Phd 2000 infusion

The in vitro model experiments were conducted in the environmental fluid mechanics laboratory located in the UCL Department of Mechanical Engineering with an additional equipment setup.
The conjunctiva layer was modelled as a thin elastic silicone sheet of uniform thickness (T = 0.8 mm) sandwiched between a lower clear acrylic plate and an upper steel plate; a circular hole (radius R = 20 mm) was cut into the upper plate so that the thin elastic sheet pursed when water was injected beneath the sheet. A PEEK tube was placed below the bleb to act as an outflow with a set resistance of 2.6*10¹⁰ Pa.s.m^-3. The inlet flow rate Q_IN was fixed at 5000, 8000 to 10000 μl/min using a syringe pump (PHD 2000 Infusion, Harvard Apparatus, Holliston, United States).
A machine vision camera (Allied Vision Technology) with a 16 mm lens was used to record the side view of the in vitro silicone bleb model at a frequency of 1.875 Hz. The bleb height was estimated using calibrated bespoke image processing algorithms written in Matlab similarly to the ex vivo approach. A 3-way luer lock connector was used to connect a calibrated pressure transducer to the inlet tube. The pressure signal was converted to volts (P8055-1 Velleman 2003) and recorded at a frequency of 1.875 Hz.

The channel geometry is shown in Figure 1, it consists of three radii of curvatures (270 μm, 50 μm and 200 μm) in a single unit with 60 μm in width and 90 μm in height. This geometry was chosen after optimization by simulation. There are two channel lengths: L (3227 μm) and 2L (6454 μm). The infusion was carried out by PHD 2000 Infusion (Harvard Apparatus, Cambridge, MA, USA), and the total flow rates (Q1 + Q2) were 175 μL/min ( $Re$ = 39), 250 μL/min ( $Re$ = 56), 525 μL/min ( $Re$ = 117), 700 μL/min ( $Re$ = 156), 875 μL/min ( $Re$ = 194) and 1050 μL/min ( $Re$ = 233) while the ratio of Q2:Q1 is always 1:2.5 to maintain the solvent to DI water ratio. The experiment was carried out under the light microscope (Olympus IX71, Tokyo, Japan) to ensure no blockages of precipitate occurred during the solution collection, an image of the setup is attached in Supplementary Materials. The collected solution was then washed and dried on diced silicon wafers on the hotplate at 40 °C until all liquid evaporated. The particles were then observed under the scanning electron microscope (SEM) (JSM-7100F, Tokyo, Japan). The SEM images were analyzed in ImageJ 1.53 t to obtain the size distribution, and the graphs were plotted in Origin 2018. And the most abundant particles’ sizes and percentage in amount are discussed.

Protoplasts in the aqueous phase were encapsulated into droplets using a flow-focusing microfluidic device: the protoplast suspension was loaded into a 500 μL Hamilton Gas-tight syringe (Hamilton Robotics, Reno, NV, USA). The fluorinated oil used as the continuous phase (3M Novec 7500 Engineered Fluid with 2.5% PicoSurf 1 surfactant, Sphere Fluidics, Cambridge, UK) was loaded in another syringe and both syringes were connected to the respective inlets of the flow-focusing device (nozzle dimensions: 40 μm x 40 μm x 50 μm) with fine bore polyethylene tubing (ID = 0.38 mm, OD = 1.09 mm, Smiths Medical International, Luton, UK). Using syringe pumps (PHD 2000 Infusion, Harvard Apparatus, Holliston, MA), the two solutions were injected simultaneously in the device. The oil phase was injected at a rate of 500 μL/h and the aqueous phase at a rate of 300 μL/h. The generated droplets were collected, through tubing connected to the outlet, into a syringe.

Leakage of the PDMS–PMMA hybrid devices was tested by injecting dye solution into the microchannels. The flow rate of the dye solution was started at 5 mL min⁻¹ and increased up to 30 mL min⁻¹ by increasing the injection rate by 5 mL min⁻¹. A syringe pump (Harvard Apparatus, PHD 2000 Infusion) was used to inject the dye and control its flow rate. These tests were performed on two types of fabricated microchannels (Fig. 1b)—one was a straight channel (total length of 30 mm, height of 0.4 mm, and width of 1 mm), and another was a serpentine channel (total length of 190 mm, height of 0.4 mm, width of 0.3 mm).

Photochromic nanofibers were prepared using a 30% w/v of PCL, in a solution containing 60:40 of THF/DMF. PEGSP2 was added in a ratio of 30% w/w. The solutions were kept under constant stirring for 2 days to ensure the complete polymer dissolution. An electrospinning apparatus consisting of a 12 mL syringe connected to a syringe pump (Harvard Apparatus PHD 2000 infusion, Holliston, MA, USA) was used to supply a steady flow of 1.8 mL h⁻¹ of solution to the tip of the needle. A high-voltage power supply (Gamma High Voltage, Ormond Beach, FL, USA) was used to apply the potential of 19 kV to the syringe needle. The target consisted of a grounded steel plate of 20 × 20 cm placed 18 cm from the needle tip to ensure that fibers were dry upon collection [17 (link)].

A total of 24 rats were randomly assigned to either a saline control (n = 12) or a DREADD experimental group (n = 12) and coded so the experimenter was blinded. A total of 9 controls and 10 DREADD animals were included in the analysis. The remainder (five animals) were excluded because postmortem histology showed off-target cannulae placement.
Unblinding occurred once all procedures and analysis were completed. Six weeks after viral transfection (see above), animals were tested in the following behavioural paradigms: auditory cued fear conditioning, beam balance, open field, and elevated plus maze. One animal was excluded from the study after the fear conditioning test because of poor health; a further three animals were excluded from the open field analysis because of technical problems with the video recording. In every animal, a volume of 500 nl of CNO (3 µM, Tocris) was infused at a rate of 0.5 µl/min to target the PAG, 15 min prior to each behavioural test (infusion pump Harvard Apparatus, PHD 2000 Infusion). Since the effect of CNO is estimated to last 60–90 min (Stachniak et al., 2014 (link); Jendryka et al., 2019 (link)), this meant that during fear conditioning the pathway under study was likely to be inhibited during both acquisition (which lasted about 10 min) and also a subsequent period of early consolidation.