Muscular and neuronal control of voice production - forgotten findings, current concepts, and new developments.

Voice production has been an area of interest in science since ancient times, and although advancing research has improved our understanding of the anatomy and function of the larynx, there is still little general consensus on these two topics. This review aims to outline the main developments in this field and highlight the areas where further research is needed. The most important hypotheses are presented and discussed highlighting the four main lines of research in the anatomy of the human larynx and their most important findings: (1) the arrangement of the muscle fibers of the thyroarytenoid muscle is not parallel to the vocal folds in the internal part (vocalis muscle), leading to altered properties during contraction; (2) the histological structure of the human vocal cords differs from other striated muscles; (3) there is a specialized type of heavy myosin chains in the larynx; and (4) the neuromuscular system of the larynx has specific structures that form the basis of an intrinsic laryngeal nervous system. These approaches are discussed in the context of current physiological models of vocal fold vibration, and new avenues of investigation are proposed.

A New Immortalized Human Lacrimal Gland Cell Line.

The lacrimal gland is crucial for maintaining ocular health by producing the aqueous component of the tear film, which hydrates and nourishes the ocular surface. Decreased production of this component results in dry eye disease, a condition affecting over 250 million people worldwide. However, the scarcity of primary human material for studying its underlying mechanisms and the absence of a cell model for human lacrimal gland epithelial cells present significant challenges. Here, we describe the generation of immortalized human lacrimal gland cell lines through the introduction of an SV40 antigen. We successfully isolated and characterized three cell clones from a female lacrimal gland donor, confirming their epithelial identity through genomic and protein analyses, including PCR, RNAseq, immunofluorescence and cultivation in a 3D spheroid model. Our findings represent a significant advancement, providing improved accessibility to investigate the molecular pathogenesis mechanisms of dry eye disease and potential therapeutic interventions. We identified the expression of typical epithelial cell marker genes and demonstrated the cells' capability to form 2D cell sheets and 3D spheroids. This establishment of immortalized human lacrimal gland cells with epithelial characteristics holds promise for future comprehensive studies, contributing to a deeper understanding of dry eye disease and its cellular mechanisms.

Historical Profiling of Dry Eye Patients - Potential Trigger Factors and Comorbidities.

PURPOSE: Dry eye syndrome (DES) is one of the most common diseases of the ocular surface. Affected persons suffer from different subjective complaints, with sometimes severe impairment in the quality of life. The aetiology and pathogenesis are multifactorial, multifaceted, and not yet fully understood. The present study is intended to provide deeper insights into possible triggering factors and correlating comorbidities. MATERIALS AND METHODS: In German ophthalmological practices, 306 persons (174 women, 132 men, age: 18 - 87 years) were interviewed by questionnaire on concomitant diseases and possible further triggering factors. DES was diagnosed by an ophthalmologist in 170 cases. The statistical comparative analysis between persons with and without DES was carried out using the chi-squared test (SPSS statistical software). RESULTS: DES occurred with significantly (p < 0.05) increased frequency in women over 40 years of age, as well as in persons exposed to screen work, air conditioning, persons with chronic ocular inflammation, myomas (hysterectomy), dry skin, arterial hypertonicity in need of medication, cardiac arrhythmias, fatty liver, gastric ulcer, appendicitis, cholecystectomy, depression, hyperlipidaemia, hyperuricaemia, osteoporosis, and nephrolithiasis. CONCLUSION: Some of the known comorbidities and DES risk factors, e.g., computer work or depression, were confirmed. In contrast, the higher prevalence of hyperlipidaemia, hyperuricaemia, osteoporosis, nephrolithiasis, and fibroids among DES patients has not previously been reported. Additional studies should be performed on causal connections between DES and specific comorbidities.

Internal vascular anatomy of the human lacrimal gland: A protocol based on cadaver dissection and three-dimensional micro-computed tomography.

