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Evidence to Suggest Morphological and Physiological Alterations of Lacrimal Gland Acini with Ageing
Exp. Eye Res. (1999) 68, 265–276 Article No. exer.1998.0605, available online at http :\\www.idealibrary.com.on
Evidence to Suggest Morphological and Physiological Alterations of Lacrimal Gland Acini with Ageing C. E. D R A P ERa, E. A. A D E G H A T E*b, J. S I N GHa D. J. P A L L OTb a b
Department of Applied Biology, University of Central Lancashire, Preston, PR1 2HE, England and the Department of Human Anatomy, United Arab Emirates, P.O. Box 17666, Al-Ain, United Arab Emirates (Received Columbia 17 July 1998 and accepted in revised form 7 August 1998) This study investigates changes in the morphology and physiology of lacrimal gland acinar cells with age. Changes in microstructural appearance of the acinar cells, the type and distribution of the different acini in glands and the secretory granules within the acini were examined in glands from animals of 3–5, 9, 12, 20, 24 and 28 month old rats. Differences in the secretory capacity of the acinar cells were also examined in animals of each age-group, with the exception of 28 months. The typical acini of young glands (3–5 months) were of the serous type. This was also true of 9 month glands, although there was a significant reduction in their overall distribution compared to young glands. The acini in the 12 month glands were predominantly of the seromucous type and appeared to be at the expense of the serous acini which were further significantly reduced compared to 3–5 and 9 month glands. This remained the prevalent acini type in 20 month glands, however by 24 months there was a significant increase in the occurrence of mucous acini and this time appeared to be at the expense of the seromucous acini which were significantly reduced in glands of this age-group. The predominant acinar cell in 28 month glands, like 24 month glands, was of the mucous variety. Qualitative EM studies revealed a progressive change in the secretory products of the lacrimal gland acini, strongly correlating to changes in acinar cell type. Typical acini of both 3–5 and 9 month glands contained numerous protein secretory granules. The seromucous acini also of these age groups contained both protein and mucous secretory granules, with the protein secretory granules in higher abundance. By 12 months the typical seromucous acini was packed with both protein and mucous secretory granules of equal proportions. However, by 20 months the predominant seromucous acini contained fewer protein secretory granules and elevated occurrence of mucous secretory granules. By 24 and 28 months the acini contained even fewer protein secretory granules and the typical acinar cell was of the mucous type containing exclusively mucous secretory granules. The secretory capacity of the acini was also altered with age. Maximum protein output in response to cholinergic stimulation resulted in an initial significant increase with ageing from 3–5 months to 9 and 12 months followed by a later significant age-dependent reduction in output. However, maximal peroxidase release from acinar cells of 3–5 and 9 month glands was the same. This was followed by a significant age-dependent reduction in peroxidase release. Furthermore, the concentrations required to evoke these responses differed with age. These results present evidence to suggest that acinar cells of the lacrimal gland undergo progressive alterations with age. The type of acini changing initially from serous to seromucous acini (intermediate phase) followed by a gradual transformation of the seromucous acini to mucous acini. This in turn changes the properties of the acini from protein producing and secreting acini to mucous producing and secreting acini. The results also suggest a reduction in the ability of the acini to synthesise proteins with age and altered responsiveness to cholinergic stimulation to secrete proteins. These findings may help in explaining the occurrence of altered protein\tear secretion with ageing. # 1999 Academic Press Key words : rat ; lacrimal gland ; acinar cells ; morphology ; physiology ; acetylcholine.
1. Introduction The aqueous component of the tear film is responsible for keeping the cornea buffered, lubricated, nourished and protected (Dilly, 1994 ; Friedlaender, 1992) and is produced and secreted from the main and accessory lacrimal glands. The exorbital gland is the main lacrimal gland of the rat and is analogous to the human lacrimal gland (Williams et al., 1989). About 80 % of the normal lacrimal gland consists of serous acini which are highly differentiated epithelial cells * Address all correspondence to : Dr Ernest Adeghate, Department of Human Anatomy, Faculty of Medicine and Health Sciences, United Arab Emirates University, P.O. Box 17666, Al-Ain, United Arab Emirates.
0014–4835\99\030265j12 $30.00\0
specialised for the synthesis, storage and exocrine secretion of proteins (Kelly et al., 1984). Water and electrolytes are also secreted from the acini. The serous acinar cell is pyramidal in shape, with a nucleus positioned in the wider basal portion of the cell. They have a well defined endoplasmic reticulum (ER), rough ER, several mitochondria and a supranuclear Golgi apparatus (Stevens and Lowe, 1993). The apical region of the acini contains granules filled with packaged protein ready for secretion by exocytosis (Herzog, Sres and Miller, 1976 ; Putney, Van De Walle and Leslie, 1978 ; Stevens and Lowe, 1993). The major protein secreted from the lacrimal acini is lipocalin. Other proteins are secreted but in lesser amounts and include ; lysozyme, peroxidase, lact# 1999 Academic Press
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oferrin, beta lysin and secretory immunoglobulins A and G (Friedlaender, 1992). Protein and fluid secretion is regulated by both autonomic and non-cholinergic, non-adrenergic nerves which innervate the acini, as well as ducts and blood vessels (Bromberg, 1981 ; Matsumoto et al., 1991). The classical neurotransmitters and neuropeptides released by these nerves are acetylcholine (ACh), noradrenaline (NA) and vasoactive intestinal polypeptides (VIP). Both cholinergic and adrenergic agonists as well as VIP can stimulate protein secretion from the lacrimal gland (Dartt et al., 1984, 1985 ; Friedman, Lowe and Selinger, 1981 ; Hussain and Singh, 1988 ; Muller, Chambaut-Guerin and Rossignol, 1985). Reduced secretion of the aqueous layer of the tear film is the most common abnormality found in the disease state Keratoconjunctivitis sicca (KCS), or dry eye syndrome (Dartt, 1994 ; Friedlaender, 1992). KCS is an ocular disorder involving local drying up of the tear film, initially causing inflammation and fibrosis of the lacrimal gland and irritation of the eyes. This, however, can lead to corneal cellular loss (Superficial Keratitis) and advanced stages of the disease include, vascularisation and scarring of the cornea causing severe visual disability (Holly and Lemp, 1977). KCS is an age-related disorder affecting mainly the middle aged and the elderly (Friedlaender, 1992). Age-related changes in both the physiological and morphological properties of the rat lacrimal gland have previously been observed (Draper et al., 1997). This included a reduction in protein secretion from aged lacrimal gland acini compared to that from young tissue. Morphologically, the aged tissue displayed structural damage, more specifically, the type of acinar cell predominant in the glands of aged animals differed from those prevalent in glands from young rats (Draper et al., 1997). However, little is known about the morphology or secretory capacity of lacrimal acini from glands of animals of intermediate age-groups. We therefore decided to perform a more comprehensive study on the effect of ageing on the lacrimal acinar cell investigating the micro- and ultra-structural morphology of the acini and their distribution in glands from rats of 3–5, 9, 12, 20, 24 and 28 months of age, using both light and electron microscopy. Total protein and peroxidase release from the lacrimal acini of glands from animals of each age-group, with the exception of 28 months, was also compared. This study was performed with the aim of understanding the influence of age on the pathological alterations of the lacrimal gland, which appear to result in decreased aqueous tear secretion, part of the aetiology of KCS.
2. Materials and Methods Animals Morphological studies were performed on isolated lacrimal glands taken from male Sprague-Dawley rats
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of ages 3–5 (n l 6), 9 (n l 6), 12 (n l 6), 20 (n l 6), 24 (n l 6) and 28 months (n l 6). These age groups span the entire range of human age. For example, 3–5 months old rats correspond to 15–18 year old humans, while 9, 12, 20 and 24 month old rats corresponds to 25–35, 35–50, 50–65, 65–75 and 75–85 year old humans, respectively. Physiological experiments were performed on lacrimal gland acini isolated from lacrimal glands of rats from each age-group with the exception of 28 months (n l 6 for each age-group). The animals were based and housed at the United Arab Emirates University in Al-Ain, United Arab Emirates and all procedures had the relevant clearance from the local ethical committee for the use of animal experiments. The aged rats used in this study were all in apparent good health. Reagents and Chemicals All reagents employed in this study were obtained from Sigma (U.K.). Morphological Studies Light microscopy Immediately after killing the animal, the lacrimal glands were removed, cut into small fragments and fixed for 24 hr in Zamboni’s solution (Adeghate et al., 1994). After fixation, the specimens were dehydrated and embedded in paraffin wax and cut into 5–7 µm-thick sections using a rotary microtome (Shandon AS 325 retraction). The sections were incubated at 50mC for 30 min prior to staining. Sections were stained with haematoxylin and eosin, dehydrated in ascending concentrations of ethanol, cleared in xylene and mounted in Cytoseal 60 (Stephens Scientific, Riverdale). Sections were viewed under a light microscope (Zeiss Axiophot, Germany). Pictures were taken using Ilford Pan F-Black and White microscope 50 ASA film. Acini counts Counts of acini type were performed using light microscopy. Acini were counted in visible sections at i40 magnification. A total of 40 random fields were taken from a total of 20 slides for each age group and these slides were masked. Because of the conspicuous changes observable with ageing, it was easy to sort out which age group each of the slide belonged to. The percentage distribution of each acini type of the glands from the 6 age-groups was calculated. Electron microscopy Lacrimal glands were cut into small pieces and fixed for 4–5 hr in 2n5 % glutaraldehyde in Karnovsky’s solution (Karnovsky, 1965). The tissue pieces were washed five times overnight at 4mC in cacodylate buffer (0n2 ). The tissue was postfixed with 1 % osmium tetroxide for 1–3 hr, washed 8 times in cacodylate buffer and dehydrated in ascending concentrations of ethanol. They were then treated
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using a diatome knife (supplied by Agar Scientific, Essex, England) mounted on 3n05 mm 20 mesh copper grid and contrast stained with saturated aqueous uranyl acetate for 30 min and Reynolds lead citrate (Reynolds, 1963) for 5 min. These were then examined and photographed in a Phillips CM10 transmission electron microscope. Pictures were taken using Kodak electron thick base 4489 film. Secretory Capacity of Lacrimal Acinar Cells Preparation of lacrimal acinar cells The lacrimal glands from animals of each age-group (6 animals for each group) were placed in Ca#+-free Krebs-Henseleit (KH) solution of the following composition (m) : NaCl 130, KCl 5n0, HEPES 20, KH PO 1n2, # % MgSO 2n0, Glucose 10 and minced (Francis and % Singh, 1990). The tissue fragments were washed extensively and placed in 10 ml KH solution containing Bovine Serum Albumin (BSA-0n2 %), trypsin inhibitor (0n01 %) and collagenase (0n025 g) and incubated at 37mC for 30 min in a shaking water bath. The cells were washed in KH solution containing BSA, trypsin inhibitor and calcium (3n0 m) and dissociated for 12 min by gently passing the tissue through flamed pipette tips of decreasing bore (3 mm, 2 mm, 1 mm) and subsequent filtering through nylon mesh (100µm-pore). The suspension of dispersed acinar cells were centrifuged twice at 800 rpm 5 min each. The supernatant was discarded and the acinar cell pellet was resuspended in KH solution containing calcium (3n0 m). Acini isolated by this technique had good morphology and a viability of 90 % when tested by trypan blue exclusion (Herzog et al., 1976).
