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.     

Catecholaminergic and acetylcholine esterase containing nerves of cranial and spinal dura mater in humans and rodents

The innervation of cranial and spinal dura mater in humans and rodents was studied by examining several dural zones (vascular, perivascular, intervascular) in different regions. Characterization and distribution of dural acetylcholinesterase-positive nerve fibers, catecholaminergic nerve fibers, and mast cells are analyzed and discussed. The results of chemical and surgical sympathectomy as well as the relationships between catecholaminergic nerve fibers and mast cells are studied. Our results are discussed in the light of possible implications in the physiopathology of dural algic syndromes including cephalalgia and spinal pain.     

Neuronal regulation of bone metabolism and anabolism: calcitonin gene-related peptide-, substance P-, and tyrosine hydroxylase-containing nerves and the bone

Bone alters its metabolic and anabolic activities in response to the variety of systemic and local factors such as hormones and growth factors. Classical observations describing abundance of the nerves fibers in bone also predict a paradigm that the nervous system influences bone metabolism and anabolism. Identification of the nerve-derived signaling molecules, capable of modulating cellular activities of the bone cells, facilitates a novel approach to study the biology of skeletal innervation. Many of the signaling molecules that may act as efferent agents on the bone cells fall into the category of neuropeptides. The present article reviews current understanding of the skeletal innervation and their proposed physiological effects on bone metabolism, with a special interest to calcitonin gene-related peptide (CGRP)-containing nerves fibers. CGRP is abundantly distributed in bone via sensory nerves, especially in the epiphyseal trabecular bones. Its in vitro actions to the cultured osteoblasts and osteoclasts, together with its in vivo localization, strongly support the paradigm that the nervous system influences bone metabolism. In addition, CGRP is recently shown to be expressed endogenously by the osteoblasts. Transgenic mice with osteoblasts overexpressing CGRP are characterized by increased bone formation rate and enhanced bone volume, suggesting that CGRP indeed acts on bone metabolism not only via nervous route but also via autocrine loop. The current article also reviews the distribution of nerve fibers containing substance P (SP), another sensory nerve-specific neuropeptide, and tyrosine hydroxylase (TH), the rate-limiting enzyme of catecholamine. The distinct effects of SP and catecholamines on the bone cells together with their in vivo influences manifested by experimental denervation studies suggest that the sensory and sympathetic nerves play important roles in bone metabolism.     

Vital confocal microscopy in bone

We wished to exploit confocal microscopy for high spatial and temporal resolution vital microscopy in bone. To this end, we evolved implants with glass windows supported in titanium, which were placed in the medial proximal tibial plateau of the rabbit, and special small, self-focussing objectives (dry 10/0.25, water immersion 20/0.45, and oil immersion 45/0.65 and 120/1.0) which mated and matched to the conical window entrance section of the metal components. At intervals of up to 21 months after implant healing, these lenses were used to study live tissue using two genera of confocal microscope: multiple aperture disc, tandem scanning, microscopes for observation in reflection, and video rate confocal laser scanning microscopes for recording, mainly in the fluorescence mode. The latter allowed the study of a variety of intravenously administered substances, including fluorescein, fluorescein-dextrans, fluorescent microspheres, acridine orange, DASPMI, calcein, and tetracycline. We were able to remove blood, stain cells with fluorescent markers, and replace them into the circulation. Calcein and tetracycline bind to the mineral front in bone: this labelling was studied in progress. We observed that both substances partition and remain for long periods (at least days) in adipocytes. Further characterisation of the system used both confocal fluorescence and scanning electron microscopy methods in the study of retrieved implants. These studies showed that the subimplant cortical bone remodelled to a less compact structure with a rich microvasculature extremely close to bone. The points of attachment of bone to glass were found to involve coarse fibres, with the matrix containing large numbers of large cells: some of this tissue was cartilage and some immature bone. An amorphous, mineralised matrix was in immediate contact with glass. The results provide further confirmation of the general utility of high-scan speed confocal methodology in physiology.     

