Digital Light Microscopy Research Articles

Sexual Reproduction in Precious Corals (Coralliidae) Collected in the Ryukyu Archipelago

Little is known about the basic life history of most species of precious corals (Octocorallia: Coralliidae). Three commercially valuable precious coral species (Paracorallium japonicum, Corallium elatius, and C. konojoi) were sampled from the Ryukyu Archipelago to study their reproductive biology. To determine features of their gamete differentiation, samples were thin-sectioned for examination with a digital light microscope, and diameters of sperm sacs and oocytes were measured. Sexual reproduction strategy in all three species was determined to be gonochoristic broadcast spawning. Almost all gonads were found to be differentiating in siphonozooids, not in autozooids. Sex ratio of C. konojoi was around 1 : 1; that of P. japonicum and C. elatius seemed biased toward females but did not deviate significantly from 1 : 1. Number of gonads per polyp in these three species was fewer and they were generally smaller in diameter in comparison with other octocorals reported elsewhere, suggesting comparatively low fecundity for these species of Coralliidae from southern Japan. Spawning of these three species appears to occur during summer, mostly from May to August.

Effect of acidic environment on the push-out bond strength of calcium-enriched mixture cement.

This laboratory study was performed to evaluate the effect of different acidic pH values on the push-out bond strength of calcium-enriched mixture (CEM) cement. Forty-eight root dentin slices were obtained from freshly extracted single rooted human teeth and their lumen were instrumented to achieve a diameter of 1.3 mm. Then, CEM cement was mixed according to manufacturers' instruction and placed in the lumens with minimal pressure. The specimens were randomly divided into four groups (n=12) which were wrapped in pieces of gauze soaked in either synthetic tissue fluid (STF) (pH=7.4) or butyric acid which was buffered at pH values of 4.4, 5.4 and 6.4. They were then incubated for 4 days at 37°C. The push-out test was performed by means of the universal testing machine. Specimens were then examined under a digital light microscope at 20× magnification to determine the nature of the bond failure. The data were analyzed using the Kruskal-Wallis test followed by Dunn's test for pairwise comparisons. The highest push-out bond strength (10.19±4.39) was seen in the pH level of 6.4, which was significantly different from the other groups (P<0.05). The values decreased to 2.42±2.25 MPa after exposure to pH value of 4.4. Lower pH value of highly acidic environments (pH=4.4), adversely affects the force needed for displacement of CEM cement; while in higher pH values (pH=6.4) the bond-strength was not affected. CEM cement is recommended in clinical situations where exposure to acidic environment is unavoidable.

Concordance between digital pathology and light microscopy in general surgical pathology: a pilot study of 100 cases

Aim(1) A pilot study to determine the accuracy of interpretation of whole slide digital images in a broad range of general histopathology cases of graded complexity. (2) To survey the...

Digital stereology in neuropathology

Two-dimensional quantitative methods have frequently been used to address questions in neuropathological research; however, they face important limitations which design-based stereology may overcome by offering a set of methods to quantify two-dimensional histological sections into three-dimensional structural knowledge. Accordingly, stereology is a science based on statistical sampling principles and geometric measures. The application of stereology to neuropathological studies allows the researcher to efficiently obtain a precise estimate of various structural quantities. This neuropathological review will therefore present the relevant stereological estimators for obtaining reliable quantitative structural data from brains and peripheral nerves when using digital light microscopy. It is discussed how to obtain brain and nerve fibre samples to fulfil the requirements for the estimators. A presentation of design-based stereological probes for obtaining global volume, total number, local volume, total length, total surface area, cross-sectional area and diameter will be followed by discussion of the error variances of the methods. No mathematical equations or calculations are shown in this review.

Histological Examination of Precious Corals from the Ryukyu Archipelago

In this paper we examined the histology of three commercially valuable species of precious corals (Paracorallium japonicum,Corallium elatius,andC. konojoi) from the Ryukyu Archipelago. In order to observe their inner structure, samples were thin sectioned and examined with a digital light microscope. Colonies ofC. konojoihad thicker coenenchyme and larger autozooids than those ofC. elatiusandP. japonicum. The sclerites of the three species tended to be concentrated in the outer layers of coenenchyme. The gastric cavities of autozooids of all three species were found to be relatively empty. Some symbiotic polychates were observed in the axis ofP. japonicum. As well, a zoanthid (Corallizoanthus tsukaharai) was often observed living on the coenenchyme surface ofP. japonicum. It is hoped our observations will provide a good foundation of future study of Japanese Coralliidae corals.

