Effect of low-level laser therapy on intramembranous and endochondral autogenous bone grafts healing

The aim of this study was to evaluate the healing process of intramembranous (IM) and endochondral (EC) bone grafts under low‐level laser therapy (LLLT). Male rabbits underwent onlay autogenous bone grafts (1 cm in diameter) retrieved from the calvaria and iliac crest and fixed on parietal bones, divided into four groups: Calvaria (C), Iliac (I), Calvaria + LLLT (C+L), and Iliac + LLLT (I+L). Animals from C+L and I+L Groups had their grafts exposed to LLLT (AlGaAs–808 nm, CW, 30 mW, 0.028 cm² average laser beam area), 15 s irradiation time (16 J/cm² per point–total of 64 J/cm² per session). After 7, 14, 30, and 60 days, grafts were retrieved and resorption pattern analyzed by means of morphometry and TRAP‐positive osteoclasts detection. Differences in the resorption levels of iliac grafts were observed, presenting 40% in I group against 8% in I+L grafts at the 14th day of evaluation (P < 0.05). After 30 days, resorption was maintained at 41% in I group, whereas I+L presented 15% in the same period (P = 0.0591). No significant differences were noted in the rates of calvaria grafts resorption in all periods. A significant higher number of osteoclasts on the grafts' surface was observed in C+L Group at day 30, in comparison with C group. In I+L Group, prevalence of osteoclasts was marked at day 7 (P < 0.05) in comparison to I Group. In general, it was concluded that biomodulative effects of LLLT did not significantly affect healing and resorption processes of autogenous bone grafts from EC and IM origins. Microsc. Res. Tech. 75:1237–1244, 2012. © 2012 Wiley Periodicals, Inc.     

Label‐free identification of antibiotic resistant isolates of living Escherichia coli: Pilot study

We introduce a label‐free spectroscopic method to classify subtypes of quinolone‐nonsusceptible Escherichia coli (E. coli ) isolates obtained from human blood cultures. Raman spectroscopy with a 30‐nm gold‐deposited, surface‐enhanced Raman scattering (SERS) substrate was used to evaluate three multilocus sequencing typing (MLST)‐predefined groups including E . coli ATCC25922, E . coli ST131:O75, and E . coli ST1193:O25b. Although there was a coffee‐ring effect, the ring zone was selected at the ideal position to screen E. coli isolates. Strong Raman peaks were present at 1001–1004 cm^?1 (C? C aromatic ring breathing stretching vibrational mode of phenylalanine), 1447–1448 cm^?1 (C? H₂ scissoring deformation vibrational mode), and 1667 cm^?1 (amide I α‐helix). Although the three MLST‐predefined E . coli isolates had similar Raman spectral patterns, a support vector machine (SVM) learning algorithm‐assisted principal component analysis (PCA) analysis had superior performance in detecting the presence of quinolone‐nonsusceptible E. coli isolates as well as classifying similar microbes, such as quinolone‐nonsusceptible E . coli ST131:O75 and E . coli ST1193:O25b isolates. Therefore, this label‐free and nondestructive technique is likely to be useful for clinically diagnosing quinolone‐nonsusceptible E. coli isolates with the MLST method.     

Chemical and morphological changes in human dentin after Er:YAGlaser irradiation: EDS and SEM analysis

