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Ultrastructural evaluation of multiple pass low energy versus single pass high energy radio-frequency treatment.

BACKGROUND AND OBJECTIVE: The radio-frequency (RF) device is a system capable of volumetric heating of the mid to deep dermis and selective heating of the fibrous septa strands and fascia layer. Clinically, these effects promote dermal collagen production, and tightening of these deep subcutaneous structures. A new technique of using multiple low energy passes has been described which results in lower patient discomfort and fewer side effects. This technique has also been anecdotally described as giving more reproducible and reliable clinical results of tissue tightening and contouring. This study will compare ultrastructural changes in collagen between a single pass high energy versus up to five passes of a multiple pass lower energy treatment. STUDY DESIGN/MATERIALS AND METHODS: Three subjects were consented and treated in the preauricular region with the RF device using single or multiple passes (three or five) in the same 1.5 cm(2) treatment area with a slight delay between passes to allow tissue cooling. Biopsies from each treatment region and a control biopsy were taken immediately, 24 hours or 6 months post treatment for electron microscopic examination of the 0-1 mm and 1-2 mm levels. Sections of tissue 1 mm x 1 mm x 80 nm were examined with an RCA EMU-4 Transmission Electron Microscope. Twenty sections from 6 blocks from each 1 mm depth were examined by 2 blinded observers. The morphology and degree of collagen change in relation to area examined was compared to the control tissue, and estimated using a quantitative scale. RESULTS: Ultrastructural examination of tissue showed that an increased amount of collagen fibril changes with increasing passes at energies of 97 J (three passes) and 122 J (five passes), respectively. The changes seen after five multiple passes were similar to those detected after much more painful single pass high-energy treatments. CONCLUSIONS: This ultrastructural study shows changes in collagen fibril morphology with an increased effect demonstrated at greater depths of the skin with multiple low-fluence passes and at lesser depths with single pass higher fluence settings. Findings suggest that similar collagen fibril alteration can occur with multiple pass low-energy treatments and single pulse high-energy treatments. The lower fluence multiple pass approach is associated with less patient discomfort, less side effects, and more consistent clinical results.

Collagen↗

Nucleolar protein allocation and ultrastructure in bovine embryos produced by nuclear transfer from granulosa cells.

In the present study immunofluorescence confocal laser scanning microscopy, autoradiography following (3)H-uridine incubation and transmission electron microscopy were used to evaluate the nucleolar protein localization, transcriptional activity, and nucleolar ultrastructure during genomic re-programming in bovine embryos reconstructed by nuclear transfer from granulosa cells into non-activated cytoplasts followed by activation. During the 1st cell cycle (1-cell embryos), no autoradiographic labelling was detected. Ultrastructurally, nucleoli devoid of a granular component were observed. During the 2nd cell cycle (2-cell embryos) autoradiographic labelling was also lacking and the embryos displayed varying degrees of nucleolar inactivation. During both the 3rd (4-cell embryos) and 4th (tentative 8-cell embryos), cell cycles autoradiographic labelling was lacking in some embryos, while others displayed labelling and associated formation of fibrillo-granular nucleoli. During the 5th cell cycle (tentative 16-cell embryos), all embryos displayed autoradiographic labelling and fibrillo-granular nucleoli. In some blastomeres, however, deviant nucleolar ultrastructure was observed. During the first cell cycle labelling of RNA polymerase I, fibrillarin, upstream binding factor (UBF) and nucleolin (C23) was localized to nuclear entities. During the 2nd cell cycle, only labelling of RNA polymerase I and fibrillarin persisted. During the 3rd and 4th cell cycle labelling of fibrillarin persisted, labelling of nucleophosmin (B23) appeared and that of nucleolin re-appeared. During the 5th cell cycle almost all embryos showed complete labelling of all proteins except for UBF, which lacked in more than half of the embryos. In conclusion, bovine granulosa cell nuclear transfer embryos showed re-modelling of the nucleoli to an inactive form followed by re-formation of fibrillo-granular nucleoli. The re-formation of fibrillo-granular nucleoli was initiated already during the 3rd cell cycle, which is one cell cycle earlier than in in vivo- and in vitro-derived bovine embryos. Moreover, in more than half of the embryos, UBF could not be immunocytochemically localized to the nucleolar compartment during the 5th cell cycle indicating lack of developmental potentials.

