Current Eye Research, 31:13–22, 2006Copyright ©c Taylor & Francis Group, LLCISSN: 0271-3683 print / 1460-2202 onlineDOI: 10.1080/02713680500428613

Unique Ultrastructure of Exorbital LacrimalGlands in Male NOD and BALB/c Mice

Chuanqing DingDepartment of Cell andNeurobiology, Keck Schoolof Medicine, Universityof Southern California, LosAngeles, California, USA

Michelle MacVeighUSC Center for Liver Diseases,Keck School of Medicine,University of SouthernCalifornia, Los Angeles,California, USA

Mike PidgeonDepartment of Cell andNeurobiology, Keck School ofMedicine, University ofSouthern California, LosAngeles, California, USA

Silvia R. da Costa,Kaijin Wu, andSarah F. Hamm-AlvarezDepartment of PharmaceuticalSciences, Keck School ofMedicine, University ofSouthern California,Los Angeles, California, USA

Joel E. SchechterDepartment of Cell andNeurobiology, Keck School ofMedicine, University ofSouthern California,Los Angeles, California, USA

ABSTRACT Lacrimal glands of male NOD and BALB/c mice have very small,pleomorphic acinar lumens. Acini contain isolated zones of highly complexcell surface interdigitations at the basal surface, sometimes occurring betweenacinar andmyoepithelial cells. In NODmice, cytological abnormalities, includ-ing mitochondrial deterioration, pleomorphic and heterogeneous cytoplasmicvacuoles, and lipid accumulation are evident within acinar cells at 1 month.Accumulation of lipid is further increased as the animal ages, accompanied bylymphocytic infiltration and destruction of acini. These results demonstrate al-terations from normal cytology as early as 1 month in NOD mice, well beforedetection of clinical signs of Sjogren syndrome.

KEYWORDS dry eye; electron microscopy; lacrimal gland; NOD mice; Sjogren syndrome

INTRODUCTIONThe non-obese diabetic (NOD) strainof mice is frequently used as a

model system to study autoimmune disease, including Sjogren syndrome.1−4

This strain was first described by Makino et al. as exhibiting pancre-atic insulitis characterized by infiltration of leukocytes into the pancreaticislets.5

Besides a prominent mononuclear cell infiltration of the islets of Langer-hans, NOD mice also show spontaneous infiltrates of lymphocytes into thesubmandibular and lacrimal glands concomitant with histological signs of tis-sue damage. Autoimmune inflammation of the submandibular and lacrimalglands is followed by secretory dysfunction; the changes being very similar tothose of human autoimmune disease, for example, Sjogren syndrome.6 There-fore, NOD mice have been used widely as a model for the investigation ofSjogren syndrome.7−9 A previous report indicated that NOD mice appear nor-mal during the postnatal period, up to about 3–4 weeks, when histologic changesindicative of insulitis become evident.10

Insulitis and submandibularitis develop in both males and females, althoughat different times, earlier in females than males. With respect to Sjogren syn-drome, the lacrimal glands of males are significantly more affected than fe-males. Lymphocytic infiltration into the lacrimal gland is first detected at about6–10 weeks in males8,11 and around 30 weeks in females.8 Clinical manifes-tations of Sjogren syndrome are generally detectable in males by 4 months.9

Received 18 March 2005Accepted 18 October 2005

Correspondence: Chuanqing Ding,M.D., Ph.D., Department of Cell andNeurobiology, University of SouthernCalifornia, Keck School of Medicine,1333 San Pablo St., BMT 304, LosAngeles, CA 90089-9112, USA; E-mail:cding@usc.edu

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By 30–40 weeks of age, dacryoadenitis is conspicuous,with extensive lymphocytic infiltration into the acinartissue and progressive destruction of acini.11 It is nowgenerally agreed that the NODmouse strain representsone of the most accurate models to investigate Sjogrensyndrome.

