Hello, dear colleagues,
We've been performing intravitreal injections in mouse eyes but in almost all cases we observe some kind of pathology developing several days after the procedure (even if we inject the PBS): the cornea of injected eye becomes slightly opaque and during the electroretinogram (ERG) recording there is a substantial decrease in b-wave amplitude but a large increase in amplitude of component originated from Muller glial cells. I would like to ask if you could suggest the nature of such pathology and give any tips about the procedure of injection itself to avoid it.
Briefly, we use the disposable 30G sharp needles to puncture the eye and a blunt 34G needle to inject the 1.5-2 microliters of solution. All instruments are desinfected with UV and ethanol, the solution and eyedrops (local anaesthetic and atropine) are filtered and, if possible, autoclaved before injection. Experimentator wears sterile gloves, the mask and operates with anesthetized mouse under a dissecting microscope. The most "suspicious" steps (in my opinion) are:
1) We storage the blunt needles and Hamilton syringe in a sealed glass with 70% ethanol. Could it be that residual ethanol somehow enters the eye together with injection solution? We rinse the needle with distilled water several times before procedure but may be it is still a possibility?
2) We use the PBS for control injection an observe the same sympthomes as described above. Could it be that PBS is not the best choice for control injection and may itself cause some damage to the eye?
3) Afrer the end of procedure we apply the tetracyclin eye ointment on the puncture location to prevent infection. In different papers describing similar procedure there are different types of antibiotics, so may be tetracyclin is not the best option? Which one would you recommend then?
Hi Alexander;
As an ophthalmologist, I have given hundreds of intravitreal injections (anti VEGF and or steroids) to my patients over the past 15 years or so. I worked in Canada, Saudi Arabia, Kuwait, and currently in Jordan. No single case of complications so far (although it's reported else where).
1) What is/are the other solution(s), other than PBS, you are injecting?
2) Why do you use 2 different gauge needles for the procedure (I couldn't get it!?)
Ayman Abdul Aziz, thank you for your reply!
As we perform injections on mice, I suppose the differences in eye structure could influence the procedure (like the larger lens size in rodents relative to human). Still, the main steps are expected to be the same, so I would really appreciate your opinion.
1) We are on the stage of establishing intravitreal injections as a method in our lab, so we mostly use the PBS to check if there is an effect of injection itself. But we also tried to inject some polypeptide-based nanoparticles (rhodamin-loaded or w/o any cargo, dissolved in PBS) in and got eventually the same results.
2) We took the idea of using two needles from published standard protocols (see for example, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7307607/). As I get it, the use of blunt needle reduces the traumatizing effect during injection, but you still need a sharp needle to puncture the sclera first. We use a blunt needle with smaller diameter than sharp one in order to facilitate its penetration through the puncture.
I can only think of 2 possibilities:
1) Most likely; direct toxicity of the PBS solution leading to corneal endothelial and retinal dysfunction. Try using another solution with better eye tolerance (in human eyes we us BSS: balanced salt solution for intraocular injection and irrigation)
2) If the eye pressure is too high after injection, this can lead to corneal edema (cloudiness and opacities) and may also result in retinal dysfunction if the retina becomes underperfused (ischemic) under the effect of the high IOP on the retinal artery. But this is expected to happen soon after the injection (within hours)
Ayman Abdul Aziz, thanks a lot for your advices!
We definetly should try BSS at some point. Although I am not aware of any studies supporting the intraocular toxicity of PBS, a better tolerance solution could at least improve our protocol, if not solve all the problems completely.
Usually some intraocular fluid flow out at the moment of scleral puncture and we do not inject really big volumes (typically 1.5-2 microliters). All the external sympthoms (like cornea opaqueness) appear after at least 12 hours after injection. Still, we should keep an eye on this problem, thank you for mentioning it.
You are welcome.
Kindly, keep me posted on how things eventually work out for you, just out of curiosity.
Good luck
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