Microscopic evidences that bone marrow mononuclear cell treatment improves sciatic nerve regeneration after neurorrhaphy

Cell therapy constitutes a possibility for improving nerve regeneration, increasing the success of nerve repair. We evaluate the use of mononuclear cells in the regeneration of the sciatic nerve after axotomy followed by end‐to‐end neurorrhaphy. Forty adult male Wistar rats (250–300 g) were divided into four groups: (1) sham, (2) neurorrhaphy: the sciatic nerve was sectioned and repaired using epineural sutures, (3) culture medium: after the suture, received an injection of 10 μL of culture medium into the nerve, and (4) mononuclear cell: after the suture, a concentration of 3 × 10⁶ of mononuclear cell was injected in epineurium region. Mononuclear cells were obtained from the bone marrow aspirates and separated by Ficoll‐Hypaque method. The histological analyses were performed at the 4th postoperative day. The sciatic functional index, histological, and morphometric analyzes were used to evaluate nerve regeneration at the 6th postoperative week. Six rats were used for immunohistochemical analysis on the 4th postoperative day. In the group 4, on the fourth day, the histological analysis demonstrated a more accelerated degenerative process and an increase of the neurotrophic factors was observed. In the 6th week, all the morphometric results of the group 4 were statistically better compared with groups 2 and 3. There was a statistically significant improvement in the sciatic functional index for group 4 compared with groups 2 and 3. Mononuclear cells stimulated nerve regeneration, most probably by speeding up the Wallerian degeneration process as well as stimulating the synthesis of neurotrophic factors. Microsc. Res. Tech., 2011. © 2010 Wiley‐Liss, Inc.     

Identification of nerve fibres in the scanning electron microscope.

The use of silver as a "stain" for nerve fibres in scanning electron microscopy (SEM) preparations has been investigated. Samples of cardiac tissues were treated according to the Bielschovsky silver impregnation method. Following embedding in paraffin, successive sections were selected for light microscopy and for SEM studies, respectively. The silver impregnated fibres, when examined in SEM preparations, appeared nearly white against a greyish background of cardiac tissue. They were therefore easy to localize even at a low magnification. These nerve fibres were identified in the same tissue, but different blocks by means of transmission electron microscopy (TEM) studies and by fluorescence microscopy.     

Histological and electrophysiological analysis of the peripheral nerve allografts using an immunosuppressive agent

In peripheral nerve allografts, use of an immunosuppressive agent is one of the ways of reducing nerve rejection. FK506 is a newly discovered substance, extracted from Streptomyces tsukubaensis, and has strong immunosuppressive effects. In the present study, immunosuppressive effects of FK 506 were examined using allografts of rat sciatic nerves. Good nerve regeneration was demonstrated through 12 weeks in this model. The immunosuppressed group gained weight over the course of the experiment. Another study was performed to observe the histological changes caused by ceasing the administration of FK506. Administration of FK506 was terminated 12 weeks after grafting. At 8 weeks after cessation, cellular infiltration and large unmyelinated axons were observed in the extended subperineurial space of grafts. At 12 weeks, histological characteristics of rejection were not observed. In the electrophysiological study, the temporal dispersions were recorded at 4 and 8 weeks. However, the normal electrophysiological waves were recorded at 12 weeks after cessation. It was concluded that FK506 is effective for preventing rejection of nerve allografts without any serious side effects on rats, and findings of total rejection of grafts were not recognized after ceasing the administration of FK 506. In peripheral nerve allografts, short-term administration of an immunosuppressive agent is sufficient to lead to good nerve regeneration.     

Making the practically impossible “Merely difficult”—Cryogenic FIB lift‐out for “Damage free” soft matter imaging

The preparation of thinned lamellae from bulk samples for transmission electron microscopy (TEM) analysis has been possible in the focussed ion beam scanning electron microscope (FIB‐SEM) for over 20 years via the in situ lift‐out method. Lift‐out offers a fast and site specific preparation method for TEM analysis, typically in the field of materials science. More recently it has been applied to a low‐water content biological sample (Rubino 2012). This work presents the successful lift‐out of high‐water content lamellae, under cryogenic conditions (cryo‐FIB lift‐out) and using a nanomanipulator retaining its full range of motion, which are advances on the work previously done by Rubino (2012). Strategies are explored for maintaining cryogenic conditions, grid attachment using cryo‐condensation of water and protection of the lamella when transferring to the TEM. Microsc. Res. Tech. 79:298–303, 2016. © 2016 Wiley Periodicals, Inc.     

