in Experiment 2B exhibited
prominentproptosis
retraction, exposing the whole cornea
and parts of the sclera. Although
there was no difference between xyla-
zine⁄ketamine groups and pentobar-
bital groups in the administration of
mydriatic eyedrops, the pupil
was slightly smaller in the pentobar-
bital groups.
Theanaesthesia
groups(Experi-
cornea wasstill
witheyelid
size
Experiment 1: anaesthetics and eyelid
position versus anterior segment reaction,
eyelids sutured
Corneal surface disturbances and
intraocular inflammation
The open eyes in both drug groups
exhibitedcorneal
30 mins and 60 mins. Corneas in ani-
mals that had undergone xylazine⁄
ketamine anaesthesia tended to show
drying atboth
Fig. 2. Experiment 2A: design. Anaesthetic, eyelid position and ultraviolet-B irradiation versus
anterior segment reaction; n ¼ 20 in each drug group.
Table 2. Corneal opacity grades (Kufoy et al. 1989).
StageDescription
0
1 +
2 +
3 +
Clear cornea
Punctate epithelial keratopathy located in the central portion of the cornea
Corneal opacity with oedema, anterior chamber structures not seen clearly
Complete corneal opacity, corneal neovascularization extending from the
temporal and nasal bulbar conjunctiva toward the corneal lesion
Fig. 3. Experiment 2B: design. Anaesthetic, eyelid position, forced proptosis and ultraviolet-B
irradiation versus anterior segment reaction; n ¼ 20 in each drug group.
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Page 4

more corneal drying than corneas in
animals that had undergone pentobar-
bital anaesthesia (Fig. 4).
There were no corneal erosions or
signs of anterior chamber inflamma-
tion in the non-anaesthetized rats.
Qualitative cataract grading
After anaesthesia with xylazine⁄keta-
mine and pentobarbital, respectively,
opacities were observed in the superfi-
cial anterior lens cortex in the open
eyes. Opacities ranged from discrete
anterior sutural involvement to dense
anterior cataracts. There was a ten-
dency towards a higher prevalence of
and more severe cataract after xyla-
zine⁄ketamine than after pentobarbital
anaesthesia (Fig. 5).
After xylazine⁄ketamine anaesthe-
sia, the cataract seemed to progress
towards more severe cataract from
30 mins to 60 mins, whereas after
pentobarbital anaesthesia, the cataract
seemed to remain constant.
Lenses in non-anaesthetized control
animals and lenses in eyes that had
been closed during anaesthesia were
all clear at examination 60 mins after
initiation of anaesthesia.
Intensity of forward light scattering
After 60 mins, the lens light scattering
in the xylazine⁄ketamine open eyes
was significantly higher than in the
closed
interval [CI] for mean paired differ-
ence excludes 0) (Fig. 6).
There was no difference in lens light
scattering between open and closed
eyes in the pentobarbital group, nor
between the open eyes in the non-
anaesthetized control
CIsformean
include 0).
The difference in lens light scatter-
ing between open and closed eyes in
the xylazine⁄ketamine group was sig-
nificantly larger than in the pentobar-
bital group (approximate t-test for
independent groups; test statistic ¼
7.19, t0.05(2):20¼ 2.09). The 95% CI
for mean paired differences between
eyes in the pentobarbital and control
groups, respectively, included 0 and
consequently there was no difference
between the two groups (Fig. 6).
eyes(the 95%confidence
group(95%
paired differences
Experiment 2A: anaesthetics, eyelid
position and UV-B irradiation versus
anterior segment reaction, eyelids sutured
Corneal surface disturbances and
intraocular reaction
Allexposedeyes
exhibited corneal drying 60 mins after
induction of anaesthesia, or about
30 mins after UV-B irradiation. Punc-
tate epithelial keratopathy located in
the central portion of the cornea was
inbothgroups
found in half the irradiated eyes in
both groups. One week after UV-B
irradiation, the prevalence of corneal
opacity in irradiated eyes in the xyla-
zine⁄ketamine group
(Experiment 2A), with hyphaema in
25% of cases. In the pentobarbital
group, 25% of the irradiated eyes
(Experiment2A)
opacity (Fig. 7).
was85%
exhibited corneal
Qualitative cataract grading
The incidence of cataract at 60 mins
after UV-B irradiation was 90% in the
xylazine⁄ketamine group and 55% in
the pentobarbital group. One week
later, 55% of the irradiated lenses in
thexylazine⁄ketamine
anterior cortical cataract. The other
45% could not be inspected due to cor-
neal opacities. In the pentobarbital
group, 20% of the lenses exhibited
cataract,whereas
remained clear.
grouphad
theother80%
Intensity of forward light scattering
One week after UV-B irradiation, 85%
of irradiated lenses in the xylazine⁄
ketamine group and 20% in the pento-
barbital group were cataractous. There
were vacuolar opacities in the equator-
ial region, and irregular opacification
in the anterior cortex (Fig. 8A).
No opacities were seen in the nuc-
lear region. All non-irradiated lenses
were clear (Fig. 8B).
In rats irradiated under xylazine⁄ke-
tamine anaesthesia with spontaneous
proptosis, the intensity of forward
light scattering was higher in irradi-
ated lenses than in non-irradiated
contralateral lenses (Fig. 9; 95% CI
for the mean
excludes 0).
