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. Eyes were not excluded based on age, race, or gender. To ensure the viability of the retina upon receipt, retinal explants were harvested from the tissue donors and cultured for 7 and 14 days (Supplemental Figure 1). These retinas were also dissociated and grew healthy RGCs in culture for 7 days with positive staining for RGC marker, RNA-binding protein with multiple splicing (RBPMS), as well as positive neurofilament light chain (NEFL) staining in their neurofilaments (Supplemental Figure 2).&#160;.
1. Preparation and sterilization of equipment and supplies
<ol>
	<li>Refer to the Table of Materials for a complete list of supplies required as well as supplier and catalogue numbers.</li>
	<li>Prior to use, sterilize all equipment and instruments by autoclaving or using ethylene oxide ampules.</li>
</ol>
2. Preparation of perfusion medium
<ol>
	<li>Add 1% penicillin streptomycin (10,000 U/mL penicillin, 10,000 &#956;g/mL streptomycin in 0.85% NaCl) and 1% L-glutamine (200 mM) to 1,000 mL high glucose Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM).</li>
	<li>Sterilize the perfusion medium by passing through a 0.22 &#956;m filter.</li>
</ol>
3. Translaminar autonomous system (TAS) setup
<ol>
	<li>Set up inflow syringes (IOP and ICP reservoirs).
	<ol>
		<li>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 &#956;m hydrophilic filter to the 3-way stopcock. Attach a 15 G Luer stub adapter to the 0.22 &#956;m hydrophilic filter.</li>
		<li>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.</li>
		<li>Label one syringe as channel 1 intracranial pressure (CH1 ICP) and the other syringe as channel 2 intraocular pressure (CH2 IOP).</li>
	</ol>
	</li>
	<li>Set up outflow syringes (IOP and ICP reservoirs).
	<ol>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
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.
<ol>
	<li>Place a whole eye into povidone-iodine solution for 2 min.</li>
	<li>Rinse the eye in sterile phosphate buffered solution (PBS) to rinse off the povidone-iodine. Repeat 2 times.</li>
	<li>Remove the adnexa from the whole eye globe using&#160;forceps and scissors. Bisect the eye at the equator to separate the anterior and posterior segments of the eye.</li>
	<li>Remove the optic nerve sheath. Remove the vitreous humor from the posterior segment.</li>
	<li>Trim additional sclera from 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.</li>
	<li>IOP (bottom) chamber setup
	<ol>
		<li>Place the human posterior segment into the IOP (bottom) chamber of the TAS over the round dome with the optic nerve facing the top.</li>
		<li>Seal the posterior segment using the epoxy resin O-ring with four screws, ensuring a tight seal.</li>
		<li>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.</li>
		<li>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 medium once both the IN and OUT tubes are void of air bubbles.</li>
		<li>Lock the stopcocks in the off position. Remove the 30 mL syringe from the IOP IN port filter assembly and refill with a total of 30 mL of medium. Replace the 30 mL syringe onto the filter assembly.</li>
	</ol>
	</li>
	<li>ICP (top) chamber setup
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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 medium once the ICP chamber as well as both the IN and OUT tubing are void of air bubbles.</li>
		<li>Lock the stopcocks in the off position. Remove the 30 mL syringe from the ICP in port filter assembly and refill with a total of 30 mL of medium. Replace the 30 mL syringe onto the filter assembly.</li>
	</ol>
	</li>
</ol>
5. Data recording system setup
NOTE: The data recording system is comprised of an 8-channel power source, multichannel bridge amplifier, hydrostatic pressure transducers, and a computer with data acquisition software (see Table of Materials). The following describes how to set up and calibrate the system.
<ol>
	<li>Connect the power cord into the back of the 8-channel power source and plug into a battery back-up device.</li>
	<li>Connect the USB cable from the 8-channel power source into the back of the computer.</li>
	<li>Connect the 8-channel power source to the multichannel bridge amplifier using the supplied I2C cord.</li>
	<li>Connect the Bayonet Neill-Concelman (BNC) cables into the channel inputs in front of the 8-channel power source and the end of the cables into the corresponding channels in the back of the multichannel amplifier.</li>
	<li>Connect the transducer cables to the front of the multichannel amplifier.</li>
	<li>Install the data acquisition software on the computer.
	<ol>
		<li>Run the data acquisition software setup installer from the supplied software CD.</li>
		<li>Follow the instructions on the computer screen.</li>
		<li>When the installation is complete, select Finish.</li>
	</ol>
	</li>
	<li>Turn on the 8-channel power source.</li>
	<li>Turn on the computer and initiate the data acquisition software.
	<ol>
		<li>Select File | New.</li>
		<li>Select Setup | Channel Settings. Select three channels (bottom left of screen). In the Channel Title column rename the channels as follows: CH1 ICP; CH2 IOP; CH3 TLPG (IOP-ICP).</li>
		<li>Select 2 mV for the Range on all channels. In the Calculation column select No Calculation for channels 1 and 2.</li>
		<li>In the Calculation column select Arithmetic for channel 3. In the Formula section: Select channels/CH2; Select arithmetic &#34;-&#34;; Select channels/CH1. In the Output section select mmHg. Select OK. Select OK again.</li>
	</ol>
	</li>
	<li>Set up and calibrate the hydrostatic pressure transducers. 
	NOTE: Hydrostatic pressure transducers must be calibrated prior to experiments using the following method.
	<ol>
		<li>Connect the hydrostatic pressure transducers to the transducer lines attached to the multichannel bridge amplifier.</li>
		<li>Attach a 30 mL syringe filled with air to the side port of the CH1 (ICP) pressure transducer. Attach the sphygmomanometer to the bottom of the CH1 (ICP) pressure transducer.</li>
		<li>On the chart, view the page of the data acquisition software, set the sampling speed by left clicking the arrow next to the sampling time and select 100. Then right click in the CH1 (ICP) area of the page.</li>
		<li>Select Bridge Amp. Select Mains Filter. Select Zero and wait for the system to zero out, taking care to not move the pressure transducer.</li>
		<li>Pinch the white tabs of the pressure transducer and push air through the transducer until 40 mmHg is obtained on the sphygmomanometer. Release the white tabs and remove the syringe and sphygmomanometer.</li>
		<li>On the Units Conversion page, select &#8216;minus (-)&#8217; sign. Highlight the highest plateau to indicate 40 mmHg. Click the Arrow for point 1 and enter 40.</li>
		<li>Highlight the lowest plateau to indicate 0 mmHg. Click the Arrow for point 2 and enter 0. Select mmHg for the units. Select OK.</li>
		<li>Select OK (Bridge Amp page). Repeat steps 9.1&#8211;9.7 for CH2 (IOP) using 100 mmHg for the highest plateau and 0 for the lowest plateau.</li>
	</ol>
	</li>
	<li>Connect the TAS/posterior segment unit onto the data acquisition system.
	<ol>
		<li>Place the TAS/posterior segment unit into an incubator (37 &#176;C, 5% CO2). Attach the ICP tubing from the OUT port to the CH1 (ICP) pressure transducer.</li>
		<li>Attach the IOP tubing from the OUT port to the CH2 (IOP) pressure transducer.</li>
		<li>Attach the syringe setups (ICP and IOP) with medium from the IN ports to the ring stand.</li>
		<li>On the Chart view page select Start Sampling. Set the sampling speed by left clicking the arrow next to the sampling time and select Slow and 1 min.</li>
		<li>Adjust the syringes on the ring stand up or down to regulate ICP and IOP pressures to protocol requirements.</li>
		<li>Perform systemic replenishment of medium in the system every 48&#8211;72 h through the push and pull method.</li>
	</ol>
	</li>
</ol>
6. Data retrieval and analysis
<ol>
	<li>Open the data file in the data acquisition software.</li>
	<li>In the Data Pad section, click on the Multiple Add to Data Pad icon. A new window will appear.
	<ol>
		<li>In the Find Using section select Time from the drop-down menu.</li>
		<li>In the Select section select 1 hr every 1 hr from the drop-down menus.</li>
		<li>In the Step Through section select Whole File then click Add.</li>
	</ol>
	</li>
	<li>In the Data Pad section click on the Data Pad View icon. Highlight all the data and copy/paste into a spreadsheet.</li>
	<li>Calculate the mean and standard deviations for IOP, ICP, and TLPG for every 24 h. Collate the data using the pivot table option in a spreadsheet program and graph.</li>
</ol>
7. Immunohistochemistry and hematoxylin and eosin staining of posterior segments
<ol>
	<li>Remove the posterior eye segments following various timepoints from the TAS model and fix in formalin prior to paraffinizing.</li>
	<li>Section the eyes to produce sagittal tissue planes.</li>
	<li>Deparaffinize the paraffin-embedded segments with a 100% xylene, 95% ethanol, 50% ethanol solution.</li>
	<li>Wash the slides with PBS for 10 min and block with a blocking buffer at room temperature for 1 h.</li>
	<li>Label sections with primary antibodies: anti-collagen IV (Extracellular Matrix (ECM) marker, NB120-6586, 1:100) and anti-laminin (ECM marker, NB300-144, 1:100, anti-RBPMS (RGC marker), GTX118619, 1:50).</li>
	<li>Detect the primary antibodies using Alexa Fluor secondary antibodies (Alexa Fluor 488 goat anti-rabbit, A11008, 1:500).</li>
	<li>Counterstain the cell nuclei using DAPI anti-fade solution.</li>
	<li>Capture images of the stained sections and phase images with 4x and 10x objective lenses using a fluorescence microscope (see Table of Materials).</li>
	<li>For the hematoxylin and eosin (H&#38;E) staining, process the sections in an automated staining system (see Table of Materials) for deparaffinization using a 100%, xylene 95% ethanol, 50% ethanol solution and stain with H&#38;E.</li>
	<li>Capture images with the 4x and 10x objective lenses using a microscope with a bright field light source.</li>
</ol>

