Unaffected contractility of diaphragm muscle fibers in humans on mechanical ventilation.

Several studies have indicated that diaphragm dysfunction develops in patients on mechanical ventilation (MV). Here, we tested the hypothesis that the contractility of sarcomeres, i.e., the smallest contractile unit in muscle, is affected in humans on MV. To this end, we compared diaphragm muscle fibers of nine brain-dead organ donors (cases) that had been on MV for 26 ± 5 h with diaphragm muscle fibers from nine patients (controls) undergoing surgery for lung cancer that had been on MV for less than 2 h. In each diaphragm specimen we determined 1) muscle fiber cross-sectional area in cryosections by immunohistochemical methods and 2) the contractile performance of permeabilized single muscle fibers by means of maximum specific force, kinetics of cross-bridge cycling by rate of tension redevelopment, myosin heavy chain content and concentration, and calcium sensitivity of force of slow-twitch and fast-twitch muscle fibers. In case subjects, we noted no statistically significant decrease in outcomes compared with controls in slow-twitch or fast-twitch muscle fibers. These observations indicate that 26 h of MV of humans is not invariably associated with changes in the contractile performance of sarcomeres in the diaphragm.

Intrinsic apoptosis in mechanically ventilated human diaphragm: linkage to a novel Fos/FoxO1/Stat3-Bim axis.

Mechanical ventilation (MV) is a life-saving measure in many critically ill patients. However, prolonged MV results in diaphragm dysfunction that contributes to the frequent difficulty in weaning patients from the ventilator. The molecular mechanisms underlying ventilator-induced diaphragm dysfunction (VIDD) remain poorly understood. We report here that MV induces myonuclear DNA fragmentation (3-fold increase; P<0.01) and selective activation of caspase 9 (P<0.05) and Bcl2-interacting mediator of cell death (Bim; 2- to 7-fold increase; P<0.05) in human diaphragm. MV also statistically significantly down-regulates mitochondrial gene expression and induces oxidative stress. In cultured muscle cells, we show that oxidative stress activates each of the catabolic pathways thought to underlie VIDD: apoptotic (P<0.05), proteasomal (P<0.05), and autophagic (P<0.01). Further, silencing Bim expression blocks (P<0.05) oxidative stress-induced apoptosis. Overlapping the gene expression profiles of MV human diaphragm and H2O2-treated muscle cells, we identify Fos, FoxO1, and Stat3 as regulators of Bim expression as well as of expression of the catabolic markers atrogin and LC3. We thus identify a novel Fos/FoxO1/Stat3-Bim intrinsic apoptotic pathway and establish the centrality of oxidative stress in the development of VIDD. This information may help in the design of specific drugs to prevent this condition.

Increased proteolysis, myosin depletion, and atrophic AKT-FOXO signaling in human diaphragm disuse.

RATIONALE: Patients on mechanical ventilation who exhibit diaphragm inactivity for a prolonged time (case subjects) develop decreases in diaphragm force-generating capacity accompanied by diaphragm myofiber atrophy. OBJECTIVES: Our objectives were to test the hypotheses that increased proteolysis by the ubiquitin-proteasome pathway, decreases in myosin heavy chain (MyHC) levels, and atrophic AKT-FOXO signaling play major roles in eliciting these pathological changes associated with diaphragm disuse. METHODS: Biopsy specimens were obtained from the costal diaphragms of 18 case subjects before harvest (cases) and compared with intraoperative specimens from the diaphragms of 11 patients undergoing surgery for benign lesions or localized lung cancer (control subjects). Case subjects had diaphragm inactivity and underwent mechanical ventilation for 18 to 72 hours, whereas this state in controls was limited to 2 to 4 hours. MEASUREMENTS AND MAIN RESULTS: With respect to proteolysis in cytoplasm fractions, case diaphragms exhibited greater levels of ubiquitinated-protein conjugates, increased activity of the 26S proteasome, and decreased levels of MyHCs and α-actin. With respect to atrophic signaling in nuclear fractions, case diaphragms exhibited decreases in phosphorylated AKT, phosphorylated FOXO1, increased binding to consensus DNA sequence for Atrogin-1 and MuRF-1, and increased supershift of DNA-FOXO1 complexes with specific antibodies against FOXO1, as well as increased Atrogin-1 and MuRF-1 transcripts in whole myofiber lysates. CONCLUSIONS: Our findings suggest that increased activity of the ubiquitin-proteasome pathway, marked decreases in MyHCs, and atrophic AKT-FOXO signaling play important roles in eliciting the myofiber atrophy and decreases in diaphragm force generation associated with prolonged human diaphragm disuse.

