Funding for this project was through discretionary funds of Dr. Colleen M. McDowell. This work was supported in part by an unrestricted grant from Research to Prevent Blindness, Inc. to the UW Madison Department of Ophthalmology and Visual Sciences. We thank Drs. Abbot F. Clark and Weiming Mao for their technical assistance with the perfusion organ culture model. We thank the Lions Eye Institute for Transplant and Research (Tampa, FL) for providing the human donor eyes.

Eyes were obtained according to the provisions of the Declaration of Helsinki for research involving human tissue.
NOTE: Eyes from reputable eye banks (e.g., Lions Eye Institute for Transplant, Research, Tampa FL) were harvested within 6&#8211;12 h of death and donor serum was tested for hepatitis B, hepatitis C, and human immunodeficiency virus 1 and 2. Once they were received, the eyes were dissected and set up in the TAS model within 24 h. Exclusion criteria included any ocular pathology. E...

The authors of the manuscript have no potential conflicts of interest to disclose.

Human postmortem tissues are an especially valuable resource for studying human neurodegenerative diseases because identification of potential drugs developed in&#160;animal models need to be translatable to humans47. The effects of human IOP elevation are well-established, but minimal research has been performed on abnormal ONH translaminar pressure changes. Even though multiple animal models and finite modeling of human ONH exist, there is no ex vivo human model to study translaminar pressure ch...

