Cholinesterase activity in the eye?

The visual system is regulated by the nervous through neurotransmitters, which play an important role in visual and ocular functions. One of those neurotransmitters is acetylcholine, a key molecule that plays a diversity of biological functions. On the other hand, acetylcholinesterase, the enzyme responsible for the hydrolysis of acetylcholine, is implicated in cholinergic function. However, several studies showed that in addition to their enzymatic functions, Acetylcholinesterase exerts non-catalytic functions. In recent years, the importance of evaluating all possible functions of acetylcholine-acetylcholinesterase has been evidenced. Nevertheless, there is evidence that suggests cholinesterase activity in the eye can regulate some biological events both in structures of the anterior and posterior segment of the eye and therefore in the visual information that is processed in the visual cortex. Hence, the evaluation of cholinesterase activity could be a possible marker of alterations in cholinergic activity not only in ocular disease but also in systemic diseases.


Introduction
The visual system is regulated by a variety of neurotransmitters among them glutamate, ácido gamma aminobutírico (GABA), adrenaline, and acetylcholine that contributed to the spread of nerve impulses in the different types of eye cells, and visual pathways, helping to control the autonomic motor and sensitive response 1-3 . Acetylcholine is a neurotransmitter (NT), that influences various biological processes as well as cell signaling, proliferation, and cellular differentiation, whereby the visual system and specifically the eye as a sensorial organ requires those NT 4,5 .
Several studies suggest the role of acetylcholine (Ach), as well as the molecules that intervene in its biogenesis and degradation, in the cortical response and cognitive functions as memory, locomotion, and perceptual learning 5 . The cholinergic signal modulates the synaptic transmission process in different groups of neurons and in the motor end-plate, for which, changes in the cholinergic activity would induce structural and anatomical changes of the cellular components 6 .
Although many studies evaluate cholinergic activity at the visual level through a functional test such as measurement of contrast sensitivity, pupil reflex, and tear secretion, few studies demonstrate the role of acetylcholinesterase (AChE) in the eye. Therefore, questions arise among them: Is it possible to evaluate the AChE in tear? What other functions could acetylcholinesterase play in the visual system? ¿Could AChE measurement serves as an indicator of neuronal condition? Therefore, this review will focus on the role of AChE in the visual system and eye (Figure 1), presenting its participation in different ocular tissues and its relevance in the diagnosis of ocular and neuronal diseases.

Cholinergic System
Acetylcholine is synthesized from acetyl Coenzyme A and choline mediated by choline acetyltransferase (ChAT), while acetylcholinesterase (AChE) is responsible for its degradation. Both ChAT and AChE are present in the site where Ach is required, such as the muscle, neurons, etc. 7,8 ; also, for acetylcholine to send the signal from one cell to another. In addition to its release, it is necessary that acetylcholine bind to specific receptors with high affinities such as muscarinic and nicotinic. Although both receptors present a great affinity for Ach, structural differences arise, being the nicotinic receptors or also called ionotropic transmembrane ion channels, while the muscarinic or metabotropic receptors are G protein-coupled receptors 9,10 .
Once Ach is bound with its receptors, it exerts an effector function in the cell and sends the signal to the other cells in a paracrine or autocrine manner 11 . For this, it requires the cholinergic activity to enter in a resting state and then reactivated again, for which the role of acetylcholinesterase is elementary for the degradation and to prevent the accumulation of Ach which could cause desensitization or over-stimulation in the receptors and thus affect biological events that depend on its activity 10,12 . However, it is noteworthy that some studies suggest the role of AChE not only for its catalytic activity but also for not-catalytic functions 13 , which makes the cholinergic system not only regulate synaptic transmission response but can modulate other functions neuronal and muscular, as we will see later.
The visual system is regulated by the nervous system and therefore, changes in the expression of certain neurotransmitter could affect vision 14 for the mammals include the human, the visually perceive requires not only optimal conditions in the media refractive of the eye and an adequate retinal response but require a visual pathway without any alteration so that the visual cortical areas that interpret the visual information. For this reason, authors such as Disney et al, have demonstrated in an animal model, the presence of nicotinic and muscarinic receptors in the primary visual cortex (V1), as well as the presence of cholinergic activity and cholinesterase in the lateral geniculate nucleus 15 . Thus, visual functions such as contrast sensitivity, color vision stereopsis, among others, could be modulated by the cholinergic system. Thus, as Han et al, experimentally demonstrated, the active visual activity leads to elevated expression of nicotinic receptors in visual cortical areas and changes in pupillary size may be related to muscarinic activity in the iris and pupillary sphincter 16 .
The visual system that includes the eye and the retina-genicular-cortical pathway requires the presence of the cholinergic activity and its action varies depending on the different nicotinic and muscarinic receptors present in each structure so that some changes in the cholinergic activity either in the synthesis or biogenesis of Ach as well as the degradation and binding of its receptors would result in a change in the answer in the visual function. Despite finding a large number of publications on the influence of the cholinergic system on visual function, very few articles report the activity of acetylcholinesterase (AChE) in the visual system, therefore this review will focus on the role of AChE in the visual system, presenting its participation in different ocular tissues and its relevance in the diagnosis of ocular and neuronal diseases. Throughout the document, an attempt will be made to explain some concepts that could solve some concerns about the enzyme responsible for degrading Ach: AChE

