Mechanical properties of the lamprey spinal cord: Uniaxial loading and physiological strain

During spinal cord injury, nerves suffer a strain beyond their physiological limits which damages and disrupts their structure. Research has been done to measure the modulus of the spinal cord and surrounding tissue; however the relationship between strain and spinal cord fibers is still unclear. In this work, our objective is to measure the stress–strain response of the spinal cord in vivo and in vitro and model this response as a function of the number of fibers. We used the larvae lamprey (Petromyzon Marinus), a model for spinal cord regeneration and animal locomotion. We found that physiologically the spinal cord is pre-stressed to a longitudinal strain of 10% and this strain increases to 15% during swimming. Tensile measurements show that uniaxial, longitudinal loading is independent of the meninges. Stress values for uniaxial strains below 18%, are homogeneous through the length of the body. However, for higher uniaxial strains the Head section shows more resistance to longitudinal loading than the Tail. These data, together with the number of fibers obtained from histological sections were used in a composite-material model to obtain the properties of the spinal cord fibers (2.4 MPa) and matrix (0.017 MPa) to uniaxial longitudinal loading. This model allowed us to approximate the percentage of fibers in the spinal cord, establishing a relationship between uniaxial longitudinal strains and spinal cord composition. We showed that there is a proportional relationship between the number of fibers and the properties of the spinal cord at large uniaxial strains.     

Immunocytochemical localization of carbonic anhydrase in the spinal cords of normal and mutant (shiverer) adult mice with comparisons among fixation methods

The peroxidase-antiperoxidase technique was used for immunocytochemical localization of carbonic anhydrase in the mouse spinal cord to detect whether this antigen was normally present in myelinated fibers, in oligodendrocytes in both white and gray matter, and in astrocytes, and to determine where the carbonic anhydrase might be localized in the spinal cords of dysmyelinating mutant (shiverer) mice. The most favorable methods for treating tissue were: 1) immersion in formalin-ethanol-acetic acid followed by paraffin embedding, or 2) light fixation with paraformaldehyde and preparation of vibratome sections. Carnoy's solution, followed by paraffin embedding, extracted myelin from the tissue, while aqueous aldehydes, when used before paraffin embedding, reduced staining everywhere except at sites of compact myelin. The latter conclusion was based, in part, on the almost complete loss of this antigen from the shiverer cord, where compact myelin is known to be virtually absent but where membrane-bound carbonic anhydrase was demonstrated enzymatically. When the optimal methods were used with normal mouse cords, carbonic anhydrase was found throughout the white matter columns and in the oligodendrocytes in gray and white matter. The staining of the white matter was attributed to myelinated fibers because of the similarity in distribution to both a histological myelin stain and the immunocytochemical staining for myelin basic protein. In the mutant mice the oligodendrocyte cell bodies and processes, which were stained in all areas of the spinal cord, were particularly numerous at the periphery of the sections. In contrast to the oligodendrocytes, the fibrous astrocytes appeared to lack carbonic anhydrase, or to have lower than detectable levels, since the astrocyte marker, glial fibrillary acidic protein, had a very different distribution from that of carbonic anhydrase. Even finer localization was obtained in vibratome sections, where the antibody against carbonic anhydrase permitted visualization of the processes connecting oligodendrocytes to myelinated fibers in the normal adult spinal cord.     

Localization of beta-endorphins in the rat spinal cord using avidin-biotin technology

The avidin-biotin-peroxidase technique is proposed for the immunological localization of beta-endorphin in the rat spinal cord. A rabbit specific antibody anti-human beta-endorphin was first obtained and then identified by immunoblotting and incubated with a quick-frozen section of young rat spinal cord. The use of a specific antibody with the immunoperoxidase reaction gave a morphological visualization of the beta-endorphin in the histological sections of the rat spinal cord.     

