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

Abstract

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.

Keywords: Aqueous humor dynamics; Conventional; Effluent; Glaucoma; Ocular perfusion.

Figure 1:. Design drawings for eye mount and images of metabolic chamber with human eyes.

(A) Side view of stand with removable insert to suspend the eye. Effluent collects at the tip of the funnel (red). (B) Top view of insert used to suspend the eye. (C) Side view of fully assembled eye mount with eye mounted, and water level in chamber is represented in transparent gray. (D) Humidified and temperature-controlled water bath (closed during perfusion) with eyes cannulated and set up on eye mounts. (B) Close up view of cannulated human eye set on removable insert. Effluent runs down surface of eye and collects at funnel tip (blue arrows).

Figure 2:. Schematic depicting perfusion setup.

Schematic depicting the perfusion setup. The syringe pump is set to constant flow (2.5μl/min), and each pressure reservoir A (closed to eye during perfusion) is set to 10mmHg (supported by stand and clamps as listed in materials) to re-inflate the eye following aqueous humor evacuation. Pressure reservoir ‘B’ is connected to both the black and the white side of the perfusions system. This reservoir is for zeroing the pressure at the height of the eyes prior to starting the perfusion. Phosphate buffered saline with calcium and magnesium (DBG) is used as perfusate. Sodium azide (NaN3) is a preservative reagent and is used to fill tubing that is not contributing to flow during the perfusion. A 25G butterfly needle is used for cannulation.

Figure 3:. Images showing system setup and diagram depicting needle placement.

(A) Image showing entire experimental setup. Eye mounts are housed in humidification chamber, tubing is arranged and connected to manifold, flow sensor, reservoirs, and perfusion needles, according to Figure 2. Syringe pump set up is also shown. (B) An image of the manifold setup in perfusion configuration. (C) An image showing the tubing setup connected to the flow sensors. (D) A schematic demonstrating the configuration of the needle prior to cannulation. The needle is bent in the middle at approximately 30°, with bevel facing up. (Inset) Following cannulation, the needle is placed in the posterior chamber, between the iris and the lens, with the bevel facing upward toward the iris.

Figure 4:. Effluent during whole eye perfusions contains abundant levels of protein.

(A) Protein concentration in the effluent samples collected from 3 pairs of human donor eyes was determined. For AH, protein concentration ranged from 2500 to 7400μg/ml for all eyes. For effluent, protein concentration ranged from 2700 to 10,000μg/ml for all eyes and timepoints. N=3. (B) Facility was calculated by averaging the pressure readings for each eye over a 3-hour period (between 1 and 4hrs). Then we used the constant flow rate (2.5μl/min) to calculate average facility over that time-period. Facilities were 0.28±0.16 and 0.33±0.11μl/minute/mmHg for OD and OS, respectively (mean±SD, n=3).

Figure 5:. MYOC and MMP2 proteins are actively secreted by conventional outflow cells into effluent during whole eye perfusions.

(A) MYOC protein was present in all samples and timepoints as determined by western blot (n=3). As a positive control for MYOC (+), lysates were prepared from dexamethasone-treated cultured human TM cells. (B) MMP2 protein was abundant in effluent at all time points, and trace amounts of MMP2 was present in AH. There was no detectable MMP2 in the dexamethasone-treated cultured human TM cell lysate (n=3). Dexamethasone-treated cultured human TM cell lysate was used as a negative control for MMP2 (−). NOTE: Dexamethasone-treated cultured human TM cell lysate was not from perfused eyes in this experiment. (C) Representative blots from A and B. Total protein analysis was used for normalization.