Rabbit Lacrimal Gland Research Articles

Developing a model for aqueous deficient dry eye secondary to periglandular cicatrizing conjunctivitis

PurposeThe current study used various techniques to develop a rabbit animal model of lacrimal gland damage caused by scarring conjunctivitis in the periglandular area. MethodsLeft eyes of New Zealand white rabbits were injected with 0.1 ml of 1M NaOH subconjunctivally around superior and inferior lacrimal gland orifices (Group 1, n = 4), touched with 1M NaOH for 100 s to the superior and inferior fornices with conjunctival denuding (Group 2; n = 4), and electrocauterization to the ductal opening area (Group 3; n = 4). The ocular surface staining, Schirmer I, lacrimal gland, and conjunctival changes were observed at baseline,1, 4, 8, and 12 weeks. The degree of glandular inflammation, conjunctival fibrosis (Masson Trichrome), and goblet cell density (PAS) were also assessed. ResultsAt 12 weeks, the lacrimal glands of group 1 rabbits with periglandular injection showed severe inflammation with mean four foci/10HPF and a significant mean reduction in the Schirmer values by 7.6 mm (P = 0.007). Lacrimal glands had diffuse acinar atrophy, loss of myoepithelial cells, and ductular dilatation. The overlying conjunctiva showed fibrosis, goblet cell loss, and corneal vascularization in the inferotemporal quadrant. No lacrimal gland or ocular surface changes were observed in groups 2 and 3 at 12 weeks, except for localized subconjunctival fibrosis. ConclusionPeriglandular injection of 0.1 ml of 1M NaOH induced extensive lacrimal gland damage with reduced secretion and scarring in the subconjunctival plane compared to direct cauterization or direct NaOH contact to the ductal orifices of the rabbit lacrimal gland.

The Role of Aquaporin 4 in Lacrimal Gland Ductal Fluid Secretion in Mice.

Earlier reports highlighted the predominant presence of aquaporin 4 (AQP4) in the duct cells of rabbit lacrimal glands (LGs). Whereas significant alterations in AQP4 mRNA levels have been observed in experimental dry eye and during pregnancy, the impact of AQP4 in LG ductal fluid production remains unclear. In our recent work, the role of AQP4 in LG ductal fluid secretion was investigated utilizing wild type (WT) and AQP4 knock out (KO) mice. Tear production was assessed in both WT and KO animals. Immunostaining was used to identify AQP4 protein. Duct segments were harvested from LGs of WT and KO mice. Fluid secretion and filtration permeability (Pf) were quantified using video-microscopy. Ductal tear production, elicited by a cell-permeable cAMP analogue (8-bromo cAMP), carbachol, vasoactive intestinal peptide (VIP), and phenylephrine (PHE), were assessed in both WT and KO ducts. A higher expression of AQP4 protein was noted in the duct cells from WT mice when compared to acinar cells. Pf did not show notable alterations between WT and AQP4 KO ducts. Carbachol elicited comparable secretory responses in ducts from both WT and KO animals. However, 8-bromo cAMP, VIP, and PHE stimulation resulted in decreased secretion in ducts from AQP4 KO LGs. Our findings underscore the functional relevance of AQP4 in the fluid production of mouse LG ducts. AQP4 seems to play different roles in fluid secretions elicited by different secretagogues. Specifically, cAMP-mediated, and adrenergic agonist-related secretions were reduced in AQP4 KO ducts.

Dry eye syndrome model established in rabbits via mitomycin C injection in the lacrimal gland.

To develop a new dry eye syndrome (DES) animal model by injecting mitomycin C (MMC) into the lacrimal glands (LGs) of rabbits evaluated by clinical examinations. A volume of 0.1 mL of MMC solution was injected in the LG and the infraorbital lobe of the accessory LG of rabbits for DES induction. Twenty male rabbits were separated into three groups, the control group, and different concentration of MMC, (MMC 0.25: 0.25 mg/mL or MMC 0.50: 0.5 mg/mL) were tested. Both MMC-treated groups received MMC twice injection on day 0 and day 7. Assessment of DES included changes in tear production (Schirmer's test), fluorescein staining pattern, conjunctival impression cytology, and corneal histological examination. After MMC injection, no obvious changes in the rabbit's eyes were noted by slit-lamp examination. Both the MMC 0.25 and the MMC 0.5 groups revealed decreased tear secretion after injection, and the MMC 0.25 group showed a continuous decrease in tear secretion up to 14 days. Fluorescent staining showed punctate keratopathy in both MMC-treated groups. In addition, both MMC-treated groups demonstrated decreased numbers of conjunctival goblet cells after injection. This model induced decreased tear production, punctate keratopathy, and decreased numbers of goblet cells, which are consistent with the current understanding of DES. Therefore, injecting MMC (0.25 mg/mL) into the LGs is an easy and reliable method to establish a rabbit DES model which can apply in new drug screening.