Animals↗

In vitro viability and ultrastructural changes in bovine oocytes treated with a vitrification solution.

Abattoir-derived oocytes were exposed to a concentrated cryoprotectant solution (DAP213: 2 M DMSO, 1 M acetamide, 3 M propanediol, and 10% FCS in TCM199) for 1.5 or 5 min at the germinal vesicle (GV) stage or after maturation in vitro (IVM). Their viability was assessed by in vitro fertilization (IVF) and culture (IVC) to blastocysts. To investigate the effect of DAP213 on the ultrastructure, GV and IVM oocytes were processed for transmission electron microscopy (TEM) before (control) or after exposure to the cryoprotectant. DAP213 induced profound ultrastructural modifications to the microvilli and mitochondria, resulted in large vesicle formation, and, most significantly, caused the premature release of the cortical granules (CG). In IVM oocytes exposed to the cryoprotectant for 5 min, exocytosis of CG into the perivitelline space was common and the IVF rate was reduced (P < .05). After exposure for 5 min, GV oocytes displayed clusters of CG comparable to controls, but after IVM-IVF, polyspermy rate was increased (P < .05). Furthermore, treated GV oocytes showed a reduced rate of cleavage and blastocyst formation and an increased percentage of oocytes exhibiting alterations in organelles, whereas the viability and ultrastructure of IVM oocytes treated for 1.5 min was not different from controls. These observations demonstrate that 1) cortical granule kinetics is one of the key elements controlling fertilizability of bovine oocytes treated with cryoprotectant, and 2) GV oocytes are more sensitive to the cryoprotectant than those that have already been matured in vitro.

Acetamides↗

Comparative study of the ultrastructural alterations in the pulmonary vessels of rats treated with alpha-naphthylthiourea (ANTU) and ammonium sulphate.

A comparative study of the ultrastructural changes in rat pulmonary vessels was made in two different situtations of increased vascular permeability: one caused by alpha-naphthylthiourea (ANTU) and the other induced by ammonium sulphate (AS). AS had a more destructive effect on the capillaries but showed little alteration in vessels larger than capillaries. ANTU provoked milder ultrastructural lesions but acted on capillaries, arterial and venous structures. In both conditions intravenously injected carbon particles adhered to the vessel wall and leakage into the interstitial space was rarely seen despite the presence of endothelial gaps. It was concluded that ANTU and AS provoke different ultrastructural alterations and that pulmonary and systemic vessels react differently when undergoing changes in permeability.

Ammonium Sulfate↗

Experimental mycobacterial granulomas in guinea pig lymph nodes: ultrastructural observations.

A systematic ultrastructural study has been performed of the mononuclear phagocytes in granulomas induced by different types of mycobacteria, e.g. live BCG (Pasteur), irradiated M. leprae and irradiated BCG (Pasteur) in guinea pig lymph nodes. Live BCG (Pasteur) induces a granuloma which peaks at 2-3 weeks and by light microscopy, a large number of the infiltrating cells have the appearance of epithelioid cells. Ultrastructurally a large proportion of these cells have a distinct appearance. They are characterised by the presence of very large nuclei, and prominent nucleoli, paucity of cytoplasmic organelles and swollen rough endoplasmic reticulum. Cells from 2 week granulomas induced by live M. kansasii and BCG (Glaxo), has a similar ultrastructural appearance. BCG (Pasteur) granulomas slowly resolved and by light and electron microscopy fibrosis and collagen deposition was seen by 7 weeks and was very extensive in some nodes at 10 weeks. The nodes from M leprae injected animals showed peak infiltration at 5 weeks and by light microscopy the infiltrating cell population was more mixed, with most of the cells having a macrophage appearance. Electron-microscopy showed these infiltrating cells to be mainly activated macrophages, containing phagocytosed organisms. Nodes from irradiated BCG (Pasteur) injected animals had peak infiltration at 1 week. The light and electron microscopic appearance showed areas consisting mainly of fibroblasts and mononuclear phagocytes. Thus, the granulomas induced by M. leprae are very different from those induced by BCG in the guinea pig. It is suggested that lymph nodes draining areas of local injection of BCG and irradiated M. leprae could respectively form a good model in the guinea pig for cellular and biochemical studies of the granulomas of tuberculoid and lepromatous leprosy.

