The Aging Lacrimal Gland: Changes in Structure and Function

Abstract

The afferent nerves of the cornea and conjunctiva, efferent nerves of the lacrimal gland, and the lacrimal gland are a functional unit that works cooperatively to produce the aqueous component of tears. A decrease in the lacrimal gland secretory function can lead to dry eye disease. Because aging is a risk factor for dry eye disease, study of the changes in the function of the lacrimal gland functional unit with age is important for developing treatments to prevent dry eye disease. No one mechanism is known to induce the changes that occur with aging, although multiple different mechanisms have been associated with aging. These fall into two theoretical categories: programmed theories of aging (immunological, genetic, apoptotic, and neuroendocrine) and error theories of aging (protein alteration, somatic mutation, etc). Lacrimal glands undergo structural and functional alteration with increasing age. In mouse models of aging, it has been shown that neural stimulation of protein secretion is an early target of aging, accompanied by an increase in mast cells and lipofuscin accumulation. Hyperglycemia and increased lymphocytic infiltration can contribute to this loss of function at older ages. These findings suggest that an increase in oxidative stress may play a role in the loss of lacrimal gland function with age. For the afferent and efferent neural components of the lacrimal gland functional unit, immune or inflammatory mediated decrease in nerve function could contribute to loss of lacrimal gland secretion with age. More research in this area is critically needed.

I. INTRODUCTION

Aging and female gender are risk factors for dry eye disease. In a single comprehensive study from the United States involving approximately 40,000 women, the prevalence of dry eye was 5.7% in women younger than 50 years of age, increasing to 9.8% in women over 75.¹ At any age, about twice as many women as men have dry eye. In eight large epidemiological studies of dry eye from around the world, the prevalence of dry eye varies from 5–30% in populations 50 years of age and older.² The considerable variation in prevalence exists, in part, because dry eye is a multifactorial disease, the criteria used to diagnose dry eye vary among studies, and a gold standard test to diagnose dry eye is not available. In an effort to standardize the study of dry eye, a revised consensus definition was recently developed,³ which defined dry eye as “a multifactorial disease of the tears and ocular surface that results in symptoms of discomfort, visual disturbance, and tear film instability with potential damage to the ocular surface. It is accompanied by increased osmolarity of the tear film and inflammation of the ocular surface.”³

Regulation of lacrimal gland secretion plays a critical role in the development of dry eye disease, as this gland is a major contributor to the tear film. The tear film has three layers: an outer lipid layer, a middle aqueous layer, and an inner mucous layer. The main lacrimal gland, along with the accessory lacrimal glands, the conjunctival epithelium, and the corneal epithelium, produce the aqueous layer. Hereafter, the main lacrimal gland will be referred to as the lacrimal gland. The conjunctival goblet cells secrete the mucous layer, and the meibomian glands secrete the lipid layer of the tear film.

Lacrimal gland secretion is stimulated by activation of sensory nerves in the cornea and conjunctiva (Figure 1). Bright light, cold temperature, mechanical stimuli, and specific chemicals stimulate the sensory nerves that send impulses to the brain. These impulses are registered as pain by the higher levels of the brain, but they also activate the efferent parasympathetic and sympathetic nerves that innervate the lacrimal gland. Nerves regulate secretion of electrolytes and water by the corneal and conjunctival epithelia and mucous by the conjunctival goblet cells. The corneal epithelium is another potential source of tears. However, the avascular cornea would most likely provide only a limited source for tears. Although the lipid-secreting meibomian glands are innervated, the role of nerves in regulating their secretion is unknown. Furthermore, the role of nerves in regulating the production and release of the membrane-spanning mucin producers in the corneal and conjunctival epithelium has yet to be studied. Overall, the evidence suggests that neural control of lacrimal gland, goblet cell, and conjunctival stratified squamous cell secretion is crucial to the production of a normal tear film and prevention of dry eye disease.

Figure 1.

Schematic of the neural regulation of lacrimal gland electrolyte, water, and protein secretion. Lacrimal gland secretion is stimulated by the sensory nerves in the cornea or conjunctiva, which, in turn, activate the efferent parasympathetic and sympathetic nerves that innervate the acini of the lacrimal gland. Lacrimal gland fluid flows onto the ocular surface through the lacrimal gland excretory ducts and is drained from the eye via the lacrimal drainage system. (Modified from Dartt DA.⁵⁵)

The amount and composition of tears is determined not only by tear secretion, but also by tear drainage and evaporation of tears from the ocular surface (Figure 1). Activation of sympathetic nerves controls tear drainage by regulating the lacrimal drainage system through the nasolacrimal duct to the nose.⁴ Nerves do not directly regulate tear evaporation, which is dependent upon lipid secretion and the external environment, but they indirectly regulate tear evaporation by controlling the amount of tear produced. Therefore, neural regulation of lacrimal gland secretion is a primary determinant of tear amount and composition.

Dry eye disease is categorized as aqueous deficiency or evaporative. According to the classical definition of dry eye, aqueous deficiency dry eye occurs when the volume or composition of the aqueous layer of tear is diminished by alteration in lacrimal gland secretion. A change in fluid secretion by the conjunctiva and cornea could also play a role in this type of dry eye. Evaporative dry eye is produced by changes in the lipid layer. Because aging produces changes that alter secretion of the lacrimal gland, it is a major risk factor for development of aqueous deficiency dry eye disease.

The lacrimal gland itself, the afferent nerves of the cornea and conjunctiva, and the efferent nerves of the lacrimal gland comprise a functional unit that works cooperatively to produce the aqueous component of tears that overlay the cornea and conjunctiva (Figure 1). All three components of this functional unit must be considered in characterizing the aging lacrimal gland, determining its effect on the ocular surface, and ascertaining its role in dry eye disease.