MicroRNA expression profile of Lacrimal Glands in rabbit autoimmune dacryoadenitis model.

Purpose: To identify the differential expression of microRNAs (miRs) and the related gene networks and signal pathways in lacrimal glands (LGs) of rabbit autoimmune dacryoadenitis.Methods: Autoimmune dacryoadenitis in rabbits was induced by transferring activated peripheral blood lymphocytes (PBLs). The LGs of normal and model group rabbits were collected for small RNA sequencing. The most differentially expressed miRs were validated by quantitative real time-polymerase chain reaction (qRT-PCR). Further, bioinformatics analysis including target gene prediction, Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed.Results: A total of 15 miRs were differentially expressed in the LGs of rabbit autoimmune dacryoadenitis relative to normal controls. GO and KEGG analysis revealed that most target genes of these dysregulated miRs were implicated in MAPK signaling pathway.Conclusion: Our results showed for the first time the differentially expressed miRs and the related pathways involved in the pathogenesis of rabbit autoimmune dacryoadenitis. These results may contribute to elucidating molecular pathogenesis of Sjögren's syndrome (SS) dry eye.

Effects of Buddlejae Flos Granules on Inflammatory Factors TGF-β1, NF-κB, IL-10 and IL-12 in Lacrimal Gland Cells of Castrated Male Rabbits

ObjectiveTo observe the effects of Buddlejae Flos (Mi Meng Hua, 密蒙花) granules on the expression of TGF-β1, NF-κB, IL-10 and IL-12 in lacrimal gland cells in castrated male rabbits. MethodsA total of 30 New Zealand male white rabbits were randomly divided into 5 groups. Group A: blank group; group B: model group; group C: Buddlejae Flos (Mi Meng Hua, 密蒙花) granules group; group D: placebo group; group E: testosterone group. Except for the group A, all rabbits were treated with bilateral testicular and epididymis resection before intervention. Rabbits in group C were administered with Buddlejae Flos (Mi Meng Hua, 密蒙花) granules (100 mg/kg), 3 times per day; Rabbits in group D were administered with normal saline, 3 times per day. Rabbits in group E were injected with testosterone propionate (0.5 mL/kg) in the thigh muscle, every 3 days. Schirmer I test (SIT) and break-up time (BUT) were measured on 1st day before modeling and 30th day after modeling. After 30 days of intervention, all rabbits were sacrificed. The removal lacrimal gland was stained in immunohistochemica staining. The expression of TGF-β1, NF-κB, IL-10 and IL-12 in lacrimal gland tissue of each group was detected, and the structure of lacrimal gland was observed. ResultsThe results of SIT and BUT showed that there was significant difference between group B and D compared with group A (P < 0.05). There were no significant differences between group C and E (P > 0.05). There were no significant differences in the comparison of inflammatory factors TGF-β1, NF-κB, IL-10 and IL-12 between group B and D (P > 0.05). There were significant differences between group B and D compared with other groups (P < 0.01). There was no significant difference between group A, C and E (P > 0.05). ConclusionsBuddlejae Flos (Mi Meng Hua, 密蒙花) granules have an inhibitory effect on the expression of TGF-β1, NF-κB, IL-10 and IL-12 in castrated male rabbit lacrimal gland cells, and alleviate the inflammation of lacrimal gland tissue by inhibiting the expression of TGF-β1, NF-κB, IL-10 and IL-12 in lacrimal gland tissue, so as to treat dry eye.

Molecular Evidence for Precursors of Sjögren's Foci in Histologically Normal Lacrimal Glands.

