Assessment of In vivo behavior of polymer tube nerve grafts simultaneously with the peripheral nerve regeneration process using scanning electron microscopy technique

In this study, scanning electron microscopy (SEM) has been applied for instantaneous assessment of processes occurring at the site of regenerating nerve. The technique proved to be especially useful when an artificial implant should have been observed but have not yet been extensively investigated before for assessment of nerve tissue. For in vivo studies, evaluation of implant's morphology and its neuroregenerative properties is of great importance when new prototype is developed. However, the usually applied histological techniques require separate and differently prepared samples, and therefore, the results are never a 100% comparable. In our research, we found SEM as a technique providing detailed data both on an implant behavior and the nerve regeneration process inside the implant. Observations were carried out during 12‐week period on rat sciatic nerve injury model reconstructed with nerve autografts and different tube nerve grafts. Samples were analyzed with haematoxylin‐eosin (HE), immunocytochemical staining for neurofillament and S‐100 protein, SEM, TEM, and the results were compared. SEM studies enabled to obtain characteristic pictures of the regeneration process similarly to TEM and histological studies. Schwann cell transformation and communication as well as axonal outgrowth were identified, newly created and matured axons could be recognized. Concurrent analysis of biomaterial changes in the implant (degradation, collapsing of the tube wall, migration of alginate gel) was possible. This study provides the groundwork for further use of the described technique in the nerve regeneration studies. SCANNING 35: 232‐245, 2013. © 2012 Wiley Periodicals, Inc.     

利用原子力显微技术对神经胶质细胞超微结构及其相互间的纳米连接结构的观察

本研究利用原子力显微技术（AFM）观察原代培养的神经胶质细胞及其相互间的纳米连接结构。选择生长良好的神经胶质细胞用戊二醛固定30分钟,固定于AFM基底上进行扫描成像,用AFM脱机软件（SPM OFFLINE 2．20）进行检测。观察到胶质细胞平铺于培养皿的底部,胞体形状不规则,表面较扁平。突起丰富,但没有极性,无轴突树突之分,还观察到两胶质细胞间存在长程纤维管状连接结构。     

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.     

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.     

Nervous system-derived chondroitin sulfate proteoglycans regulate growth cone morphology and inhibit neurite outgrowth: a light, epifluorescence, and electron microscopy study

Proteoglycans influence aging and plasticity in the nervous system. Particularly prominent are the chondroitin sulfate proteoglycans (CSPGs), which are generally inhibitory to neurite outgrowth. During development, CSPGs facilitate normal guidance, but following nervous system injury and in diseases of aging (e.g., Alzheimer's disease), they block successful regeneration, and are associated with axon devoid regions and degenerating nerve cells. Whereas previous studies used non-nervous system sources of CSPGs, this study analyzed the morphology and behavior of sensory (dorsal root ganglia) neurons, and a human nerve cell model (SH-SY5Y neuroblastoma cells) as they contacted nervous system-derived CSPGs, using a variety of microscopy techniques. The results of these qualitative analyses show that growth cones of both nerve cell types contact CSPGs via actin-based filopodia, sample the CSPGs repeatedly without collapse, and alter their trajectory to avoid nervous system-derived CSPGs. Turning and branching are correlated with increased filopodial sampling, and are common to both neurons and Schwann cells. We show that CSPG expression by rat CNS astrocytes in culture is correlated with sensory neuron avoidance. Further, we show for the first time the ultrastructure of sensory growth cones at a CSPG-laminin border and reveal details of growth cone and neurite organization at this choice point. This type of detailed analysis of the response of growth cones to nervous system-derived CSPGs may lead to an understanding of CSPG function following injury and in diseases of aging, where CSPGs are likely to contribute to aberrant neurite outgrowth, failed or reduced synaptic connectivity, and/or ineffective plasticity.     

Ultrastructural neurobiology of the olfactory mucosa of the brown trout, Salmo trutta.