PURPOSE: To evaluate the feasibility of studying the vascular supply of the orbital and palpebral lobes of the human lacrimal gland using micro-computed tomography (micro-CT) and microscopic dissection. METHODS: The lacrimal gland artery of a fresh parasagittalized cadaver head (male, aged 76 years) was infused with a lead oxide-latex mixture near the occipital pole of the gland. The entire lacrimal gland was imaged using micro-CT and 3D cinematic rendering (CR) and then dissected under a surgical microscope. RESULTS: Micro-CT and CR images showed well-demarcated internal vascular branches of the lacrimal artery and their distribution within the orbital and palpebral lobes. The entire course of the artery and its branches could be visualized by CR and microscopic dissection, with the former showing better spatial orientation and finer branching. The main artery runs along the free edge of the aponeurosis of the levator palpebrae superior muscle and lies in the isthmus portion of the gland (between the orbital and palpebral lobes). The branches of the main lacrimal artery include one branch to the orbital adipose tissue just before entering the gland, two branches to the orbital lobe (medial and lateral), and two branches to the palpebral lobe (medial and lateral). The main artery terminates as palpebral and orbital lobe branches in the lateral half of the lacrimal gland. CONCLUSION: Latex and contrast-enhanced micro-CT is very well suited to visualize the vascular anatomy of the lacrimal artery within the gland. A large number of lacrimal gland examinations using the method presented here are required to demonstrate and understand the variability of the vascular anatomy of the human lacrimal gland.

Inverse identification of region-specific hyperelastic material parameters for human brain tissue.

The identification of material parameters accurately describing the region-dependent mechanical behavior of human brain tissue is crucial for computational models used to assist, e.g., the development of safety equipment like helmets or the planning and execution of brain surgery. While the division of the human brain into different anatomical regions is well established, knowledge about regions with distinct mechanical properties remains limited. Here, we establish an inverse parameter identification scheme using a hyperelastic Ogden model and experimental data from multi-modal testing of tissue from 19 anatomical human brain regions to identify mechanically distinct regions and provide the corresponding material parameters. We assign the 19 anatomical regions to nine governing regions based on similar parameters and microstructures. Statistical analyses confirm differences between the regions and indicate that at least the corpus callosum and the corona radiata should be assigned different material parameters in computational models of the human brain. We provide a total of four parameter sets based on the two initial Poisson's ratios of 0.45 and 0.49 as well as the pre- and unconditioned experimental responses, respectively. Our results highlight the close interrelation between the Poisson's ratio and the remaining model parameters. The identified parameters will contribute to more precise computational models enabling spatially resolved predictions of the stress and strain states in human brains under complex mechanical loading conditions.

New insights into lacrimal gland anatomy using 7T MRI and electron microscopy: Relevance for lacrimal gland targeted therapies and bioengineering.

PURPOSE: To study the tissue architecture, isthmus (connection between two lobes) of the lacrimal gland using preclinical 7T MRI in combination with histology and electron microscopy. METHODS: Ten lacrimal glands from Caucasian body donors (mean age 78.7 years) were studied using 7T-MRI (N = 5; scanned at 75-µm intervals), histology, and electron microscopy (N = 5) and 3D cinematic rendering (CR) techniques. RESULTS: 3D CR images showed uniform-sized lobules (widest lobule diameter, 1.68 ± 0.19 mm in orbital lobe, 1.68 ± 0.17 mm in palpebral lobe) in both lobes, separated by septae (size, 0.29 ± 0.09 mm). The internal framework of the gland resembled a honeycoomb pattern. In CR and histology, the isthmus contained glandular acini, large blood vessels, nerves, and no more than two ducts having a tortuous course towards the conjunctival surface. On assigning a color display to the rendered lacrimal gland, all glands showed a blood vessel originating from the main lacrimal artery just 5 mm beyond the hilum and making it course to the palpebral lobe via isthmus. The distance between the conjunctiva and the central substance of the orbital and palpebral lobe was 9.4 ± 0.2 mm and 2.8 ± 0.7 mm, respectively. Electron microscopy of the palpebral lobe revealed compact subepithelial layer in the overlying conjunctiva, followed by loosely scattered collagen bundles that contained the gland lobules. CONCLUSION: 3D-CR can be used to study the lacrimal gland microstructure, help fabricate a 3D scaffold for lacrimal gland bioprinting, and serve as guide for transconjunctival lacrimal gland targeted therapies i.e., 2.9 & 9 mm long needle to reach the orbital and palpebral lobe center, respectively in normal-size glands.