F. 1. Light micrographs showing the progressive change in the acinar cell appearance of 3–5 and 9 months glands [A] to glands of 12 and 20 months [B] and to glands of 24 and 28 months [C] ; from serous to seromucous (intermediate phase) and to mucous acini, respectively. These micrographs are typical of 6 such samples from 6 animals of each agegroup. s l serous acini ; sm l seromucous acini ; m l mucous acini. i262. Scale bar l 50 µm.
with propylene oxide ; 2 changes of 15 min and placed in propylene oxide and resin (1 : 1) for 1 hr followed by pure resin overnight. The samples were embedded and polymerised at 60mC for 24 hr. Thin sections were cut
Stimulation of lacrimal acinar cell secretion Acinar cell aliquots (250 µl) were placed into microfuge tubes containing 750 µl of KH buffer solution. To each tube 110 µl of varying concentrations of ACh (10−"$– 10−( ) was added. In control aliquots an equivalent of 110 µl of KH buffer solution was added to each tube. The suspension in each tube was gently vortexed and incubated in a shaking waterbath at 37mC for 30 min. The suspensions were spun in a microfuge for 5 min. The supernatant was removed, collected and analysed for protein and peroxidase. The residue was resuspended in a known amount of distilled water, sonicated and analysed for cell protein content. Total protein in supernatant and residue suspensions were measured spectrophometrically (Beckman DU2-70Spectrophotometer), using a Bio-Rad protein assay (Sigma, U.K.) based on the Lowry method (Lowry et al., 1951). Bovine serum albumin was used as standard. Peroxidase in the supernatant was also measured spectrophometrically using a colorimetric method previously described by Herzog and Fahimi (1973) incorporating diaminobenzidine as a hydrogen donor. Commercial horseradish peroxidase was used as standard. Protein output was expressed as µg ml−"
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F. 2. For legend see facing page.
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(mg cell protein)−" while peroxidase release was expressed as ηg ml−" (mg cell protein)−". Statistical Analysis All data provided are expressed as meanpstandard error of the mean (...). Data were compared using analysis of variance (ANOVA) and only values with P 0n05 were accepted as significant. 3. Results Light Microscopy Light microscopical studies of the lacrimal glands from animals of 3–5, 9, 12, 20, 24 and 28 months of age revealed the presence of the 3 distinct types of acini ; serous (protein secreting) acini, seromucous (protein and mucous secreting) acini and mucous acini (Fig. 1). The glands from the 3–5 month old rats (regarded as normal, control rats) were composed of tubulo-acinar tissue mainly of the serous type (Fig. 1(A)]. At 9 months the majority of acini were also of the serous type. However, with ageing significant changes in the size and shape of the acini were observed. By 12 and 20 months the majority of the acini were of the seromucous variety [Fig. 1(B)]. Moreover, by 24 and 28 months most of the gland consisted of mucous acini (Fig. 1(C)]. Identification of the mucous acini have previously been confirmed (Draper et al., 1998) by staining of sections with Alcian blue at pH 1n0. Acinar Cell Type Distribution A quantitative analysis was performed to determine the distribution of each acinar cell type within the glands of the animals from the 6 age-groups. Acini were counted from random sections at i40 magnification using light microscopy and the percentage distribution of each acinar cell type was calculated. Effect of ageing on distribution of serous acini In young glands 81n2p4n7 % (meanp...) of the acini were serous (Fig. 2(A)]. By 9 months this was significantly reduced to 50n5p9n4 % (ANOVA ; P 0n05) and by 12 months further significantly (P 0n01) reduced to 19n5p5n2 %. The proportion of serous acini remained unchanged by 20 and 24 months, however, by 28 months there was a further significant (P 0n05) reduction to 7n6p4n3 %. Effect of ageing on distribution of seromucous acini In young glands 17n2p2n2 % of the acini were sero-
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mucous [Fig. 2(B)]. By 9 months this significantly (P 0n05) increased to 32n5p3n3 % and by 12 months further significantly (P 0n01) increased to 65n7p4n4 %. By 20 months there was a slight decrease in seromucous acini to 53n3p6n0 %, moreover, by 24 months a significant (P 0n01) reduction to 29n3p 3n7 % with a small increase at 28 months to 37n1p 3n2 %. Effect of ageing on distribution of mucous acini Only 1n6p0n4 % of the acini in young glands were mucous [Fig. 2(C)]. By 9 and 12 months this increased significantly (P 0n05) to 17n0p1n9 % and 15n8p2n3 %, respectively and there was no significant change by 20 months. However, by 24 and 28 months there were significant (P 0n01) increases in mucous acini to 46n6p5n6 % and 53n3p5n0 %, respectively. Electron Microscopy Electron microscopic studies were performed to investigate the influence of age on the secretory products of the lacrimal gland acini. Again glands from young animals (3–5 months) were regarded as normal, controls. Typical acini of the glands from the young animals contained numerous protein (electron dense) secretory granules [Fig. 3(A)]. There was also the presence of a few acini containing both protein and mucous (electron lucent) secretory granules [Fig. 3(B)] the protein granules being in higher abundance. Both types of acini were typical of the 9 month lacrimal glands. However, by 12 months the typical acini was packed with both protein and mucous secretory granules of similar proportions [Fig. 3(C)]. The predominant acini of 20 month glands also contained both protein and mucous secretory granules [Fig. 3(D)], however the mucous secretory granules were in higher abundance to the protein granules which were markedly reduced compared to the typical acini of the 12 month glands. This was elevated further by 24 and 28 months where the typical acini contained exclusively mucous secretory granules [Fig. 3(E)]. Total Protein Output and Peroxidase Release Isolated lacrimal acinar cells of glands from rats of 3–5, 9, 12, 20 and 24 months of age were incubated with ACh at varying concentrations (10−"$–10−( ) to establish the secretory capacity of the acini and to determine the concentrations required to achieve maximum secretion of total protein output and peroxidase release with ageing.
F. 2. Histograms showing the percentage distribution of the 3 types of secretory acini ; serous [A], seromucous [B] and mucous [C] in rat lacrimal glands from animals of 3–5, 9, 12, 20, 24 and 28 months of age. Each point is meanp... A total of 40 random sections typical of 6 samples from 6 animals of each age-group. Note the age-related changes in the distribution of each acinar cell type.
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F. 3. Electron micrographs showing the progressive change in the secretory products of the typical acinar cells of 3–5 and 9 month glands (A) and (B) to glands of 12 months (C) to glands of 20 months (D) and to glands of 24 and 28 months of age (E). Serous acini from 3–5 and 9 months old animals with many protein-containing secretory granules (arrow), and seromucous acini from these glands with both protein-containing secretory ; electron dense granules and mucous-containing, electron lucent secretory granules (arrow head). Note the age-related reduction in protein-secretory granules and progressive increase in mucous-secretory granules of the seromucous acini of both the 12 and 20 month glands to the acini containing only mucous-secretory granules of the 24 and 28 month glands. These micrographs are typical of 6 such samples from 6 agegroups. (A) serous acini of 3–5 month glands ; (B) seromucous acini of 3–5 and 9 month glands ; (C) seromucous acini of 12 month glands ; (D) seromucous acini of 20 month glands ; (E) mucous acini of 24 and 28 months. Magnification : A l i3120 ; B l i4800 ; C l i7840 ; D l i5288 and E l i6480. Scale bar l 2 µm.
