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.

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.

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

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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.

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.

Ocular adenovirus gene transfer varies in efficiency and inflammatory response.

PURPOSE: To study the effects of adenoviral gene transfer to the tissues of the anterior segment in vitro by rat and monkey lens organ cultures and in vivo by single injection into the anterior chamber of rabbits. METHODS: In vitro, intact lens cultures were exposed to 1 to 4 x 10(8) pfu Av1LacZ4 and Av1Luc1 in TC199 medium containing no serum or growth factors. Av1LacZ4 and Av1Luc1 are replication-deficient adenovirus vectors, carrying the reporter genes Escherichia coli LacZ and firefly luciferase, respectively. In vivo, the anterior chambers of eight rabbits were injected once with 20 mumol Av1LacZ4 (8 x 10(8) pfu) and evaluated 48 hours after injection. Enzyme activity of the reporter genes was measured biochemically and histochemically. RESULTS: In organ cultures, adenovirus delivers reporter genes efficiently to the ciliary processes but penetrates poorly into the capsulated lenses. Viral receptors, however, are present in rat lens epithelium, as in primary trabecular meshwork and other lens cell lines. In vivo, gene transfer was evident in corneal endothelium, iris anterior surface, and trabecular meshwork. Presence of the virus did not affect lens transparency or provoke external discomfort signs. Infected corneal endothelial cells were swollen and partly detached; 3 of 8 infected eyes showed a severe inflammatory response in chamber angle, anterior uvea, and limbal conjunctiva. CONCLUSIONS: These findings reveal the distinct gene transfer potential of each of the tissues of the anterior segment and emphasize the need to address the inflammatory response to these first-generation adenoviral vectors.

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.

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.

Human and monkey trabecular meshwork accumulate alpha B-crystallin in response to heat shock and oxidative stress.

PURPOSE: Oxidative stress and other forms of injury to trabecular meshwork (TM) cells may contribute to changes seen with age and primary open-angle glaucoma. This study was designed to investigate if TM expresses alpha B-crystallin, a small heat-shock protein with chaperone activity, and whether it might be overexpressed under stress conditions. METHODS: The TM from human and monkey eyes, as well as organ and primary cell cultures derived from these eyes, were investigated for alpha B-crystallin by immunohistochemistry, two-dimensional gel electrophoresis, Northern and Western blot analysis. The TM cell cultures were stressed by heat shock (44 degrees C for 15 minutes) or hydrogen peroxide (200 mumol for 1 hour). Semiquantitation of alpha B-crystallin messenger RNA (mRNA) or protein was obtained by densitometry. RESULTS: In both species, alpha B-crystallin could be detected in fresh and cultured TM by two-dimensional gel electrophoresis in conjunction with Western blot analysis. Immunohistochemistry of fresh samples showed that alpha B-crystallin was expressed predominantly in the cribriform area. Protein expression was enhanced in 4- to 7-day organ cultures. Primary cultures from human TM cells expressed two sizes (approximately 0.8 and 1.1 kb) of alpha B-crystallin mRNA in Northern blots. In monkey TM cultures, a 0.8-kb band was observed, which comigrated with lens alpha B-crystallin. In both species, heat shock caused a significant increase in alpha B-crystallin mRNA with a peak after 4 hours. An increase in alpha B-crystallin mRNA also was observed after oxidative stress; however, the onset of mRNA induction was slower. After heat shock, but not after oxidative stress, a transient change in mRNA mobility was observed. Western dot blot analysis showed a 3.4-fold increase in protein 24 hours after heat shock and a 20-fold increase after 48 hours. No constitutive mRNA expression and only a minimal increase 4 hours after heat shock could be observed in simian virus 40 transformed cell lines from human TM. CONCLUSIONS: Overexpression of alpha B-crystallin might be an important mechanism for TM to prevent cellular damage associated with various stress conditions.

Nerve endings with structural characteristics of mechanoreceptors in the human scleral spur.

PURPOSE: The innervation of the scleral spur region was investigated to learn whether mechano-receptors are present in this region. METHODS: Serial tangential sections and whole-mount preparations of the scleral spur region of 18 human eyes of different ages were investigated with electronmicroscopic and immunohistochemical methods. For immunohistochemistry antibodies against neurofilament-proteins, synaptophysin, substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY), tyrosine-hydroxylase, dopamine-beta-hydroxylase, and acetylcholinesterase were used. RESULTS: Club- or bulb-shaped nerve endings with a diameter of 5 microns to 25 microns were identified in the scleral spur region throughout the whole circumference of the eyes. The terminals derive from myelinated axons with a diameter of approximately 3 microns and stain with antibodies against neurofilament-proteins and synaptophysin but do not stain for tyrosine-hydroxylase, dopamine-beta-hydroxylase, acetylcholinesterase, NPY, VIP, SP, or CGRP. Electronmicroscopically, the endings contain abundant neurofilaments, granular and agranular vesicles of different sizes, numerous mitochondria, and lysosome-like lamellated structures. The endings are incompletely ensheathed by Schwann cells. Those areas of the cell membrane of the endings that are not covered by Schwann cells are in intimate contact with the fibrillar connective tissue elements of the scleral spur. CONCLUSION: These structural features are highly characteristic for mechanoreceptive nerve endings in other tissues of the human body. The authors therefore hypothesize that the club-or bulb-shaped nerve endings in the human scleral spur are afferent mechanoreceptors that measure stress or strain in the connective tissue elements of the scleral spur. Such changes might be induced by ciliary muscle contraction and/or by changes in intraocular pressure.