Identification of the neuromuscular junction transcriptome of extraocular muscle by laser capture microdissection.

PURPOSE: To examine and characterize the profile of genes expressed at the synapses or neuromuscular junctions (NMJs) of extraocular muscles (EOMs) compared with those expressed at the tibialis anterior (TA). METHODS: Adult rat eyeballs with rectus EOMs attached and TAs were dissected, snap frozen, serially sectioned, and stained for acetylcholinesterase (AChE) to identify the NMJs. Approximately 6000 NMJs for rectus EOM (EOMsyn), 6000 NMJs for TA (TAsyn), equal amounts of NMJ-free fiber regions (EOMfib, TAfib), and underlying myonuclei and RNAs were captured by laser capture microdissection (LCM). RNA was processed for microarray-based expression profiling. Expression profiles and interaction lists were generated for genes differentially expressed at synaptic and nonsynaptic regions of EOM (EOMsyn versus EOMfib) and TA (TAsyn versus TAfib). Profiles were validated by using real-time quantitative polymerase chain reaction (qPCR). RESULTS: The regional transcriptomes associated with NMJs of EOMs and TAs were identified. Two hundred seventy-five genes were preferentially expressed in EOMsyn (compared with EOMfib), 230 in TAsyn (compared with TAfib), and 288 additional transcripts expressed in both synapses. Identified genes included novel genes as well as well-known, evolutionarily conserved synaptic markers (e.g., nicotinic acetylcholine receptor (AChR) alpha (Chrna) and epsilon (Chrne) subunits and nestin (Nes). CONCLUSIONS: Transcriptome level differences exist between EOM synaptic regions and TA synaptic regions. The definition of the synaptic transcriptome provides insight into the mechanism of formation and functioning of the unique synapses of EOM and their differential involvement in diseases noted in the EOM allotype.

Gene expression in the lamellar dermis-epidermis during the developmental phase of carbohydrate overload-induced laminitis in the horse.

OBJECTIVE: Gene expression in the lamellar dermis and epidermis was compared between healthy horses and horses in the developmental phase of carbohydrate overload-induced laminitis, in order to better understand the local biochemical and cellular events involved in the pathogenesis of laminitis. ANIMALS: Six healthy adult horses, with no history or clinical evidence of laminitis. PROCEDURES: Horses were randomly divided into two groups: control (n=3) and laminitis (n=3). Control horses received no treatment and were humanely euthanatized at the same time as the laminitis group. Horses in the laminitis group were given oligofructose (10g/kg bwt by nasogastric tube) and humanely euthanatized 24-30h later, before any clinical signs of laminitis were apparent. Sections of lamellar dermis and epidermis were harvested from the dorsal hoof wall of each horse immediately after death and cryopreserved until analysis. A bovine microarray chip, comprising approximately 15,000 genes, was used to compare gene expression between laminitis and control groups. RESULTS: A total of 155 genes were up-regulated in the laminitis group. No genes were down-regulated. Genes coding for the production of pro-inflammatory biochemical or cellular processes and those involved in protein degradation/turnover predominated. Several regulatory or anti-inflammatory genes were also up-regulated. CONCLUSIONS AND CLINICAL RELEVANCE: Generation of inflammatory mediators within the lamellar tissues occurred before the development of substantial dermal-epidermal separation, inflammatory infiltrate, or vascular changes, and before the horses began showing signs of foot pain. While further studies are needed, early and targeted anti-inflammatory therapy may halt or prevent the development of laminitis in at-risk individuals.

Influence of hyperthyroid conditions on gene expression in extraocular muscles of rats.