Total protein output Stimulation of 3–5, 9, 12, 20 month old rat lacrimal acinar cells with ACh resulted in significant (P 0n05) increases in protein output when compared to basal. In contrast, ACh evoked only a small but not significant increase in total protein output from 24-month old rat lacrimal acinar cells when compared to basal. Basal total protein outputs from the acini of glands from the 5 age-groups were 0n69p0n06 (n l 10), 0n84p0n03 (n l 12), 0n76p 0n03 (n l 12), 0n41p0n03 (n l 10) and 0n37p 0n02 µg ml−" (mg cell protein)−" (meanp... ; n l 10), respectively. Note the age-related changes in basal secretory responses. A small, but not significant increases in basal protein output were observed in 9 and 12 month old lacrimal gland cells, whereas, there was a significant (P 0n05) decrease in basal protein output from 20 and 24 month olds [Fig. 4(A)]. The maximal secretory effect exerted by ACh on total
protein output from acini of the glands from 3–5, 9, 12, 20 and 24 month animals is presented in Fig. 4(B). This figure shows the mean peak (p...) protein output above basal level for the concentrations of ACh (10−"", 10−"", 10−"#, 10−"# and 10−* ) that produced the maximal secretory effect on lacrimal acinar cells of glands from the 3–5, 9, 12, 20 and 24 month old animals, respectively. Note the age-related change in the concentration of ACh required to achieve the maximal secretory response. There is an inverse correlation between the concentration of ACh and maximal protein release. This may be due to the alterations in the number and intactness of the membrane-bound ACh receptors with ageing leading to reduced sensitivity and reduced protein secretion. ACh evoked significant (ANOVA ; ... ; P 0n01) increases in protein output from lacrimal gland acini of young animals compared to basal. At 9 and 12
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F. 4. Family of histograms showing mean peak (p... ; n l 6) basal protein output (A) and mean peak (p... ; n l 6) total protein release (B) above basal levels from the lacrimal acinar cells of glands from 3–5, 9, 12, 20 and 24 month old rats, following stimulation with a concentration of ACh that elicited maximal response in each age-group. Note the age-related changes in secretion of both basal and ACh-evoked total protein output.
months ACh evoked a further significant (P 0n05) increase in protein output. However, maximal secretion at 9 months was significantly (P 0n05)
greater than at both 3–5 and 12 month rats. In contrast, by 20 months total protein output was significantly (P 0n01) reduced compared to that
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F. 5. Family of histograms showing mean peak (p... ; n l 6) basal peroxidase output (A) and mean peak (p... ; n l 6) peroxidase release (B) above basal levels from the lacrimal acinar cells of glands from 3–5, 9, 12, 20 and 24 month old rats, following stimulation with a concentration of ACh that elicited maximal response in each age-group. Note the age-related changes in secretion of both basal and ACh-evoked peroxidase release.
from acini of glands from 3–5, 9 and 12 month old rats. Moreover, there was a further significant (P 0n05) reduction in the amount of protein secreted from acini of glands from animals of 24 months of age when compared to 20 month glands.
Peroxidase release In comparison with basal peroxidase release, there was a significant increase (P 0n05) in peroxidase release when 3–5, 9, 12 and 20 month old rat lacrimal acinar cells were incubated with ACh. At 24 months, ACh evoked only a small but
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not significant increase in peroxidase output when compared to basal. Basal peroxidase release from the acini of glands from the 3–5, 9, 12, 20 and 24 month animals were 0n34p0n01 (n l 10), 0n31p0n01 (n l 12), 0n26p0n01 (n l 12), 0n13p0n01 (n l 10) and 0n10p0n01 ηg ml−" (mg cell protein)−" (meanp... ; n l 10), respectively. Note the age-related changes in basal secretory responses. At 9 and 12 months, there was a small but not significant reduction in basal peroxidase release when compared to 3–5 month old lacrimal gland cells. However, the basal peroxidase release was further reduced significantly (P 0n01) at 20 and 24 months when compared to 3–5 month olds [Fig. 5(A)]. Figure 5(B) shows the maximal secretory effect exerted by ACh on peroxidase release from acini of glands from the 5 age-groups. This figure shows the mean peak (p...) peroxidase release above basal level for the concentrations of ACh (10−"", 10−"!, 10−"!, 10−"! and 10−* ) that produced the maximal secretory effect on lacrimal acinar cells from glands of the 5 age-groups, respectively. Again note the agerelated changes in the concentration of ACh required to achieve the maximal response. It is evident that there is an inverse correlation between the concentration of ACh and maximal peroxidase release. This may be due to the alterations in the number and intactness of the membrane-bound ACh receptors with ageing leading to reduced sensitivity and reduced peroxidase secretion. ACh evoked significant (ANOVA ; ..., P 0n01) increases in peroxidase release from lacrimal gland acini of young animals compared to basal. There was no change in secretion at 9 months, however, by 12 months peroxidase release was significantly (P 0n01) reduced compared to that from 3–5 and 9 month acini. There was a further significant (P 0n01) decrease in peroxidase release from acini of 20 month glands compared to 12 month glands and by 24 months a further significant (P 0n01) reduction in release where only a very small amount of peroxidase was released from these acini. 4. Discussion The results of this study have demonstrated that lacrimal gland acini change with age both morpho logically and physiologically. The acinar cell type of young glands were predominantly serous (protein producing and secreting cells). This was also true of the 9 month glands although there was a significant (P 0n01) decrease in the overall distribution of serous acini when compared to the young glands. In consequence there were increases in both seromucous (protein and mucous producing and secreting cells) and mucous acini. By 12 months there was a significant (P 0n01) increase in the relative occurrence of the seromucous acini and appeared to be at the expense of the serous acini which were further significantly (P 0n01) reduced when compared to 9 month glands. The distribution of mucous acini in
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glands from this age-group remained unchanged. Seromucous acini remained the prominent acinar cell type in glands from 20 month old animals and the distribution of both serous and mucous acini remained unchanged when compared to 12 month glands. However, by 24 months there was a significant (P 0n01) increase in the overall prevalence of mucous acini and now appeared to be at the expense of the seromucous acini which were significantly (P 0n01) reduced when compared to 20 month glands. The distribution of serous acini again remained unchanged at this age. Nevertheless, in 28 month glands the distribution of serous acini was further significantly (P 0n05) reduced when compared to 24 month glands. In consequence there was a very small but not statistically significant increase in seromucous acini and no change in the distribution of mucous acini. However, like glands from 24 month old animals the predominant acinar cell type in 28 month glands were of the mucous type. The acinar cell dominating the glands from young animals contained many protein secretory granules. A few acini also contained both protein and mucous secretory granules. Both types of acini were typical of 9 month glands, however the serous acini were the prevalent type. The predominant seromucous acini of the 12 month glands differed compared to the seromucous acini of the 3–5 and 9 month glands. The acini were packed with both protein and mucous secretory granules of equal proportions, whereas in the acini of the young and 9 month tissue the protein secretory granules were in higher abundance. By 20 months the dominant seromucous acini differed from those of the 3–5, 9 and 12 month tissues, where the presence of protein secretory granules was reduced and the presence of mucous secretory granules was markedly increased. Moreover, the presence of protein secretory granules in acini from 24 and 28 month tissue was further reduced and the typical acini was a mucous cell containing exclusively mucous secretory granules. These results indicate progressive alterations in the specialised protein-secreting cells of the lacrimal gland with age, initially from serous to seromucous (intermediate stage) and gradually with time from seromucous to mucous acini. The progression from serous to mucous acini is a new and important finding, however, the causes and mechanisms of these changes remain elusive. However, it is tempting to speculate that these changes are due to the normal process of cellular ageing and senescence with involves loss of cellular homeostasis and maintenance over time (Kirkwood, 1992 ; Rattan, 1995). The major modes of cellular maintenance include ; processes of cellular repair, cell division, cell replacement, neuronal and hormonal responsiveness, immune response, detoxification, free-radical scavenging, stress-protein synthesis, macromolecular turn-over and maintenance of the fidelity of genetic information transfer
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(Rattan, 1995). A loss of one or a number of these mechanisms may be responsible for the changes in the acinar cells observed in this study. Altered cellular responsiveness to growth factors, hormones and other factors have been found to be reduced significantly during cellular ageing of cultured diploid cells (Cristofalo, Pignolo and Rotenberg, 1992 ; Rattan and Derventzi, 1991). This resulted in a significant reduction in both protein and DNA synthesis and reduced induction of several cell-cycle related genes. Certainly, in these studies the reduced presence of protein secretory granules of the acini indicate a reduction in protein synthesis. In addition, the changes which have occurred in this study may be due to increased sensitivity to toxic agents such as phorbol esters, free-radical inducers, irradiation and heat shock which have also been observed in ageing cells (Rattan and Derventzi, 1991). Reduced stimulation of the acini by neurotransmitters due to denervation of the autonomic nerves (Williams, Singh and Sharkey, 1994) may also be responsible for reduced synthesis of protein secretory products of the acini. Consequently increased stimulation by other factors for example, mast cell mediators may be responsible for stimulation of synthesis of the mucous secretory products of the acini. Increased infiltration of mast cells to the lacrimal gland with ageing have previously been observed (Draper et al., 1997 ; Williams et al., 1994). In this study stimulation of protein and peroxidase secretion from isolated lacrimal gland acinar cells differed considerably with age. ACh evoked significant (P 0n01) increases in total protein output from the acinar cells of young glands above basal and the maximal secretory response was achieved at 10−"" . The same concentration evoked maximal protein output from acinar cells of 9 month glands, however, the maximum amount of protein secreted by significantly (P 0n05) higher than that from young glands. Protein output from acinar cells of 12 month glands was significantly (P 0n05) reduced compared to that from 9 month glands but was still greater than that from young glands (P 0n05). Furthermore, the concentration required to achieve the maximum response was 10−"# compared to 10−"" required for acini of both young and 9 month glands. Maximum secretory response from acinar cells of 20 month glands was also achieved at 10−"# , however, the maximum amount of protein secreted was significantly (P 0n01) reduced compared to that from 3–5, 9 and 12 month old rats. Moreover, there was a further significant (P 0n05) reduction in the amount of protein secreted from acini of glands from rats of 24 months of age when compared to 20 month glands and the concentration required to produce this effect was 10−* . In another set of experiment, ACh also evoked significant (P 0n01) increases in peroxidase release from acinar cells compared to basal at a concentration of 10−"" . By 9 months there was no change in the
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amount of peroxidase released, however, the concentration required to evoke the maximum response was 10−"! . The same concentration was required to produce the maximum response from acinar cells of 12 month glands, however, the amount of peroxidase released was significantly (P 0n01) reduced compared to that from acinar cells of both 3–5 and 9 month glands. There was a further significant (P 0n01) decrease in peroxidase release from cells of 20 month glands compared to 12 month glands with stimulation of the same concentration of ACh. The maximum peroxidase released from acinar cells of 24 month glands was further significantly (P 0n01) reduced when compared to that from acinar cells of 20 month glands. Only a very small amount of peroxidase was released from these cells. Furthermore, the concentration of ACh required to achieve this response was 10−* . Earlier studies observed age-related reductions in total protein output from rat lacrimal acinar cells (Draper et al., 1997) and intact rat lacrimal tissue segments (Bromberg and Welch, 1985). This study, however, has revealed that protein output initially increases with ageing followed by an age-related reduction. The cellular mechanism(s) involved in the alterations in protein output with age is still unknown. It is possible that more than one mechanism may be associated with ageing and include ; changes in acinar cell morphology, alterations in the ability of the acini to synthesise and secrete proteins, elevations or reductions in the number of acinar plasma membrane receptors for the secretagogues and\or changes to the signal transduction mechanism. The reduction in serous acini at 9 months may initiate a compensatory mechanism to increase protein output. It is unlikely that a change in the responsiveness to ACh would result in this increased secretion since maximum output was achieved at the same concentration as for young glands. Since the abundance of protein secretory granules in acini of both 3–5 and 9 month glands were similar it is also unlikely that protein synthesis is elevated at 9 months. However, it may be possible that the signal transduction mechanism is altered to increase output. The further reduction in serous acini at 12 months results in a decrease in protein output, but which is still greater than that from young glands (P 0n05). Since maximum stimulation is achieved at a weaker concentration it is possible that there is an increase in the number of membrane bound receptors for ACh. By 20 months the same concentration is required for maximum output suggesting, like 12 month acini, elevated receptors for ACh. However, the significant reduction in protein output coupled with the reduction in protein secretory granules of the acini from 20 month animals suggests a reduction in protein synthesis. The stimulation of protein synthesis in response to various extracellular stimuli has been seen to be significantly reduced in ageing cells (Cristofalo et al., 1992).