A multiparametric assay for quantitative nerve regeneration evaluation

We introduce an assay for the semi-automated quantification of nerve regeneration by image analysis. Digital images of histological sections of regenerated nerves are recorded using an automated inverted microscope and merged into high-resolution mosaic images representing the entire nerve. These are analysed by a dedicated image-processing package that computes nerve-specific features (e.g. nerve area, fibre count, myelinated area) and fibre-specific features (area, perimeter, myelin sheet thickness). The assay's performance and correlation of the automatically computed data with visually obtained data are determined on a set of 140 semithin sections from the distal part of a rat tibial nerve from four different experimental treatment groups (control, sham, sutured, cut) taken at seven different time points after surgery. Results show a high correlation between the manually and automatically derived data, and a high discriminative power towards treatment. Extra value is added by the large feature set. In conclusion, the assay is fast and offers data that currently can be obtained only by a combination of laborious and time-consuming tests.     

Ultrastructure of motor nerve terminals in the anterior third of wistar rat tongue

The nerve terminals of intrinsic muscular fibers of the tongue of adult wistar rats was studied by using silver impregnation techniques, transmission electron microscopy (TEM), and high resolution scanning electron microscopy (HRSEM) to observe the nerve fibers and their terminals. Silver impregnation was done according to Winkelman and Schmit, 1957 . For TEM, small blocks were fixed in modified Karnovsky solution, postfixed in 1% buffered osmium tetroxide solution, and embedded in Spurr resin. For HRSEM, the parts were fixed in 2% osmium tetroxide solution with 1/15 M sodium phosphate buffer (pH 7.4) at 4°C for 2 h, according to the technique described by Tanaka, 1989 . Thick myelinated nerve bundles were histologically observed among the muscular fibers. The intrafusal nerve fiber presented a tortuous pathway with punctiform terminal axons in clusters contacting the surface of sarcolemma. Several myelinated nerve fibers involved by collagen fibers of the endoneurium were observed in HRSEM in three-dimensional aspects. The concentric lamellae of the myelin sheath and the axoplasm containing neurofilaments interspersed among the mitochondria were also noted. In TEM, myofibrils, mitochondria, rough endoplasmic reticulum, Golgi's apparatus, and glycogen granules were observed in sarcoplasm. It is also noted that the sarcomeres constituted by myofilaments with their A, I, and H bands and the electron dense Z lines. In areas adjacent to muscular fibers, there were myelinated and unmyelinated nerve fibers involved by endoneurium and perineurium. In the region of the neuromuscular junction, the contact with the sarcolemma of the muscular cell occurs forming several terminal buttons and showing numerous evaginations of the cell membrane. In the terminal button, mitochondria and numerous synaptic vesicles were observed. Microsc. Res. Tech., 2009. © 2009 Wiley-Liss, Inc.     

Feasibility of detecting trabecular bone around percutaneous titanium implants in rabbits by in vivo microfocus computed tomography

Objectives: The goal of this study was to examine the feasibility of in vivo imaging of trabecular bone around titanium implants by means of microfocus computed tomography (micro‐CT) and the use of rabbits for this purpose. Materials and Methods: Ten male rabbits type Hollander, received a titanium implant (1.7 mm diameter and 10 mm length) in the trabecular bone of the left tibia. Seven weeks later a micro‐CT scan was taken. Four rabbits were used to monitor potential harmful effects from X‐ray absorption until 4 weeks after scanning. A second group of six rabbits was used for testing the hypothesis that a good correlation exists between in vivo micro‐CT images and histological images of trabecular bone around titanium implants. The six rabbits were scanned and sacrificed immediately. The tibias were extracted and submitted to standard histological procedures. This resulted in a total of 12 histological sections and their corresponding 12 micro‐CT images. Bone area measurements were performed at the left and right side of the implant in three regions: 0–500, 500–1000 and 1000–1500 μm distance from the implant interface. Intra‐class correlations (ICC) were calculated between both techniques. Results: The four rabbits did not show any sign of radiodermatitis 4 weeks after scanning. In the micro‐CT images of the group of six rabbits, trabeculae are visible, but not well defined, due to the presence of noise in the image. The ICC for the right implant side were 0.44 for zone 0–500 μm, 0.48 for zone 500–1000 μm and 0.40 for zone 1000–1500 μm. The ICC for the left implant side could not be calculated. Conclusion: A low agreement was found between the bone measurements from histology and in vivo micro‐CT images. The use of the in vivo micro‐CT for trabecular bone imaging around metallic implants should be restricted to track tendencies in follow‐up studies.     

