In this study, we developed a working protocol to analyze ultrasound RF data obtained from multiple cardiac cycles to quantify and spatially map the cornea's mechanical response (i.e. CAS) to the ocular pulse. Our main finding is that keratoconus corneas, on average, deformed two times more than the normal corneas in response to IOP fluctuations at each heartbeat. This biomechanical weakening was more pronounced at higher grade keratoconus. We also detected a spatial variation of responses in keratoconus corneas that the cone center had a significantly larger deformation than its surrounding tissue in grades 3 and 4 corneas. In addition, both the cone and the surrounding regions were significantly weaker than the normal cornea.
The CAS is the through-thickness compression of the cornea in response to a change in IOP. The compression associated with the ocular pulse is small in magnitude because of the typically small magnitude of the ocular pulse (on average about 3 mm Hg
25). A much larger CAS would be expected during larger IOP fluctuations, such as those associated with eye rubbing (80–150 mm Hg
26). Although the physiological consequences of these IOP-related mechanical strains are unclear, it is reasonable to expect a level of impact on the extracellular matrix (e.g. tear and wear over time) and active mechano-stimulation of cellular responses.
27 To our best knowledge, our study is one of the first to quantify the in vivo mechanical deformation of the cornea in response to natural fluctuations of IOP. Most other approaches utilize external mechanical disturbances, such as an air puff,
28 a compression plate,
29 or shear waves.
30 Our OPE method does not require an external mechanical excitation and this brings two advantages. First, the application of an external mechanical load, such as an air puff or a compression plate, alters the instantaneous IOP at the time deformation is measured. Our method does not induce a change in IOP and thus avoids the challenge to account for this change. Second, the application of mechanical disturbances requires additional apparatus and increases the complexity of the overall approach. In terms of ultrasound data acquisition, our method is as straightforward as a clinical B-mode ultrasound. The only difference is that we record a 10-second video of RF data and images of the pulsating cornea for offline analysis. In addition, our method obtains both the loading (i.e. OPA) and the resultant deformation (i.e. CAS). OPA is the pulsating component of the IOP measured by DCT, one of the most reliable devices for measuring OPA
31,32 by averaging over multiple cardiac cycles. Together with the CAS measured by ultrasound, we can define an elastic modulus-like parameter as the ratio of the load (i.e. OPA) and the deformation (i.e. CAS) to characterize corneal mechanical properties in the axial direction.
Our results showed a nearly two times higher deformation in the keratoconic cornea as compared to a normal cornea. In the current study, the age, diastolic IOP, and OPA were statistically different between the normal and the keratoconus subjects. Patients were consecutively recruited at our institute, and we did not control the match of any parameters between groups. Age was higher in the normal group (46 ± 16 years old) than the keratoconus group (37 ± 12 years old). Older age may contribute to tissue stiffening due to glycation-induced crosslinking. The diastolic IOP was also higher in the normal group (17.4 ± 2.93 mm Hg) than the keratoconus group (15.0 ± 3.7 mm Hg). Lower IOP readings in patients with keratoconus were often reported in previous studies,
33,34 but it is believed that the readings were artificially low because the altered corneal properties (thinner and more compliant) in keratoconus are known to cause lower tonometric readings.
35 We used DCT in this study, which was designed to reduce dependence on corneal properties, but it is unclear whether tonometric errors have contributed to the observed difference in IOP. The higher baseline IOP in the normal group may result in a slightly stiffer response due to stress-related stiffening. On the other hand, the normal group had a larger OPA (2.57 ± 1.02 mm Hg) than the keratoconus group (2.06 ± 0.72 mm Hg), which would induce a larger deformation, creating an opposing effect of the older age and larger diastolic IOP. Importantly, CAS remained significantly different between the two groups after considering these covariates (see the
Table). In addition, CAS was not correlated with any of these parameters in either the normal or the keratoconus eyes (see
Fig. 9), indicating that age, diastolic IOP, and OPA differences were not the main contributors to the CAS difference observed in this study. In contrast, CAS was strongly correlated with K
max and thinnest pachymetry in keratoconus, both clinically accepted indicators of keratoconus progression.
36,37 These observations suggest that the CAS difference was more associated with the keratoconus diagnosis and may have potential diagnostic value as indicators of mechanical weakening in keratoconus corneas.
We also observed a trend of increasing CAS at higher grade of keratoconus, consistent with our previous findings of an increasing CAD in higher grades.
18 Grade 4 had the largest CAS, more than 3 times on average than normal, indicating substantial biomechanical compromise as the severest stage. It is noted that although the trend was statistically significant, the current sample size is small and future studies with a larger sample size in each grade are needed to verify this trend. Brillouin imaging showed an increased spatial variance at higher grades.
38 These results together support the “progressive mechanical weakening” hypothesis underlying keratoconus progression.
39,40 We further analyzed the spatial variance of CAS in grades 3 and 4 keratoconus eyes and found that CAS at the cone center (defined as the 500-µm region around the thinnest point in the ultrasound cross-sectional scan of the cornea) was approximately 1.6 times greater than its surrounding tissue in the same eye. Brillouin imaging studies also reported greater mechanical weakening at the cone center.
10,38 The ability to detect regional differences may be useful for planning customized corneal collagen crosslinking (CXL) treatment and monitoring patient outcome. We previously have demonstrated in human donor eyes the feasibility of quantifying CXL-associated stiffening using the OPE technique.
16
A few limitations of the present study are noted. First, the cone center was defined from ultrasound images based on one B-mode scan through the corneal apex, which is likely not the actual cone center, because the cone is most commonly observed slightly inferotemporal to the apex.
41 Although our current analysis showed a substantial regional biomechanical variation in the keratoconus eye, three-dimensional imaging is needed to fully analyze the extent of spatial variation and to develop sensitive biomechanical markers for keratoconus detection. Second, we only analyzed the axial strains, which characterizes the through-thickness compression of the cornea. Future studies are needed to develop sensitive and robust methods to characterize the cornea's lateral response (i.e. in-plane stretch), which will add sensitivity to changes in collagen structure. We did not observe an age effect in through-thickness compression in the present study, which may be due to the limited age range, or may be because through-thickness compressive properties are less sensitive to age-associated stiffening. It is noted that the Brillouin frequency shift also showed a minimal age association (
r2 = 0.08).
38
In conclusion, high-frequency ultrasound OPE provides noninvasive, real-time, spatially resolved characterization of the biomechanical responses of the cornea to intrinsic IOP fluctuations. Each measurement takes less than 1 minute to complete. Our results showed the ability of this method to detect and quantify the overall weakening as well as the local variances in keratoconus corneas. High-resolution ultrasound may provide a sensitive tool for quick mapping of corneal biomechanics to aid keratoconus detection and diagnosis.