Cellulolytic environment in the midgut of the wood-feeding higher termite Nasutitermes takasagoensis

Unlike lower termites, xylophagous higher termites thrive on wood without the aid of symbiotic protists. In the higher termite Nasutitermes takasagoensis, both endogenous endo-β-1,4-glucanase and β-glucosidase genes are expressed in the midgut, which is believed to be the main site of cellulose digestion. To further explore the detailed cellulolytic system in the midgut of N. takasagoensis, we performed immunohistochemistry and digital light microscopy to determine distributions of cellulolytic enzymes in the salivary glands and the midgut as well as the total cellulolytic activity in the midgut. Although cellulolytic enzymes were uniformly produced in the midgut epithelium, the concentration of endo-β-1,4-glucanase activity and luminal volume in the midgut were comparable to those of the wood-feeding lower termite Coptotermes formosanus, which digests cellulose with the aid of hindgut protists. However, the size of ingested wood particles was considerably larger in N. takasagoensis than that in C. formosanus. Nevertheless, it is possible that the cellulolytic system in the midgut of N. takasagoensis hydrolyzes highly crystalline cellulose to a certain extent. The glucose produced did not accumulate in the midgut lumen. Therefore, the present study suggests that the midgut of the higher termite provides the necessary conditions for cellulolysis.

Preparation and Properties of Biodegradable Foams

Foam extrusion of biodegradable polyester [poly(butylene adipate-co-terephthalate) (PBAT)] and its blends with maleated thermoplastic starch (MTPS) using a chemical blowing agent was performed. The effect of MTPS and percentage of chemical blowing agent on various foam properties is discussed. In general, an increased amount of PBAT in the foams improves the properties of the foams. The foam samples were characterized by measurements of density, expansion ratio, specific length, compressive strength, resiliency, moisture sorption, and imaging using digital light microscopy. Density, expansion ratio, and specific length measurements show that the best characteristics of lowest density, highest expansion ratio, and highest specific length are exhibited by the PBAT samples. The compressive strength and foam density exhibit a power-law relationship. Greater amounts of PBAT in the samples increase the resiliency and decrease the steady state weight gain during moisture sorption. All samples show regions of unfoamed material when only 3% chemical blowing agent is used, but when 5 and 7% chemical blowing agent is used, the samples exhibit cells throughout the matrix.

The fine structure of human germ layers in vivo: clues to the early differentiation of embryonic stem cells in vitro

The fine structure of the three germ layers in human ectopic embryos (stage 7) have been documented by digital light and electron microscopy. The formation of ectoderm, endoderm and mesoderm and notochordal cells, and also the extraembryonic membranes, amnion and yolk sac, are imaged. The germ layers give rise to all the cells and tissues of the human body. Possible clues to the early differentiation of embryonic stem cells (ESC) in vitro were obtained, since these events are more or less mimicked in cultures of ESC derived from the inner cell mass of human blastocysts. The findings are discussed with reference to previous studies on the fine structure of ESC using the same technique. The fine structure of the three germ layers of the week three human embryo has been documented. These germ layers give rise to all the cells and tissues of the human body. They are the blueprints from which embryonic stem cells are formed later on in development and would be useful to those who are involved in stem cell biotechnology.

Surface investigation on prototype cavities for the European X-ray Free Electron Laser

The accelerating gradient Eacc of X-ray Free Electron Laser (XFEL) prototype cavities manufactured at the industry and treated at DESY demonstrates wide-range scattering from 15 to $41\text{ }\text{ }\mathrm{MV}/\mathrm{m}$. Most cavities satisfy the XFEL specification. Few cavities with low performance ($15--17\text{ }\text{ }\mathrm{MV}/\mathrm{m}$) are limited by quench without field emission. The T-map analysis detected quench areas mainly close to the equator. Optical control by a high resolution camera has been applied and allowed to monitor the defects in some cases with good correlation to T-map observation. In order to understand the cause of reduced performance and get more detailed information of the origin of defects, some samples have been extracted from two cavities and investigated by light microscope, digital light microscope with 3D profile measurement, scanning electron microscope SEM, energy dispersive x-ray analysis, and Auger spectroscopy. The electron backscattered diffraction method in a SEM is used to make localized measurements of the lattice curvature. Several surface flaws with sizes from a few $\ensuremath{\mu}\mathrm{m}$ to hundreds of $\ensuremath{\mu}\mathrm{m}$ detected by microscopy. The defects can be separated into two categories. The first category of defects consists of foreign elements (often an increased content of carbon). Inclusions with increased content of carbon adhere on the surface and presumably have a hydrocarbon nature. Deviation from a smooth surface profile characterizes the second type of defects (holes, bumps, and pits). Some holes and bumps were found directly in the welding seam. The hot spots in the heat-affected zone (HAZ) of the equator welds have been partially associated with pits too. The study correlates the location of pits with the presence of plastic strain found to remain after welding. Pits near the HAZ were found either coincident with or near areas of high strain. Pits away from the weld were often found at grain boundary triple junctions.