This paper describes four investigations of the olfactory mucosa of the brown trout: 1) the ultrastructure of the olfactory mucosa as revealed by scanning (SEM), conventional transmission (TEM), and high voltage (HVEM) electron microscopy; 2) light and electron-microscopic investigations of retrograde transport of the tracer macromolecule horseradish peroxidase (HRP) when applied to the cut olfactory nerve; 3) SEM and TEM investigations of the effects of olfactory nerve transection on cell populations within the olfactory epithelium; and 4) ultrastructural investigations of reversible degeneration of olfactory receptors caused by elevated copper concentrations. The trout olfactory epithelium contains five cell types: ciliated epithelial cells, ciliated olfactory receptor cells, microvillar olfactory receptor cells, supporting cells, and basal cells. The ciliated and microvillar olfactory receptor cells and a small number of basal cells are backfilled by HRP when the tracer is applied to the cut olfactory nerve. When the olfactory nerve is cut, both ciliated and microvillar olfactory receptor cells degenerate within 2 days and are morphologically intact again within 8 days. When wild trout are taken from their native stream and placed in tanks with elevated copper concentrations, ciliated and microvillar cells degenerate. Replacement of these trout into their stream of origin is followed by morphologic restoration of both types of olfactory receptor cells. Ciliated and microvillar receptor cells are primary sensory bipolar neurons whose dendrites make contact with the environment; their axons travel directly to the brain. Consequently, substances can be transported directly from the environment into the brain via these "naked neurons." Since fish cannot escape from the water in which they swim, and since that water may occasionally contain brain-toxic substances, the ability to close off--and later reopen--this anatomic gateway to the brain would confer a tremendous selective advantage upon animals that evolved the "brain-sparing" capacity to do so. Consequently, the unique regenerative powers of vertebrate olfactory receptor neurons may have their evolutionary origin in fishes.     

Two-photon fluorescent microlithography for live-cell imaging

Fluorescent dyes added to UV-cure resins allow the rapid fabrication of fluorescent micropatterns on standard glass coverslips by two-photon optical lithography. We use this lithographic method to tailor fiduciary markers, focal references, and calibration tools, for fluorescence and laser scanning microscopy. Fluorescent microlithography provides spatial landmarks to quantify molecular transport, cell growth and migration, and to compensate for focal drift during time-lapse imaging. We show that the fluorescent patterned microstructures are biocompatible with cultures of mammalian cell lines and hippocampal neurons. Furthermore, the high-relief topology of the lithographed substrates is utilized as a mold for poly(dimethylsiloxane) stamps to create protein patterns by microcontact printing, representing an alternative to the current etching techniques. We present two different applications of such protein patterns for localizing cell adhesion and guidance of neurite outgrowth.     

A microfluidic positioning chamber for long-term live-cell imaging

We report a microfluidic positioning chamber (MPC) that can rapidly and repeatedly relocate the same imaging area on a microscope stage. The "roof" of the microfluidic chamber was printed with serials of coordinate numbers that act as positioning marks for mammalian cells that grow attached to the "floor" of the microfluidic chamber. MPC cell culture chamber provided a simple solution for tracking the same cell or groups of cells over days or weeks. The positioning marks were used to register time-lapse images of the same imaging area to single-pixel accuracy. Using MPC cell culture chamber, we tracked the migration, division, and differentiation of individual PC12 cells for over a week using bright field and fluorescence imaging.     

New ways to look at axons in Caenorhabditis elegans

In the nematode Caenorhabditis elegans, a well-established model organism for the analysis of nervous system development and function, nerve processes can be labelled in the intact animal with markers based on the "Green Fluorescent Protein" (GFP). The generation of GFP variants with improved brightness and modified emission spectra potentiated the use of this marker for in vivo labelling of subcellular structures. This made it possible to label different groups of neurons and their axons in the same animal with GFP variants of different spectral characteristics. Here I show with double labelling experiments that spatial relationships of axons in small axon bundles can now be resolved at the light microscopic level. In the future this will largely circumvent the need for time-consuming electron microscopic reconstructions to detect local defects in axon outgrowth. Furthermore, I demonstrate that neuronal processes can now be traced even in the head ganglia, an area of the nervous system that was previously almost inaccessible for analysis due to the compact arrangement of cell bodies and axons.     

