Development and testing of a metabolic chamber for effluent collection during whole eye perfusions.

Ocular hypertension is caused by dysregulated outflow resistance regulation by the conventional outflow (CO) pathway. The physiology of the CO pathway can be directly studied during ex vivo ocular perfusions. In addition to measuring outflow resistance generation by the CO tissues, perfusion media that is conditioned by CO pathway cells can be collected upon exiting the eye as effluent. Thus, contents of effluent include factors contributed by upstream cells that report on the (dys)functionality of the outflow tissues. Two methods have been used in the past to monitor effluent contents from perfused eyes, each with their limitations. To overcome these limitations, we designed and printed a metabolic chamber to accommodate eyes of different sizes during perfusions. To test this new chamber, human eyes were perfused for 4 h at constant flow rate of 2.5 μl/min, while pressure was continuously monitored and effluent was collected every hour. Facility was 0.28 ± 0.16 μl/min/mmHg for OD eyes and 0.33 ± 0.11 μl/min/mmHg for OS eyes (n = 3). Effluent samples were protein rich, with protein concentration ranging from 2700 to 10,000 μg/ml for all eyes and timepoints (N = 3). Effluent samples expressed proteins that were actively secreted by the TM and easily detectible including MYOC and MMP2. Taken together, our model provides a reliable method to collect effluent from ex vivo human eyes, while maintaining whole globe integrity.

Duke Scholars

Author W Daniel Stamer Ophthalmology, Glaucoma