II. DEFINITION OF AGING

The National Institute on Aging of the National Institutes of Health (USA) described aging as follows. “Aging is a complex natural process involving every molecule, cell, and organ in the body. In its broadest sense, aging merely refers to changes that occur during the lifespan.”⁵ Many changes are superficial manifestations of aging, but are not harmful, eg, gray hair and wrinkles. Other age-related changes increase the risk of disease. It is important to make the distinction between the aging process and age-associated disease.⁶ Hayflick believes that research into age-related disease will not provide insight into the biology of age changes and that the fundamental question to be answered is: “What changes occur in biomolecules that lead to the manifestation of aging at higher orders of complexity and then increase vulnerability to all age-associated pathology?”⁶ This leads us to consider that age-related changes in lacrimal gland structure and function are not necessarily the same as those leading to aqueous deficiency dry eye, even though the prevalence of dry eye is age-dependent.^1,7 Thus, animal models of lacrimal gland aging are different from animal models of lacrimal gland-induced dry eye, although insight can be gained by comparing findings from both models. Furthermore, aqueous deficiency dry eye disease has a defined pathology that differs from that of aging.⁷

III. THEORIES OF AGING

Theories of aging can be classified as programmed theories and damage or error theories⁵ (Table 1)^8–35

Table 1.

Evidence to support aging theories in exocrine gland dysfunction

	Origin of change	Theoretical premise	Exocrine glands
	Genetic (programmed longevity)	Senescence is regulated by determined genetic pathways.	Pancreas, mammary, prostate, sebaceous glands8–11
Programmed theories	Neuroendocrine	Failure of integrative inputs lead to organ senescence.	Lacrimal, mammary, prostate, sweat, sebaceous, salivary glands10,12–16
	Immunological	Organ function depends on immune system action, including regulation of neuroendocrine system.	Lacrimal, mammary, prostate, salivary, sweat glands17–19
	Apoptosis	Programmed cell suicide is induced by extracellular signals.	Lacrimal, prostate, sweat, sebaceous, salivary glands14,20,23
Error theories	Protein alterations (wear and tear)	Time-dependent change in a molecule related to conformational or enzyme activity.	Lacrimal, mammary, prostate, salivary glands9,19,24,27
	Somatic mutations	Alterations in genetic information and decrease in cell efficiency.	Mammary, prostate, salivary glands8,11,28,30
	Oxidative stress (free radicals)	Lower oxidative damage and/or antioxidant capacity increase longevity.	Lacrimal, pancreas, mammary, prostate, salivary, sebaceous glands10,27,31
	Metabolic (oxidative stress)	Hyperglycemia and free radicals induce inflammation and oxidative damage.	Pancreas, lacrimal, mammary, salivary glands26,27,32,33
	DNA damage	DNA repair efficiency decreases with aging.	Pancreas, mammary, prostate, salivary, sweat glands28,31,34,35
	Posttranslational	Modification of structural molecules impairs tissue function.	Mammary, prostate, salivary, sweat glands

Programmed theories include:

1. Programmed longevity: Aging is the result of the sequential switching on and off of certain genes.

2. Endocrine theory: Biological clocks act through hormones to control the pace of aging.

3. Immunological theory: A programmed decline in immune system functions leads to an increased vulnerability to infectious disease and, thus, aging and death.

Components of error theories include:

1. Wear and tear: Cells and tissues have vital parts that wear out.

2. Rate of living: The greater the organism’s rate of oxygen basal metabolism, the shorter the life span.

3. Crosslinking: An accumulation of crosslinked proteins damages cells and tissues, slowing down bodily processes.

4. Free radicals: Accumulated damage caused by oxygen free radicals causes cells and eventually organs to stop functioning.

5. Somatic DNA damage: Genetic mutations occur and accumulate with increasing age, causing cells to deteriorate and malfunction. In particular, damage to mitochondrial DNA might lead to mitochondrial dysfunction.

Although substantial research has been done in each of the areas mentioned above, most of it is correlative, and there is no consensus that the process described in one or more of these theories is the one responsible for induction of aging. Considerable evidence has demonstrated that many of these changes occur during aging, but aging can occur in the absence of these changes, and these changes can occur with age but not lead to aging. Lacrimal gland research has demonstrated that endocrinological changes, immunological alterations, increased protein crosslinking, and increased free radical-mediated oxidative stress occur during the aging process. These processes lead to alterations in lacrimal gland structure and function. The most important alteration appears to be the decrease with age of neural activation of cellular signaling pathways, leading to loss of lacrimal gland secretion of protein, electrolytes, and water, and resulting in aqueous deficiency dry eye disease.

In the present review, we will: 1) describe the aging tear film and how this reflects changes in lacrimal gland secretion; 2) describe the normal structure and function of the lacrimal gland and how this changes with age; 3) compare age-dependent changes in the lacrimal gland with those in a model of aqueous-deficiency dry eye (Sjogren syndrome); and 4) discuss evidence for the different theories of aging in the context of the lacrimal gland.

IV. THE AGING TEAR FILM

With such a complex system regulating tear secretion, it is not surprising that the evidence for the effect of age on the tear film and ocular surface is inconsistent. One important reason for the inconsistent results is that decreases in function of one component can be compensated for by the other regulatory components.³⁶ For example, a decrease in tear secretion can be compensated for by a decrease in tear drainage, which would mitigate the decrease in tear volume. A second reason for inconsistency is the inaccuracy or variability of the methods used to measure tear function. Finally, results can be complicated by use of different protocols for the same technique, ^36,37 although efforts at standardization have been made.

Although wide discrepancies may exist in absolute values of the same measurement from multiple studies, for some parameters of the tear film, the trends in change are consistent. There is consensus that reflex tear secretion, as measured by the Schirmer test in the unanesthetized eye, decreases with increasing age when investigated in the 20–80-year-old age range.³⁶ Reflex tear secretion is predominantly the product of neural stimulation of lacrimal gland fluid secretion; thus, changes in the neural reflex arc and the lacrimal gland itself are expected to occur with aging and could account for the decrease in the unanesthetized Schirmer test. Along with a decrease in the rate of tear secretion, there is an age-related decrease in tear volume and in the concentration of the tear proteins lysozyme and lactoferrin.^38–44 Because lysozyme and lactoferrin are proteins secreted by the lacrimal gland in response to neural stimulation, it appears that their decrease with age indicates a decrease in neural stimulation of the lacrimal gland.

