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it has been applied to treat animal models with ocular diseases , including diabetic retinopathy   and choroid neovascularization .
good performance of intravitreous injection has 3 aspects : 1 ) the injection site should not disrupt retina structure , 2 ) bleeding should be avoided to reduce the risk of infection , and 3 ) the lens should be untouched to avoid traumatic cataract . as a typical microsurgery , intravitreous injection has to be performed with a surgical microscope . since the refractive power for an adult rat s eye is close to + 70 d , it is impossible to capture the images of fundus structures directly by the microscope unless performing rat funduscopy .
typically , images of the ocular fundus of rats can be obtained by using a modified fundus camera , indirect ophthalmoscopy , confocal scanning laser ophthalmoscopy , or optical coherence tomography , all of which are noncontact methods that relay the fundus image several centimeters in front of the cornea . a contact fundus camera based on rol s
grin lenses or topical endoscopy especially designed to examine rats was introduced recently . however , these instruments are costly , bulky , and difficult to align with the surgical microscope in the course of intravitreous injection of rats . at present , a more economical and general solution of observing rat fundus is to planish the cornea with a transparent disc or a cover - glass until the retina , vitreous , and lens can be discerned .
then the operator should manipulate a microsyringe to puncture the eyeball and then inject cells or drugs slowly .
the entire injection procedure often lasts for at least 10 min per eye and complications such as lens injury and retinal vascular injury have been reported .
use of corneal contact lenses during clinical vitreous surgery is popular because they afford excellent visualization of the fundus . in 2004 , chalam et al .
manufactured an acrylic , self - containing corneal contact lens for improving the efficiency of vitrectomy .
the contact surface curvature almost equals the curvature of the anterior corneal surface , eliminating the refractive power of the cornea .
this does not change the image formation but eliminates the reflection on the inferior surface of the lens and the cornea , which increases the image resolution and stereopsis .
its geometric profile and material property enabled it to maintain position steadily on the cornea by negative suction and present a clear view with no need to change the shape of the cornea during the operation . in this paper , we demonstrate its utility in the course of intravitreous injection in rats .
the contour of the rat corneal contact lens was designed using solidworks 2010 software ( dassault systmes solidworks corp . , ma , usa ) ( figure 1 ) .
the lens is made of pmma ( polymethylmethacrylate , also known as acrylic ) with a refractive index of 1.5 at 550 nm and weight of 0.05 g. in size , the lens is 3.0 mm in length ; 6.0 mm in diameter , the superior prism surface of the lens is 10 , and the radius of the curvature of the inferior concave surface is 3.0 mm . to evaluate the efficiency of this lens for intraocular observation and surgery in rats , an intravitreal injection experiment was performed .
according to previous methods reported , 40 male sprague - dawley ( sd ) rats were induced to be diabetic with streptozotocin ( stz , sigma - aldrich , st .
forty diabetic rats were randomly divided into 2 groups and received the intravitreous injection of 10 l of a cell suspension containing bone marrow - derived stroma cells ( bmsc ) . in group a we used a microsyringe and a self - retaining rat contact lens ( n=20 ) . in group
b we used the same microsyringe and a traditional cover - slip ( n=20 ) .
the animals were cared for in accordance with the arvo statement for the use of animals in ophthalmic and vision research .
all experimental procedures used aseptic techniques and were approved by the animal care and use committee of he university .
the diabetic rats to be anesthesia were placed in a box ventilated with mixed gas containing 5 mac ( maximum alveolar concentration ) isoflurane ( beijing pharmaceutical co. , ltd . , china ) , and 0.5 l / min o2 by a matrx vip 3000 anesthesia machine ( midmark co. , usa ) .
after sedation , the diabetic rat was transferred out of the box and isoflurane was continuously supplied at the rate of 11.5 mac through a mask made of the protective sleeve of a disposable 10-ml syringe .