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 changes41,54,55,56,57. A current unmet need exists for a new preclinical human model that can target disease etiology ex vivo using IOP and ICP and&#160;can identify various pathogenic paradigms related to glaucoma pathogenesis. Understanding pressure-related pathological changes at the ONH will be crucial to preventing RGC death. The combined use of IOP, ICP, and TLPG within the TAS model is a unique approach to study pressure-dependent degeneration in a preclinical manner utilizing human posterior segment tissue. In the TAS model, we can culture posterior segments of human eye cups to study changes of translaminar pressure through autonomous regulation of the IOP and ICP chambers. It provides a foundation for developing a new range of therapeutics that focus on translaminar pressure as a mechanism of degeneration.
Setting up the TAS model requires attention to detail in many aspects: the correct dissection of the human posterior segments, ensuring that the retina is in intact and spread over the posterior cup, proper placement of the segment over the dome of the IOP chamber, accurately situating the ICP chamber over the ON, effective sealing of both chambers, and maintenance of hydrostatic pressures independently by regulating height of IOP and ICP reservoirs. Dissection needs to be performed in eyes that are no more than 24&#8211;36 h postmortem, because the retina progressively deteriorates if effective culture medium is not replenished. Systemic replenishment of medium was performed in our system every 48&#8211;72 h. Another crucial aspect of the system is the length of the ON. It is critical to ensure that at least 0.5&#8211;1 cm of ON is left on the cadaveric eye. Donor eyes should not be used if they have short ONs, the ON is damaged, the globe is compromised and deflated, or the ON sheath is detached. Further, when placing the posterior segment over the dome, the O-ring must be tightly fit in place and the top ICP chamber correctly sealed with screws. The pushpin fittings where the tubing attaches on each side of the top and bottom base of the model also need to be tested to ensure that the tubing fits and locks in place. If the tubing is not properly in place, air bubbles will be observed within the tubing and compromise the pressure measurements within each chamber.
Maintenance and viability of the postmortem posterior segments in our TAS model was a critical concern for this protocol. Human postmortem tissue has previously been extensively studied48,49, with a recent RNA analysis study of 1,068 postmortem donor tissues confirming that postmortem human brain tissue collected over decades can serve as high-quality material for study of human disorders47. In addition, previously successful&#160;expression profiling of ocular human donor eye tissues postmortem has been performed58. Gene expression PLIER values for apoptosis genes were minimal or nonexistent in this dataset for retinal tissue 6 h postmortem58. Furthermore, it has been shown that hypothermic storage of eye tissue can be performed effectively59. It has been shown that ganglion cell activity is maintained for 50 h when minipig eyes are stored at ischemic and hypothermic conditions41,60. Therefore, we used the 6 h time point as our inclusion criteria for donor eyecup collection. The speed of postmortem deterioration of posterior segments and retinal detachment is lacking in the literature, but our enucleation within 6 h, delivery over ice, and culture setup of maximal 36 h is well within the range of tissue viability as depicted in Supplemental Figure 1 and Supplemental Figure 2. Using the TAS model, we successfully achieved healthy maintenance of tissue for 30 days.
Another limitation of the TAS model is our current inability to model the cyclic circadian rhythms of ICP and IOP that are observed under normal physiological conditions. This can be addressed in the future by using a pump that can regulate rhythmic IOP and ICP infusion. Further, another caveat to the model is the lack of blood circulation within the cadaveric eye. Thus, the effects of blood pressure cannot be studied, but this also allows us to specifically delineate the pathogenic effects of only TLPG changes, including IOP and ICP.
A future scope of the model would incorporate automation of the reservoir systems for hydrostatic changes and perfusion of medium through an infusion pump with an exit empty syringe on the transducer instead of the multiple rounds of medium change that were implemented in this protocol. The fluid from the IOP and ICP reservoir could also be collected and analyzed. Medium can be collected for biomarker expression to target future therapies. We can also identify pathways or molecules that can be treated with drugs or gene therapy and test these therapies in various animal models of ICP before translation to human clinical trials.
In conclusion, our model not only provides a human basis of testing, but it can also be utilized to validate therapies that can target translaminar pressure changes in the eye. It opens an avenue to perform precision medicine through transplantation of patient stem cells on human donor eyecups and pressurize them in the TAS model. This allows us to test therapies ex vivo with the capacity to be translatable to the clinic and relatable to living individuals. With our model we can now assess the changes occurring in translaminar pressure and how it plays a crucial role in the pathogenesis associated with various traumatic and neurodegenerative diseases. This will lead to a better understanding of pathogenic molecular mechanisms in the ONH that are associated with IOP and ICP.