V. LACRIMAL GLAND STRUCTURE AND FUNCTION

A. Structure

The lacrimal gland consists predominantly of acinar cells that form grape-like structures or tubules, depending upon the species. About 80% of the lacrimal gland is acinar cells, 12% ductal cells, and the remainder plasma cells, blood vessels, and nerves. The types of acinar cell varies among species.⁴⁵ In some species, they are all serous, and in others, they are mixed serous and mucous.⁴⁶ The acinar cells are linked to each other and are polarized. Nerves and blood vessels surround the basolateral side of the acinar cells and initiate secretion. The stellate-shaped myoepithelial cells are in closest apposition to the basal membranes of both acinar and duct cells and surround the basal area. Polarized secretion of protein, electrolytes, and water occurs across the luminal (apical) membrane into the ductal system. Secreted lacrimal gland fluid flows through the ductal system and is modified by the single layer of cuboidal duct cells. Lacrimal gland fluid exits the gland onto the surface of the eye through the lacrimal gland excretory duct, the terminal portion of the duct system.⁴⁵

There are considerable gender-based differences in lacrimal gland structure. After puberty, female lacrimal glands are smaller than male glands, but represent a larger portion of body weight than do male glands.⁴⁷ Female lacrimal gland acini are smaller in area than male glands, but they have more acinar cells per acinus than males. When studying aging of the lacrimal gland, it is important to take gender-based differences into consideration.

B. Function

1. Mechanism of Protein Secretion

Proteins are synthesized and secreted by the lacrimal gland in three ways: regulated protein secretion, constitutive protein secretion, and ectodomain shedding. Proteins secreted by the regulated pathway are synthesized in the endoplasmic reticulum, modified in the Golgi apparatus, and stored in secretory granules in the apical portion of the acinar cells. Upon the appropriate stimulus, the secretory granule membranes fuse with the apical membrane and release the contents of the secretory granule into the apical lumen. Thus, regulated protein secretion is controlled by regulation of protein release (exocytosis). Proteins secreted by the regulated mechanism in the lacrimal gland include peroxidase, lysozyme, and lactoferrin, whose release is stimulated by activation of parasympathetic and sympathetic nerves.⁴⁵

Proteins secreted by the constitutive pathway also are synthesized in the endoplasmic reticulum, modified by the Golgi apparatus, and packaged in secretory granules. These secretory granules are not stored, but rather they immediately fuse with the luminal membranes, releasing the proteins into the lumen. In this way, secretion is controlled by regulation of protein synthesis. A protein secreted by the constitutive pathway in the lacrimal gland is secretory IgA, which is secretory component bound to polymeric IgA. Secretory component is synthesized by lacrimal acinar cells, whereas polymeric IgA is synthesized by plasma cells and transported into the acinar cells.⁴⁸ Synthesis of secretory component is regulated by the steroid hormone androgens.⁴⁹ Cholinergic agonists also have a complex effect on secretory component synthesis and secretion.

An important component of the constitutive pathway is trancytosis, ie, the transport of material across an epithelium by uptake on one face into a coated vesicle, which then can be transported to the opposite face in another vesicle Many tear proteins, such as IgA (through its transport by the polymeric IgA receptor), transferrin, and albumin, are trancytosed. The newly synthesized receptors first traffic to the basolateral membrane and then to the apical membrane, where they may pick up protein cargo.

Proteins secreted by ectodomain shedding are synthesized in the endoplasmic reticulum and inserted into cellular membranes, as they contain a transmembrane domain. They are trafficked to the basolateral and apical membranes of both acinar and duct cells. These proteins consist of an extracellular domain that extends into the extracellular space, a transmembrane domain, and an intracellular domain that reaches into the cytoplasm. The extracellular domain of the protein is released from the cell by enzymatic cleavage at a specific extracellular cleavage site in the protein. Stimulation of intracellular signaling pathways activates membrane-bound matrix metalloproteases to cleave the membrane-bound proteins.⁵⁰ Proteins secreted by ectodomain shedding in the lacrimal gland include epidermal growth factor (EGF) and the other members of its family of growth factors whose release is regulated by activation of sympathetic nerves.⁵¹ Also secreted by ectodomain shedding is the polymeric immunoglobulin receptor (pIgR). When pIgR does not bind to IgA to form secretory IgA (sIgA), its cleaved extracellular domain is released by ectodomain shedding and is known as secretory component (Figure 2).⁵²

Figure 2.

Polymeric immunoglobulin receptor (pIgR) trafficking and secretory component (SC) secretion in lacrimal gland acini, pIgR is synthesized in the endoplasmic reticulum (ER). pIgR exits from the trans-Golgi network (TGN) in two groups. One group exits via the regulated secretory vesicles (SVs), and the other group is packaged into vesicles to be inserted into the basolateral membrane. Additionally, pIgR inserted into the basolateral membrane may be endocytosed and transported through a series of endosomal compartments along the constitutive transcytotic pathway (red arrows). BE, basolateral endosomes; dlgA, dimeric IgA; sIgA, secretory IgA. (Modified from Evans et al.⁵²)

Lacrimal gland secretory proteins play a pivotal role in maintaining the health of the ocular surface and protecting it from the extracellular environment. For example, the EGF family of growth factors regulates proliferation of the cornea and conjunctiva, especially after injury, and some of this EGF may come from the lacrimal gland. Also, peroxidase, lysozyme, and lactoferrin protect the ocular surface from bacterial infection. Similarly, secretory IgA is part of the adaptive immune system and also protects from infection.