AGE-RELATED CHANGES OF THE RAT LACRIMAL ACINI
Problems with signal transduction and\or exocytosis may also contribute to this reduced output. The secretory responses evoked by cholinergic agonists are associated with an increase in cellular calcium mobilisation (Dartt, 1989, 1994 ; Putney et al., 1978). Recent studies have shown that the intracellular calcium concentration either remains the same (Brooks-Frederick et al., 1993 ; Takahashi, Yoshida and Takaswima, 1992) or decreases during ageing and the defect may be at the level of the cells capacity to mobilize calcium in response to extracellular stimuli. The further significant reduction in protein output from acini of 24 month glands may largely be due to the acini changing to mucous cells containing exclusively mucous secretory granules. Other acini of this age-group displayed a further reduction in the presence of protein secretory granules also suggesting reduced protein synthesis. A far stronger concentration of ACh was required to produce the maximum secretory response suggesting a reduction in the number of plasma membrane receptors. A decline in the synthesis and activity of cholinergic system components and variations in receptor response with ageing has been reported (Rattan, 1995). Alterations to the signal transduction mechanisms and\or exocytosis may also be responsible in part for the reduced protein output at this age. The results of this study also suggest that the type of proteins secreted may change with age. Although at 9 months more protein is secreted from the acini compared to that from 3–5 month glands, the amount of peroxidase released is the same. At 12 months peroxidase release is significantly reduced while total protein output is higher than that from young glands. This may be due to reduced synthesis or a problem with packaging and secretion. By 20 and 24 months there is an age-related reduction in peroxidase release which correlates with reduced total protein output. Again this may be due to reduced synthesis. Experiments are currently in progress to determine which of the parameters are involved in the age-related changes in the protein synthesis and secretory capacity of the acini. In conclusion, the results of this study put forward evidence to suggest that acinar cells of the lacrimal gland undergo progressive alterations with age. Initially from serous to seromucous acini followed by gradual transformation of seromucous acini to mucous acini. Considerable changes to the secretory capacity of the lacrimal acini with age were also observed and strongly correlated to the changes in acini type and changes to the secretory product of the acini. In addition, a reduction in the ability of the acini to synthesise proteins with ageing coupled to alterations in membrane bound receptors for cholinergic agonists were also implicated in altering the physiological response of the lacrimal acini. These altered morphological and physiological properties of the lacrimal gland acini may help to explain the oc-
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currence of reduced aqueous tear secretions with age in human, since the five age groups investigated in this study span the entire range of human age. References Adeghate, E., Singh, J., Burrows, S., Howarth, F. C. and Donath, T. (1994). Secretory responses and peptidergic and aminergic innervation of rat lacrimal glands. Biogenic Amines 10 (2), 487–98. Bromberg, B. B. (1981). Autonomic control of lacrimal protein secretion. Invest. Ophthalmol. Vis. Sci. 20, 110–16. Bromberg, B. B. and Welch, M. H. (1985). Lacrimal protein secretion : Comparison of young and aged rats. Exp. Eye Res. 40, 313–20. Brooks-Frederich, K. M., Cianciarulo, F., Rittling, S. and Cristofalo, V. J. (1993). Cell cycle-dependent regulation of Ca#+ in young and senescent W1-38 cells. Exp. Cell. Res. 205, 412–15. Cristofalo, V. J., Pignolo, R. J. and Rotenberg, M. O. (1992). Molecular changes with in vitro cellular senescence. Ann. N.Y. Acad. Sci. 663, 187–94. Dartt, D. A. (1989). Signal transduction and control of lacrimal gland protein : A review. Curr. Eye Res. 8, 619–36. Dartt, D. A. (1994). Regulation of tear secretion. In (David A. Sullivan’s Ed.). Lacrimal gland, tear film and dry eye syndromes. Vol. 350, Pp. 1–9. Adv. Exp. Med. Biol. Plenum Press : New York, U.S.A. Dartt, D. A., Baker, A. K., Vaillant, C. and Rose, P. E. (1984). Vasoactive intestinal polypeptide stimulation of protein secretion from rat lacrimal gland acini. Am. J. Physiol. 247, G502–9. Dartt, D. A., Rose, P. E., Joshi, V. M., Donowitz, M. and Sharp, G. W. G. (1985). Role of calcium in cholinergic stimulation of lacrimal gland protein secretion. Curr. Eye Res. 4, 475–83. Dilly, P. N. (1994). Structure and Function of the tear film. In (David A. Sullivan’s ed.) Lacrimal gland, tear film and dry eye syndromes. Plenum Press : New York, U.S.A. Adv. Exp. Med. Biol. 350, 239–47. Draper, C. E., Singh, J., Pallot, D. and Adeghate, E. A. (1997). The effect of age on protein and peroxidase secretion and morphological changes in the isolated rat lacrimal gland. J. Physiol. 501.P, 155 P. Draper, C. E., Singh, J., Lawrence, P. A. and Adeghate, E. A. (1997). Light and electron microscopic investigations of age-related morphological changes of male rat lacrimal glands. J. Physiol. 504.P, 34 P. Draper, C. E., Adeghate, E. A., Lawrence, P. A., Pallot, D. J., Garner, A. and Singh, J. (1998). Age-relates changes in morphology and secretory responses of male rat lacrimal gland. J. Aut. Nerv. Syst. 69, 173–83. Francis, L. P. and Singh, J. (1990). Effects of phorbol esters and related compounds on acetylcholine evoked [$H]Leucine-labelled protein secretion in permeabilised rat pancreatic acini. Exp. Physiol. 75, 537–46. Friedlaender, M. H. (1992). Ocular manifestations of Sjogrens-syndrome, Keratoconjunctivitis sicca. Rheum. Dis. Clin. North Am. 18 (3), 591–608. Friedman, Z. Y., Lowe, M. and Selinger, Z. (1981). βadrenergic receptor stimulated peroxidase secretion from rat lacrimal gland. Biochimica. et Biophysica. 675, 40–5. Herzog, V. and Fahimi, H. D. (1973). A new and sensitive colorimetric assay of peroxidase using 3h,3h-diaminobenzidine as a hydrogen donor. Anal. Biochem. 5, 554–62.
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Herzog, V., Sies, H. and Miller, F. (1976). Exocytosis in secretory cells of rat lacrimal gland. J. Cell Biol. 70, 692–706. Holly, F. J. and Lemp, M. A. (1977). Tear physiology and dry eyes. Surv. Ophthalmol. 22, 69–87. Hussain, M. and Singh, J. (1988). Is VIP the putative noncholinergic, non-adrenergic neurotransmitter controlling protein secretion in rat lacrimal glands ? Quart. J. Exp. Physiol. 73, 135–8. Karnovsky, M. J. (1965). A formaldehyde-glutaraldehyde fixative of light osmolarity for the use in electron microscopy. J. Cell Biol. 27, 137A. Kelly, D. E., Wood, R. L. and Enders, A. C. (1984). Bailey’s textbook of microscopic anatomy. 18th ed. Williams & Wilkins : Baltimore, U.S.A. Kirkwood, T. B. L. (1992). Biological origins of ageing. In Oxford textbook of geriatric medicine (Evans, J. G. and Williams, T. F. Eds). Pp. 35–40. Oxford University Press : Oxford, U.K. Lowry, O. H., Roseborough, N. J., Farr, A. L. and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–75. Matsumoto, Y., Tanabe, T., Ueda, S. and Kawata, M. (1991). Immunohistochemical and enzyme histochemical studies of peptidergic, aminergic and cholinergic innervation of the lacrimal gland of the monkey (Macaca fuscaca). J. Aut. Nerv. Syst. 37, 207–14.
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Mu$ ller, P., Chambaut-Guerin, A. and Rossignol, B. (1985). Comparative effects of cytochalisin D on protein discharge induced by α- and β-adrenergic or cholinergic agonists in rat exorbital lacrimal glands. Biochem. Biophys. Acta. 884, 158–66. Putney, J. W., van De Walle, C. and Leslie, B. A. (1978). Stimulus-Secretion Coupling in the rat lacrimal gland. Am. J. Physiol. 235, 188–98. Rattan, S. I. S. (1995). Ageing : a biological perspective. Mol. Aspects Ageing 16, 439–508. Rattan, S. I. S. and Derventzi, A. (1991). Altered cellular responsiveness during ageing. BioEssays 13, 601–6. Reynolds, E. S. (1963). The use of lead citrate at high pH as an electron-opaque staining in electron microscopy. J. Cell Biol. 17, 208. Stevens, A. and Lowe, J. (1993). Histology. Mosby-Year Book Europe Limited. Takahashi, Y., Yoshida, T. and Takashima, S. (1992). The regulation of intracellular calcium ion and pH in young and old fibroblast cells (W1-38). J. Gerontol. 47, B65–70. Williams, P. L., Warwick, R., Dyson, M. and Bannister, L. H. (1989). Grays Anatomy. 37th ed. pp. 1216–18. Churchill Livingstone : Edinburgh, U.K. Williams, R. M., Singh, J. and Sharkey, K. A. (1994). Innervation and mast cells of the rat exorbital lacrimal gland : the effect of age. J. Aut. Nerv. Syst. 47, 95–108.
