Author Correction: Rapid and Precise Semi-Automatic Axon Quantification in Human Peripheral Nerves.

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Rapid and Precise Semi-Automatic Axon Quantification in Human Peripheral Nerves.

We developed a time-efficient semi-automated axon quantification method using freeware in human cranial nerve sections stained with paraphenylenediamine (PPD). It was used to analyze a total of 1238 facial and masseteric nerve biopsies. The technique was validated by comparing manual and semi-automated quantification of 129 (10.4%) randomly selected biopsies. The software-based method demonstrated a sensitivity of 94% and a specificity of 87%. Semi-automatic axon counting was significantly faster (p < 0.001) than manual counting. It took 1 hour and 47 minutes for all 129 biopsies (averaging 50 sec per biopsy, 0.04 seconds per axon). The counting process is automatic and does not need to be supervised. Manual counting took 21 hours and 6 minutes in total (average 9 minutes and 49 seconds per biopsy, 0.52 seconds per axon). Our method showed a linear correlation to the manual counts (R = 0.944 Spearman rho). Attempts have been made by several research groups to automate axonal load quantification. These methods often require specific hard- and software and are therefore only accessible to a few specialized laboratories. Our semi-automated axon quantification is precise, reliable and time-sparing using publicly available software and should be useful for an effective axon quantification in various human peripheral nerves.

Perfusion maintains functional potential in denervated mimic muscles in early persistent facial paralysis which requires early microsurgical treatment - the histoanatomic basis of the extratemporal facial nerve trunk assessing axonal load in the context of possible nerve transfers.

BACKGROUND AND OBJECTIVES: Early persistent facial paralysis is characterized by intact muscles of facial expression through maintained perfusion but lacking nerve supply. In facial reanimation procedures aiming at restoration of facial tone and dynamics, neurotization through a donor nerve is performed. Critical for reanimating target muscles is axonal capacity of both donor and recipient nerves. In cases of complete paralysis, the proximal stump of the extratemporal facial nerve trunk may be selected as a recipient site for coaptation. To further clarify the histological basis of this facial reanimation procedure we conducted a human cadaver study examining macro and micro anatomical features of the facial nerve trunk including its axonal capacity in human cadavers. Axonal loads, morphology and morbidity of different donor nerves are discussed reviewing literature in context of nerve transfers. METHODS: From 6/2015 to 9/2016 in a group of 53 fresh frozen cadavers a total of 106 facial halves were dissected. Biopsies of the extratemporal facial nerve trunk (FN) were obtained at 1 cm distal to the stylomastoid foramen. After histological processing and digitalization of 99 specimens available, 97 were selected eligible for fascicle counts and 87 fulfilled quality criteria for a semi-automated computer-based axon quantification software using ImageJ/Fiji. RESULTS: An average of 3.82 fascicles (range, 1 to 9) were noted (n = 97). 6684±1884 axons (range, 2655- 12457) were counted for the entire group (n = 87). Right facial halves showed 6364±1904 axons (n = 43). Left facial halves demonstrated 6996±1833 axons (n = 44) with no significant difference (p = 0.73). Female cadavers featured 6247±2230 (n = 22), male showed 6769±1809 axons (n = 40). No statistical difference was seen between genders (p = 0.59). A comparison with different studies in literature is made. The nerve diameter in 82 of our specimens could be measured at 1933±424 µm (range, 975 to 3012). CONCLUSIONS: No donor nerve has been described to match axonal load or fascicle number of the extratemporal facial nerve main trunk. However, the masseteric nerve may be coapted for neurotization of facial muscles with a low complication rate and good clinical outcomes. Nerve transfer is indicated from 6 months after onset of facial paralysis if no recovery of facial nerve function is seen.

The nerve supply of zygomaticus major: Variability and distinguishing zygomatic from buccal facial nerve branches.