Sixty samples of human dentin were divided into six groups (n = 10) and were irradiated with Er:YAG laser at 100 mJ–19.9 J/cm², 150 mJ–29.8 J/cm², 100 mJ–35.3 J/cm², 150 mJ–53.0 J/cm², 200 mJ–70.7 J/cm², and 250 mJ–88.5 J/cm², respectively, at 7 Hz under a water spray. The atomic percentages of carbon, oxygen, magnesium, calcium, and phosphorus and the Ca‐to‐P molar ratio on the dentin were determined by energy dispersive X‐ray spectroscopy. The morphological changes were observed using scanning electron microscopy. A paired t‐test was used in statistical analysis before and after irradiation, and a one‐way ANOVA was performed (P ≤ 0.05). The atomic percent of C tended to decrease in all of the groups after irradiation with statistically significant differences, O and Mg increased with significant differences in all of the groups, and the Ca‐to‐P molar ratio increased in groups IV, V, and VI, with statistically significant differences between groups II and VI. All the irradiated samples showed morphological changes. Major changes in the chemical composition of dentin were observed in trace elements. A significant increase in the Ca‐to‐P ratio was observed in the higher energy density groups. Morphological changes included loss of smear layer with exposed dentinal tubules. The changes produced by the different energy densities employed could have clinical implications, additional studies are required to clarify them. Microsc. Res. Tech. 78:1019–1025, 2015. © 2015 Wiley Periodicals, Inc.     

Label‐free monitoring of inflammatory tissue conditions using a carrageenan‐induced acute inflammation rat model

Although the confirmation of inflammatory changes within tissues at the onset of various diseases is critical for the early detection of disease and selection of appropriate treatment, most therapies are based on complex and time‐consuming diagnostic procedures. Raman spectroscopy has the ability to provide non‐invasive, real‐time, chemical bonding analysis through the inelastic scattering of photons. In this study, we evaluate the feasibility of Raman spectroscopy as a new, easy, fast, and accurate diagnostic method to support diagnostic decisions. The molecular changes in carrageenan‐induced acute inflammation rat tissues were assessed by Raman spectroscopy. Volumes of 0 (control), 100, 150, and 200 µL of 1% carrageenan were administered to rat hind paws to control the degree of inflammation. The prominent peaks at [1,062, 1,131] cm^?1 and [2,847, 2,881] cm^?1 were selected as characteristic measurements corresponding to the C–C stretching vibrational modes and the symmetric and asymmetric C–H (CH₂) stretching vibrational modes, respectively. Principal component analysis of the inflammatory Raman spectra enabled graphical representation of the degree of inflammation through principal component loading profiles of inflammatory tissues on a two‐dimensional plot. Therefore, Raman spectroscopy with multivariate statistical analysis represents a promising method for detecting biomolecular responses based on different types of inflammatory tissues.     

Single-Frequency TEA CO<Subscript>2</Subscript> Laser for Experiments on Non-Resonant Radiation Interaction with Matter

The design philosophy and output radiation parameters of single frequency TEA CO₂ laser with bleaching intracavity longitudinal modes selector (cell filled with SF₆) are described. At cavity tuning to 10P(16) line and choosing optimum SF₆ pressure in the cell the stable single frequency lasing is realized with scatter of radiation peak power in a series of “shots” less than ±7% of average value. The radiation energy density and intensity gradually tuned in the ranges 0.36–12.5 J/cm² and 2.9–100 MW/cm² correspondingly were realized in the focal plane of a lens with f = 127 mm.     

Chronic alcohol consumption promotes alterations on salivary gland regeneration process

The aim of this study is to investigate the histological effect of alcohol ingestion on the regeneration of the submandibular gland (SMG) in rats. Twelve 60‐day‐old male Wistar rats were randomized into two experimental groups. Test group (TG) animals ingested 40° GL of alcohol for 45 days before surgery, being its concentration gradually increased 10° GL/week for 4 weeks to achieve the final concentration of 40° GL. The control group (CG) received water during the whole experimental period. One‐third of the left SMG lobe was removed. Three and seven days after, the whole gland was excised and analyzed. In the TG, the inflammatory process was pronounced when comparing the CG on day 3. The inverse aspect was observed on day 7, associated with an advanced parenchyma development. Changes in laminin expression and glycoproteins production were observed in the TG, causing advanced morphogenesis and delay in cytodifferentiation during the salivary gland regeneration, probably due to alcohol effects. Animals who received ethanol showed alterations in the pattern of glandular regeneration. Microsc. Res. Tech. 76:1125–1130, 2013. © 2013 Wiley Periodicals, Inc.     