Animals↗

Histological and ultrastructural studies of the human juxtaglomerular apparatus in Bartter's syndrome and renal artery stenosis.

Histological and ultrastructural studies of the juxtaglomerular apparatus (JGA) were performed on renal biopsy material from three cases of Bartter's syndrome and two cases of renal artery stenosis. These cases of known JGA hyperfunction were compared with the apparatus in a presumed inactive or basal state as in minimal lesion glomerulonephritis. Light microscopic assessment of the ease of JGA identification, its size and cellularity, provides a valuable reflection of activity and indicates those cases which require ultrastructural assessment of granule turnover. Granule stains often failed to demonstrate any significant increase in granule number in the two conditions studied, and only ultrastructural studies allowed evaluation of secretory activity, rhomboid granule formation and granule release. From these investigations, a proposed mechanism of granule turnover is suggested. The morphological findings in both Bartter's syndrome and renal artery stenosis support the concept of hyperfunction of the apparatus. They may help to elucidate the pathogenesis of Bartter's syndrome and to predict the outcome of surgical intervention in renal artery stenosis. Such studies could profitably be performed on renal biopsies in other disease states, so augmenting our understanding of the contribution of the JGA to the pathogenesis of a variety of glomerular and vascular lesions.

Bartter Syndrome↗

Non-isotopic in situ hybridization at the ultrastructural level.

Non-isotopic in situ hybridization techniques are becoming increasingly widely used at the ultrastructural level, permitting rapid localization of nucleic acid targets with a high degree of resolution. Technical considerations dictate that the great specificity of the method cannot be matched by a similar degree of sensitivity; the value of non-isotopic ultrastructural in situ hybridization lies in its unique ability to localize nucleic acid targets in relation to submicroscopic cellular structures. This article presents an overview of non-isotopic ultrastructural hybridization methods and applications.

Animals↗

Pharmacologically induced ultrastructural and immunohistochemical changes in the prostate of the castrated dog.

The effects of an aromatase inhibitor and of an antiandrogen on the ultrastructure and the expression of a secretory protein (acid phosphatase) and marker proteins for basal cells (keratin) and fibroblasts (vimentin) were studied in the prostate of castrated, androstenedione-treated dogs. Androstenedione treatment partially restored the normal appearance of the gland and also some secretory activity. In the central portion of the gland, basal cell hyperplasia developed instead of secretory activity after androstenedione treatment. Administration of the aromatase inhibitor reduced the number of secretory cells but did not completely suppress the latter. There was some proliferation of the connective tissue surrounding the atrophic acini. Combined treatment with aromatase inhibitor and antiandrogen resulted in a general atrophy of prostatic acini that was less intense relative to the changes observed after castration. Residual secretory activity, detected in specimens treated exclusively with aromatase inhibitor, were lacking after combined treatment. The influence of all regimens on the ultrastructure of smooth muscle cells was comparably discrete, whereas regional differences in the arrangement pattern of the epithelium and the fibromuscular stroma were impressive. The ultrastructural findings support previous results of a synergic inhibitory effect of aromatase inhibitor and antiandrogens on the canine prostate.

Acid Phosphatase↗

Using the atomic force microscope to observe and study the ultrastructure of the living BIU-87 cells of the human bladder cancer.

In this study, the ultrastructure of living BIU-87 cells of human bladder cancer was mapped using atomic force microscopy to reveal the dynamic change of single cancerous cell division. Simultaneously, the feasibility and functional reliability of the atomic force microscope (AFM) were established and a laboratory model using AFM to study living cancerous cells was created. In this experiment, BIU-87 cells of human bladder cancer were cultured by conventional methods and grown in gelatin-treated dishes. A thermostat was used for preserving the cell's living temperature. Scanning of these cells using AFM was carried out in physiologic condition. The AFM images of the ultrastructure of living BIU-87 cells as well as those of the cell's membrane and cytoskeleton were very clear. The dynamic phenomenon of single cell division was observed. It was concluded that the AFM was able to observe and depict the ultrastructure of living cells of human bladder cancer directly and in real time. This experimental model is expected to play an important role in elucidating the cancerous mechanism of bladder normal cells at the atomic or nanometer level.