Species differences in choroidal vasodilative innervation: evidence for specific intrinsic nitrergic and VIP-positive neurons in the human eye.

PURPOSE: There is evidence that vasodilation of choroidal vessels results from facial nerve stimulation. To obtain more information about the role of this innervation, the authors examined the presence and spatial organization of nitrergic and vasoactive intestinal peptide (VIP) immunoreactive nerves in the human choroid. For comparison, the choroid of rabbit and rat eyes, with different types of retinal vascularization and no fovea, were studied. METHODS: Whole mounts of five human, nine rat, and two rabbit choroids were stained for NADPH-diaphorase. In addition, immunocytochemical staining was carried out on tangential frozen sections of two human choroids using antibodies against nitric oxide synthase (NOS), synaptophysin, and VIP. RESULTS: In all species, a perivascular network of diaphorase-positive nerve fibers with varicose terminals accompanied the arteries and arterioles of the choroidal stroma. A striking difference to rat and rabbit choroids was the presence of numerous positively stained ganglion cells in human choroids. Positively stained axons connected the neurons with each other and with the perivascular network. Most of the ganglion cells were concentrated in the temporal-central region, adjacent to the fovea. Immunocytochemically, the choroidal ganglion cells were immunoreactive for NOS. Some ganglion cells stained for VIP. Staining for synaptophysin demonstrated varicose terminals innervating the perikarya of the ganglion cells. Many of these terminals stained for NOS and VIP. CONCLUSIONS: The presence of an intrinsic nerve cell plexus that is specifically localized in human eyes in the temporal-central portion of the choroid indicates a functional significance of the nitrergic choroidal innervation for the fovea.

Nerve cells in the human ciliary muscle: ultrastructural and immunocytochemical characterization.

PURPOSE: Intrinsic nerve cells in the human ciliary muscle were identified and characterized by immunohistochemical and ultrastructural methods. METHODS: Serial sections through the ciliary muscle of 10 human donors (age range, 53 to 91 years) were investigated by electron microscopy, NADPH-diaphorase (NADPH-d) staining, and immunohistochemistry. Antibodies against nitric oxide synthase (NOS), protein gene product 9.5 (PGP 9.5), neurofilament proteins, tyrosine hydroxylase (TH), neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), substance P (SP) and calcitonin gene-related peptide (CGRP) were used. Nerve cell density per millimeter of circumferential width was analyzed in three eyes, and in one eye the total number of neurons in the entire ciliary muscle was evaluated. RESULTS: Small (70% of the total; longitudinal diameter 10 to 14 microns) and large (longitudinal diameter 20 to 30 microns) ganglion cells were identified in the inner parts of the muscles' reticular and circular portions. No nerve cells were observed in the anterior longitudinal portion. The cells were in contact with unmyelinated axons and synaptic boutons containing small agranular and large granular vesicles. Axo-somatic and axo-dendritic synapses were observed. Histochemically and ultrahistochemically, the neurons stained intensely for NADPH-d. Both cell types were multipolar and expressed long filamentous processes. Axonal processes with periodic swellings suggesting varicosities ran close and parallel to neighboring muscle bundles. Some nerve cells were connected with each other by axonal processes. No perivascular NADPH-d-positive nerves were seen around ciliary muscle vessels, but they were present in the wall of the major arterial circle of the iris. A small number of ganglion cells contributed to this perivascular network. NADPH-d-positive neurons stained for PGP 9.5 and NOS. No TH, NPY, or VIP-positive ciliary muscle neurons were observed. In double labeling experiments, 70% of the nerve cells were in contact with nerve endings expressing SP-like and CGRP-like immunoreactivity. Seventeen to 32 NADPH-d-positive neurons were counted per millimeter of ciliary muscle circumferential width, with 923 in the entire ciliary muscle of one donor eye. CONCLUSIONS: The presence of intrinsic NOS-positive nerve cells concentrated in the inner parts of the ciliary muscle might indicate a physiological role of nitric oxide for disaccommodation or fluctuations during accommodation.

Innervation of myofibroblast-like scleral spur cells in human monkey eyes.

