Expression of high-molecular-mass neurofilament protein in horse (Equus caballus) spinal ganglion neurons

Spinal ganglion (SG) neurons are subdivided, on the basis of their cytoplasmic aspect at light and electron microscopy, into dark (D) and light (L) neurons. Numerous efforts have been made to find specific markers able to identify D and L neuronal cytotypes. The isolectin B4 (IB4), utilized to identify nonpeptidergic D neurons in mice, unfortunately, has not proved as effective in other species. The 200-kDa neurofilament protein (NF200) is considered as a typical marker of L neurons in the rat, cat, and chick. The aim of this study was to analyze the histological, morphometric, and neurochemical characteristic of NF200-immunoreactive (IR) horse SG neurons, to better characterize them morphologically and functionally. NF200-IR neurons showed two levels (strong and weak) of staining intensity. Most (84%) strongly stained NF200-IR neurons corresponded to L neurons, and showed similar bimodality as in the size distribution study, which seems to indicate a third population of neurons, in addition to the two populations (small and large) previously identified. In triple-staining experiments where NF200 was colocalized with IB4, substance P (SP), and neuronal nitric oxide synthase (nNOS) neuronal markers, most NF200-IR neurons were single stained. On the contrary, most IB4-, SP-, and nNOS-stained neurons were triple labeled and almost equally subdivided between strong and weak NF200-IR with the latter being always smaller in size than strong NF200-IR neurons. In conclusion, horse SG neurons display significant morphometric and neurochemical differences compared with those of rodents.     

Innervation of the carotid body: Immunohistochemical,denervation, and retrograde tracing studies

This review presents information about multiple neurochemical substances in the carotid body. Nerve fibers around blood vessels and glomus cells within the chemoreceptive organ contain immunoreactivities (IR) for tyrosine hydroxylase (TH), calcitonin gene-related peptide (CGRP), substance P (SP), galanin (GAL), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY), calretinin (CR), calbindin D-28k (CB), parvalbumin (PV), and nitric oxide synthase (NOS). Parasympathetic neurons scattered around the carotid body contain VIP, choline acetyltransferase, and vanilloid receptor 1-like receptor. In the mammalian carotid body, transection of the carotid sinus nerve (CSN) causes the absence or decrease of CGRP-, SP-, and NOS-immunoreactive (IR) nerve fibers, whereas all NPY-IR nerve fibers disappear after removal of the superior cervical ganglion. Most VIP-IR nerve fibers disappear but a few persist after sympathetic ganglionectomy. In addition, the CSN transection appears to cause the acquisition of GAL-IR in originally immunonegative glomus cells and nerve fibers within the rat carotid body. On the other hand, 4%, 25%, 17%, and less than 1% of petrosal neurons retrogradely labeled from the rat CSN contain TH-, CGRP-, SP-, and VIP-IR, respectively. In the chicken carotid body, many CGRP- and SP-IR nerve fibers disappear after vagus nerve transection or nodose ganglionectomy. GAL-, NPY-, and VIP-IR nerve fibers mostly disappear after removal of the 14th cervical ganglion of the sympathetic trunk. The origin and functional significance of the various neurochemical substances present in the carotid body is discussed.     