Global estimates in recent surveys suggest that 285 million people live with visual impairment, including 39 million who are blind1. In 2010, the World Health Organization documented that three of the nine listed leading causes of blindness occur in the posterior segment of the eye1. Posterior segment eye diseases involve the retina, choroid, and optic nerve2. The retina and optic nerve are central nervous system (CNS) extensions of the brain. The retinal ganglion cell (RGC) axons are vulnerable to damage because they exit the eye through the optic nerve head (ONH) to form the optic nerve3. The ONH remains the most vulnerable point for the RGC&#160;axons because of the 3D meshwork of connective tissue beams called the lamina cribrosa (LC)4. The ONH is the initial site of insult to RGC axons in glaucoma5,6,7, and gene expression changes within the ONH have been studied in ocular hypertension and glaucoma models8,9,10. The RGC axons are susceptible at the ONH due to pressure differentials between the intraocular compartment, called the intraocular pressure (IOP), and within the external perioptic subarachnoid space, called the intracranial pressure (ICP)11. The LC region separates both areas, maintaining normal pressure differentials, with IOP ranging from 10&#8211;21 mmHg and ICP from 5&#8211;15 mmHg12. The pressure difference through the lamina between the two chambers is called the translaminar pressure gradient (TLPG)13. A major risk factor of glaucoma is elevated IOP14.
Increased IOP increases the strain within and across the laminar region6,15,16. Experimental observations in humans and animal models present the ONH as being the initial site of axonal damage17,18. The biomechanical paradigm of IOP-related stress and strain causing glaucomatous damage at the ONH also influences the pathophysiology of glaucoma19,20,21. Even though in humans pressure-induced changes mechanically damage RGC axons22, rodents lacking collagenous plates within the lamina can also develop glaucoma7,23. In addition, elevated IOP remains the most prominent risk factor in primary open angle glaucoma patients, while normal tension glaucoma patients develop glaucomatous optic neuropathy even without elevated IOP. Furthermore, there are also a subset of ocular hypertensive patients that show no optic nerve damage. It has also been suggested that cerebrospinal fluid pressure (CSFp) may play a role in glaucoma pathogenesis. Evidence indicates that ICP is lowered to ~5 mmHg in glaucoma patients compared to normal individuals, thereby causing increased translaminar pressure and playing a crucial role in disease24,25. Previously, it was demonstrated in a canine model, that by controlling IOP and CSFp changes, there can be large displacements of the optic disc26. Elevating CSFp in porcine eyes has also shown increased principal strain within the LC region and retrolaminar neural tissue. Increased strain on the RGCs and the LC region contributes to axonal transport blockage and loss of RGCs27. Progressive degeneration of RGCs has been associated with loss of trophic support28,29, stimulation of inflammatory processes/immune regulation30,31, and apoptotic effectors29,32,33,34,35. Additionally, axonal injury (Figure 3) causes detrimental effects on the RGCs, triggering regenerative failure36,37,38,39. Even though the effects of IOP have been well studied, minimal research has been performed on abnormal translaminar pressure changes. Most treatments for glaucoma focus on stabilizing IOP. However, even though lowering of IOP slows the progression of the disease, it does not reverse visual field loss and prevent complete loss of RGCs. Understanding pressure-related neurodegenerative changes in glaucoma will be crucial to preventing RGC death.
Current evidence indicates that translaminar pressure modulations due to various mechanical, biological, or physiological changes in patients suffering from traumatic or neurodegenerative visual impairments can cause significant vision loss. Currently, no true preclinical human posterior segment model exists that can allow the study of glaucomatous biomechanical damage within the ex vivo human ONH. Observation and treatment of the posterior segment of the eye is a huge challenge in ophthalmology27. There are physical and biological barriers to target the posterior eye, including high elimination rates, blood-retinal barrier, and potential immunological responses40. Most efficacy and safety tests for novel drug targets are accomplished utilizing in vitro cellular and in vivo animal models41. Ocular anatomy is complex, and in vitro studies do not accurately mimic the anatomical and physiological barriers presented by tissue model systems. Even though animal models are a necessity for pharmacokinetic studies, the ocular physiology of the human posterior eye may vary between various animal species, including cellular anatomy of the retina, vasculature, and ONH41,42.
The use of living animals requires intensive and detailed ethical regulations, high financial commitment, and effective reproducibility43. Recently,&#160;multiple other guidelines have ensued for the ethical use of animals in experimental research44,45,46. An alternative to animal testing is the use of ex vivo human eye models to investigate disease pathogenesis and potential analysis of drugs for protecting ONH damage. Human postmortem tissue is a valuable resource for studying human disease paradigms, especially in the case of human neurodegenerative diseases, because identification of potential drugs developed in&#160;animal models require the need to be translatable to humans47. The ex vivo human donor tissue has been extensively utilized for the study of human disorders47,48,49, and human anterior segment perfusion organ culture systems have previously provided a unique ex vivo model to study the pathophysiology of elevated IOP50,51,52.
To study translaminar pressure related to IOP and ICP in human eyes, we successfully designed and developed a two-chamber translaminar autonomous system (TAS) that can independently regulate IOP and ICP using posterior segments from human donor eyes. It is the first ex vivo human model to study translaminar pressure and exploit the biomechanical effects of TLPG on the ONH.
This ex vivo human TAS model can be used to discover and classify cellular and functional modifications that occur due to chronic elevation of IOP or ICP. In this report, we detail the step-by-step protocol of dissecting, setting up, and monitoring the TAS human posterior segment model. The protocol will allow other researchers to effectively reproduce this novel ex vivo pressurized human posterior segment model to study biomechanical disease pathogenesis.