In spite of the wide usage, we could find no lit-erature on the ultrastructural features of the murinelacrimal gland, although there are numerous citationsdescribing the histology of the murine lacrimal glandat the light microscopic level.12−14 Our studies of malemurine lacrimal glands, including bothNODand a nor-mal strain, BALB/c, revealed some unique features. Ourdata also demonstrated morphological alterations fromnormal in lacrimal glands of NOD mice as early as 4weeks of age, suggesting that cellular dysfunction of thelacrimal glands of NODmice occurs well before the on-set of clinical findings and that these observations needto be considered in interpreting data obtained from thismouse strain.

MATERIALS AND METHODSMaleNOD (Taconic Farms, Germantown,NY,USA)

andBALB/cmice (Jackson Labs, BarHarbor,ME,USA)were used throughout the study. The mice were main-tained in our vivarium facility on a 12-hr light-dark cycleand given food and water ad libitum. Animals were nar-cotized with a mixture of ketamine and xylazine andsacrificed with carbon dioxide. Careful visual inspec-tion of the lacrimal glands was performed before andafter removal.

All studies conformed to the standards and proce-dures for the proper care and use of animals as describedin the Declaration of Helsinki and the Guiding Princi-ples in the Care and Use of Animals (DHEW publica-tion, NIH 80-23). Mice used were four 1-month andfourteen 4-month NOD, and nine 1-month and four4-month BALB/c, all males.

Electron MicroscopyExorbital lacrimal glands were removed, and some

tissue fragments were placed directly in OCT (SakuraFinetek USA, Torrance, CA, USA), rapidly frozen withliquid nitrogen, and cryosectioned at 6–8 µm in thick-ness. Other fragments were fixed and processed forepoxy embedment for light and electron microscopyas previously described.15 Processing of some of the tis-sue samples included use of 1% tannic acid in 0.1 M

cacodylate buffer, pH 7.0, as a stain enhancer after os-mification. Tissue samples were embedded in LRWhite(London Resin, London, UK) for light and electronmicroscopy. Analysis of 0.75-µm-thick sections stainedwith toluidine blue always preceded collection of thinsections for electron microscopic study.

Oil Red O Staining and ConfocalImmunofluorescence ObservationAdditional fragments of lacrimal glands were fixed

in 4% paraformaldehyde/4% sucrose in PBS at 4◦C for4 hr, then transferred to 30% sucrose in PBS at 4◦C forat least overnight. Tissue fragments were embedded inOCT, rapidly frozen with liquid nitrogen, and cryosec-tioned at 5 µm and 10 µm. The 5-µm sections werestained with Oil Red O stain, a histochemical stain forlipids.

Ten-micrometer sections were used for confocal im-munofluorescence studies. These sections were stainedwith rhodamine-conjugated phalloidin (Sigma, St.Louis, MO, USA), dilution 1:200, at room tempera-ture for 30 min. Phalloidin labels actin, which is mostprominent in the apical (luminal) and basal sides ofacinar cells. The slides were examined with a confocallaser scanning microscope (LSM 510 Meta, Carl Zeiss,Thornwood, NY, USA) and analyzed on a PC computerusing Photoshop 7.0 software (Adobe Systems, Moun-tain View, CA, USA).

Ocular Surface EvaluationTear production and corneal fluorescein staining

were performed to evaluate lacrimal secretion and theintegrity of corneal epithelium. While the animals wereunder general anesthesia with ketamine and xylazinemixture, tear production was measured on 4-month-oldBALB/c and NOD mice with cotton threads (Zone-quick, Oasis, CA, USA), which were held with forcepsand applied to the lateral canthus for 5min. The wettinglength of the thread was measured by using a magnifierand expressed in millimeters. For fluorescein staining,1 µl of 1% fluorescein was applied to the ocular sur-face and photographed with a built-in digital camera ina Motic microscope (VWR International, Bristol, CT,USA) by using a cobalt blue light.