Automated identification of neurons and their locations

Individual locations of many neuronal cell bodies (>10⁴) are needed to enable statistically significant measurements of spatial organization within the brain such as nearest‐neighbour and microcolumnarity measurements. In this paper, we introduce an Automated Neuron Recognition Algorithm (ANRA) which obtains the (x, y) location of individual neurons within digitized images of Nissl‐stained, 30 μm thick, frozen sections of the cerebral cortex of the Rhesus monkey. Identification of neurons within such Nissl‐stained sections is inherently difficult due to the variability in neuron staining, the overlap of neurons, the presence of partial or damaged neurons at tissue surfaces, and the presence of non‐neuron objects, such as glial cells, blood vessels, and random artefacts. To overcome these challenges and identify neurons, ANRA applies a combination of image segmentation and machine learning. The steps involve active contour segmentation to find outlines of potential neuron cell bodies followed by artificial neural network training using the segmentation properties (size, optical density, gyration, etc.) to distinguish between neuron and non‐neuron segmentations. ANRA positively identifies 86 ± 5% neurons with 15 ± 8% error (mean ± SD) on a wide range of Nissl‐stained images, whereas semi‐automatic methods obtain 80 ± 7%/17 ± 12%. A further advantage of ANRA is that it affords an unlimited increase in speed from semi‐automatic methods, and is computationally efficient, with the ability to recognize ～100 neurons per minute using a standard personal computer. ANRA is amenable to analysis of huge photo‐montages of Nissl‐stained tissue, thereby opening the door to fast, efficient and quantitative analysis of vast stores of archival material that exist in laboratories and research collections around the world.     

Noninvasive assessment of the iridial microcirculation in rats using sidestream dark field imaging

Sidestream dark field imaging represents a novel, noninvasive method to study the microcirculation in humans and animals. To‐date, it has been used extensively in various peripheral tissues (e.g. sublingual area, intestinal mucosa), however no data for the ocular vasculature, including the iridial microcirculation, are currently available. Therefore, the aim of this study was to examine the reliability and reproducibility of sidestream dark field imaging within the iridial microcirculation in experimental animals. Male Lewis rats were anaesthetized and the iris microvasculature was observed using an sidestream dark field probe gently placed against a cover slip covering the right eye. All video sequences recorded were analysed off‐line by using AVA 3.0 software (MicroVision Medical, Amsterdam, The Netherlands). Results are expressed as mean (±SE) or median (interquartile range). Clear images were recorded from each animal and the total number of analysable video sequences was 50. All raw data for selected vessel density parameters passed normality test. The total all and small vessel density (in mm mm^‐2) were 22,6 (±0,58) and 19,6 (±0,68), respectively. The perfused all and small vessel density were 20,9 (±0,61) and 19,1 (±0,65), respectively. The mean values of all iris vessel density parameters are shown in Figure 4 . The DeBacker Score (n/mm) was 15,2 (±0,45), the proportion of perfused vessel was 94,5% (89,8–99,1%), and the MFI was 3 points (3–3). Taken together, these results indicate that SDF imaging provides a reliable and noninvasive method to examine the iridial microvascular bed in vivo and, thus, may provide unique opportunities for the study of the iridial vascular network in various experimental and clinical settings and disease models.

Figure 4 Open in figure viewer PowerPoint Vessel Density. TAVD = Total all vessel density; PAVD = Perfused all vessel density; TSVD = Total small vessel density; PSVD = Perfused small vessel density. Data are shown as a mean (±SE), small vessels are referred to diameter ≤ 25 um.     

In situ assessment of the renal microcirculation in mechanically ventilated rats using sidestream dark‐field imaging

For microcirculation research there is a need for baseline data and feasibility protocols describing microcirculation of various organs. The aim of our study was to examine the reliability and reproducibility of sidestream dark‐field (SDF) imaging within the renal cortical microcirculation in rats. Renal microcirculation was observed using SDF probe placed on the exposed renal surface via the upper midline laparotomy. Video sequences recorded intermittently in short apneic pauses were analyzed off‐line by using AVA 3.0 software (MicroVision Medical, Amsterdam, the Netherlands). Results are expressed as mean (SD) or median (25–75% percentiles). We obtained 60 clear sequences from all recorded analyzable videos from all the animals. The total small vessel and all vessel density (in mm.mm^–2) were (28.79 ± 0.40) and (28.95 ± 0.40), respectively. The perfused small and all vessel density were (28.79 ± 0.40) and (28.95 ± 0.40), respectively. The DeBacker Score was (19.14 ± 0.43), the proportion of perfused vessels was 100% (100–100%) and the microvascular flow index was 3.49 (3–3.75). We conclude SDF imaging provides a reliable method to examine the renal microvascular bed in vivo and thus can be used for the study of the renal cortical vascular network in various experimental diseases models and clinical settings.     