Middle ear of human and pig: a comparison of structures and mechanics

Animal studies are an essential method to evaluate implants for the middle ear prior to clinical studies. New Zealand rabbits, guinea pigs and chinchillas are, among other small mammals, well established animal models, but their auditory system is significantly smaller compared to human. Hence, the suitability of the domestic pig (sus scrofa domesticus) as a new animal model for research on the middle ear (ME), that would match the human in size, was investigated. Thirty halved pig heads were obtained from the butcher and each middle ear was dissected. Using a digital light microscope, several anatomical magnitudes were determined for 24 specimens, namely the planar projected area of the tympanic membrane (TM), in relation to the stapes footplate as well as the dimensions and weight of the ossicles, in order to determine the effective lever ratios. Using normal and micro computed tomography (CT), six porcine temporal bones were scanned and the geometric data obtained were transferred into a finite element model (FEM) simulation of the porcine middle ear. The transfer function was determined and compared to those from humans determined by measurements and simulations, respectively. The anatomy investigated presented itself as highly comparable to that of the human. Differing from literature, no fourth ossicle could be found. The porcine dimensions and lever ratios determined match the humans far better than those of all established animal models. The obtained transfer function was congruent to the human one. This underlines the suitability of the pig as an animal model for middle ear implants.

Preliminary Studies of a Stearylamine-Based Cationic Liposome

Cationic liposomes are the leading drug delivery systems and are gaining increasing importance in gene therapy as an alternative to recombinant viruses. This article is a preliminary work on the characterization and in vitro study of a stearylamine-based cationic liposome. Multilamellar vesicles were prepared using the lipid film hydration technique. Vesicle morphology was evaluated by digital light microscopy. Particle size, zeta potential and polydispersity were determined using photon correlation spectroscopy. Susceptibility of Staphylococcus aureus to amoxicillin encapsulated in stearylamine-based cationic liposome was compared to 1,2-dioleoyl-3-trimethylammonium propane (DOTAP)-based cationic liposome and the marketed drug. The vesicles of the stearylamine based cationic liposome were spherical and concentric with a core. Particle size distribution had a peak at 94 nm, zeta potential was 34 mV and polydispersity was greater than 0.3. The clinical isolate of Staphylococcus aureus was most susceptible to stearylaminecationic liposome with an inhibitory zone diameter (IZD) of 25.00 mm+0.58>DOTAP-based cationic liposome with IZD of 24.67mm+0.67>tablet amoxicillin with IZD of 23.00 mm+0.58.

Digital Light Microscopy of Macroporous Ceramics Processed by Consolidation Casting with Mixtures of Native Starches

Abstract Porosity in starch consolidation casting technique is rightly related to original size and morphology of starch granules, leaving a pore structure after burning out. This work reports the results for the addition of different native potato and corn starch proportions in suspensions with TiO2 (rutile) powder. Gelling temperature have been defined after observation under light microscopy using a heating stage. Analysis of porous network and isolated pores have been done from images of samples surfaces obtained by depth from focus reconstruction, revealing a qualitative correlation of pores characteristics and starches additions in suspensions, suggesting that the presence of isolated or interconnected pores can be handled by starches selection to control the amylopectin and amylose contents in slurries. Also, the analysis of porous fraction distribution shows no consistent pattern through specimens’ volume according to starches in mixtures.