The chemicals between us-First results of the cluster analyses on anatomy embalming procedures in the German-speaking countries.

Hands-on courses utilizing preserved human tissues for educational training offer an important pathway to acquire basic anatomical knowledge. Owing to the reevaluation of formaldehyde limits by the European Commission, a joint approach was chosen by the German-speaking anatomies in Europe (Germany, Austria, Switzerland) to find commonalities among embalming protocols and infrastructure. A survey comprising 537 items was circulated to all anatomies in German-speaking Europe. Clusters were established for "ethanol"-, formaldehyde-based ("FA"), and "other" embalming procedures, depending on the chemicals considered the most relevant for each protocol. The logistical framework, volumes of chemicals, and infrastructure were found to be highly diverse between the groups and protocols. Formaldehyde quantities deployed per annum were three-fold higher in the "FA" (223 L/a) compared to the "ethanol" (71.0 L/a) group, but not for "other" (97.8 L/a), though the volumes injected per body were similar. "FA" was strongly related to table-borne air ventilation and total fixative volumes ≤1000 L. "Ethanol" was strongly related to total fixative volumes >1000 L, ceiling- and floor-borne air ventilation, and explosion-proof facilities. Air ventilation was found to be installed symmetrically in the mortuary and dissection facilities. Certain predictors exist for the interplay between the embalming used in a given infrastructure and technical measures. The here-established cluster analysis may serve as decision supportive tool when considering altering embalming protocols or establishing joint protocols between institutions, following a best practice approach to cater toward best-suited tissue characteristics for educational purposes, while simultaneously addressing future demands on exposure limits.

The Potential Role of SP-G as Surface Tension Regulator in Tear Film: From Molecular Simulations to Experimental Observations.

The ocular surface is in constant interaction with the environment and with numerous pathogens. Therefore, complex mechanisms such as a stable tear film and local immune defense mechanisms are required to protect the eye. This study describes the detection, characterization, and putative role of surfactant protein G (SP-G/SFTA2) with respect to wound healing and surface activity. Bioinformatic, biochemical, and immunological methods were combined to elucidate the role of SP-G in tear film. The results show the presence of SP-G in ocular surface tissues and tear film (TF). Increased expression of SP-G was demonstrated in TF of patients with dry eye disease (DED). Addition of recombinant SP-G in combination with lipids led to an accelerated wound healing of human corneal cells as well as to a reduction of TF surface tension. Molecular modeling of TF suggest that SP-G may regulate tear film surface tension and improve its stability through specific interactions with lipids components of the tear film. In conclusion, SP-G is an ocular surface protein with putative wound healing properties that can also reduce the surface tension of the tear film.

Unraveling the Local Relation Between Tissue Composition and Human Brain Mechanics Through Machine Learning.