Region‐specific localization of NOS isoforms and NADPH‐diaphorase activity in the intratesticular and excurrent duct systems of adult domestic cats (Felis catus)

Nitric oxide (NO) is produced by nitric oxide synthases (NOSs) and plays an important role in all levels of reproduction from the brain to the reproductive organs. Recently, it has been discovered that all germ cells and Leydig cells in the cat testis exhibit stage‐dependent nuclear and cytoplasmic endothelial (eNOS) and inducible (iNOS)‐NOS immunoreactivity and cytoplasmic nicotinamide adenine dinucleotide phosphate‐diaphorase (NADPH‐d) reactivity. As a continuation of this finding, in this study, cellular localization of NADPH‐d and immunolocalization and expression of all three NOS isoforms were investigated in the intratesticular (tubuli recti and rete testis), and excurrent ducts (efferent ductules, epididymal duct and vas deferens) of adult cats using histochemistry, immunohistochemistry and western blotting. NADPH‐d activity was found in the midpiece of the spermatozoa tail and epithelial cells of all of ducts, except for nonciliated cells of the efferent ductules. Even though the immunoblotting results revealed similar levels of nNOS, eNOS and iNOS in the caput, corpus and cauda segments of epididymis and the vas deferens, immunostainings showed cell‐specific localization in the efferent ductules and region‐ and cell‐specific localization in the epididymal duct. All of three NOS isoforms were immunolocalized to the nuclear membrane and cytoplasm of the epithelial cells in all ducts, but were found in the tail and the cytoplasmic droplets of spermatozoa. These data suggest that NO/NOS activity might be of importance not only for the functions of the intratesticular and excurrent ducts but also for sperm maturation. Microsc. Res. Tech. 79:192–208, 2016. © 2016 Wiley Periodicals, Inc.     

Micromorphology analysis of TiO2 thin films by atomic force microscopy images: The influence of postannealing

This work describes an analysis of titanium dioxide (TiO₂) thin films prepared on silicon substrates by direct current (DC) planar magnetron sputtering system in O₂/Ar atmosphere in correlation with three‐dimensional (3D) surface characterization using atomic force microscopy (AFM). The samples were grown at temperatures 200, 300, and 400°C on silicon substrate using the same deposition time (30 min) and were distributed into four groups: Group I (as‐deposited samples), Group II (samples annealed at 200°C), Group III (samples annealed at 300°C), and Group IV (samples annealed at 400°C). AFM images with a size of 0.95 μm × 0.95 μm were recorded with a scanning resolution of 256 × 256 pixels. Stereometric analysis was carried out on the basis of AFM data, and the surface topography was described according to ISO 25178‐2:2012 and American Society of Mechanical Engineers (ASME) B46.1‐2009 standards. The maximum and minimum root mean square roughnesses were observed in surfaces of Group II (Sq = 7.96 ± 0.1 nm) and Group IV (Sq = 3.87 ± 0.1 nm), respectively.     

Comparison of methods for quantifying dental wear caused by erosion and abrasion

Various methods have been applied to evaluate the effect of erosion and abrasion. So, the aim of this study was to check the applicability of stylus profilometry (SP), surface hardness (SH) and focus‐variation 3D microscopy (FVM) to the analysis of human enamel and dentin subjected to erosion/abrasion. The samples were randomly allocated into four groups (n = 10): G1‐enamel/erosion, G2‐enamel/erosion plus abrasion, G3‐dentin/erosion, and G4‐dentin/erosion plus abrasion. The specimens were selected by their surface hardness, and they were subjected to cycles of demineralization (Coca‐Cola®‐60 s) and remineralization (artificial saliva‐60 min). For groups G2 and G4, the remineralization procedures were followed by toothbrushing (150 strokes). The above cycle was repeated 3×/day during 5 days. The samples were assessed using SH, SP, and FVM. For each substrate, the groups were compared using an unpaired t‐test, and Pearson correlation coefficients were calculated (α = 5%). For enamel, both profilometry technique showed greater surface loss when the erosion and abrasion processes were combined (P <0.05). The correlation analysis did not reveal any relationships among SH, SP, and FVM to G2 and G4. There were significant correlation coefficients (–0.70 and –0.67) for the comparisons between the FVM and SH methods in enamel and dentin, respectively, in G1 and G3. Choosing the ideal technique for the analysis of erosion depends on the type of dental substrate. SP was not sufficiently sensitive to measure the effects on dentin of erosion or erosion/abrasion. However, SP, FVM and SH were adequate for the detection of tissue loss and demineralization in enamel. Microsc. Res. Tech., 2013. © 2012 Wiley Periodicals, Inc.     

