Name: translaminar autonomous system model for the modulation of intraocular and intracranial pressure in human donor posterior segments
Uploaded: 2020-04-24T15:00:00
Description: 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

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

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

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

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

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translaminar autonomous system model for the modulation of intraocular and intracranial pressure in human donor posterior segments

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

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

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

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

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

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

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