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><tbody><tr><td>#122, 1-1/8&quot; Inside x 1-5/16&quot; Outside Diam, Viton O-Ring, 3/32&quot; Thick,&lt;br/&gt; 755 Durometer 50 Pack</td><td>Amazon</td><td>B07DRGPPZJ</td><td></td></tr><tr><td>114 Buna-N O-Ring, 70A Durometer, Black, 5/8&quot; ID, 13/16&quot; OD, 3/32&quot; Width (Pack of 100)</td><td>Amazon</td><td>B000FMYRHK</td><td></td></tr><tr><td>30 mL Syringes without Needle</td><td>Vitality Medical</td><td>302832</td><td></td></tr><tr><td>3-Way Stopcock, 2 Female Luer Locks, Swivel Male Luer Lock, Vented Cap</td><td>QOSINA</td><td>2C6201</td><td></td></tr><tr><td>4-40 X 1/2 PH PAN MS SS/CHROME &amp;amp; appropriate sized phillips screwdriver</td><td>Brikksen Stainless Steel Fastners</td><td>PPMSSSCH4C.5</td><td>&lt;strong&gt;&amp;nbsp;&lt;/strong&gt;</td></tr><tr><td>ANPROLENE 16 LARGE AMPULE</td><td>Fisher Scientific</td><td>NC9085343</td><td>&lt;strong&gt;&amp;nbsp;&lt;/strong&gt;</td></tr><tr><td>Betadine</td><td>Purdue</td><td>PUR1815001EACH</td><td>&lt;strong&gt;&amp;nbsp;&lt;/strong&gt;</td></tr><tr><td>Corning 100 x 20mm tissue-culture treated culture dishes</td><td>Sigma-Aldrich</td><td>CLS430167-100EA</td><td>&lt;strong&gt;&amp;nbsp;&lt;/strong&gt;</td></tr><tr><td>Corning&amp;nbsp;L-glutamine Solution</td><td>Fisher Scientific</td><td>MT25005CI</td><td></td></tr><tr><td>Covidien 3033 Curity Gauze Sponge, 4&quot; x 4&quot;, 12-Ply, Sterile, 1200/CS</td><td>Med Plus Medical Supply</td><td>COV-3033-CS</td><td></td></tr><tr><td>Dressing Forceps Delicate Curved (serrated)</td><td>Katena</td><td>K5-4010</td><td></td></tr><tr><td>Dumont #5 - Fine Forceps</td><td>F.S.T.</td><td>11254-20</td><td></td></tr><tr><td>Eye Scissors Standard Curved</td><td>Katena</td><td>K4-7410</td><td></td></tr><tr><td>Falcon 150 x 15mm Plain Sterile Disposable Petri Dishes</td><td>Capitol Scientific</td><td>351058</td><td></td></tr><tr><td>Fisherbrand 4 oz. Specimen Containers</td><td>Fisher Scientific</td><td>16-320-730</td><td></td></tr><tr><td>Fisherbrand Instant Sealing Sterilization Pouches</td><td>Fisher Scientific</td><td>01-812-54</td><td></td></tr><tr><td>Fisherbrand Instant Sealing Sterilization Pouches</td><td>Fisher Scientific</td><td>01-812-55</td><td></td></tr><tr><td>Fisherbrand Instant Sealing Sterilization Pouches</td><td>Fisher Scientific</td><td>01-812-58</td><td></td></tr><tr><td>HyClone Dulbecco&#039;s Modified Eagles Medium</td><td>Fisher Scientific</td><td>SH3024302</td><td></td></tr><tr><td>HyClone Penicillin Streptomycin 100X Solution</td><td>Fisher Scientific</td><td>SV30010</td><td></td></tr><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 &amp;reg; II, Catalog # DPT-200 with a 3CC/HR flow rate</td><td>AD instruments</td><td>DPT-200</td><td></td></tr><tr><td>JG15-0.5HPX 15 Gauge 0.5&quot; NT Premium Series Dispensing Tip 50/Box</td><td>Jenson Global</td><td>JG15-0.5HPX 15</td><td></td></tr><tr><td>Keyence B2?X710 microscope</td><td>Keyence</td><td>B2-X710</td><td></td></tr><tr><td>LabChart 8</td><td>AD instruments</td><td>LabChart 8</td><td></td></tr><tr><td>Leica ST5020 Multi-stainer</td><td>Leica</td><td>ST5020</td><td></td></tr><tr><td>Non-Vented Universal Luer Lock Cap, White</td><td>QOSINA</td><td>65811</td><td></td></tr><tr><td>Octal Bridge Amp (Model # FE228)</td><td>AD instruments</td><td>FE228</td><td></td></tr><tr><td>Pharmco Products ETHYL ALCOHOL, 200 PROOF</td><td>Fisher Scientific</td><td>NC1675398</td><td></td></tr><tr><td>Phosphate Buffered Solution (PBS)</td><td>Sigma-Aldrich</td><td>D8537-500ML</td><td></td></tr><tr><td>PowerLab 8/35 (Model # PL3508)</td><td>AD instruments</td><td>PL3508</td><td></td></tr><tr><td>ProLong Gold Antifade Mountant with DAPI</td><td>ThermoFisher</td><td>P36935</td><td></td></tr><tr><td>Push-to-Connect Tube Fitting for Air and Water&amp;nbsp;Straight Adapter, 1/8&quot; Tube OD x 1/8 NPT Male</td><td>McMAster-Carr</td><td>7880T113</td><td></td></tr><tr><td>Push-to-Connect Tube Fitting with Universal Thread&amp;nbsp;for Air and Water, Adapter, 1/8&quot; Tube OD x 1/8 Pipe</td><td>McMAster-Carr</td><td>51235K101</td><td></td></tr><tr><td>Saint-Gobain Tygon S3 E-3603 Flexible Tubing 500 ft.</td><td>Fisher Scientific</td><td>14-171-268</td><td></td></tr><tr><td>Superblock T20</td><td>Fisher Scientific</td><td>PI37536</td><td></td></tr><tr><td>Surgical Scissors - Sharp-Blunt</td><td>F.S.T.</td><td>14001-14</td><td></td></tr><tr><td>Tissue Forceps Delicate 1x2 Teeth Curved</td><td>Katena</td><td>K5-4110</td><td></td></tr><tr><td>Translaminar Autonomous System (TAS)</td><td>University of North Texas Health Science Center</td><td>N/A</td><td></td></tr><tr><td>USA Size 030 O-ring Buna-N, B1000, 70 Durometer, Black, Buna-N&lt;br/&gt; (NBR, Nitrile, Buna)</td><td>Marco Rubber &amp;amp; Plastics</td><td>B1000-030</td><td></td></tr></tbody></table>