The zygomaticus major (ZM) is important for the human smile. There are conflicting data about whether the zygomatic or buccal branches of the facial nerve are responsible for its motor innervation. The literature provides no precise distinction of the transition zone between these two branch systems. In this study, a definition to distinguish the facial nerve branches at the level of the body of the zygoma is proposed. In the light of this definition, we conducted an anatomical study to determine how the source of innervation of the ZM was distributed. A total of 96 fresh-frozen cadaveric facial halves were dissected under loupe magnification. A hemiparotidectomy was followed by antegrade microsurgical dissection. Any branch topographically lying superficial to the zygoma or touching it was classed as zygomatic, and any neighboring inferior branch was considered buccal. The arborization of the facial nerve was diffuse in all cases. In 64 out of 96 specimens (67%, 95% CI: 56% to 76%), zygomatic branches innervated the ZM. Buccal branches innervated ZM in the other 32 facial halves (33%, 95% CI: 24% to 44%). There were no differences in respect of sex or facial side. All facial halves displayed additional branches, which crossed the muscle on its inner surface without supplying it. In 31 specimens, a nerve branch ran superficial to ZM in its cranial third. According to our classification, the zygomaticus major is innervated by zygomatic branches in 67% of cases and by buccal branches in 33%. Clin. Anat. 31:560-565, 2018. © 2018 Wiley Periodicals, Inc.

Cre recombinase expression or topical tamoxifen treatment do not affect retinal structure and function, neuronal vulnerability or glial reactivity in the mouse eye.

Mice with a constitutive or tamoxifen-induced Cre recombinase (Cre) expression are frequently used research tools to allow the conditional deletion of target genes via the Cre-loxP system. Here we analyzed for the first time in a comprehensive and comparative way, whether retinal Cre expression or topical tamoxifen treatment itself would cause structural or functional changes, including changes in the expression profiles of molecular markers, glial reactivity and photoreceptor vulnerability. To this end, we characterized the transgenic α-Cre, Lmop-Cre and the tamoxifen-inducible CAGG-CreER™ mouse lines, all having robust Cre expression in the neuronal retina. In addition, we characterized the effects of topical tamoxifen treatment itself in wildtype mice. We performed morphometric analyses, immunohistochemical staining, in vivo ERG and angiography analyses and realtime RT-PCR analyses. Furthermore, the influence of Cre recombinase or topical tamoxifen exposure on neuronal vulnerability was studied by using light damage as a model for photoreceptor degeneration. Taken together, neither the expression of Cre, nor topical tamoxifen treatment caused detectable changes in retinal structure and function, the expression profiles of investigated molecular markers, glial reactivity and photoreceptor vulnerability. We conclude that the Cre-loxP system and its induction through tamoxifen is a safe and reliable method to delete desired target genes in the neural retina.

What is the nature of the RGC-5 cell line?

The immortalized RGC-5 cell line has been widely used as a cell culture model to study the neurobiology of retinal ganglion cells (RGCs). The cells were originally introduced as derived from rat RGC showing expression of various neuronal markers, in particular the RGC-characteristic proteins Brn3 and Thy1. Recent data gave rise to concerns regarding the origin and nature of the cells. RGC-5 cells were identified to be of mouse origin and their expression of RGC characteristics was questioned by some laboratories. This article summarizes the available data on the properties of RGC-5, discusses common protocols for their differentiation and is aimed at providing researchers some guidelines on the benefits and limitations of RGC-5 for research.

[Functional morphology of the outflow pathways of aqueous humor and their changes in open angle glaucoma]. / Funktionelle Morphologie der Abflusswege des Kammerwassers und ihre Veränderungen beim Offenwinkelglaukom.

