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Treatment of retinal vein occlusion in rabbits with traditional Chinese medicine
Fufang XueShuan Tong
Keywords: experimental, retinal vein occlusion, Fufang XueShuan Tong
Background Retinal vein occlusion (RVO) is one of the most common causes of
visual loss. Many approaches have been tried to treat central retinal vein occlusion (CRVO),
and branch retinal vein occlusion (BRVO) with various results. However, there is no
defined protocol and limited evidence to support the interventions currently used. The aim of
this study was to assess the efficacy of the traditional Chinese medicine Fufang XueShuan
Tong (FXST) in treating experimentally created RVO.
Method RVO model was first induced in forty-four pigmented rabbits through
photocoagulation following injection of rose Bengal. The rabbits were divided into four
groups based on the dose of FXST administered (212mg/kg, 424mg/kg 848mg/kg and control
group). The rabbits were observed for four weeks after the procedure, using color fundus
photography, fundus fluorescein angiography (FFA) and electroretinogram (ERG)
examination; VEGF, IL-6, NO levels in the vitreous and histopathologic evaluation were also
monitored.
Results
The obstructed vessels in the treatment groups reopened or anastomosed faster
than those in the control group (P<0.05). The amplitude of max b-mave and the oscillatory
potential were significantly higher in the treatment groups than in the control group (p<0.01).
At both two weeks and four weeks, VEGF and IL-6 levels in the vitreous were significantly
decreased in the treatment groups (P<0.01), while NO levels were significantly elevated
(P<0.01). At the same time, histopathologic evaluation showed different retinal
neuroepithelium structures in the different groups. Immunoreactivity of VEGF was greater in
the control group than in the treatment groups.
Conclusion
FXST was helpful in reconstructing retinal vessels in the RVO model,
protecting retinal structures and improving visual function, and could inhibit the NV factor.
Retinal vein occlusion is the second most common sight-threatening vascular disorder,
behind only diabetic retinopathy. The prevalence of branch retinal vein occlusion (BRVO)
and central retinal vein occlusion (CRVO) is 0.6% and 0.1%, respectively.1
Until recently, many different interventions were advocated, focusing mainly on the
sequelae
of
the
occluded
veinssuch
as
macular
edema,
epiretinal
membrane,
neovascularization, vitreous hemorrhage and traction retinal detachment. Treatment options
include anticoagulation, thrombolysis, steroid, isovolaemic haemodilution, radial optic
neurotomy, chorioretinal venous anastomosis, arteriovenous crossing sheathotomy,
pan-retinal photocoagulation, and vitrectomy. However, most visual outcomes were
unsatisfactory.2-3 Although new interventions continue to emerge, there is still no definitive
treatment for RVO.
Fufang Xue Shuan Tong (FXST) capsules which contain SanQi , HuangQi, DanShen
and XuanShen have been used for more than twenty years to treat retinal diseases in China. It
is a traditional Chinese medicine compound. FXST is composed of SanQi, in which Panax
notoginseng saponin （PNGS） is believed to be the active ingredient, and it can particularly
block receptor-operated calcium-channel in vascular smooth muscle and reduce blood
viscosity4, improve tissue anti-anoxia potential ; DanShen, which can enhance fibrinolysis
and anti-coagulation; HuangQi, a vasodilatation agent; and XuanShen, which is made up of
harpagide, harpagoside ,angoroside and cistanosis5-7 Previous studies have shown that FXST
can promote blood circulation, thus accelerating dissolution of a microthrombus.8
Furthermore, FXST effectively prevents changes in the microangium in diabetic rats.9
Whether FXST affects the course of RVO or cures the disorder by reversing the main
pathology is still unclear, however. The rabbit is often used as a model for studies involving
eye disease10, researchers have used methods such as laser photocoagulation and intravitreal
thrombin injection11-12 among others to induce retinal vein occlusion in rabbits. The aim of
this study is to investigate how FXST affects vascular structure, cytokine and function in
experimental retinal vein occlusion in rabbits.
METHODS
Animals and creation of RVO
Forty-four pigmented rabbits weighing 2-3 kg were used in accordance with the
Association for Research in Vision and Ophthalmology Statement for the Use of Animals in
Ophthalmic and Vision Research.
In all animals, retinal vein occlusion (RVO) was induced using a krypton green laser
(Varia, Lumenis, USA) mounted on a slit lamp. Only the right eye of each animal was used.