Statistical AnalysisData were expressed as mean ± SEM, wherever

appropriate. Student’s t test was performed with

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Microsoft Excel software. p < 0.05 was considered tobe significant.

RESULTSLacrimal glands in both strains of mice at 1 month

of age demonstrated the histotypical dense clusters ofsecretory acini with scattered ducts (Fig. 1). Acinar cellswere rich in secretory granules with the nuclei locatedbasally. The presence and contours of the acinar lu-mens were often difficult to determine in light micro-scopic sections, and frequently it appeared as thougha lumen either was not present or multiple small pro-

FIGURE 1 Lightmicrograph of lacrimal glands of 1-monthmicestained with toluidine blue. (A) NOD. Bar, 50 µm. (B) BALB/c. Bar,100 µm. Multiple profiles (arrows) of the acinar lumen are charac-teristic of the mouse lacrimal gland, and many of these profilesproject nearly to the basal lamina enveloping the acinar epithe-lium, as was confirmed by electron microscopy. Darkly stainedcells in (A) proved not to have features of apoptosis when viewedat the electron microscopic level, although they may suggest aprelude of apoptosis. D, duct; SG, secretory granules; N, nucleus.

files of lumens were present at various heights withinthe acinar epithelium. However, electron microscopyconfirmed this observation, that is, lumens were gen-erally small and extended narrow projections towardthe basal lamina (Fig. 2). In many sections, the luminalprojections reached nearly to the basal lamina with tightjunctions connecting adjacent cells just above the basallamina.

Many of the acini in NOD mice contained numer-ous lipid inclusions (Figs. 2B, 3C, and 3D), whereasmuch fewer were observed in BALB/c mice (Figs. 2C,3A, and 3B). In 1-month NOD mice, the lipid inclu-sions were already a dominant feature in the cytoplasmof acinar cells, most prominent in the basal side of thesecells. As the animal ages, the number of lipid dropletsincreased, and these droplets tend to coalesce to formlarger aggregates in the basal cytoplasm. Although thenumber of lipid droplets also appeared to increase inBALB/c mice as the animals aged, the number andsize of these droplets were much less than those ob-served in the NOD mice, particularly the large lipidaccumulations.

The accumulation of lipids in the lacrimal glands ofNOD mice was even readily apparent at necropsy, thatis, the glands had a foamy, greasy appearance in contrastwith the deeper pink coloration of the glands in BALB/cmice.

In contrast with BALB/c mice, the lacrimal glands ofNOD mice were observed to have enormous infiltra-tions of lymphocytes into the glandular parenchyma.Proliferation of lymphocytes around the acini was evi-dent in 4-month NOD mice with disruption of manyof the acini (Fig. 4A). Lymphocytic infiltration was notuniform throughout all of the lobules, whereas cytolog-ical changes appeared to bemore uniformly distributed.Although lymphocytes dominated the immune cellinfiltrates, macrophages and mast cells also were fre-quently identifiable as well.

The organelles within the acinar cells of NOD miceshowed atypical and deteriorating changes even at theage of 1month, and these changes worsened as these an-imals aged. Mitochondria in these cells appear enlargedwith fragmentation and disruption of internal struc-ture (Fig. 4). The endoplasmic reticulum of 4-month-old NOD mice was frequently vesiculated rather thanorganized in parallel cisternae as observed in normalmice, although the basal cytoplasm of acinar cells inboth strains at 1 month contained densely packed, par-allel cisternae of rough endoplasmic reticulum (Fig. 4).