2. Mechanism of Electrolyte and Water Secretion

For lacrimal gland acinar cells, the Na⁺,K⁺-ATPase (Na⁺ pump), located in the basolateral membrane of acinar and ductal cells, hydrolyzes ATP to produce energy to cause the efflux of Na⁺ from the cell and the influx of K⁺ into the cell across the basolateral membrane against their electrochemical gradients (Figure 3).⁵³ Na⁺/H⁺ exchangers, C1⁻/HCO₃⁻ exchangers, and Na⁺, K⁺, 2C1⁻ co-tranporters use the energy from the Na⁺ pump to induce Cl⁻ influx into the cell across the basolateral membrane and an outwardly directed Cl⁻ efflux across that apical membrane. Cl⁻ channels in the apical membrane facilitate Cl⁻ efflux, which produces a negative potential difference in the lumen that causes a secretory flux of Na⁺ through the paracellular pathway into the lumen. K⁺-selective channels let K⁺ ions recycle across the basolateral membrane, so that the acini produce a Na⁺-Cl⁻ rich fluid. For duct cell secretion, the K⁺ and Cl⁻ channels are both in the apical membrane, so that a K⁻-Cl⁻rich fluid is secreted.⁵⁴ Activation of parasympathetic and sympathetic nerves stimulates electrolyte and water secretion by activating the K⁺ and Cl⁻ channels in the basolateral and apical membranes of the acinar and ductal cells.

Figure 3.

Electrolyte transport mechanisms driving water secretion across lacrimal gland acinar epithelium. NKA, Na⁺,K⁺-ATPase; AE, anion (CI⁻/HCO3⁻) exchanger; NHE, Na⁺/H⁺ exchanger; NKCC, Na⁺-K⁺-2CI⁻ cotransporter. (Reprinted from Selvin et al⁵³ with permission of the authors and Am J Physiol Cell Physiol.)

3. Regulation of Secretion

Reflex activation of the efferent parasympathetic and sympathetic nerves that innervate the lacrimal gland causes secretion of proteins, electrolytes, and water. Parasympathetic nerves are the major stimuli for secretion of both regulated protein and electrolyte/water. Parasympathetic nerves release the neurotransmitters acetylcholine (Ach) and vasoactive intestinal peptide (VIP). In brief, Ach causes secretion by binding to M3 muscarinic Ach receptors that activate phospholipase C to increase the intracellular Ca²⁺ concentration and stimulate protein kinase C activity. VIP binds to VIPRI (VIPAC1) receptors that activate adenyl cyclase to produce cAMP. cAMP activates protein kinase A to stimulate secretion. VIP also induces the influx of extracellular Ca²⁺.

Sympathetic nerves release norepinephrine, which binds to α_1D-adrenergic receptors and β-adrenergic receptors. Activation of α_1D-adrenergic receptors stimulates endothelial nitric oxide (NO) synthase to produce NO. NO activates guanylate cyclase to produce cGMP, which stimulates regulated secretion. Stimulation of α_1D-adrenergic receptors also causes an influx of extracellular Ca²⁺ without stimulation of phospholipase C. Stimulation of β-adrenergic receptors causes regulated secretion by producing cAMP, as does VIP. α_1D-Adrenergic receptors are more effective stimuli than β-adrenergic receptors. Detailed discussion of these signaling pathways is provided in a recent review by Dartt.⁵⁵

Ectodomain shedding of EGF is stimulated by α_1D-adrenergic receptors by activation of the matrix metalloproteinase ADAM17.⁵¹ The released EGF functions by binding to the EGF receptor that activates phospholipase Cγ. This activation increases the intracellular Ca²⁺ concentration and stimulates protein kinase C activity, which then induces regulated protein secretion.⁵⁶ The details of the regulation of EGF ectodomain shedding and the signaling pathway activated by the shed EGF are described by Tepavcevic et al⁵⁶ and Chen et al.⁵¹

4. Gender-Based Differences

Significant gender-related differences exist in the anatomy, physiology, and pathophysiology of the lacrimal gland.⁵⁷ These disparities are found in multiple species, although there are significant differences between species. These gender-based disparities represent differences in lacrimal gland structure and function that could influence how the lacrimal gland ages in each sex.

Most studies on the functional differences in lacrimal gland secretion between males and females have been performed in the rat or rabbit. Measurement of regulated protein secretion indicates that cholinergic agonist stimulation of total protein and peroxidase secretion did not differ between male and female rats,⁵⁸ nor did the activity of β-adrenergic receptors.⁵⁹ An important distinction is that total protein consists of protein secreted by the regulated, constitutive, and ectodomain shedding mechanisms; however, peroxidase is secreted by regulated secretion only. In contrast to total protein secretion, peroxidase secretion, and activity of β-adrenergic receptors, the activity of Na⁺,K⁺-ATPase, cholinergic receptors, galactosyltransferase, and alkaline phosphatase was demonstrated to be greater in female rabbits than in males.⁵⁹

From the existing evidence, a clear effect of gender on lacrimal gland protein and fluid secretion cannot be drawn. It appears that some functions, such as secretory IgA production, are altered by gender, but other functions, such as neurally stimulated protein secretion, appear not to be gender-related. For additional functions, such as fluid secretion, the role of gender has not been directly studied.

Knowledge of the pathways that control lacrimal gland secretion and the cellular mechanisms used by these individual pathways provide the basis for determining the dysfunction that could occur in the aging process. Studies of the gender effects on lacrimal gland structure and function, although not definitive, suggest that gender differences must be taken into account in studies of the aging lacrimal gland. This is particularly critical, as female sex and androgen deficiency, along with aging, are “mostly consistent” risk factors for dry eye disease.²

VI. EFFECT OF AGING ON LACRIMAL GLAND STRUCTURE AND FUNCTION

A. Structure

Aging alterations in lacrimal gland structure begin in middle age in both humans and experimental animals. In humans, acinar atrophy and fibrosis first occur in younger patients, but increase with age.⁶⁰ This is accompanied by dilation and increased tortuosity of secretory ducts, suggesting ductal obstruction. These ductal and acinar changes occur in the same region, suggesting that the ductal obstruction could account for the acinar atrophy.⁶¹ Thus, with middle age there is a gradual decline in acinar mass that begins in early adulthood.