First, the rabbits were anesthetized with an intramuscular injection of a ketamine
hydrochloride (30mg/kg) and diazepam (5 mg/kg).The pupils were dilated with a topical
application of Tropicamide Phenylephrine Eye Drops (Mydrin®-P, Japan). A contact lens
(Ocular® , Bellevue, USA) was then placed on the cornea and, a few seconds after
intravenous injection of rose Bengal (50 mg/kg), light from the krypton laser delivered to the
retinal veins around the optic disk margin. Nasal and temporal major veins were treated with
the laser. The laser spot size was 200 μm, and the duration of each shot was 0.3 second.
Thirty to forty shots were applied to each spot. The laser power was 200- 250 mW. To
prevent immediate reopening of the vein, a length of vein nearly 1/2 the disc diameter was
treated, and one side adjacent the arteries was damaged as well. Laser treatment was stopped
when definite obstruction segments were observed in the major veins on both sides.
The day after RVO was induced, rabbits were divided into four groups, including three
treated groups and one control group. Group A received a daily oral dose of FXST (212
mg/kg); Group B received a daily oral dose of FXST (424 mg/kg); Group C received a daily
oral dose of FXST (848mg/kg); Group D, the control group, received a daily oral dose of
placebo (filtered water). Weighed FXST was mixed up with some daily animal feeds together,
and all rabbits were found had taken all these feeds up every day.
Follow-up examinations
Follow-up examinations in the four weeks following the procedure included color fundus
photography, fundus fluorescein angiography (FFA) and electroretinograms (ERG).The
rabbits were anesthetized and the pupils dilated as described above.
Color fundus photos were then taken using a digital fundus camera system (Topcon
TRC50EX , Japan). At least three images were acquired in each eye: images of the optic disc,
the temporal medullary wing and the nasal medullary wing.
FFA was then performed using either the same digital fundus camera system or the
HRAⅡ (The Heidelberg Retina AngiographⅡ; Heidelberg Engineering GmbH, Dossenheim,
Germany). An intravenous line was established on the marginal ear vein and （10mg/kg）of
10% fluorescein (Alcon, USA) was injected. Sequential fundus photos were taken
immediately after fluorescein injection. Photos were also taken at 3 and 5 min.
ERGs were recorded using the commercial RetiPort device (Roland Consult Systems,
Germany) . All procedures were performed by the two same technicians each time. After a
dark adaptation period of 40 minutes, Jet contact lens electrodes attached to the cornea of the
eyes served as working electrodes. Stainless steel needle electrodes served as reference and
grounding electrodes, respectively, and were inserted in the skin between the eyes and behind
the ears. All of these manipulations were performed under dim red light. Max-response of
scotopic flash ERG was recorded with the flash of 3.0 cd.s.m2, and an additional run for
scotopic oscillatory potentials was performed. The measurement time was 150 msec; the
sample rate was 3.4 kHz; and the frequency range was 1-300Hz for scotopic flash ERG,
100-500Hz for oscillatory potentials.
Measurement of VEGF, IL-6 and NO levels
Samples of undiluted vitreous fluid (300–500 μl) were obtained from experimental eyes
by puncturing the pars plana with sterile tuberculosis (TB) needle at two and four weeks after
RVO was induced. This procedure caused slight lens opacity and vitreous hemorrhage which
did not interfere with fundus observation in some rabbits and there were no retinal
detachment. The samples were collected in sterile tubes and rapidly frozen at −80°C.
The concentrations of Vascular Endothelial Growth Factor (VEGF) and IL-6 were
measured by enzyme-linked immunosorbent assay using human VEGF and IL-6
immunoassays (R&D Systems, Minneapolis, Minnesota, USA). The concentrations of NO
were measured by nitrate reduction according to agent instruction (Jianchen technology,
China)
Histopathology
At the mid-point of the follow-up examination, one eye in each group was enucleated
and at the end of the follow-up period, all experimental eyes were enucleated (the rabbit were
sacrificed by intravenous overdose injection of pentobarbital), fixed in 10% formaldehyde for
24 hours and then embedded in paraffin. Blocks were obtained from cuts through the whole
globe oriented perpendicular to the medullary wings. Five-μm-thick sections obtained using a
microtome were stained with Hematolxylin and Eosin (H&E) for light microscopic (LM)
examination of retinal structure.
