In this study, we developed a custom experimental platform that enables precise application of measurable fluid pressures in the anterior chamber of intact eye explants, as well as a novel ISSI method that together allow for assessment of CEC damage triggered by fluid pressure alone. This approach was used to determine if surgically relevant magnitudes of fluid pressures applied for surgically relevant durations resulted in acute CEC injury/death in porcine corneas. Contrary to our hypothesis, we found no significant differences in mean PCI values between eyes that were perfused to a physiological pressure (15 mm Hg) versus those that were perfused to surgical magnitudes of pressure, for both short (5 minutes) and long (45 minutes) surgical durations. Notably, the mean PCI values measured in all of the surgical pressure groups, even including eyes perfused to 400 mm Hg (>25 times higher than physiological IOP), were less than the low end of CEC loss observed clinically within 1 year after cataract surgery (2% to 42%).
5–15 The positive control eyes that were perfused with DI-H
2O did have significantly higher PCI values than all other groups, demonstrating that our staining assay was capable of detecting CEC injury/death in our experimental setup. Interestingly, the mean PCI value for the positive control group (49.5%) closely resembled the high end of CEC loss observed clinically within 1 year after cataract surgery (42%),
5–15 allowing the DI-H
2O treatment to serve as a benchmark for extreme cases of iatrogenic CEC loss (
Fig. 3). These results strongly suggest that fluid pressure alone does not result in clinically significant acute iatrogenic CEC damage.
To our knowledge, this was the first study to isolate the effects of surgical fluid pressures, with magnitudes and durations specific to cataract surgery, on acute CEC loss. Numerous studies measuring CEC loss after cataract surgery report differences in ECD as a result of different surgical techniques,
6–9,11,12 but they are not able to distinguish how each of the individual factors that may affect CEC health during cataract surgery (e.g., ultrasound energy, free radical production, contact with lens fragments and/or instruments and fluid flow) contribute to the total iatrogenic CEC damage. Wenzel et al.
42 used a research model that eliminated some of these factors by investigating the influence of bottle height during irrigation and aspiration (I/A) on CEC loss in porcine eyes. They found that short durations (10 minutes) of I/A without ultrasound energy led to significant CEC loss when compared to controls, with more CEC loss occurring at higher bottle heights. Although bottle height is closely related to IOP (i.e., a higher bottle height results in larger hydrostatic pressure), Wenzel et al.
42 acknowledged that bottle height also directly influences turbulent fluid flow within the anterior chamber. Thus, their experimental approach did not isolate the effects of fluid pressure on CEC viability. We believe that the results of our study expand on the work by Wenzel et al. by delineating the specific contribution of IOP to CEC loss by (1) eliminating the confounding variable of pressure-dependent turbulent fluid flow, and (2) actively measuring the static fluid pressure applied to the endothelium (the ACP).
Compared with previous studies, we investigated a broader range of pressure magnitudes (15–400 mm Hg) and durations (5–45 minutes). This approach should account for any level of pressure conceivably encountered during cataract surgery, even in extreme cases. For example, Khng et al.
28 measured mean IOPs as high as 220 mm Hg and transient spikes exceeding 400 mm Hg during simulated cataract surgeries, although these values are much higher than other values reported in the literature.
29,43–45 Similarly, it is difficult to clearly identify the duration for which CECs experience high pressures during a normal cataract surgery due to differences in how surgical durations are measured and reported.
28,29,44–50 Consequently, we chose to pressurize eyes to 60 mm Hg for 45 minutes, which surpasses the International Council of Ophthalmology (ICO)–Ophthalmology Surgical Competency Assessment Rubrics (OSCARs) resident/surgeon training competent criteria of 30 minutes (for a total surgery).
51,52 Therefore, 45 minutes at an elevated pressure may represent what could occur during a complicated surgery or a surgery completed by a less experienced surgical resident.
46,49,50
A key benefit of our platform is that it allows for in situ viability staining that reduces the risk of measuring CEC damage that occurs as a result of sample preparation/manipulation. Specifically, in contrast with other studies,
34,42 our ISSI approach allows for assessment of CEC loss without dissecting/removing the cornea from the globe. Additionally, the stain enters the anterior chamber of the intact eye using the same needle that is used to pressurize the eye earlier in the experiment. Hence, there is no need to remove and reinsert this needle, make any new incisions, or introduce any other instruments into the anterior chamber. We believe all of these considerations strengthen our conclusion that the fluid pressure used during cataract surgery alone is not a primary contributor to CEC loss during cataract surgery.
Our findings are consistent with previous clinical studies. For example, it has been reported that LASIK surgery has no long-term effect on CEC loss.
53,54 These findings are notable because IOP can be as high as 82 mm Hg to >140 mm Hg during LASIK surgery,
55,56 but CECs in eyes undergoing this procedure do not experience other possible contributors to CEC loss including turbulent fluid flow, lens fragmentation, and ultrasound cavitation.
Although our data appear to suggest that high surgical IOP levels are harmless, our findings should be interpreted with caution, as high IOP imparted during eye surgery could lead to other severe complications outside of CEC viability. For example, studies have found that acute IOP elevation common to cataract surgery can lead to impairment of ocular blood flow, inhibit transport of neurotrophins from the brain to the retina, and cause cellular and molecular retinal injuries.
29,44,57 We also acknowledge that, although our study provides a direct indication of immediate CEC death that is valuable for understanding acute CEC loss that occurs during an intraocular surgery, it may not capture the full extent of CEC damage. Li et al.
