In this study, we use a wide variety of techniques to describe for the first time the chronic ocular surface pathology caused by acute/subacute mercury poisoning in workers accidentally exposed to toxic doses of mercury. Briefly, we showed that most patients were highly symptomatic and had increased tear osmolarity, corneal hypoesthesia, altered corneal sub-basal nerve and dendritic cell parameters, and altered tear levels of some inflammation-related cytokines. We concluded that the pathology encountered 1–2 years after the acute/subacute event is consistent with a neurogenic-based DE disease that was more severe in patients with higher urine levels of mercury.
The chief target organ of mercury vapor is the brain, where it causes apoptosis and ischemia of nerve fibers.[4] The eye and visual pathways are especially susceptible to neurologically-driven diseases, and the ocular effects of poisoning due to mercury exposure are not unexpected because of the extraordinarily abundant innervation of the eye. This is especially true in the cornea, the most highly innervated tissue in the whole human body. This innervation is sensitive and is delivered by the ophthalmic branch of the trigeminal or V cranial nerve.[34] Because the damage caused by mercury poisoning could target this rich innervation, we evaluated corneal sensitivity and the morphology of the sub-basal corneal nerves by non-contact esthesiometry and IVCM, respectively. Both techniques are minimally invasive, and although not regularly performed in the clinical setting, they can provide invaluable information about some ocular surface diseases. We have accumulated experience with these techniques in contact lens-related discomfort [18] and stem cell therapy for corneal pathology.[35]
Mercury poisoning, which could be responsible for the neurotoxicity and subsequent damage to the corneal nerves, could also be why the vast majority of the patients had DE-related symptoms, most of which were strongly experienced. Aside from changes in some tear cytokines (as discussed below), there were no signs of alteration in tear production and/or tear quality that can cause epithelial damage to the ocular surface as would be typical in tear-deficient and/or evaporative DE. In fact, a disparity between signs visualized with the slit lamp and symptoms is one of the most striking aspects of DE disease and has been reported in many types of DE patients.[12, 34, 36] This is especially true after corneal refractive surgery (the so-called “pain without stain”) in which there is an unavoidable lesion to the corneal nerves as part of the required laser treatment.[37] In post-refractive surgery patients, and most likely in our mercury-intoxicated patients, neurogenic inflammation due to corneal nerve damage results in the release of the inflammatory mediators.[37] This inflammation could cause the patients to have DE symptoms without manifestation of compromised tear production, and therefore not causing an obvious ocular surface integrity problem. It is not clear why corneal nerve damage and hypoesthesia caused by refractive surgery or poisons like mercury develop into neurogenic DE without epithelial involvement. This problem is especially confounding because other deleterious causes, e.g., neurotrophic keratitis, also have corneal nerve damage and reduction of corneal sensation but result in epithelial damage. We hypothesize that neurogenic DE represents either another stage or another cause of neurotrophic keratitis. Clearly, more research is needed to better understand these conditions at the molecular and genetic levels.[38]
Tear osmolarity was elevated in 19 of the 22 patients. Although tear hyperosmolarity has been implicated in the pathogenesis of DE,[12] there is no obvious explanation as to why the osmolarity was high in these patients, especially since there was no accompanying damage to the integrity of the ocular surface. Consistent with our findings, Yi et al.[39] reported a significant positive correlation between tear osmolarity and ocular symptoms, including cold sensitivity, foreign body sensation, and light sensitivity; however in our patients, T-BUT, corneal staining, eyelid hyperemia, and tear secretion volume were not significantly correlated with tear osmolarity. Similarly, Gjerdrum et al.[40] also found tear hyperosmolarity with normal tear production in patients after nerve alteration caused by corneal laser surgery.
Using Belmonte’s gas esthesiometer to measure corneal sensitivity thresholds, we found an increase in the mechanical threshold and in the heat and cold thermal thresholds in the mercury-intoxicated patients. This means that the overall corneal sensitivity was diminished. Benitez del Castillo et al.[36] found similar results for mechanical and thermal sensitivities in Sjögren syndrome-associated DE disease. Likewise, Bourcier et al.[41] reported corneal hypoesthesia with mechanical and thermal stimuli in a more mixed sample of DE patients.
The changes in corneal sensitivity are probably due to the nerve damage that we detected by IVCM. Corneal nerves not only protect the ocular surface through the mechanism of sensation, but they also release trophic factors that regulate wound healing, epithelial integrity, and cell proliferation.[19, 34, 42] Thus, nerve damage caused by mercury intoxication could be responsible for an alteration in neuronal stimulation and a delay in the transmission of nerve impulses of the affected fibers. This would explain the decrease in mechanical and thermal sensitivity that we found.
Regarding nerve morphology, corneal nerve density and nerve branch density were significantly lower in the mercury-intoxicated patients, and these changes were associated with higher levels of mercury in the urine of Cluster 2 patients. These results are in agreement with most studies in which there was a significant reduction in the sub-basal nerve density in DE patients compared with controls.[23, 43] There are, however, two studies[44, 45] that show no difference in sub-basal nerve density, but instead, the DE patients had abnormal nerve morphology. Finally, one study of patients with aqueous-deficient DE disease found increased sub-basal nerve density, suggesting the possibility of corneal nerve regeneration in this form of DE.[46] In general, regenerative activity is manifested by nerve branches from endbulbs, and in our patients, the density of branches was diminished. All of these findings support the notion that mercury intoxication adversely affects nerve function and also the capacity for nerve regeneration.