We performed VEGF immunohistochemistry on above sections with a two-step staining
procedure. Antigen retrieval was performed using the steamer method. Slides were washed
with 0.1 mol/L phosphate buffered saline (PBS) three times, and endogenous peroxidase
activity was quenched with 1% H2O2; all sections were blocked by 10% goat Serum after
PBS washing. Incubation for 1 hour at 370C with mouse monoclonal VEGF（Thermo
Scientific, USA）
followed. A secondary antibody (anti-mouse antibody labeled with HRP,
DAKO, EnVision) was applied in humid chambers for 30 min at room temperature after
washing the slides with PBS. The staining result was detected using a 3, 3'-diaminobenzidine
tetrahydrochloride solution (DAB) (DAKO Co., Glostrup, Denmark) for 10 min.
Statistical analysis
All analyses were performed using SPSS System 13.0 software (Lead technologies,
USA). The data are presented as the frequencies or means (±SD), and the results are
compared by chi square test and analysis of variance. A two-tailed P value of less than 0.05
was considered statistically significant.
Results
Immediately following laser application, it was observed, in all cases, that there was no
blood flow in the treated segments of the retinal veins. In addition, in most cases, the arterial
blood flow either slowed or stopped as well.
Color fundus photography and fluorescein angiography
On day 2, following laser photothrombosis, most of the cases showed retinal whitening and
hemorrhage around the medullary wings (Fig.1) and did not exhibit any retinal blood flow in
either arteries or veins on FFA. From week 1, hemorrhage started to absorb while vessels
gradually reopened (Fig.2) and anastomosed (Fig.3). Treatment groups changed faster than
control group. Table 1 shows the FA results at week 1, week 2, and week 4 following
inducement of vein occlusion.
ERG
Laser photothrombosis resulted in a significant decrease in the ERG response, including in
the scotopic and photopic a, b wave and oscillatory potentials. In the first two weeks, there
was no significant difference between these four groups with respect to the
electrophysiological parameters; after two weeks the responses for each group recovered
variably. Table 2 shows the results for the amplitude of max b and oscillatory potentials wave
in week 4. The amplitude of max-b wave and oscillatory potentials in the treatment groups
was significantly higher than in the control group. When group A, B, C was compared with
group D respectively, all the difference between them was significant ( P=0.000~0.008) .
And the difference within treatment groups was also significant (P=0.000~0.008).
Vitreous levels VEGF, IL-6 and NO levels
Vitreous fluid levels for VEGF and IL-6 decreased while NO increased in the treatment
groups, with respect to the control group. At weeks 2 and 4, there were significant differences
between the four groups in the levels of VEGF, IL-6 and NO, as shown in Table 3. When
group A, B, C was compared with group D respectively, all the difference of (VEGF:
P=0.000~0.002; IL-6:P=0.000~0.001; NO:P=0.000~0.018) between them was significant .
And the differences within some treatment groups were not significant : VEGF between
group A and B at week 2 (P=0.084 ) ; between group B and C at week 4 (P=0.442), IL-6
between
group
B
and
C
significant( VEGF:P=0.000~0.045 ,
at
week
2
(P=0.080)
while
others
were
IL-6 : P=0.000~0.013: ;NO:P=0.000).
Histological aspects
The optical microscopic examination of the serial sections of impact areas demonstrated
morphology changes in the RVO rabbits. At weeks 2 and 4, compared to the normal rabbit
retina, neuroretinal layers were markedly disorganized and became thicker in control group,
especially the inner layers (At weeks 2 , as shown in Fig 4, neuro retinal layers in group
A,B,C,D was 119.9µm, 98.3µm, 69.0µm, 139.8µm, respectively; at weeks 4 , as shown in Fig
5 , neuro retinal layers in group A,B,C,D was 83.4µm, 75.3µm, 68.6µm, 110.1µm,
respectively; while it was 44.0µm in normal rabbit ) . Cells were swollen, and numerous
vacuoles could be seen. In the treatment groups, these histological changes were less severe,
differing in extent according to the dose of FXST administrated. (Fig.4 -5) At the same time,
VEGF immunohistochemistry revealed positive VEGF staining in ganglion cell, inner nuclear,
outer plexiform and photoreceptor in all RVO retina. Also, VEGF expression was higher in
the control group than in the treatment groups, especially group C. (Fig.6-7)
DISCUSSION
In rabbits, retinal veins leave the eye separately and, unlike in humans, do not converge
into a single central retinal vein.13 This anatomical difference makes it impossible to create
with a laser experimental RVO (in which we induced multiple BRVO at the optic disk
margin) identical to that of human CRVO or BRVO. However, occlusion of all retinal veins
at the optic disc, which blocks all outflow drainage, is expected to have similar pathological
effects and may be assumed a CRVO.10
In this study, a traditional medicine has been proposed as a potential treatment for RVO.