In humans, lymphocytic infiltrates containing lymphocytes and plasma cells are detected in the lacrimal gland. Lymphocytic infiltrates were found in about 63% of the lacrimal glands, and the incidence of lymphocytic infiltration was higher in individuals 40 years of age and older.⁶² The infiltrates occurred mostly in the periductal and periacinar areas and extended into the interacinar areas. Both Damato et al⁶⁰ and Nasu et al⁶² suggested that lymphocytic infiltration is related to fibrosis as a chronic, perhaps episodic, inflammatory disease. However, not all glands that are fibrotic have evidence of inflammatory lesions.

Use of animal models, such as the aging mouse or rat, can be used to determine the natural history of age-related changes in lacrimal gland structure and to compare structural changes with functional ones. Structural changes that paralleled those detected in the aging human lacrimal gland occurred in the mouse at 12 months of age (middle age) and progressed with age up to 32 months (elderly). These changes were increased acinar atrophy, acinar fibrosis, periductular fibrosis, interlobular ductal dilation, and interlobular proliferation.⁶³ In male rats, Draper et al^64,65 found that the lacrimal gland acini were predominantly serous in young animals, whereas older animals had an increased percentage of mucous acini. Structural changes in aging mouse and rat lacrimal glands parallel those in human, except for the serous to mucous acinar differentiation.

B. Function

The first functional studies on the effect of aging on lacrimal gland secretion used the male rat as the animal model and measured the effect of several agonists on total protein and peroxidase secretion.⁶⁶ The cholinergic agonist carbachol stimulated secretion of total protein, which was decreased in 24-month-old rats compared to 4-month-old rats. In contrast, stimulated peroxidase secretion was the same in 24-month old compared to 4-month old rats. When induced by the α₁-adrenergic agonist phenylephrine, neither total protein nor peroxidase secretion differed in aged vs. younger rat lacrimal glands.⁵⁸ Finally, when the β-adrenergic agonist isoproterenol plus a cAMP phosphodiesterase inhibitor, which increases cellular cAMP levels, was used, aged animals secreted less total protein and peroxidase than did younger ones.

The effect of agonists on secretion of total protein and of peroxidase could differ, as they are secreted by different mechanisms. Peroxidase secretion is produced by regulated secretion, but total protein is produced by all three types of secretion: regulated, constitutive, and ectodomain shedding. The effect of the three different agonists on secretion could differ, as each agonist activates a separate, distinct signaling pathway to stimulate regulated protein secretion. Cholinergic agonists use phospholipase C, Ca²⁺ and protein kinase C^67–69; α₁-adrenergic agonists use NO and cGMP⁷⁰; and β-adrenergic agonists plus a cAMP phosphodiesterase inhibitor use cAMP.⁷¹ Thus, in these first experiments, the effect of aging on lacrimal gland protein secretion depended upon the type of secretion being measured and the cellular signaling pathway activated.

A second study by Draper et al⁶⁵ used male rat lacrimal glands at 3–5, 9, 12, 20, 24, and 28 months of age and obtained results that were somewhat inconsistent with the first study by Bromberg and Welch.⁶⁶ In the Draper study, acetylcholine-stimulated peroxidase and total protein secretion were both decreased in older compared to younger animals.⁶⁵ However, acetylcholine is a muscarinic agonist similar to carbachol, but acetylcholine has a much shorter duration of action, as it is inactivated by cholinesterases. Peroxidase secretion was decreased at an earlier age (12 months) than total protein secretion, which was decreased at 20 months of age.⁶⁵ Furthermore, the concentration of acetylcholine that caused maximum secretion was about 100-fold less for total protein than for peroxidase secretion.⁶⁵ Secretion induced by agonists other than cholinergic agonists was also altered with increasing age.⁷³ The parasympathetic neuropeptide VIP, the sensory neuropeptide substance P, histamine, and serotonin each caused decreases in total protein and peroxidase secretion in older (24-month-old) compared to younger (3–5-month-old) rats.⁷³

In contrast to the findings of Bromberg and Welch,⁶⁶ the experiments of Draper et al⁶⁵ and Williams et al⁷² found that aging decreased secretion of protein independent of the mechanism used for secretion or the signaling pathway activated. Aging decreased protein secretion by the regulated and constitutive pathways. The effect of aging on ectodomain shedding was not measured. Aging also decreased secretion stimulated by the phospholipase C, intracellular Ca²⁺, and protein kinase C pathway (acetylcholine, Sub P, histamine, serotonin) and the cAMP and Ca²⁺ pathway (VIP).^58,63,73 Although the signaling pathways used by Sub P, histamine, and serotonin have not been investigated in the lacrimal gland, all three agonists are likely to use phospholipase C, Ca²⁺, and protein kinase C to stimulate secretion.

In a third study using male mice, Rios et al⁶³ used high KCl levels (which depolarizes nerves to activate release of their neurotransmitters), the cholinergic agonist carbachol, or the α₁-adrenergic agonist phenyleprhine to stimulate peroxidase secretion. Secretion induced by all three stimuli plummeted at 8 months compared to 3 months of age and remained decreased as long as 24 months of age (Table 2). High KCl stimulation releases neurotransmitters from all three types of nerves in the lacrimal gland—parasympathetic, sympathetic and sensory-- but as parasympathetic nerves predominate, the primary effect on secretion should be by acetylcholine and VIP. Zoukhri et al⁷⁴ demonstrated that acetylcholine was the predominant agonist released, as atropine blocked high KCl stimulated secretion by about 70%. The experiments of Rios et al⁶³ suggest that the signaling pathways activated by cholinergic agonists (phospholipase C, Ca²⁺, and protein kinase C) and α₁-adrenergic agonists (NO and cGMP) are decreased relatively early (middle age) in the aging process.

Table 2.