Understanding the formation of Sjogren’s lymphocytic infiltrates could permit earlier diagnosis and better outcomes. We submitted gene transcript abundances in histologically normal rabbit lacrimal glands to principal component analysis. The analysis identified a cluster of transcripts associated with Sjögren’s foci, including messenger RNAs (mRNAs) for C–X–C motif chemokine ligand 13 (CXCL13) and B-cell activating factor (BAFF), which dominated the major principal component. We interpreted the transcript cluster as the signature of a cluster of integrally functioning cells. Pregnancy and dryness increased the likelihood that the cluster would develop to high levels, but responses were subject to high levels of stochasticity. Analyzing microdissected samples from high- and low-cluster-level glands, we found that certain transcripts, including mRNAs for C–C motif chemokine ligand 21 (CCL21), CXCL13, cluster of differentiation 4 (CD4), CD28, CD25, BAFF, and interleukin 18 (IL-18) were significantly more abundant in immune cell clusters (ICs) from the high-cluster-level gland; mRNAs for CCL2, CD25, and IL-1RA were significantly more abundant in acinus-duct axis samples; mRNAs for CCL4, BAFF, IL-6, and IL-10 were more abundant in some acinus-duct samples; cells with high prolactin immunoreactivity were more frequent in interacinar spaces. In conclusion, integrated functional networks comprising Sjögren’s infiltrates, such as ICs, acinar cells, ductal cells, and interacinar cells, can form in histologically normal glands, and it is feasible to detect their molecular signatures.

Characterization of Na+-K+-2Cl- Cotransporter Activity in Rabbit Lacrimal Gland Duct Cells.

PurposeWe recently reported that isolated duct segments from rabbit lacrimal gland (LG) were able to secrete fluid in response to secretagogues, which were blocked completely by bumetanide. This suggests the functional involvement of Na+-K+-2Cl− cotransporter (NKCC1) in ductal fluid secretion. Therefore, the aim of this study was to investigate the activity profile of NKCC1 in isolated rabbit LG duct segments.MethodsInterlobular ducts were isolated from fresh rabbit LG tissue. Microfluorometry with the ammonium (NH4+)–pulse technique was used to elicit pH changes in duct cells, and the rate of bumetanide-sensitive cytosolic acidification after addition of NH4+ was used to quantify the activity of NKCC1.ResultsWhile basal activity of NKCC1 was undetectable, low cytosolic chloride (Cl−) level and hyperosmotic challenge (390 mOsm) were able to increase the activity of NKCC1. Carbachol (100 μM) had no significant effect on NKCC1 activity. Elevation of cytosolic calcium (Ca2+) level with Ca2+-ionophore (A 23187, 1 μM) did not cause any alteration in the activity of the cotransporter while direct activation of protein kinase C (phorbol myristate acetate, 100 nM) increased its activity slightly but in a significant manner. Addition of either forskolin (10 μM), cell-permeable cAMP analogue (8-bromo cAMP, 100 μM) or vasoactive intestinal peptide (200 nM) resulted in a significant increase in the activity of NKCC1.ConclusionsThese results highlight the functional involvement of NKCC1 in LG duct secretion. These findings may facilitate our understanding of LG function and may contribute to the development of targeted pharmacologic interventions in case of dry eye disease.

Three-Dimensional Culture of Functional Adult Rabbit Lacrimal Gland Epithelial Cells on Decellularized Scaffold.

Aqueous tear-deficient dry eye disease is a multifactorial chronic disorder, in which the lacrimal gland fails to produce enough tears to maintain a healthy ocular surface. Some severe cases may develop corneal damage and significant vision loss. Treatment primarily involves palliation using ocular surface lubricants, but can only provide temporary relief. Construction of a bioengineered lacrimal gland having functional secretory epithelial cells is a potentially promising option for providing long-term relief to severe dry eye patients. Using sphere-forming culture techniques, we cultured adult rabbit lacrimal gland progenitor cells and prepared a lacrimal gland scaffold by decellularization. When progenitor cells were seeded onto the decellularized scaffold, they formed duct- and acinar-like structures in the three-dimensional culture system. Lacrimal gland epithelial cells showed good cell viability, cell differentiation, and secretory function in decellularized lacrimal gland matrix, as indicated by morphology, immunostaining, and β-hexosaminidase secretion assay. This study demonstrated the potential suitability of utilizing tissue-specific progenitor cells and a tissue-derived bioscaffold for lacrimal gland restoration.

Vitamin D in Tear Fluid.