Cell Division↗

Ultrastructural alterations in the liver and kidney of white sea bass, Lates calcarifer, in acute and subchronic cadmium exposure.

Ultrastructural alterations in the liver and kidney of 3-month-old white sea bass, Latescalcarifer, after cadmium exposure were studied by transmission electron microscopy (TEM). One group of fish was exposed to a cadmium concentration of 10 mg/L (acute) for 96 h in a static system, and another group was exposed to cadmium concentrations of 0.8 and 3 mg/L cadmium (subchronic) for 3 months in a recirculation closed system. Ultrastructural alterations observed in the hepatocytes included mitochondrial condensation, swelling, and lysis. The rough endoplasmic reticulum (RER) showed dilation, fragmentation, and vesiculation. After subchronic exposure there were numerous large lipid droplets and abundant stored glycogen. Ultrastructural alterations observed in the proximal tubules of the kidney included nuclear degeneration, condensation, and massive swelling of the mitochondria; RER fragmentation and vesiculation. Disorganized brush borders and increased numbers of large hydropic vacuoles and lysosomes were also observed.

Animals↗

Ultrastructure of IVM-IVF bovine blastocysts vitrified after equilibration in glycerol 1,2-propanediol using 2-step and 16-step procedures.

Two experiments were conducted to investigate the relationship between survival rates and ultrastructural appearance, using the freeze-replica technique, of bovine blastocysts after vitrification. In experiment 1, blastocysts obtained from in vitro-matured and in vitro-fertilized (IVM-IVF) bovine oocytes were either equilibrated in a stepwise manner in a vitrification solution (VS; 22.5% glycerol + 22.5% 1,2-propanediol) using 16 steps for 18 min in total (the 16-step method) or equilibrated with 10% glycerol + 20% 1,2-propanediol for 10 min and then exposed to VS (the 2-step method). The blastocysts were then vitrified by plunging them into liquid nitrogen. All samples were subsequently thawed in a water bath at 37 degrees C and cultured in vitro with a monolayer of cumulus cells. The survival rate obtained for blastocysts equilibrated by the 16-step method was 83.3% (25/30). In contrast, no blastocysts survived by the 2-step method (0/30). In experiment 2, freeze-replica observations were carried out on blastocysts vitrified by the 16-step method and the 2-step method. In all the blastocysts, no ice crystals were observed in the cytoplasm, blastocoelic cavity, or extracellular areas, which confirmed the occurrence of complete vitrification. Little ultrastructural change was observed in the plasma membrane of the blastocysts equilibrated by the 16-step method. In contrast, small vesicles and distinct intramembrane particle (IMP) aggregation were frequently observed in the plasma membranes of blastocysts equilibrated by the 2-step method. These results indicate that the successful cryopreservation of blastocysts following the 16-step equilibration requires not only complete vitrification, but also minimization of ultrastructural damage to the plasma membrane.

Animals↗

Ultrastructure and phagocytic activity of rat peritoneal macrophages exposed to low temperatures in vitro.

Hypothermia affects various components of the immune system, leading to impaired immune resistance. To examine the in vitro effect of low temperature on the ultrastructure and phagocytic function of rat peritoneal macrophages, cells were incubated at 4, 10, 24, and 37 degrees C for 60 min. Subsequently, their ultrastructure and capacity to engulf latex particles and generate superoxide anions were evaluated. The results showed a close inverse relationship between incubation temperature and ultrastructural changes, i.e., the lower the temperature, the higher the number of altered cells. In addition, at lower temperatures the number of cells capable of phagocytosis was reduced; the cells engulfed fewer particles per cell and generated less superoxide anions. These findings may be relevant for explaining the increased susceptibility to bacterial infections under hypothermic conditions.

Animals↗

Ultrastructural nucleolar alterations induced by an ametantrone/polyr(A-U) complex.

