**3. Results**

#### *3.1. Participant Demography*

Table 2 contains the socio-demographic characteristics of the patients with the CSLM assessment of the SNP.


**Table 2.** Socio-demographic characteristics according to the treatment arm (*n* = 16).

#### *3.2. Confocal Microscopy Results*

Five CSLM images of eight patients of the control group and eight patients of the Comfort Shield group taken at the end of the baseline visit and at the end of the week 8 visit were analyzed (see examples in Figure 2).

**Figure 2.** Typical SNP images of subjects from the control and study group, as well as a schematic representation of detected nerve fibers used for characterization of the SNP at baseline and after 8 weeks of treatment.

We found a statistically significant difference in CNFL between baseline and the eight weeks follow-up visit; the Comfort Shield group showed a significant difference in CNFL (*p* = 0.030) contrary to the control group (*p* = 0.294). CNFL was comparable for Comfort Shield and control at baseline (*p* = 0.793) and showed a significant difference after eight weeks (*p* = 0.031). Possibly due to the small number of patients, we did not find significant differences for the other SNP parameters (CNFD, CNBD, CNFTo, CNCP, CNSFL, CNFTh). Moreover, patients suffering from severe dry eye generally do not have a well-developed SNP, and there was a lot of foreign tissue in the vicinity of the SNP that complicated the image analysis. Figure 3 summarized the CNFL findings of the Comfort Shield group and the control group at baseline and eight weeks visit.

**Figure 3.** CNFL of the Comfort Shield group and the control group at baseline and eight weeks.

## **4. Discussion**

Due to the heterogeneous, multicausal nature of DED, particularly in patients suffering from severe, chronic DED, a personalized clinical managemen<sup>t</sup> resulting in an individualized optimum therapy is required [57]. Only patients under stable therapy had been included in the HYLAN M study, and their optimum individual therapy served as a control in comparison to patients in which 0.15% HMWHA eye drops were tested. The assessment of the subbasal corneal nerve plexus was an optional test in addition to the standard diagnostic test battery of the study. Four out of 11 study centers provided CSLM images from 16 per-protocol patients, eight each in the Comfort Shield group and in the control group. The SNP is usually not well structured in severe dry eye disease [58]. Due to the small number of patients, only the results of CNFL showed a significant difference between the two study arms. This is in accordance with other studies reporting that CNFL is the most reproducible parameter in the evaluation of IVCM images of the subbasal nerve plexus [28,59–65].

Until recently, HA eye drops had been applied as a lubricating, hydrating, and mechanically buffering tear substitute [66]. It was known that the apical surface of the superficial epithelial cells of the cornea and conjunctiva have HA receptors (CD44 and HARE), which can bind HA and thus support the antiadhesive properties of the glycocalyx [67–71]. HMWHA, but not LMWHA, can also adhere to the membrane-bound mucins of the glycocalyx, thus strengthening the cellular barrier of the ocular surface [72]. HA is an essential part of the extracellular matrix (ECM) and plays an important molecular weight dependent role in wound healing and immunoregulation [4,73–78]. Disturbed immunoregulation involving chronic inflammation, which triggers a vicious circle, is currently considered the characteristics of severe DED [10]. HMWHA enables cross-bridging between the HA receptors of adjacent cells and can thus contribute to the mechanical stabilization of the wing cell layers of the corneal epithelium [79–81]. Reactive oxygen species (ROS) formed during inflammatory processes effectively cleave HMWHA, which in turn enhances the inflammatory process and weakens the cross-bridging function of HA between epithelial cells [82]. So far, it had been unknown whether HMWHA from topically applied eye drops can, in a situation of chronic ocular inflammation, pass the ocular surface barrier to recover the homeostatic HA weight distribution in the extracellular matrix. The first evidence came from an animal experiment where 0.15% HMWHA eye drops were compared with 0.1% and 0.3% LMWHA eye drops with respect to their ability to prevent and treat DED caused by environmental stress [6]. The 0.15% HMWHA eye drops proved superiority with respect to the prevention and treatment of inflammation and stabilization of aqueous tear secretion and mucin production [6].