osaka , japan ) was administered and the pupil was dilated with 1% tropicamide ( 1% mydriacyl ; alcon laboratories , hempstead , uk ) . with a drop of viscoelastic material ( healon ; pharmacia and upjohn , uppsala , sweden ) ,
the contact lens was kept in place on the cornea by negative suction ( figure 2 ) . upon visualizing the fundus with a surgical microscope ,
next , a 27-gauge needle ( hamilton , reno , nv , usa ) was inserted into the vitreous cavity , and 10 l of the cell suspension containing 1.110 bone marrow - derived stroma cells ( bmsc ) was injected .
the needle had to be left in the vitreous cavity for at least 1 min after injection to reduce reflux .
fort worth , tx , usa ) was applied to prevent infection and the inhalant anesthesia of isoflurane was stopped . in matched group b , a coverslip ( boxinda co. ltd . ,
one exception was the hand - held coverslip used in observation of the ocular fundus , but anesthesia , animal eye preparation , and injection procedures in group b were similar to that of group a. the duration of the intravitreous injection course and the success rate of intravitreous injection were observed .
operation time was analyzed using the 2 independent samples t test , and success rate was analyzed using the fisher s exact test .
the contour of the rat corneal contact lens was designed using solidworks 2010 software ( dassault systmes solidworks corp . , ma , usa ) ( figure 1 ) .
the lens is made of pmma ( polymethylmethacrylate , also known as acrylic ) with a refractive index of 1.5 at 550 nm and weight of 0.05 g. in size , the lens is 3.0 mm in length ; 6.0 mm in diameter , the superior prism surface of the lens is 10 , and the radius of the curvature of the inferior concave surface is 3.0 mm .
to evaluate the efficiency of this lens for intraocular observation and surgery in rats , an intravitreal injection experiment was performed . according to previous methods reported , 40 male sprague - dawley ( sd ) rats were induced to be diabetic with streptozotocin ( stz , sigma - aldrich , st .
forty diabetic rats were randomly divided into 2 groups and received the intravitreous injection of 10 l of a cell suspension containing bone marrow - derived stroma cells ( bmsc ) . in group a we used a microsyringe and a self - retaining rat contact lens ( n=20 ) . in group
b we used the same microsyringe and a traditional cover - slip ( n=20 ) .
the animals were cared for in accordance with the arvo statement for the use of animals in ophthalmic and vision research .
all experimental procedures used aseptic techniques and were approved by the animal care and use committee of he university .
the diabetic rats to be anesthesia were placed in a box ventilated with mixed gas containing 5 mac ( maximum alveolar concentration ) isoflurane ( beijing pharmaceutical co. , ltd . , china ) , and 0.5 l / min o2 by a matrx vip 3000 anesthesia machine ( midmark co. , usa ) .
after sedation , the diabetic rat was transferred out of the box and isoflurane was continuously supplied at the rate of 11.5 mac through a mask made of the protective sleeve of a disposable 10-ml syringe .
a , a topical anaesthetic ( 0.5% tetracaine hydrochloride ; santen , inc . , osaka , japan ) was administered and the pupil was dilated with 1% tropicamide ( 1% mydriacyl ; alcon laboratories , hempstead , uk ) . with a drop of viscoelastic material ( healon ; pharmacia and upjohn , uppsala , sweden ) ,
the contact lens was kept in place on the cornea by negative suction ( figure 2 ) . upon visualizing the fundus with a surgical microscope ,
next , a 27-gauge needle ( hamilton , reno , nv , usa ) was inserted into the vitreous cavity , and 10 l of the cell suspension containing 1.110 bone marrow - derived stroma cells ( bmsc ) was injected .
the needle had to be left in the vitreous cavity for at least 1 min after injection to reduce reflux .
