Trabecular Meshwork Motion Profile from Pulsatile Pressure Transients: A New Platform to Simulate Transitory Responses in Humans and Nonhuman Primates

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The newly developed platform permits the study of the trabecular meshwork motion profile induced by ocular transients. Trabecular meshwork motion is an important profile for aqueous circulation in the eye. Its abnormality is the cause of primary open angle glaucoma, which is the leading cause of irreversible blindness in the world. Our research platform permitted a phase-sensitive optical coherence tomography system to quantitate TM motion responses to experimentally controlled pulse transients.

Abstract

Trabecular meshwork (TM) motion abnormality is the leading cause of glaucoma. With technique limitations, how TM moves is still an enigma. This study describes a new laboratory platform to investigate TM motion responses to ocular transients in ex vivo eyes. The anterior segments of human cadaver and primate eyes were mounted in a perfusion system fitting. Perfusion needles were placed to establish mean baseline pressure. A perfusion pump was connected to the posterior chamber and generated an immediate transient pressure elevation. A phase-sensitive optical coherent tomography system imaged and quantified the TM motion. The peak-to-peak TM displacements (ppTMD) were determined, a tissue relaxation curve derived, and a time constant obtained. This study showed that the ppTMD increased with a rise in the pulse amplitude. The ppTMD was highest for the lowest mean pressure of 16 mmHg and decreased with mean pressure increase. The pulse frequency did not significantly change ppTMD. With a fixed pulse amplitude, an increase in mean pressure significantly reduced the time constant of recoil from maximum distension. Our research platform permitted quantitation of TM motion responses to designed pulse transients. Our findings may improve the interpretation of new TM motion measurements in clinic, aiding in understanding mechanisms and management.

1. Introduction

Glaucoma is a leading cause of irreversible blindness in the world [1]. Primary open-angle glaucoma (POAG) is the most common type with characteristics of optic neuropathy and visual field defects. Intraocular pressure (IOP) elevation is widely accepted as a major cause and the only treatable factor for POAG [2,3].

Similar to blood pressure, IOP constantly varies. Many IOP transients occur over a day in response to the ocular pulse [4,5,6], blinking, and eye movements [4,7]. Telemetry in monkeys shows that each waking hour, the eye experiences ~10,000 transient IOP fluctuations larger than 0.6 mmHg above the mean baseline pressure with ~5000 transient IOP fluctuations greater than 5 mmHg [8,9]. Pulsatile aqueous flow from Schlemm’s canal (SC) into the episcleral veins occurs in synchrony with the cardiac pulse. Pulsatile cyclic mechanical wall and shear stresses induce signals that confer physiological benefits important to IOP homeostasis. IOP transients also provide forces necessary for the pulsatile discharge of aqueous.

In humans, peak-to-peak IOP oscillations with the ocular pulse are about 1.5 mmHg above baseline IOP [5], providing a 3 mmHg pulse amplitude. IOP fluctuations above the baseline of 10 to 14 mmHg occur with saccadic eye movements and with blinking that happens about every 20 s [4]. These oscillatory and transient pressure spikes cause the mobile trabecular meshwork (TM) lamellae to deform outward into SC, which acts as a collapsible chamber. TM distension into SC forces aqueous into collector channels (CC) entrances and aqueous veins. As the pressure spike decays, the TM lamellae return to their original configuration with a resultant reduction of SC pressure, permitting aqueous flow into the canal [10,11].

Pressure-induced deformation of the TM changes SC dimensions, inducing oscillatory pulsations in SC. TM biomechanics determine TM stiffness and the ability to induce pumplike pulsatile flow [11,12,13,14]. Clinical and laboratory evidence has shown that the pulsatile pattern of aqueous flow decreases and even stops in advanced glaucoma, thought to result from increased TM stiffness [10,14].

Optical coherence tomography (OCT) imaging is shedding new light on TM biomechanics. The technology provides unprecedented details of structures, relationships, and motion that provide an evolving picture of functional behavior. OCT coupled with the ability to change pressure gradients in SC [12,15,16], and the anterior chamber (AC) [17] permits studying TM biomechanics under steady-state conditions. However, no technique has been available to measure TM responses to rapid physiological ocular transients.