Extraocular muscles (EOMs) are a highly specialized type of tissue with a wide range of unique properties, including characteristic innervation, development, and structural proteins. Even though EOMs are frequently and prominently affected by thyroid-associated diseases, little is known about the direct effects of thyroid hormone on these muscles. To create a comprehensive profile of changes in gene expression levels in EOMs induced by thyroid hormone, hyperthyroid conditions were simulated by treating adult Sprague-Dawley rats with intraperitoneal injections of the thyroid hormone 3,3',5-triiodo-L-thyronine (T(3)); subsequently, microarray analysis was used to determine changes in mRNA levels in EOMs from T(3)-treated animals relative to untreated control animals. The expression of 468 transcripts was found to be significantly altered, with 466 of these transcripts downregulated in EOMs from T(3)-treated animals. The biological processes into which the affected genes could be grouped included cellular metabolism, transport, biosynthesis, protein localization, and cell homeostasis. Moreover, 15 distinct biochemical canonical pathways were represented among the genes with altered transcription levels. Strikingly, myostatin (Gdf8), a potent negative regulator of muscle growth, was found to be strongly downregulated in EOMs from T(3)-treated animals. Together, these findings suggest that pathological concentrations of thyroid hormone have a unique effect on gene expression in EOMs, which is likely to play a hitherto neglected role in thyroid-associated ophthalmopathies.

Transcriptional and functional differences in stem cell populations isolated from extraocular and limb muscles.

The extraocular muscles (EOMs) are a distinct muscle group that displays an array of unique contractile, structural, and regenerative properties. They also have differential sensitivity to certain diseases and are enigmatically spared in Duchenne muscular dystrophy (DMD). The EOMs are so distinct from other skeletal muscles that the term "allotype" has been coined to highlight EOM group-specific properties. We hypothesized that increased and distinct stem cells may underlie the continual myogenesis noted in EOM. The side population (SP) stem cells were isolated and studied. EOMs had 15x higher SP cell content compared with limb muscles. Expression profiling revealed 348 transcripts that define the EOM-SP transcriptome. Over 92% of transcripts were SP specific, because they were absent in previous whole muscle microarray studies. Cultured EOM-SP cells revealed superior in vitro proliferative capacity. Finally, assays of the committed progenitors or satellite cells performed on myofibers isolated from EOM and limb muscles independently validated the increased proliferative capacity of these muscles. We suggest a model in which unique EOM stem cells contribute to the continual myogenesis noted in EOM and consistent with a role for their sparing in DMD. We believe the greater numbers of stem cells, their unique transcriptome, the greater proliferative capacity of EOM stem cells, and the greater number of satellite cells also offer clues for novel cell-based therapeutic strategies.

Rapid disuse atrophy of diaphragm fibers in mechanically ventilated humans.

BACKGROUND: The combination of complete diaphragm inactivity and mechanical ventilation (for more than 18 hours) elicits disuse atrophy of myofibers in animals. We hypothesized that the same may also occur in the human diaphragm. METHODS: We obtained biopsy specimens from the costal diaphragms of 14 brain-dead organ donors before organ harvest (case subjects) and compared them with intraoperative biopsy specimens from the diaphragms of 8 patients who were undergoing surgery for either benign lesions or localized lung cancer (control subjects). Case subjects had diaphragmatic inactivity and underwent mechanical ventilation for 18 to 69 hours; among control subjects diaphragmatic inactivity and mechanical ventilation were limited to 2 to 3 hours. We carried out histologic, biochemical, and gene-expression studies on these specimens. RESULTS: As compared with diaphragm-biopsy specimens from controls, specimens from case subjects showed decreased cross-sectional areas of slow-twitch and fast-twitch fibers of 57% (P=0.001) and 53% (P=0.01), respectively, decreased glutathione concentration of 23% (P=0.01), increased active caspase-3 expression of 100% (P=0.05), a 200% higher ratio of atrogin-1 messenger RNA (mRNA) transcripts to MBD4 (a housekeeping gene) (P=0.002), and a 590% higher ratio of MuRF-1 mRNA transcripts to MBD4 (P=0.001). CONCLUSIONS: The combination of 18 to 69 hours of complete diaphragmatic inactivity and mechanical ventilation results in marked atrophy of human diaphragm myofibers. These findings are consistent with increased diaphragmatic proteolysis during inactivity.