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FIGURE 2 Electron micrographs of lacrimal glands of 1-month mice to show the multiple luminal profiles. (A) NOD. Luminal profiles (L)project very close to the basal lamina (arrowhead) surrounding the acini. This specimen, and (C), have been counterstained with tannicacid to increase the contrast of intercellular spaces. Tannic acid staining stops just below the apical surface (arrow), the zones at whichtight junctions are located. Bar, 0.5 µm. (B) NOD. Projections of the lumen (L) are evident just above the basal lamina (arrowhead). At leftcenter is an isolated zone (arrow) into which acinar cells project microvilli and small processes. Numerous lipid droplets (asterisks) arefrequently observed in the basal cytoplasm of acinar cells in NOD mice as early as 4 weeks, and less frequently in BALB/c mice. Bar,1 µm. (C) BALB/c. Like those found in NOD mice, multiple luminal profiles are evident and frequently project down nearly to the basallamina (arrowhead). In contrast with the numerous lipid droplets found in NOD mice, few or none were observed in the BALB/c mice, asshown in this micrograph. Bar, 2 µm.

Although evidence of apoptosis is often reported inlacrimal glands of NOD mice, we did not see clearevidence of it in our tissue samples. In some sec-tions, isolated acinar and ductal epithelial cells tookup more stain than adjacent cells, possibly suggesting

a prelude to apoptosis. However, no cells could beclearly identified in our EM studies having features ofapoptosis.

In addition to the accumulation of lipid dropletswithin the acinar cells in NODmice as the animal ages,

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FIGURE 3 Lightmicrographsof lacrimal glands stainedwithOil RedO to show the lipid contents. These slideshavebeencounterstainedwith toluidine blue. (A) one-month BALB/c. Only limited numbers and small lipid droplets were visible in the acinar cells and were locatedin the basal cytoplasm (arrows). (B) Four-month BALB/c. Many more lipid droplets were visible in the acinar cells compared with thoseobserved in 1-month-old mice, and these droplets appear to coalesce into larger ones (arrows) aggregated in the basal cytoplasm. (C)One-month NOD. Compared with the BALB/c mice, more lipid droplets were observed, and these droplets coalesced into even largerdroplets (arrows), primarily within the basal cytoplasm of acinar cells, although not uniformly distributed throughout the entire gland. (D)Four-month NOD. Lipid droplets were numerous and formed large aggregates (arrows), located primarily within the basal cytoplasm. Bar,25 µm.

the basal cytoplasm of many of the 4-month-old NODmice was dominated by large, heterogeneous vacuoles.Lipid inclusions frequently had a heterogeneous ap-pearance, containing membranous whorls and fine par-ticles (Fig. 4D).

In both strains of mice, isolated zones of aggregatesof small cell processes were apparent within the acini ad-jacent to the basal lamina (Figs. 4D and 5A). They wereoften well outlined by the tannic acid component ofour staining method. In some instances, it was evidentthat these zones were composed of interdigitations be-tween acinar cells and myoepithelial cells (Fig. 5B). We

could not detect any differences between these zonesof interdigitations comparing NOD and BALB/c miceat either 1 month or 4 months.

By using rhodamine-conjugated phalloidin to labelthe actin components, where the apical and basal sidesof the acini are most rich in actin and stained intensely,three-dimensional reconstruction images by confocalmicroscopy showed essentially the same features as ob-served in the electron micrograph (Fig. 6), that is, lu-minal profiles were highly pleomorphic and frequentlyramified down to the basal side of the acini. The basalsurface of the acini often showed a beaded appearance,

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FIGURE 4 Electron micrographs of lacrimal glands of NOD mice showing infiltrated lymphocytes among acini with accumulated lipiddroplets and atypically shaped and deteriorated organelles within acinar cells. (A) Four-month. The basal cytoplasm of the acinar cellsis filled with lipid droplets (LP) and vesiculated endoplasmic reticulum (vER). The periphery of the acinus is infiltrated (at right) bynumerous lymphocytes. Bar, 2 µm. (B) One-month. Mitochondria (M) frequently assume bizarre shapes with disrupted or fragmentedinternal structure. Discretely isolated zones of complexly folded cell surface projections (arrow) are evident just beneath the basal lamina,see also (C) and Fig. 6A. Bar, 1 µm. (C) Four-month. Fragmented and deteriorated mitochondria were more numerous in 4-month mice,and vesiculated endoplasmic reticulum. Bar, 0.4 µm. (D) Four-month. Large, heterogeneous vacuoles (V) accumulate within the basalcytoplasm of the acinar cells, and the endoplasmic reticulum is increasingly vesiculated rather than lamellar. As in (B) complexly foldedcell surface projections (arrows) are also evident beneath the basal lamina. Bar, 2 µm.