The regional mechanical properties of brain tissue are not only key in the context of brain injury and its vulnerability towards mechanical loads, but also affect the behavior and functionality of brain cells. Due to the extremely soft nature of brain tissue, its mechanical characterization is challenging. The response to loading depends on length and time scales and is characterized by nonlinearity, compression-tension asymmetry, conditioning, and stress relaxation. In addition, the regional heterogeneity-both in mechanics and microstructure-complicates the comprehensive understanding of local tissue properties and its relation to the underlying microstructure. Here, we combine large-strain biomechanical tests with enzyme-linked immunosorbent assays (ELISA) and develop an extended type of constitutive artificial neural networks (CANNs) that can account for viscoelastic effects. We show that our viscoelastic constitutive artificial neural network is able to describe the tissue response in different brain regions and quantify the relevance of different cellular and extracellular components for time-independent (nonlinearity, compression-tension-asymmetry) and time-dependent (hysteresis, conditioning, stress relaxation) tissue mechanics, respectively. Our results suggest that the content of the extracellular matrix protein fibronectin is highly relevant for both the quasi-elastic behavior and viscoelastic effects of brain tissue. While the quasi-elastic response seems to be largely controlled by extracellular matrix proteins from the basement membrane, cellular components have a higher relevance for the viscoelastic response. Our findings advance our understanding of microstructure - mechanics relations in human brain tissue and are valuable to further advance predictive material models for finite element simulations or to design biomaterials for tissue engineering and 3D printing applications.

Polytetrafluoroethylene (PTFE) suture vs fiberwire and polypropylene in flexor tendon repair.

BACKGROUND: In this study, we evaluate the value of novel suture material based on monofilamentous-extruded polyfluoroethylene (PTFE) compared to polypropylene (PPL) and Fiberwire (FW). MATERIALS AND METHODS: 60 flexor tendons were harvested from fresh cadaveric upper extremities. 4-0 sutures strands were used in the PPL, FW and PTFE group. Knotting properties and mechanical characteristics of the suture materials were evaluated. A 4-strand locked cruciate (Adelaide) or a 6-strand (M-Tang) suture technique was applied as core sutures for a tendon repair. Two-way ANOVA tests were performed with the Bonferroni correction. RESULTS: Stable knotting was achieved with 5 throws with the PPL material, 7 throws for FW and 9 throws for PTFE. In the PPL group, linear tensile strength was 45.92 ± 12.53 N, in the FW group 80.11 ± 18.34 N and in the PTFE group 76.16 ± 29.10 N. FW and PTFE are significantly stronger than PPL but show no significant difference among each other. Similar results were obtained in the subgroup comparisons for different repair techniques. The Adelaide and the M-Tang knotting technique showed no significant difference. CONCLUSION: Fiberwire showed superior handling and knotting properties in comparison to PTFE. However, PTFE allows easier approximation of the stumps. In both, M-Tang and Adelaide repairs, PTFE was equal to FW in terms of repair strength. Both PTFE and FW provide for a robust tendon repair so that early active motion regimens for rehabilitation can be applied.

[Is there a new salivary gland? - Rather not!] / Gibt es eine neue Kopfspeicheldrüse? ­ Eher nicht!

In October 2020, the lay press, but also some medical journals and websites reported the putative discovery of a new salivary gland in the nasopharynx based on prostate-specific membrane antigen positron emission tomography computed tomography (PSMA-PET/CT) examinations. As an interdisciplinary group from the fields of anatomy, pathology, nuclear medicine and otorhinolaryngology, we come to the view that an accumulation of minor salivary glands has been described here. Minor salivary glands in the nasopharynx and in the peritubar region have been described at least since 1866. The current description in PSMA-PET/CT does not justify the definition of a new, independent salivary gland. The PSMA-PET/CT could, however, be suitable to better protect salivary glands in the nasopharynx when planning radiation therapy. This should be evaluated in clinical trials.

New insights into the lacrimal pump.