Evidence to Suggest Morphological and Physiological Alterations of Lacrimal Gland Acini with Ageing C. E. D R A P ERa, E. A. A D E G H A T E*b, J. S I N GHa D. J. P A L L OTb a b
Department of Applied Biology, University of Central Lancashire, Preston, PR1 2HE, England and the Department of Human Anatomy, United Arab Emirates, P.O. Box 17666, Al-Ain, United Arab Emirates (Received Columbia 17 July 1998 and accepted in revised form 7 August 1998) This study investigates changes in the morphology and physiology of lacrimal gland acinar cells with age. Changes in microstructural appearance of the acinar cells, the type and distribution of the different acini in glands and the secretory granules within the acini were examined in glands from animals of 3–5, 9, 12, 20, 24 and 28 month old rats. Differences in the secretory capacity of the acinar cells were also examined in animals of each age-group, with the exception of 28 months. The typical acini of young glands (3–5 months) were of the serous type. This was also true of 9 month glands, although there was a significant reduction in their overall distribution compared to young glands. The acini in the 12 month glands were predominantly of the seromucous type and appeared to be at the expense of the serous acini which were further significantly reduced compared to 3–5 and 9 month glands. This remained the prevalent acini type in 20 month glands, however by 24 months there was a significant increase in the occurrence of mucous acini and this time appeared to be at the expense of the seromucous acini which were significantly reduced in glands of this age-group. The predominant acinar cell in 28 month glands, like 24 month glands, was of the mucous variety. Qualitative EM studies revealed a progressive change in the secretory products of the lacrimal gland acini, strongly correlating to changes in acinar cell type. Typical acini of both 3–5 and 9 month glands contained numerous protein secretory granules. The seromucous acini also of these age groups contained both protein and mucous secretory granules, with the protein secretory granules in higher abundance. By 12 months the typical seromucous acini was packed with both protein and mucous secretory granules of equal proportions. However, by 20 months the predominant seromucous acini contained fewer protein secretory granules and elevated occurrence of mucous secretory granules. By 24 and 28 months the acini contained even fewer protein secretory granules and the typical acinar cell was of the mucous type containing exclusively mucous secretory granules. The secretory capacity of the acini was also altered with age. Maximum protein output in response to cholinergic stimulation resulted in an initial significant increase with ageing from 3–5 months to 9 and 12 months followed by a later significant age-dependent reduction in output. However, maximal peroxidase release from acinar cells of 3–5 and 9 month glands was the same. This was followed by a significant age-dependent reduction in peroxidase release. Furthermore, the concentrations required to evoke these responses differed with age. These results present evidence to suggest that acinar cells of the lacrimal gland undergo progressive alterations with age. The type of acini changing initially from serous to seromucous acini (intermediate phase) followed by a gradual transformation of the seromucous acini to mucous acini. This in turn changes the properties of the acini from protein producing and secreting acini to mucous producing and secreting acini. The results also suggest a reduction in the ability of the acini to synthesise proteins with age and altered responsiveness to cholinergic stimulation to secrete proteins. These findings may help in explaining the occurrence of altered protein\tear secretion with ageing. # 1999 Academic Press Key words : rat ; lacrimal gland ; acinar cells ; morphology ; physiology ; acetylcholine.
1. Introduction The aqueous component of the tear film is responsible for keeping the cornea buffered, lubricated, nourished and protected (Dilly, 1994 ; Friedlaender, 1992) and is produced and secreted from the main and accessory lacrimal glands. The exorbital gland is the main lacrimal gland of the rat and is analogous to the human lacrimal gland (Williams et al., 1989). About 80 % of the normal lacrimal gland consists of serous acini which are highly differentiated epithelial cells * Address all correspondence to : Dr Ernest Adeghate, Department of Human Anatomy, Faculty of Medicine and Health Sciences, United Arab Emirates University, P.O. Box 17666, Al-Ain, United Arab Emirates.
0014–4835\99\030265j12 $30.00\0
specialised for the synthesis, storage and exocrine secretion of proteins (Kelly et al., 1984). Water and electrolytes are also secreted from the acini. The serous acinar cell is pyramidal in shape, with a nucleus positioned in the wider basal portion of the cell. They have a well defined endoplasmic reticulum (ER), rough ER, several mitochondria and a supranuclear Golgi apparatus (Stevens and Lowe, 1993). The apical region of the acini contains granules filled with packaged protein ready for secretion by exocytosis (Herzog, Sres and Miller, 1976 ; Putney, Van De Walle and Leslie, 1978 ; Stevens and Lowe, 1993). The major protein secreted from the lacrimal acini is lipocalin. Other proteins are secreted but in lesser amounts and include ; lysozyme, peroxidase, lact# 1999 Academic Press
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oferrin, beta lysin and secretory immunoglobulins A and G (Friedlaender, 1992). Protein and fluid secretion is regulated by both autonomic and non-cholinergic, non-adrenergic nerves which innervate the acini, as well as ducts and blood vessels (Bromberg, 1981 ; Matsumoto et al., 1991). The classical neurotransmitters and neuropeptides released by these nerves are acetylcholine (ACh), noradrenaline (NA) and vasoactive intestinal polypeptides (VIP). Both cholinergic and adrenergic agonists as well as VIP can stimulate protein secretion from the lacrimal gland (Dartt et al., 1984, 1985 ; Friedman, Lowe and Selinger, 1981 ; Hussain and Singh, 1988 ; Muller, Chambaut-Guerin and Rossignol, 1985). Reduced secretion of the aqueous layer of the tear film is the most common abnormality found in the disease state Keratoconjunctivitis sicca (KCS), or dry eye syndrome (Dartt, 1994 ; Friedlaender, 1992). KCS is an ocular disorder involving local drying up of the tear film, initially causing inflammation and fibrosis of the lacrimal gland and irritation of the eyes. This, however, can lead to corneal cellular loss (Superficial Keratitis) and advanced stages of the disease include, vascularisation and scarring of the cornea causing severe visual disability (Holly and Lemp, 1977). KCS is an age-related disorder affecting mainly the middle aged and the elderly (Friedlaender, 1992). Age-related changes in both the physiological and morphological properties of the rat lacrimal gland have previously been observed (Draper et al., 1997). This included a reduction in protein secretion from aged lacrimal gland acini compared to that from young tissue. Morphologically, the aged tissue displayed structural damage, more specifically, the type of acinar cell predominant in the glands of aged animals differed from those prevalent in glands from young rats (Draper et al., 1997). However, little is known about the morphology or secretory capacity of lacrimal acini from glands of animals of intermediate age-groups. We therefore decided to perform a more comprehensive study on the effect of ageing on the lacrimal acinar cell investigating the micro- and ultra-structural morphology of the acini and their distribution in glands from rats of 3–5, 9, 12, 20, 24 and 28 months of age, using both light and electron microscopy. Total protein and peroxidase release from the lacrimal acini of glands from animals of each age-group, with the exception of 28 months, was also compared. This study was performed with the aim of understanding the influence of age on the pathological alterations of the lacrimal gland, which appear to result in decreased aqueous tear secretion, part of the aetiology of KCS.
2. Materials and Methods Animals Morphological studies were performed on isolated lacrimal glands taken from male Sprague-Dawley rats
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of ages 3–5 (n l 6), 9 (n l 6), 12 (n l 6), 20 (n l 6), 24 (n l 6) and 28 months (n l 6). These age groups span the entire range of human age. For example, 3–5 months old rats correspond to 15–18 year old humans, while 9, 12, 20 and 24 month old rats corresponds to 25–35, 35–50, 50–65, 65–75 and 75–85 year old humans, respectively. Physiological experiments were performed on lacrimal gland acini isolated from lacrimal glands of rats from each age-group with the exception of 28 months (n l 6 for each age-group). The animals were based and housed at the United Arab Emirates University in Al-Ain, United Arab Emirates and all procedures had the relevant clearance from the local ethical committee for the use of animal experiments. The aged rats used in this study were all in apparent good health. Reagents and Chemicals All reagents employed in this study were obtained from Sigma (U.K.). Morphological Studies Light microscopy Immediately after killing the animal, the lacrimal glands were removed, cut into small fragments and fixed for 24 hr in Zamboni’s solution (Adeghate et al., 1994). After fixation, the specimens were dehydrated and embedded in paraffin wax and cut into 5–7 µm-thick sections using a rotary microtome (Shandon AS 325 retraction). The sections were incubated at 50mC for 30 min prior to staining. Sections were stained with haematoxylin and eosin, dehydrated in ascending concentrations of ethanol, cleared in xylene and mounted in Cytoseal 60 (Stephens Scientific, Riverdale). Sections were viewed under a light microscope (Zeiss Axiophot, Germany). Pictures were taken using Ilford Pan F-Black and White microscope 50 ASA film. Acini counts Counts of acini type were performed using light microscopy. Acini were counted in visible sections at i40 magnification. A total of 40 random fields were taken from a total of 20 slides for each age group and these slides were masked. Because of the conspicuous changes observable with ageing, it was easy to sort out which age group each of the slide belonged to. The percentage distribution of each acini type of the glands from the 6 age-groups was calculated. Electron microscopy Lacrimal glands were cut into small pieces and fixed for 4–5 hr in 2n5 % glutaraldehyde in Karnovsky’s solution (Karnovsky, 1965). The tissue pieces were washed five times overnight at 4mC in cacodylate buffer (0n2 ). The tissue was postfixed with 1 % osmium tetroxide for 1–3 hr, washed 8 times in cacodylate buffer and dehydrated in ascending concentrations of ethanol. They were then treated
AGE-RELATED CHANGES OF THE RAT LACRIMAL ACINI
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using a diatome knife (supplied by Agar Scientific, Essex, England) mounted on 3n05 mm 20 mesh copper grid and contrast stained with saturated aqueous uranyl acetate for 30 min and Reynolds lead citrate (Reynolds, 1963) for 5 min. These were then examined and photographed in a Phillips CM10 transmission electron microscope. Pictures were taken using Kodak electron thick base 4489 film. Secretory Capacity of Lacrimal Acinar Cells Preparation of lacrimal acinar cells The lacrimal glands from animals of each age-group (6 animals for each group) were placed in Ca#+-free Krebs-Henseleit (KH) solution of the following composition (m) : NaCl 130, KCl 5n0, HEPES 20, KH PO 1n2, # % MgSO 2n0, Glucose 10 and minced (Francis and % Singh, 1990). The tissue fragments were washed extensively and placed in 10 ml KH solution containing Bovine Serum Albumin (BSA-0n2 %), trypsin inhibitor (0n01 %) and collagenase (0n025 g) and incubated at 37mC for 30 min in a shaking water bath. The cells were washed in KH solution containing BSA, trypsin inhibitor and calcium (3n0 m) and dissociated for 12 min by gently passing the tissue through flamed pipette tips of decreasing bore (3 mm, 2 mm, 1 mm) and subsequent filtering through nylon mesh (100µm-pore). The suspension of dispersed acinar cells were centrifuged twice at 800 rpm 5 min each. The supernatant was discarded and the acinar cell pellet was resuspended in KH solution containing calcium (3n0 m). Acini isolated by this technique had good morphology and a viability of 90 % when tested by trypan blue exclusion (Herzog et al., 1976).