, fort worth , tx , usa ) was applied to prevent infection and the inhalant anesthesia of isoflurane was stopped . in matched group b , a coverslip ( boxinda co. ltd . ,
one exception was the hand - held coverslip used in observation of the ocular fundus , but anesthesia , animal eye preparation , and injection procedures in group b were similar to that of group a.
the duration of the intravitreous injection course and the success rate of intravitreous injection were observed .
operation time was analyzed using the 2 independent samples t test , and success rate was analyzed using the fisher s exact test .
after inhalation inducing anesthesia , all diabetic rats fell into sleep placidly and slept during the whole experiment , and no animal showed stress reactions such as squealing or struggling . in group
a , typical fundus images of the rats including , crystalline lens , vitreous body , and retina , were instantly shown through the self - retaining contact lens , and then the operator used 2-hand technique to puncture the rat eyeball and inject the cell suspension . finally , the cells adhering to the posterior capsule as oil droplets could be observed after the bmsc liquid was smoothly injected into the vitreous cavity ( figure 3 ) . in group
b , the operator had to use single - hand technique to puncture the rat eyeball and inject cell suspension and another hand was needed to planish the cornea with a cover - slip .
the average time of intravitreous injection was 4.650.53 min in group a and 12.332.79 min in group b. the difference was statistically significant ( p<0.01 ) between group a and group b. compared with the traditional cover - slip used in group b , the using of the newly - designed self - containing rat lens reduced operation time by an average of 7.68 min .
moreover , there was no lens injury or retinal vascular injury in group a and the success rate was 100% ( 20/20 ) , whereas 2 of 20 group b rats had retinal bleeding , 1 of 20 had lens injury , and the success rate was 85% ( 17/20 ) . between the groups a and b , the difference was no statistically significant ( p>0.05 ) .
because the inferior surface curvature approximately matches the curvature of the anterior corneal surface of rats , the refractive power of the cornea would be compensated without planishing the cornea and then the image resolution and stereopsis would be also improved , using the same principle as that used in the chalam kv s prism lens . in terms of stability ,
the new - designed lens is made of a high refractive acrylic material instead of quartz or optical crown glass , which gives the lens a weight advantage as it is lighter than 0.05 g , allowing the self - containing lens to attach to the cornea firmly with the aid of the viscoelastic material . as we shown in this paper ,
the results in the experimental group achieved the criterion of good intravitreous injection , and this lens might provide a friendly - used instrument with good versatility and stability for researchers .
compared to previous rat funduscopies , the newly designed rat lens could conveniently and inexpensively match the surgical microscope in the course of intraocular operation . as one of the commonest experimental techniques in ophthalmic research , the repeated frequency of intravitreal injection of rats usually exceeds 60 times . in this study
, the data suggest that using the self - containing rat contact lens could reduce the operation time and minimize complications .
although the time saved is 7 min per time , but total time saving of multiple operates will by several hours , which will obviously increase the coefficient of performance . additionally , the lower complication rate in the rat will improve animal welfare .
in conclusion , the self - retaining rat contact lens is a potentially powerful instrument that allows high - quality observation of the rat fundus and simplifies the course of intravitreal injection , with important benefits for researchers .
backgroundthe aim of this study was to design a self - retaining rat contact lens to simplify intravitreous injection in rats.material/methodsa self - retaining , plane - concave prism contact lens customized for rats was designed . 
 forty diabetic rats were randomly divided into 2 groups and received the intravitreous injection of 10 l of a cell suspension containing bone marrow - derived stroma cell ( bmsc ) . 
 group a : used a microsyringe and a rat contact lens ( n=20 ) . 
 group b : used the same microsyringe and a traditional cover - slip ( n=20 ) . 
 the duration of the intravitreous injection course and the success rate of intravitreous injection were observed.resultswith the use of a self - retaining rat contact lens , a clear and stable view of the rat fundus was provided and the intravitreous injection course of rats quickly achieved , averaging 4.650.53 min in group a and 12.332.79 min in group b. the difference was statistically significant , and the time saved averaged 7.68 min . 
 none of the group a rats had retinal bleeding or lens injury ; whereas 2 of 20 group b rats had bleeding and 1 of 20 had lens injury . 
 there was no significant difference between the rats in group a and group b.conclusionsa self - retaining rat contact lens is a potentially powerful instrument that allows high - quality observation of the rat fundus and simplifies the course of intravitreal injection .