Aqueous humor exits the eye through the trabecular and uveoscleral outflow pathways. Under normal conditions intraocular pressure is maintained in the trabecular outflow pathways in which aqueous humor passes through the trabecular meshwork into Schlemm's canal. Intraocular pressure is generated through an outflow resistance in the juxtacanalicular region which consists of juxtacanalicular tissue and the inner wall endothelium of Schlemm's canal. The resistance of this region is under the influence of two contractile systems, the anterior longitudinal portion of the ciliary muscle and the contractile myofibroblast-like cells in the trabecular outflow pathways. Resistance is lowered through contraction of the ciliary muscle or relaxation of the contractile cells in the trabecular outflow pathways. In primary open angle glaucoma, resistance in the juxtacanalicular region is abnormally high. The cause of the increase is related to an increased activity in transforming growth factor beta and connective tissue growth factor signaling. The cells of the trabecular meshwork outflow pathways are stimulated to form a stronger contractile phenotype involving both an increase in the actin cytoskeleton and the surrounding fibrillar extracellular matrix. As a result there is an increase in cellular tone in the trabecular outflow pathways leading to an increase in rigidity and outflow resistance.

[Development of the human eye]. / Entwicklung des menschlichen Auges.

The development of the human eye requires a coordinated interplay between cells from different origins. The optic cup which is neuroectodermal in origin and derives from the neural tube, gives rise to the neuronal retina, the retinal pigmented epithelium, the epithelial layers of ciliary body and iris, and the iris musculature. The lens which is displaced into the optic cup during development originates from the surface ectoderm. Cells of the neural crest provide the ocular mesenchyme while ocular blood vessels are of mesodermal origin. The basic morphogenetic processes of eye development are completed at the end of the second month of embryonic life. However, for correct functioning further maturation processes are required which are not completed before birth or several months after. Examples are aqueous humor circulation, maturation of cones in the foveola, myelination of optic nerve axons and completion of the retinal vasculature.

[Development of the iridocorneal angle and congenital glaucoma]. / Entwicklung des Kammerwinkels und kongenitales Glaukom.

The trabecular meshwork originates from cells of the neural crest which migrate to the iridocorneal angle during embryonic and fetal development of the eye. Correct morphogenesis of trabecular outflow pathways requires the differentiation of the cells to a porous and lamellate meshwork as well as the ingrowth of Schlemm's canal and posterior movement of the iris root. A failure in these processes is responsible for primary congenital or infantile glaucoma which presents with increased resistance to aqueous humor outflow resulting in increased intraocular pressure. Most cases appear to be of a sporadic nature but hereditary cases are often caused by mutations in the CYP1B1 gene which encodes for the enzyme cytochrome P450 1B1. Mutations cause a reduction in enzymatic activity which probably leads to diminished turnover of an as yet unidentified metabolite taking part in the signaling processes essential for formation of the trabecular meshwork and Schlemm's canal. More rarely, mutations in latent transforming growth factor beta binding protein 2 (LTBP2) or in the transcription factor FOXC1 have been described as causative for primary congenital glaucoma.

[The role of myocilin in the pathogenesis of primary open-angle glaucoma]. / Die Rolle von Myocilin bei der Pathogenese des primären Offenwinkelglaukoms.

Mutations in myocilin are responsible for autosomal-dominant inherited juvenile open-angle glaucoma and some subgroups of adult-onset glaucoma (POAG). Myocilin is a secreted glycoprotein with a molecular weight of approximately 55-57 kDa that forms dimers and multimers. Structural motives are domains that have similarities with myosin and olfactomedin, a signalling sequence as well as a leucine zipper at the N-terminus of myocilin. Most mutations that were identified in patients with POAG are localized in the olfactomedin domain which is highly conserved between species. In the eye, myocilin is highly expressed in the trabecular meshwork (TM), the sclera, the ciliary body and the iris, lower amounts are seen in the retina and the optic nerve. In addition, secreted myocilin is detected in the aqueous humor. In the chamber angle, myocilin is associated with fibrillar components of the extracellular matrix in the cribriform portion of the TM. Myocilin binds specifically to the HepII domain of fibronectin. In organ cultures of perfused anterior eye segments, recombinant myocilin increases outflow resistance. In cultured TM cells, myocilin is induced by treatment with dexamethasone in a similar time course as observed in steroid-induced glaucoma. Myocilin is also induced by transforming growth factor-beta (TGF-beta) and mechanical stress. Mice with a targeted disruption of the myocilin gene do not express a phenotype. Experimental data indicate that mutated myocilin is not secreted and accumulates in cells which might critically impair the function of the TM.