Detailed characterisation of sulphur and nitrogen components in a multifunctional,ashless lubricant additive by spectroscopic and chromatographic techniques

The work reported here used infrared (IR), multinuclear nuclear magnetic resonance (NMR), and chromatographic techniques to identify and characterise a commercial sulphur‐ and nitrogen‐containing ashless multifunctional additive. The proper assignment of signals in the ¹H/¹³C NMR and IR spectra of the sample has facilitated the identification of different types of sulphur‐ and nitrogen‐containing components. The methodology involves investigation by NMR and IR spectroscopies, thin‐layer chromatography (TLC) and high‐performance liquid chromatogaphy (HPLC) techniques to elucidate the types of components present. This requires the generation of NMR and IR spectral data for standard compounds of alkyl sulphides with different sulphur content and alkyl chain, and nitrogen and sulphur components such as thiadiazole, imidazoline, triazole, etc., and spectral comparison with the spectra of the unknown sample. Further, these components have been separated by silica/alumina column chromatographic and preparative TLC techniques and subsequently analysed for their exact chemical structure by spectroscopic techniques. The combined spectroscopic and chromatographic analyses have yielded the presence of four types of components: di‐t‐octyl polysulphide, di‐t‐octyl thiadiazole, a component containing amine ether functionality, and alkyl amine salts of mono/dialkyl phosphoric acid.     

弱激光对神经元瞬时外向钾通道电流特性的影响

应用全细胞膜片钳技术,研究了急性分离的大鼠海马CA3区锥体神经细胞在波长670 nm、功率5 mW的半导体激光器照射时,其瞬时外向钾通道电流特性。实验发现:弱激光对瞬时外向钾电流I_A有抑制作用,5 min激光抑制作用达到稳定,去极化至+50 mV时抑制百分比为(40.13±5.19)%(n=10);弱激光对I_A的抑制作用呈现电压依赖性和可逆性,对照组、照射组和恢复组的最大激活电流密度分别为(398.55±36.49)pA/pF、(238.62±30.78)pA/pF(n=10,P<0.01)和(354.08±35.16)pA/pF(n=10,P>0.05);激光作用可显著地影响瞬时外向钾通道电流的稳态激活和失活过程,对照组和激光照射组通道的半数激活电压分别为(-27.05±4.53) mV和(-2.10±3.14) mV(n=10, P<0.01),斜率因子分别为(-26.71±6.15) mV和(-20.70±4.38) mV(n=10,P<0.05),半数失活电压分别为(-70.49±7.21) mV和(-81.27±6.26) mV(n=10, P<0.01),斜率因子分别为(9.47±3.54) mV和(9.58±3.02) mV(n=10,P>0.05)。结果表明:弱激光作用海马神经元可以改变其瞬时外向钾通道特性,从而影响动作电位的形成和发放,调节神经元的生理功能,有利于受损神经元的恢复和再生。     

Reflectance confocal‐laser‐scanning microscopy in vivo assessments of cigarette‐induced dynamic alterations of cutaneous microcirculation on histomorphological level