Form-deprivation myopia in chick induces limited changes in retinal gene expression.

PURPOSE: Evidence has implicated the retina as a principal controller of refractive development. In the present study, the retinal transcriptome was analyzed to identify alterations in gene expression and potential signaling pathways involved in form-deprivation myopia of the chick. METHODS: One-week-old white Leghorn chicks wore a unilateral image-degrading goggle for 6 hours or 3 days (n = 6 at each time). Total RNA from the retina/(retinal pigment epithelium) was used for expression profiling with chicken gene microarrays (Chicken GeneChips; Affymetrix, Santa Clara, CA). To identify gene expression level differences between goggled and contralateral nongoggled eyes, normalized microarray signal intensities were analyzed by the significance analysis of microarrays (SAM) approach. Differentially expressed genes were validated by real-time quantitative reverse transcription-polymerase chain reaction (qPCR) in independent biological replicates. RESULTS: Small changes were detected in differentially expressed genes in form-deprived eyes. In chickens that had 6 hours of goggle wear, downregulation of bone morphogenetic protein 2 and connective tissue growth factor was validated. In those with 3 days of goggle wear, downregulation of bone morphogenetic protein 2, vasoactive intestinal peptide, preopro-urotensin II-related peptide and mitogen-activated protein kinase phosphatase 2 was validated, and upregulation of endothelin receptor type B and interleukin-18 was validated. CONCLUSIONS: Form-deprivation myopia, in its early stages, is associated with only minimal changes in retinal gene expression at the level of the transcriptome. While the list of validated genes is short, each merits further study for potential involvement in the signaling cascade mediating myopia development.

Ets-2 repressor factor silences extrasynaptic utrophin by N-box mediated repression in skeletal muscle.

Utrophin is the autosomal homologue of dystrophin, the protein product of the Duchenne's muscular dystrophy (DMD) locus. Utrophin expression is temporally and spatially regulated being developmentally down-regulated perinatally and enriched at neuromuscular junctions (NMJs) in adult muscle. Synaptic localization of utrophin occurs in part by heregulin-mediated extracellular signal-regulated kinase (ERK)-phosphorylation, leading to binding of GABPalpha/beta to the N-box/EBS and activation of the major utrophin promoter-A expressed in myofibers. However, molecular mechanisms contributing to concurrent extrasynaptic silencing that must occur to achieve NMJ localization are unknown. We demonstrate that the Ets-2 repressor factor (ERF) represses extrasynaptic utrophin-A in muscle. Gel shift and chromatin immunoprecipitation studies demonstrated physical association of ERF with the utrophin-A promoter N-box/EBS site. ERF overexpression repressed utrophin-A promoter activity; conversely, small interfering RNA-mediated ERF knockdown enhanced promoter activity as well as endogenous utrophin mRNA levels in cultured muscle cells in vitro. Laser-capture microscopy of tibialis anterior NMJ and extrasynaptic transcriptomes and gene transfer studies provide spatial and direct evidence, respectively, for ERF-mediated utrophin repression in vivo. Together, these studies suggest "repressing repressors" as a potential strategy for achieving utrophin up-regulation in DMD, and they provide a model for utrophin-A regulation in muscle.

Comparative analysis of human conjunctival and corneal epithelial gene expression with oligonucleotide microarrays.

