Uptake Of Reactive Oxygen Species And Amniotic Membrane: Further Evidence For Therapeutic Use In Ocular Chemical Injury?

1. David Lockington¹
2. Pankaj Agarwal¹
3. Anne Lewis²
4. Josephine Cooney²
5. Muriel Caslake²
6. Kanna Ramaesh¹

¹Tennent Institute of Ophthalmology, Glasgow, UK
²Division of Cardiovascular and Medical Sciences, University of Glasgow, UK

Purpose
Amniotic membrane is increasingly used in ocular surface disease due to its' many anti-inflammatory properties. This includes chemical injuries. We wanted to evaluate if these properties were more than simply a mechanical biological bandage and basement membrane substrate. Amniotic membrane may have scavenging properties of free radicals and reactive oxygen species (ROS) from the grafted environment.

Methods
Uniform amniotic membrane sections were incubated at 37ºC in sealed containers with concentrations of hydrogen peroxide (range 50micromol/l to 500micromol/l). On completion the tissue was sonicated. Levels of ROS were evaluated by using Amplex Red reagent (Invitrogen) in the presence of peroxidase.

Results
There was a trend for ROS uptake by the tissue at all tested concentrations.
At 1 hour there was 18% uptake by tissue for 200micromol/l and 45% uptake for 100micromol/l. For the 50micromol/l concentration there was an 85% uptake at 1hr, 93% uptake at 2 hrs and 97.7% update at 3 hrs. This was confirmed on tissue testing.

Conclusions
Some of the therapeutic properties of amniotic membrane are due to intrinsic anti-oxidant activity. We have demonstrated that amniotic membrane can act as a sponge, absorbing free radicals. However, there may also be a saturation level where amniotic membrane is no longer effective in absorbing ROS. These findings further support the role of amniotic membrane in the management of chemical injuries in the acute setting.