To determine the source(s) of vitamin D in tear fluid and examine the expression of the endocytic proteins and putative vitamin D transporters megalin and cubilin in lacrimal and Harderian glands. Wild-type, heterozygous, and vitamin D receptor (VDR) knockout C57BL/6 mice were used, with a subset of knockout mice fed a replenishment diet for some studies. Mouse lacrimal and Harderian glands from each group were used to measure megalin and cubilin by RT-PCR, Western blot, and immunohistochemistry. New Zealand white rabbits were used to collect lacrimal and accessory gland fluid for vitamin D mass spectroscopy measurements. Ten-week-old knockout mice were significantly (P < 0.05) smaller than wild-type mice. Real-time PCR and Western blot showed decreased expression of megalin and cubilin in select VDR knockout mouse groups. Immunohistochemistry showed apical duct cell megalin staining and weaker megalin staining in VDR knockout mice compared with controls. Vitamin D2 was more prevalent in rabbit lacrimal and accessory gland fluid than vitamin D3, and greater amounts of Vitamin D2 were found in in tear fluid obtained directly from lacrimal and accessory glands as compared with plasma concentrations. This is the first study to demonstrate the presence of megalin and cubilin in lacrimal and accessory glands responsible for producing tear fluid. The results strengthen the hypothesis that megalin and cubilin are likely involved in the secretory pathway of vitamin D into tear fluid by the duct cells.

ENaC in the Rabbit Lacrimal Gland and its Changes During Sjögren Syndrome and Pregnancy.

Epithelial sodium channel (ENaC) plays a critical role in the control of Na(+) balance and the development and progression of exocrine gland pathologic condition. The aim of the present study was to investigate the presence of ENaC in the rabbit lacrimal gland (LG) and its potential changes during induced autoimmune dacryoadenitis (IAD) and pregnancy. Total messenger RNA (mRNA) of α, β, and γ subunits was extracted from whole LG, acinar cells, and ductal cells by laser capture microdissection (LCM) for real-time reverse-transcriptase polymerase chain reaction. Lacrimal glands were processed for Western blot and immunofluorescence. Messenger RNA for both α and γ was expressed in whole LG lysates, whereas β was undetectable. In rabbits with IAD, the levels of mRNA for α and γ were 20.9% and 58.9% lower (P<0.05), whereas no significant changes were observed in term-pregnant rabbits (P=0.152). However, we were unable to detect mRNA of any subunit in LCM specimens of ductal cells because of their low levels. Western blot demonstrated bands for both α (90 kDa) and γ (85 kDa) but β was undetectable. In rabbits with IAD, densitometry analysis showed that expression of α decreased 22%, whereas γ decreased 26% (P<0.05). In pregnant rabbits, however, α expression was 31% lower, whereas γ expression was 34% lower (P<0.05). From immunofluorescence studies, all subunits were present in ductal cells, whereas virtually no immunoreactivity was detected in acini. No noticeable changes of their distribution pattern and intensity were found in rabbits with IAD or during pregnancy. The present study demonstrated the presence of ENaC in the rabbit LG and its alterations in IAD and pregnancy, suggesting that ENaC may contribute to the pathogenesis of altered LG secretion and ocular surface symptoms in these animals.

A thermo-responsive protein treatment for dry eyes

Millions of Americans suffer from dry eye disease, and there are few effective therapies capable of treating these patients. A decade ago, an abundant protein component of human tears was discovered and named lacritin (Lacrt). Lacrt has prosecretory activity in the lacrimal gland and mitogenic activity at the corneal epithelium. Similar to other proteins placed on the ocular surface, the durability of its effect is limited by rapid tear turnover. Motivated by the rationale that a thermo-responsive coacervate containing Lacrt would have better retention upon administration, we have constructed and tested the activity of a thermo-responsive Lacrt fused to an elastin-like polypeptide (ELP). Inspired from the human tropoelastin protein, ELP protein polymers reversibly phase separate into viscous coacervates above a tunable transition temperature. This fusion construct exhibited the prosecretory function of native Lacrt as illustrated by its ability to stimulate β-hexosaminidase secretion from primary rabbit lacrimal gland acinar cells. It also increased tear secretion from non-obese diabetic (NOD) mice, a model of autoimmune dacryoadenitis, when administered via intra-lacrimal injection. Lacrt ELP fusion proteins undergo temperature-mediated assembly to form a depot inside the lacrimal gland. We propose that these Lacrt ELP fusion proteins represent a potential therapy for dry eye disease and the strategy of ELP-mediated phase separation may have applicability to other diseases of the ocular surface.