Pulse-transient-induced TM distention has not been studied in the aqueous outflow system, although it has been reported in the prelaminar regions of the optic nerve [18,19]. The time course of TM recoil after transients also remains unknown. The ability of the TM configuration to respond to mean pressure changes and pulse-transients is vital for IOP homeostasis. Loss of TM motion responsiveness to pressure changes might be a crucial factor leading to pressure abnormalities in glaucoma. Our recently developed OCT technologies permit exploring TM biomechanical responses to transients that can add to the understanding of the physiology and pathophysiology of glaucoma.

Our goal in this pilot study is to describe a newly developed platform that permits the study of the TM motion profile induced by ocular transients. We continually monitor TM responses in real-time with OCT during the experiments. We experimentally control mean baseline IOP, pulse speed, pulse amplitude, and pulse frequency. We use our newly developed peak-to-peak TM displacement (ppTMD) parameter to analyze TM responses to distention and a TM relaxation constant to assess recoil responses after distention.

Our first aim is to assess the effects of stepwise increases in the amplitude of pulse transients while maintaining a stable baseline IOP. Our second aim is to assess how the changes in the baseline IOP loading force affect the TM responses to pulse transients. We do this by maintaining a constant pulse amplitude while inducing stepwise increases in IOP. A third aim is to assess how the frequency of pulse transients changes TM responses. A final aim is to explore the TM recoil response after stress-induced deformation of the TM by the pulse transient loading force.

2. Materials and Methods

Figure 1 is a schematic of the experimental study’s setup used to image and quantify dynamic TM motion in response to pressure transient [20]. The setup has four components: (1) a PhS-OCT system for imaging the TM motion, (2) an anterior segment perfusion system for controlling the mean pressures, (3) a perfusion pump controlling pulse transients, (4) a Powerlab^TM processing unit for real-time pressure monitoring and synchronization with the PhS-OCT imaging system during data acquisition.

(1)

Eyes and anterior segment perfusion setup

A nonhuman primate eye (Macaca nemestrina, female, 9-year-old) was obtained from the University of Washington Primate Center and harvested within 0.5 h of experiment initiation. A normal postmortem human eye (Caucasian, female, 70-year-old, Caucasian) with no history of ocular disease was provided by the Sightlife^TM eye bank within 24 h after the donor’s death.

The eyes were hemisected along the equator, followed by removing the vitreous, retina, and choroid leaving the crystalline lens and ciliary body intact. The prepared anterior segments were mounted on the eye perfusion fitting. Two 23-gauge perfusion needles were inserted into the AC—one connected by a cannula to a pressure transducer, the other to a reservoir controlling AC pressure. An additional cannula led from a perfusion fitting communicating with the posterior chamber (PC) to a valve that switched between a pressure reservoir and a perfusion pump.

Baseline mean AC and PC pressure was initially experimentally controlled by establishing identical chamber pressures. The posterior chamber cannula was switched to the perfusion pump, and the pulse transient was immediately initiated. Pressure transient elevations simulating blink and eye movement were generated by controlling the perfusion pump infusion rate and duration. An anterior chamber needle connected to a pressure transducer (PowerLab, ML866) continuously monitored the mean pressure and pulse amplitudes in the anterior chamber. The mean baseline or resting pressures tested were 16, 24, 32, and 40 mmHg. The pulse amplitudes tested were 2.5, 5-, 7.5-, and 10-mmHg. The appropriate transient pulse amplitude was determined by maintaining a steady 3 mL/min infusion rate and experimentally varying the infusion volume. Controlled pulse frequencies of 30, 60, and 90 pulses/min were experimentally induced with the above variables.

(2)

Imaging system and scanning procedures

A spectrometer-based, phase-sensitive optical coherent tomography (PhS-OCT) system was used for imaging the TM region of the ex vivo anterior segments. The system was coupled with a broad bandwidth light source centered at 1340 nm (~7.2 μm axial resolution in air) and used a ~92 kHz A-line rate InGaAs array sensor. Tracings were acquired using 1500 repeated B-scans of 360 A-lines and 512 axial pixels at the rate of 200 f/s from the surface overlying the TM.

(3)

Data analysis

The data were processed using a PhS-OCT Matlab algorithm to extract the structure and phase of each B-scan. The average of 5 phase frames (Δφ) was used to reduce the effect of phase noise on the final result. The instantaneous depth-resolved tissue velocity, the total displacement, and the ppTMD values were extracted as previous report [20] (Figure 2).The time constant, determined by tissue recoil in response to the removal of the IOP loading force, was calculated by the fitted exponential decay function.