Histomicroscopy and normal anatomy of the adult killifish Aphanius hormuzensis (Teleostei; Aphaniidae) from the Persian Gulf coastal environment

The histomicroscopy and normal anatomy of the major body organ systems were investigated in the adult killifish, Aphanius hormuzensis using histological examination, X‐ray imaging, double staining, light microscopy and scanning electron microscopy (SEM). Based on the histomicroscopic observations, the kidney, liver and swim bladder in the studied species were comparable to other fish models. The anterior portion of the kidney is bulbous, while the posterior portion is narrow and elongated; the liver has a single lobe and the swim bladder is a single‐chambered organ with no connection to the digestive tract (physoclistous). X‐ray imaging and double staining examination showed 12 abdominal and 15 caudal vertebrae and a single hypural plate in the caudal skeleton. According to light microscopy, the scales were rounded to pentagonal in shape with three types of radii (primary, secondary and tertiary), and the urohyal bone was elongated. SEM microscopy showed a single row of tricuspid teeth on the upper and lower jaw, respectively, each tooth has two lateral cusps that are shorter than the middle one. The number of teeth was 17–18 in the upper jaw and 19–20 in the lower jaw. The saccular otoliths were rounded‐trapezoid in shape with a moderately incised and V‐shaped excisura. The members of killifishes are an important group for biologists because of their evolutionary properties, regeneration capacity and usefulness as biological control and also for the ecotoxicological assessment of environmental pollution. The outcomes of this study may provide a useful basis for future research on the genus Aphanius.     

Capsaicin-sensitive nerve fibers: a potential extra-ACTH mechanism participating in adrenal regeneration in rats

Pituitary-derived factors, including ACTH, have been widely implicated in initiating adrenal regeneration. However, recent work has demonstrated that adrenal regeneration is also modulated by adrenal nerves that extensively reinnervate the regenerating adrenal. Moreover, transection of the splanchnic nerve removes sensory calcitonin gene-related peptide (CGRP) and preganglionic sympathetic vesicular acetylcholine transporter (VAChT)-positive fibers from the regenerating gland and delays regeneration. However, it is not known whether the splanchnic nerve effects on adrenal regeneration are mediated by the CGRP-positive or VAChT-positive innervation. The present studies use the drug capsaicin, a neurotoxin selective for a subset of primary afferent neurons, to specifically remove CGRP-positive fibers from the adrenal gland and assess subsequent effects on the recovery of adrenal mass and function after surgical enucleation. Male, Sprague-Dawley rats were anesthetized and treated with capsaicin (vs. vehicle) periaxonally to the thoracic splanchnic nerve (33 mM, 15 minutes) or systemically (30-100 mg/kg for 4 days, s.c.). After 7-12 days of recovery, rats received right adrenalectomy and left adrenal enucleation. At 14 and 21 days postenucleation, prestress and poststress plasma and adrenals glands were collected; adrenals were weighed and fixed for immunolabeling of CGRP-positive nerve fibers. Periaxonal capsaicin treatment decreased adrenal CGRP content prior to surgical enucleation; however, reinnervation by CGRP-positive fibers was not prevented and regeneration was not affected. Systemic capsaicin treatment attenuated the reinnervation by CGRP-positive fibers and increased the rate, but not extent, of adrenal regeneration. These results support the hypothesis that adrenal innervation represents an extra-ACTH mechanism to modulate the rate of adrenal regeneration.     