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 translaminar pressure in the human eye, we designed, created, and validated the TAS model. Our novel ex vivo human model gives a unique preclinical system to independently study ICP and IOP-associated pathogenic changes. To address human preclinical applications, our model provides an ex vivo paradigm of studying pathogenesis due to translaminar pressure changes. The sealed model design is depicted with solid front and transparent views (Figure 1A, 1B) with a detailed diagrammatic view of the model to depict all the inflow and outflow ports (Figure 1C). The color transparent view with a human posterior segment in an actual 3D printed model is shown (Figure 1D, 1E).
Translaminar Autonomous System: A novel ex vivo human translaminar pressure model 
We generated the TAS model with two autonomous chambers (i.e., IOP and ICP chambers). In the bottom base of the model, the human posterior cup was placed over the top of the round dome with the optic nerve facing the top. Once the posterior cup was placed and sealed in the IOP chamber, we placed the ICP chamber on top of the nerve. We maintained the independence of both chambers and a perfect seal using O-rings that fit each chamber precisely (Figure 2A). The bottom chamber or the IOP chamber filled and regulated pressure in the cup, while the top chamber fit around the optic nerve and regulated the ICP around the nerve through hydrostatic pressure reservoirs. Using the model, we independently regulated IOP and ICP using hydrostatic pressure. The difference between both chambers was identified as a change in translaminar pressure gradient (Figure 2B). The model depicted with all final fittings in place including the inflow and outflow reservoir syringes connected is shown in Figure 2C.
Successful culture and pressure maintenance in the translaminar autonomous system 
To ensure that both chambers worked independently in the system, we regulated several pressure differentials by keeping the IOP chamber and ICP at different average pressure differentials (normal TLPG: IOP: ICP, 15:5 mmHg; elevated TLPG &#62;10 mmHg; elevated TLPG &#62;20 mmHg). We initially tested the maintenance of average normal pressure differentials in both chambers (normal IOP/ICP) through various different parameters of IOP and ICP conditions: 1) normal IOP: decreased ICP (Figure 3A); 2) elevated IOP: decreased ICP (Figure 3B); and 3) elevated IOP: elevated ICP (Figure 3C). The average normal IOP ranges from 10&#8211;21 mmHg (episcleral venous pressure factored in) and normal ICP from 5&#8211;15 mmHg. In lieu of the limitation of not having vascular pressure, we still maintained the pressure to these rates, as the idea was to exert the maximal pressure at the ONH. We independently regulated various pressure levels in both chambers (ICP, 5&#8211;10 mmHg; IOP, 20&#8211;40 mmHg). To ensure pressure maintenance between both chambers, we kept IOP under normal conditions (15 mmHg) and decreased ICP (4 mmHg) to sustain a TLPG (IOP-ICP) between the LC of 11 mmHg (Figure 3A). We then elevated IOP (43 mmHg) and decreased ICP (3 mmHg) (Figure 3B) and finally elevated pressures in both (IOP, 64 mmHg; ICP, 9 mmHg) to generate the largest level of TLPG at 55 mmHg (Figure 3C). To ensure the viability of the tissue (Figure 4), the medium in the tissues was exchanged every 48 h by attaching an empty syringe to the outflow stopcock and slowly pushing approximately 5 mL of perfusion medium through the inflow port using the push/pull method. Minimal pressure increases occurred at the time of medium exchange (Figure 4G) and did not affect the morphology of the ONH as shown in the 14- and 30 day immunohistochemistry data (Figure 4A&#8211;F). To confirm that we could culture posterior segments for extended timeframes with effective viability within the TAS model, we analyzed cross sections of the ONH after maintenance of normal IOP and ICP for 14 and 30 days. We were able to successfully culture these segments in the model for 14 days (Figure 4A, 4B) with healthy ONH cells and extracellular matrix expression of collagen IV (COLIV) at the optic nerve head (Figure 4C). Similar viability and maintenance of the posterior segment was also observed for 30 days (Figure 4D, 4E) with expression of COLIV and DAPI (Figure 4F). The graphical representation of TLPG (IOP-ICP) values (Figure 4G) depicts a constant maintenance of IOP values over time at 15.6 &#177; 4.6 mmHg and ICP mean at 11.0 &#177; 4.6 mmHg for 30 days with a TLPG of 4.6 &#177; 1.3 mmHg (Table 1).
Morphological changes to the ONH post elevated translaminar pressure gradient 
A common clinical feature of the age-related neurodegenerative disease glaucoma is ONH cupping. Prelaminar cupping is distinguished by progressive loss of prelaminar neural tissues, which increases both the depth and width of the cup and thus increases the cup-to-disk ratio. Laminar cupping is connective tissue-based, with the LC moving posteriorly progressively and excavating. Glaucomatous cupping is a combination of these two components, reflecting both damage and remodeling of laminar connective tissues. Elevation in IOP leads to LC thickening due to an increase in collagen fibril mass53. Utilizing the TAS model, we created an elevated TLPG by increasing IOP or decreasing ICP over various timepoints. We maintained a range of elevated TLPG for 7 days with average IOP values over time at 22.8 &#177; 18.6 mmHg and ICP mean at 6.9 &#177; 7.6 mmHg with a TLPG of 15.9 &#177; 11.8 mmHg (Table 2). The highest TLPGs were documented at 36 mmHg. Human posterior segments were then analyzed morphologically for progressive thickening of laminar beams and cupping at the ONH in H&#38;E stained sections as time progressed between control, 1 day, 3 days, and 7 days under elevated TLPG (Figure 5A&#8211;D). Cupping and thickening were observed at 7 days of elevated TLPG (Figure 5D). Further, COLIV expression over time between control, 1 day, 3 days, and 7 days showed thickened beams and increased expression by 7 days (Figure 5E&#8211;H). Phase images comparing control tissue not cultured in the TAS model (Figure 5I) and 7 days (Figure 5J) of elevated TLPG within the TAS model show healthy RGCs within the GCL (Figure 5I) with no cupping (Figure 5I) for the control, while under conditions of elevated TLPG the images show extensive cupping with no remaining RGCs (RBPMS-RGC marker) in the RNFL (Figure 5J) and increased remodeling of ECM as shown by elevated COLIV within the ONH (Figure 5J).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61006/61006fig01.jpg" /> 
Figure 1: Translaminar autonomous system.&#160;Model depiction. (A) Solid front view. (B) Transparent view. (C) Diagrammatic view. (D) Color transparent view. (E) Actual 3D printed model. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61006/61006fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61006/61006fig02.jpg" /> 
Figure 2: Mechanics of the translaminar autonomous system.&#160;(A) The TAS model with ICP and IOP chambers for regulating translaminar pressure differentials. (B) Depiction of the TAS model with autonomous regulation of hydrostatic pressure in both chambers through elevation of reservoirs. (C) Image of the TAS model with all the fittings in place and representation of the inflow and outflow reservoir syringes. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61006/61006fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61006/61006fig03.jpg" /> 