An interacting multiple model filter-based autofocus strategy for confocal time-lapse microscopy

Gene expression and other cellular processes are stochastic, thus their study requires observing multiple events in multiple cells. Therefore, confocal microscopy cell imaging has recently gained much interest. In time-lapse imaging, adjustments are needed at short intervals to compensate for focus drift. There are several automated methods for this purpose. In general, before acquiring higher resolution images, software-based autofocus algorithms require a set of low-resolution images along the z-axis to determine the plane for which a predefined focusing function is maximized. These algorithms require 10-100 z-slices each time, and there is no fixed number or upper limit of required z-slices that ensures optimal focusing. The higher is this number, the stronger is photo bleaching, hampering the feasibility of long-time series measurements. We propose a new focusing strategy in time-lapse imaging. The algorithm relies on the nature and predictability of the focus drift. We first show that the focus drift curve is predictable within a small error bound in standard experimental setups. We, then, exploit the interacting multiple model filter algorithm to predict the drift at time, t, based on the measurement at time t-1. This allows a drastic reduction of the number of required z-slices for focus drift correction, largely overcoming the problem of photo bleaching. In addition, we propose a new set of functions for focusing in time-lapse imaging, derived from preexisting ones. We demonstrate the method's efficiency in time-lapse imaging of Escherichia coli cells expressing MS2d-GFP tagged RNA molecules.     

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.     

Some methods for more efficient processing of SEM specimens

Three techniques are described for increasing the quantity of specimens which may be processed for SEM while maintaining the quality of the final product. They are: a simple stainless steel holder for safe manipulation of coverslips during incubation and fixation; a coverslip carrier which permits four or more coverslips to be dehydrated and critically point dried at the same time; and a simple pattern for making baskets to hold relatively large but delicate specimens during processing for SEM. Processing soft tissues for scanning electron microscopy (SEM) presents many problems depending on the size, shape and type of specimen. Cohen (1974) presented an overall view of this problem. Methods for handling cells in suspension have been suggested by Baker & Princen (1975), Newell & Roath (1975) and Rostgaard & Christensen (1975). Tissue culture specimens are also difficult to process for SEM. These may be long-term cultures such as fibroblasts or kidney cells, or short-term preparations such as the collecting of white blood cells or bacteria on coverslips. Nemanic (1972) described a method using Tygon tubing with slits to hold the coverslips after incubation. Perecko et al. (1973) gave details of a holder for processing six small (6 mm) coverslips after incubation. Rice et al. (1976) designed several complex multipurpose carriers for 9 × 22 mm coverslips and for cells on silver or cellulose membrane filters. Broers et al. (1975) used a mesh receptacle for bacteria attached to silicon dioxide slivers. Many investigators simply process pieces of plastic cut from the tissue culture dish itself. Using the plastic directly avoids the problems of incubating cells on glass coverslips. Coverslips are fragile and difficult to remove from the culture dish or flask. Each coverslip must be processed individually, increasing the risk of breakage and limiting the number which can be done at one time. For processing larger tissues there are commercial baskets available. Cohen (1974) illustrated mesh baskets for odd sizes and shapes of soft tissues. We have devised a simple holder which facilitates the use of 12 mm 0 grade coverslips in several types of culture dishes. We have developed a multipurpose carrier which increases the number of coverslips which can be processed for SEM and reduces the number of manipulations needed for each individual coverslip. This carrier can be used for histological and histochemical studies as well as SEM processing. We have modified Cohen's technique (1974) for mesh baskets for larger soft tissue specimens.     

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.     