F. 1. Light micrographs showing the progressive change in the acinar cell appearance of 3–5 and 9 months glands [A] to glands of 12 and 20 months [B] and to glands of 24 and 28 months [C] ; from serous to seromucous (intermediate phase) and to mucous acini, respectively. These micrographs are typical of 6 such samples from 6 animals of each agegroup. s l serous acini ; sm l seromucous acini ; m l mucous acini. i262. Scale bar l 50 µm.
with propylene oxide ; 2 changes of 15 min and placed in propylene oxide and resin (1 : 1) for 1 hr followed by pure resin overnight. The samples were embedded and polymerised at 60mC for 24 hr. Thin sections were cut
Stimulation of lacrimal acinar cell secretion Acinar cell aliquots (250 µl) were placed into microfuge tubes containing 750 µl of KH buffer solution. To each tube 110 µl of varying concentrations of ACh (10−"$– 10−( ) was added. In control aliquots an equivalent of 110 µl of KH buffer solution was added to each tube. The suspension in each tube was gently vortexed and incubated in a shaking waterbath at 37mC for 30 min. The suspensions were spun in a microfuge for 5 min. The supernatant was removed, collected and analysed for protein and peroxidase. The residue was resuspended in a known amount of distilled water, sonicated and analysed for cell protein content. Total protein in supernatant and residue suspensions were measured spectrophometrically (Beckman DU2-70Spectrophotometer), using a Bio-Rad protein assay (Sigma, U.K.) based on the Lowry method (Lowry et al., 1951). Bovine serum albumin was used as standard. Peroxidase in the supernatant was also measured spectrophometrically using a colorimetric method previously described by Herzog and Fahimi (1973) incorporating diaminobenzidine as a hydrogen donor. Commercial horseradish peroxidase was used as standard. Protein output was expressed as µg ml−"
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F. 2. For legend see facing page.
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(mg cell protein)−" while peroxidase release was expressed as ηg ml−" (mg cell protein)−". Statistical Analysis All data provided are expressed as meanpstandard error of the mean (...). Data were compared using analysis of variance (ANOVA) and only values with P 0n05 were accepted as significant. 3. Results Light Microscopy Light microscopical studies of the lacrimal glands from animals of 3–5, 9, 12, 20, 24 and 28 months of age revealed the presence of the 3 distinct types of acini ; serous (protein secreting) acini, seromucous (protein and mucous secreting) acini and mucous acini (Fig. 1). The glands from the 3–5 month old rats (regarded as normal, control rats) were composed of tubulo-acinar tissue mainly of the serous type (Fig. 1(A)]. At 9 months the majority of acini were also of the serous type. However, with ageing significant changes in the size and shape of the acini were observed. By 12 and 20 months the majority of the acini were of the seromucous variety [Fig. 1(B)]. Moreover, by 24 and 28 months most of the gland consisted of mucous acini (Fig. 1(C)]. Identification of the mucous acini have previously been confirmed (Draper et al., 1998) by staining of sections with Alcian blue at pH 1n0. Acinar Cell Type Distribution A quantitative analysis was performed to determine the distribution of each acinar cell type within the glands of the animals from the 6 age-groups. Acini were counted from random sections at i40 magnification using light microscopy and the percentage distribution of each acinar cell type was calculated. Effect of ageing on distribution of serous acini In young glands 81n2p4n7 % (meanp...) of the acini were serous (Fig. 2(A)]. By 9 months this was significantly reduced to 50n5p9n4 % (ANOVA ; P 0n05) and by 12 months further significantly (P 0n01) reduced to 19n5p5n2 %. The proportion of serous acini remained unchanged by 20 and 24 months, however, by 28 months there was a further significant (P 0n05) reduction to 7n6p4n3 %. Effect of ageing on distribution of seromucous acini In young glands 17n2p2n2 % of the acini were sero-
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mucous [Fig. 2(B)]. By 9 months this significantly (P 0n05) increased to 32n5p3n3 % and by 12 months further significantly (P 0n01) increased to 65n7p4n4 %. By 20 months there was a slight decrease in seromucous acini to 53n3p6n0 %, moreover, by 24 months a significant (P 0n01) reduction to 29n3p 3n7 % with a small increase at 28 months to 37n1p 3n2 %. Effect of ageing on distribution of mucous acini Only 1n6p0n4 % of the acini in young glands were mucous [Fig. 2(C)]. By 9 and 12 months this increased significantly (P 0n05) to 17n0p1n9 % and 15n8p2n3 %, respectively and there was no significant change by 20 months. However, by 24 and 28 months there were significant (P 0n01) increases in mucous acini to 46n6p5n6 % and 53n3p5n0 %, respectively. Electron Microscopy Electron microscopic studies were performed to investigate the influence of age on the secretory products of the lacrimal gland acini. Again glands from young animals (3–5 months) were regarded as normal, controls. Typical acini of the glands from the young animals contained numerous protein (electron dense) secretory granules [Fig. 3(A)]. There was also the presence of a few acini containing both protein and mucous (electron lucent) secretory granules [Fig. 3(B)] the protein granules being in higher abundance. Both types of acini were typical of the 9 month lacrimal glands. However, by 12 months the typical acini was packed with both protein and mucous secretory granules of similar proportions [Fig. 3(C)]. The predominant acini of 20 month glands also contained both protein and mucous secretory granules [Fig. 3(D)], however the mucous secretory granules were in higher abundance to the protein granules which were markedly reduced compared to the typical acini of the 12 month glands. This was elevated further by 24 and 28 months where the typical acini contained exclusively mucous secretory granules [Fig. 3(E)]. Total Protein Output and Peroxidase Release Isolated lacrimal acinar cells of glands from rats of 3–5, 9, 12, 20 and 24 months of age were incubated with ACh at varying concentrations (10−"$–10−( ) to establish the secretory capacity of the acini and to determine the concentrations required to achieve maximum secretion of total protein output and peroxidase release with ageing.
F. 2. Histograms showing the percentage distribution of the 3 types of secretory acini ; serous [A], seromucous [B] and mucous [C] in rat lacrimal glands from animals of 3–5, 9, 12, 20, 24 and 28 months of age. Each point is meanp... A total of 40 random sections typical of 6 samples from 6 animals of each age-group. Note the age-related changes in the distribution of each acinar cell type.
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F. 3. Electron micrographs showing the progressive change in the secretory products of the typical acinar cells of 3–5 and 9 month glands (A) and (B) to glands of 12 months (C) to glands of 20 months (D) and to glands of 24 and 28 months of age (E). Serous acini from 3–5 and 9 months old animals with many protein-containing secretory granules (arrow), and seromucous acini from these glands with both protein-containing secretory ; electron dense granules and mucous-containing, electron lucent secretory granules (arrow head). Note the age-related reduction in protein-secretory granules and progressive increase in mucous-secretory granules of the seromucous acini of both the 12 and 20 month glands to the acini containing only mucous-secretory granules of the 24 and 28 month glands. These micrographs are typical of 6 such samples from 6 agegroups. (A) serous acini of 3–5 month glands ; (B) seromucous acini of 3–5 and 9 month glands ; (C) seromucous acini of 12 month glands ; (D) seromucous acini of 20 month glands ; (E) mucous acini of 24 and 28 months. Magnification : A l i3120 ; B l i4800 ; C l i7840 ; D l i5288 and E l i6480. Scale bar l 2 µm.