Figure 3: Independent pressure maintenance within the translaminar autonomous system.&#160;Graphical representation of pressures being independently modulated, and stable pressures being maintained in the top (ICP) and bottom (IOP) chambers with (A) normal IOP/decreased ICP (B) elevated IOP/decreased ICP, and (C) elevated IOP/elevated ICP. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61006/61006fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61006/61006fig04.jpg" /> 
Figure 4: Maintenance and viability of posterior segments within the translaminar autonomous system.&#160;Human posterior segments were cultured using the TAS model for 14 and 30 days under normal conditions of IOP and ICP. H&#38;E stained cross sections of human ONH at 14 days in (A) low magnification (40x) and (B) high magnification (100x). (C) COLIV immunostaining with DAPI expression (100x). Similar depictions of H&#38;E staining at 30 days in (D) 40x and (E) 100x micrographs and (F) COLIV immunostaining with DAPI expression (100x). G) Graphical presentation of &#916; in mmHg of IOP-ICP (TLPG) for human posterior segments maintained for 30 days in culture. COLIV = green; DAPI = blue; (A, inset B); (D, inset E); H&#38;E = hematoxylin and eosin stain. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61006/61006fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61006/61006fig05.jpg" /> 
Figure 5: Morphological restructuring of the optic nerve head after elevated translaminar pressure gradient in the Translaminar Autonomous System.&#160;Human posterior segments were cultured using the TAS model for various time points under elevated TLPG conditions. Cross sections of human ONH depicting H&#38;E staining of (A) control (B) 1 day in TAS (C) 3 days in TAS, and (D) 7 days of culture. Expression of COLIV with DAPI in the ONH of (E) control (F) 1 day in TAS (G) 3 days in TAS, and (H) 7 days in culture. Phase contrast ONH cross section images of (I) control ONH depicting (I&#8217;) retina staining of RBPMS and (I&#8217;&#8217;) ONH staining with COLIV and DAPI. Phase contrast of (J) 7 days of elevated TLPG in TAS with insets depicting (J&#8217;) retina staining of RBPMS and (J&#8217;&#8217;) ONH staining with COLIV and DAPI. COLIV, RBPMS = green; DAPI = blue; (A&#8211;D) 40x magnification; (E&#8211;H) 100x Magnification; (I and J) 200x magnification; (J&#8217;) 400x magnification; (J&#8217;&#8217;) 100x magnification; (J, inset J&#8217; and J&#8217;&#8217;); H&#38;E = hematoxylin and eosin stain; TAS = Translaminar Autonomous System. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61006/61006fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Days</td>
			<td>Mean IOP of 24 h</td>
			<td>Mean ICP of 24 h</td>
			<td>Mean TLPG (IOP-ICP)</td>
		</tr>
		<tr>
			<td>1</td>
			<td>17.7</td>
			<td>12.1</td>
			<td>5.7</td>
		</tr>
		<tr>
			<td>2</td>
			<td>20.0</td>
			<td>15.0</td>
			<td>5.0</td>
		</tr>
		<tr>
			<td>3</td>
			<td>13.4</td>
			<td>9.6</td>
			<td>3.7</td>
		</tr>
		<tr>
			<td>4</td>
			<td>15.1</td>
			<td>10.5</td>
			<td>4.5</td>
		</tr>
		<tr>
			<td>5</td>
			<td>11.6</td>
			<td>8.3</td>
			<td>3.3</td>
		</tr>
		<tr>
			<td>6</td>
			<td>14.0</td>
			<td>9.5</td>
			<td>4.5</td>
		</tr>
		<tr>
			<td>7</td>
			<td>17.2</td>
			<td>13.8</td>
			<td>3.4</td>
		</tr>
		<tr>
			<td>8</td>
			<td>19.3</td>
			<td>16.1</td>
			<td>3.2</td>
		</tr>
		<tr>
			<td>9</td>
			<td>17.7</td>
			<td>15.0</td>
			<td>2.8</td>
		</tr>
		<tr>
			<td>10</td>
			<td>10.9</td>
			<td>8.0</td>
			<td>2.9</td>
		</tr>
		<tr>
			<td>11</td>
			<td>16.3</td>
			<td>10.2</td>
			<td>6.1</td>
		</tr>
		<tr>
			<td>12</td>
			<td>14.7</td>
			<td>11.8</td>
			<td>2.9</td>
		</tr>
		<tr>
			<td>13</td>
			<td>7.5</td>
			<td>4.5</td>
			<td>3.0</td>
		</tr>
		<tr>
			<td>14</td>
			<td>5.5</td>
			<td>1.4</td>
			<td>4.1</td>
		</tr>
		<tr>
			<td>15</td>
			<td>13.5</td>
			<td>8.3</td>
			<td>5.2</td>
		</tr>
		<tr>
			<td>16</td>
			<td>15.4</td>
			<td>10.3</td>
			<td>5.1</td>
		</tr>
		<tr>
			<td>17</td>
			<td>11.7</td>
			<td>4.5</td>
			<td>7.3</td>
		</tr>
		<tr>
			<td>18</td>
			<td>13.3</td>
			<td>9.3</td>
			<td>4.0</td>
		</tr>
		<tr>
			<td>19</td>
			<td>23.5</td>
			<td>19.7</td>
			<td>3.8</td>
		</tr>
		<tr>
			<td>20</td>
			<td>20.3</td>
			<td>14.5</td>
			<td>5.7</td>
		</tr>
		<tr>
			<td>21</td>
			<td>12.8</td>
			<td>5.8</td>
			<td>7.0</td>
		</tr>
		<tr>
			<td>22</td>
			<td>25.8</td>
			<td>19.9</td>
			<td>5.9</td>
		</tr>
		<tr>
			<td>23</td>
			<td>19.3</td>
			<td>13.5</td>
			<td>5.8</td>
		</tr>
		<tr>
			<td>24</td>
			<td>18.8</td>
			<td>15.1</td>
			<td>3.7</td>
		</tr>
		<tr>
			<td>25</td>
			<td>14.4</td>
			<td>8.9</td>
			<td>5.5</td>
		</tr>
		<tr>
			<td>AVG</td>
			<td>15.6</td>
			<td>11.0</td>
			<td>4.6</td>
		</tr>
		<tr>
			<td>STD</td>
			<td>4.6</td>
			<td>4.6</td>
			<td>1.3</td>
		</tr>
	</tbody>
</table>
Table 1: Maintenance of normal TLPG maintained for 30 days. Tabular values depicting IOP, ICP, and TLPG values every 24 h with average and standard deviation over the complete time course.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Days</td>
			<td>Mean IOP of 24 h</td>
			<td>Mean ICP of 24 h</td>
			<td>Mean TLPG of 24 h</td>
		</tr>
		<tr>
			<td>1</td>
			<td>4.1</td>
			<td>-1.0</td>
			<td>5.1</td>
		</tr>
		<tr>
			<td>2</td>
			<td>6.3</td>
			<td>1.1</td>
			<td>5.3</td>
		</tr>
		<tr>
			<td>3</td>
			<td>13.4</td>
			<td>4.0</td>
			<td>9.4</td>
		</tr>
		<tr>
			<td>4</td>
			<td>19.0</td>
			<td>1.1</td>
			<td>17.9</td>
		</tr>
		<tr>
			<td>5</td>
			<td>55.6</td>
			<td>19.5</td>
			<td>36.2</td>
		</tr>
		<tr>
			<td>6</td>
			<td>39.5</td>
			<td>12.8</td>
			<td>26.7</td>
		</tr>
		<tr>
			<td>7</td>
			<td>21.5</td>
			<td>10.8</td>
			<td>10.6</td>
		</tr>
		<tr>
			<td>AVG</td>
			<td>22.8</td>
			<td>6.9</td>
			<td>15.9</td>
		</tr>
		<tr>
			<td>STD</td>
			<td>18.6</td>
			<td>7.6</td>
			<td>11.8</td>
		</tr>
	</tbody>
</table>
Table 2: Maintenance of a range of elevated TLPG maintained for 7 days. Tabular values depicting IOP, ICP, and TLPG values every 24 h with average and standard deviation over the complete time course.
Supplemental Figure 1: Ex vivo human retinal explant culture. Phase contrast, RGC positive stained (RBPMS-green), and cellular (DAPI-blue) stained images of retinal explants in culture for (A&#8211;C) 7 days and (D&#8211;F) 14 days (200x magnification). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61006/61006supfig01.jpg" target="_blank">Please click here to download this figure.</a>
Supplemental Figure 2: Human adult RGC cultures. RGC marker (RBPMS-green) and DAPI (blue) stained RGCs 7 days in culture (A) 200x (B) 400x magnification. (C) RGCs stained for NEFL (green) and DAPI (blue) at 400x magnification. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61006/61006supfig02.jpg" target="_blank">Please click here to download this figure.</a>