Objective: Until now, high resolution reflectance confocal‐laser‐scanning microscopy (CLSM) was used for observation of cutaneous morphology in vivo and in real time. We hypothesized that CLSM also allows observation of dynamic processes of cutaneous microcirculation. Methods: Reflectance CLSM (Vivascope1500; Lucid, Rochester, NY) was performed in 24 young male habitual smokers (23 years, range: 19–26, body mass index 23.9 ± 4.04) with relatively limited cigarette exposure (mean: 3.1 ± 2.4 pack‐years). Eight matched nonsmokers served as controls. The quantitative blood cell flow and the diameter of capillary loops were determined prior (baseline), during, as well as 5 and 10 min after smoking. Results: Baseline value for blood cell flow was 55.50 ± 2.33 cells/min, and decreased over 45% during smoking (30.43 ± 3.76/min; P = 0.02). They were still 22% lower (43.33 ± 2.45/min; P = 0.01) 5 min after smoking and exceeded baseline values 10 min after smoking by 13% (63.00 ± 3.10/min; P > 0.05). The baseline values for capillary loop diameter (9.03 ± 0.22 μm) decreased by 21% (7.18 ± 0.28 μm; P = 0.03) during smoking, remained about 9% (8.23 ± 0.18 μm; P = 0.01) lower 5 min after smoking and exceeded baseline values insignificantly by 4% (9.38 ± 0.28 μm; P > 0.05) 10 min after smoking. There were no significant differences to the controls. Conclusion: Reflectance CLSM enables qualitative and quantitative observation of dynamic processes of cutaneous microcirculation on histomorphological level. Microsc. Res. Tech., 2009. © 2008 Wiley‐Liss, Inc.     

Dentin erosion by whitening mouthwash associated to toothbrushing abrasion: A focus variation 3D scanning microscopy study

The aim of this study was to determine the erosive potential of hydrogen peroxide (HP) containing mouthwash on dentin assessed by Focus variation three‐dimensional (3D) microscopy. Twenty dentin slabs were selected and randomly allocated into two groups (n = 10): DW—Distilled water (pH = 7.27) and HP—1.5% (pH = 3.78). Each specimen was cyclically demineralized (4 × 60 s/day, 10 days) with HP or DW and brushed 3×/day (200 g, 150 strokes—toothpaste with 1,450 ppmF as NaF). Between the challenges, the specimens were exposed to artificial saliva. Afterward, dentin loss was analyzed using focus variation 3D microscopy, and the data were submitted to unpaired t‐test (α = 0.05). Statistically significant difference was found between the mean wear rate (μm, ±SD) of HP (1.98 ± 0.51) and DW (1.45 ± 0.39). The results suggest that the use of HP‐containing mouthwash associated to brushing may increase the risk of tissue loss and focus variation 3D microscopy may be used as a technique for quantifying dental wear. Microsc. Res. Tech. 76:904–908, 2013. © 2013 Wiley Periodicals, Inc.     

Morphometric,quantitative, and three‐dimensional analysis of the heart muscle fibers of old rats: Transmission electron microscopy and high‐resolution scanning electron microscopy methods

This research investigated the morphological, morphometric, and ultrastructural cardiomyocyte characteristics of male Wistar rats at 18 months of age. The animals were euthanized using an overdose of anesthesia (ketamine and xylazine, 150/10 mg/kg) and perfused transcardially, after which samples were collected for light microscopy, transmission electron microscopy, and high‐resolution scanning electron microscopy. The results showed that cardiomyocyte arrangement was disposed parallel between the mitochondria and the A‐, I‐, and H‐bands and their M‐ and Z‐lines from the sarcomere. The sarcomere junction areas had intercalated disks, a specific structure of heart muscle. The ultrastructural analysis revealed several mitochondria of various sizes and shapes intermingled between the blood capillaries and their endothelial cells; some red cells inside vessels are noted. The muscle cell sarcolemma could be observed associated with the described structures. The cardiomyocytes of old rats presented an average sarcomere length of 2.071 ± 0.09 μm, a mitochondrial volume density (Vv) of 0.3383, a mitochondrial average area of 0.537 ± 0.278 μm², a mitochondrial average length of 1.024 ± 0.352 μm, an average mitochondrial cristae thickness of 0.038 ± 0.09 μm and a ratio of mitochondrial greater length/lesser length of 1.929 ± 0.965. Of the observed mitochondrial shapes, 23.4% were rounded, 45.3% were elongated, and 31.1% had irregular profiles. In this study, we analyzed the morphology and morphometry of cardiomyocytes in old rats, focusing on mitochondria. These data are important for researchers who focus the changes in cardiac tissue, especially changes owing to pathologies and drug administration that may or may not be correlated with aging. Microsc. Res. Tech., 2013. © 2012 Wiley Periodicals, Inc.     