Laminins and human disease

The laminin protein family has diverse tissue expression patterns and is involved in the pathology of a number of organs, including skin, muscle, and nerve. In the skin, laminins 5 and 6 contribute to dermal-epidermal cohesion, and mutations in the constituent chains result in the blistering phenotype observed in patients with junctional epidermolysis bullosa (JEB). Allelic heterogeneity is observed in patients with JEB: mutations that results in premature stop codons produce a more severe phenotype than do missense mutations. Gene therapy approaches are currently being studied in the treatment of this disease. A blistering phenotype is also observed in patients with acquired cicatricial pemphigoid (CP). Autoantibodies targeted against laminins 5 and 6 destabilize epithelial adhesion and are pathogenic. In muscle cells, laminin alpha 2 is a component of the bridge that links the actin cytoskeleton to the extracellular matrix. In patients with laminin alpha 2 mutations, the bridge is disrupted and mature muscle cells apoptose. Congenital muscular dystrophy (CMD) results. The role of laminin in diseases of the nervous system is less well defined, but the extracellular protein has been shown to serve an important role in peripheral nerve regeneration. The adhesive molecule influences neurite outgrowth, neural differentiation, and synapse formation. The broad spatial distribution of laminin gene products suggests that laminin may be involved in a number of diseases for which pathogenic mechanisms are still being unraveled.     

Microglia in neuroregeneration

Microglia has the potential to produce and release a range of factors that directly and/or indirectly promote regeneration in the injured nervous system. The overwhelming evidence indicates, however, that this potential is generally not expressed in vivo. Activated microglia may enhance neuronal degeneration following axotomy, thereby counteracting functional recovery. Microglia does not seem to contribute significantly to axonal outgrowth after peripheral nerve injury, since this process proceeds uneventful even if perineuronal microglia is eliminated. The phagocytic phenotype of microglia is highly suppressed during Wallerian degeneration in the central nervous system. Therefore, microglia is incapable of rapid and efficient removal of myelin debris and its putative growth inhibitory components. In this way, microglia may contribute to regeneration failure in the central nervous system. Structural and temporal correlations are compatible with participation by perineuronal microglia in axotomy-induced shedding of presynaptic terminals, but direct evidence for such participation is lacking. Currently, the most promising case for a promoting effect on neural repair by activated microglia appears to be as a mediator of collateral sprouting, at least in certain brain areas. However, final proof for a critical role of microglia in these instances is still lacking. Results from in vitro studies demonstrate that microglia can develop a regeneration supportive phenotype. Altering the microglial involvement following neural injury from a typically passive or even counterproductive state and into a condition where these cells are actively supporting regeneration and plasticity is, therefore, an exciting challenge and probably a realistic goal.     

Regulatory cell interactions between retinal ganglion cells and radial glia during axonal and dendritic outgrowth

Neuronal differentiation and the formation of cell polarity are crucial events during the development of the nervous system. Cell polarity is a prerequisite for directed information flux within neuronal networks. In this article, we focus on neuro-glial cell interactions that influence the establishment of neural cell polarity and the directed outgrowth of axons versus dendrites. The cellular model discussed in detail is the retinal ganglion cell (RGC) of the chick retina, which is investigated by a comprehensive set of in vitro assays. The experiments demonstrate that retinal microenvironment determines axon vs. dendrite formation of RGCs. The instructive differences in different retinal microenvironments are substantially influenced by radial glia. Different glial domains support or inhibit axon vs. dendrite outgrowth. The data support the notion that neuro-glial interactions are crucial for directed neurite outgrowth.     