Spinal Morphine Administration Reduces the Fatty Acid Contents in Spinal Cord and Brain by Increasing Oxidative Stress

It is well known that oxidative stress damages bimolecules such as DNA and lipids. No study is available on the morphine-induced oxidative damage and fatty acids changes in brain and spinal tissues. The aim of this work was to determine the effects of morphine on the concentrations and compositions of fatty acid in spinal cord segments and brain tissues in rabbits as well as lipid peroxidation (LP) and glutathione (GSH) levels in cortex brain. Twelve New Zealand albino rabbits were used and they were randomly assigned to two groups of 6 rabbits each. First group used as control although morphine administrated to rats in second group. Cortex brain and (cervical, thoracic, lumbar) samples were taken. The fatty acids between n:18.0 and 21.0 were present in spinal cord sections and n:10 fatty acids in control animals were present in the brain tissues. Compared to n:20.0–24.0 fatty acids in spinal cord sections and 8.0 fatty acids in the brain tissues of drug administered animals. The concentration and composition of the fatty acid methyl esters in spinal cord and brain tissues was decreased by morphine treatments. LP levels in the cortex brain were increased although GSH levels were decreased by the morphine administration. In conclusion, unsaturated fatty acids contents in brain and spinal cord sections and GSH were reduced by administrating spinal morphine although oxidative stress as LP increased. The inhibition oxidative damage may be a useful strategy for the development of a new protection for morphine administration as well as opiate abuse.     

A Rapid Marchi Technic

By varying the thickness of the nervous tissue immersed in chlorate-osmic-formalin staining fluid (Swank and Davenport, 1935) it was found that a section 1 mm. thick can be completely and adequately stained in 24 hours. Thicker sections require a proportionately longer time. The quality of the Marchi stain in the rapidly prepared section is as good as that in the material stained for 10 days although the background is slightly lighter in the latter preparations. This method can be used where time is an important element and is especially applicable to spinal cord, small animal brains, or portions of larger brains in which serial sections are not required.     

A Rapid Marchi Technic

By varying the thickness of the nervous tissue immersed in chlorate-osmic-formalin staining fluid (Swank and Davenport, 1935) it was found that a section 1 mm. thick can be completely and adequately stained in 24 hours. Thicker sections require a proportionately longer time. The quality of the Marchi stain in the rapidly prepared section is as good as that in the material stained for 10 days although the background is slightly lighter in the latter preparations. This method can be used where time is an important element and is especially applicable to spinal cord, small animal brains, or portions of larger brains in which serial sections are not required.     

Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal cord of the majority of these fiber systems have not been thoroughly investigated in any species. Serotonergic fibers, which project to the spinal cord, have been studied in rats and opossums on histological sections and their functional significance has been deduced based on their fiber termination pattern in the spinal cord. With the development of CLARITY and CUBIC techniques, it is possible to investigate this fiber system and its distribution in the spinal cord, which is likely to reveal previously unknown features of serotonergic supraspinal pathways. Here, we provide a detailed protocol for imaging the serotonergic fibers in the mouse spinal cord using the combined CLARITY and CUBIC techniques. The method involves perfusion of a mouse with a hydrogel solution and clarification of the tissue with a combination of clearing reagents. Spinal cord tissue was cleared in just under two weeks, and the subsequent immunofluorescent staining against serotonin was completed in less than ten days. With a multi-photon fluorescent microscope, the tissue was scanned and a 3D image was reconstructed using Osirix software.     

Chromatographic identification of Met- and Leu-enkephalin in the human fetal spinal cord

Using the indirect immunofluorescence method, enkephalin-like immunoreactivity was visualized on human fetus spinal cord sections (gestational age from 17 to 25 weeks). Immunolabeled varicose fibers and terminal-like structures were seen through the whole length fetal spinal cord principally in the dorsal gray, in the intermediate gray and in the lateral funiculus. A few enkephalin-like immunoreactive cells were sometimes detected in the intermediate gray. Finally, some immunolabeled fibers were also visible in the ventral spinal cord especially proximate to the motor nuclei areas at the sacral level. Fetal spinal cord tissue extracts from the cervical thoracic and lumbosacral region were chromatographically analyzed using high pressure liquid chromatography in combination with the radioimmunoassay. This biochemical analysis indicates that authentic pentapeptides Met- and Leu-enkephalin may account for a large part (more than 90%) of the enkephalin-like immunoreactivity detected in the fetal spinal cord investigated. Taken together our results suggest that the biosynthetic processing of Met- and Leu-enkephalin in this tissue might be functional early before birth.     

A brachial glycogen body in the spinal cord of the domestic chicken.