Age-dependence of structural and functional changes in the mouse lacrimal gland

	Age (months)
	3	8	12	24	32
Structural changes	−	−	+/−	+	+
Decrease in peroxidase secretion	−	+	+	+	ND
Decrease in innervation	−	−	−	+	+
Decrease in Ach release	−	ND	−	+	ND
Increase in number of lymphocytes	−	−	−	+	+
Increase in number of mast cells	−	+	+	+	+
Increase in lipofuscin accumulation	−	+	+	+	+

ND = Not done

− = No change

+ = Change

A consensus appears to have emerged that regulated protein secretion (peroxidase) decreases as a function of age, the loss of function is independent of the signaling pathway activated, and the decrease occurs during middle age. Thus, alteration in lacrimal gland structure and decrease in function both occur at middle age.

Rios et al further investigated the mechanisms responsible for the inhibition of neural induction of secretion that was detected at 8 months of age.⁶³ Changes in the structure and function of efferent (parasympathetic and sympathetic) nerves were first investigated (Table 2). The following possibilities could have accounted for the decrease in neural stimulation of secretion: 1) a decrease in the number of efferent nerves in the lacrimal gland parenchyma; 2) prevention of neurotransmitter release from the efferent nerves; 3) blockage of neurotransmitter binding to its receptor; or 4) inhibition of the intracellular signaling pathways. The number of efferent parasympathetic and sympathetic nerves that surround the lacrimal gland acini does decrease with age, but the alteration was not apparent until 24–32 months of age^63,72 and thus could not account for the loss of secretion at 8 months of age. The amount of acetylcholine released by high KCl stimulation also decreases with age, but the inhibition did not occur until 24 months of age. Thus, neither loss of nerves nor inhibition of neurotransmitter release could account for the decrease in secretion at 8 months of age. Secretion stimulated by exogenous cholinergic and α₁-adrenergic agonists decreases at the same age as KCl-stimulated secretion. All the findings suggest that it is a change in either the binding of the agonists to their receptors or in the signaling pathways activated by the receptors, but not the efferent nerves, that causes the early loss of secretion in the lacrimal gland. Loss of efferent nerves or nerve function could, however, play a role in the sustained loss of secretion at older ages.

To determine the mechanism for the loss of nerve and agonist-stimulated lacrimal gland secretion, changes in lacrimal gland itself were investigated. Two alterations that occurred at an early age were an increase in intracellular lipofuscin inclusions and parenchymal mast cell numbers detected at 8 months of age⁶³ (Table 2). An increase in mast cells was also documented by Williams et al,⁷² who detected an increase in lacrimal gland mast cell infiltration at 24 months compared to 3–5 months of age. In this latter study, no intermediate ages were studied.

Because an age-dependent increase in lymphocytic infiltration was reported for human lacrimal glands,⁶¹ lymphocyctic infiltration was evaluated in the aging mouse. In the mouse lacrimal gland, a detectable increase in lymphocytes was first documented at 24 months of age.⁶³ Thus, an increase in intracellular lipofuscin accumulation and mast cell, but not lymphocyte, infiltration could contribute to the early age-dependent loss of neural and agonist-stimulated lacrimal gland secretion. Lipofuscin accumulation suggests that oxidative stress could be damaging lacrimal gland secretion. Mast cell accumulation suggests that their release of histamine, trophic factors, and proinflammatory cytokines could destroy lacrimal gland secretion. That lymphocyte accumulation occurs later than the loss in secretion implies that lymphocyte-mediated inflammation is not playing a role in the early loss of secretion, but, along with loss of efferent nerve and nerve function, could play a role in the later sustained inhibition of secretion.

The afferent sensory nerves of the cornea drive lacrimal gland secretion by activating the efferent parasympathetic and sympathetic nerves that innervate the lacrimal gland. An age-dependent decrease in the number or function of afferent nerves could be responsible for the early loss of lacrimal gland secretion, even though the number and function of efferent nerves were not. The structure and function of afferent nerves has been investigated in both humans and rats. In humans, a decrease in corneal sensitivity with increasing age, as measured with a non-contact corneal esthesiometer, has been reported.⁷³ The change in sensitivity in humans did not appear to be due to a change in the density or orientation of subbasal nerves in the central cornea.⁷⁶ When the density of corneal nerves was more closely examined in the rat cornea, the density of nerve terminals decreased linearly with increasing age from 6 to 12 months, but the density of subbasal nerves increased. Because pain receptors are localized to the nerve terminals, the age-dependent decrease in nerve terminals correlates with the age-dependent decrease in corneal sensitivity. Thus, the driving force for lacrimal gland secretion could decline as a function of age. A defect in the afferent sensory neural regulation of lacrimal gland secretion could potentially account for a portion of the loss of function that occurs with aging, but substantial further investigation is required to correlate alterations in sensory nerve terminals, sensory nerve sensitivity, and lacrimal gland secretion with age.

C. Comparison of Mouse Models for Aging and Sjogren Syndrome Dry Eye

Several murine models exist for the autoimmune form of dry eye known as Sjogren syndrome. One model, the MRL/MpJ-Faslpr/J (MRL/lpr) mouse, has a spontaneous mutation in Fas that causes an age-dependent accumulation of lymphocytic foci, the hallmark of Sjogren syndome, in the female lacrimal gland. The changes in lacrimal gland structure and function occur more rapidly in the MRL/lpr mice compared to normal mice, and the MRL/lpr mice usually die at about 5–6 months of age compared to about 36 months for normal mice. Lymphocytes first appear at about 2 months of age in the MRL/lpr mouse lacrimal gland. In the 4.5-month old mice, high KCl-induced, but not α₁-adrenergic agonist-induced, peroxidase secretion was blocked compared to control mice (Table 3).⁷⁴ The KCl-induced secretion was predominantly from release of cholinergic agonist from parasympathetic nerves. Further experimentation showed that the number of efferent nerves in the acinar and ductal areas of the lacrimal gland did not decrease, even at 4.5 months of age. The areas of lymphocytic infiltration did not contain nerves and were not examined. Thus, a loss of efferent nerves surrounding the acini and ducts did not account for the loss of neural stimulation of secretion. However, the infiltrating lymphocytes synthesized and secreted the inflammatory cytokines IL-1β and TNF-α that prevented the nerves from releasing their neurotransmitters.⁷⁷ Thus, a decrease in nerve function occurred at the same time as the blockage of secretion. The nerve impairment led to a denervation-like state, in which neural activation of secretion was blocked, but the response to exogenous addition of agonists was increased. This is the hallmark of denervation supersensitivity.