Three‐dimensional morphology of cerebellar climbing fibers. A study by means of confocal laser scanning microscopy and scanning electron microscopy

The intracortical pathway of cerebellar climbing fibers have been traced by means of scanning electron microscpy (SEM) and confocal laser scanning microscopy (CLSM) to study the degree of lateral collateralization of these fibers in the granular Purkinje cell and molecular layers. Samples of teleost fish were processed for conventional and freeze‐fracture SEM. Samples of hamster cerebellum were examined by means of CLSM using FM4–64 as an intracellular stain. High resolution in lens SEM of primate cerebellar cortex was carried out using chromium coating. At scanning electron and confocal laser microscopy levels, the climbing fibers appeared at the white matter and granular layer as fine fibers with a typical arborescence or crossing‐over branching pattern, whereas the mossy fibers exhibited a characteristic dichotomous bifurcation. At the granular layer, the parent climbing fibers and their tendrils collaterals appeared to be surrounding granule and Golgi cells. At the interface between granule and Purkinje cell layers, the climbing fibers were observed giving off three types of collateral processes: those remaining in the granular layer, others approaching the Purkinje cell bodies, and a third type ascending directly to the molecular layer. At this layer, retrograde collaterals were seen descending to the granular layer. By field emission high‐resolution SEM of primate cerebellar cortex, the climbing fiber terminal collaterals were appreciated ending by means of round synaptic knobs upon the spines of secondary and tertiary Purkinje cell dendrites.     

High dose glucocorticoid hampers bone formation and resorption after bone marrow ablation in rat

We analyzed the effect of glucocorticoid on bone regeneration after bone marrow ablation in tibiae of 8-week-old rats. Methylprednisolone sodium succinate (MPSS) was injected intramuscularly at a dose of 100 mg/kg/day for 3 days. Tibiae on days 1, 3, 5, 7, 10, 12, and 14 after ablation were subjected to tartrate-resistant acid phosphatase staining, immunohistochemistry, in situ hybridization, and transmission electron microscopy (TEM), and measurement of the volume of newly-formed bone and the osteoclast number. MPSS significantly decreased the newly-formed bone volume on day 7, and immature bone still remained on day 10 in the MPSS-treated group. The volume of this bone was significantly higher than that in the control group. However, there were no differences between the groups in the osteoclast number, the expression of mRNAs for osteoblast differentiation markers, and alkaline phosphatase and cathepsin K judged by immunohistochemistry. TEM findings showed no difference in the form of osteoblasts, whereas osteoclasts in the MPSS-treated group had less developed ruffled borders, compared to those in the control group. These results suggest that MPSS treatment affects neither the differentiation nor the shape of osteoblasts, and does not change the osteoclast number or the cathepsin K level. However, high dose MPSS inhibits both bone formation and resorption during bone regeneration after rat tibial bone marrow ablation, and inhibits ruffled border formation in osteoclasts. These data will be useful to develop bone regenerative therapies for bone diseases due to high dose steroid administration.     

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.     

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.     

Calcitonin gene-related peptide (CGRP)-containing nerve fibers in bone tissue and their involvement in bone remodeling

Bone remodeling is a process of bone renewal accomplished by osteoclastic bone resorption and osteoblastic bone formation. These two activities are regulated by systemic hormones and by local cytokines and growth factors. Moreover, the nervous system and certain neuropeptides seem to be involved in regulation of bone remodeling. In this paper, we focus on the distribution of CGRP-containing nerve fibers and their dynamics, and discuss the role of these fibers as a possible mechanism for nervous system involvement in regulation of bone remodeling. CGRP-immunoreactive nerve fibers are widely distributed in bone tissue, such as periosteum and bone marrow, and show apparent regional distribution with different densities. They are often associated with blood vessels and show a beaded appearance. The wide distribution of CGRP-immunoreactive nerve fibers in bone tissue and the changes in distribution during bone development and regeneration suggest the involvement of these fibers in bone remodeling. The effect of CGRP on bone remodeling could partly be through its action on blood vessels, thereby regulating local blood flow. Moreover, in vitro biochemical data and the localization of CGRP-immunoreactive nerve fibers in the vicinity of bone cells suggest that they are directly involved in local regulation of bone remodeling by elevating the concentration of CGRP in the microenvironment around bone cells, especially during bone growth or repair.     