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 ...

translaminar-autonomous-system-model-for-modulation-intraocular

Nanyang Technological University

Research

JoVE Journal

Neuroscience

3.0K Views.  University of North Texas Health Science Center. We beschrijven en detailleren het gebruik van het translaminaire autonome systeem. Dit systeem maakt gebruik van het menselijke posterieure segment om onafhankelijk de druk in het segment (intraoculair) en rond de oogzenuw (intracranieel) te reguleren om een translaminaire drukgradiënt te genereren die kenmerken van glaucomateuze optische neuropathie nabootst.

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

Trabecular Meshwork Response to Pressure Elevation in the Living Human Eye

The mechanical characteristics of the trabecular meshwork (TM) are linked to outflow resistance and intraocular pressure (IOP) regulation. The rationale behind this technique is the direct observation of the mechanical response of the TM to acute IOP elevation. Prior to scanning, IOP is measured at baseline and during IOP elevation. The limbus is scanned by spectral-domain optical coherence tomography at baseline and during IOP elevation (ophthalmodynamometer (ODM) applied at 30 g force). Scans are processed to enhance visualization of the aqueous humor outflow pathway using ImageJ. Vascular landmarks are used to identify corresponding locations in baseline and IOP elevation scan volumes. Schlemm canal (SC) cross-sectional area (SC-CSA) and SC length from anterior to posterior along its long axis are measured manually at 10 locations within a 1 mm segment of SC. Mean inner to outer wall distance (short axis length) is calculated as the area of SC divided by its long axis length. To examine the contribution of adjacent tissues to the effect IOP elevations, measurements are repeated without and with smooth muscle relaxation with instillation of tropicamide. TM migration into SC is resisted by TM stiffness, but is enhanced by the support of its attachment to adjacent smooth muscle within the ciliary body. This technique is the first to measure the living human TM response to pressure elevation in situ under physiological conditions within the human eye.

Trabecular meshwork (TM) migration into Schlemm&#8217;s canal space can be induced by acute pressure elevation by ophthalmodynamometer, and observed by spectral domain optical coherence tomography. The goal of this method is to quantify the morphometric response of the living outflow tract to acute pressure elevation in living tissues in situ.

The mechanical characteristics of the trabecular meshwork (TM) are linked to outflow resistance and intraocular pressure (IOP) regulation. The rationale ...