Connexin43 antisense oligodeoxynucleotide treatment down-regulates the inflammatory response in an in vitro interphase organotypic culture model of optic nerve ischaemia

Using a model of optic nerve ischaemia, this study investigated oxygen-glucose deprivation (OGD) on isolated rat optic nerve segments cultured in vitro. Thereafter, the effect of antisense oligodeoxynucleotides (ASODN) specific to the gap junction protein connexin43 (Cx43) was evaluated in this same model. Following exposure to OGD for 2 hours, optic nerves were maintained in interphase organotypic culture with and without exposure to Cx43 ASODN. Optic nerves were sectioned at 2 hours, 6 hours, and at days 1, 2, 3 and 6 following culture. Cell death was quantified using propidium iodide (PI) staining and specific markers for Cx43, capillaries (von Willebrand factor), astrocytes (glial fibrillary acidic protein), microglia and endothelial cells (isolectin B4) were used to evaluate these parameters in conjunction with digital light and confocal microscopy. In this model, up-regulation of Cx43 was seen at 2 hours following exposure of the optic nerve to OGD and peaked at day 3. Cx43 ASODN treatment dampened this up-regulation. Additionally, more PI labeled cells were found in the centre of control optic nerve segments than in treated nerves ( p < 0.01). Controls also showed evidence of capillary breakdown and increased numbers of astrocytes and activated microglia compared to Cx43 ASODN treated nerves ( p < 0.05). Thus, the application of Cx43 ASODN to post-ischaemic optic nerve segments significantly reduced the up-regulation of Cx43 and, subsequently, the spread of injury and a resultant inflammatory response. Cx43 up-regulation may play an important role in optic nerve injury, offering a potential avenue for treatment in optic neuropathy.

A comparative study of microstructural development in paired human hepatic and gallbladder biles

Cholesterol gallstones usually develop in the gallbladder and rarely form in bile ducts even in patients with highly lithogenic bile. Bile concentration and proteins (e.g. mucin) may affect crystallization, but the exact nature of this effect, especially in relation to crystallization pathways and microstructural evolution remains unclear. We examined lipid microstructures in paired hepatic and gallbladder biles to reveal ones that are essential for crystallization. Combining digital light microscopy with cryogenic-temperature transmission electron microscopy we are able to directly visualize and compare the time evolution of lipid microstructures in paired hepatic, gallbladder and diluted gallbladder biles of gallstone patients and controls, without drying or separating. Gallbladder bile exhibited several multilamellar vesicles and spheroidal micelles preceding and throughout crystallization. Vesicle morphology changed before crystallization was observed. In contrast, hepatic bile revealed almost no crystallization and while a variety of unilamellar vesicles and spheroidal micelles existed throughout the examination, multilamellar vesicles were rare. Diluted gallbladder bile was different from native gallbladder bile, as well as the paired hepatic bile, yielding occasional crystallization. Our findings suggest that maturing multilamellar vesicles precede (and at least partially initiate) crystallization in gallbladder bile. Although microstructural development seems to be concentration dependent, dilution of gallbladder bile to hepatic bile concentrations neither makes it identical to hepatic bile, nor prevents crystallization.

The effect of intermittent composite curing on marginal adaptation

The aim of this research was to study the effect of intermittent polymerization on marginal adaptation by comparing the marginal adaptation of intermittently polymerized composite to that of continuously polymerized composite. The materials used for this study were Pyramid (Bisco Inc., Schaumburg, U.S.A.) and Heliomolar (Ivoclar Vivadent, Liechtenstein). The experiment was carried out in class II MOD cavities prepared in 48 extracted human maxillary premolars. The samples were divided into 4 groups by light curing method; group 1- continuous curing (60s light on with no light off); group 2- intermittent curing (cycles of 3s with 2s light on & 1s light off for 90s); group 3- intermittent curing (cycles of 2s with 1s light on & 1s light off for 120s); group 4- intermittent curing (cycles of 3s with 1s light on & 2s light off for 180s). Consequently the total amount of light energy radiated was same in all the groups. Each specimen went through thermo-mechanical loading (TML) which consisted of mechanical loading (720,000 cycles, 5.0 kg) with a speed of 120 rpm for 100hours and thermocycling (6000 thermocycles of alternating water of 50℃ and 55℃). The continuous margin (CM) (%) of the total margin and regional margins, occlusal enamel (OE), vertical enamel (VE), and cervical enamel (CE)) was measured before and after TML under a × 200 digital light microscope. Three-way ANOVA and Duncan’ s Multiple Range Test was performed at 95% level of confidence to test the effect of 3 variables on CM (%) of the total margin: light curing conditions, composite materials and effect of TML. In each group, One-way ANOVA and Duncan’ s Multiple Range Test was additionally performed to compare CM (%) of regions (OE, VE, CE). The results indicated that all the three variables were statistically significant (p < 0.05). Before TML, in groups using Pyramid, groups 3 and 4 showed higher CM (%) than groups 1 and 2, and in groups using Heliomolar, groups 3 and 4 showed higher CM (%) than group 1 (p < 0.05). After TML, in both Pyramid and Heliomolar groups, group 3 showed higher CM (%) than group 1 (p < 0.05). CM (%) of the regions are significantly different in each group (p < 0.05). Before TML, no statistical