We applied finger printing technology to ensure the uniformity and stability of FXST. Finger
printing technology can show every ingredient and the respective amounts, based on the
height and area of the color spectrum.
In follow-up examinations with fundus fluorescein angiography, the extent to which
vessels reopened or anastomosed was used to define the status of blood flow. Reopening of
laser sites is a common phenomenon in rabbit RVO models.14 Reopening or anastomosis of
vessels occurred earlier in the treatment groups than in the control groups and were
significantly different at week 2. Reopening of the precise obstruction spot or nearby
anastomosed vessels suggests reperfusion of certain parts of the retina. This was probably due
to the anticoagulation and thrombolysis effect of FXST. One ingredient is DanShen, which
can enhance fibrinolysis and anti-coagulation; HuangQi which is other ingredient is a
vasodilatation agent5-8. It has been postulated that dissolving the thrombus should effectively
restore retinal blood flow and visual function and prevent further visual loss due to chronic
complications of CRVO.2 However, systemic anticoagulants (for example, oral aspirin,
subcutaneous heparin and intravenous thrombolysis) have not been demonstrated to be
effective treatments for vein occlusions. In addition, use of such anticoagulants causes
unacceptable risks of intraocular, intracerebral and gastrointestinal haemorrhage.15 Local
routes of administration for thrombolytic agents such as tissue plasminogen activator (t-PA)
have been investigated. Some data suggest that intravitreal t-PA injection may have a
beneficial role in the management of CRVO when performed within a few days of the onset
of symptoms in patients with no angiographic evidence of severe capillary nonperfusion.16 In
contrast, Hossein concluded that intravitreal or subretinal injection of t-PA did not improve
reperfusion of retinal vessels in experimentally induced RVO in rabbits with respect to a
control group.12
The intravitreal levels of VEGF and IL-6 are elevated in patients with BRVO and are
significantly correlated with the size of the nonperfusion area and the severity of macular
edema. Therefore, they may play a role in the duration of BRVO17-18. However, the
experimental branch vein occlusion impairs the release of constitutive NO in the affected
retina and induces an arteriolar constriction, which in turn contributes to the development of
tissue hypoxia and neuronal swelling.19 These data are consistent with our results. In our
study, vitreous levels of VEGF and IL-6 were significantly higher in the control group than in
the treatment groups, while NO levels were lower. Also, immunochemistry results have
revealed decreased VEGF expression in the treatment groups. Luhai et al.20 reported that
VEGF mRNA expression was detected in inner ischemic retina and retinal neovascular issue
in a rabbit RVO model and
corresponded to the distribution of retinal ischemia. This
indicates that FXST may improve retinal ischemia after RVO and prevent neovascularization.
FXST is believed to protected blood vascular structure and then promote blood circulation
after thrombosis 4 .Other thrombolysis agents are not associated with a lower risk of ocular
neovascularization.21 Intravitreal injection of triamcinolone acetonide or bevacizumab can
also inhibit VEGF expression, but the risks of steroid administration and intravitreal injection
must also be considered. Antcliff et al
22
reported that intraocular pressure in the eyes
increased by 25% after intravitreal steroid administration, with other complications including
infection, cataract, haemorrhage, and retinal detachment.
Full-field ERG is an objective method used in pharmacological studies or studies of
degenerative disorder in humans. In rabbits, the separate b-wave amplitudes measured at the
highest peak in single flash recordings with the different stimulations were both detectable
and reproducible. The oscillatory potentials were also prominent and reproducible.23 Our
study demonstrated significant differences between the control group and the treatment
groups in the b-wave and the oscillatory potentials but no significant differences in the
a-wave. This is because animal model for RVO mainly produced ischemic damage to the
inner layer retina, which normally receives its blood supply from the retinal central artery
while the outer layer retina obtain some blood supply from the choroidal vasculature. The
ERG amplitudes was higher in treated groups was probably because FXST helped with
reperfusion to the retina, improved tissue anti-anoxia potential8. Then histological exams
showed that ischemic damage of the retina was less severe in the treatment groups than in the
group.
In conclusion, in an experimental RVO model, FXST was effective in restoring blood
flow to the retina, protecting retina structure and visual function and inhibiting the
neovascular factor. Although our animal RVO model does not exactly replicate human
CRVO or BRVO—thrombus formation, the main component of human RVO, was not
present, and the follow-up time was not as long as clinical course—the conclusions drawn
from this study could have implications for FXST in clinical application.