The HYLAN M study indicated that topically applied HMWHA could pass the intercellular barrier of the corneal epithelium. By changing the extracellular matrix in the proximity of the subbasal

nerve plexus, this could result in trophic effects reflected in the significant regeneration of compromised nerves. This also provided evidence that HMWHA becomes available in the ECM in all cell layers of the corneal epithelium and thus can contribute to regaining ocular surface homeostasis in eyes with chronic inflammation. Further methods detecting specific anti-inflammatory and neurotrophic factors such as nerve growth factor (NGF) in the tear film or in the ocular surface will provide valuable additional information in future clinical studies. On the other hand, the study showed that within eight weeks of treatment, simultaneously with nerve growth, the symptoms of patients with severe DED improved significantly. According to the results of the HYLAN M study, in combination with the animal study [6], we may conclude that 0.15% HMWHA eye drops gran<sup>t</sup> a holistic approach in the treatment of DED, simultaneously addressing the various and complex interacting pathomechanisms of the disease: lubrication, hydration, stabilization of glycocalyx and barrier function, downregulation of inflammation, trophic support to corneal nerves, increasing goblet cell counts and expression of MUC5AC [83], support of aqueous tear production, and reduction of pain.

It needs to be emphasized that the effect of HMWHA on nerve regeneration has only been investigated in a very small number of eyes. Hence, conclusions on significance in numbers cannot be given. Nevertheless, the high incidence of nerve regeneration during treatment with HMWHA was clearly different from the unchanged situation in the control group. Future studies with a higher number of eyes and a primary focus on nerve regeneration will provide further details.

As the HYLAN M study included dry eye patients with any disease etiology, it seems likely that patients with corneal nerve injury or degeneration as an underlying cause for ocular surface disease or neurotrophic keratopathy would benefit from treatment with HMWHA eye drops [11,84]. The causes may include acute nerve injury like in ocular surgery, refractive surgery, corneal cross-linking, chemical burns, or ocular trauma [85,86]. Similarly, HMWHA eye drops may also be effective in promoting neuroregeneration in progressing peripheral neuropathies associated with ocular infections, keratoconus, small-fiber neuropathy, diabetes mellitus, or simply aging [87–90].

The progressive loss of corneal sensory innervation of any etiology may result in neurotrophic keratopathy (NK) [15,16,35,91–94]. NK is characterized by corneal anesthesia and is a condition that is very difficult to treat, especially as for the required regeneration of trigeminal terminal nerve fibers, no such treatment is currently available [35,93,95]. Medical managemen<sup>t</sup> with lubricating eye drops, anti-inflammatory agents, and anti-proteases provide unspecific temporary relief in NK but do not prevent disease progression [93,96]. Whereas some degree of inflammation promotes nerve regeneration, excessive inflammation may lead to a loss of corneal innervation and subsequent development of NK [15]. As corneal nerve regeneration and inflammation are intertwined, the therapeutic strategy must consider the interaction of both pathways [15]. HMWHA eye drops seem to offer a promising treatment option in this situation.

According to the International Diabetes Federation (IDF), more than 400 million people worldwide suffer from diabetes mellitus (DM). DM is associated with a progressive loss of peripheral nerves. Corneal nerve damage may serve as an early indicator in DM [63,97–99]. The prevalence of corneal neuropathy in diabetic patients is approximately 50% [32,100–103]. However, corneal neuropathy, as a manifestation of DM, is underrated due to the absence of ocular discomfort and pain [33,98,103,104]. Persistent corneal epithelial erosions, superficial punctate keratopathy, delayed epithelial regeneration, and decreased sensitivity are associated with diabetic keratopathy [32,34,105,106]. Diabetic keratopathy is a significant clinical problem and a progressing disease, and currently, no effective treatment is available [34]. IVCM has proven to be a valuable and reliable diagnostic tool to assess nerve fiber damage and assess improvement of risk factors for diabetic neuropathy, thus allowing visualizing treatment success [59,107,108]. Having shown that 0.15% HMWHA eye drops support corneal nerve regeneration allows the assumption that these eye drops will also prove as an effective preventive therapy against the progression of diabetic keratopathy. This may contribute to lower the enormous global economic burden of DM [109]. The results reported here were obtained from a small number of patients. This report is, therefore, intended to encourage further clinical research rather than to provide comprehensive answers or interpretation.