Expression and regulatory role of receptors for vasoactive intestinal peptide in bone cells

An intense network of nerve fibers can be demonstrated in skeletal tissues, not only in the periosteum but also within cortical bone, growth plate, and bone marrow. This neuro-osteogenic network expresses a restricted number of signalling molecules, including neuropeptides, neurotransmitters, and neurotrophins. Several lines of evidence indicate that receptors for these molecules are present on bone cells and that activation of these receptors leads to changes in bone cell activities. In addition, deletion of signalling molecules has been shown to alter bone metabolism. In the present review, these studies are summarized with a focus on distribution and effects of vasoactive intestinal peptide.     

Improved algorithms for automatic bone histomorphometry on a numerized image analysis system

Quantitative histological methods have proved to be the most effective methods in bone disease research. Faster and more accurate techniques are currently needed. We have developed a simple digitized image analysis system which allows accurate measurements of trabecular bone mass. The algorithm is based on the 'four-connected sets' mathematical theory. Given a numerized image displayed by a CCD camera, the algorithm recognizes all possible four-connected sets and provides area measurements. The first procedure automatically eliminates small, irrelevant profiles (wrinkles, cell nuclei, etc.) while larger profiles are erased interactively. The second procedure similarly erases the artefactual defects within the trabecule (artefactual cracks or empty osteocytic lacunae). The method was shown to be very accurate and time-saving.     

Histological evaluation on bone regeneration of dental implant placement sites grafted with a self-setting alpha-tricalcium phosphate cement

This study aimed to evaluate the histological characteristics of the new bone formed at dental implant placement sites concomitantly grafted with a self-setting tricalcium phosphate cement (BIOPEX-R). Standardized defects were created adjacent to the implants in maxillae of 4-week-old male Wistar rats, and were concomitantly filled with BIOPEX-R. Osteogenesis was examined in two sites of extreme clinical relevance: (1) the BIOPEX-R-grafted surface corresponding to the previous alveolar ridge (alveolar ridge area), and (2) the interface between the grafting material and implants (interface area). At the alveolar ridge area, many tartrate-resistant acid phosphatase (TRAPase)-reactive osteoclasts had accumulated on the BIOPEX-R surface and were shown to migrate toward the implant. After that, alkaline phosphatase (ALPase)-positive osteoblasts deposited new bone matrix, demonstrating their coupling with osteoclasts. On the other hand, the interface area showed several osteoclasts initially invading the narrow gap between the implant and graft material. Again, ALPase-positive osteoblasts were shown to couple with osteoclasts, having deposited new bone matrix after bone resorption. Transmission electron microscopic observations revealed direct contact between the implant and the new bone at the interface area, although few thin cells could still be identified. At both the alveolar ridge and the interface areas, newly formed bone resembled compact bone histologically. Also, concentrations of Ca, P, and Mg were much alike with those of the preexistent cortical bone. In summary, when dental implant placement and grafting with BIOPEX-R are done concomitantly, the result is a new bone that resembles compact bone, an ideal achievement in reconstructive procedures for dental implantology.     

Three-dimensional wall structure and the innervation of dental pulp blood vessels.

The involvement of neural components in plasma extravasation and blood flow in the dental pulp has been established by pharmacological and physiological studies. We review here the segmental constitution of pulp vessels and the possible involvement of neural components in both the contractility and permeability of the pulp vessels from a morphological viewpoint. Six vascular segments can be identified based on the morphology of peri-endothelial cells, such as smooth muscle cells and pericytes. These are: muscular arterioles, terminal arterioles, precapillary arterioles, capillaries, postcapillary venules, and collecting or muscular venules. The perivascular nerve forms a mesh with numerous terminal varicosities, some of which attach directly to arteriolar smooth muscle cells. This mesh can be seen by scanning electron microscopy, and indicates the important role of neural components in regulating the pulpal circulation. After administering norepinephrine (0.2 mg/kg/dog), the surface texture of the smooth muscle cells of pulp arterioles reveals marked irregularities, which are correlated with arteriolar contraction. The pericytes in larger postcapillary venules (diameter 20 microm or larger) also show irregularities, whereas no changes are seen in the pericytes of either smaller postcapillary venules or capillaries. The intercellular spaces of pericytes in the postcapillary venules are wide enough for leukocytes to pass through, and the occasional extravasation of leukocytes through venule walls can be seen under electron microscopy. The microvessels of healthy human dental pulp react weakly to selectins, indicating that apparently healthy dental pulp may be weakly inflamed. In rat dental pulp, CGRP-immunoreactive nerves and nerve terminals containing many granular vesicles supply the postcapillary venules more densely than the arterioles, which suggests the involvement of postcapillary venules in neurogenic inflammation in the dental pulp.     

