EFFECTS OF DEXAMETHASONE IN A RAT MODEL … · Title: EFFECTS OF DEXAMETHASONE IN A RAT MODEL OF...
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Effects of Dexamethasone in a Rat Model of Choroidal Neovascularization L. Feraille, S. Grillo-Antonelli, N. Cimbolini, T. Caillaud, P.P. Elena Iris Pharma, La Gaude, France. a b c d e f • Fluorescein angiography (FA) FA was performed on days 7, 14, 21 after the laser induction with Heidelberg Retinal Angiograph. Anesthetized animal received subcutaneous injection of Fluorescein Sodium (10%, 250µl/100g body weight. Photographies were taken in the late phase (10min). The intensity of fluorescein staining of each lesion was graded by two independent examiners using the leakage score (0 = no leakage to 3 = strong leakage) established by Takehana et al (3). • Histology Twenty one days after the laser burn, the rats were euthanazed with an overdose of sodium pentobarbital. Eyeballs were fixed in Bouin Hollande solution and embedded in paraffin. Six µm sagital sections were performed and stained with hematoxylin and eosin. Result : At day 21, compared to the vehicle groups, CNV lesions were reduced by 79% with oral administration of dexamethasone, by 65% with subconjunctival treatment and only by 33% with intravitreal administration. Figure 1 : Late phase fluorescein Angiograms (FA) carried out 21 days after the laser-induced rupture of the Bruch membrane. Right eye received twice weekly 40µg dexamethasone (a), or vehicle (b) by intravitreal injection; 120µg dexamethasone (c), or vehicle (d) by subconjunctival injection; Rat were daily treated by oral gavage of 500 μg/kg dexamethasone (e) or vehicle (f). Graph 1 : Decreased of FA leakage in dexamethasone treated rats. FA was performed on days 7, 14, 21 after the laser induction. The intensity of leakage in late phase FA were graded using the scale 0 = no leakage to 3 = strong leakage. Histograms represent the means + SD of scores per treatment and per time-point. Figure 2 : Light microscopy of retinal lesion 21 days after the laser-induced rupture of the Bruch membrane. (a) typical section of control rat showing the marked membrane proliferation and thickening in subretinal space, (b) in treated rats (oral gavage of 500 μg/kg/day dexamethasone ) the thickness of the lesion was decreased. Conclusions : In our experimental condition, subconjunctival and oral administration of dexamethasone efficiently impaired CNV development. There was a trend indicating an inhibition of CNV with intravitreal injection of dexamethasone but not significant. References : (1)Tobe T, Takahashi K, Ohkuma H, Uyama M. Experimental choroidal neovascularization in the rat; Nippon Ganka Gakkai Zasshi. 1994 98(9):837- 45. (2)Dobi ET, Puliafito CA, Destro M. A new model of experimental choroidal neovascularization in the rat. Arch Ophthalmol. 1989 107(2):264-9. (3)Takehana Y, Kurokawa T, Kitamura T, Tsukahara Y, Akahane S, Kitazawa M, Yoshimura N. Suppression of laser-induced choroidal neovascularization by oral tranilast in the rat. Invest Ophthalmol Vis Sci. 1999 40(2):459-66. 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 ivt DEX 40µg ivt VEH subconj DEX 120µg Subconj VEH per os DEX 500µg/kg/day per os VEH D7 D14 D21 FA score Introduction : Choroidal neovascularisation (CNV) is a major cause of severe central vision loss in patient with exudative age related macular degeneration (ARMD). In response to an increase of locally produced angiogenic factors, such as VEGF, vessels grow between Bruch’s membrane and the RPE as well as into the subretinal space. Various strategies were developed, including eradication of CNV by laser photocoagulation, obstruction of CNV by photodynamic therapy (PDT), prevention of angiogenesis by treatment with steroids or anti VEGF agents. To demonstrate the success or failure of these therapeutic strategies, visualization and quantification of CNV are crucial. Although there are no direct animal models of ARMD, the rat laser trauma model was developed (1)(2) . The laser trauma induces fibrovascular proliferation (FVP) araising from the choroid. Purpose : The aim of this study was to evaluate the efficacy of intravitreal, subconjunctival and oral administration of dexamethasone, and to demonstrate the success of this reference compound in a rat model of choroidal neovascularization. Methods : • Animals Sixty (60) adult male Brown Norway rats were allocated in six groups. • Treatment/Administration Group1 = intravitreal injection (ivt) of 40µg dexamethasone phosphate in saline solution twice weekly in the right eye. Group2 = ivt of vehicle, saline solution, twice weekly in the right eye. Group3 = subconjunctival injection (subconj ) of 120µg dexamethasone phosphate in saline solution twice weekly in the right eye. Group4 = subconj l injection of vehicle, saline solution, twice weekly in the right eye. Group5 = oral administration of 500µg/kg dexamethasone in olive oil daily. Group6 = oral administration of vehicle (olive oil) daily. Treatment started the day of the laser injury and continued through Day 21. • Induction All animal were anesthetized with intramuscular injection of 25 mg/kg ketamine and 5 mg/kg xylasine, and the pupils were dilated with tropicamide. With a contact lens, eight laser spots (150mW, 100ms, 75µm, Argon laser 532nm, Viridis) were applied to each right eye fundus in a circle around the optic disc Visit us on the web at www.iris-pharma.com a b
Transcript of EFFECTS OF DEXAMETHASONE IN A RAT MODEL … · Title: EFFECTS OF DEXAMETHASONE IN A RAT MODEL OF...