Swimming and cleaning in the free‐swimming phase of Argulus larvae (crustacea, branchiura)—Appendage adaptation and functional morphology

The free-swimming early larval stages of Argulus foliaceus (Linneaus) (Branchiura) are studied using digital video, light microscopy, and SEM. We analyze and document the mode of swimming in the hatching stage of A. foliaceus and the subsequent juvenile stages with fully developed thoracopods. We present new observations and an analysis of the functional morphology of a cleaning behavior in the first stage. This stage swims very efficiently using the large exopods of the second antennae in concert with the mandibular palp (naupliar limbs), while the subsequent stages use the now developed thoracopods for propulsion. This posterior shift in propulsion is similar to--but independent from--what is seen in other crustaceans. The hatching stage has previously been referred as a "metanauplius" but as the first and second maxillae are developed and active, and buds of all four thoracopods are present, it is too advanced to be included in the naupliar phase. The hooks of the first antennae and the distal hooks of the maxillae are demonstrated to function not only as attachment organs (to the host), but also to play a significant role in the cleaning of the naupliar swimming appendages. A digital video-based analysis of the swimming mode is provided. The larval swimming pattern is generally similar to that of other crustaceans such as Branchiopoda and Cirripedia, but autapomorphies of the Branchiura include the following: 1) While actively swimming, the naupliar appendages are almost straight during the recovery stroke and 2) they have a relatively small deflection during movement ( approximately 25 degrees or approximately 35 degrees for mandible and second antenna respectively), 3) the larval mandible has a uniramous palp which is the retained exopod. The morphological implications of the transition from the possibly nonfeeding pelagic, or free-swimming, first larval stage to the feeding, parasitic second stage are discussed and compared with other crustaceans.

In vitro human corneal model to investigate stromal epithelial interactions following refractive surgery

To develop an in vitro human corneal model to evaluate stromal epithelial interactions following corneal refractive surgical procedures. Department of Academic Ophthalmology, Rayne Institute, St. Thomas' Hospital, London, United Kingdom. Fifty-six human donor corneas procured from the eye bank were placed in a specially designed acrylic corneal holder and were cultured using the air-interface organ culture technique for up to 4 weeks. Corneal refractive surgical procedures such as a simple epithelial defect, 4 diopter (D) and 9 D photorefractive keratectomy (PRK), 4 D and 9 D laser-assisted subepithelial keratectomy (LASEK), and 9 D laser in situ keratomileusis (LASIK) were performed on the model. Temporal events in epithelial and keratocyte cell kinetics were evaluated using digital imaging, confocal microscopy, and light microscopy. Two-way analysis of variance and Student t tests were used to assess statistical significance. Epithelial healing following PRK was completed by 92 hours +/- 10 (SD) at a rate of 0.58 +/- 0.45 mm2/hour. In LASEK, the epithelial flap was replaced by regenerating peripheral epithelium that showed significant delay in epithelial closure (120 +/- 5 hours) with prolonged latency (24 +/- 4 hours, P<.0001) in comparison with PRK. The magnitude of keratocyte loss corresponded to ablation depth, and keratocyte regeneration was dependent on epithelial closure. In comparison, LASIK corneas showed a lesser percentage of keratocyte loss with poor recovery of keratocyte density in the stromal flap. Epithelial viability and keratocyte density were well preserved in the in vitro human model as observed in control corneas for up to 4 weeks. The temporal events in stromal epithelial interactions in the in vitro human model closely mimicked in vivo observations. The human model further avoided species-specific variations and provided a suitable test bed for evaluating newer algorithms and therapeutic regimens following refractive surgery.

