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  • Podsumowanie
  • Streszczenie
  • Wprowadzenie
  • Protokół
  • Wyniki
  • Dyskusje
  • Ujawnienia
  • Podziękowania
  • Materiały
  • Odniesienia
  • Przedruki i uprawnienia

Podsumowanie

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.

Streszczenie

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 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.

Wprowadzenie

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 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 10–21 mmHg and ICP from 5–15 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, 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 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.

Protokół

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 6–12 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). .

1. Preparation and sterilization of equipment and supplies

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

2. Preparation of perfusion medium

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

3. Translaminar autonomous system (TAS) setup

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

4. Preparation of human whole eye globe

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

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

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.

  1. Connect the power cord into the back of the 8-channel power source and plug into a battery back-up device.
  2. Connect the USB cable from the 8-channel power source into the back of the computer.
  3. Connect the 8-channel power source to the multichannel bridge amplifier using the supplied I2C cord.
  4. 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.
  5. Connect the transducer cables to the front of the multichannel amplifier.
  6. Install the data acquisition software on the computer.
    1. Run the data acquisition software setup installer from the supplied software CD.
    2. Follow the instructions on the computer screen.
    3. When the installation is complete, select Finish.
  7. Turn on the 8-channel power source.
  8. Turn on the computer and initiate the data acquisition software.
    1. Select File | New.
    2. 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).
    3. Select 2 mV for the Range on all channels. In the Calculation column select No Calculation for channels 1 and 2.
    4. In the Calculation column select Arithmetic for channel 3. In the Formula section: Select channels/CH2; Select arithmetic "-"; Select channels/CH1. In the Output section select mmHg. Select OK. Select OK again.
  9. Set up and calibrate the hydrostatic pressure transducers.
    NOTE: Hydrostatic pressure transducers must be calibrated prior to experiments using the following method.
    1. Connect the hydrostatic pressure transducers to the transducer lines attached to the multichannel bridge amplifier.
    2. 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.
    3. 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.
    4. Select Bridge Amp. Select Mains Filter. Select Zero and wait for the system to zero out, taking care to not move the pressure transducer.
    5. 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.
    6. On the Units Conversion page, select ‘minus (-)’ sign. Highlight the highest plateau to indicate 40 mmHg. Click the Arrow for point 1 and enter 40.
    7. Highlight the lowest plateau to indicate 0 mmHg. Click the Arrow for point 2 and enter 0. Select mmHg for the units. Select OK.
    8. Select OK (Bridge Amp page). Repeat steps 9.1–9.7 for CH2 (IOP) using 100 mmHg for the highest plateau and 0 for the lowest plateau.
  10. Connect the TAS/posterior segment unit onto the data acquisition system.
    1. Place the TAS/posterior segment unit into an incubator (37 °C, 5% CO2). Attach the ICP tubing from the OUT port to the CH1 (ICP) pressure transducer.
    2. Attach the IOP tubing from the OUT port to the CH2 (IOP) pressure transducer.
    3. Attach the syringe setups (ICP and IOP) with medium from the IN ports to the ring stand.
    4. 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.
    5. Adjust the syringes on the ring stand up or down to regulate ICP and IOP pressures to protocol requirements.
    6. Perform systemic replenishment of medium in the system every 48–72 h through the push and pull method.

6. Data retrieval and analysis

  1. Open the data file in the data acquisition software.
  2. In the Data Pad section, click on the Multiple Add to Data Pad icon. A new window will appear.
    1. In the Find Using section select Time from the drop-down menu.
    2. In the Select section select 1 hr every 1 hr from the drop-down menus.
    3. In the Step Through section select Whole File then click Add.
  3. In the Data Pad section click on the Data Pad View icon. Highlight all the data and copy/paste into a spreadsheet.
  4. 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.

7. Immunohistochemistry and hematoxylin and eosin staining of posterior segments

  1. Remove the posterior eye segments following various timepoints from the TAS model and fix in formalin prior to paraffinizing.
  2. Section the eyes to produce sagittal tissue planes.
  3. Deparaffinize the paraffin-embedded segments with a 100% xylene, 95% ethanol, 50% ethanol solution.
  4. Wash the slides with PBS for 10 min and block with a blocking buffer at room temperature for 1 h.
  5. 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).
  6. Detect the primary antibodies using Alexa Fluor secondary antibodies (Alexa Fluor 488 goat anti-rabbit, A11008, 1:500).
  7. Counterstain the cell nuclei using DAPI anti-fade solution.
  8. Capture images of the stained sections and phase images with 4x and 10x objective lenses using a fluorescence microscope (see Table of Materials).
  9. For the hematoxylin and eosin (H&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&E.
  10. Capture images with the 4x and 10x objective lenses using a microscope with a bright field light source.