3D‐quantum interferometer using silicon microring‐embedded gold grating circuit

A 3D (three‐dimensional) quantum interferometer consisting of a silicon microring circuit proposed. The interferometer based on the electron spin cloud projections generated by microring‐embedded gold grating. The electron cloud oscillations result from the excitation of the gold grating at the center of the silicon microring by the dark soliton pulse of 1.50 μm center wavelength. The electron cloud spin‐down, spin‐up automatically formed in the two axes (x, y, respectively) and propagated along the z‐axis. In this proposal, the sensing mechanism of the circuit is manipulated by varying the reflector gold lengths of the sensing arm. The electron cloud spin coupled and changed by changing the gold lengths. The sensitivity measurement of the 3D quantum interferometer for three gold layer lengths of 100 nm, 500 nm, and 1,000 nm is (47.62 nm fs^?1, ±0.4762 fs^?1, ±0.01 nm^?1), (238.10 nm fs^?1, ±0.4762 fs^?1, ±0.002 nm^?1), (476.20 nm fs^?1, ±0.4762 fs^?1, ±0.001 nm^?1), respectively. The used circuit parameters are the real ones that can be fabricated by the currently available technology. Moreover, the silicon micro ring circuit acts as a plasmonic antenna, which can apply for wireless quantum communication. The electron cloud spin projection space–time control can apply for quantum cellular automata.     

A rabbit maxillary sinus model with simultaneous customized‐implant placement: Comparative microscopic analysis for the evaluation of surface‐treated implants

We describe the use of a rabbit maxillary sinus model, characterized by thin osseous tissue and low bone density, for the evaluation of surface‐treated implants by histologically and histomorphometrically comparing the osseointegration patterns depending on the surface treatment methods. Twenty rabbits were randomly assigned to two groups of 10 animals, one receiving 5 × 3 mm customized implants (machined, MA or sandblasted and acid etched, SLA) placed in sinus and the other receiving implants placed in a tibia. Histological observation of the implant placed in sinus shows relatively more active new bone formation, characterized by trabecular bone pattern underneath the cortical bone in sinus as compared with that in tibia. Histomorphometric analysis in the rabbits receiving implants in a tibia, the NBIC (%) associated with the SLA surface implant was greater than that associated with the MA implant at 2 weeks (55.63 ± 8.65% vs. 47.87 ± 10.01%; P > 0.05) and at 4 weeks (61.76 ± 9.49% vs. 42.69 ± 10.97%; P < 0.05). Among rabbits receiving implants in a sinus, the NBIC (%) associated with the SLA surface implant was significantly greater than that associated with the MA surface implant both at 2 weeks (37.25 ± 7.27% vs. 20.98 ± 6.42%; P < 0.05) and at 4 weeks (48.82 ± 6.77% vs. 31.51 ± 9.14%; P < 0.05). As a result, we suggest that the maxillary sinus model is an appropriate animal model for assessing surface‐treated implants and may be utilized for the evaluation of surface‐treated implants in poor bone quality environment. Microsc. Res. Tech. 78:697–706, 2015. © 2015 Wiley Periodicals, Inc.     