PURPOSE: To determine global mRNA expression levels in corneal and conjunctival epithelia and identify transcripts that exhibit preferential tissue expression. METHODS: cDNA samples derived from human conjunctival and corneal epithelia were hybridized in three independent experiments to a commercial oligonucleotide array representing more than 22,000 transcripts. The resultant signal intensities and microarray software transcript present/absent calls were used in conjunction with the local pooled error (LPE) statistical method to identify transcripts that are preferentially or exclusively expressed in one of the two tissues at significant levels (expression >1% of the beta-actin level). EASE (Expression Analysis Systematic Explorer software) was used to identify biological systems comparatively overrepresented in either epithelium. Immuno-, and cytohistochemistry was performed to validate or expand on selected results of interest. RESULTS: The analysis identified 332 preferential and 93 exclusive significant corneal epithelial transcripts. The corresponding numbers of conjunctival epithelium transcripts were 592 and 211, respectively. The overrepresented biological processes in the cornea were related to cell adhesion and oxiredox equilibria and cytoprotection activities. In the conjunctiva, the biological processes that were most prominent were related to innate immunity and melanogenesis. Immunohistochemistry for antigen-presenting cells and melanocytes was consistent with these gene signatures. The transcript comparison identified a substantial number of genes that have either not been identified previously or are not known to be highly expressed in these two epithelia, including testican-1, ECM1, formin, CRTAC1, and NQO1 in the cornea and, in the conjunctiva, sPLA(2)-IIA, lipocalin 2, IGFBP3, multiple MCH class II proteins, and the Na-Pi cotransporter type IIb. CONCLUSIONS: Comparative gene expression profiling leads to the identification of many biological processes and previously unknown genes that are potentially active in the function of corneal and conjunctival epithelia.

Identification of the A3 adenosine receptor in rat retinal ganglion cells.

PURPOSE: Adenosine can protect retinal ganglion cells from the death that accompanies a general ischemic challenge as well as excitotoxic death. In other tissues, both A1 and A3 adenosine receptor subtypes can mediate protection. While a role for the A1 adenosine receptor in ganglion cell protection has been established, a potential for the A3 receptor has only recently been proposed. Although the pharmacology is promising, the molecular identity of the responsible receptor is unclear as previous studies were unable to detect message for the A3 receptor in retinal ganglion cells. We combined laser capture microdisection (LCM) and immunopurification with traditional and real-time PCR to unequivocally demonstrate the presence of the A3 receptor message in rat retinal ganglion cells. METHODS: Retinal ganglion cells of Long-Evans rat pups were retrograde labeled with aminostilbamidine. Eyeballs were enucleated, embedded, frozen, sectioned, and fluorescent cells in the ganglion cell layer were collected with LCM. Purified ganglion cells were also isolated with a two-step panning procedure. cDNA for the A3 receptor obtained from the microdissected ganglion cell layer, immunopurified ganglion cells, whole retina and testis was amplified using RT-PCR, confirmed by DNA sequencing and compared with published sequences. A3 receptor message was also amplified using real-time PCR. Ca2+ levels in immunopanned ganglion cells were measured ratiometrically with fura-2. RESULTS: RNA from immunopurified ganglion cells and from dye-loaded cells in the ganglion cell layer contained message for the A3 receptor when amplified with either traditional RT-PCR or real-time PCR. The entire encoding region was sequenced and found to be 99% identical to the published code. The sequence closely resembled the consensus form of the gene, with other sequences deviating from this default code. Molecular identification was functionally confirmed in purified ganglion cells as the A3 receptor agonist Cl-IB-MECA prevented the excessive Ca2+ rise triggered by P2X7 agonist BzATP. CONCLUSIONS: Retinal ganglion cells express A3 adenosine receptor mRNA. Stimulation of this receptor can reduce the Ca2+ overload following excessive activation of P2X7 receptors.

Ocular surface epithelia contain ABCG2-dependent side population cells exhibiting features associated with stem cells.

When cell populations are incubated with the DNA-binding dye Hoechst 33342 and subjected to flow cytometry analysis for Hoechst 33342 emissions, active efflux of the dye by the ABCG2/BCRP1 transporter causes certain cells to appear as a segregated cohort, known as a side population (SP). Stem cells from several tissues have been shown to possess the SP phenotype. As the lack of specific surface markers has hindered the isolation and subsequent biochemical characterization of epithelial stem cells this study sought to determine the existence of SP cells and expression of ABCG2 in the epithelia of the ocular surface and evaluate whether such SP cells had features associated with epithelial stem cells. Human and rabbit limbal-corneal and conjunctival epithelial cells were incubated with Hoechst 33342, and analyzed and sorted by flow cytometry. Sorted cells were subjected to several tests to determine whether the isolated SP cells displayed features consistent with the stem cell phenotype. Side populations amounting to <1% of total cells, which were sensitive to the ABCG2-inhibitor fumitremorgin C, were found in the conjunctival and limbal epithelia, but were absent from the stem cell-free corneal epithelium. Immunohistochemistry was used to establish the spatial expression pattern of ABCG2. The antigen was detected in clusters of conjunctival and limbal epithelia basal cells but was not present in the corneal epithelium. SP cells were characterized by extremely low light side scattering and contained a high percentage of cells that: showed slow cycling prior to tissue collection; exhibited an initial delay in proliferation after culturing; and displayed clonogenic capacity and resistance to phorbol-induced differentiation; all features that are consistent with a stem cell phenotype.