PURPOSE: To date, there are many theories about tear transport through the canaliculi of the draining lacrimal system into the lacrimal sac but only few with supportive data. It is certain that the function of the lacrimal part of orbicularis oculi muscle (Horner-Duverney's muscle) is indispensable for the transport of "used" tears. However, the muscle's exact structure and the mechanisms of its functions are as yet unclear. To obtain deeper insights we undertook the present study. METHODS: Upper and lower canaliculi (including the entrance into the lacrimal sac) from donor cadavers were dissected. Some of the specimens were prepared for scanning electron microscopy (SEM) to analyze the course of muscle fibers surrounding the canaliculi. Others were sectioned for enzyme- (EHC) and immunohistochemistry (IHC) to learn about the distribution of slow and fast reacting muscle fibers in Horner-Duverney's muscle as well as to analyze the distribution of different neurotransmitters to learn more about the innervation of Horner-Duverney's muscle. Four tear duct systems taken from body donors were cut out en bloc after formalin fixation, serially sectioned and reconstructed using a newly developed technology for 3D reconstruction of histological serial sections named HiD® (Chimaera GmbH, Germany). Patients that had undergone dacryocystorhinostomy (DCR) were video-analyzed endonasally during active blinking, focusing on viewing the temporal wall of the lacrimal sac movement where the canaliculi penetrated the lacrimal sac. RESULTS: SEM revealed that muscle fibers of Horner-Duverney's muscle surround the vertical parts of the upper and lower canaliculus in a scissor like pattern whereas they ran in parallel to the first two thirds of the horizontal parts surrounding the respective canaliculus. Here, the muscle fibers were embedded in dense connective tissue forming a unique network. At the nasal third, muscle fibers left the canaliculi and ran to the posterior part of the fascia of the lacrimal sac and the lacrimal bone. EHC revealed that Horner-Duverney's muscle contained nearly an equal distribution of type I and type IIb muscle fibers compared to the superior rectus muscle which contains more type I and the masseter and iliopsoas muscles with more type IIb muscle fibers. IHC indicated presence of trigeminal, catecholaminergic and cholinergic nerve endings. 3D reconstructions supported the SEM data. Endonasal video analysis of patients after DCR with a nasally open lacrimal sac revealed bulging of the temporal wall of the lacrimal sac during blinking. On the basis of these findings, a modified lacrimal pump theory is proposed. CONCLUSION: The results support the hypothesis that contraction of Horner-Duverney's muscle leads to closure of the canaliculi in their first two thirds based on the special arrangement of muscle fibers and connective tissue fibers. This causes the tear fluid in the canaliculi to be pressed/transported towards the lacrimal sac. The medial third of the vertical portions of the canaliculi, the canaliculus communis and the intrasaccal portion of the canaliculus are compressed by the shortening and thickening of the Horner-Duverney muscle from dorsal, which leads to a compression of the canaliculi lumens in this part of the system, thereby pushing the lacrimal fluid further towards the lacrimal sac. The mix of fast contracting and fatigue resistant muscle fibers is ideally suited for the blink mechanism that is complexly regulated by the nervous system.

Altered Surfactant Protein Expression in Primary Acquired Nasolacrimal Duct Obstruction.

PURPOSE: To investigate the presence and distribution patterns of 6 surfactant proteins in lacrimal drainage tissues of patients with primary acquired nasolacrimal duct (NLD) obstruction. METHODS: The presence and distribution of surfactant proteins (SP)-G and SP-H was first assessed in normal cadaveric lacrimal systems. The study was then performed in 10 samples of lacrimal sac and the respective NLDs obtained from patients suffering from primary acquired NLD obstruction who underwent either a dacryocystorhinostomy or a dacryocystectomy. The lacrimal sac samples were further divided into fundus and body, soon after their removal. Immunohistochemical labeling was performed for assessing the presence and distribution of SPs: SP-A, SP-B, SP-C, SP-D, SP-G/SFTA2, and SP-H/SFTA3. The results were then scored as positive or negative and the distribution pattern, if any, within the lacrimal sac and NLDs was assessed. Human lung tissues were used as controls. RESULTS: SP-H was demonstrated in the lining epithelia of the normal lacrimal drainage systems, whereas SP-G was uniformly negative. Immunohistochemical labeling revealed wide variations in the staining patterns of different SPs in different regions of the lacrimal sac and the NLD. SP-D and SP-G revealed uniformly negative immunoreactivity. Variable staining patterns were also noted between the superficial and basal layers of the lining epithelia. However, the goblet cells and intraepithelial mucous glands did not express any of the SPs. CONCLUSIONS: This study provides a proof of principle for the presence of SP-H and absence of SP-G in the normal lacrimal drainage systems. In cases of primary acquired nasolacrimal duct obstruction, there were alterations or loss of SP expression in the lining epithelia of the lacrimal sac and NLDs, reflecting their possible role in the etiopathogenesis of primary acquired nasolacrimal duct obstruction.In cases of primary-acquired nasolacrimal duct obstruction, the expression of multiple surfactant proteins was either deranged or lost in the lining epithelium of the lacrimal sac and nasolacrimal ducts.