Wyniki

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...

Dyskusje

Human postmortem tissues are an especially valuable resource for studying human neurodegenerative diseases because identification of potential drugs developed in 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...

Ujawnienia

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

Podziękowania

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.

Materiały

NameCompanyCatalog NumberComments
#122, 1-1/8" Inside x 1-5/16" Outside Diam, Viton O-Ring, 3/32" Thick,
755 Durometer 50 Pack		AmazonB07DRGPPZJ
114 Buna-N O-Ring, 70A Durometer, Black, 5/8" ID, 13/16" OD, 3/32" Width (Pack of 100)AmazonB000FMYRHK
30 mL Syringes without NeedleVitality Medical302832
3-Way Stopcock, 2 Female Luer Locks, Swivel Male Luer Lock, Vented CapQOSINA2C6201
4-40 X 1/2 PH PAN MS SS/CHROME & appropriate sized phillips screwdriverBrikksen Stainless Steel FastnersPPMSSSCH4C.5 
ANPROLENE 16 LARGE AMPULEFisher ScientificNC9085343 
BetadinePurduePUR1815001EACH 
Corning 100 x 20mm tissue-culture treated culture dishesSigma-AldrichCLS430167-100EA 
Corning L-glutamine SolutionFisher ScientificMT25005CI
Covidien 3033 Curity Gauze Sponge, 4" x 4", 12-Ply, Sterile, 1200/CSMed Plus Medical SupplyCOV-3033-CS
Dressing Forceps Delicate Curved (serrated)KatenaK5-4010
Dumont #5 - Fine ForcepsF.S.T.11254-20
Eye Scissors Standard CurvedKatenaK4-7410
Falcon 150 x 15mm Plain Sterile Disposable Petri DishesCapitol Scientific351058
Fisherbrand 4 oz. Specimen ContainersFisher Scientific16-320-730
Fisherbrand Instant Sealing Sterilization PouchesFisher Scientific01-812-54
Fisherbrand Instant Sealing Sterilization PouchesFisher Scientific01-812-55
Fisherbrand Instant Sealing Sterilization PouchesFisher Scientific01-812-58
HyClone Dulbecco's Modified Eagles MediumFisher ScientificSH3024302
HyClone Penicillin Streptomycin 100X SolutionFisher ScientificSV30010
Hydrophilic Filter with Female Luer Lock Inlet, Male Luer Slip Outlet, Blue and ClearQosina28217
Hydrostatic pressure transducers, DELTRAN ® II, Catalog # DPT-200 with a 3CC/HR flow rateAD instrumentsDPT-200
JG15-0.5HPX 15 Gauge 0.5" NT Premium Series Dispensing Tip 50/BoxJenson GlobalJG15-0.5HPX 15
Keyence B2?X710 microscopeKeyenceB2-X710
LabChart 8AD instrumentsLabChart 8
Leica ST5020 Multi-stainerLeicaST5020
Non-Vented Universal Luer Lock Cap, WhiteQOSINA65811
Octal Bridge Amp (Model # FE228)AD instrumentsFE228
Pharmco Products ETHYL ALCOHOL, 200 PROOFFisher ScientificNC1675398
Phosphate Buffered Solution (PBS)Sigma-AldrichD8537-500ML
PowerLab 8/35 (Model # PL3508)AD instrumentsPL3508
ProLong Gold Antifade Mountant with DAPIThermoFisherP36935
Push-to-Connect Tube Fitting for Air and Water Straight Adapter, 1/8" Tube OD x 1/8 NPT MaleMcMAster-Carr7880T113
Push-to-Connect Tube Fitting with Universal Thread for Air and Water, Adapter, 1/8" Tube OD x 1/8 PipeMcMAster-Carr51235K101
Saint-Gobain Tygon S3 E-3603 Flexible Tubing 500 ft.Fisher Scientific14-171-268
Superblock T20Fisher ScientificPI37536
Surgical Scissors - Sharp-BluntF.S.T.14001-14
Tissue Forceps Delicate 1x2 Teeth CurvedKatenaK5-4110
Translaminar Autonomous System (TAS)University of North Texas Health Science CenterN/A
USA Size 030 O-ring Buna-N, B1000, 70 Durometer, Black, Buna-N
(NBR, Nitrile, Buna)		Marco Rubber & PlasticsB1000-030

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