Morphological changes produced by acid dissolution in Er:YAG laser irradiated dental enamel

Several scientific reports have shown the effects of Er:YAG laser irradiation on enamel morphology. However, there is lack of information regarding the morphological alterations produced by the acid attack on the irradiated surfaces. The aim of this study was to evaluate the morphological changes produced by acid dissolution in Er:YAG laser irradiated dental enamel. Forty‐eight enamel samples were divided into four groups (n = 12). GI (control); Groups II, III, and IV were irradiated with Er:YAG at 100 mJ (12.7 J/cm²), 200 mJ (25.5 J/cm²), and 300 mJ (38.2 J/cm²), respectively, at 10 Hz without water irrigation. Enamel morphology was evaluated before‐irradiation, after‐irradiation, and after‐acid dissolution, by scanning electron microscopy (SEM). Sample coating was avoided and SEM analysis was performed in a low‐vacuum mode. To facilitate the location of the assessment area, a reference point was marked. Morphological changes produced by acid dissolution of irradiated enamel were observed, specifically on laser‐induced undesired effects. These morphological changes were from mild to severe, depending on the presence of after‐irradiation undesired effects. Microsc. Res. Tech. 77:410–414, 2014. © 2014 Wiley Periodicals, Inc.     

Effect of Er:YAG laser irradiation on deciduous enamel roughness and bacterial adhesion: An in vitro study

Laser irradiation has been proposed as a preventive method against dental caries since it is capable to inhibit enamel demineralization by reducing carbonate and modifying organic matter, yet it can produce significant morphological changes. The purpose of this study was to evaluate the influence of Er:YAG laser irradiation on superficial roughness of deciduous dental enamel and bacterial adhesion. Fifty‐four samples of deciduous enamel were divided into three groups (n = 18 each). G1_control (nonirradiated); G2_100 (7.5 J/cm²) and G3_100 (12.7 J/cm²) were irradiated with Er:YAG laser at 7.5 and 12.7 J/cm², respectively, under water irrigation. Surface roughness was measured before and after irradiation using a profilometer. Afterwards, six samples per group were used to measure bacterial growth by XTT cell viability assay. Adhered bacteria were observed using confocal laser scanning microscopy (CLSM) and a scanning electron microscopy (SEM). Paired t‐, one‐way analysis of variance (ANOVA), Kruskal‐Wallis and pairwise Mann–Whitney U tests were performed to analyze statistical differences (p < .05). Before treatment, samples showed homogenous surface roughness, and after Er:YAG laser irradiation, the surfaces showed a significant increase in roughness values (p < .05). G3_100 (12.7 J/cm²) showed the highest amount of Streptococcus mutans adhered (p < .05). The increase in the roughness of the tooth enamel surfaces was proportional to the energy density used; the increase in surface roughness caused by laser irradiation did not augment the adhesion of Streptococcus sanguinis; only the use of the energy density of 12.7 J/cm² favored significantly the adhesion of S. mutans.     

Effect of Aloe vera application on the content and molecular arrangement of glycosaminoglycans during calcaneal tendon healing

Although several treatments for tendon lesions have been proposed, successful tendon repair remains a great challenge for orthopedics, especially considering the high incidence of re‐rupture of injured tendons. Our aim was to evaluate the pharmacological potential of Aloe vera on the content and arrangement of glycosaminoglycans (GAGs) during tendon healing, which was based on the effectiveness of A. vera on collagen organization previously observed by our group. In rats, a partial calcaneal tendon transection was performed with subsequent topical A. vera application at the injury site. The tendons were treated with A. vera ointment for 7 days and excised on the 7^th, 14^th, or 21^st day post‐surgery. Control rats received ointment without A. vera. A higher content of GAGs and a lower amount of dermatan sulfate were detected in the A. vera‐treated group on the 14^th day compared with the control. Also at 14 days post‐surgery, a lower dichroic ratio in toluidine blue stained sections was observed in A. vera‐treated tendons compared with the control. No differences were observed in the chondroitin‐6‐sulfate and TGF‐β1 levels between the groups, and higher amount of non‐collagenous proteins was detected in the A. vera‐treated group on the 21^st day, compared with the control group. No differences were observed in the number of fibroblasts, inflammatory cells and blood vessels between the groups. The application of A. vera during tendon healing modified the arrangement of GAGs and increased the content of GAGs and non‐collagenous proteins. Microsc. Res. Tech. 77:964–973, 2014. © 2014 Wiley Periodicals, Inc.     