Recommendations of the working group of the Anatomische Gesellschaft on reduction of formaldehyde exposure in anatomical curricula and institutes.

The practice of human and veterinary medicine is based on the science of anatomy and dissection courses are still irreplaceable in the teaching of anatomy. Embalming is required to preserve body donors, for which process formaldehyde (FA) is the most frequently used and well characterized biocidal substance. Since January 2016, a new occupational exposure limit (OEL) for FA of 0.37mg/m3 issued by the European Committee on Hazardous Substances is obligatory since FA has been classified as a human 1B carcinogen. The anatomical institutes in the German-speaking region are called upon to consolidate efforts to reduce use of FA in anatomical curricula and body donations. As a result, the Anatomische Gesellschaft (AG) has formed a "Working Group for Reduction of Formaldehyde Exposure in Dissection Courses" tasked with discussion and recommendation of measures to reduce FA. Based on the assessment of the Working Group, the AG has issued an official opinion to the effect that, at this point in time, embalming of body donors without FA completely is not feasible. Therefore, a combination of approaches are to be used to reduce FA exposure, including technical and structural (architectural) adaptations, modification of protocols for fixation and preservation as well as organizational measures. One structural measure considered unavoidable is the integration of air supply and exhaust of individual dissecting tables into the ventilation system of the anatomy building. To embalm human body donors, intra-arterial perfusion fixation with up to 4% FA and a total fluid volume of 150mL/kg body weight will suffice. For animals where body weights and biology of bodies vary widely (i.e. special needs of fixation for ruminants, large animals as horses) perfusion fixation with up to 4% FA and a quantity of fixative solution of 10-15% of the body weight may be required. Preservation of body donors in storage (immersion) can be done with 40% ethanol or in a full bath preservation containing up to 2% FA. Corpse humidification in the dissecting room is possible with 2% phenoxyethanol, in each case without FA. In veterinary anatomy, microbiological burden is often higher and therefore might lead to a need of FA in long-time storage. Compliance with the current OEL in all institutes would appear to be feasible in combination with various organizational measures.

Examining the role of the surfactant family member SFTA3 in interneuron specification.

The transcription factor NKX2.1, expressed at high levels in the medial ganglionic eminence (MGE), is a master regulator of cortical interneuron progenitor development. To identify gene candidates with expression profiles similar to NKX2.1, previous transcriptome analysis of human embryonic stem cell (hESC)-derived MGE-like progenitors revealed SFTA3 as the strongest candidate. Quantitative real-time PCR analysis of hESC-derived NKX2.1-positive progenitors and transcriptome data available from the Allen Institute for Brain Science revealed comparable expression patterns for NKX2.1 and SFTA3 during interneuron differentiation in vitro and demonstrated high SFTA3 expression in the human MGE. Although SFTA3 has been well studied in the lung, the possible role of this surfactant protein in the MGE during embryonic development remains unexamined. To determine if SFTA3 plays a role in MGE specification, SFTA3-/- and NKX2.1 -/- hESC lines were generated using custom designed CRISPRs. We show that NKX2.1 KOs have a significantly diminished capacity to differentiate into MGE interneuron subtypes. SFTA3 KOs also demonstrated a somewhat reduced ability to differentiate down the MGE-like lineage, although not as severe relative to NKX2.1 deficiency. These results suggest NKX2.1 and SFTA3 are co-regulated genes, and that deletion of SFTA3 does not lead to a major change in the specification of MGE derivatives.