Total protein output Stimulation of 3–5, 9, 12, 20 month old rat lacrimal acinar cells with ACh resulted in significant (P 0n05) increases in protein output when compared to basal. In contrast, ACh evoked only a small but not significant increase in total protein output from 24-month old rat lacrimal acinar cells when compared to basal. Basal total protein outputs from the acini of glands from the 5 age-groups were 0n69p0n06 (n l 10), 0n84p0n03 (n l 12), 0n76p 0n03 (n l 12), 0n41p0n03 (n l 10) and 0n37p 0n02 µg ml−" (mg cell protein)−" (meanp... ; n l 10), respectively. Note the age-related changes in basal secretory responses. A small, but not significant increases in basal protein output were observed in 9 and 12 month old lacrimal gland cells, whereas, there was a significant (P 0n05) decrease in basal protein output from 20 and 24 month olds [Fig. 4(A)]. The maximal secretory effect exerted by ACh on total
protein output from acini of the glands from 3–5, 9, 12, 20 and 24 month animals is presented in Fig. 4(B). This figure shows the mean peak (p...) protein output above basal level for the concentrations of ACh (10−"", 10−"", 10−"#, 10−"# and 10−* ) that produced the maximal secretory effect on lacrimal acinar cells of glands from the 3–5, 9, 12, 20 and 24 month old animals, respectively. Note the age-related change in the concentration of ACh required to achieve the maximal secretory response. There is an inverse correlation between the concentration of ACh and maximal protein release. This may be due to the alterations in the number and intactness of the membrane-bound ACh receptors with ageing leading to reduced sensitivity and reduced protein secretion. ACh evoked significant (ANOVA ; ... ; P 0n01) increases in protein output from lacrimal gland acini of young animals compared to basal. At 9 and 12
AGE-RELATED CHANGES OF THE RAT LACRIMAL ACINI
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F. 4. Family of histograms showing mean peak (p... ; n l 6) basal protein output (A) and mean peak (p... ; n l 6) total protein release (B) above basal levels from the lacrimal acinar cells of glands from 3–5, 9, 12, 20 and 24 month old rats, following stimulation with a concentration of ACh that elicited maximal response in each age-group. Note the age-related changes in secretion of both basal and ACh-evoked total protein output.
months ACh evoked a further significant (P 0n05) increase in protein output. However, maximal secretion at 9 months was significantly (P 0n05)
greater than at both 3–5 and 12 month rats. In contrast, by 20 months total protein output was significantly (P 0n01) reduced compared to that
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C. E. D R A P E R E T A L.
F. 5. Family of histograms showing mean peak (p... ; n l 6) basal peroxidase output (A) and mean peak (p... ; n l 6) peroxidase release (B) above basal levels from the lacrimal acinar cells of glands from 3–5, 9, 12, 20 and 24 month old rats, following stimulation with a concentration of ACh that elicited maximal response in each age-group. Note the age-related changes in secretion of both basal and ACh-evoked peroxidase release.
from acini of glands from 3–5, 9 and 12 month old rats. Moreover, there was a further significant (P 0n05) reduction in the amount of protein secreted from acini of glands from animals of 24 months of age when compared to 20 month glands.
Peroxidase release In comparison with basal peroxidase release, there was a significant increase (P 0n05) in peroxidase release when 3–5, 9, 12 and 20 month old rat lacrimal acinar cells were incubated with ACh. At 24 months, ACh evoked only a small but
AGE-RELATED CHANGES OF THE RAT LACRIMAL ACINI
not significant increase in peroxidase output when compared to basal. Basal peroxidase release from the acini of glands from the 3–5, 9, 12, 20 and 24 month animals were 0n34p0n01 (n l 10), 0n31p0n01 (n l 12), 0n26p0n01 (n l 12), 0n13p0n01 (n l 10) and 0n10p0n01 ηg ml−" (mg cell protein)−" (meanp... ; n l 10), respectively. Note the age-related changes in basal secretory responses. At 9 and 12 months, there was a small but not significant reduction in basal peroxidase release when compared to 3–5 month old lacrimal gland cells. However, the basal peroxidase release was further reduced significantly (P 0n01) at 20 and 24 months when compared to 3–5 month olds [Fig. 5(A)]. Figure 5(B) shows the maximal secretory effect exerted by ACh on peroxidase release from acini of glands from the 5 age-groups. This figure shows the mean peak (p...) peroxidase release above basal level for the concentrations of ACh (10−"", 10−"!, 10−"!, 10−"! and 10−* ) that produced the maximal secretory effect on lacrimal acinar cells from glands of the 5 age-groups, respectively. Again note the agerelated changes in the concentration of ACh required to achieve the maximal response. It is evident that there is an inverse correlation between the concentration of ACh and maximal peroxidase release. This may be due to the alterations in the number and intactness of the membrane-bound ACh receptors with ageing leading to reduced sensitivity and reduced peroxidase secretion. ACh evoked significant (ANOVA ; ..., P 0n01) increases in peroxidase release from lacrimal gland acini of young animals compared to basal. There was no change in secretion at 9 months, however, by 12 months peroxidase release was significantly (P 0n01) reduced compared to that from 3–5 and 9 month acini. There was a further significant (P 0n01) decrease in peroxidase release from acini of 20 month glands compared to 12 month glands and by 24 months a further significant (P 0n01) reduction in release where only a very small amount of peroxidase was released from these acini. 4. Discussion The results of this study have demonstrated that lacrimal gland acini change with age both morpho logically and physiologically. The acinar cell type of young glands were predominantly serous (protein producing and secreting cells). This was also true of the 9 month glands although there was a significant (P 0n01) decrease in the overall distribution of serous acini when compared to the young glands. In consequence there were increases in both seromucous (protein and mucous producing and secreting cells) and mucous acini. By 12 months there was a significant (P 0n01) increase in the relative occurrence of the seromucous acini and appeared to be at the expense of the serous acini which were further significantly (P 0n01) reduced when compared to 9 month glands. The distribution of mucous acini in
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glands from this age-group remained unchanged. Seromucous acini remained the prominent acinar cell type in glands from 20 month old animals and the distribution of both serous and mucous acini remained unchanged when compared to 12 month glands. However, by 24 months there was a significant (P 0n01) increase in the overall prevalence of mucous acini and now appeared to be at the expense of the seromucous acini which were significantly (P 0n01) reduced when compared to 20 month glands. The distribution of serous acini again remained unchanged at this age. Nevertheless, in 28 month glands the distribution of serous acini was further significantly (P 0n05) reduced when compared to 24 month glands. In consequence there was a very small but not statistically significant increase in seromucous acini and no change in the distribution of mucous acini. However, like glands from 24 month old animals the predominant acinar cell type in 28 month glands were of the mucous type. The acinar cell dominating the glands from young animals contained many protein secretory granules. A few acini also contained both protein and mucous secretory granules. Both types of acini were typical of 9 month glands, however the serous acini were the prevalent type. The predominant seromucous acini of the 12 month glands differed compared to the seromucous acini of the 3–5 and 9 month glands. The acini were packed with both protein and mucous secretory granules of equal proportions, whereas in the acini of the young and 9 month tissue the protein secretory granules were in higher abundance. By 20 months the dominant seromucous acini differed from those of the 3–5, 9 and 12 month tissues, where the presence of protein secretory granules was reduced and the presence of mucous secretory granules was markedly increased. Moreover, the presence of protein secretory granules in acini from 24 and 28 month tissue was further reduced and the typical acini was a mucous cell containing exclusively mucous secretory granules. These results indicate progressive alterations in the specialised protein-secreting cells of the lacrimal gland with age, initially from serous to seromucous (intermediate stage) and gradually with time from seromucous to mucous acini. The progression from serous to mucous acini is a new and important finding, however, the causes and mechanisms of these changes remain elusive. However, it is tempting to speculate that these changes are due to the normal process of cellular ageing and senescence with involves loss of cellular homeostasis and maintenance over time (Kirkwood, 1992 ; Rattan, 1995). The major modes of cellular maintenance include ; processes of cellular repair, cell division, cell replacement, neuronal and hormonal responsiveness, immune response, detoxification, free-radical scavenging, stress-protein synthesis, macromolecular turn-over and maintenance of the fidelity of genetic information transfer
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(Rattan, 1995). A loss of one or a number of these mechanisms may be responsible for the changes in the acinar cells observed in this study. Altered cellular responsiveness to growth factors, hormones and other factors have been found to be reduced significantly during cellular ageing of cultured diploid cells (Cristofalo, Pignolo and Rotenberg, 1992 ; Rattan and Derventzi, 1991). This resulted in a significant reduction in both protein and DNA synthesis and reduced induction of several cell-cycle related genes. Certainly, in these studies the reduced presence of protein secretory granules of the acini indicate a reduction in protein synthesis. In addition, the changes which have occurred in this study may be due to increased sensitivity to toxic agents such as phorbol esters, free-radical inducers, irradiation and heat shock which have also been observed in ageing cells (Rattan and Derventzi, 1991). Reduced stimulation of the acini by neurotransmitters due to denervation of the autonomic nerves (Williams, Singh and Sharkey, 1994) may also be responsible for reduced synthesis of protein secretory products of the acini. Consequently increased stimulation by other factors for example, mast cell mediators may be responsible for stimulation of synthesis of the mucous secretory products of the acini. Increased infiltration of mast cells to the lacrimal gland with ageing have previously been observed (Draper et al., 1997 ; Williams et al., 1994). In this study stimulation of protein and peroxidase secretion from isolated lacrimal gland acinar cells differed considerably with age. ACh evoked significant (P 0n01) increases in total protein output from the acinar cells of young glands above basal and the maximal secretory response was achieved at 10−"" . The same concentration evoked maximal protein output from acinar cells of 9 month glands, however, the maximum amount of protein secreted by significantly (P 0n05) higher than that from young glands. Protein output from acinar cells of 12 month glands was significantly (P 0n05) reduced compared to that from 9 month glands but was still greater than that from young glands (P 0n05). Furthermore, the concentration required to achieve the maximum response was 10−"# compared to 10−"" required for acini of both young and 9 month glands. Maximum secretory response from acinar cells of 20 month glands was also achieved at 10−"# , however, the maximum amount of protein secreted was significantly (P 0n01) reduced compared to that from 3–5, 9 and 12 month old rats. Moreover, there was a further significant (P 0n05) reduction in the amount of protein secreted from acini of glands from rats of 24 months of age when compared to 20 month glands and the concentration required to produce this effect was 10−* . In another set of experiment, ACh also evoked significant (P 0n01) increases in peroxidase release from acinar cells compared to basal at a concentration of 10−"" . By 9 months there was no change in the
