Developing a Translaminar Pressure Model Using a Translaminar Autonomous System

Samenvatting

Source: Sharma, T. P., et al., Translaminar Autonomous System Model for the Modulation of Intraocular and Intracranial Pressure in Human Donor Posterior Segments. J. Vis. Exp. (2020).

This video demonstrates the procedure of preparing a human eye to study the effects of translaminar pressure on retinal ganglion cell signaling by simulating intraocular and intracranial pressures in a translaminar autonomous system.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Preparation and sterilization of equipment and supplies

1. Refer to the Table of Materials for a complete list of supplies required as well as supplier and catalogue numbers.
2. Prior to use, sterilize all equipment and instruments by autoclaving or using ethylene oxide ampules.

2. Preparation of perfusion medium

1. Add 1% penicillin-streptomycin (10,000 U/mL penicillin, 10,000 μg/mL streptomycin in 0.85% NaCl) and 1% L-glutamine (200 mM) to 1,000 mL high glucose Dulbecco’s modified Eagle’s medium (DMEM).
2. Sterilize the perfusion medium by passing through a 0.22 μm filter.

3. Translaminar autonomous system (TAS) setup

1. Set up inflow syringes (intraocular pressure (IOP) and intracranial pressure (ICP) reservoirs).
   1. Add 30 mL of the perfusion medium (section 2) to a 30 mL syringe. Attach a 3-way stopcock to the 30 mL syringe. Attach a 0.22 μm hydrophilic filter to the 3-way stopcock. Attach a 15 G Luer stub adapter to the 0.22 μm hydrophilic filter.
   2. Remove air bubbles from the syringe setup. Attach tubing to the 15 G Luer stub adapter. Close the side port of the stopcock with an unvented universal lock cap. Repeat for a total of two setups.
   3. Label one syringe as channel 1 intracranial pressure (CH1 ICP) and the other syringe as channel 2 intraocular pressure (CH2 IOP).
2. Set up outflow syringes (IOP and ICP reservoirs).
   1. Attach a 3-way stopcock to a 30 mL syringe. Attach a 15 G Luer stub adapter to the 3-way stopcock. Attach tubing to the 15 G Luer stub adapter.
   2. Close the side port of the stopcock with an unvented universal lock cap. Repeat for a total of two setups. Label one syringe as CH1 ICP and the other syringe as CH2 IOP.

4. Preparation of human whole eye globe

NOTE: If whole eyes are received, follow the procedure below to separate the anterior segment from the posterior segment of the eye. If the eyes are received bisected, start at step 4.4.