Histometric and histomorphologic comparison of combustion and ambustion using in vivo reflectance‐confocal microscopy

Background: When combustion and ambustion induce a superficial injury, they are summarized as superficial burns, regardless of the underlying cause. Reflectance‐confocal microscopy (RCM) allows noninvasive imaging of the human skin on morphological features. We hypothesized that combustion and ambustion have different histomorphological effects on the human skin. Methods: Superficial burns caused by combustion (CO‐group, five females, three males; aged 26.8 ± 14.2 years) and caused by ambustion (AM‐group, four females, four males; aged 28.1 ± 13.8 years) were evaluated 24 h after injury. The following parameters were obtained using RCM on injured and noninjured (control) site: horny layer thickness, epidermal thickness, granular cell size, basal layer thickness. Results: Compared with the controls (12.8 ± 2.5 μm), horny layer thickness decreased significantly to 10.6 ± 2.1 μm in the CO‐group, whereas it increased significantly to 17.8 ± 2.8 μm in the AM‐group. The epidermal thickness did not differ significantly in CO‐group (47.9 ± 2.1 μm) and AM‐group (49.0 ± 3.1 μm), however, both increased significantly compared with the controls (42.7 ± 1.6 μm). The basal layer thickness increased more in AM‐group (17.0 ± 1.2 μm) compared to CO‐group (15.4 ± 1.1 μm). Both differed significantly compared with their controls (13.9 ± 0.9 μm). The granular cell size increased significantly in both groups ompared to the controls (721 ± 42 μm), however, a significantly higher increase was observed in CO‐group compared to AM‐group (871 ± 55 μm vs. 831 ± 51 μm). Conclusions: RCM evaluates significant histomorphological differences in superficial burns caused by combustion and ambustion. The term “superficial burn” should consider the underlying cause and thus supplemented by the term “combustion” or “ambustion.” Microsc. Res. Tech., 2010. © 2009 Wiley‐Liss, Inc.     

Leucine enkephalin—A mass spectrometry standard

The present article reviews the mass spectrometric fragmentation processes and fragmentation energetics of leucine enkephalin, a commonly used peptide, which has been studied in detail and has often been used as a standard or reference compound to test novel instrumentation, new methodologies, or to tune instruments. The main purpose of the article is to facilitate its use as a reference material; therefore, all available mass spectrometry‐related information on leucine enkephalin has been critically reviewed and summarized. The fragmentation mechanism of leucine enkephalin is typical for a small peptide; but is understood far better than that of most other compounds. Because ion ratios in the MS/MS spectra indicate the degree of excitation, leucine enkephalin is often used as a thermometer molecule in electrospray or matrix‐assisted laser desorption ionization (ESI or MALDI). Other parameters described for leucine enkephalin include collisional cross‐section and energy transfer; proton affinity and gas‐phase basicity; radiative cooling rate; and vibrational frequencies. The lowest‐energy fragmentation channel of leucine enkephalin is the MH⁺ → b₄ process. All available data for this process have been re‐evaluated. It was found that, although the published E_a values were significantly different, the corresponding Gibbs free energy change showed good agreement (1.32 ± 0.07 eV) in various studies. Temperature‐ and energy‐dependent rate constants were re‐evaluated with an Arrhenius plot. The plot showed good linear correlation among all data (R² = 0.97), spanned over a 9 orders of magnitude range in the rate constants and yielded 1.14 eV activation energy and 10^11.0 sec^?1 pre‐exponential factor. Accuracy (including random and systematic errors, with a 95% confidence interval) is ±0.05 eV and 10^±0.5 sec^?1, respectively. The activation entropy at 470 K that corresponds to this reaction is ?38.1 ± 9.6 J mol^?1 K^?1. We believe that these re‐evaluated values are by far the most accurate activation parameters available at present for a protonated peptide and can be considered as “consensus” values; results on other processes might be compared to this reference value. © 2010 Wiley Periodicals, Inc., Mass Spec Rev 30:298–320, 2011     