In rats irradiated under pentobar-
bital anaesthesia with slight proptosis
induced by suture on the upper eyelid
and adhesive tape on the lower eyelid,
there was no difference in lens light
scattering between exposed and con-
tralateral non-exposed lenses (Fig. 9;
95% CI for the mean paired differ-
ence includes 0).
The mean paired difference in lens
light scattering was higher in the
UV-B irradiated
groupwithspontaneous
than in the UV-B irradiated pento-
barbital group with slight proptosis
inducedbysuture
eyelid andadhesive
lower eyelid (approximate t-test for
paired difference
xylazine⁄ketamine
proptosis
ontheupper
ontapethe
<
30
60
100
50
Prev. corneal
drying (%)
Prev. corneal
drying (%)
Prev. corneal
drying (%)
Post-exposure time (min.)
Xylazine/ketamin
Pentobarbital
0
;
30
60
100
50
Post-exposure time (min.)
0
0
0
30
60
>
100
50
Post-exposure time (min.)
]
[
(A)
(B)
(C)
Fig. 4. Prevalence of corneal drying in open rat eyes at 30 mins and 60 mins after xylazine⁄
ketamine (black columns) or pentobarbital (white columns); n ¼ 20 in each drug group. (A)
Corneas with <25% dry area. (B) Corneas with 25)75% dry area. (C) Corneas with >75%
dry area.
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Page 5

independent groups; test statistic ¼
6.10; t0.05(2):21¼ 2.08).
Experiment 2B: anaesthetics, eyelid
position and UV-B irradiation versus
anterior segment reaction, eyelids taped
Corneal surface disturbances and
intraocular reaction
One week after UV-B irradiation, the
incidence of corneal opacity in the
UV-B irradiated eyes in the xyla-
zine⁄ketamine group was 90% (Fig. 7;
Experiment2B).
found in 30% of cases. In the pento-
barbital group, 70% of irradiated eyes
exhibitedcorneal
Experiment 2B). The incidence of hyp-
haema was 15%.
Hyphaemawas
opacity(Fig.7;
Qualitative cataract grading
At 60 mins after in vivo UV-B irradi-
ation under xylazine⁄ketamine anaes-
thesia, the prevalence of cataract was
95%. At the same post-exposure inter-
val after in vivo UV-B irradiation
under pentobarbital anaesthesia with
proptosis induced by adhesive tape
around the eye, the prevalence of
cataract was 70%.
One week after UV-B irradiation
under xylazine⁄ketamine anaesthesia,
55% of the irradiated lenses had
anterior cortical cataract, whereas the
remaining 45% could not be inspected
due to severe corneal opacities and
hyphaema. At the same post-exposure
interval after in vivo UV-B irradiation
under pentobarbital anaesthesia with
enforced eyelid opening, cataract was
found in 55% of eyes, whereas 30%
were clear and the remaining 15%
could not be inspected due to hyp-
haema.
Intensity of forward light scattering
One week after UV-B irradiation under
xylazine⁄ketamine
spontaneous proptosis, 90% of irradi-
ated lenses had anterior cortical and
equatorial cataract. In the pentobarbit-
al anaesthesia group with proptosis
induced by adhesive tape around the
eye, 55% of irradiated lenses had anter-
ior cortical and equatorial cataract.
Forbothtreatment
intensity of forward light scattering
was higher in the irradiated lenses
than in the non-irradiated lenses (95%
CIsfor mean
between irradiated and contralateral
non-irradiated lenses exclude 0).
There was no statistically significant
differencein lens
between the xylazine⁄ketamine and
the pentobarbital groups, comparing
the paired mean differences (approxi-
mate t-test for independent groups;
test statistic ¼ 1.72; t0.05(2):30¼ 2.04)
(Fig. 10).
anaesthesia with
groups the
paireddifferences
light scattering
Discussion
This study was designed to quantita-
tively examine the effect of xyla-
zine⁄ketamineand
anaesthesia, respectively, on lens light
scattering. It also aimed to quantita-
tively study the impact of each of
these anaesthetic drugs on in vivo
UVR-induced cataract development.
In Experiments 1 and 2A, the prop-
tosis occurring during xylazine⁄keta-
mine anaesthesia was simulated in the
pentobarbital anaesthetized
by eyelid sutures. However, it was
noted in these experiments that eyes
sutured open did not present the same
amount of proptosis as eyes under
xylazine⁄ketamine anaesthesia. Hence,
in Experiment 2B the proptosis in the
pentobarbitalanaesthetized
was increased by using adhesive tape
around the eye. This increased the
proptosis, but the eye opening was
still less than that observed under xyl-
azine⁄ketamine anaesthesia.
That there was a higher prevalence
ofand morewidespread
pentobarbital
animals
animals
surface
30
60
0
2
4
0
(A)
(B)
20
40
60
80
100
Cataract type prevalence (%)
Cataract type prevalence (%)
1
3
30
60
0
2
4
0
20
40
60
80
100
Post exp. int. (min)
Post exp. int. (min)
1
3
Cataract grade
Cataract grade
Fig. 5. Evolution of cataract stage distribution (0 ¼ no cataract, 4 ¼ dense opacity; Kufoy
et al. 1989) in open eyes after (A) xylazine⁄ketamine or (B) pentobarbital anaesthesia; n ¼ 20
in each drug group.
Acta Ophthalmologica Scandinavica 2007
749