AlphaB-crystallin in lens development and muscle integrity: a gene knockout approach.

PURPOSE: To study the role of alphaB-crystallin (alphaB) in the developing lens and its importance in lens structure and function. METHODS: Gene targeting in embryonic stem cells was used to generate mouse lines in which the alphaB gene and its protein product were absent. Gene structure and expression were characterized by genomic Southern blot, immunoblot, and Northern blot analyses, and two-dimensional gel electrophoresis. The gene knockout mice were screened for cataract with slit lamp biomicroscopy, and dissected lenses were examined with dark-field microscopy. Lenses and other tissues were analyzed by standard histology and immunohistochemistry. Chaperone activity was determined by heating lens homogenate supernatants and measuring absorbance changes. RESULTS: In an unexpected result, lenses in the alphaB gene knockout mice developed normally and were remarkably similar to wild-type mouse lenses. All the other crystallins were present. The thermal stability of a lens homogenate supernatant was mildly compromised, and when oxidatively stressed in vivo with hyperbaric oxygen, the knockout lenses reacted similarly to wild type. In targeting the alphaB gene, the adjacent HSPB2 gene, which is not expressed in the lens, was also disrupted. Loss of alphaB and/or HSPB2 function leads to degeneration of some skeletal muscles. CONCLUSIONS: AlphaB is not essential for normal development of a transparent lens in the mouse, and therefore is more dispensable to the lens than the closely related alphaA-crystallin. It may play a small role in maintaining transparency throughout life. alphaB and/or the closely related HSPB2 is required to maintain muscle cell integrity in some skeletal muscles.

Expression of cyclooxygenase-1 and -2 in normal and glaucomatous human eyes.

PURPOSE: Primary open-angle glaucoma (POAG) is the predominant form of chronic glaucoma, but the underlying pathologic mechanisms are largely unknown. Because prostaglandins (PGs) have been introduced into POAG treatment with remarkable success, this study was undertaken to investigate whether a change in the expression of the PG-synthesizing enzymes cyclooxygenase (COX)-1 and -2 might be involved in the pathogenesis of POAG. METHODS: Expression of COX-1 and -2 was assessed by confocal laser microscopy, immunohistochemistry, Western blot analysis, and real-time RT-PCR in human eyes with different forms of glaucoma (primary open-angle, angle-closure, congenital juvenile, and steroid-induced), as well as in age-matched control eyes. Additionally, PGE2 was measured in aqueous humor by means of an enzyme-linked immunoassay as a product of COX activity. RESULTS: In normal eyes, ocular COX-1 and -2 expression were largely confined to the nonpigmented secretory epithelium of the ciliary body. By immunohistochemistry and real-time RT-PCR, COX-2 expression was completely lost in the nonpigmented secretory epithelium of the ciliary body of eyes with end-stage POAG, whereas COX-1 expression was unchanged. By immunohistochemistry, in the ciliary bodies of eyes in five patients with diagnosis of early POAG, eyes in two had complete loss of COX-2 expression and in three showed only a few remaining scattered COX-2-expressing cells. COX-2 expression in the ciliary body was also lost in patients with steroid-induced glaucoma and was reduced in patients receiving topical steroid treatment. Eyes of patients with either congenital juvenile or angle-closure glaucoma showed COX-2 expression indistinguishable from control eyes. Aqueous humor of eyes with POAG contained significantly less PGE2 than control eyes. CONCLUSIONS: Both cyclooxygenase isoforms are constitutively expressed in the normal human eye. Specific loss of COX-2 expression in the nonpigmented secretory epithelium of the ciliary body appears to be linked to the occurrence of POAG and steroid-induced glaucoma.

The role of myocilin/TIGR in glaucoma: results of the Glaucoma Research Foundation catalyst meeting in Berkeley, California, March 2000.