Figure 1 (a)
Figure 1 (b)
Figure 1 (a) normal rabbit retinal fundus photography
(b) 2 days after laser photothrombosis on the same eye , retina hemorrhage
looks like a flame around the two medullary wings (black arrow)
Figure 2 (a)
Figure 2 (b)
Figure 2
(a) FFA (one eye in group D) of obstructed vessel 1 day after RVO was
induced (the white arrow indicates the obstruction spot)
(b)FFA (the same eye) of reopened vessel 1 week after RVO was induced
(the black arrow indicates the reopened obstruction spot)
Figure 3 (a)
Figure 3 (b)
Figure 3
(a) FFA (one eye in group D) of obstructed vessel 1 day after RVO was
induced (the white arrow indicates the obstruction spot)
(b) FFA (the same eye) of anastomosed vessels after RVO has been induced (the black arrow
indicates the anastomosed part around previously obstructed segment)
(a)
(c)
(b)
(d)
(e)
Figure 4
Light microscopic examination of rabbit retina (stain, hematoxylineosin;
magnification, 400x). (a)-(d) are from 2 weeks after RVO was induced in Groups A,B,C,D,
respectively, and show swollen cells in the inner nuclear layer (black arrows) and the thick
internal retina layer from the nerve fiber layer to the inner plexiform layer (asterisks), (e)
shows a normal retina.
(a)
(c)
(b)
(d)
Figure 5
Light microscopic examination of rabbit retina (stain, hematoxylineosin;
magnification, 400x). (a)-(d) are from 4 weeks after RVO was induced in Groups A,B,C,D,
respectively, and show swollen cells in the inner nuclear layer (black arrows) and the thick
internal retina layer from the nerve fiber layer to the inner plexiform layer (asterisks)
(a)
(b)
(c)
(d)
(e)
Figure 6
Immunohistochemistry stain of VEGF in a rabbit retina (magnification,
400x). (a)-(d) are from 2 weeks after RVO was induced in Groups A,B,C,D, respectively, and
show positive (less than two weeks) immunostaining (appears brown) of cytoplasm, cell
surface and extracelluar matrix. Group D exhibited higher VEGF expression than did the
treatment groups. (e) shows a normal retina.
(a)
(b)
(c) (d)
Figure 7
Immunohistochemistry stain of VEGF in a rabbit retina (magnification, 400x).
(a)-(d) are from 4 weeks after RVO was induced in Groups A,B,C,D, respectively, and show
positive (less than two weeks) immunostaining (appears brown) of cytoplasm, cell surface
and extracelluar matrix. Group D exhibited higher VEGF expression than did the treatment
groups.
Table 1 Comparisons of revascularization between the control group and the treatment
groups.
week 1
(p=0.078)
controlled group
treated group
A
2
18
B
20
48
week 2
(p=0.023)
A
4
30
B
18
36
week 4
(p=0.168)
A
11
43
B
9
17
“A”= the amount of reopened or anastomosed vessels
“B”= the amount of obstructed vessels
Table 2
Comparisons of ERG amplitude parameters between the four groups.
max-b wave
(μv)
(p=0.000)
oscillatory
potentials (μv)
(p=0.000)
group A
173.35±24.36
78.41±13.40
group B
207.10±20.69
92. 27±12.94
group C
241.80±37.59
93. 53±19.68
group D
Table 3
groups.
136. 10±32.77
Comparisons of
59. 50±17.47
lintravitreal VEGF, IL-6 and NO levels between the four
VEGF
(pg/ml)
IL-6
(pg/ml)
NO
(μmol/ml)
week 2 week 4
(p=0.000) (p=0.000)
week 2 week 4
(p=0.000) (p=0.000)
week 2 week 4
(p=0.000) (p=0.000)
group A
13.67± 7. 81±
1.76
4.24
42.55± 23. 91±
13.99
6.20
2. 09± 4. 02±
0.52
1.38
group B
9.47± 3. 26±
1.59
0.72
4. 55± 2.75±
2.43
1.12
22.32±
5.57
13.56±
2.27
4. 52± 7. 77±
1.23
1.48
9. 59± 12.83±
1.70
3.16
24.67± 10. 05±
9.86
1.82
56.49±
17.27
group C
group D
15.34±
2.83
9. 06±
0.96
32. 30±
8.16
0.89± 1.53±
0.71
0.62
Acknowledgements
The authors would like to thank Li Bin, Zhang ZiRong, Li LiaoQing ,Gao Fei, Xu
XiaoLin for their technical support.
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