<table>
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			<td>#122, 1-1/8&#34; Inside x 1-5/16&#34; Outside Diam, Viton O-Ring, 3/32&#34; Thick, 
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			<td>114 Buna-N O-Ring, 70A Durometer, Black, 5/8&#34; ID, 13/16&#34; OD, 3/32&#34; Width (Pack of 100)</td>
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			<td>30 mL Syringes without Needle</td>
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			<td>NC9085343</td>
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			<td>Corning 100 x 20mm tissue-culture treated culture dishes</td>
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			<td>Corning&#160;L-glutamine Solution</td>
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			<td>Covidien 3033 Curity Gauze Sponge, 4&#34; x 4&#34;, 12-Ply, Sterile, 1200/CS</td>
			<td>Med Plus Medical Supply</td>
			<td>COV-3033-CS</td>
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			<td>Dressing Forceps Delicate Curved (serrated)</td>
			<td>Katena</td>
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			<td>Dumont #5 - Fine Forceps</td>
			<td>F.S.T.</td>
			<td>11254-20</td>
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			<td>Eye Scissors Standard Curved</td>
			<td>Katena</td>
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			<td>Falcon 150 x 15mm Plain Sterile Disposable Petri Dishes</td>
			<td>Capitol Scientific</td>
			<td>351058</td>
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			<td>Fisherbrand 4 oz. Specimen Containers</td>
			<td>Fisher Scientific</td>
			<td>16-320-730</td>
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			<td>Fisherbrand Instant Sealing Sterilization Pouches</td>
			<td>Fisher Scientific</td>
			<td>01-812-54</td>
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			<td>Fisherbrand Instant Sealing Sterilization Pouches</td>
			<td>Fisher Scientific</td>
			<td>01-812-55</td>
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			<td>Fisherbrand Instant Sealing Sterilization Pouches</td>
			<td>Fisher Scientific</td>
			<td>01-812-58</td>
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			<td>HyClone Dulbecco&#39;s Modified Eagles Medium</td>
			<td>Fisher Scientific</td>
			<td>SH3024302</td>
			<td></td>
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			<td>HyClone Penicillin Streptomycin 100X Solution</td>
			<td>Fisher Scientific</td>
			<td>SV30010</td>
			<td></td>
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		<tr>
			<td>Hydrophilic Filter with Female Luer Lock Inlet, Male Luer Slip Outlet, Blue and Clear</td>
			<td>Qosina</td>
			<td>28217</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrostatic pressure transducers, DELTRAN &#174; II, Catalog # DPT-200 with a 3CC/HR flow rate</td>
			<td>AD instruments</td>
			<td>DPT-200</td>
			<td></td>
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			<td>JG15-0.5HPX 15 Gauge 0.5&#34; NT Premium Series Dispensing Tip 50/Box</td>
			<td>Jenson Global</td>
			<td>JG15-0.5HPX 15</td>
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		<tr>
			<td>Keyence B2&#8208;X710 microscope</td>
			<td>Keyence</td>
			<td>B2-X710</td>
			<td></td>
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			<td>LabChart 8</td>
			<td>AD instruments</td>
			<td>LabChart 8</td>
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			<td>Leica ST5020 Multi-stainer</td>
			<td>Leica</td>
			<td>ST5020</td>
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			<td>Non-Vented Universal Luer Lock Cap, White</td>
			<td>QOSINA</td>
			<td>65811</td>
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			<td>Octal Bridge Amp (Model # FE228)</td>
			<td>AD instruments</td>
			<td>FE228</td>
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			<td>Pharmco Products ETHYL ALCOHOL, 200 PROOF</td>
			<td>Fisher Scientific</td>
			<td>NC1675398</td>
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			<td>Phosphate Buffered Solution (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>D8537-500ML</td>
			<td></td>
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			<td>PowerLab 8/35 (Model # PL3508)</td>
			<td>AD instruments</td>
			<td>PL3508</td>
			<td></td>
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			<td>ProLong Gold Antifade Mountant with DAPI</td>
			<td>ThermoFisher</td>
			<td>P36935</td>
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			<td>Push-to-Connect Tube Fitting for Air and Water&#160;Straight Adapter, 1/8&#34; Tube OD x 1/8 NPT Male</td>
			<td>McMAster-Carr</td>
			<td>7880T113</td>
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			<td>Push-to-Connect Tube Fitting with Universal Thread&#160;for Air and Water, Adapter, 1/8&#34; Tube OD x 1/8 Pipe</td>
			<td>McMAster-Carr</td>
			<td>51235K101</td>
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			<td>Saint-Gobain Tygon S3 E-3603 Flexible Tubing 500 ft.</td>
			<td>Fisher Scientific</td>
			<td>14-171-268</td>
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			<td>Superblock T20</td>
			<td>Fisher Scientific</td>
			<td>PI37536</td>
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			<td>Surgical Scissors - Sharp-Blunt</td>
			<td>F.S.T.</td>
			<td>14001-14</td>
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			<td>Tissue Forceps Delicate 1x2 Teeth Curved</td>
			<td>Katena</td>
			<td>K5-4110</td>
			<td></td>
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		<tr>
			<td>Translaminar Autonomous System (TAS)</td>
			<td>University of North Texas Health Science Center</td>
			<td>N/A</td>
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			<td>USA Size 030 O-ring Buna-N, B1000, 70 Durometer, Black, Buna-N 
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			<td>Marco Rubber &#38; Plastics</td>
			<td>B1000-030</td>
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translaminar autonomous system model for the modulation of intraocular and intracranial pressure in human donor posterior segments