Approximately 3 years ago, the first major (biochemical, molecular biologic, and biologic) insight into primary open-angle glaucoma was the finding that mutations in the myocilin (MYOC/TIGR) gene were related to certain forms of juvenile onset of this disease. Since then, a great deal of work has been done to determine the possible mechanisms by which MYOC/TIGR might cause not only juvenile but also adult-onset primary open-angle glaucoma. To assess the current knowledge and those areas in which more research is necessary, a meeting of scientists was held by the Glaucoma Research Foundation of San Francisco, California in the spring of 2000. This meeting attempted to concentrate on the MYOC/TIGR protein rather than the genetics of this gene. Possible functions and roles of this protein intracellularly and extracellularly were critically examined and discussed. Normal transcriptional and translational events and the effect of mutations on these events were explored. The discussions yielded insight not only in those areas in which important information is known but also in vital areas in which little is currently understood. This review attempts to summarize the current knowledge regarding MYOC/TIGR and to elucidate the points that the people attending the meeting thought needed further study.

The presence and properties of myocilin in the aqueous humor.

PURPOSE: To determine whether myocilin is present in the aqueous humor (AH) and to examine certain properties of this protein. METHODS: Human AH was obtained at the time of either glaucoma surgery or cataract extraction. Monkey AH was obtained at the time of death, and bovine aqueous was obtained from eyes delivered from an abattoir. Column chromatography was performed on aqueous samples to determine the approximate size of the myocilin present. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis were performed using antibody prepared against a peptide sequence in myocilin. Analysis of the bovine proteins present in AH that were retained by a microporous filter was also performed using western blot analysis. RESULTS: By western blot analysis, myocilin was present in human, monkey, and bovine AH. The apparent molecular size of the myocilin present in the AH were greater than 250,000 Da, when quantified with a gel filtration column. Myocilin appeared to be hydrophobic and was one of the proteins that was retained on microporous filters that were obstructed by AH. CONCLUSIONS: Myocilin is a constituent in the AH. It appears that myocilin is a hydrophobic protein that may exist in an oligomeric state or in association with other proteins. Myocilin is retained by microporous filters and may be involved in the obstruction of these filters that occurs when AH is perfused through them.

Omega -crystallin of the scallop lens. A dimeric aldehyde dehydrogenase class 1/2 enzyme-crystallin.

While many of the diverse crystallins of the transparent lens of vertebrates are related or identical to metabolic enzymes, much less is known about the lens crystallins of invertebrates. Here we investigate the complex eye of scallops. Electron microscopic inspection revealed that the anterior, single layered corneal epithelium overlying the cellular lens contains a regular array of microvilli that we propose might contribute to its optical properties. The sole crystallin of the scallop eye lens was found to be homologous to Omega-crystallin, a minor crystallin in cephalopods related to aldehyde dehydrogenase (ALDH) class 1/2. Scallop Omega-crystallin (officially designated ALDH1A9) is 55-56% identical to its cephalopod homologues, while it is 67 and 64% identical to human ALDH 2 and 1, respectively, and 61% identical to retinaldehyde dehydrogenase/eta-crystallin of elephant shrews. Like other enzyme-crystallins, scallop Omega-crystallin appears to be present in low amounts in non-ocular tissues. Within the scallop eye, immunofluorescence tests indicated that Omega-crystallin expression is confined to the lens and cornea. Although it has conserved the critical residues required for activity in other ALDHs and appears by homology modeling to have a structure very similar to human ALDH2, scallop Omega-crystallin was enzymatically inactive with diverse substrates and did not bind NAD or NADP. In contrast to mammalian ALDH1 and -2 and other cephalopod Omega-crystallins, which are tetrameric proteins, scallop Omega-crystallin is a dimeric protein. Thus, ALDH is the most diverse lens enzyme-crystallin identified so far, having been used as a lens crystallin in at least two classes of molluscs as well as elephant shrews.

Regulation of human myocilin/TIGR gene transcription in trabecular meshwork cells and astrocytes: role of upstream stimulatory factor.