A simple protocol for paraffin-embedded myelin sheath staining with osmium tetroxide for light microscope observation

Experimental investigation of peripheral nerve fiber regeneration is attracting more and more attention among both basic and clinical researchers. Assessment of myelinated nerve fiber morphology is a pillar of peripheral nerve regeneration research. The gold standard for light microscopic imaging of myelinated nerve fibers is toluidine blue staining of resin-embedded semithin sections. However, many researchers are unaware that the dark staining of myelin sheaths typically produced by this procedure is due to osmium tetroxide postfixation and not due to toluidine blue. In this article, we describe a simple pre-embedding protocol for staining myelin sheaths in paraffin-embedded nerve specimens using osmium tetroxide. The method involves immersing the specimen in 2% osmium tetroxide for 2 h after paraformaldehyde fixation, followed by routine dehydration and paraffin embedding. Sections can then be observed directly under the microscope or counterstained using routine histological methods. Particularly good results were obtained with Masson's trichrome counterstain, which permits the imaging of connective structures in nerves that are not detectable in toluidine blue-stained resin sections. Finally, we describe a simple protocol for osmium etching of sections, which makes further immunohistochemical analysis possible on the same specimens. Taken together, our results suggest that the protocol described in this article is a valid alternative to the conventional resin embedding-based protocol: it is much cheaper, can be adopted by any histological laboratory, and allows immunohistochemical analysis to be conducted.     

Influence of nanostructure composition on its morphometric characterization by different techniques

Morphometric characterization of nanoparticles is crucial to determine their biological effects and to obtain a formulation pattern. Determining the best technique requires knowledge of the particles being analyzed, the intended application of the particles, and the limitations of the techniques being considered. The aim of this article was to present transmission (TEM) and scanning (SEM) electron microscopy protocols for the analysis of two different nanostructures, namely polymeric nanoemulsion and poly(lactic‐co‐glycolic acid) (PLGA) nanoparticles, and to compare these results with conventional dynamic light scattering (DLS) measurements. The mean hydrodynamic diameter, the polydispersity index, and zeta potential of the nanostructures of polymeric nanoemulsion were 370.5 ± 0.8 nm, 0.133 ± 0.01, and ?36.1 ± 0.15 mV, respectively, and for PLGA nanoparticles were 246.79 ± 5.03 nm, 0.096 ± 0.025, and ?4.94 ± 0.86 mV, respectively. TEM analysis of polymeric nanoemulsion revealed a mean diameter of 374 ± 117 nm. SEM analysis showed a mean diameter of 368 ± 69 nm prior to gold coating and 448 ± 70 nm after gold coating. PLGA nanoparticles had a diameter of 131 ± 41.18 nm in TEM and 193 ± 101 nm in SEM. Morphologically, in TEM analysis, the polymeric nanoemulsions were spherical, with variable electron density, very few showing an electron‐dense core and others an electron‐dense surface. PLGA nanoparticles were round, with an electron‐lucent core and electron‐dense surface. In SEM, polymeric nanoemulsions were also spherical with a rough surface, and PLGA nanoparticles were round with a smooth surface. The results show that the “gold standards” for morphometric characterization of polymeric nanoemulsion and PLGA nanoparticles were, respectively, SEM without gold coating and TEM with negative staining. Microsc. Res. Tech. 77:691–696, 2014. © 2014 Wiley Periodicals, Inc.     

Bacterial biofilm on successful and failed orthodontic mini‐implants—a scanning electron microscopy study

Mini‐implants have been extensively used in Orthodontics as temporary bone anchorage devices. However, early failure of mini‐implants due to mobility might occur and the colonization of their surfaces by pathogenic bacteria has been referred to as one of the contributing factors. In this study, scanning electron microscopy (SEM) was used to assess the presence of microorganisms adhered to the surface of mini‐implants that failed due to loss of stability. Twelve self‐drilling titanium mini‐implants (1.6 mm diameter × 9.0 mm long) were collected from 12 patients undergoing orthodontic treatment—7 successful and 5 failed mini‐implants. The mean time of permanence in the mouth was 15.8 and 2.4 months for successful and failed mini‐implants, respectively. The devices were placed in the maxilla and/or mandible and removed by the same surgeon and were processed for SEM analysis of the presence of microorganisms on their surfaces (head, transmucosal profile, and body). Extensive bacterial colonization on mini‐implant head and transmucosal profile was observed in all successful and failed mini‐implants. None of the failed mini‐implants exhibited bacteria on its body and only one mini‐implant belonging to the successful (stable) group exhibited bacteria on its body. The results did not suggest a relationship between failure and presence of bacterial colonies on mini‐implant surfaces. Microsc. Res. Tech. 78:1112–1116, 2015. © 2015 Wiley Periodicals, Inc.     