Mineral content changes in bone associated with damage induced by the electron beam

Energy-dispersive x-ray (EDX) spectroscopy and backscattered electron (BSE) imaging are finding increased use for determining mineral content in microscopic regions of bone. Electron beam bombardment, however, can damage the tissue, leading to erroneous interpretations of mineral content. We performed elemental (EDX) and mineral content (BSE) analyses on bone tissue in order to quantify observable deleterious effects in the context of (1) prolonged scanning time, (2) scan versus point (spot) mode, (3) low versus high magnification, and (4) embedding in poly-methylmethacrylate (PMMA). Undemineralized cortical bone specimens from adult human femora were examined in three groups: 200x embedded, 200x unembedded, and 1000x embedded. Coupled BSE/EDX analyses were conducted five consecutive times, with no location analyzed more than five times. Variation in the relative proportions of calcium (Ca), phosphorous (P), and carbon (C) were measured using EDX spectroscopy, and mineral content variations were inferred from changes in mean gray levels ("atomic number contrast") in BSE images captured at 20 keV. In point mode at 200x, the embedded specimens exhibited a significant increase in Ca by the second measurement (7.2%, p < 0.05); in scan mode, a small and statistically nonsignificant increase (1.0%) was seen by the second measurement. Changes in P were similar, although the increases were less. The apparent increases in Ca and P likely result from decreases in C: -3.2% (p < 0.05) in point mode and -0.3% in scan mode by the second measurement. Analysis of unembedded specimens showed similar results. In contrast to embedded specimens at 200x, 1000x data showed significantly larger variations in the proportions of Ca, P, and C by the second or third measurement in scan and point mode. At both magnifications, BSE image gray level values increased (suggesting increased mineral content) by the second measurement, with increases up to 23% in point mode. These results show that mineral content measurements can be reliable when using coupled BSE/EDX analyses in PMMA-embedded bone if lower magnifications are used in scan mode and if prolonged exposure to the electron beam is avoided. When point mode is used to analyze minute regions, adjustments in accelerating voltages and probe current may be required to minimize damage.     

Quantification and visualization of anisotropy in trabecular bone

A number of methods for measuring anisotropy in trabecular bone using high‐resolution X‐ray computed tomography exist, which give different answers but have not been compared in detail. In this study, we examine the mean‐intercept length (MIL), star volume distribution (SVD) and star length distribution (SLD) methods, their algorithmic implementation for three‐dimensional (3D) data, and how their results relate to each other. A uniform ordered sampling scheme for determining which orientations to sample during analysis enhances the reproducibility of anisotropy and principal component direction determinations, with no evident introduction of biasing. This scheme also facilitates the creation of a 3D rose diagram that can be used to gain additional insights from the data. The directed secant algorithm that is frequently used for traversing pixel and voxel grids for these calculations is prone to bias unless a previously unreported normalization is used. This normalization ameliorates the bias present when using cubic voxels, and also permits calculations on data sets in which the slice spacing is not equal to the pixel spacing. Overall, the three methods for quantification of anisotropy give broadly similar results, but there are systematic divergences that can be traced to their differences in data and processing, and which may impact on their relative utility in estimating mechanical properties. Although discussed in the context of computed tomography of trabecular bone, the methods described here may be applied to any 3D data set from which fabric information is desired.     

Fluorescence in the tandem scanning microscope

Our studies have shown that the fluorescence mode can be used to good effect in both tandem scanning microscopes (TSM: direct view confocal microscopes) as well as confocal scanning laser microscopes (CSLM). Applications are presented which show that the two great advantages of TSM are real-time viewing and real colour, which allow faster use and interpretation. CSLM are complementary, not competitive, being currently more sophisticated for low-level fluorescence work. This is equally possible with available TSM, but requires further development using CCD cameras and image-processing systems.