Differential interference contrast‐photothermal microscopy in nanospace: impacts of systematic parameters

Differential interference contrast‐photothermal microscopy (DIC‐PTM), as a promising tool for trace analysis of nonfluorescent compounds, suffered low sensitivity in nanospace especially for aqueous samples, due to the poor thermophysical property of water and the unoptimised configuration. To improve its performance, a five‐layer DIC‐PTM model is built and influences of different parameters on the photothermal signal are investigated. The initial phase shift φ₀ between two branches of the probe beam is found to be a key factor determining the detection sensitivity and response linearity: at a large φ₀ (≤π/2) both a high sensitivity and a good linearity can be achieved, while a high signal‐to‐noise ratio occurs at a small φ₀. The steady‐state photothermal phase shift φ_dc has little impact on the linearity, which, however, is greatly influenced by the range of periodic photothermal phase shift φ_ac. By introducing two coatings into a nanospace to confine the photothermal effect within and around the sample, the sensitivity can be enhanced from a few times to over 100 times. On an optimised DIC‐PTM configuration and chip structure, detection limit down to 10^?3 cm^?1 (or 40 molecules in a detection volume of 0.2 fL) was achieved in a 300‐nm‐thick nanospace. This work paves a way for optimising the DIC‐PTM and chip structure for sensitive detection of analytes in nanospaces.     

Label‐free optical detection of age‐related and diabetic oxidative damage in human aqueous humors

In this study, we investigate the biochemical characteristics of oxidative stress in age‐related macular degeneration (AMD) and diabetic retinopathy (DR) by analyzing aqueous humors. Nondiabetic cataract aqueous humor was used as the control. The level of oxidative damage was evaluated based on changes in Raman spectral intensity. Seven prominent peaks were detected at 1002, 1043, 1062, 1352, 1419, 1454, and 1656 cm^?1. We proposed four multimodal biomarkers to distinguish these peaks based on the ratios of Raman intensities in two wavelengths, including CHO (C–O stretching or C–O–H bending modes), AG (adenine and guanine), PRO‐AG (protein and AG), and PHEα (phenylalanine symmetric ring breath and amide I α‐helix) markers. The presence of oxidative damage was detected by CHO and AG markers associated with C–O stretching, C–O–H bending modes in carbohydrates (1043 cm^?1), and the nucleic acids adenine and guanine (1352 cm^?1), respectively. DR‐related oxidative damage was identified by PRO‐AG and PHEα markers associated with adenine, guanine, and protein components (1419 and 1454 cm^?1) and amide I α‐helix protein structure (1656 cm^?1), respectively. AMD‐related oxidative damage was identified by four biomarkers. Four multimodal biomarkers with simple linear threshold values achieved high sensitivity of 100% and high specificity of 100% for classifying oxidative stress‐induced AMD and DR diseases. Therefore, Raman‐based label‐free optical detection is effective for detecting the presence of age‐related or diabetic oxidative damage in aqueous humor.     