Morphological,microscopic, and physicochemical studies of Diospyros montana

Adulteration is the root cause of producing not only a chemically and pharmacologically inferior but also in some instances hazardous or poisonous drug. Despite availability of several techniques, microscopy and physicochemical analyses are the most practical approaches for crude drug authentication. Hence, the present study aimed to evaluate morphological, microscopic, and physicochemical properties of root, bark, leaf, and fruit of Diospyros montana Morphological properties were determined by sensory organs, whereas microscopic features of cross‐sections and powders were determined by light and scanning electron microscopy. The proximate and fluorescence analyses were performed using the standard guidelines. The physical examination of fresh, shade‐dried, and powdered material showed no significant change in color. The identifying cellular structures included cuboidal cork, pitted tracheids, scalariform, reticulate and spiral xylary vessels, and rosettes, raphide, and cuboidal calcium oxalate crystals. The stomatal number, stomatal index, vein‐islet and vein‐termination number, and palisade ratio in the leaf were found to be 293.91 ± 32.68 mm^?2, 64.18 ± 3.42%, 22.00 ± 3.81 mm^?2 and 38.40 ± 5.81 mm^?2, and 3.85 ± 0.60, respectively. Total ash, acid insoluble ash, water soluble ash and sulfated ash of leaf (9.00 ± 0.50%, 1.67 ± 0.23%, 2.00 ± 0.22% and 14.50 ± 0.99%, respectively), foaming index of bark and root (111.11 ± 2.11), and swelling index of fruit (19.00 ± 3.45) were higher than the other parts. The powder of different parts showed characteristic colors in the daylight and UV light upon treatment with various regents. The plant was found to be rich in saponins, fibers, and flavonoids. The results of the present study may serve as identifiers of different parts of Diospyros montana.     

Origin and Co-localization of nitric oxide synthase, CGRP, PACAP, and VIP in the cerebral circulation of the rat

The origin of perivascular nerve fibres storing nitric oxide synthase (NOS) and co-localisation with perivascular neuropeptides were examined in the rat middle cerebral artery (MCA) by retrograde tracing with True Blue (TB) in combination with immunocytochemistry. Application of TB to the proximal part of the middle cerebral artery labelled nerve cell bodies ipsilaterally in the trigeminal, sphenopalatine, otic, and superior cervical ganglia. A few labelled cell bodies were seen contralaterally, suggesting bilateral innervation. In the parasympathetic sphenopalatine and otic ganglia, numerous TB-labelled cell bodies contained neuronal NOS (C- and N-terminal), vasoactive intestinal peptide (VIP), and pituitary adenylate cyclase activating peptide (PACAP). In the trigeminal ganglion, almost all TB-labelled cell bodies contained calcitonin gene-related peptide (CGRP) but only a few cells contained NOS. In the superior cervical ganglion, the majority of the TB-labelled nerve cells contained neuropeptide Y (NPY) but none of them contained NOS. Removal of the ipsilateral sphenopalatine ganglion caused a slight reduction in the number of perivascular VIP-, PACAP-, and NOS-containing fibres after 3 days in the MCA while there was no difference at 2 and 4 weeks after the denervation as compared to control. This indicates that the parasympathetic VIP-, PACAP-, and NOS-immunoreactive nerve fibres in the rat MCA originate from several sources.     

Presence of voids after warm vertical compaction and single‐cone obturation in band‐shaped isthmuses using micro‐computed tomography: A phantom study

The present micro‐computed tomography (micro‐CT) study compared the presence of voids in the band‐shaped isthmuses obturated by warm vertical compaction (WVC) and single‐cone (SC) techniques. Twenty mesial roots from mandibular first molar teeth showing a band‐shaped isthmus were selected and assigned into two groups based on their preoperative anatomical dimensions (n = 10), according to the filling technique: WVC or SC. Post‐filling micro‐CT scanning was performed. The percentage volume of root canal filling materials and voids were calculated and data were analyzed using the Mann–Whitney U test with a significance level of 5 and 95% confidence interval. Mean percentage volumes of filling materials and voids were 86.88 ± 8.53 and 13.11 ± 8.53 for the WVC group, 84.39 ± 8.30 and 15.60 ± 8.30 for the SC group, respectively, with no significant difference between them (p > .05). Neither WVC nor SC produced void‐free root canal fillings in the band‐shaped isthmuses and both techniques resulted in a similar quality of root canal filling.