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 JoVE Biology

Intravitreous Injection for Establishing Ocular Diseases Model

1, 1, 1

1The University of Hong Kong - HKU

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    Summary

    Intravitreous injection is a widely used technique in visual sciences research for ocular diseases or as direct application of local treatment. This video demonstrated a protocol for intravitreous injection using a 1ml syringe with glass pipette. Useful tips about avoiding massive bleeding and lens damage are given.

    Date Published: 10/01/2007, Issue 8; doi: 10.3791/313

    Cite this Article

    Chiu, K., Chang, R. C., So, K. Intravitreous Injection for Establishing Ocular Diseases Model. J. Vis. Exp. (8), e313, doi:10.3791/313 (2007).

    Abstract

    Intravitreous injection is a widely used technique in visual sciences research. It can be used to establish animal models with ocular diseases or as direct application of local treatment. This video introduces how to use simple and inexpensive tools to finish the intravitreous injection procedure. Use of a 1 ml syringe, instead of a hemilton syringe, is used. Practical tips for how to make appropriate injection needles using glass pipettes with perfect tips, and how to easily connect the syringe needle with the glass pipette tightly together, are given.

    To conduct a good intravitreous injection, there are three aspects to be observed: 1) injection site should not disrupt retina structure; 2) bleeding should be avoided to reduce the risk of infection; 3) lens should be untouched to avoid traumatic cataract. In brief, the most important point is to reduce the interruption of normal ocular structure. To avoid interruption of retina, the superior nasal region of rat eye was chosen. Also, the puncture point of the needle was at the par planar, which was about 1.5 mm from the limbal region of the rat eye. A small amount of vitreous is gently pushed out through the puncture hole to reduce the intraocular pressure before injection. With the 45° injection angle, it is less likely to cause traumatic cataract in the rat eye, thus avoiding related complications and influence from lenticular factors. In this operation, there was no cutting of the conjunctiva and ocular muscle, no bleeding. With quick and minor injury, a successful intravitreous injection can be done in minutes.

    The injection set outlined in this particular protocol is specific for intravitreous injection. However, the methods and materials presented here can also be used for other injection procedures in drug delivery to the brain, spinal cord or other organs in small mammals.

    Protocol

    1. Prepare the glass pipettes using pipettes puller, connect it to a 1ml syringe, and seal the connection with parafilm. Withdraw 2 ml of solution into the tip of the pipettes and get ready for injection.
    2. Anaesthetize the rat by intra-peritoneal injection of ketamine (80mg/kg) and xylazine (8mg/kg) (volume ratio at 2:1).
    3. Apply one drop of 0.5% alcaine to the rat eyes as topical anesthetics before intravitreous injection.
    4. Position the rat and expose the superior nasal region of the eye.
    5. Use a 30 gauge needle to puncture the superior nasal sclera at the level of the par plana. Avoid touching the ocular muscle and vessels.
    6. Give the eye ball mild pressure to get rid of a small amount of vitreous through the puncture hole from the posterior chamber.
    7. Insert the tip of the glass pipette through the puncture hole at a 45° angle through the sclera into the vitreous body. Inject 2 ml of solution into the posterior chamber and keep in place for about a few seconds, then gently remove the needle. Avoid touching the lens and intraocular vessels.
    8. After intravitreous injection, apply ophthalmic Tobrex ointment on the rat eye to prevent infection.

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    Discussion

    Good intravitreous injection is useful for establishing animal model as well as direct intraocular treatment. First, injection site should not disrupt retina structure. Second, bleeding should be avoided to reduce the risk of infection. Third, lens should be untouched to avoid traumatic cataract. Most important is to reduce the interruption of normal ocular structure.

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    Disclosures

    The animals were handled according to the protocol for the use of animal in research approved by the Committee on the Use of Live Animals in Teaching and Research (CULATR) of the University of Hong Kong and the Association for Research in Vision and Ophthalmology Statements for the use of animals in Ophthalmic and Vision Research.

    Acknowledgements

    The study of glaucoma in this laboratory is supported by National Glaucoma Research from American Health Assistant Foundation.

    Materials

    Name Company Catalog Number Comments
    Operating Microscope Olympus Corporation OME
    Glass Pipettes World Precision Instruments, Inc.
    Pipette Puller David Kopf Instruments
    1ml syringes single use
    30G needles single use
    Ketamine Aldasan anesthetic
    Xylazine Aldasan anesthetic
    Alcaine Alcon 0.5% proparacaine hydrochloride
    Tobrex Alcon ointment, 3% tobramyxin.

    References

    1. Chiu, K., Lam, T. T., Ying Li, W. W., Caprioli, J., Kwong Kwong, J. M., 2005. Calpain and N-methyl-d-aspartate (NMDA)-induced excitotoxicity in rat retinas. Brain Research. 1046, 207-215.

    2. Fisher,D., Pavlidis, M., Thanos, S., Cataractogenic lens injury prevents traumatic ganglion cell death and promotes axonal regeneration both in vivo and in culture. Investigative Ophthalmology & Visual Science. Vol. 41, 2000, pp. 3943-3954.