In the present study we examined the ultrastructural modifications as well as the precise distribution of DNA and RNA in RT4 cell nucleoli following a 3-h exposure to nontoxic or toxic doses of ametantrone (AMT), poly(adenylate-uridylate) (polyr(A-U), or an AMT/polyr(A-U) combination. While distribution of nucleic acids within the various nucleolar components is not modified following all treatments, the nucleoli exhibit several ultrastructural changes: redistribution of the nucleolar components, decrease in the number of fibrillar centers, and increase in the size of the fibrillar centers. The relative frequencies of the test agents to induce the apparition of nucleoli of compact type are AMT/polyr(A-U) > AMT approximately polyr(A-U) > sham-treated, while the abilities of the test agents to induce the nucleolar segregation are AMT/polyr(A-U) approximately AMT > polyr(A-U) > sham-treated cells. These ultrastructural changes are characteristics of drugs that intercalate into DNA and inhibit rDNA transcription as well as rRNA processing and release of nascent preribosomes from the nucleolus.

Antineoplastic Agents↗

Histones and DNA ultrastructural distribution in plant cell nucleus: a combination of immunogold and cytochemical methods.

In this work we report for the first time the ultrastructural distribution of histones and DNA in the nuclear compartments in two different plant cell types: Allium cepa L. root meristems and Capsicum annuum L. microspores and pollen grains, by using antibodies against histones H2B and H4 and anti-DNA. Immunolocalizations were combined with ultrastructural cytochemistry for nucleic acids (methylation-acetylation method), DNA (NAMA-Ur) and RNPs (EDTA), to relate the subcellular location of histones and DNA with the chemical subcompartmentalization of the cell nucleus. This is particularly interesting concerning the presence of histones or not on fibers of the interchromatin region and on the fibrillar components of the nucleolus, nuclear subcompartments where transcription has been shown to take place at some regions. Our methodological approach permitted to define precisely the structures where histones were detected in relation to the ultrastructural localization of chromatin in various structural condensation levels. Concerning the localization of DNA and histones on the different components of the nucleolus, the combination of immunogold labeling with the methylation-acetylation cytochemical method, developed in our laboratory, was very useful, thus permitting a clear recognition of the nucleolar components and a correct assignment of labeling, which is not always evident on uranyl-lead-stained Lowicryl sections. Double immunogold assays were also done for a simultaneous visualization of histones and DNA. Our results show a coincident distribution of histones and DNA on the same nuclear compartments revealing the presence of both antigens on condensed chromatin, fibers of the interchromatin region, principally located at the periphery of the condensed chromatin, and in the fibrillar components of the nucleolus.

Acetic Anhydrides↗

Ultrastructural changes in the rat phrenic nucleus developing within 2 h after cervical spinal cord hemisection.

The present study was conducted to describe the ultrastructural changes which occur in the young adult rat phrenic nucleus within 2 h after an ipsilateral C2 spinal cord hemisection. The main objective was to determine if there is a temporal relationship between specific ultrastructural changes in the phrenic nucleus and a significant augmentation of crossed phrenic nerve activity which occurs as early as 2 h after hemisection. Phrenic motoneurons were identified at electron microscopic levels by retrograde HRP labeling. Ultrastructural features in the phrenic nucleus of control and experimental rats were qualitatively analyzed and then quantitated. At 2 h posthemisection, there was a significant increase in the mean percentage of phrenic dendrodendritic appositions. In the control rats, 4.73 +/- 0.18% of phrenic dendrites were in apposition, and this percentage increased significantly to 8.58 +/- 0.54% at 2 h after injury. Furthermore, the mean lengths of asymmetrical and symmetrical synaptic active zones increased significantly at 2 h posthemisection from control lengths of 0.372 +/- 0.009 microns and 0.404 +/- 0.007 microns to 0.410 +/- 0.011 microns and 0.513 +/- 0.032 microns, respectively, in experimental rats. The phrenic nucleus is therefore capable of morphological plasticity as early as 2 h after spinal cord hemisection and this plasticity coincides temporally with the physiological augmentation of crossed phrenic nerve activity at 2 h. The data further suggest that these morphological changes may be part of the substrate for the unmasking of ineffective synapses during the crossed phrenic phenomenon.

Animals↗

Laser effects on myelinated and nonmyelinated fibers in the rat peroneal nerve: a quantitative ultrastructural analysis.