Descriptive statistics calculated the mean and standard deviations (mean ± SDs). The significant difference of the TM recoil time constant was determined using ANOVA, while the significant differences of time constant between different pressure modalities were analyzed using a paired t-test. p < 0.05 is considered as statistically significant.

3. Results

Figure 3 depicts the ppTMD measured in response to pairs of increasing mean pressures and pulse amplitudes in human and nonhuman primate eyes. Mean baseline pressures were experimentally controlled at 16, 24, 32, and 40 mmHg. Pulse amplitudes were set to 2.5, 5, 7.5, and 10 mmHg for each mean pressure. The pulse frequency was maintained at 30 pulses/min. In both human and monkey eyes, when mean pressure remained constant, the ppTMD increased along with the experimentally controlled increase in pulse amplitude. The ppTMD is at its highest value at the lowest mean pressure of 16 mmHg at each pulse amplitude.

A systematic increase in mean baseline pressure reduces the ppTMD response at each pulse amplitude. The ppTMD values for the 4 mean pressures at lower pulse amplitudes are similar. As the mean pressure increases, the relative effect of the pulse amplitude on ppTMD decreases. The increased separation of the curves is consistent with movement along the length–tension curve as pressure rises. As the baseline stress from pressure rises, the TM experiences greater baseline strain. Increased baseline strain deformation results in progressively reduced TM deformation in response to the same pulse transient.

Figure 4 shows how pulse frequency affects the ppTMD response to stepwise increases in mean pressure and pulse transient parameters in a nonhuman primate eye. The ppTMD curves showed quite limited differences in response to changes in frequency. At a fixed mean pressure, the ppTMD increased with the rise in pulse amplitude. When mean pressure was 16 or 24 mmHg, the ppTMD of the higher frequencies decreased slightly compared with the 30 pulse/min, made more evident with higher pulse amplitudes (7.5 mmHg and 10 mmHg). With higher mean pressure (32 mmHg or 40 mmHg), when pulse amplitude was 2.5 mmHg and 5 mmHg, the ppTMD responses to pulse frequency were similar. When pulse amplitude was 7.5 mmHg and 10 mmHg, the ppTMD was at its highest value for the lowest frequency of 30 pulse/min.

Figure 5 shows the normalized TM recoil time constant for various mean pressures and pulse amplitudes in human and monkey eyes. Using a fixed pulse amplitude while increasing mean pressure, the statistical analysis revealed significant time constant differences. The mean pressure increase resulted in a decrease in the time constant while maintaining the same pulse amplitude. The p values for the TM recoil time constant over different mean pressures and pulse amplitudes are listed in

Table 1. p-value for TM recoil time constant over different mean pressures and pulse amplitudes.

	Human Eye				Monkey Eye
MP (mmHg)	PA (2.5)	PA (5)	PA (7.5)	PA (10)	PA (2.5)	PA (5)	PA (7.5)	PA (10)
16–24	0.975	0.005 **	0.868	0.492	0.258	0.978	0.109	0.074
16–32	0.153	0.145	0.036 *	0.005 **	0.008 **	0.007 **	0.054	0.011 **
16–40	0.002 **	<0.001 **	0.01 *	<0.001 **	0.003 **	0.004 **	0.001 **	0.002 **
24–32	0.284	0.272	0.009 **	<0.001 **	0.222	0.014 *	0.975	0.712
24–40	0.004 **	0.02 *	0.003 **	<0.001 **	0.078	0.008 **	0.076	0.167
32–40	0.104	<0.001 **	0.891	0.051	0.913	0.991	0.151	0.655

MP: mean pressure, PA: pulse amplitude (mmHg); * indicates p < 0.05, ** indicates p < 0.01.

. A Multiple Comparison test indicated that in all cases, the TM showed significantly faster recoil (i.e., shorter time constant) at 40 mmHg IOP compared with 16 mmHg IOP (p < 0.05).