Table 3.

Comparison of age-dependent structural and functional changes in the lacrimal gland in murine models of autoimmunity and aging

	MRL/MpJ-Faslpr/J1		BalbC2
Age	2 wks	18 wks	3 mo	18 mo
Decrease KCI secretion	−	+74	−	+63
Decrease in α1D-adrenergic agonist-stimulated secretion	−	−74	−	+63
Lymphocytic accumulation	−	+79	−	−63
Decrease in innervation	−	−79	−	−63
Decrease in Ach secretion	−	+74	−	−63

¹Model of autoimmunity

²Model of aging

Evidence that denervation supersensitivity had occurred in the MRL/lpr mice was a profound increase in the intracellular Ca²⁺ response to the cholinergic agonist carbachol or the α₁-adrenergic agonist phenylephrine that correlated with an increase in lymphocytic infiltration.⁷⁸ None of the changes described for the MRL/lpr mice were detected in the control MRL/++ mice. Thus, in the mouse model of Sjogren syndrome, the development of inflammation, as indicated by the lymphocytic infiltration and production of pro-inflammatory cytokines, accounted for the loss of neurally-induced lacrimal gland secretion (Table 3).⁷⁹ Agonist-induced secretion was not lost in the MRL/lpr mouse lacrimal gland.

The results in the mouse model of autoimmune disease are in contrast to those in the mouse model of aging, in which an increase in oxidative stress (lipofuscin accumulation) and mast cell infiltration led to an early loss of both neural and exogenous agent stimulation of secretion (Table 3). No increased intracellular Ca²⁺ response to agonists occurred in the aging mouse lacrimal gland, as occurred in the MRL/lpr mice (Rios, unpublished results). Furthermore, lymphocyte-mediated inflammation and loss of nerve function occurred much later than the loss of lacrimal gland secretion in the aging mice as compared to a simultaneous loss of nerve function, lymphocyte infiltration, and loss of secretion in the autoimmune mice. Finally, in the aging mice, agonist-stimulated secretion was inhibited at the same time as neural-stimulated secretion, but in the MRL/lpr mice, agonist-stimulated secretion was unchanged. Thus, the loss of lacrimal gland function that occurs with aging is mediated by processes different from those that mediate lacrimal gland secretory impairment in the autoimmune dry eye disease Sjogren syndrome.

VII. THE ROLE OF OXIDATIVE STRESS IN LACRIMAL GLAND AGING

The oxidative stress theory of aging suggests that damage from free radicals, such as reactive oxygen species (ROS), plays a role.⁸⁰ The current oxidative stress theory is: “A chronic state of oxidative stress exists in cells of aerobic organisms even under normal physiological conditions because of a balance of pro-oxidants and antioxidants. This imbalance results in a steady-state accumulation of oxidative damage in a variety of macromolecules. Oxidative damage increases during aging, which results in a progressive loss in the functional efficiency of various cellular processes.”⁸¹

This theory has recently been expanded to include the mitochondrial theory of aging, which identifies a vicious cycle in which mutations in mitochondrial DNA result in respiratory chain dysfunction that leads to increased ROS production, which, in turn, causes further mutations in mitochondrial DNA.⁸² Substantial correlative data shows that oxidative stress and increased mitochondrial mutations occur with aging, but these data do not exclude the possibility that mitochondrial damage and ROS production are consequences of aging, rather than the driving force for aging.

An increase in oxidative stress is correlated with lacrimal gland aging, and the progressive accumulation of cellular lipofuscin granules in the lacrimal gland with increasing age suggests a role for oxidative stress.³⁷ Lipofuscin is composed of highly oxidized and cross-linked proteins.⁸³ It is insoluble, not degradable by lysosomal enzymes, and its accumulation shortens the life of the cell. Lipofuscin accumulation depends on the rate of oxidative damage to proteins and on how well mitochondrial repair systems, the proteosomal system, and lysosomes are functioning. In addition to an increase in lipofuscin accumulation, the aging mouse lacrimal gland shows an elevation in cellular indicators of oxidative stress, such as lipid peroxidation, reduced glutathione levels, and inducible nitric oxide synthase (Rios et al. IOVS 48:2007 ARVO Eabstract 2007, #5659). Because peroxidase is synthesized and stored in the lacrimal gland at particularly high levels in the rat and mouse compared to the rabbit and human lacrimal gland and is an oxidizing agent, peroxidase measurement cannot be considered as a parameter in any study of oxidative stress.

Increased oxidative stress can result from hyperglycemia and cellular resistance to insulin in the setting of elevated blood insulin levels. Rocha et al^26,84 demonstrated that older rats (20-month-old) compared to 2-month-old rats were significantly hyperinsulinemic and had a reduced rate of blood glucose disappearance in response to insulin. Aging was found to impair one branch of the insulin-signaling pathways in the rat lacrimal gland.²⁷ Insulin binds to its receptor (IR) and, via the insulin receptor substrate (IRS), induces the phosphorylation of the adapter proteins Shc and Grb2. Shc and Grb2 can activate p44/p42 mitogen-activated protein kinase and Janus kinase (JAK), which stimulates the transcription factors STAT (JAK/STAT pathway).⁸⁵ In the 20-month-old rats, insulin phosphorylation of IR was reduced compared to the younger rats, although Shc and STAT-1 phosphorylation were unchanged. These results suggest that older rats are chronically exposed to higher insulin levels, and this is accompanied by insulin resistance, but not with a decrease in amounts of IR, Shc, and STAT-1 protein. Further study of the insulin and IGF-1 signaling pathways in the aging lacrimal gland is necessary to determine if an alteration in these pathways leads to dysfunction in the older animals.