1. Place a whole eye into the povidone-iodine solution for 2 min.
2. Rinse the eye in a sterile phosphate-buffered solution (PBS) to remove the povidone-iodine. Repeat two times.
3. Remove the adnexa from the whole eye globe using forceps and scissors. Bisect the eye at the equator to separate the anterior and posterior segments of the eye.
4. Remove the optic nerve sheath. Remove the vitreous humor from the posterior segment.
5. Trim additional sclera from the posterior segment, if needed, to ensure a good fit on the round dome of the IOP (bottom) chamber. Using forceps, ensure that the retina is spread evenly over the posterior of the segment.
6. IOP (bottom) chamber setup
   1. Place the human posterior segment into the IOP (bottom) chamber of the TAS over the round dome with the optic nerve facing the top.
   2. Seal the posterior segment using the epoxy resin O-ring with four screws, ensuring a tight seal.
   3. Insert the tubing into the IN and OUT ports of the IOP (bottom) chamber. The IOP inflow syringe with tubing containing medium is inserted into the IN port and the empty IOP outflow syringe with tubing is inserted into the OUT port.
   4. Use the push/pull method to slowly infuse the perfusion medium into the inflow port to fill the posterior eye cup while simultaneously slowly pulling the perfusion medium out through the outflow syringe to remove any air bubbles from the lines. Stop infusing the medium once both the IN and OUT tubes are void of air bubbles.
   5. Lock the stopcocks in the off position. Remove the 30 mL syringe from the IOP IN port filter assembly and refill it with 30 mL of medium. Then, replace the syringe with the filter assembly.
7. ICP (top) chamber setup
   1. Place the ICP (top) chamber/lid over the back of the posterior segment. Make certain that the optic nerve is within the top chamber. Seal the top chamber with four screws.
   2. Insert the tubing into the IN and OUT ports of the ICP (top) chamber. The ICP inflow syringe with tubing containing medium is inserted into the IN port and the empty ICP outflow syringe with tubing is inserted into the OUT port.
   3. Gently and slowly infuse the medium into the IN port to fill the ICP chamber and remove air bubbles from the lines using the push/pull method. Stop infusing the medium once the ICP chamber and both the IN and OUT tubing are void of air bubbles.
   4. Lock the stopcocks in the off position. Remove the 30 mL syringe from the ICP in port filter assembly and refill it with 30 mL of medium. Then, replace the syringe with the filter assembly.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
#122, 1-1/8" Inside x 1-5/16" Outside Diam, Viton O-Ring, 3/32" Thick, 755 Durometer 50 Pack	Amazon	B07DRGPPZJ
114 Buna-N O-Ring, 70A Durometer, Black, 5/8" ID, 13/16" OD, 3/32" Width (Pack of 100)	Amazon	B000FMYRHK
30 mL Syringes without Needle	Vitality Medical	302832
3-Way Stopcock, 2 Female Luer Locks, Swivel Male Luer Lock, Vented Cap	QOSINA	2C6201
4-40 X 1/2 PH PAN MS SS/CHROME & appropriate sized phillips screwdriver	Brikksen Stainless Steel Fastners	PPMSSSCH4C.5
ANPROLENE 16 LARGE AMPULE	Fisher Scientific	NC9085343
Betadine	Purdue	PUR1815001EACH
Corning 100 x 20mm tissue-culture treated culture dishes	Sigma-Aldrich	CLS430167-100EA
Corning L-glutamine Solution	Fisher Scientific	MT25005CI
Covidien 3033 Curity Gauze Sponge, 4" x 4", 12-Ply, Sterile, 1200/CS	Med Plus Medical Supply	COV-3033-CS
Dressing Forceps Delicate Curved (serrated)	Katena	K5-4010
Dumont #5 - Fine Forceps	F.S.T.	11254-20
Eye Scissors Standard Curved	Katena	K4-7410
Falcon 150 x 15mm Plain Sterile Disposable Petri Dishes	Capitol Scientific	351058
Fisherbrand 4 oz. Specimen Containers	Fisher Scientific	16-320-730
Fisherbrand Instant Sealing Sterilization Pouches	Fisher Scientific	01-812-54
Fisherbrand Instant Sealing Sterilization Pouches	Fisher Scientific	01-812-55
Fisherbrand Instant Sealing Sterilization Pouches	Fisher Scientific	01-812-58
HyClone Dulbecco's Modified Eagles Medium	Fisher Scientific	SH3024302
HyClone Penicillin Streptomycin 100X Solution	Fisher Scientific	SV30010
Hydrophilic Filter with Female Luer Lock Inlet, Male Luer Slip Outlet, Blue and Clear	Qosina	28217
Hydrostatic pressure transducers, DELTRAN ® II, Catalog # DPT-200 with a 3CC/HR flow rate	AD instruments	DPT-200
JG15-0.5HPX 15 Gauge 0.5" NT Premium Series Dispensing Tip 50/Box	Jenson Global	JG15-0.5HPX 15
Keyence B2‐X710 microscope	Keyence	B2-X710
LabChart 8	AD instruments	LabChart 8
Leica ST5020 Multi-stainer	Leica	ST5020
Non-Vented Universal Luer Lock Cap, White	QOSINA	65811
Octal Bridge Amp (Model # FE228)	AD instruments	FE228
Pharmco Products ETHYL ALCOHOL, 200 PROOF	Fisher Scientific	NC1675398
Phosphate Buffered Solution (PBS)	Sigma-Aldrich	D8537-500ML
PowerLab 8/35 (Model # PL3508)	AD instruments	PL3508
ProLong Gold Antifade Mountant with DAPI	ThermoFisher	P36935
Push-to-Connect Tube Fitting for Air and Water Straight Adapter, 1/8" Tube OD x 1/8 NPT Male	McMAster-Carr	7880T113
Push-to-Connect Tube Fitting with Universal Thread for Air and Water, Adapter, 1/8" Tube OD x 1/8 Pipe	McMAster-Carr	51235K101
Saint-Gobain Tygon S3 E-3603 Flexible Tubing 500 ft.	Fisher Scientific	14-171-268
Superblock T20	Fisher Scientific	PI37536
Surgical Scissors - Sharp-Blunt	F.S.T.	14001-14
Tissue Forceps Delicate 1x2 Teeth Curved	Katena	K5-4110
Translaminar Autonomous System (TAS)	University of North Texas Health Science Center	N/A
USA Size 030 O-ring Buna-N, B1000, 70 Durometer, Black, Buna-N (NBR, Nitrile, Buna)	Marco Rubber & Plastics	B1000-030