BACKGROUND: Mutations in the myocilin (MYOC)/TIGR gene are responsible for autosomal-dominant juvenile primary open-angle glaucoma (POAG). In patients with non-autosomal-dominant POAG, such mutations are rare, but the expression of MYOC/TIGR in the trabecular meshwork (TM) of the eye is considerably higher than in normals. We performed transfection, DNAse I footprinting, mutagenesis and electrophoretic mobility shift assays (EMSA) to identify elements responsible for the basal transcription of MYOC/TIGR in TM cells and astrocytes. RESULTS: DNAse I footprinting experiments of the human MYOC/TIGR promoter showed a major protected area between nt -106 to -77, which was not conserved in the homologous region of the mouse myoc/tigr promoter. In addition, the TATA-box was protected, as well as at least three downstream sites, including an AP-1-like sequence. Deletion of the -106 to -77 region caused a substantial loss of functional promotor activity in all cell types. Site-directed mutagenesis and EMSA experiments revealed the presence of two regulatory elements in the -106 to -77 region. Each of these cis-elements is essential for minimal promoter activity. The 5'-half of the region contains a sequence with similarities to NF-kappaB-related sites, however, binding of NF-kappaB could not be confirmed by EMSA. The 3'-half contains a canonical E-box sequence. EMSA experiments showed that the upstream regulatory factor (USF) was binding to the E-box sequence and that the binding can be supershifted by specific antibodies. CONCLUSIONS: Several DNA-protein binding elements contribute to a transcription of MYOC/TIGR, and USF is critically required for its basal transcription in trabecular meshwork cells and astrocytes.

Localization of myocilin/trabecular meshwork--inducible glucocorticoid response protein in the human eye.

PURPOSE: To study distribution and cellular localization of myocilin/trabecular meshwork-inducible glucocorticoid response protein (TIGR) in the human eye. METHODS: A peptide antibody against a portion of the myosin-like domain of myocilin/TIGR was developed. Different ocular tissues from three human donors were investigated by one- and two-dimensional gel electrophoresis and Western blot analysis. Immunohistochemistry was performed on 25 human eyes enucleated because of posterior choroidal melanoma and on 7 normal human donor eyes. RESULTS: By Western blot analysis, a band at approximately 57 kDa was visualized in cornea, trabecular meshwork, lamina cribrosa, optic nerve, retina, iris, ciliary body, and vitreous humor. By immunohistochemistry, immunoreactivity for myocilin/TIGR was observed in cells of the corneal epi- and endothelium and extracellularly in the corneal stroma and sclera. In the trabecular meshwork, cells of the uveal and corneoscleral meshwork were stained, as was the cribriform area directly adjacent to Schlemm's canal. Positive staining was seen in cells of the ciliary epithelium, ciliary muscle, lens epithelium, and in stromal and smooth muscle cells of the iris. Throughout the entire vitreous body, fine filamentous material was positively labeled. In the retina, staining was seen along the outer surface of rods and cones, in neurons of the inner and outer nuclear layer, and in the axons of optic nerve ganglion cells. Optic nerve axons were stained in the prelaminar, laminar, and postlaminar parts of the nerve. In the region of the lamina cribrosa, astrocytes in the glial columns and cribriform plates were positively labeled. CONCLUSIONS: Myocilin TIGR is expressed in almost every ocular tissue. Depending on the respective tissue, it is observed extra- or intracellularly. The presence of myocilin/TIGR in optic nerve axons and lamina cribrosa astrocytes indicates that the trabecular meshwork might not be the only target of abnormal myocilin/TIGR in GLC1A-linked open-angle glaucoma.

Modulation of myocilin/TIGR expression in human trabecular meshwork.