Light exposure and cell viability in fluorescence microscopy

Test systems for measuring cell viability in optical microscopy (based on colony formation ability or lysosomal integrity) were established and applied to native cells as well as to cells incubated with fluorescence markers or transfected with genes encoding for fluorescent proteins. Human glioblastoma and Chinese hamster ovary cells were irradiated by various light doses, and maximum doses where at least 90% of the cells survived were determined. These tolerable light doses were in the range between 25 J cm^?2 and about 300 J cm^?2 for native cells (corresponding to about 250?3000 s of solar irradiance and depending on the wavelength as well as on the mode of illumination, e.g. epi‐ or total internal reflection illumination) and decreased to values between 50 J cm^?2 and less than 1 J cm^?2 upon application of fluorescent markers, fluorescent proteins or photosensitizers. In high‐resolution wide field or laser scanning microscopy of single cells, typically 10?20 individual cell layers needed for reconstruction of a 3D image could be recorded with tolerable dose values. Tolerable light doses were also maintained in fluorescence microscopy of larger 3D samples, e.g. cell spheroids exposed to structured illumination, but may be exceeded in super‐resolution microscopy based on single molecule detection.     

Influence of fluorescent dye on physical–mechanical properties of luting cements for confocal microscopy analysis

Aims: To evaluate the influence of a fluorescent dye (rhodamine B) on the physical and mechanical properties of three different luting cements: a conventional adhesive luting cement (RelyX ARC, 3M/ESPE), a self‐adhesive luting cement (RelyX U‐200, 3M/ESPE), and a self‐etching and self‐adhesive luting cement (SeT PP, SDI). Materials and Methods: The cements were mixed with 0.03 wt% rhodamine B, formed into bar‐shaped specimens (n = 10), and light cured using an LED curing unit (Radii, SDI) with a radiant exposure of 32 J/cm². The Knoop hardness (KHN), flexural strength (FS), and Young's modulus (YM) analyses were evaluated after storage for 24 h. Results: Outcomes were subjected to two‐way ANOVA and Tukey's test (P = 0.05) for multiple comparisons. No significant differences in FS or YM were observed among the tested groups (P ≥ 0.05); the addition of rhodamine B increased the hardness of the luting cements tested. Conclusion: The addition of a fluorescent agent at 0.03 wt% concentration does not negatively affect the physical–mechanical properties of the luting cement polymerization behavior. Microsc. Res. Tech. 77:986–988, 2014. © 2014 Wiley Periodicals, Inc.     

Dislocations in nanostructured two‐phase Fe30Ni20Mn20Al30

In a previous study, the dislocations in Fe₃₀Ni₂₀Mn₂₅Al₂₅ (at. %), which consist of 50 nm wide alternating b.c.c. and B2 phases, were shown to have a/2<111> Burgers vectors after room temperature deformation. The dislocations were found to glide in pairs on both {110} and {112} slip planes and were relatively widely separated in the b.c.c. phase, where the dislocations were uncoupled, and closely spaced in the B2 phase, where the dislocations were connected by an anti‐phase boundary. In this article, we analyze the dislocations in the two ～5 nm‐wide B2 phases in a related two‐phase alloy Fe₃₀Ni₂₀Mn₂₀Al₃₀, with compositions Fe‐23Ni‐21Mn‐24Al and Fe‐39Ni‐12Mn‐34Al, compressed to ～3% strain at a strain rate 5 × 10^?4 s^?1 at 873 K (the lowest temperature at which substantial plastic flow was observed). It is shown that slip occursby the glide of a<100> dislocations. A review of the literature suggests that the differences in the observed slip vector between these B2 phases could be due to the differences in composition, differences in deformation temperature, or possibly both. Microsc. Res. Tech. 76:263–267, 2013. © 2013 Wiley Periodicals, Inc.     

Developing of N‐(4‐methylpyrimidine‐2‐yl)methacrylamide Langmuir–Blodgett thin film chemical sensor via quartz crystal microbalance technique

In the present work, the characterization and gas sensing properties of newly synthesized N‐(4‐methylpyrimidine‐2‐yl)methacrylamide ( N‐MPMA ) monomer Langmuir–Blodgett (LB) thin films were investigated. The UV–visible spectroscopy, quartz crystal microbalance (QCM), and atomic force microscopy were utilized to characterize N‐MPMA LB thin films. The surface behavior of N‐MPMA monolayer was stable and allowed an effective transfer at a surface pressure of 14 mN/m. The mass change/unit area value of the N‐MPMA LB thin film deposited quartz crystal surfaces was investigated. The amount of N‐MPMA LB thin film deposited on the substrate for bilayer was calculated as 228.72 ng (86.31 ng/mm²) and 12.5 Hz frequency shift was observed for each layer of the N‐MPMA film. The kinetic responses of N‐MPMA LB film against chloroform, dichloromethane, benzene, and toluene were measured via QCM system at room temperature. N‐MPMA QCM sensor results displayed that chloroform has the largest frequency shifts compared with the other vapors used in the present work and these results can be illuminating in terms of physical properties of organic vapors.     