Definition of the unique human extraocular muscle allotype by expression profiling.

The extraocular muscles (EOMs) are a unique group of specialized muscles that are anatomically and physiologically distinct from other skeletal muscles. Perhaps the most striking characteristic of the EOMs is their differential sensitivity to disease. EOMs are spared in Duchenne's muscular dystrophy (DMD) despite widespread involvement of other skeletal muscles. Conversely, they are early and prominent targets in myasthenia gravis and mitochondrial myopathies. It is unclear how EOMs achieve such specialization or a differential response to diseases; however, this has been attributed to a unique, group-specific pattern of gene expression or "allotype." To begin to address these issues as well as define the human EOM allotype, we analyzed the human EOM transcriptome using oligonucleotide-based expression profiling. Three hundred thirty-eight genes were found to be differentially expressed in EOM compared with quadriceps femoris limb muscle, using a twofold cutoff. Functional characterization revealed expression patterns corresponding to known metabolic and structural properties of EOMs such as expression of EOM-specific myosin heavy chain (MYH13) and high neural, vascular, and mitochondrial content, suggesting that the profiling was sensitive and specific. Genes related to myogenesis, stem cells, and apoptosis were detected at high levels in normal human EOMs, suggesting that efficient and continuous regeneration and/or myogenesis may be a mechanism by which the EOMs remain clinically and pathologically spared in diseases such as DMD. Taken together, this study provides insight into how human EOMs achieve their unique structural, metabolic, and pathophysiological properties.

Ocular surface epithelial and stem cell development.

Phenotypic features and developmental events involved in the genesis of the limbo-corneal and conjunctival epithelia are described. Together, these two epithelia define the ocular surface. They derive from a small cohort of optic vesicle-induced PAX6+ head ectodermal cells that remain on the surface following lens vesicle formation by the main PAX6+ cell cohort. Both epithelia are stratified, and display wet, non-keratinizing phenotypes. The most significant spatial feature of the limbo-corneal epithelium is the segregation of its supporting stem and early precursor cells to the limbus, the outer vascularized rim separating the cornea from the conjunctiva. These stem cells express ABCG2, a xenobiotic transporter present in stem cells from other organs. ABCG2 transport activity excludes the DNA dye Hoechst 33342, allowing the isolation of the ocular stem cells by flow cytometry, as a unique cohort known as a side 'side population'. Limbal stem cells do not form gap junctions and exist as metabolically isolated entities. Tracking of expression changes in Cx43, the main gap junction protein expressed in both the pre-epithelial ectoderm and in the mature central corneal epithelium, indicates that a limbal stem cell phenotype starts developing very soon after lens vesicle invagination, in advance of the appearance of any recognizable anatomical sub-epithelial limbal feature. Differences in Cx43 expression also reveal the very early nature of the divergence in limbo-corneal and conjunctival lineages. The putative involvement of several early genes, including gradients of PAX6 and differences in expression patterns for members of the Id or msh gene expression regulators are reviewed.

Layer-specific differences of gene expression in extraocular muscles identified by laser-capture microscopy.