    3. Ji, J.-Z., Elyaman, W., Yip, H. K., Lee, V. W. H., Yick, L.-W., Hugon, J., So, K.-F., 2004. CNTF promotes survival of retinal ganglion cells after induction of ocular hypertension in rats: the possible involvement of STAT3 pathway. European Journal of Neuroscience. 19, 265-272.

    4. Lam, T. T., Abler, A. S., Kwong, J. M. K., Tso, M. O. M., N-Methyl-D-Aspartate (NMDA)-Induced Apoptosis in Rat Retina. Investigative Ophthalmology & Visual Science. Vol. 40, 1999, pp. 2391-2397.

    Comments

    16 Comments

    Hello ma'am,
    Video is really good.
    i want to ask the folll questions:
    1. After how much time interval we can do intravitreal injection
    ². what dose should be ideally given?
    3. How much vitreous is present in rat?
    As in few papers they have gven 60 microlitre and in some 1²0 microlitre of vitreous is present in rat.

    Reply

    Posted by: AnonymousOctober 9, 2010, 3:36 AM

    1. Normally we do not do multiple injections. I believe time interval
    depends on your experiment design. However, frequent injections will
    lead to higher infection rate and there will be leaking of the
    vitreous.
    ². If you mean volume, I give ² microliter. This is the volume will
    not affect the IOP. About the dose you want to give, that depends on
    the concentration of the drug.
    3. I am not sure about the posterior chamber size of rats, but for
    sure more than 5 microliter injection will result in sudden increase
    in IOP and the outflow of the drug.
    Reply

    Posted by: AnonymousOctober 25, 2010, 10:02 PM

    Dear Madam,
    I would like to ask if this protocal is feasible in feline or rabit and whether this intravitreous injection can be used to establish a retinal detachment model for further trials of certain medical devices or drugs?
    Thank you.
    Reply

    Posted by: AnonymousJanuary 6, 2011, 12:30 AM

    Hi, I believe this method is feasible in feline or rabbit. Dependents on the position and deepth of the injection site, you can use this method to establish the retinal detachment model.
    Good luck.
    Reply

    Posted by: AnonymousJanuary 6, 2011, 9:07 PM

    Dear Madam,
    Thank you for your prompt reply, and a further question is how is the time for the retina to reattached after performing this procedure. Could the detached status be maintained for about 10 weeks?
    Thanks again sincerely,
    LS
    Reply

    Posted by: AnonymousJanuary 7, 2011, 2:07 AM

    Sorry that I just see your question today. I think that you need to do the sub- retinal injection instead of an intravitreous injection. The time of detech depends on the amount of solution you injected at the position and also ocular reaction of different animals.
    Reply

    Posted by: AnonymousMay 21, 2011, 4:22 AM

    Hi Madam, I am trying to do intravitreous injection in mice with a 33 gauge needle, thus I am asking you whether it is possible for me acheive this just with this needle, and will 1.5 microlitter increase the IOP in sudden (for mice)? And one more question, are there any side effects when intravitreally inject DMSO as a solvent control?
    Reply

    Posted by: AnonymousMay 21, 2011, 3:50 AM

    Hi, I think 33G needle should be OK for the injection. To avoid sudden increase in IOP, you should release some of the vitrous before the injection, and do the injection nice and slowly for a prolonged duration.
    Reply

    Posted by: AnonymousMay 21, 2011, 4:28 AM

    For the DMSO, I believe you can try different dilution and then analyze the effect. Hope you can find an ideal dilution works well for your drug and do not affect the retinal as a solvent control.
    Good luck.
    Reply

    Posted by: AnonymousMay 21, 2011, 4:32 AM

    Im very appreciated for your suggestion.
    Reply

    Posted by: AnonymousMay 22, 2011, 1:29 AM

    Hi Madam, I am trying to do posterior chamber injections in rats with alpha-chymotrypsin. Do you have any recommended volumes and concentrations? Some papers showed ²0 microliter is an acceptable volume. I just failed the trail.
    Reply

    Posted by: AnonymousMay 30, 2011, 6:21 AM

    Hi, normally I do the intravitrous injection at ² microliter, 3 microliter is the most. I do not want to cause any dramatic changes of the intraocular pressure. As you may know that high ocuar pressure either acute or chronic sustained, will damage the retinal neurons. The concerntration depends on the solubility and toxicity of your drug. Good luck.
    Reply

    Posted by: kin c.May 30, 2011, 9:03 AM

    hi madam kin chiu, could you kindly provide me your email address? thanks..
    Reply

    Posted by: ooi y.June 30, 2011, 3:14 PM

    hi, it is ckirisli@gmail.com
    Reply

    Posted by: AnonymousJuly 1, 2011, 3:39 AM

    Sir/Mam,

    It would be greatly helpful for my thesis work if i get this protocol and video. This is my email id rupadevim@gmail.com.

    Thanking you with anticipation
    Reply

    Posted by: Rupadevi M.March 29, 2014, 2:50 AM

    Hi, thanks for the video...it is really helpful.
    I am trying to do the same injections but in a neonatal mouse. What kind of needle would you recommend for that? And does 1ul injection of the drug sounds reasonable amount to you?
    It would really appreciate if you could please let me know,
    Thanks again.
    Reply

    Posted by: Arpita m.June 3, 2014, 2:05 PM

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