The density of dendritic cells in the corneal stroma of our mercury-intoxicated patients was decreased. Dendritic cells are in contact with the sensory nerve fibers, and play an important role in corneal homeostasis.[23, 47, 48] Elevated density of dendritic cells is a common finding in inflammatory disorders such as DE disease,[23] after refractive surgery, [49] in diabetic neuropathy,[49] and in infectious keratitis.[23] Consequently, we initially expected to find a higher density of these cells in the cornea of our patients. However, where more centralized nerve damage occurs, such as in patients with fibromyalgia syndrome where the corneal sub-basal nerve plexus is also damaged, corneal dendritic cell density is similaly decreased.[47] In animals, after trigeminal denervation, there is a depletion of dendritic cells, and corneal sensitivity is significantly reduced, delaying corneal recovery during wound healing.[48] So the decrease in the density of dendritic cells in our mercury-intoxicated patients could be due to the damage we demonstrated in corneal sub-basal nerve plexus.
Lastly, we found alterations in some tear cytokine levels in the mercury-intoxicated patients. Damage caused by mercury intoxication could be responsible for the alterations in nerve stimulation and impulse transmission. It could also cause nerve inflammation, resulting in liberation of several inflammatory cytokines. Indeed, neuro-inflammation is one of the main pathways of methyl mercury-induced central nervous system impairment.[2] Furthermore, in addition to affecting the nervous system, there is accumulating evidence that exposure to mercury alters immunomodulation, although with differences in the mechanism of action depending on the specific form of mercury (inorganic or organic), the species, and even the cell type or tissue.[2, 50] Also, other cell types apart from nerves, such as ocular epithelial and/or immune cells, could participate in the ocular surface inflammatory response to mercury exposure. These responses are related to interactions of metals, such as mercury, with electrophilic groups that are not solely restricted to the central nervous system, but are also ubiquitously present in several systems and organs.[2, 6, 7, 50]
Other studies have already shown tear molecule alterations in several ocular pathologies.[10, 11, 13, 15, 23–30] Additionally, some studies reported alteration of tear cytokine levels after corneal refractive surgery.[16, 31–33] While there are several published studies regarding serum and/or tissue cytokine/chemokine levels or gene expression in mercury-intoxicated patients,[2, 21, 22] to our knowledge, this study is the first to address tear cytokine levels in these patients.
We found that mercury-intoxicated patients had significantly increased tear levels of lL-12p70, IL-1RA, RANTES, and VEGF. Similar findings have been described in DE patients[13, 26, 51] and in tears from advanced surface ablation refractive surgery patients.[16] The increase in these molecules is in agreement with the increase in serum cytokines in mercury-exposed patients, [2, 21, 22] and it reflects an inflammatory response at the ocular surface of these patients.
On the other hand. EGF, IP-10, and IL-6 tear levels were significantly decreased in mercury-intoxicated patients. EGF tear levels usually decrease in DE patients, particularly in the more severe forms.[10, 27, 29, 30] A decrease in tear IP-10 levels has also been described by our group in patients with severe DE associated with ocular graft vs host disease[30] and by others in primary Sjögren syndrome and in Stevens-Johnson syndrome patients.[52]
The finding of decreased tear IL-6 levels is in contrast to the increased concentration in DE patients.[24, 26, 27, 29] However, a decrease in serum cytokines, including IL-6, has been reported among anti-nuclear antibody-positive subjects after mercury exposure. Nyland et al. suggested that the decrease in cytokine concentration was a specific phenotype of mercury susceptibility.[21] A recent study in mice intoxicated with mercury showed increased levels of IL-6 in serum but not in the cerebellum.[53] IL-6 is a pleiotropic cytokine that acts as a neurotrophic factor and is crucial in the differentiation of oligodendrocytes and regeneration of peripheral nerves.[54] The decrease in IL-6 levels could be a factor contributing to the lower density of corneal nerves, branching density, and dendritic cell density observed in our patients.
In addition to the measurement of tear cytokine levels in the mercury-intoxicated patients, we performed PC and hierarchical agglomerative cluster analyses to explore correlation patterns among cytokine tear levels and the associations with the clinical findings. Based on the tear cytokine levels, we identified two patient clusters. Patients in Cluster 2 had significantly increased tear levels for 18 out of the 23 assayed cytokines, indicating a higher degree of ocular surface inflammation in this group. In agreement with this, the Cluster 2 patients also had significantly decreased tear lysozyme levels, indicating reduced tear production, compared to the patients in Cluster 1. Interestingly, in the same group, the nerve branching density and dendritic cell density were also lower than in Cluster 1. Because the maximum urine mercury levels were significantly higher in patients belonging to Cluster 2, this probably indicates a more intense mercury intoxication in dose and/or exposure time, and/or a higher susceptibility to mercury toxicity.[21]
Regarding our control groups, like the mercury-intoxicated patients, they were composed of males whose ages were not significantly different from that of the patients. IVCM was the only parameter compared with control values taken from three publications (two of them from our group), that included females and the ages were significantly different (see Table 4). While acknowledging that this is a limitation, we however feel that the comparisons are reliable because the corneal IVCM parameters that we analyzed do not change with age and are not associated with sex. [55–57]
Another potential limitation is that our patients were taking psychotropic drugs for their erethism mercurialis. Because all of the patients had a similar clinical picture and were taking similar drugs, it is unlikely that this unavoidable potential bias had any effect of the results of our evaluations.
In summary, we described a range of unreported ocular surface pathologies produced by mercury poisoning. We hypothesize that the DE-related symptoms experienced by the patients are due to mercury-related damage to the corneal innervation, corneal sensitivity, and tear cytokine disturbances. Thus, this DE could be described as neurogenic in origin, in contrast to the more classic tear-deficient and/or evaporative-DE subtypes.