Evaluation of surfactant proteins A, B, C, and D in articular cartilage, synovial membrane and synovial fluid of healthy as well as patients with osteoarthritis and rheumatoid arthritis.

OBJECTIVE: Surfactant Proteins (SPs) are well known from lung and form, along with phospholipids, a surface-active-layer at the liquid-air-interface of the alveolar lining. They play a major protective role by lowering surface tension, activating innate and adaptive immune defense at the lung mucosal interface, especially during infection. We analyzed the regulation of SPs in human and mouse articular chondrocytes, synoviocytes, and synovial fluid under healthy and inflammatory conditions, as well as in tissues of patients suffering from osteoarthritis and rheumatoid arthritis. METHODS: Immunohistochemistry, RT-PCR, qRT-PCR, ELISA, Western blotting were performed in cell cultures and tissue samples to determine localization, regulation, and concentration of SPs. RESULTS: All four SPs, were expressed by healthy human and mouse articular chondrocytes and synoviocytes and were also present in synovial fluid. Treatment with inflammatory mediators like IL-1ß and TNF-α led to short-term upregulation of individual SPs in vitro. In tissues from patients with osteoarthritis and rheumatoid arthritis, protein levels of all four SPs increased significantly compared to the controls used. CONCLUSION: These results show the distribution and amount of SPs in tissues of articular joints. They are produced by chondrocytes and synoviocytes and occur in measurable amounts in synovial fluid. All four SPs seem to be differently regulated under pathologic conditions. Their physiological functions in lowering surface tension and immune defense need further elucidation and make them potential candidates for therapeutic intervention.

Detection of intrinsic cholinergic system in the human lacrimal drainage system: evidence and potential implications.

PURPOSE: To investigate the presence and distribution of epithelial and non-epithelial cholinergic system and cholinergic brush cells in the human lacrimal drainage system. METHODS: The study was performed on fresh frozen human cadaveric samples of the lacrimal drainage system. Immunohistochemistry was performed for assessing the presence and distribution of cholinergic brush cell proteins-villin, acetylcholine synthesizing enzyme, choline acetyltransferase (ChAT); vesicular acetylcholine transporter (VAChT); components of canonical taste transduction signaling cascade, phospholipase C ß2 (PLCß2), and transient receptor potential cation channel, subfamily M, and member 5 (TRPM5). In addition, immunoreactivity to carbonic anhydrase 4 (CA4) was assessed. The immunoreactivity was scored as positive or negative and the distribution patterns in the canaliculi, lacrimal sac, and nasolacrimal duct were investigated. In addition, ultrastructural analysis was performed to ascertain the presence of brush cells by means of scanning electron microscopy (SEM). RESULTS: Villin revealed immunoreactivity in the superficial epithelial cells of lacrimal sac and nasolacrimal ducts. Positive immunoreactivity was also found for ChAT, VAChT, TRPM5, and PLCß2. ChAT expression was limited to the superficial epithelial layers of the lacrimal sac epithelium. TRPM5 and PLCß2 were expressed on the cell membranes, cytoplasm, and basolateral surfaces of the lacrimal sac epithelium and also showed strong expression in the submucosal glandular acinar cells. VAChT showed strong expression in the canaliculus and lacrimal sac and was expressed on the surface of the superficial epithelial cells and the submucosal glandular acinar cells and lining of the blood vessels. There was a uniformly negative immunoreactivity for CA4. SEM revealed single epithelial cells with dense tuft of rigid apical microvilli in the lacrimal sac and nasolacrimal ducts. CONCLUSIONS: This study provides a proof of principle for the presence of an intrinsic epithelial cholinergic mechanism in the lacrimal drainage system.