## 4 Discussion
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 \(\leq\)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.