which was most prominent in BALB/c mice, and ap-peared to correspond to the electron microscopic ob-servations of surface projections or interdigitating pro-cesses (Figs. 4D and 5A). The basolateral sides of theacini in NOD mice often appeared to be thickeneddramatically relative to BALB/c mice, thus obscuringthe beaded appearance (Fig. 6).

The apparent ultrastructural differences betweenBALB/c and NOD mice at the age of 4 months weresupported by the findings from ocular surface evalua-tions. The wetting length of cotton thread was 1.28±0.2 mm in NOD mice and 2.87 ± 0.57 mm in BALB/cmice, which represents a significant difference (p <

0.05). Fluorescein staining also demonstrated that thecorneas of NOD mice showed numerous punctatestaining indicative of disruption of epithelial integrity,in contrast with the BALB/c mice that showed no stain-ing (Fig. 7).

DISCUSSIONOur ultrastructural studies revealed the unique his-

toarchitecture of the lacrimal glands in both BALB/cand NOD mice. Acinar lumens in the lacrimal glandsof both strains are generally small and highly pleo-morphic, that is, invaginations from the central lumenfrequently project nearly to the basement membrane

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FIGURE 5 Electron micrographs of lacrimal glands of 4-monthBALB/c. Tannic acid has been employed in these specimens toincrease the staining of intercellular spaces. (A) Dense clusters(arrows) of interdigitating cell processes were observed withinthe basal cytoplasm of acinar cells. Isolated zones such as thesewere present in both strains of mice. M, mitochondria; N, nucleus.Bar, 1.5 µm. (B) Areas at which surface projections (arrows) fromacinar cells and myoepithelial cells interdigitate are evident, andnumerous caveolae (arrowheads) are present within the myoep-ithelial cell (Myo), appearing dark because of uptake of tannic acid.Bar, 0.8 µm.

enclosing the acini. Tight junctions mark the most dis-tal extent of the luminal invaginations.

Our data indicate that the pathogenesis of thelacrimal glands of male NODmice begins very early inpostnatal development. Hunger et al. reported the ap-pearance of immune cells infiltrating into the lacrimalglands of NODmice at approximately 6–8 weeks of age

FIGURE 6 Confocal microscopy images of three-dimensionalreconstructions of lacrimal glands from 4-monthBALB/c andNODmice. (A) BALB/c. Luminal profiles of the acini are highly pleo-morphic, frequently projecting to the basal sides of the acinarcells (arrows), confirming our electron microscopic observations.Many of the basal membranes showed a characteristic beaded ap-pearance (arrowhead), corresponding to the surface projectionsor interdigitating processes observed with electron microscopy.(B) Essentially identical luminal profiles (arrows) were observedin NOD mice, that is, ramifying extensively and projecting to thebasal side of the acini. In this composite micrograph, the basalmembranes (arrowhead) are densely stained with the phalloidin,obscuring the characteristic beaded appearance as observed inBALB/c mice. Asterisks: lymphocytes.

and rapidly accelerating after 8 weeks, although theydid not comment on cytological features of the acinarcells.8 Even in 1-month-old NOD mice, we observe anabundance of lipid accumulating within the basal com-partment of acinar cells, whereas few were observed inBALB/c mice. In 4-month old NOD mice, large areasof cytoplasm are filled with lipid droplets and pleomor-phic, heterogeneous vacuoles containing membranewhorls and lipid fragments. Lipid droplets within the