Insulin resistance can lead to hyperglycemia and oxidative stress, which produce advanced glycation end products (AGE). AGE bind to their receptor RAGE, which activates stress-activated signaling pathways, including nuclear factor-κB (NF-κB), p38MAPK, NH₂-terminal Jun kinases/stress activated protein kinases (JNK/SAPK⁸⁶ [Figure 4]). In turn, NF-κB regulates the expression of a large number of genes, including growth factors, inflammatory cytokines, and RAGE. Many of these products regulate NF-κB, leading to a positive feed-forward cycle. The role of the NF-κB in regulating lacrimal gland secretion is unknown. It could affect the acinar cells directly, or it could induce cytokine production, which affects lacrimal cells or inflammatory cells, such as macrophages, lymphocytes, and mast cells. However, changes in insulin with age and the resultant oxidative stress could impair lacrimal gland secretion. Further experimentation is necessary to link insulin levels, insulin resistance, and activation of AGE and RAGE with the blockage of lacrimal gland secretion that occurs as a function of age.

Figure 4.

Schematic of insulin resistance leading to hyperglycemia and oxidative stress by activating I kappa kinase and NF-KB that leads to the production of inflammatory cytokines and the receptor for advanced glycation end products (RAGE). Advanced Glycation End products (AGE); nuclear factor-KB (NF-KB), p38MAPK, NH2-terminal Jun kinases/stress activated protein kinases (JNK/SAPK); I kappa kinase (I-KK); I kappa Beta (1-KB).

In the lacrimal gland, the increased insulin levels in older rats was accompanied by an increase in AGE, RAGE, and NF-κB in the lacrimal gland.²⁷ This was accompanied by an increase in IL-1β in tears and a trend to an increase in TNFα. The role of NF-κB in lacrimal gland cell survival was not investigated. Thus, the metabolic effects of insulin may lead to lacrimal gland dysfunction in aging through increased oxidative stress. The role of longevity and pro-aging genes remain to be investigated. As with other tissues in the body, a causative link between insulin, oxidative stress, and aging remains to be demonstrated.

Although there are limited studies to date in the lacrimal gland, available data indicate that oxidative stress increases with aging in the lacrimal gland and that this could be correlated with the loss of lacrimal gland secretion.

IX. ROLE OF LACRIMAL GLAND SECRETORY IMMUNE SYSTEM IN AGING

The immunological theory of aging suggests that a decline in immunity and immune system function leads to increased susceptibility to infectious disease, thus predisposing to aging and death.⁵ The secretory immune system of the lacrimal gland produces sIgA and protects the ocular surface against infection.^87,88 Aging decreases sIgA in mucosal secretions. As discussed in Section V.B.1, there are substantial gender-based differences in lacrimal gland and tear sIgA. The tear sIgA and free secretory component increase with age in male, but not female, rats.⁸⁸ In contrast, lacrimal gland secretion of free secretory component peaks at 4 months in male rats and then deceases with age. In female rats, it peaks at about 2 months of age and remains constant with increasing age. The number of IgA-containing cells in the lacrimal gland peaks at 3 months of age in both males and females and thereafter remains essentially constant.⁸⁹ In contrast, the number of IgM-containing cells remains constant from birth through aging.

The types of T cells, which affect the secretory immune system, were analyzed in human male and female lacrimal glands.⁸⁹ In females, around puberty, the number of total, helper/inducer, and suppressor/cytotoxic T cells in the lacrimal gland decreased with age. In males, around puberty, these cell numbers decreased or remained constant. Thus, the numbers of cells and tear sIgA content in the secretory immune system of the eye, does not appear to change with increasing age once the puberty-induced changes have occurred. The function of the secretory immune system in protecting the ocular surface from infection might be altered, but this remains to be investigated. The decreased or unchanged T cell content found by Hann et al⁸⁹ is consistent with the findings of Rios et al⁶³ and Williams et al⁷² that mast cell infliltration with increasing age is independent of T cell infiltration in the rodent lacrimal gland. Most aging studies on the lacrimal gland have used rodents, and their conclusions may not be entirely applicable to the human lacrimal gland.

XI. SUMMARY

In both the human and rodent lacrimal gland, functional unit structure and secretory function begin to change in middle age. Evidence from studies on the aging mouse and rat lacrimal gland support the hypothesis that with increasing age, a combination of events occurs in the lacrimal gland that lead to impaired secretion. Lacrimal gland alteration starts with an increase in lipofuscin-like material (indicates increase oxidative stress) and mast cell infiltration that coincides with a decrease in neural and agonist-stimulated secretion. The infiltrating mast cells could recruit lymphocytes that secrete cytokines, which cause an immune damage to the efferent nerves and impair neurotransmitter release. This impairment could exacerbate the earlier changes, leading to a continued decrease in secretion, resulting in aqueous deficiency dry eye disease. Age-dependent changes occur in the afferent corneal nerves, and the number of corneal nerve endings decreases as a function of age. Underlying the reduction in secretion could be the destruction of acinar cells associated with increased oxidative stress and chronic inflammation from mast cell and lymphocyte infiltration, as well as from an age-dependent hyperglycemia. As in other tissues, however, all evidence to date is correlative. No age-dependent-changes in lacrimal gland structure or function described to date are causative.

Although progress has been made in describing the effects of aging on lacrimal gland function, there are many areas in need of research. First, no studies on the effect of age on lacrimal gland fluid secretion have been performed. Only protein secretion has been studied. As both protein and fluid secretion contribute to the aqueous layer of tears, studies on a critical component are missing. Second, there are no studies on the gender differences in the aging lacrimal gland. As there are considerable gender-based differences in lacrimal gland structure and function, male and female lacrimal glands could age differently. Third, although the effect of age on sIgA amount has been investigated, the function of the secretory immune system of the lacrimal gland as a function of age has not been investigated. Fourth, the effect of age on tear amount and composition and on corneal and conjunctival structure and function has not been determined except to a limited extent in the human. Finally, there are limited studies on transgenic mouse models of aging that could identify specific molecules or molecular pathways altered by aging.