We have recently shown that Nd:YAG laser irradiation of rat peripheral nerve differentially impairs action potential transmission in small, slowly conducting sensory fibers compared to fast conducting afferents. In addition, the number of small sensory neurons of the A-delta- and C-fiber group labeled with HRP is significantly reduced after laser irradiation, while the number of labeled large sensory neurons and motoneurons was not affected. To further evaluate this laser-induced injury, we examined three distinct regions of the laser-irradiated rat peroneal nerve using ultrastructural morphometric methods. These regions were the site of laser irradiation and zones 10 mm proximal and 5 mm distal to the injury. The contralateral nerve was sham treated. Our results indicate that for the small nonmyelinated fibers, there was a significant increase in both mean fiber size and the number of microtubules per fiber, but a decrease in the number of neurofilaments. In contrast, the number of myelinated and nonmyelinated fibers is not significantly altered at 7 days following laser irradiation, and the mean diameter and frequency distribution of myelinated nerve fibers was unchanged. This study demonstrates that selective functional alterations in laser-irradiated nerves (nerve conduction velocity, HRP transport properties) are accompanied by ultrastructural changes of axonal organelles in nonmyelinated fibers. Nd:YAG laser light might ultimately prove to be a powerful tool to selectively alter functional properties in small, slowly conducting afferent fibers, without causing degeneration at the ultrastructural level at the site of irradiation. We hypothesize further that the laser-induced functional alterations might be related to differential thermally mediated changes.

Animals↗

Impact of experimental acute hyponatremia on severe traumatic brain injury in rats: influences on injuries, permeability of blood-brain barrier, ultrastructural features, and aquaporin-4 expression.

The effects of acute hyponatremia on severe traumatic brain injury (TBI) in 35 adult male Sprague-Dawley rats were studied in a replicated focal and diffuse injury rat model. Such effects were assessed by the cerebral contusion volume and axonal injury (AI) densities, determined by quantitative immunoreactivity of beta-amyloid precursor protein, by blood-brain barrier (BBB) permeability based on endogenous IgG immunostaining, and by ultrastructural features. Significant increase of contusion volume (P < 0.05) and of AI in the segment of corpus callosum beneath the contusion (P < 0.05) and ipsilateral thalamus (P < 0.05) were observed at 4 h postinjury during the hyponatremic phase. No change in BBB permeability was observed in the hyponatremia + TBI (HT) groups. Significant swelling of perivascular astrocytic foot processes in the HT groups was seen at 4 h (P < 0.01) and 1 day postinjury (P < 0.01) by quantitative image analysis of ultrastructures. However, attenuated swelling of perivascular astrocytic foot processes in severely edematous medulla oblongata with simultaneous swelling of perikaryal astrocytic processes was observed in the HT 1-day group. The ultrastructural features were also correlated with the down-regulation of aquaporin-4 (AQP4) mRNA expression (P < 0.05). Results suggest that acute hyponatremia acts as one of the secondary insults following severe TBI. Such exacerbation may not be attributable to further disruption of BBB permeability, but rather to the ischemia resulting from the swelling of perivascular astrocytic foot processes impeding microcirculation. Down-regulated AQP4 mRNA expression may be one of the molecular mechanisms maintaining water homeostasis in diffusely injured brain exposed to acute hyponatremia.

Acute Disease↗

Ultrastructural localization of CD36 in human hepatic sinusoidal lining cells, hepatocytes, human hepatoma (HepG2-A16) cells, and C32 amelanotic melanoma cells.

Ultrastructural localization of CD36 in human hepatic sinusoidal lining cells, hepatocytes, human hepatoma (HepG2-A16) cells, and C32 amelanotic melanoma cells. Experimental Parasitology 79, 383-390. CD36 is expressed in the endothelial cells of some human organs, but the ultrastructural localization of this molecule in the sinusoidal lining cells of human liver is not well established. We report the ultrastructural localization of CD36 in the liver using a novel murine monoclonal antibody against CD36, namely MO30, as a primary antibody. Immunocytochemistry by the postembedding method showed that CD36 was localized in endothelial cells of sinusoids and in hepatocyte microvilli protruding into the space of Disse. Moreover, in cultured human hepatoma (HepG2-A16) cells and C32 amelanotic melanoma cells, MO30 reacted with microvilli. Hence, CD36 expressed on these cells may be involved in recognition and/or entry of these cells by malaria sporozoites.

Amino Acid Sequence↗