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FIGURE 7 Corneal fluorescein staining of 4-month BALB/c andNOD mice. (A) BALB/c. No obvious staining was observed. (B)NOD. Significant punctate staining (arrows) was demonstrated inthe corneas of NODmice, suggesting pathological changes in theepithelium.

basal cytoplasm of acinar cells of 4-month-old BALB/cmice are very few when compared with NODmice, andheterogeneous vacuoles are not present. Mitochondrialabnormalities are also noted in the NOD mice, thatis, mitochondria are swollen and their internal struc-ture is often fragmented or disrupted even at 1 month.These mitochondrial changes are more pronounced inthe 4-month-old NOD mice and are accompanied bya dramatic vesiculation of the endoplasmic reticulum.The increased lipid accumulation in lacrimal glandsof NOD mice was even evident by visual inspectionat necropsy. Lacrimal glands in NOD mice appearedto be bigger than those obtained from the BALB/cmice at corresponding ages, and the glands were foamyand whitish in appearance, in contrast with the pinkercoloration in BALB/c mice. Functionally, NOD miceshowed less tear production than BALB/c mice and re-

vealed fluorescein staining in the corneal epithelium,corroborating the ultrastructural findings.

The early ultrastructural and functional changes, re-ported here, are also supported by findings in an ad-ditional recent report by our group.16 We showed inthat report that the basolateral actin filaments werediscontinuous in nature, with a “beads-on-a-string” ap-pearance, in both BALB/c and NOD mice at 1 and 4months. These likely correspond to the complex cellmembrane interdigitations presented here. Also, weshowed that NOD mice as young as 1 month exhibitaberrant secretory vesicles with altered effector mecha-nisms such that they undergo premature cytoplasmicfusion. The M3 muscarinic receptor, which is a keysignaling effector of exocytosis, underwent redistribu-tion away from its normal basolateral locale in lacrimalglands of BALB/c mice, to intracellular aggregates inglands from NOD mice.16

Some of our observations correlate with those ofother investigators using different animal models of di-abetes or Sjogren syndrome. An ultrastructural studyby Chomette et al. reported an accumulation of lipidwithin lacrimal acinar cells of a 78-year-old female withSjogren syndrome.17 The authors also commented on“large phagosomes,” which look very similar to the het-erogeneous vacuoles seen in our study. Swelling andfragmentation of mitochondria was reported in β-cellsof the pancreas in a mouse model of maturity-onsetdiabetes.18 Mitochondrial abnormalities and vesicula-tion of the endoplasmic reticulum were also noted inpancreatic exocrine cells in a rabbit model of diabetes.19

Although not demonstrating the pathology histologi-cally, a sixfold increase in triglyceride content withinpancreatic islet cells in Zucker diabetic fatty (fa/fa) ratswas reported,20 and overaccumulation of lipid withinthese cells was associated with a reduced ability of β-cells to maintain insulin secretion, that is, a lipotoxi-city followed by functional loss.21 Mitochondrial de-rangement was also associated with these pathologicalchanges in pancreatic islet cells.22 Very similar degener-ative changes appear to be occurring within the acinarcells in lacrimal glands of male NOD mice.

The pathological changes evident within the lacrimalglands of NOD mice appear similar to pathologicalchanges associated with lysosomal storage diseases.23,24

Altered use of lipid components of membranes may bereflected in the dramatic increase in cytoplasmic lipidand buildup of heterogeneous vacuoles. Alterations inmembrane composition andmembrane trafficking then

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may contribute to regurgitation and presentation ofpeptide fragments that normally are kept sequestered,thus helping to provoke an autoimmune response char-acteristic of Sjogren syndrome. The increase in lipid de-position in the older NODmice is an important obser-vation in this regard, as alterations in lipid metabolismare associated with other defects in intracellular traffick-ing, such as cholesterol, which may result in enhancedlipid deposition in liposomes accompanied by neuronaldegeneration.24