Functional presence of Na+‐K+‐2Cl‐ cotransporter in rabbit lacrimal gland ductal epithelial cells

AbstractPurpose Capability of fluid secretion of rabbit lacrimal gland duct epithelial cells was recently demonstrated by the authors using isolated duct segment model developed earlier. Inhibition of basolateral Na+‐K+‐2Cl‐ cotransporter (NKCC1) with bumetanide resulted in a complete stoppage of fluid secretion. Aim of the present work was to determine the activity and the regulation of NKCC1.Methods Experiments were performed on isolated rabbit lacrimal gland interlobular duct segments. Intracellular fluorophotometry with ammonium pulse technique was used to measure cotransporter activity. The rate of bumetanide sensitive cytosolic acidification after addition of NH4‐ can be used to quantify the activity of the cotransporter. Role of various potentially stimulatory effects were investigated.Results Unstimulated activity of NKCC1 was determined. Elevated intracellular cAMP level evoked by forskolin stimulation resulted in marked increase in NKCC1 activity. Beside increased intracellular cAMP levels, low cytosolic Cl‐ concentration and cell shrinkage evoked by hyperosmotic environment were significant determinants of increased cotransporter activity.Conclusion Our earlier results suggested the dominant role of Cl‐ dependent mechanisms in lacrimal gland ductal fluid secretion. The presented experimental work proves the substantial role of NKCC1 in basolateral Cl‐ uptake. Intracellular regulation of Cl‐ handling and luminal Cl‐ secretion needs further investigation.

Experimental Evidence of Fluid Secretion of Rabbit Lacrimal Gland Duct Epithelium

Experimental Evidence of Fluid Secretion of Rabbit Lacrimal Gland Duct Epithelium

Experimental evidence of fluid secretion of rabbit lacrimal gland duct epithelium.

To investigate the osmotic water permeability of lacrimal gland (LG) duct epithelium by means of calculation of filtration permeability and to investigate LG ductal fluid secretion. Experiments were performed on isolated rabbit LG duct segments maintained in short-term culture. Osmotically determined fluid movement or fluid secretion into the closed intraluminal space of cultured LG interlobular ducts was analyzed using video microscopic technique. The end of the LG ducts sealed after overnight incubation forming a closed luminal space. For the calculation of osmotic water permeability, ducts were initially perfused with isotonic HEPES buffered solution, and then with hypotonic HEPES buffered solution. Filtration permeability was calculated from the initial slope of the relative volume increase. Secretory responses to carbachol or to forskolin stimulation were also investigated. Forskolin stimulation resulted in a rapid and sustained secretory response in both solutions. Forskolin-stimulated fluid secretion was completely inhibited by bumetanide both in HEPES buffered and in HCO3 (-)/CO2 buffered solutions, suggesting the central role of Na(+)-K(+)-2Cl(-) cotransporter type 1 (NKCC1). Administration of carbachol initiated a rapid but short secretory response in both HEPES buffered and in HCO3 (-)/CO2 buffered solutions. Atropine completely abolished the carbachol-evoked fluid secretion. A new method was introduced to investigate LG duct function. Water permeability of rabbit LG duct epithelium was measured by calculating filtration permeability. Fluid secretion of LG duct cells induced by carbachol or forskolin was also demonstrated. These results provide calculated values of lacrimal duct osmotic permeability and direct experimental evidence of LG duct fluid secretion.

Functional and Histologic Changes in the Lacrimal Gland After Botulinum Toxin Injection