Medicine

A Mouse Model of Retinal Ischemia-Reperfusion Injury Through Elevation of Intraocular Pressure

Retinal ischemia-reperfusion (I/R) is a pathophysiological process contributing to cellular damage in multiple ocular conditions, including glaucoma, diabetic retinopathy, and retinal vascular occlusions. Rodent models of I/R injury are providing significant insights into mechanisms and treatment strategies for human I/R injury, especially with regard to neurodegenerative damage in the retinal neurovascular unit. Presented here is a protocol for inducing retinal I/R injury in mice through elevation of intraocular pressure (IOP). In this protocol, the ocular anterior chamber is cannulated with a needle, through which flows the drip of an elevated saline reservoir. Using this drip to raise IOP above systolic arterial blood pressure, a practitioner temporarily halts inner retinal blood flow (ischemia). When circulation is reinstated (reperfusion) by removal of the cannula, severe cellular damage ensues, resulting ultimately in retinal neurodegeneration. Recent studies demonstrate inflammation, vascular permeability, and capillary degeneration as additional elements of this model. Compared to alternative retinal I/R methodologies, such as retinal arterial ligation, retinal I/R injury by elevated IOP offers advantages in its&#160;anatomical specificity, experimental tractability, and technical accessibility, presenting itself as a valuable tool for examining neuronal pathogenesis and therapy in the retinal neurovascular unit.

This article describes a procedure for inducing retinal ischemia-reperfusion injury by elevated intraocular pressure in mice. Retinal ischemia-reperfusion injury by elevated intraocular pressure serves to model human pathologies characterized by compromised oxygen and nutrient delivery in the retina, enabling researchers to examine potential cellular mechanisms and treatments for human diseases of the retinal neurovascular unit.

Retinal ischemia-reperfusion (I/R) is a pathophysiological process contributing to cellular damage in multiple ocular conditions, including glaucoma, ...

Anterior Segment Organ Culture Platform for Tracking Open Globe Injuries and Therapeutic Performance

Open globe injuries have poor visual outcomes, often resulting in permanent loss of vision. This is partly due to an extended delay between injury and medical intervention in rural environments and military medicine applications where ophthalmic care is not readily available. Untreated injuries are susceptible to infection after the eye has lost its watertight seal, as well as loss of tissue viability due to intraocular hypotension. Therapeutics to temporarily seal open globe injuries, if properly developed, may be able to restore intraocular pressure and prevent infection until proper ophthalmic care is possible. To facilitate product development, detailed here is the use of an anterior segment organ culture open globe injury platform for tracking therapeutic performance for at least 72 h post-injury. Porcine anterior segment tissue can be maintained in custom-designed organ culture dishes and held at physiological intraocular pressure. Puncture injuries can be created with a pneumatic-powered system capable of generating injury sizes up to 4.5 mm in diameter, similar to military-relevant injury sizes. Loss of intraocular pressure can be observed for 72 h post-injury confirming proper injury induction and loss of the eye&#39;s watertight seal. Therapeutic performance can be tracked by application to the eye after injury induction and then tracking intraocular pressure for multiple days. Further, the anterior segment injury model is applicable to widely used methods for functionally and biologically tracking anterior segment physiology, such as assessing transparency, ocular mechanics, corneal epithelium health, and tissue viability. Overall, the method described here is a necessary next step toward developing biomaterial therapeutics for temporarily sealing open globe injuries when ophthalmic care is not readily available.

Open globe eye injuries may go untreated for multiple days in rural or military-relevant scenarios, resulting in blindness. Therapeutics are needed to minimize loss of vision. Here, we detail an organ culture open globe injury model. With this model, potential therapeutics for stabilizing these injuries can be properly evaluated.

Open globe injuries have poor visual outcomes, often resulting in permanent loss of vision. This is partly due to an extended delay between injury and ...

Bioengineering

A Model of Glaucoma Induced by Circumlimbal Suture in Rats and Mice

The circumlimbal suture is a technique for inducing experimental glaucoma in rodents by chronically elevating intraocular pressure (IOP), a well-known risk factor for glaucoma. This protocol demonstrates a step-by-step guide on this technique in Long Evans rats and C57BL/6 mice. Under general anesthesia, a "purse-string" suture is applied on the conjunctiva, around the equator and behind the limbus of the eye. The fellow eye serves as an untreated control. Over the duration of our study, which was a period of 8 weeks for rats and 12 weeks for mice, IOP remained elevated, as measured regularly by rebound tonometry in conscious animals without topical anesthesia. In both species, the sutured eyes showed electroretinogram features consistent with preferential inner retinal dysfunction. Optical coherence tomography showed selective thinning of the retinal nerve fiber layer. Histology of the rat retina in cross-section found reduced cell density in the ganglion cell layer, but no change in other cellular layers. Staining of flat-mounted mouse retinae with a ganglion cell specific marker (RBPMS) confirmed ganglion cell loss. The circumlimbal suture is a simple, minimally invasive and cost-effective way to induce ocular hypertension that leads to ganglion cell injury in both rats and mice.

Chronic ocular hypertension is induced by applying a circumlimbal suture in rats and mice, leading to functional and structural deterioration of the retinal ganglion cells consistent with glaucoma.

The circumlimbal suture is a technique for inducing experimental glaucoma in rodents by chronically elevating intraocular pressure (IOP), a well-known ...

A Reversible Silicon Oil-Induced Ocular Hypertension Model in Mice

Elevated intraocular pressure (IOP) is a well-documented risk factor for glaucoma. Here we describe a novel, effective method for consistently inducing stable IOP elevation in mice that mimics the post-operative complication of using silicone oil (SO) as a tamponade agent in human vitreoretinal surgery. In this protocol, SO is injected into the anterior chamber of the mouse eye to block the pupil and prevent inflow of aqueous humor. The posterior chamber accumulates aqueous humor and this in turn increases the IOP of the posterior segment. A single SO injection produces reliable, sufficient, and stable IOP elevation, which induces significant glaucomatous neurodegeneration. This model is a true replicate of secondary glaucoma in the eye clinic. To further mimic the clinical setting, SO can be removed from the anterior chamber to reopen the drainage pathway and allow inflow of aqueous humor, which is drained through the trabecular meshwork (TM) at the angle of the anterior chamber. Because IOP quickly returns to normal, the model can be used to test the effect of lowering IOP on glaucomatous retinal ganglion cells. This method is straightforward, does not require special equipment or repeat procedures, closely simulates clinical situations, and may be applicable to diverse animal species. However, minor modifications may be required.

Here, we present a protocol to induce ocular hypertension and glaucomatous neurodegeneration in mouse eyes by intracameral injection of silicone oil and the procedure for silicone oil removal from the anterior chamber to return elevated intraocular pressure to normal.

Elevated intraocular pressure (IOP) is a well-documented risk factor for glaucoma. Here we describe a novel, effective method for consistently inducing ...

Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic nerve and retinal ganglion cell (RGC) degeneration, resulting in visual dysfunction by interrupting the transmission of visual information from the eye to the brain. Elevated intraocular pressure is the most important risk factor; thus, several models of ocular hypertension have been developed in rodents by either genetic or experimental approaches to investigate the causes and effects of the disease. Among those, some limitations have been reported such as surgical invasiveness, inadequate functional assessment, requirement of extensive training, and highly variable extension of retinal damage. The present work characterizes a simple, low-cost, and efficient method to induce ocular hypertension in rodents, based on low-temperature, full-circle cauterization of the limbal vascular plexus, a major component of aqueous humor drainage. The new model provides a technically easy, noninvasive, and reproducible subacute ocular hypertension, associated with progressive RGC and optic nerve degeneration, and a unique post-operative clinical recovery rate that allows in vivo functional studies by both electrophysiological and behavioral methods.

The goal of this protocol is to characterize a novel model of glaucomatous neurodegeneration based on 360&#176; thermic cauterization of limbal vascular plexus, inducing subacute ocular hypertension.

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic ...

Retinal Pigment Epithelium Transplantation in a Non-human Primate Model for Degenerative Retinal Diseases

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These conditions include retinitis pigmentosa (RP) and advanced forms of age-related macular degeneration (AMD), such as geographic atrophy (GA). Together, these disorders represent a significant proportion of currently untreatable blindness globally. These unmet medical needs have generated heightened academic interest in developing methods of RPE replacement. Among the animal models commonly utilized for preclinical testing of therapeutics, the non-human primate (NHP) is the only animal model that has a macula. As it shares this anatomical similarity with the human eye, the NHP eye is an important and appropriate preclinical animal model for the development of advanced therapy medicinal products (ATMPs) such as RPE cell therapy.
This manuscript describes a method for the submacular transplantation of an RPE monolayer, cultured on a polyethylene terephthalate (PET) cell carrier, underneath the macula onto a surgically created RPE wound in immunosuppressed NHPs. The fovea-the central avascular portion of the macula-is the site of the greatest mechanical weakness during the transplantation. Foveal trauma will occur if the initial subretinal fluid injection generates an excessive force on the retina. Hence, slow injection under perfluorocarbon liquid (PFCL) vitreous tamponade is recommended with a dual-bore subretinal injection cannula at low intraocular pressure (IOP) settings to create a retinal bleb. 
Pretreatment with an intravitreal plasminogen injection to release parafoveal RPE-photoreceptor adhesions is also advised. These combined strategies can reduce the likelihood of foveal tears when compared to conventional techniques. The NHP is a key animal model in the preclinical phase of RPE cell therapy development. This protocol addresses the technical challenges associated with the delivery of RPE cellular therapy in the NHP eye.

The non-human primate (NHP) is an ideal model for studying human retinal cellular therapeutics due to the anatomical and genetic similarities. This manuscript describes a method for submacular transplantation of retinal pigment epithelial cells in the NHP eye and strategies to prevent intraoperative complications associated with macular manipulation.

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These ...

Transpupillary-Guided Trans-Scleral Transplantation of Subretinal Grafts in a Retinal Degeneration Mouse Model

Transplantation of photoreceptor cells and retinal pigment&#160;epithelial (RPE) cells provide a potential therapy for retinal degeneration diseases. Subretinal transplantation of therapeutic donor cells into mouse recipients is challenging due to the limited surgical space allowed by the small volume of the mouse eye. We developed a trans-scleral surgical transplantation platform with direct transpupillary vision guidance to facilitate the subretinal delivery of exogenous cells in mouse recipients. The platform was tested using retinal cell suspensions and three-dimensional retinal sheets collected from rod-rich Rho::EGFP mice and cone-rich OPN1LW-EGFP;NRL-/- mice, respectively. Live/dead assay showed low cell mortality for both forms of donor cells. Retinal grafts were successfully delivered into the subretinal space of a mouse model of retinal degeneration, Rd1/NS, with minimum surgical complications as detected by multimodal confocal scanning laser ophthalmoscope (cSLO) imaging. Two months post-transplantation, histological staining demonstrated evidence of advanced maturation of the retinal grafts into &#39;adult&#39; rods and cones (by robust Rho::EGFP, S-opsin, and OPN1LW:EGFP expression, respectively) in the subretinal space. Here, we provide a surgical platform that can enable highly accurate subretinal delivery with a low rate of complications in mouse recipients. This technique offers precision and relative ease of skill acquisition. Furthermore, the technique could be used not only for studies of subretinal cell transplantation but also for other intraocular therapeutic studies including gene therapies.

This protocol presents a transpupillary vision-guided trans-scleral approach to safely and precisely deliver subretinal cellular grafts, with a low rate of surgical complications, in mouse recipients with or without retinal degeneration.

Transplantation of photoreceptor cells and retinal pigment&#160;epithelial (RPE) cells provide a potential therapy for retinal degeneration diseases. ...

Establishing a Subarachnoid Hemorrhage Rat Model Induced by Autologous Blood Injection

Source: B&#246;mers, J. et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/62567">Pre-Chiasmatic, Single Injection of Autologous Blood to Induce Experimental Subarachnoid Hemorrhage in a Rat Model.</a> J. Vis. Exp. (2021)
This video demonstrates the procedure for establishing a subarachnoid hemorrhage rat model. An anesthetized rat is implanted with probes to measure intracranial pressure and cerebral blood flow. The target site is identified, and the needle is inserted at a pre-chiasmatic position. Autologous blood is then injected into the subarachnoid space, increasing intracranial pressure and decreasing cerebral blood flow, establishing the model.

Source: B&#246;mers, J. et al. Pre-Chiasmatic, Single Injection of Autologous Blood to Induce Experimental Subarachnoid Hemorrhage in a Rat Model. J. Vis. ...

JoVE Encyclopedia of Experiments

Neuropathology

Cerebrovascular Diseases

 Induction of Elevated Intraocular Pressure in a Rat Model

<a href='https://app-jove-com.remotexs.ntu.edu.sg/v/63442/full-circle-cauterization-limbal-vascular-plexus-for-surgically'>Source: Lani-Louzada, R., et al., Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents. J. Vis. Exp. (2022)</a>This video demonstrates the induction and monitoring of elevated intraocular pressure (IOP) in a rat model. The process involves cauterizing the limbal blood vessels, resulting in vessel occlusion and elevated IOP. The rat is then allowed to recover, and IOP is regularly monitored in both the experimental and control eyes for comparison.

Source: Lani-Louzada, R., et al., Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents. J. Vis. Exp. (2022)This ...

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

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