4. Discussion

This study demonstrates our research platform’s ability to explore TM motion responses to intraocular pulse transients in human and nonhuman primate eyes. Pulse transients are a crucial component of the forces the TM experiences because blinking and eye movement are frequent [9]. Studying responses to transient ocular spikes can provide insights into the normal TM tissue biomechanical properties and provide a means to compare the findings with those of glaucoma patients.

Our PhS-OCT study is limited to measuring bulk responses of the trabecular beams because the resolution is not adequate to assess the movement of smaller structural features such as SC inner wall endothelium [20]. In vivo, IOP induces a constant loading force or prestress on the TM tissues [10,12]. SC is a compressible chamber, and IOP loading forces continuously drive the TM tissues outward toward SC external wall, tending to reduce the relative size of the chamber lumen.

Schwalbe’s line anteriorly combined with the scleral spur and ciliary body posteriorly anchor the TM between the AC and SC. Ciliary body tension and elastance of the trabecular beams determine the ability of the TM to maintain its optimal position suspended in 3D space between the AC and the canal. Elastance, also called stiffness, is the property that determines the ability of a tissue to store elastic energy controlling the ability to distend and recoil [12,21,22].

The loading force of IOP causes stresses that induce TM strain. TM strain deformation causes the SC lumen to narrow. The IOP loading force on the TM must be constantly balanced by the combined forces of ciliary muscle tension and trabecular lamellar elastance. In the absence of optimized elastance, the TM would distend into SC, resulting in persistent canal closure and occlusion of collector channel entrances.

SC collapse is widely recognized by clinical and laboratory studies as a causal factor resulting in increased resistance in glaucoma [23,24]. Drugs such as pilocarpine increase ciliary body tension, increasing the tensile load, thereby altering the TM elastance/stiffness. The altered elastance increases pulsatile aqueous outflow and reduces pressure in glaucoma patients [25,26]. Such considerations emphasize the clinical importance of understanding the mechanics of the TM tissues.

When we induce an experimental pulse amplitude increase, the ppTMD markedly increases at each of the mean pressures tested in both the human and NHP eyes (Figure 3). For example, at a mean pressure of 16 mmHg, ppTMD rose from 0.3 µm to 1.6 µm, demonstrating a profound effect of pulse amplitude on ppTMD.

Increases in baseline IOP decreased the ppTMD pulse amplitude response (Figure 3). At a pulse amplitude of 2.5 mmHg, minimal differences were present at a baseline IOP of 16 mmHg. However, the relative ppTMD markedly decreased as baseline IOP increased. The baseline IOP of 32 and 40 mmHg reduced ppTMD by 50% or more compared with the 16 mmHg baseline IOP in both the human and NH primate eyes.

The ppTMD was affected by both baseline IOP and pulse amplitude, indicating that the TM shows a nonlinear force–displacement relationship (hyperelastic) that is typical in ocular tissues. Specifically, at a relatively lower baseline IOP, the TM’s stiffness is lower than that of a higher baseline IOP. Mean IOP and pulse amplitude increases are both parameters that alter the load the TM experiences. Our findings indicate that the two forces function synergistically to alter tissue stiffness. The findings indicate that changes in ppTMD induced by baseline and pulsatile changes in IOP reflect increases in stiffness in response to stepwise increases in pressure (different stretch levels).

We asked how the frequency of oscillatory pressures or pulse transients impacts the biomechanical properties of the TM by using a physiologic pulse of 60 beats per minute (bpm) as the reference frequency, then bracketed it with 30 and 90 bpm. Although there were modest tracing differences when comparing the lower and higher frequencies, the curves demonstrate a minimal frequency-dependent impact on the ppTMD. Our study suggests the tissues are well-adapted to respond rapidly over the frequency range tested.

For the exploration of TM recoil properties, the time constant following a single pulse was fitted to an exponential decay function to obtain a time constant T [27]. A smaller T indicates a faster recoil after unloading. The resulting normalized TM displacement curve corresponded well with the normalized pressure recorded by the transducer when the two were overlaid. The time constant provides a quantitative expression of the intrinsic recoil properties of the TM tissues.

The time constants for TM recoil between the human and NH primate eyes were similar, although the NP primate results were slightly higher at most time points. The time constant decrease between 16 mm and 40 mmHg was significant at all pulse amplitudes in each eye. The well-recognized force-displacement relationships may explain the reduction in the time constant in response to increasing baseline pressures. The increased stiffness with an increasing tension or load is a behavior that occurs in many ocular tissues [28,29,30,31]. At a higher baseline IOP, the force–displacement curve is steeper, and thus, the recoil speed at unloading is faster, which leads to a reduced time constant, as seen in our study.