The purpose of this study was to evaluate changes in tear volume, epidermal growth factor (EGF), and histology of the rabbit lacrimal gland after intraglandular application of botulinum toxin. Ten New Zealand rabbits were separated into 3 groups. Eight rabbits received botulinum toxin injection (2.5 U per 0.1 mL) into the right lacrimal gland and saline injection (0.1 mL) into the left lacrimal gland as a sham control. Two rabbits were untreated to serve as normal controls. Tear volume was measured using cotton thread every 2 weeks, and 4 rabbits were killed after 2 and 4 weeks. The lacrimal glands were surgically excised and sectioned or lysed for gene expression analysis. Epidermal growth factor expression and concentration were evaluated by real-time polymerase chain reaction and enzyme-linked immunosorbent assay; morphometric and histologic analyses were performed. The rabbits that were killed 2 weeks after the injection showed decreased tear volume and increased EGF expression and concentration, but differences were not statistically significant. The rabbits that were killed 4 weeks after the injection exhibited similar results. When all 8 rabbits were considered, we detected a significant decrease in tear volume and increased EGF expression and concentration (P = 0.012, P = 0.011, and P = 0.012, respectively). The EGF level was not significantly correlated with the tear volume. There were no prominent histologic changes between the glands, and the lumen versus fibrosis ratio in the interlobular ducts showed no statistically significant difference. The use of botulinum toxin in patients with epiphora is effective, safe, and repeatable because it reduces tear volume and increases the EGF level to prevent corneal damage while causing no histologic changes.

Experimental evidence of fluid secretion of rabbit lacrimal gland ductal epithelia

AbstractPurpose Authors developed a novel lacrimal gland (LG) duct isolation technique earlier (IOVS, 2007; 48:3746‐3755) which makes possible to obtain more information on the function and regulation of LG ductal cells. The role of LG ductal epithelium on fluid secretion is unknown. The aim of the present study was to investigate LG ductal fluid secretion by means of measurement of fluid secretion evoked by different agonists.Methods Experiments were performed on isolated rabbit LG interlobular duct segments maintained in short term culture. The ends of the ducts sealed after overnight incubation forming a closed luminal space. Fluid secretion into closed intraluminal space as swelling response was analyzed using bright field videomicroscopy technique.Results The secretory response to carbachol or to forskolin stimulation was measured in HEPES buffered and in HCO3‐/CO2‐ buffered solutions. Carbachol initiated ductal swelling. This result proves the cholinergic effect‐evoked ductal fluid secretion. Fluid secretion was biphasic consisting of a continuous swelling in the first 5 minutes followed by a plateau phase. Secretory rates did not show a substantial difference measured in HEPES and HCO3‐/CO2 ‐buffered solutions. In contrast to the biphasic effect of carbachol, forskolin stimulation resulted in a rapid, continuous swelling response. The secretory effect of forskolin did not differ in HEPES and HCO3‐/CO2‐buffered solutions.Conclusion Fluid secretion of LG ductal cells induced by carbachol and by forskolin stimulations strongly supports the hypothesis, that LG ductal system is actively involved in lacrimal fluid secretion. Our results provide the first experimental evidence of secretory function of this epithelium.

Differential expression and localization of transient receptor potential vanilloid 1 in rabbit and human eyes.

The superfamily of transient receptor potential (TRP) cation channels is involved in nociception. Members of this family, such as the vanilloid receptor type 1 (TRPV1) channel, are activated by a wide range of stimuli including heat (⟩43°C), low pH (⟨6.5), hypoxia, and hypertonicity. Here we report TRPV1 expression in rabbit and human eyes. We analyzed the expression of TRPV1 mRNA by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and protein by immunohistochemistry in eyes of New Zealand White rabbits and humans. In rabbit and human eyes, TRPV1 protein was present in all layers of the corneal epithelium, but only in the basal layer of the conjunctiva. It was also in the ciliary and lens epithelia of both species as well as in the secretory cells of the rabbit lacrimal gland. The retinal pigment epithelium was positive for this protein in both species. TRPV1 was also present in rabbit Müller cells, where it had a similar pattern of expression to vimentin intermediate filaments. Analysis by qRT-PCR showed that TRPV1 mRNA was found in all of the structures where the protein was present. The highest level was in the lens and the lowest in the retina. TRPV1 is expressed in cells that are particularly active in Ca²⁺ exchange as well as in cells with significant water transport activity. Because TRPV1 is a Ca²⁺ channel, it probably functions in the regulation of both water and Ca²⁺ movements in ocular tissues.