PURPOSE: To study the innervation of the presumably contractile, myofibroblast-like scleral spur cells in human and cynomolgus monkey eyes. METHODS: Serial tangential sections of the scleral spur region of the eyes of 16 human donors and 6 cynomolgus monkeys were investigated with immunocytochemical methods. Antibodies against acetylcholinesterase, synaptophysin, alpha-smooth muscle actin, calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY), nitric oxide synthase (NOS), substance P (SP), tyrosine hydroxylase (TH), and vasoactive intestinal peptide (VIP) were used. In addition, sections were processed for glyoxylic acid-induced catecholamine fluorescence (CF) and for NADPH-diaphorase (NADPH-d). RESULTS: In the eyes of both species, circumferentially oriented varicose axons were observed in the scleral spur region of all quadrants. Double labeling showed that most of these scleral spur axons were in close contact with the alpha-smooth muscle actin-positive, myofibroblast-like scleral spur cells. In human eyes, the axons showed like-immunoreactivity (LI) for SP, CGRP, NPY, VIP, and NOS. In addition, numerous scleral spur axons stained for NADPH-d. Most SP-LI scleral spur axons were double-labeled for CGRP-LI, and none for VIP-LI. All NPY-LI scleral spur axons were double labeled for VIP-LI but lacked immunoreactivity to TH. Some VIP-LI axons were not labeled for NPY-LI. Nerve fibers immunoreactive (IR) for TH or positively stained for CF were not observed in association with scleral spur cells. In contrast, in cynomolgus monkey eyes, circumferentially oriented TH-IR and CF-positive varicose axons were observed frequently in the scleral spur region. In addition, SP-LI, CGRP-LI, and NPY-LI/TH-IR axons were present in the chamber angle of monkey eyes, whereas VIP-LI, VIP-LI/NPY-LI, NOS-positive, or NADPH-d-positive nerve fibers were absent. In both species, positive staining for acetylcholinesterase was seen only in the ciliary muscle, not in the scleral spur region. CONCLUSIONS: The close association of varicose axons with the myofibroblast-like scleral spur cells indicates that nervous signals modulate scleral spur cell tone. A sympathetic scleral spur cell innervation is present only in cynomolgus monkeys but seems to be absent in humans. Conversely, scleral spur axons of presumably parasympathetic origin (NOS-IR or NADPH-d-positive, VIP-LI, and VIP-LI/NPY-LI) are absent in the cynomolgus monkeys but present in humans. In both species, a cholinergic innervation of the scleral spur cells seems to be rare or absent.

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.

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.

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.

Immunohistochemical localization of neuropeptides in the human ciliary ganglion.

In human ciliary ganglia, 18% of neurons were in contact with substance P (SP) and 12% with calcitonin gene-related peptide (CGRP) like-immunoreactive (LI) varicose axons. CGRP was colocalized with SP. Numerous SP-LI and CGRP-LI non-varicose nerve fibers were found between the ganglion cells and in nerve trunks that entered the ganglia. Axons immunoreactive for neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), tyrosine hydroxylase (TH) or dopamine-beta-hydroxylase (DBH) never contacted neuronal cell bodies. Perikarya of ciliary neurons neither stained for any of the neuropeptides nor for DBH. 23% of ciliary perikarya were TH-immunoreactive. These observations suggest an innervation of human ciliary ganglion neurons by peptidergic primary afferent collaterals presumably of trigeminal origin.

Characterization of Meibomian gland innervation in the cynomolgus monkey (Macaca fascicularis).

To characterize the innervation of the cynomolgus monkey (Macaca fascicularis) Meibomian (tarsal) glands, upper lids of six cynomolgus monkeys were investigated with electronmicroscopical and double-labeling immunocytochemical methods. Antibodies against calcitonin gene-related peptide (CGRP), dopamine-beta-hydroxylase (DBH), neuropeptide Y (NPY), nitric oxide synthase (NOS), protein gene product 9.5 (PGP 9.5), substance P (SP), tyrosine hydroxylase (TH), and vasoactive intestinal peptide (VIP) were used. In addition, sections were processed for NADPH-diaphorase (NADPH-d) histochemistry. Staining for PGP 9.5 and electron microscopy showed that Meibomian gland acini were surrounded by a network of unmyelinated nerves and terminal varicose axons. The terminals contained small agranular (30-60 nm) and large granular vesicles (65-110 nm), and were observed in close contact with the basal lamina of the acini, but never internally to the basal lamina. Meibomian axons showed like-immunoreactivity (LI) for the neuropeptides SP, CGRP, NPY, and VIP. In addition, the axons stained for TH, DBH, NOS, and NADPH-d. VIP-LI, NOS- and NADPH-d-positive axons appeared to be more numerous, TH- and DBH-positive axons more rare than others. Most SP-LI axons were double-labelled for CGRP-LI, some for VIP-LI or NPY-LI. In addition, some VIP-LI axons were double-labeled for NPY-LI. NPY/VIP-LI and NPY/SP-LI axons were only observed close to the Meibomian acini. Conversely, NPY-LI colocalized with TH-IR or DBH-IR predominated in perivascular nerves of Meibomian gland vasculature. The close association of varicose axons with the acini of Meibomian glands indicates that nervous signals modulate meibomian secretion. Meibomian gland nerve fibers in the cynomolgus monkey appear to utilize various neuropeptides, catecholamines and nitric oxide as transmitter substances, and seem to derive from the pterygopalatine, superior cervical and trigeminal ganglion respectively.