Effects of Dexamethasone in a Rat Model of Choroidal NeovascularizationL. Feraille, S. Grillo-Antonelli, N. Cimbolini, T. Caillaud, P.P. Elena
Iris Pharma, La Gaude, France.
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• Fluorescein angiography (FA)FA was performed on days 7, 14, 21 after the laser induction with HeidelbergRetinal Angiograph. Anesthetized animal received subcutaneous injection ofFluorescein Sodium (10%, 250µl/100g body weight. Photographies were takenin the late phase (10min). The intensity of fluorescein staining of each lesionwas graded by two independent examiners using the leakage score (0 = noleakage to 3 = strong leakage) established by Takehana et al(3).
• HistologyTwenty one days after the laser burn, the rats were euthanazed with anoverdose of sodium pentobarbital. Eyeballs were fixed in Bouin Hollandesolution and embedded in paraffin. Six µm sagital sections were performed andstained with hematoxylin and eosin.
Result :At day 21, compared to the vehicle groups, CNV lesions werereduced by 79% with oral administration of dexamethasone, by 65%with subconjunctival treatment and only by 33% with intravitrealadministration.
Figure 1 : Late phase fluorescein Angiograms (FA) carried out 21days after the laser-induced rupture of the Bruch membrane.Right eye received twice weekly 40µg dexamethasone (a), or vehicle(b) by intravitreal injection; 120µg dexamethasone (c), or vehicle (d)by subconjunctival injection; Rat were daily treated by oral gavage of500 µg/kg dexamethasone (e) or vehicle (f).
Graph 1 : Decreased of FA leakage in dexamethasone treatedrats. FA was performed on days 7, 14, 21 after the laser induction.The intensity of leakage in late phase FA were graded using the scale0 = no leakage to 3 = strong leakage. Histograms represent themeans + SD of scores per treatment and per time-point.
Figure 2 : Light microscopy of retinal lesion 21 days after thelaser-induced rupture of the Bruch membrane. (a) typical sectionof control rat showing the marked membrane proliferation andthickening in subretinal space, (b) in treated rats (oral gavage of 500µg/kg/day dexamethasone ) the thickness of the lesion wasdecreased.
Conclusions :In our experimental condition, subconjunctival and oral administrationof dexamethasone efficiently impaired CNV development. There wasa trend indicating an inhibition of CNV with intravitreal injection ofdexamethasone but not significant.
References :(1)Tobe T, Takahashi K, Ohkuma H, Uyama M. Experimental choroidalneovascularization in the rat; Nippon Ganka Gakkai Zasshi. 1994 98(9):837- 45.(2)Dobi ET, Puliafito CA, Destro M. A new model of experimental choroidalneovascularization in the rat. Arch Ophthalmol. 1989 107(2):264-9.
(3)Takehana Y, Kurokawa T, Kitamura T, Tsukahara Y, Akahane S, Kitazawa M,Yoshimura N. Suppression of laser-induced choroidal neovascularization by oraltranilast in the rat. Invest Ophthalmol Vis Sci. 1999 40(2):459-66.
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
ivt DEX 40µg
ivt VEH subconj DEX 120µg
Subconj VEH
per os DEX500µg/kg/day
per os VEH
D7 D14 D21
FA
sco
re
Introduction :Choroidal neovascularisation (CNV) is a major cause of severecentral vision loss in patient with exudative age related maculardegeneration (ARMD). In response to an increase of locally producedangiogenic factors, such as VEGF, vessels grow between Bruch’smembrane and the RPE as well as into the subretinal space. Variousstrategies were developed, including eradication of CNV by laserphotocoagulation, obstruction of CNV by photodynamic therapy(PDT), prevention of angiogenesis by treatment with steroids or antiVEGF agents. To demonstrate the success or failure of thesetherapeutic strategies, visualization and quantification of CNV arecrucial. Although there are no direct animal models of ARMD, the ratlaser trauma model was developed(1)(2). The laser trauma inducesfibrovascular proliferation (FVP) araising from the choroid.
Purpose :The aim of this study was to evaluate the efficacy of intravitreal,subconjunctival and oral administration of dexamethasone, and todemonstrate the success of this reference compound in a rat modelof choroidal neovascularization.
Methods :• Animals
Sixty (60) adult male Brown Norway rats were allocated in six groups.
• Treatment/Administration
Group1 = intravitreal injection (ivt) of 40µg dexamethasone phosphate in salinesolution twice weekly in the right eye.
Group2 = ivt of vehicle, saline solution, twice weekly in the right eye.
Group3 = subconjunctival injection (subconj ) of 120µg dexamethasonephosphate in saline solution twice weekly in the right eye.
Group4 = subconj l injection of vehicle, saline solution, twice weekly in the righteye.
Group5 = oral administration of 500µg/kg dexamethasone in olive oil daily.
Group6 = oral administration of vehicle (olive oil) daily.
Treatment started the day of the laser injury and continued through Day 21.
• Induction
All animal were anesthetized with intramuscular injection of 25 mg/kg ketamineand 5 mg/kg xylasine, and the pupils were dilated with tropicamide. With acontact lens, eight laser spots (150mW, 100ms, 75µm, Argon laser 532nm,Viridis) were applied to each right eye fundus in a circle around the optic disc
Visit us on the web at www.iris-pharma.com
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