The ppTMD tracings demonstrate a rapid change in the loading force the TM experiences in response to pulse transients. The tissue response to the abrupt change in load is different from the increase experienced in response to a steady-state mean load because of the property termed viscoelasticity [32,33]. Under the same loading amplitude, a higher pulse frequency results in a smaller ppTMD (Figure 4). The response is typical viscoelastic behavior, in which tissue resistance increases with increasing loading speed. Our study found that the TM shows viscoelastic behavior, though the viscous effects are very small under an ocular pulse of ≤5 mmHg at all baseline IOP levels.

Our study finds that sudden changes associated with pulse amplitude differences alter the time constant for TM recoil. When the pulse amplitude rose above 16 mmHg, the time constant regularly decreased in the human and NH primate eyes. The findings suggest that at a change from 2.5- and 5-mmHg pulse amplitude, there is a nonlinear change in the biomechanics of the tissues. Perhaps, the amplitude increase creates a phase shift related to the uncrimping of the linear and crosslinked collagen and elastin in the trabecular beams with associated changes in recovery after loading. Biomechanical properties change in response to load changes and the rapidity of change. Our data suggest that the ppTMD biomechanical response to a pulse-dependent oscillatory load may differ substantially from that associated with blinking and eye movement.

The major limitations of our study are the same ones associated with all ex vivo studies. Normal EVP and ciliary body tone are absent, and each factor is widely recognized as playing an essential role in aqueous outflow regulation [34,35]. We are aware that findings in vivo may differ from those of our study. However, using ex vivo eyes, we can definitively control the experimental conditions associated with mean pressures, pulse amplitudes, and frequency without the many confounding factors present in vivo.

Ex vivo human eyes are subject to autolysis that, over time, may substantially alter the mechanical properties of tissues. Acott’s work has shown that eyes placed in media recover functional properties remarkably similar to those present in vivo [36,37]. We begin experiments in primate eyes immediately after death and quickly immerse them in media, reducing the likelihood of autolysis-related issues. We are reassured by the similarity between the findings in the human and nonhuman primate eyes. The properties we are exploring are those of the TM lamellae composed primarily of collagen and elastin. The components are less susceptible to postmortem artifact and the autolysis of the cellular elements of the system, which may help explain the similarities between the human and primate tissue responses.

In summary, our newly developed platform permits the introduction of pulse transients in ex vivo eyes while permitting real-time measurement of TM motion with PhS-OCT. We demonstrated that while maintaining a steady mean IOP, increasing the amplitude of pulse transients caused a significant increase in TM deformation. We found that maintaining a steady pulse amplitude while increasing mean IOP resulted in decreased pulse-dependent TM deformation. We identified only limited effects of pulse frequencies within the physiologic range. We conclude that forces including the mean IOP, the ocular pulse, blinking, and eye movements are determinants of TM motion responses, while the cardiac pulse rate is less likely to be clinically relevant.

We demonstrated an exponential decay function after the introduction of a pulse transient that provides a time constant. The time constant decreases as mean IOP increases, consistent with the concept that increasing stresses introduce increasing strain in TM tissue; more rapid tissue recoil was results in. The amplitude of the transient pulse alters the subsequent time constant of recovery, indicating that not only mean but also the amplitude of sudden changes in pressure alters the biomechanical responses of the tissues.

5. Conclusions

The TM stiffness properties are crucial to the maintenance of optimal SC lumen dimensions. In glaucoma, those properties become abnormal, leading to lumen collapse and CC entrance occlusion. Mean baseline pressure is an essential determinate of TM distention into SC. However, the eye also faces constant ocular transients that the TM tissues must withstand to maintain an optimal configuration.

The ability of our system to introduce ocular transients and quantitatively assess real-time TM responses provides a means of assessing this clinically relevant issue. Our study of pulse transients adds another dimension to the understanding of outflow system biomechanics. Future studies can explore alterations in biomechanical properties in glaucomatous eyes. Such studies should help unravel the elusive cause of IOP dysregulation and help find a solution to the problem of pressure elevation in glaucoma.