Acetylcholinesterase
It is an enzyme with hydrolase esterase function for Ach, being hydrolase in the ester type bond, structurally it appears as a homomeric assembly with three catalytic subunits H, T, and S, being different in the C-terminal region. The soluble form can found in the cell membrane and extracytoplasmatic regions 17,18 . Human AChE is encoded on chromosome 7q22 and three subunits of AChE are produced through alternative splicing, which differs in the site of cell localization, taking into account that the places where this enzyme is expressed are in cholinergic neurons, attached to the erythrocyte membrane and in the membrane of postsynaptic neurons including the neuromuscular junctions 17 .
The catalytic site of AChE allows the specific binding to Ach in an irreversible way, achieving the transformation into acetate and choline after hydrolysis, the latter can be reuptained by a choline transporter within the terminal nerve fibers 19 .

The non-enzymatic activity of AChE
AChE has been attributed important functions from neurodevelopmental to the modulation of various higher cognitive functions; from developmental, AChE acts as a nerve growth factor, stimulating the process of neurogenesis and synaptogenesis 13,20,21 . Indeed, it plays an important role in the development of neuroectoderm structures such as the ciliary body, retina, and optic nerve. In some in vitro models, the presence of AChE in the plasma region has been suggested where various non-catalytic motifs interact with laminin, collagen IV and intervene in cell adhesion, migration, and neuronal growth 22 , for which different ocular tissues of the neural origin may include such functions. Thus, Slotkin et al, observed in an in vivo model, that AChE intervenes in the formation of neurites and stability of neural networks 13 , suggesting that events that induce changes in the expression of AChE as exposure to pesticides, including Organophosphates and carbamates, could induce changes in the formation and growth of neuritis and thus the development of neurocognitive functions such as learning and memory 23 .

AChE activity in the posterior segment of the eye:
Retina and optic nerve: The characteristics of both the retina and optic nerve make them structures like windows of the nervous system (NS); in fact, some authors describe these structures as prolongations of the NS 24 . Consequently, the retinal neuronal activity includes the participation not only of NTs such as glutamate and GABA [25][26][27] . Besides, several studies show that retinal functionality requires autonomic activity. Indeed, some researchers have reported the presence of muscarinic receptors in cells as photoreceptors and amacrine, as well as the presence of nicotinic receptors in retinal glial cells (Müller Cell), bipolar and retinal ganglion cells 3,28,29 .
Although some studies focus on the evaluation of the parasympathetic cholinergic activity by determining the presence of Ach receptors 25,26 , other studies suggest instead, the evaluation of cholinesterase activity in retinal tissue as part of the parasympathetic evaluation. For instance, Nichols et al, demonstrated high esterase activity in rabbit retina, in different cell types of the internal layers, such as the amacrine and ganglion retinal cells 30 , which suggest that in intraretinal areas, nerve conduction requires cholinergic activity 31 . In addition, Glow & Rose suggest that changes in cholinesterase activity may be related to efferent pupillary response defects that may occur to retinal and optic nerve injury 32,33 .
The combination of clinical, histological, and molecular studies has allowed us to better characterize the cholinergic activity in the different layers of the retina, finding a relationship between retinal functional changes with parasympathetic dysfunctions, the foregoing by evaluating retinal cholinesterase activity 30

AChE activity in the Anterior segment of the eye:
Some research suggests the role of parasympathetic autonomic activity in the anterior segment to the presence of muscarinic receptors in the iris sphincter muscle, ciliary muscle, ciliary processes, trabecular meshwork, and lacrimal gland 37 . However, other studies demonstrate that ocular surface structures may be cholinergically regulated mediated by AChE activation as described below.