C. E. D R A P E R E T A L.
amount of peroxidase released, however, the concentration required to evoke the maximum response was 10−"! . The same concentration was required to produce the maximum response from acinar cells of 12 month glands, however, the amount of peroxidase released was significantly (P 0n01) reduced compared to that from acinar cells of both 3–5 and 9 month glands. There was a further significant (P 0n01) decrease in peroxidase release from cells of 20 month glands compared to 12 month glands with stimulation of the same concentration of ACh. The maximum peroxidase released from acinar cells of 24 month glands was further significantly (P 0n01) reduced when compared to that from acinar cells of 20 month glands. Only a very small amount of peroxidase was released from these cells. Furthermore, the concentration of ACh required to achieve this response was 10−* . Earlier studies observed age-related reductions in total protein output from rat lacrimal acinar cells (Draper et al., 1997) and intact rat lacrimal tissue segments (Bromberg and Welch, 1985). This study, however, has revealed that protein output initially increases with ageing followed by an age-related reduction. The cellular mechanism(s) involved in the alterations in protein output with age is still unknown. It is possible that more than one mechanism may be associated with ageing and include ; changes in acinar cell morphology, alterations in the ability of the acini to synthesise and secrete proteins, elevations or reductions in the number of acinar plasma membrane receptors for the secretagogues and\or changes to the signal transduction mechanism. The reduction in serous acini at 9 months may initiate a compensatory mechanism to increase protein output. It is unlikely that a change in the responsiveness to ACh would result in this increased secretion since maximum output was achieved at the same concentration as for young glands. Since the abundance of protein secretory granules in acini of both 3–5 and 9 month glands were similar it is also unlikely that protein synthesis is elevated at 9 months. However, it may be possible that the signal transduction mechanism is altered to increase output. The further reduction in serous acini at 12 months results in a decrease in protein output, but which is still greater than that from young glands (P 0n05). Since maximum stimulation is achieved at a weaker concentration it is possible that there is an increase in the number of membrane bound receptors for ACh. By 20 months the same concentration is required for maximum output suggesting, like 12 month acini, elevated receptors for ACh. However, the significant reduction in protein output coupled with the reduction in protein secretory granules of the acini from 20 month animals suggests a reduction in protein synthesis. The stimulation of protein synthesis in response to various extracellular stimuli has been seen to be significantly reduced in ageing cells (Cristofalo et al., 1992).
AGE-RELATED CHANGES OF THE RAT LACRIMAL ACINI
Problems with signal transduction and\or exocytosis may also contribute to this reduced output. The secretory responses evoked by cholinergic agonists are associated with an increase in cellular calcium mobilisation (Dartt, 1989, 1994 ; Putney et al., 1978). Recent studies have shown that the intracellular calcium concentration either remains the same (Brooks-Frederick et al., 1993 ; Takahashi, Yoshida and Takaswima, 1992) or decreases during ageing and the defect may be at the level of the cells capacity to mobilize calcium in response to extracellular stimuli. The further significant reduction in protein output from acini of 24 month glands may largely be due to the acini changing to mucous cells containing exclusively mucous secretory granules. Other acini of this age-group displayed a further reduction in the presence of protein secretory granules also suggesting reduced protein synthesis. A far stronger concentration of ACh was required to produce the maximum secretory response suggesting a reduction in the number of plasma membrane receptors. A decline in the synthesis and activity of cholinergic system components and variations in receptor response with ageing has been reported (Rattan, 1995). Alterations to the signal transduction mechanisms and\or exocytosis may also be responsible in part for the reduced protein output at this age. The results of this study also suggest that the type of proteins secreted may change with age. Although at 9 months more protein is secreted from the acini compared to that from 3–5 month glands, the amount of peroxidase released is the same. At 12 months peroxidase release is significantly reduced while total protein output is higher than that from young glands. This may be due to reduced synthesis or a problem with packaging and secretion. By 20 and 24 months there is an age-related reduction in peroxidase release which correlates with reduced total protein output. Again this may be due to reduced synthesis. Experiments are currently in progress to determine which of the parameters are involved in the age-related changes in the protein synthesis and secretory capacity of the acini. In conclusion, the results of this study put forward evidence to suggest that acinar cells of the lacrimal gland undergo progressive alterations with age. Initially from serous to seromucous acini followed by gradual transformation of seromucous acini to mucous acini. Considerable changes to the secretory capacity of the lacrimal acini with age were also observed and strongly correlated to the changes in acini type and changes to the secretory product of the acini. In addition, a reduction in the ability of the acini to synthesise proteins with ageing coupled to alterations in membrane bound receptors for cholinergic agonists were also implicated in altering the physiological response of the lacrimal acini. These altered morphological and physiological properties of the lacrimal gland acini may help to explain the oc-
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currence of reduced aqueous tear secretions with age in human, since the five age groups investigated in this study span the entire range of human age. References Adeghate, E., Singh, J., Burrows, S., Howarth, F. C. and Donath, T. (1994). Secretory responses and peptidergic and aminergic innervation of rat lacrimal glands. Biogenic Amines 10 (2), 487–98. Bromberg, B. B. (1981). Autonomic control of lacrimal protein secretion. Invest. Ophthalmol. Vis. Sci. 20, 110–16. Bromberg, B. B. and Welch, M. H. (1985). Lacrimal protein secretion : Comparison of young and aged rats. Exp. Eye Res. 40, 313–20. Brooks-Frederich, K. M., Cianciarulo, F., Rittling, S. and Cristofalo, V. J. (1993). Cell cycle-dependent regulation of Ca#+ in young and senescent W1-38 cells. Exp. Cell. Res. 205, 412–15. Cristofalo, V. J., Pignolo, R. J. and Rotenberg, M. O. (1992). Molecular changes with in vitro cellular senescence. Ann. N.Y. Acad. Sci. 663, 187–94. Dartt, D. A. (1989). Signal transduction and control of lacrimal gland protein : A review. Curr. Eye Res. 8, 619–36. Dartt, D. A. (1994). Regulation of tear secretion. In (David A. Sullivan’s Ed.). Lacrimal gland, tear film and dry eye syndromes. Vol. 350, Pp. 1–9. Adv. Exp. Med. Biol. Plenum Press : New York, U.S.A. Dartt, D. A., Baker, A. K., Vaillant, C. and Rose, P. E. (1984). Vasoactive intestinal polypeptide stimulation of protein secretion from rat lacrimal gland acini. Am. J. Physiol. 247, G502–9. Dartt, D. A., Rose, P. E., Joshi, V. M., Donowitz, M. and Sharp, G. W. G. (1985). Role of calcium in cholinergic stimulation of lacrimal gland protein secretion. Curr. Eye Res. 4, 475–83. Dilly, P. N. (1994). Structure and Function of the tear film. In (David A. Sullivan’s ed.) Lacrimal gland, tear film and dry eye syndromes. Plenum Press : New York, U.S.A. Adv. Exp. Med. Biol. 350, 239–47. Draper, C. E., Singh, J., Pallot, D. and Adeghate, E. A. (1997). The effect of age on protein and peroxidase secretion and morphological changes in the isolated rat lacrimal gland. J. Physiol. 501.P, 155 P. Draper, C. E., Singh, J., Lawrence, P. A. and Adeghate, E. A. (1997). Light and electron microscopic investigations of age-related morphological changes of male rat lacrimal glands. J. Physiol. 504.P, 34 P. Draper, C. E., Adeghate, E. A., Lawrence, P. A., Pallot, D. J., Garner, A. and Singh, J. (1998). Age-relates changes in morphology and secretory responses of male rat lacrimal gland. J. Aut. Nerv. Syst. 69, 173–83. Francis, L. P. and Singh, J. (1990). Effects of phorbol esters and related compounds on acetylcholine evoked [$H]Leucine-labelled protein secretion in permeabilised rat pancreatic acini. Exp. Physiol. 75, 537–46. Friedlaender, M. H. (1992). Ocular manifestations of Sjogrens-syndrome, Keratoconjunctivitis sicca. Rheum. Dis. Clin. North Am. 18 (3), 591–608. Friedman, Z. Y., Lowe, M. and Selinger, Z. (1981). βadrenergic receptor stimulated peroxidase secretion from rat lacrimal gland. Biochimica. et Biophysica. 675, 40–5. Herzog, V. and Fahimi, H. D. (1973). A new and sensitive colorimetric assay of peroxidase using 3h,3h-diaminobenzidine as a hydrogen donor. Anal. Biochem. 5, 554–62.
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Mu$ ller, P., Chambaut-Guerin, A. and Rossignol, B. (1985). Comparative effects of cytochalisin D on protein discharge induced by α- and β-adrenergic or cholinergic agonists in rat exorbital lacrimal glands. Biochem. Biophys. Acta. 884, 158–66. Putney, J. W., van De Walle, C. and Leslie, B. A. (1978). Stimulus-Secretion Coupling in the rat lacrimal gland. Am. J. Physiol. 235, 188–98. Rattan, S. I. S. (1995). Ageing : a biological perspective. Mol. Aspects Ageing 16, 439–508. Rattan, S. I. S. and Derventzi, A. (1991). Altered cellular responsiveness during ageing. BioEssays 13, 601–6. Reynolds, E. S. (1963). The use of lead citrate at high pH as an electron-opaque staining in electron microscopy. J. Cell Biol. 17, 208. Stevens, A. and Lowe, J. (1993). Histology. Mosby-Year Book Europe Limited. Takahashi, Y., Yoshida, T. and Takashima, S. (1992). The regulation of intracellular calcium ion and pH in young and old fibroblast cells (W1-38). J. Gerontol. 47, B65–70. Williams, P. L., Warwick, R., Dyson, M. and Bannister, L. H. (1989). Grays Anatomy. 37th ed. pp. 1216–18. Churchill Livingstone : Edinburgh, U.K. Williams, R. M., Singh, J. and Sharkey, K. A. (1994). Innervation and mast cells of the rat exorbital lacrimal gland : the effect of age. J. Aut. Nerv. Syst. 47, 95–108.