In vivo coherent anti-Stokes Raman scattering imaging of sciatic nerve tissue

We report in vivo nonlinear optical imaging of mouse sciatic nerve tissue by epidetected coherent anti‐Stokes Raman scattering and second harmonic generation microscopy. Following a minimally invasive surgery to open the skin, coherent anti‐Stokes Raman scattering imaging of myelinated axons and second harmonic generation imaging of the surrounding collagen fibres were demonstrated with high signal‐to‐background ratio, three‐dimensional spatial resolution, and no need for labelling. The underlying contrast mechanisms of in vivo coherent anti‐Stokes Raman scattering were explored by three‐dimensional imaging of fat cells that surround the nerve. The epidetected coherent anti‐Stokes Raman scattering signals from the nerve tissues were found to arise from interfaces as well as back reflection of forward coherent anti‐Stokes Raman scattering.     

Microstructural characterization of Ti‐6Al‐4V alloy subjected to the duplex SMAT/plasma nitriding

In this study, microstructural characterization of Ti‐6Al‐4V alloy, subjected to the duplex surface mechanical attrition treatment (SMAT)/nitriding treatment, leading to improve its mechanical properties, was carried out through novel and original samples preparation methods. Instead of acid etching which is limited for morphological characterization by scanning electron microscopy (SEM), an original ion polishing method was developed. Moreover, for structural characterization by transmission electron microscopy (TEM), an ion milling method based with the use of two ions guns was also carried out for cross‐section preparation. To demonstrate the efficiency of the two developed methods, morphological investigations were done by traditional SEM and field emission gun SEM. This was followed by structural investigations through selected area electron diffraction (SAED) coupled with TEM and X‐ray diffraction techniques. The results demonstrated that ionic polishing allowed to reveal a variation of the microstructure according to the surface treatment that could not be observed by acid etching preparation. TEM associated to SAED and X‐ray diffraction provided information regarding the nanostructure compositional changes induced by the duplex SMAT/nitriding process. Microsc. Res. Tech. 76:897–903, 2013. © 2013 Wiley Periodicals, Inc.     

A vision-based, 3D reconstruction technique for scanning electron microscopy: direct comparison with atomic force microscopy

High-resolution, detailed 3D reconstructions of biological specimens obtained from scanning electron microscopy stereo-micrographs and proprietary software were compared with Tapping-Mode AFM datasets of the same fields. The reconstruction software implements several original solutions including a neural adaptive point-matching technique, the ability to build an irregular triangulated mesh rather than a regular orthogonal grid, and the ability to re-map one of the original images exactly onto the reconstructed surface. The technique was applied to human nerve tissue to obtain 1,424 x 968-pixel, texture-mapped datasets, which were subsequently compared against 512 x 512-pixel AFM datasets from the same viewfields. Accounting for the inherent differences of the two techniques, direct comparison revealed an excellent visual match. The correspondence was also quantified by calculating the cross-correlation coefficient between corresponding altimetric profiles in SEM and AFM data, which consistently exceeded a figure of 0.9, with a rate of point mismatch in the order of 0.01%. Research is still underway to improve the robustness of the technique when applied to arbitrary images     

A novel scale‐down cell culture and imaging design for the mechanistic insight of cell colonisation within porous substrate

At the core of translational challenges in tissue engineering is the mechanistic understanding of the underpinning biological processes and the complex relationships among components at different levels, which is a challenging task due to the limitations of current tissue culture and assessment methodologies. Therefore, we proposed a novel scale‐down strategy to deconstruct complex biomatrices into elementary building blocks, which were resembled by thin modular substrate and then evaluated separately in miniaturised bioreactors using various conventional microscopes. In order to investigate cell colonisation within porous substrate in this proof‐of‐concept study, TEM specimen supporters (10–30 μm thick) with fine controlled open pores (100～600 μm) were selected as the modular porous substrate and suspended in 3D printed bioreactor systems. Noninvasive imaging of human dermal fibroblasts cultured on these free‐standing substrate using optical microscopes illustrated the complicated dynamic processes used by both individual and coordinated cells to bridge and segment porous structures. Further in situ analysis via SEM and TEM provided high‐quality micrographs of cell–cell and cell–scaffold interactions at microscale, depicted cytoskeletal structures in stretched and relaxed areas at nanoscale. Thus this novel scaled‐down design was able to improve our mechanistic understanding of tissue formation not only at single‐ and multiple‐cell levels, but also at micro‐ and nanoscales, which could be difficult to obtain using other methods.     