There is a current unmet need for a new preclinical human model that can target disease etiology ex vivo using intracranial pressure (ICP) and intraocular pressure (IOP) which&#160;can identify various pathogenic paradigms related to the glaucoma pathogenesis. Ex vivo human anterior segment perfusion organ culture models have previously been successfully utilized and applied as effective technologies for the discovery of glaucoma pathogenesis and testing of therapeutics. Preclinical drug screening and research performed on ex vivo human organ systems can be more translatable to clinical research. This article describes in detail the generation and operation of a novel ex vivo human translaminar pressure model called the translaminar autonomous system (TAS). The TAS model can independently regulate ICP and IOP using human donor posterior segments. The model allows for studying pathogenesis in a preclinical manner. It can reduce the use of living animals in ophthalmic research. In contrast to in vitro experimental models, optic nerve head (ONH) tissue structure, complexity, and integrity can also be maintained within the ex vivo TAS model.

Design and creation of the translaminar autonomous system 
Translaminar pressure differential is a potential key mechanism in the pathogenesis of various diseases, including glaucoma. Uses for the model described include, but are not limited to, the study of glaucoma (elevated IOP, perhaps decreased ICP), traumatic brain injury (elevated ICP), and long-term exposure to microgravity-associated visual impairment (elevated ICP, elevated IOP). To help discover molecular pathogenesis targeting translamin...

Watch this Scientific Journal Video about Translaminar Autonomous System Model for the Modulation of Intraocular and Intracranial Pressure in Human Donor Posterior Segments at JoVE.com

Translaminar Autonomous System Model for the Modulation of Intraocular and Intracranial Pressure in Human Donor Posterior Segments

We describe and detail the use of the translaminar autonomous system. This system utilizes the human posterior segment to independently regulate the pressure inside the segment (intraocular) and surrounding the optic nerve (intracranial) to generate a translaminar pressure gradient that mimics features of glaucomatous optic neuropathy.

There is a current unmet need for a new preclinical human model that can target disease etiology ex vivo using intracranial pressure (ICP) and intraocular ...