When cervical segments 14 to 15 of the chicken spinal cord are cut transversely and studied by routine histological and histochemical methods, an onion-shaped region, filled with thread-like fibers, is seen to surround the ependymal cells of the central canal and to be bounded laterally by the neural elements of the spinal gray matter. This area is negative for succinic dehydrogenase, beta-hydroxybutyrate dehydrogenase and cholinesterase activity, but very strongly periodic acid-Schiff positive. Diastase controls show the positive material to be glycogen. Parasagittal sections through this cervical region and into the upper thoracic cord, show the glycogen-rich region to extend longitudinally throughout the region. Because of its location and histochemical characterization, which are similar to that of the ventral portion of the glycogen body, the term brachial glycogen body is proposed for this structure.     

Standardization of Size,Shape and Internal Structure of Spinal Cord Images: Comparison of Three Transformation Methods

Functional fluorescence imaging has been widely applied to analyze spatio-temporal patterns of cellular dynamics in the brain and spinal cord. However, it is difficult to integrate spatial information obtained from imaging data in specific regions of interest across multiple samples, due to large variability in the size, shape and internal structure of samples. To solve this problem, we attempted to standardize transversely sectioned spinal cord images focusing on the laminar structure in the gray matter. We employed three standardization methods, the affine transformation (AT), the angle-dependent transformation (ADT) and the combination of these two methods (AT+ADT). The ADT is a novel non-linear transformation method developed in this study to adjust an individual image onto the template image in the polar coordinate system. We next compared the accuracy of these three standardization methods. We evaluated two indices, i.e., the spatial distribution of pixels that are not categorized to any layer and the error ratio by the leave-one-out cross validation method. In this study, we used neuron-specific marker (NeuN)-stained histological images of transversely sectioned cervical spinal cord slices (21 images obtained from 4 rats) to create the standard atlas and also to serve for benchmark tests. We found that the AT+ADT outperformed other two methods, though the accuracy of each method varied depending on the layer. This novel image standardization technique would be applicable to optical recording such as voltage-sensitive dye imaging, and will enable statistical evaluations of neural activation across multiple samples.     

小鼠脊髓损伤模型的建立及其评价

通过对模型的制备模拟脊髓损伤,研究其病理和影像的变化及脊髓组织的病理分析,为后期的唔疗提供了实验信息。使用7～8周龄小鼠,咬除T9～T10棘突及相应椎板,用重物压迫脊髓,缝合皮肤,制成脊髓损伤模型。分不同的时间进行行为学评分及病理和影像学的检测。结果显示对照组在不同时间行为学评分较高,而实验组评分较低。脊髓损伤区出现明显的病理改变和影像学的改变。可见在实验组中小鼠脊髓损伤区无脊髓组织残留,且出现明显的组织和影像改变,在行为学上两组相比具有显著差异,适用于脊髓再生的研究,从而为进一步研究脊髓损伤提供了较为可靠的模型。     

Mucopolysaccharides in the dog spinal cord and dorsal root ganglia. A histochemical study.

A histochemical study of mucopolysaccharides in the dog spinal cord and dorsal root ganglia is reported in this paper. The histochemical techniques used were the following: PAS, colloidal iron, toluidine blue (pH 5.4 and 3.5), thionine (pH 5.4 and 3.5) and alcian blue 8GX (pH 1 and 2.5). Some histological stains were used also. Two types of neurons could be observed in spinal cord sections stained with colloidal iron techniques. In some neuron a border line of mucosubstances could be seen. In the dorsal root ganglia, different patterns of Nissl bodies distribution in neurons were described. This different distribution of Nissl bodies is associated with different metachromatic colorations of neurons. By using the colloidal iron method, two types of neurons were also revealed in the dorsal root ganglia: some neurons are of a yellow, small-sized and star-shaped type and others are of a light green, larger and round-shaped type. Mucosubstances in the endoneurium and perineurium of nerve fibers, in the Ranvier nodules and in the Schmidt-Lantermann incisures were observed. The possibility that the functional rhythm in some cases might be responsible for the difference in coloration between the dorsal root ganglia neurons is suggested.     