Polymeric immunoglobulin receptor traffics through two distinct apically targeted pathways in primary lacrimal gland acinar cells

The polymeric immunoglobulin receptor (pIgR) mediates transcytosis of dimeric immunoglobulin A (dIgA) and its release into mucosal secretions. The present study reveals the complexity of the trafficking of pIgR to the apical plasma membrane in epithelial cells with exocrine secretory functions; in rabbit lacrimal gland acinar cells (LGACs), trafficking of pIgR involves both the transcytotic pathway and one arm of the regulated secretory pathway. By specifically tracking pIgR endocytosed from the basolateral membrane, we show here that the Rab11a-regulated transcytotic pathway mediates the basal-to-apical transport of pIgR, and that pIgR sorted into the transcytotic pathway does not access the regulated secretory pathway. However, previous work in LGACs expanded in the present study has shown that some pIgR is localized to Rab3D-enriched mature secretory vesicles (SVs). Myosin Vb and myosin Vc motors modulate release of proteins from the Rab11a-regulated transcytotic pathway and the Rab3D-enriched secretory pathway in LGACs, respectively. Confocal fluorescence microscopy and biochemical assays showed that inhibition of myosin Vb and myosin Vc activity by overexpression of their dominant-negative mutants each significantly but differentially impaired aspects of apically targeted pIgR trafficking and secretory component release, suggesting that these motors function to regulate pIgR trafficking in both the transcytotic and exocytotic pathways. Intriguingly, a second mature SV population enriched in Rab27b was devoid of pIgR cargo, suggesting the specialization of Rab3D-enriched mature SVs to carry a particular subset of cargo proteins from the trans-Golgi network to the apical plasma membrane.

Na+/K+-ATPase Expression Changes in the Rabbit Lacrimal Glands During Pregnancy

Purpose: To investigate the expressional changes of Na+/K+-ATPase subunits in the lacrimal glands (LG) of term pregnant rabbits.Methods: LG were obtained from term pregnant rabbits and age-matched female control rabbits for laser capture microdissection (LCM), real time RT-PCR, western blot, and immunofluorescence. The mRNA and proteins of α1, α2, β1, β2, and β3 subunits of Na+/K+-ATPase were detected and quantified.Results: Although only the mRNA for β3 from whole LG of pregnant rabbits was significantly different from that of normal controls, many mRNA levels for α1, α2, β1, β2, and β3 from acini and epithelial cells from various duct segments that were collected by LCM were significantly different from those of normal control rabbits. Western blots demonstrated that the expressions of all three β subunits were significantly higher in pregnant rabbits, while both α subunits remained unchanged during pregnancy. Interestingly, immunofluorescence results showed that the distribution patterns of all Na+/K+-ATPase subunits during pregnancy were similar to those of the control rabbits.Conclusions: Changes were found in mRNA and protein expressions of Na+/K+-ATPase subunits in LG from term pregnant rabbits and these changes suggest a role in the pregnancy-related LG secretion changes and dry eye symptoms observed in these animals.

CFTR-Mediated Cl− Transport in the Acinar and Duct Cells of Rabbit Lacrimal Gland

Purpose: We investigated the role that the cystic fibrosis transmembrane conductance regulator (CFTR) may play in Cl− transport in the acinar and ductal epithelial cells of rabbit lacrimal gland (LG).Methods: Primary cultured LG acinar cells were processed for whole-cell patch-clamp electrophysiological recording of Cl− currents by using perfusion media with high and low [Cl−], 10 µM forskolin and 100 µM 3-isobutyl-1-methylxanthine (IBMX), the non-specific Cl− channel blocker 4,4′-disothiocyanostilbene-2, 2′ sulphonic acid (DIDS; 100 µM) and CFTRinh-172 (10 µM), a specific blocker for CFTR. Ex vivo live cell imaging of [Cl−] changes in duct cells was performed on freshly dissected LG duct with a multiphoton confocal laser scanning microscope using a Cl− sensitive fluorescence dye, N-[ethoxycarbonylmethyl]-6-methoxy-quinolinium bromide.Results: Whole-cell patch-clamp studies demonstrated the presence of Cl− current in isolated acinar cells and revealed that this Cl− current was mediated by CFTR channel. Live cell imaging also showed the presence of CFTR-mediated Cl− transport across the plasma membrane of duct cells.Conclusions: Our previous data showed the presence of CFTR in all acinar and duct cells within the rabbit LG, with expression most prominent in the apical membranes of duct cells. The present study demonstrates that CFTR is actively involved in Cl− transport in both acinar cells and epithelial cells from duct segments, suggesting that CFTR may play a significant role in LG secretion.