Uveal tract:
Parasympathomimetic activity regulates a large part of the iris functions, allowing to induce modifications in the pupillary response, and therefore in the amount of light stimulus that leads to the retina. Studies in rabbits and cats have found that the cholinesterase activity in the iris structure is increased in visual activities 38 . Interestingly, Hayakawa et al, found in a murine model that exposure to organophosphates whose objective is to inhibit cholinesterase, decreases AChE activity in the Edinger Westphal nucleus, iris, and pretectal nucleus, aspects that correlated with the pupillary miotic response 39 .
On the other hand, a high cholinesterase activity has been reported in pars plana and in the pigment epithelium of the ciliary body 40 , this has been demonstrated by analysis of the ciliary muscle before and after ciliary ganglionectomy, where cholinesterase activity is affected later to the surgical act 41 . The active presence of cholinesterase, both in the ciliary muscle and ciliary body could explain the association between uveitic processes associated with autonomic dysregulation 42 and in some cases with changes in the accommodative functions in individuals exposed to organophosphates, whose cholinesterase is decreased 43 .
Regarding the ciliary body, some differences between the results may be due to the animal model and the biochemical techniques used for the evaluation of AChE activity, for example, Gabelt et al, found in experiments with macaques a higher AChE activity that could be related to a greater parasympathetic demand on the ciliary muscle 44 . Another study found that inhibition of AChE activity reduces blood flow in the anterior uvea and induces pupillary miosis 45 . This suggests that cholinesterase activity plays a role in functions in the uveal tract to maintain intraocular homeostasis.

Anterior Chamber
Some studies suggest the presence of AChE in the anterior chamber, this being the one that contributes greatly to the nutrition and metabolism of the iris, ciliary and crystalline bodies. Previous studies in cats have described the activity of AChE from studies with paracentesis, where it is possible to obtain aqueous humor analysis 46 , this could explain to some extent, why autonomic dysregulation plays an important role in glaucoma diseases and how AChE inhibitors could be included in glaucoma pharmacotherapeutics 47 .
Consequently, for several decades it has been mentioned that from paracentesis it is possible to extract aqueous humor and evaluate cholinesterase activity. However, Appleyard et al. found that cholinesterase activity in ocular fluids is attributed more to true cholinesterase than to butyrylcholinesterase using Ellman´s method. Curiously, higher cholinesterase activity was observed in the vitreous humor and posterior cavities, so the authors suggest that the origin of this activity can occur from retinal tissue 35 . Despite, underestimating BuChE activity for years as a cholinergic indicator and recent studies in fluids such as saliva, and serum, attribute its importance in the measurement of cholinergic activity 48,49 . Therefore, the fact that there is cholinesterase activity of BuChE could be associated with the parasympathetic state.

Cornea:
The cornea is an avascular structure, which is in communication with the tear film and aqueous humor, this structure presents a sensitive response accompanied by somatosensory activity due to the presence of thermoreceptors, polymodal receptors, and mechanoreceptors, responding to sensations such as pain, temperature and chemical changes 50 . The reception of the visual stimulus generates a series of cellular and molecular events in the cornea, of which certain NTs such as substance P and acetylcholine 51 . In an animal model, cholinesterase activity has been described in chicken corneal extracts, as well as the AChE reaction in corneal layers such as corneal epithelium and stroma in rats and rabbits 52,53 , which could suggest a regulatory element of corneal sensoriality. Lasys et al. found cholinesterase activity in the corneal nerve plexus 54 and a study in corneal epithelial cell culture showed that the epithelium expresses mAchR, and AChE, which contribute to cell migration and survival 55 .
Consequently, some studies have evaluated the activity of both the specific cholinesterase and pseudocholinesterase in corneal nerve fibers in different species of animals, and changes in the corneal sensory response, could affect the functionality of AChE. Thus, raising the possibility of find changes in AchE activity in corneal alterations such as herpetic keratitis, diabetic keratopathy, dry eye, corneal surgery, these changes are related to changes in the morphology of corneal nerve fibers using in vivo microscopy 56 . Interestingly, a study suggests, the participation of the cholinergic system in the modulation of the immune response and corneal repair, taking into account the presence of Ach and its binding with receptors present in different types of immune cells such as macrophages and T lymphocytes 57 and therefore the consequent cholinesterase activity to allow the action of Ach.

Lacrimal Gland and conjunctiva
Although the expression of acetylcholine and choline acetyltransferase may be greater than cholinesterase, both in conjunctival and corneal epithelium, the role of their activity is unknown. Some reports, such as Wilson & McKean, associate the cholinergic activity in these epithelia in cellular repair processes, where the activity increases together with glycogen and oxidative enzymes 58 . On the other hand, the use of pesticides such as carbamates seeks cholinesterase inhibitors, can affect the cholinergic terminals in conjunctiva tissue 59 and this can be associated with the over-stimulation that occur in the muscarinic receptors located in conjunctival tissue, which generates a change in the effector response, resulting in the presence of symptoms such as eye irritation, burning, and redness. This may be associated with the activation of intracellular signals, such as the MAPK pathway, which can occur when faced with excessive cholinergic stimulation 60 .
In the case of Globet cells of the conjunctiva, in the pathophysiological process such as dry eye and allergic conjunctivitis, it can increase mucin production through the interaction of vasoactive intestinal peptide (VIP) and acetylcholine with muscarinic receptors M2 and M3, which can generate the activation of intracellular signaling cascades that induce the secretion of inflammatory mediators 61,62 . In this sense, it could be thought that cholinesterase is part of the regulatory elements to know when the binding of receptors with Ach must continue and when it must exert its hydrolysis function so that Ach is reuptained and the function is changed.
In the lacrimal gland, cholinergic activity can be mediated by the interaction between ACh and M3 receptors, located in secretory globular acinal cells and in myoepithelial cells to stimulate tear secretion 63 . Nevertheless, this secretion process must be counteracted with elimination mainly via the nasolacrimal duct. Therefore, excessive lacrimation generates an imbalance between tear secretion and excretion process, something typical in cholinergic syndrome associated with the use of pesticides such as cholinesterase inhibitors 64 . The above suggests that cholinesterase activity is necessary to maintain tear homeostasis.