Entropy of corneal nerve fibers distribution observed by laser scanning confocal microscopy: A noninvasive quantitative method to characterize the corneal innervation in Sjogren's syndrome patients

Sjogren's syndrome (SS) is a progressive autoimmune condition mainly affecting the salivary and lacrimal glands with an incidence of primary SS between 1/100 and 1/1,000. SS implies an alteration in the epithelium and subepithelium innervation, with consequent reduction of corneal sensitivity. It is necessary to have noninvasive quantitative methods to characterize the status of the corneal nerve fibers of the patients in order to choose and follow the best therapy. Entropy (information dimension) of the nerve corneal fibers distribution observed by confocal microscopy was evaluated in patients with primary SS (n = 30, 6 males, 24 females, 21–81 years), diagnosed by biopsy of salivary gland and blood tests and in sex‐ age‐matched healthy subjects (n = 12). Corneal nerve fiber density, Langerhans cell count, and cell density in the nerve plexus images were also evaluated. In selected patients salivary gland atrophy degree was also evaluated. Nerve corneal distribution observed by confocal microscopy is fractal. Entropy of the corneal nerve distribution statistically distinguishes between SS patients and healthy subjects: patients present a lower value of information dimension of the corneal nerve fibers distribution than healthy individuals (P < 0.001). Percentage of grouped cases classified by entropy according to the subjects (selected patients vs. healthy) showed a 100% sensitivity and 96% specificity, P < 0.0001 with a low value of coefficient of variation among the individuals (6–7 times lower than the other morphometric indexes). Entropy correlated with the severity of the disease (salivary gland atrophy degree, P < 0.01). Evaluation of entropy of the corneal nerve distribution observed by a laser confocal microscopy appears to quantitatively and noninvasively characterize an aspect of the SS patients in relation to the recognition of an impairment of their ocular surface, giving us for the first time a method to objectively and precisely characterize the corneal innervation status in the SS patients. Microsc. Res. Tech. 78:1069–1074, 2015. © 2015 Wiley Periodicals, Inc.     

Correlated light optical and scanning electron microscopy of Gram smears of bacteria and paraffin sections of cardiac muscle

Light-microscope slides (3 in. × 1 in.) bearing Gram smears of Erysipelothrix rhusiopathiæ, or Staphylococcus aureus, after preliminary examination under the light-optical microscope (LM), were cut down in size, glued onto specimen stubs, coated with gold and examined in the scanning electron microscope (SEM). These preparations served as a control for investigations into bacteria-cell junctions in tissue. Cover-slips from stained sections of staphylococcal or swine erysipelas endocarditis mounted on 3 in. × 1 in. microscope slides (which had been intensively studied previously with conventional light microscopy) were floated off by immersing the slides in xylol. After dehydration of the tissues on the slides, the preparations were treated similarly to the Gram smears, and were examined with the SEM. Lesions of endocarditis were thus examined, and the information gained from these preliminary examinations shed new light on the pathogenesis of the disease. This information had not previously been available by any other technique. Because of this, and in view of the simplicity of preparing sections for scanning electron microscopy, it is suggested that the SEM might be a useful tool to be applied to routine histological sections.     

Effects of the Ar ions pre‐amorphization of Si substrateon interface mixing of Fe/Si bilayers

Ion beam mixing of Fe/Si bilayers, induced by 100 keV ⁴⁰Arions at room temperature was investigated. Rutherford backscattering spectroscopy (RBS), atomic force microscopy (AFM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were applied for structural characterization. The main focus of this study was on the influence of the substrate structure on interface mixing. The influence of the substrate structure is due to the two classes of irradiated bilayers, Fe thin films deposited on crystalline or pre‐amorphized Si substrates. An about 76% higher efficiency of atomic transport across the pre‐amorphized Fe/a‐Si interface as compared to that of Fe/c‐Si bilayers was observed.