This novel ocular translaminar autonomous system uses the human posterior segment to independently regulate intraocular and intracranial pressures to generate a translaminar pressure gradient. The TAS model allows the evaluation of human intracranial pressure in an ex vivo, preclinical manner that previously could not be studied. This model could potentially be used to study diseases such as glaucoma, traumatic brain injury, idiopathic intracranial hypertension, and spaceflight-associated neuro-ocular syndrome.To set up the inflow syringes, load 30-milliliter syringe with 30 milliliters of the perfusion fluid of interest and attach a three-way stop cock to the syringe. Attach a 0.22-micrometer hydrophilic filter to the stop cock, and attach a 15-gauge luer stub adapter to the 0.22-micrometer hydrophilic filter. After removing air bubbles from the syringe setup, attach tubing to the luer stub adapter and close the side port of the stop cock with an unvented universal lock cap.After setting up a second inflow syringe as just demonstrated, label one syringe channel one ICP, and label the other syringe channel two IOP. Then set up the outflow syringes as just demonstrated, but without loading the syringes with the infusion medium. To prepare a human whole eye globe sample, remove the optic nerve sheath and remove the vitreous humor from the posterior segment.To ensure good fit on the round dome of the IOP chamber, trim additional sclera from the posterior segment as necessary, and use forceps to ensure that the retina is spread evenly over the human posterior of the segment. Then place the segment into the IOP chamber of the TAS over the round dome with the optic nerve facing up, and use an epoxy resin O-ring and four screws to seal the posterior segment. To set up the IOP chamber, insert the tubing into the in and out ports of the chamber and insert the IOP inflow syringe into the in port.Insert the empty IOP outflow syringe setup into the out port and 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. Once both the in and out tubes are void of air bubbles, stop the infusion and lock the stop cocks in the off position. Remove the syringe from the IOP in port filter assembly and refill the syringe with 30 additional millimeters of medium, then reinsert the syringe setup into the filter assembly.To set up the ICP chamber, place the chamber over the back of the posterior segment, taking care that the optic nerve is within the top chamber, and seal the top chamber with four screws then insert the tubing into the in and out ports of the ICP chamber, the ICP input syringe into the in port, and the empty ICP outflow syringe into the out port to flush the system with medium as just demonstrated. To set up the data recording system, turn on the eight-channel power source and the computer, and initiate the data acquisition software. Select File and New and select Setup and Channel Settings.Select three channels, and rename the channels as indicated. Select two millivolts for the Range on all if the channels and in the Calculation column, select No Calculation for channels one and two. Select Arithmetic for channel three In the Formula window, under Channels, select Ch 2.Under Function, select Math and Minus, and under Channels, Select Ch 1. To set up and calibrate the hydrostatic pressure transducers, connect the hydrostatic pressure transducers to the transducer lines attached to the multi-channel bridge amplifier, and attach an empty 30-milliliter syringe to the side port of the channel one ICP pressure transducer. Attach a sphygmomanometer to the bottom of the channel one ICP pressure transducer, and click the arrow next to the sampling time to set the sampling speed to 100.Select Bridge Amp, Mains filter, and Zero, and wait for the system to zero out, taking care to not move the pressure transducer. Next, pinch the white tabs of the pressure transducer and push air through the transducer until 40 millimeters of mercury are obtained on the sphygmomanometer, then release the white tabs and remove the syringe and sphygmomanometer. On the Units Conversion page, select minus and highlight the highest plateau to indicate 40 millimeters of mercury.Click the arrow for 0.1, enter 100, and highlight the lowest plateau to indicate zero millimeters of mercury. Click the arrow for 0.2, enter zero, select millimeters of mercury for the units, and click Okay. In the Bridge Amp window, click Okay, and calibrate the hydrostatic pressure transducers for channel two IOP as just demonstrated, using 100 millimeters of mercury for the highest plateau, and zero for the lowest plateau.To connect the TAS posterior segment unit to the data acquisition system, place the TAS posterior segment unit into a 37 degree Celsius, 5%carbon dioxide incubator, and attach the ICP tubing from the out port of the channel one ICP pressure transducer. Attach the IOP tubing from the out port to the channel two IOP pressure transducer. On the Chart View page, select Start Sampling and set the sampling speed to Slow and one minute.Then adjust the syringes on the ring stand up or down to regulate ICP and IOP pressures according to the protocol requirements, or place them on perfusion pumps for regulating pressure. In this representative analysis, the maintenance of the average normal pressure differentials in both chambers were tested through various parameters of IOP and ICP conditions. To ensure the viability of the tissue, the medium in the tissue was exchanged every 48 hours.Minimal pressure increases occurred at the time of medium exchange, and did not affect the viability or morphology of the optic nerve head as observed by immunohistochemical analysis and collagen IV expression on days 14 and 30 of the experiment. Increasing the IOP or decreasing the ICP over various time points allows the maintenance of a range in elevated translaminar pressure gradient for seven days. At seven days of elevated translaminar pressure gradient, cupping and thickening were observed in posterior segments, and collagen IV expression demonstrates thickened beams with an increased signal expression over the experimental period.Phase imaging reveals healthy retinal ganglion cells within the ganglion cell layer with no cupping in the control segments, while images from elevated translaminar pressure gradient cultures exhibit extensive cupping with no retinal ganglion cells remaining within the retinal nerve fiber layer, and increased remodeling of the extra cellular matrix. This technique will allow researchers to effectively study biomechanical disease paradigms, and discover molecular pathogenesis-targeting translaminar pressure in the human eye.

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Neuroscience

2.9K 조회수.  University of North Texas Health Science Center. This novel ocular translaminar autonomous system uses the human posterior segment to independently regulate intraocular and intracranial pressures to generate a translaminar pressure gradient. The TAS model allows the evaluation of human intracranial pressure in an ex vivo, preclinical manner that previously could not be studied. This model could potentially be used to study diseases such as glaucoma, traumatic brain injury, idiopathic intracranial hypertension, and spaceflight-associated neu...