Is it possible to find AChE activity in tear?
Tear fluid, in recent decades, has had great clinical importance, in the application of molecular and cellular biology techniques. From this fluid, its small volume has not impeded for it to search for biomolecules such as proteins, mRNA, enzymes, and even for small non-coding RNAs, that contribute to gene expression and are involved in the development of various ocular disorders such as dry eye, keratoconus, glaucoma, etc. 65-68 . Therefore, it is possible to think that from the tear fluid, information can be obtained on biomolecules that intervene not only in ocular homeostasis but also affect systemic diseases such as metabolic and neurological conditions. For this reason, ocular fluids could be a source of biomarkers [69][70][71][72] . This can be applied to the case of AChE, although some studies suggest that the activity of the enzyme occurs in different ocular structures that have parasympathetic activity, other studies, provide information on the cholinergic influence on tear secretion, therefore it is possible to think that both enzyme activity and its expression could eventually participate in the regulation of various functions in the anterior segment of the eye.
Several studies show that the measurement of AChE is possible in body fluid such as blood, serum, urine, saliva, and cerebrospinal fluid, so it could be speculated that through the tear fluid the activity can be evaluated by enzymatic assay of AChE or BuChE 49,73 , considering the volumes required for the reaction 74 . For example, Ellman´s method is one of the most used assays to evaluate BuChE, and although some authors describe its activity as not specified, others show that BuChE activity in saliva can provide relevant information on cholinergic alterations such as the area in two particular cases: Alzheimer´s disease and neurotoxicity due to pesticides, where it occurs as a loss of cholinergic neurons, two situations that area of the public health problems worldwide 48,75,76 .
Furthermore, Thiphom et al. demonstrated that in farmers exposed to pesticides, the plasma and saliva levels of BuChE are correlated in the two fluids and decreases in the population exposed to pesticides 49 , in this sense, the possibility of evaluating the enzymatic activity in tear fluids, is highly interesting due to its low cost and less invasiveness. Therefore, I suggest, research to evaluated non only AChE in saliva but also in tears using a test with greater sensitivity, even though the concentrations of some biomolecules that are evidenced in blood, in the tear show a lower concentration, and additional evaluation is correlated with other visual and ocular functions with cholinergic activity.

Future Directions
From the applications of molecular biology to health science, it has been possible to estimate several biological events that intervene in ocular diseases of clinical importance, such as Keratoconus, Dry eye, and Glaucoma 67,77,78 . These advances have allowed the application of new technological tools for the diagnosis and monitoring of ocular or systemic alterations from ocular structures 79 . Although to data, the publications on AChE in ocular structure is evident, the presence of the enzymatic activity of AChE in tear is unknown, so it could be a new research door that will not only be applied to visual professionals, but it could be a good source of research from the area of Neuroscience applied to scenarios such as neurotoxicity by pesticides, and Alzheimer´s disease, taking into account that the AChE evaluation in various fluids including "tears" it could be used as a biomarker of the disease and correlate it with visual and ocular functions that require cholinergic regulation.

Conclusions
The cholinesterase activity in the eye is very important in visual and structural functions, its necessary for manteinance and regulation in the cholinergic system, from enzymatic activity to not enzymatic. For this reason, its possible to think that in cholinergic disease (upregulation or downregulation), the AChE in eye affects its role and the structures of the anterior and posterior segment of the eye such as cornea, conjunctiva, lacrimal gland, uveal tract and retina. Ultimately, novel researchs suggest evaluate a variety of biomarkers in ocular and systemic disease, therefore, the AChE as part of cholinergic system could be evaluate through of tear film.
Author Contributions: S.C.D.C conceived the review paper.
Funding: This work was funded by Facultad Ciencias de la Salud, Universidad de la Salle (Bogotá, Colombia).
Ethical Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Conflicts of Interest:
The Autor declare no conflict of interest.