In mammals, separate muscles are typically specialized as a whole to provide distinct functional roles leading to well-recognized adaptations. This is exemplified in the lower limb by the slow, fatigue-resistant soleus, which provides a postural role vs. the fast, fatiguable tibialis anterior (TA), which provides rapid movements. A unique characteristic of extraocular muscles (EOMs) is their compartmentalization into two distinct layers, the orbital layer (OL) and global layer (GL), presumably to subserve diverse functions within the same muscle. However, molecular evidence of this diversity has been limited. We used laser-capture microscopy coupled with microarray-based expression profiling to identify molecular differences between the OL and GL of rat EOMs. We found that 210 genes were differentially regulated between these layers at a twofold expression cutoff. Differences in genes related to metabolic pathways and related to structural elements of muscle and nerve formed the largest functional clusters. Layer-specific differential expression was validated at both mRNA and protein level for MYH3, MYH6, and ACTN3. The expected layer-specific differences among genes encoding vascular elements were not evident by profiling; morphometric analysis demonstrated that the differences exist, but at a magnitude below the cutoff level established by our statistical methods. Comparison of these results with previous results comparing whole EOMs and TA suggest evolutionary mechanisms may play a role in achieving functional distinctions between OL and GL.

Different cell sizes in human limbal and central corneal basal epithelia measured by confocal microscopy and flow cytometry.

PURPOSE: In the epidermis, the highest clonogenicity, a feature of stem cells (SCs), is found in the smallest keratinocyte. In the limbal-corneal (LC) epithelium the SCs are exclusively localized in the basal epithelial layer of the limbal domain. The current study was conducted to determine whether this spatial SC arrangement is reflected in differences in the cell size between limbal and corneal cells. METHODS: In vivo confocal microscopy was used to scan and measure the size of the cells of the central cornea and the superior limbus in five normal subjects, from the superficial to the basal cell layer. Limbal and corneal pure epithelial sheets were isolated by dispase digestion from human tissues and dissociated into single cells by trypsin digestion. The forward (FSC; a relative measure of cell size) and side (SSC; a relative measure of cytoplasmic complexity) light-scattering properties of these cells were determined by flow cytometry. RESULTS: Confocal microscopy showed that diameters of the basal cells of the limbal and corneal zones were 10.1 +/- 0.8 and 17.1 +/- 0.8 micro m, respectively. The corresponding values for the superficial layers were 19.9 +/- 1.6 and 36.6 +/- 1.6 microm, respectively (P < 0.0001). The mean FSC and SSC of the limbal cells amounted to 65.7% +/- 8.7% and of the corneal cells, 74.4% +/- 4.6%. Furthermore, only 1.40% +/- 0.83% and 0.69% +/- 0.37% of the corneal cells had FSC and SSC equal to the lowest 15% of FSC and SCC of the limbal cells, respectively, indicating that the limbus contained a substantial proportion of very low FSC and SSC cells for which there was no corneal counterpart. CONCLUSIONS: The data collectively demonstrate that the smallest cells are located in the limbal basal epithelium. This feature may help isolate corneal SCs located in the limbus.

Changes in connexin43 in early ocular surface development.

PURPOSE: In the limbo-corneal epithelium the stem and early precursor epithelial cell pool is confined to the limbal rim. Among the features associated with this spatial segregation is the general paucity of connexin43 (Cx43) within the limbal basal cell population and its complete absence in resident stem cells. The limbo-corneal epithelial lineage derives from a Cx43-positive (Cx43+) embryonic outer ectoderm. Accordingly, as a means of identifying the process through which limbal cell phenotypes emerge, we investigated the expression of Cx43 in the ocular surface of embryonic rats. METHODS: Ocular surface expression of Cx43 or K12 was determined in cryostat sections of rat embryos and eyes using immunohistological methods. RESULTS: Changes in Cx43 expression revealed the early phenotypic divergence of three main epithelial cell phenotypes of the ocular surface. An analysis of the level and distribution pattern of Cx43 puncta lead to the identification of two distinct domains by embryonic day 10 (E10), a stage that occurs soon after formation of the lens vesicle. Additionally, at E12, ectodermal cells directly adjacent to the edges of the developing retina no longer express connexin. A comparison of anatomical and expression changes throughout embryonic development demonstrated that the two early zones represent the rudiments for the epithelia of the central cornea and conjunctiva, respectively, and that the isolated Cx43-negative (Cx43-) cells represent the precursors of the basal and, putatively, stem cells of the limbal epithelium. CONCLUSIONS: Changes in Cx43 expression revealed that the phenotypic divergence of ocular surface epithelial cells and the generation of limbo-corneal stem cell precursors takes place at a very early stage in ocular development, ahead of the establishment of any identifiable anatomical or differentiation features for these domains.