39 demonstrated that in rats sudden increases in IOP (∼83 mm Hg for 2 hours) disturbed the apical junctional complex (AJC) integrity of the corneal endothelium and the expression of Na, K-ATPase, thereby compromising the endothelial barrier function. Thus, it is possible that the CEC plasma membrane can tolerate larger mechanical loads than the endothelial AJC, and CECs might be functionally damaged prior to membrane rupture. It is also possible that acutely exposing CECs to high IOP might result in delayed/long-term CEC damage that was not captured in this study. For example, subjecting CECs to high fluid pressure may activate apoptotic pathways that trigger cell death over many hours or days, but our cell viability assessments were made <3 hours after pressurization. Nevertheless, we believe the platform we have developed is amenable to use in conjunction with other assays, such as the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) apoptosis assay or immunostaining of flatmounted whole corneas.
58 Our approach can also be adapted to facilitate assessment of CEC changes after several hours or days (e.g., by integrating perfusion/exchange of media into the eye with optimized eye storage conditions) to enable longer term evaluation of CEC health after anterior chamber pressurization. However, another animal model (e.g., rabbit or rat) would likely be required to confirm that acute application of surgical fluid pressure does not contribute to an increased rate of CEC loss over a longer duration (1 months or more).
Although results obtained from animal models should not be overgeneralized to make definitive conclusions about human surgeries, we believe that the porcine eye was an appropriate model for this study due to its physiological and anatomical similarities to the human eye.
59 In particular, pigs have a similar CEC density to humans (∼2800–3400 cells/mm
2 in pigs 5 to 10 months old
60 versus ∼2500–3000 cells/mm
2 in adult humans),
61 both porcine CECs and human CECs lack regenerative capacity in vivo,
60 and porcine eyes have the same general pattern of age-related CEC decline as human eyes.
60 Furthermore, as an accessible and cost effective model with similar macroscopic size and anatomy compared to human eyes, porcine eyes are the most frequently used model in surgical wet lab training.
59,62 This feature was an important consideration because developing our method in the porcine eye model demonstrates its utility for use in the wet lab setting for clinical training purposes and/or preclinical testing of new phacoemulsification devices. However, there are limitations to using porcine eyes for studies related to corneal biomechanics. For example, at the tissue level, porcine corneas are thicker
59,63,64 and less stiff
65 and exhibit different viscoelastic behavior when compared to human corneas.
66 It is also plausible that porcine CECs experience different cell-level stresses when subjected to external mechanical loads (such as fluid pressure) than human CECs because they have more intrinsic morphological heterogeneity than human CECs.
60 Although we do not expect the conclusions of this study to be model specific, repeating these experiments in human tissue is warranted. To this end, we believe that our experimental platform can be readily applied to human cadaver eyes in future studies.
Even though contact pressure is a distinct loading environment from fluid pressure, it is striking that porcine CECs can withstand 400 mm Hg of fluid pressure but immediately die with 43 mm Hg of contact pressure during indentation.
34 These contrasting findings demonstrate that the out-of-plane compressive and in-plane tensile stresses triggered by contact pressure are likely not directly responsible for acute CEC injury/death during contact indentation, and they suggest that revisiting how and why CECs are harmed during contact indentation is warranted.
To our knowledge, this study was the first to use the combination of HI and SYTOX Green to assess CEC viability. Although HI is typically used to identify Gram-positive bacteria,
67,68 we demonstrated that HI stain is highly effective at labeling CEC nuclei in porcine corneas (see
Supplementary Fig. S16). Although the seminal studies of CEC damage after simulated surgeries by Terry and others have established robust CEC viability measurements using dyes such as Calcein AM and Trypan Blue,
69–78 we believe that our combination of stains can overcome some drawbacks of these traditional viability dyes. In particular, in our experience, Calcein AM and Trypan Blue live stains often do not define discernable borders between cells and can exhibit poor contrast between live and dead cells when not thoroughly washed. However, HI (in combination with SYTOX Green) allows for clear identification of CECs and easy quantification of CEC death at single-cell resolution, even when imaging is performed through the full thickness of the cornea. Interestingly, we have found that HI can also be used to stain and image corneal and lens epithelial nuclei in intact porcine eyes (see
Supplementary Fig. S17). The primary challenge we encountered using this combination of stains was that the SYTOX Green stain had a tendency to diffuse into the stroma and stain keratocytes, sometimes with a fluorescence intensity similar to that of the CECs. This made it difficult, even when applying filtering algorithms (see Methods), to avoid counting keratocytes when assessing CEC death. Fortunately, in most cases it was easy to visually distinguish keratocytes (due to their unique nuclear morphology) and remove them from analysis during the manual adjustment step.
Collectively, the results of this study strongly suggest that surgical fluid pressure alone is not responsible for acute CEC loss during cataract surgery. Our findings, along with the novel experimental platform and ISSI method introduced in this study, open new avenues for future work pinpointing the key factors that drive iatrogenic CEC injury in cataract and other surgeries. For example, our experimental approach can be readily deployed to assess how different surgical techniques or devices (e.g., new phacoemulsification handpieces) impact CEC injury/death in simulated surgeries with porcine or human eyes, ultimately paving the way toward our long-term goal of developing novel surgical techniques and medical devices that avoid iatrogenic CEC injury and improve intraocular surgical outcomes.