Effects of Er:YAG laser on bond strength of self‐etching adhesives to caries‐affected dentin

The erbium:yttrium–aluminum–garnet (Er:YAG) laser may be effective the bond strength of adhesive systems on dentine surfaces, the chemical composition and aggressiveness of adhesive systems in clinical practice. The purpose of this study was to evaluate the effects of the Er:YAG laser system with the bonding ability of two different self‐etching adhesives to caries‐affected dentine in primary molars. Ninety mid‐coronal flat dentine surfaces obtained from sound and caries‐affected human primary dentine were treated with an Er:YAG laser or a bur. The prepared surfaces were restored with an adhesive system (Xeno V; Clearfil S³) and a compomer (Dyract Extra). The restored teeth were sectioned with a low‐speed saw and 162 samples were obtained. The bond strength of the adhesive systems was tested using the micro‐tensile test method. The data were statistically analyzed. A restored tooth in each group was processed for scanning electron microscopy evaluation. The values of the highest bond strength were obtained from the Clearfil S³‐Er:YAG laser‐sound dentine group in all groups. (24.57 ± 7.27 MPa) (P > 0.05). The values of the lowest bond strength were obtained from the Xeno V‐Er:YAG laser‐sound dentine group in all groups (11.01 ± 3.89 MPa). It was determined that the Clearfil S³ increased the bond strength on the surface applied with Er:YAG laser according to the Xeno V. Microsc. Res. Tech. 77:282–288, 2014. © 2014 Wiley Periodicals, Inc.     

Aged garlic extract ameliorates the oxidative stress,histomorphological, and ultrastructural changes of cisplatin‐induced nephrotoxicity in adult male rats

Aim: Aged garlic extract (AGE) is a natural dietary substance having different antioxidant free‐radical‐scavenger compounds that ameliorates the toxicity of the oxidative stress. This study aimed to investigate the effect of AGE on cisplatin (CP)‐induced nephrotoxicity in rats. Materials and Methods: Twenty‐four, adult male Wistar albino rats were randomly divided into four groups namely control, AGE‐treated (a single oral dose of 250 mg/kg/day for 21 days), CP‐treated (a single intraperitoneal dose of 7.5 mg/kg on Day 16), and AGE + CP‐treated (AGE at a dose of 250 mg/kg/once daily for 21 days and a single dose of CP of 7.5 mg/kg intraperitoneally on Day 16). Body weight and absolute and relative kidney weights of each rat were calculated. Serum creatinine, uric acid, and urea levels were determined. Level of malondialdehyde and reduced glutathione and activity of superoxide dismutase and catalase of renal tissues were measured. Renal specimens from each rat were prepared for both light and electron microscopic examinations. Results: Interstitial cell infiltration, hemorrhage, glomerular atrophy, necrosis, and tubular degeneration were observed after CP treatment. Superoxide dismutase and catalase activities and glutathione level were significantly decreased and malondialdehyde level was significantly increased in CP‐treated rats compared with AGE + CP‐treated animals. A remarkable improvement in the histopathological and ultrastructural changes induced by CP in renal tissues was observed in AGE + CP‐treated rats. Conclusion: AGE exhibited antioxidant effect that could ameliorate the nephrotoxic effects of CP. Microsc. Res. Tech. 78:452–461, 2015. © 2015 Wiley Periodicals, Inc.     

