The basic protocol for each perfusion comprises three sections: calibration/testing of the system, cannulation of the eye and acclimatisation, and a multi-step perfusion regimen.
For each day of experiments, the pressure sensor was calibrated using an automated 8-point calibration with the automated linear actuator, which has a resolution of 1.25 μm per step. Subsequently, the resistance of the flow sensor was measured to ensure that it was blockage free. Prior to each perfusion, the resistance of a glass capillary of known resistance (comparable to a mouse eye) was measured to confirm that the system was functioning properly. The perfusion tubing (downstream of the flow sensor) and needle were then filled with perfusate and the resistance of the needle was measured before cannulating the eye. Note that, as the fluid in the system was water, there was a water-perfusate interface in the perfusion tubing. However, given that the volume of the tubing (≈2ml) was large compared to the total volume perfused in an experiment (≈25μl), neither advection nor diffusion could have altered the perfusate entering the eye. If any of the readings from the tests were unexpected, for example if the system time-response was too long or measured resistances were not correct, leaks/bubbles/blockages were identified and removed before the ocular perfusion commenced.
All animal experiments were done ex vivo in accordance with the Animals (Scientific Procedure) Act with the authority of a UK Home Office project licence (PPL 70/7306). All perfusions were carried out on 10–14 week old male C57BL/6J (B6) mice (Charles River UK, Ltd.), that were euthanised via cervical dislocation. All mice were fed ad libitum and housed in clear cages at 21°C with a 12 hour light-dark cycle (lights on at 7AM).
After enucleation, the eyes were stored in PBS at room temperature to await perfusion. Each eye was then affixed to a platform inside the heated bath with a small amount of cyanoacrylate glue (Loctite, UK). An XYZ micromanipulator (World Precision Instruments, USA) was used to cannulate the eye via the anterior chamber with a 33 gauge needle (Nanofil, World Precision Instruments, USA) under a stereomicroscope. The control perfusate was DBG: Dulbecco’s PBS containing divalent cations, supplemented with 5.5 mM glucose and passed through a 0.2 μm filter. After cannulation, the bath was filled with PBS to fully immerse the eye and the temperature was raised to 35°C. The applied pressure was held at 9 mmHg for a period of 30–45 minutes to allow the eye to acclimatise to the pressure and temperature environment.
After acclimatisation, a nine-step perfusion protocol was carried out, consisting of applied pressures of 4.5, 6, 7.5, 9, 10.5, 12, 15, 18 and 21 mmHg. A sample perfusion tracing is shown inFig 1b and 1c .
In order to avoid a subjective element in the definition of steady state at each pressure step, a steady state condition was defined and automatically monitored by the perfusion software. A parameter Γ(t) = Q(t)/P(t) was continuously evaluated and dΓ/dt was estimated by linear regression over a moving window of 5 minutes. When |dΓ/dt| was continuously less than 0.1 nl/min/mmHg/min for one minute, the system was considered to be at steady state. The measured flow and pressure were then averaged over the four previous minutes to yield Qj and Pj respectively, for pressure step j. The red lines inFig 1b and 1c indicate these averaging periods. This approach ensures that the measured facility changes by less than 0.4 nl/min/mmHg over the averaging period. For the eyes in the present study, the median change in facility over the averaging window was 0.16 nl/min/mmHg.
For each day of experiments, the pressure sensor was calibrated using an automated 8-point calibration with the automated linear actuator, which has a resolution of 1.25 μm per step. Subsequently, the resistance of the flow sensor was measured to ensure that it was blockage free. Prior to each perfusion, the resistance of a glass capillary of known resistance (comparable to a mouse eye) was measured to confirm that the system was functioning properly. The perfusion tubing (downstream of the flow sensor) and needle were then filled with perfusate and the resistance of the needle was measured before cannulating the eye. Note that, as the fluid in the system was water, there was a water-perfusate interface in the perfusion tubing. However, given that the volume of the tubing (≈2ml) was large compared to the total volume perfused in an experiment (≈25μl), neither advection nor diffusion could have altered the perfusate entering the eye. If any of the readings from the tests were unexpected, for example if the system time-response was too long or measured resistances were not correct, leaks/bubbles/blockages were identified and removed before the ocular perfusion commenced.
All animal experiments were done ex vivo in accordance with the Animals (Scientific Procedure) Act with the authority of a UK Home Office project licence (PPL 70/7306). All perfusions were carried out on 10–14 week old male C57BL/6J (B6) mice (Charles River UK, Ltd.), that were euthanised via cervical dislocation. All mice were fed ad libitum and housed in clear cages at 21°C with a 12 hour light-dark cycle (lights on at 7AM).
After enucleation, the eyes were stored in PBS at room temperature to await perfusion. Each eye was then affixed to a platform inside the heated bath with a small amount of cyanoacrylate glue (Loctite, UK). An XYZ micromanipulator (World Precision Instruments, USA) was used to cannulate the eye via the anterior chamber with a 33 gauge needle (Nanofil, World Precision Instruments, USA) under a stereomicroscope. The control perfusate was DBG: Dulbecco’s PBS containing divalent cations, supplemented with 5.5 mM glucose and passed through a 0.2 μm filter. After cannulation, the bath was filled with PBS to fully immerse the eye and the temperature was raised to 35°C. The applied pressure was held at 9 mmHg for a period of 30–45 minutes to allow the eye to acclimatise to the pressure and temperature environment.
After acclimatisation, a nine-step perfusion protocol was carried out, consisting of applied pressures of 4.5, 6, 7.5, 9, 10.5, 12, 15, 18 and 21 mmHg. A sample perfusion tracing is shown in
In order to avoid a subjective element in the definition of steady state at each pressure step, a steady state condition was defined and automatically monitored by the perfusion software. A parameter Γ(t) = Q(t)/P(t) was continuously evaluated and dΓ/dt was estimated by linear regression over a moving window of 5 minutes. When |dΓ/dt| was continuously less than 0.1 nl/min/mmHg/min for one minute, the system was considered to be at steady state. The measured flow and pressure were then averaged over the four previous minutes to yield Qj and Pj respectively, for pressure step j. The red lines in
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