Investigation of morphological and functional changes during neuronal differentiation of PC12 cells by combined hopping probe ion conductance microscopy and patch-clamp technique

PC12 cells derived from rat pheochromocytoma can differentiate into sympathetic-neuron-like cells in response to nerve growth factor (NGF). These cells have been proved to be a useful cell model to study neuronal differentiation. NGF induces rapid changes in membrane morphology, neurite outgrowth, and electrical excitability. However, the relationship between the 3D morphological changes of NGF-differentiated PC12 cells and their electrophysiological functions remains poorly understood.In this study, we combined a recently developed Hopping Probe Ion Conductance Microscopy (HPICM) with patch-clamp technique to investigate the high-resolution morphological changes and functional ion-channel development during the NGF-induced neuronal differentiation of PC12 cells. NGF enlarged TTX-sensitive sodium currents of PC12 cells, which associated with cell volume, membrane surface area, surface roughness of the membrane, and neurite outgrowth. These results demonstrate that the combination of HPICM and patch-clamp technique can provide detailed information of membrane microstructures and ion-channel functions during the differentiation of PC12 cells, and has the potential to become a powerful tool for neuronal research.     

Dynamic three-dimensional visualization of collagen matrix remodeling and cytoskeletal organization in living corneal fibroblasts

The remodeling of extracellular matrices by cells plays a defining role in developmental morphogenesis and wound healing, as well as in tissue engineering. Three-dimensional (3-D) type I collagen matrices have been used extensively as an in vitro model for studying cell-induced matrix reorganization at the macroscopic level. However, few studies have directly assessed the dynamic process of 3-D matrix remodeling at the cellular and subcellular level. We recently developed an experimental model for investigating cell-matrix mechanical interactions by plating green fluorescen protein (GFP)-zyxin transfected cells inside fibrillar collagen matrices and performing high-magnification time-lapse differential interference microscopy (DIC) and wide-field fluorescent imaging. In this study, we extend this experimental model by performing four-dimensional (4-D) reflected light and fluorescent confocal imaging (using either visible light or multiphoton excitation) of living corneal fibroblasts transfected to express GFP-zyxin or GFP-alpha-actinin, 18 h after plating inside 3-D collagen matrices. Reflected light confocal imaging allowed detailed visualization of the cells and the fibrillar collagen surrounding them. By overlaying maximum intensity projections of reflected light and GFP-zyxin or GFP-alpha-actinin images and generating stereo pair reconstructions, 3-D interactions between focal adhesions and collagen fibrils in living cells could be visualized directly. Focal adhesions were generally oriented parallel to the direction of collagen fibril alignment in front of the cell. Killing the cells induced relaxation of transient cell-induced tension on the matrix; however, significant permanent remodeling always remained. Time-lapse 3-D imaging demonstrated an active response to the Rho-kinase inhibitor Y-27632, as indicated by cell elongation, extracellular matrix relaxation, and extension of pseudopodial processes. It is interesting that, at higher cell densities, groups of collagen fibrils were compacted and aligned into straps between neighboring cells. Overall, the continued development and application of this new approach should provide important insights into the basic underlying biochemical and biomechanical regulatory mechanisms controlling matrix remodeling by corneal fibroblasts.     

Ciliated and microvillar receptor cells degenerate and then differentiate in the olfactory epithelium of rainbow trout following olfactory nerve section.

We used scanning (SEM) and transmission (TEM) electron microscopy to examine ultrastructural changes in the olfactory epithelium (OE) of rainbow trout following unilateral olfactory nerve section. Both ciliated receptor cells (CRC) and microvillar receptor cells (MRC) degenerated and subsequently differentiated from unidentified precursor cells. The following changes took place in fish that were held at 10 degrees C at the stated period following olfactory nerve section: on day 7, MRC and CRC contained intracellular vacuoles; on day 12, the olfactory knobs appeared disrupted; by day 26, olfactory receptor cells were absent from the OE; on day 42, there were receptor cell bodies and a few CRC with short cilia at the apical surface; and on day 55, a small number of both CRC and MRC had differentiated. By day 76, both CRC and MRC repopulated the OE. Degenerative changes in the cytoplasm of the sustentacular cells (SC) and ciliated nonsensory cells (CNC) were observed in the first 26 days following olfactory nerve section, but these cells remained intact throughout the experiment. The degeneration and subsequent differentiation of CRC and MRC supports and extends previous observations that both cell types are olfactory receptor neurons with axons that extend along the olfactory nerve to the olfactory bulb.