Depletion of enteroendocrine and mucus‐secreting cells is associated with colorectal carcinogenesis severity and impaired intestinal motility in rats

This study investigated the relationship between enteroendocrine and mucus‐secreting cells distribution, the severity of colonic mucosal injury and intestinal motility in experimental colorectal carcinogenesis. Using a standardized murine model of colorectal carcinogenesis, eight‐weeks‐old female Wistar rats weighting 147.30 ± 29.15g were randomized into two groups receiving a subcutaneous injection of 0.9% saline (control) or the chemical carcinogen 1,2‐dimethylhydrazine (DMH) at 20 mg/kg per week during 10 weeks. Aberrant crypt foci (ACF) were more frequent in DMH group compared to control group (P < 0.001). The number of enteroendocrine and mucus‐secreting cells, and intestinal motility was reduced in DMH animals (P < 0.05). The distribution of enteric neurons was similar in both groups. In DMH animals there was a direct correlation between colonic motility and distribution of enteroendocrine (R² = 0.68, P < 0.05) and mucus‐secreting cells (R² = 0.77, P < 0.05). Inverse correlation between the number of ACF, mucus‐secreting cells (R² = ?0.57, P < 0.05), and enteroendocrine cells (R² = ?0.74, P < 0.05) was also observed. There was inverse correlation between the severity of the mucosal lesion, the number of mucus‐secreting cells (R² = ?0.83, P < 0.05) and enteroendocrine cells (R² = ?0.96, P < 0.05). There was a direct correlation between nucleolar organizer regions (AgNOR) and ACF number (R² = 0.62; P < 0.01). Inverse correlation was also found between AgNOR, the number of mucus‐secreting cells (R² = ?0.76; P < 0.001), and enteroendocrine cells (R² = ?0.86; P < 0.001). Taken together, the results indicated that colonic malignant transformation is related to depletion of mucus‐secreting and enteroendocrine cells, which was a useful indicator of the evolutionary status of intestinal neoplasm, partially explaining the intestinal motility disorders in the early stages of colorectal carcinogenesis. Microsc. Res. Tech. 79:3–13, 2016. © 2015 Wiley Periodicals, Inc.     

Nitric oxide regulates mitochondrial re‐modelling in interscapular brown adipose tissue: ultrastructural and morphometric‐stereologic studies

As a complex, cell‐specific process that includes both division and clear functional differentiation of mitochondria, mitochondriogenesis is regulated by numerous endocrine and autocrine factors. In the present ultrastructural study, in vivo effects of l ‐arginine‐nitric oxide (NO)‐producing pathway on mitochondriogenesis in interscapular brown adipose tissue (IBAT) were examined. For that purpose, adult Mill Hill hybrid hooded rats were receiving l ‐arginine, a substrate of NO synthases (NOSs), or N^ω‐nitro‐l ‐arginine methyl ester (l ‐NAME), an inhibitor of NOSs, as drinking liquids for 45 days. All experimental groups were divided into two sub‐groups – acclimated to room temperature and cold. IBAT mitochondria were analyzed by transmission electron microscopy and stereology. l ‐Arginine treatment acted increasing the number of mitochondrial profiles per cell profile, as well as volume fraction of mitochondria per cell volume in animals maintained at room temperature. Cold‐induced enhancement of number of mitochondrial profiles per cell profile was additionally increased in l ‐arginine‐treated rats. Ultrastructural examinations of l ‐arginine‐treated cold‐acclimated animals clearly demonstrated thermogenically active mitochondria (larger size, lamellar, more numerous and well‐ordered cristae in their profiles), which however were inactive in l ‐arginine‐receiving animals kept at room temperature (small mitochondria, tubular cristae). By contrast, l ‐NAME treatment of rats acclimated to room temperature induced mitochondrial alterations characterized by irregular shape, short disorganized cristae and megamitochondria formation. These results showed that NO is a necessary factor for mitochondrial biogenesis and that it acts intensifying this process, but NO alone is not a sufficient stimulus for in vivo induction of mitochondriogenesis in brown adipocytes.