Many age-related degenerative diseases are associatedwith lysosomal abnormalities, accompanied by accu-mulations of age-related pigment, such as lipofuscin,25

and subsequent evidence of cellular damage.26−28

Other lysosomal storage diseases, such as Chediak-Higashi syndrome, which causes severe defects in mul-tiple systems in the body and early lethality, is character-ized by vacuolization and accumulation of abnormallyenlarged lysosomes in many cell types.29

Recent studies in NOD and NOD/scid mice suggestthat apoptosis may play a role in damaging salivary andlacrimal glands, resulting in impairment of their secre-tory function.30 However, we found no clear evidenceof apoptotic changes in the lacrimal glands of NODmice. Although some intermittent darkly stained cellswere present in our light microscopic sections, bothacinar and ductal, they did not have features of apop-tosis when viewed by electron microscopy, such as thecharacteristic changes of condensation of the nuclearchromatin to form one or more dark-staining massesfound against the nuclear membrane, no obvious cellshrinkage of neighboring acinar epithelial cells with lossof cell-cell contacts, no fragmentation of nuclear ma-terials, and no cytoplasmic blebs breaking away fromthe cell surface. However, we cannot rule out the pos-sibility that they may represent a prelude to apoptosisthat may follow as the animals age or that cells withapoptotic signs were not included in the sections weobserved.

Corrons et al. reported on the presence of complexmembrane interdigitations in β-cells of the pancreasin an induced insulin-dependent model of diabetesin transgenic mice.31 The authors suggested that thesemembrane interdigitations were a manifestation of cel-lular injury related to the induced diabetes. We foundessentially the same complex interdigitations of cellularprocesses in the acinar cells of NOD and BALB/c mice.Their presence in the control BALB/cmice argues for aninterpretation other than cellular damage, that is, that

they represent a previously unreported cellular special-ization unique to mice. Our finding that the interdigi-tations occurred between acinar cells and myoepithelialcells prompts us to propose that these interdigitationsmay represent a mechanism of communication or ex-change between myoepithelial and acinar cells. Further-more, we found no difference in these interdigitationsbetween NOD and BALB/c mice and no significantchanges from 1 month to 4 months of age, that is, theyappear to be a consistent feature of the murine lacrimalgland, at least as exemplified by these two strains. Analternate consideration for future study is based on theobservation that mitochondria often were noted to beintimately aligned adjacent to the complex interdigita-tions, a phenomenon suggesting energy production atmembrane sites of active transport as is characteristic ofthe renal proximal tubule epithelium.

An additional report described complex membraneinterdigitations in a study of lymphocytic submandibu-litis in NOD mice that appeared to be between ductalcells and lymphocytes.32 However, we have not seenevidence to date of this structural specialization asso-ciated with lacrimal ductal cells in NOD or BALB/cmice.

In summary, our ultrastructural observations re-vealed that the lacrimal glands of both BALB/c andNOD mice exhibit some unique features comparedwith other species, such as rabbit. Our findings alsoindicate that although clinical manifestations mimick-ing Sjogren syndrome are evident in NOD mice by 4months of age, alterations from normal cytology, suchas accumulations of lipid, presence of vesicular endo-plasmic reticulum, atypically shaped and deterioratedmitochondria, in lacrimal acinar cells suggest cellulardysfunction occurring as early as 1 month of age inthe NOD mice. These novel findings will help us tobetter understand the unique morphological featuresof the murine lacrimal gland and to better interpretthe experimental results obtained from studies of theseanimals.

ACKNOWLEDGMENTSThis work was supported by NIH-EY 10550 (J.E.S.),

EY 11386 (S.H.A.), DK 48522 (Confocal MicroscopyCore, USC Center for Liver Disease), and a grant fromthe Sjogrens Syndrome Foundation (CD). We thankSamuel Yiu, M.D. (Department of Ophthalmology,USC) for helping with the ocular surface evaluation.

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