PURPOSE: To study factors that modulate myocilin/trabecular meshwork inducible glucocorticoid response protein (TIGR) mRNA expression in human trabecular meshwork (TM). METHODS: mRNA from fresh TM of four human donors, from perfused anterior segment organ cultured TM of three donors, and from four primary TM cell lines of different donors was isolated. The full length cDNA of myocilin/TIGR was cloned from TM mRNA using a polymerase chain reaction approach and used as probe for northern blot analysis hybridization. Trabecular meshwork cell cultures were treated with transforming growth factor (TGF)-beta1 (1 ng/ml), dexamethasone (10(-7) M), and mechanical stretch (10%). RESULTS: mRNA for myocilin/TIGR could be readily detected by northern blot analysis hybridization in 2 to 3 microg of total RNA from all fresh and all organ-cultured TM samples. In contrast, no mRNA for myocilin/TIGR could be detected in 20 microg of total RNA isolated from three different primary TM cell lines. Only one TM cell line had a baseline expression of myocilin/TIGR, which was 35- to 55-fold lower than that of fresh or organ-cultured TM samples. Treatment of TM cell cultures with dexamethasone for 1 day markedly increased expression of myocilin/TIGR mRNA, an effect that was even more pronounced after 3 days of treatment. Treatment with TGF-beta1 for 24 hours had no effect; however, after 3 and 12 days of treatment a 3.8- and 4-fold increase in myocilin/TIGR mRNA expression was observed. Expression of myocilin/TIGR mRNA was also increased after 10% mechanical stretch; however, in contrast to the effects of TGF-beta-1, this effect was observed much earlier (8-24 hours) after treatment. CONCLUSIONS: Dynamic mechanical stimuli maintain myocilin/TIGR expression in TM in situ and lack of these stimuli in monolayer cell cultures might be involved in downregulation of myocilin/TIGR expression.

Development of characterization of a immortal and differentiated murine trabecular meshwork cell line.

PURPOSE: To study mouse trabecular meshwork (TM) and to develop a murine TM cell line. METHODS: Mouse TM in situ was studied by light and electron microscopy (EM). In addition, TM was isolated from the H-2K(b)-tsA58 transgenic mouse strain in which promoter sequences of the major histocompatibility complex H-2Kb class 1 gene are fused to sequences of the SV40 mutant temperature-sensitive (ts) strain tsA58. The promoter is inducible by interferon (IFN)-gamma, and the tsA58 gene product is active at 33 degrees C (permissive conditions), but not at 37 degrees C (nonpermissive conditions). The TM explant was cultured in permissive conditions. Outgrowing cells were passaged through two rounds of single-cell cloning. One clonal cell line (MUTM-NEI/1) was characterized in nonpermissive conditions by EM, immunohistochemistry, reverse transcription-polymerase chain reaction (RT-PCR), and northern blot hybridization. In addition, MUTM-NEI/1 cells were transfected with plasmid DNA. RESULTS: The mouse eye has a circumferentially oriented outflow vessel and a TM that is subdivided in an outer juxtacanalicular or cribriform part and an inner lamellated or trabecular part. From the TM of the H-2Kb-tsA58 mouse, a clonal cell line (MUTM-NEI/1) was established. In permissive conditions, MUTM-NEI/1 cells remained proliferative through at least 80 generations without change in phenotype. In nonpermissive conditions, proliferation was slower, and MUTM-NEI/1 cells differentiated and synthesized collagen types I, III, IV, and VI; laminin; and fibronectin. MUTM-NEI/1 cells were immunoreactive for vimentin, alphaB-crystallin, and neural cell adhesion molecule (NCAM), but not for desmin or cytokeratin. Less than 10% of MUTM-NEI/1 cells stained for alpha-smooth muscle actin, whereas after 3 days of treatment with transforming growth factor-beta1 almost all cells were positive. MUTM-NEI/1 cells expressed mRNA for NCAM, aquaporin 1, myocilin/trabecular meshwork glucocorticoid-inducible protein, and alphaB-crystallin, which was increased after oxidative stress. MUTM-NEI/1 cells could be successfully transfected with plasmid DNA. CONCLUSIONS: The architecture of the murine outflow system is comparable to that in primates. The MUTM-NEI/1 cell line is a clonal, immortal, and differentiated TM cell line that will be an important tool for study of the expression of TM genes.