Biliary cholesterol crystallization characterized by single-crystal cryogenic electron diffraction

Cholesterol crystals are the building blocks of cholesterol gallstones. The exact structure of early-forming crystals is still controversial. We combined cryogenic-temperature transmission electron microscopy with cryogenic-temperature electron diffraction to sequentially study crystal development and structure in nucleating model and native gallbladder biles. The growth and long-term stability of classic cholesterol monohydrate (ChM) crystals in native and model biles was determined. In solutions of model bile with low phospholipid-to-cholesterol ratio, electron diffraction provided direct proof of a novel transient polymorph that had an elongated habit and unit cell parameters differing from those of classic triclinic ChM. This crystal is exactly the monoclinic ChM phase described by Solomonov and coworkers (Biophysical J., In press) in cholesterol monolayers compressed on the air-water interface. We observed no evidence of anhydrous cholesterol crystallization in any of the biles studied. In conclusion, classic ChM is the predominant and stable form in native and model biles. However, under certain (low phospholipid) conditions, transient intermediate polymorphs may form. These findings, documenting single-crystal analysis in bulk solution, provide an experimental approach to investigating factors influencing biliary cholesterol crystal nucleation and growth as well as other processes of nucleation and crystallization in liquid systems.

Human Saphenous Vein Coronary Artery Bypass Graft Morphology, Geometry and Hemodynamics

Coronary artery bypass graft (CABG) failure has been linked to graft hemodynamics, in particular wall shear stress. This study characterizes the morphology, geometry and wall shear stress patterns in human CABGs. The intimal thickness (IT) in 49 human saphenous vein CABGs was measured by digital light microscopy. The geometry of six saphenous vein CABGs was replicated by post-mortem infusion of Batson's #17 anatomical corrosion casting compound. Graft hemodynamics were evaluated in two flow models, fabricated from the casts, under steady (Re = 110) and pulsatile flow (Re = 110, alpha = 2) conditions. Saphenous vein CABGs in situ for more than 2 months had, on average, the greatest IT on the hood and suture sites of the distal anastomosis. Floor thickening was highly variable and significantly less than IT at the hood, suture site and graft body. All casts showed an indentation along the floor and 5/6 casts displayed a sharp local curvature on the hood. In both flow models, a large increase in wall shear rate occurred on the hood, just proximal to the toe. The local geometry of the hood created this large spatial gradient in wall shear stress which is a likely factor in hood intimal hyperplasia.

L-NAME Prevents Stretch-induced Increases in Myotube Size and Nuclear Number

1037 The myotube stretch model allows for isolation and study of cell signaling pathways intrinsic to stretch-induced myocyte growth in the absence of humoral responses inherent to in vivo studies. PURPOSE: To determine if inhibition of nitric oxide synthase (NOS) activity via L-NAME attenuates stretch-induced hypertrophy of myotubes. METHODS: C2C12 myoblasts were cultured on silastic membranes suspended between concave metal clamps attached to a computer-controlled linear stepper motor. Cultures were maintained in growth medium (DMEM supplemented with 10% fetal bovine serum) in a humidified incubator at 37°C, 5% CO2 until 80% confluency was reached, then switched to differentiation media (DM; DMEM supplemented with 2% horse serum). Hypertrophy was induced in some cultures by an intermittent linear stretch protocol. The protocol involved three 60-min bouts of stretch each consisting of 12 sets of 10 stretch cycles (± 10% length change applied at a rate of 20%/s). One set was completed every 5 min for the 60 min period. Stretch bouts were completed at 12h intervals beginning at 96h post serum withdrawal. DM was refreshed for all groups (stretched and non-stretched) before each stretching bout. Cultures were divided into four treatment groups (n = 3 cultures/group): 1) Control, 2) L-NAME (100μM in DM), 3) Stretch only, and 4) L-NAME + Stretch. Four hours after the last stretching bout (124 hrs), cultures were fixed with 10% methanol and stained with hematoxylin and eosin (H&E). Microphotometric analysis was performed on digital light microscopy images calibrated with a stage micrometer. Myotube width (calculated as the average of ten transverse measurements taken along the length of each myotube), and the number of myonuclei/myotube were recorded in three separate fields of view for each culture. RESULTS: The stretch protocol increased average myotube width (+17%, P < 0.05) and number of myonuclei/myotube (+28%, P < 0.05). L-NAME treatment alone reduced myotube area 18% compared to control cultures (P < 0.05). Further, L-NAME treatment during the stretch protocol eliminated stretchinduced myotube growth (i.e no difference between L-NAME+Stretch and L-NAME only; P > 0.05). Myonuclei content in cultures treated with L-NAME did not differ (P > 0.05) from controls with or without stretch. CONCLUSIONS: Stretch-induced hypertrophy and myonuclear accretion in myotubes was attenuated with L-NAME treatment. Additionally, L-NAME treatment alone caused a reduction in myotube size and nuclei number. Thus, myotube growth and stretch-induced hypertrophy is dependent upon intrinsic NOS activity.