Changes in progenitor populations and ongoing neurogenesis in the regenerating chick spinal cord

The chick spinal cord can regenerate following injury until advanced developmental stages. It is conceivable that changes in stem/progenitor cell plasticity contribute to the loss of this capacity, which occurs around E13. We investigated the contribution of proliferation, phenotypic changes in radial glia progenitors, and neurogenesis to spinal cord regeneration. There was no early up-regulation of markers of gliogenic radial glia after injury either at E11 or E15. In contrast, increased proliferation in the grey matter and up-regulation of transitin expression following injury at E11, but not E15, suggested high levels of plasticity within the E11 spinal cord progenitor population that are lost by later stages. Changes in neural progenitors with development were also supported by a higher neurosphere forming ability at E11 than at E15. Co-labelling with doublecortin and neuron-specific markers and BrdU in spinal cord sections and dissociated cells showed that neurogenesis is an ongoing process in E11 chick spinal cords. This neurogenesis appeared to be complete by E15. Our findings demonstrate that the regeneration-competent chick spinal cord is less mature and more plastic than previously believed, which may contribute to its favourable response to injury, and suggest a role for neurogenesis in maintaining regenerative capacity.     

Thoracic intramedullary sarcoidosis mimicking an intramedullary tumor

Sarcoidosis is a chronic, systemic granulomatous reticulosis of unknown origin, characterized by formation of hard tubercles and noncaseatinggranulomas. Since other infectious diseases such as berylliosis, mycobacterium and fungal infections may present with a noncaseating granulomas, histological diagnosis of sarcoidosis is made using the elimination method. Central nervous system manifestations of sarcoidosis may be present in 5-10% of the cases involving cranial nerves, leptomeninges and third ventricle respectively. Any part of the central nervous system can be affected. Involvement of spinal cord in sarcoidosis is extremely rare and presents with only 0.3-0.4% in patients with systemic sarcoidosis. Intramedullary sarcoidosis is a rare first manifestation of the disease and it can mimic an intramedullary tumor, which is often manifested with symptoms that initiate from spinal cord compression, resulting in paraparesis, sensory disorders and sphincter dysfunction. We present a case of intramedullary sarcoidosis that mimics a tumor of the thoracic spinal cord. Clinical features, neuroradiological, pathohistological findings, laboratory analysis and surgical treatment of such a rare entity are being discussed.     

Distribution of label after intrathecal administration of 125I-substance P in the rat

Despite the widespread use of the intrathecal route for the administration of neuroactive agents, little is known about the penetration of these agents into the spinal cord. In the present study, 125I-substance P was injected via a spinal catheter to the thoracic or sacro-coccygeal spinal cord in the rat (350-400 g) anesthetized with urethane (2.5 g/kg). Spinal cords were removed rapidly at 1 or 10 min after injection and immediately frozen in CCl2F2. Frozen sections, 20 micron thick, were cut and mounted for autoradiography. Autoradiographs of transverse sections demonstrated that the label penetrated 700 to 1800 micron from the surface of the spinal cord at both levels. In longitudinal sections, this penetration extended about 0.5 cm rostrally and caudally from the site of injection. Serial autoradiographs of transverse sections showed a similar penetration rostro-caudally. In addition, venous blood samples were taken at 1, 6, 11 and 16 min after injection of the labelled peptide. Quantification of the radioactivity in the samples revealed that 0.8 to 3.5% of the total CPM injected had passed into the general circulation at these times. These data indicate that after intrathecal administration of radiolabelled substance P, the label penetrates into the grey matter of the spinal cord to presumed sites of action. They also suggest that the rostro-caudal extent of penetration is more localized than suggested from earlier studies which looked only at levels of radioactivity in pieces of whole spinal cord. Finally, our study has indicated that passage of label into the circulation is negligible at least for substance P.(ABSTRACT TRUNCATED AT 250 WORDS)     

A method for preparing 2- to 50-µm-thick fresh-frozen sections of large samples and undecalcified hard tissues

This article describes a method for preparing 2- to 50-micron-thick fresh-frozen sections from large samples and completely calcified tissue samples. In order to perform the more routine work involved, a tungsten carbide disposable blade was installed to a heavy-duty sledge cryomicrotome. An entire 10-day-old rat and bone and tooth samples from a 7-month-old rat were rapidly frozen. The frozen samples were attached to the cryomicrotome stage. The cutting surface of the samples was covered with a polyvinylidene chloride film coated with synthetic rubber cement and cut at -25 degrees C. The soft tissues and the hard tissues were satisfactorily preserved and all tissue cells were easily identifiable. Enzymatic activity in the fresh sections was much stronger than that in chemically fixed and/or decalcified sections. The sections permitted histological and histochemical studies without trouble. In addition, the sections can be used for multiple experiments such as immunohistochemistry, in situ hybridization, and electron microprobe X-ray micro-analysis. This method can be used with conventional cryomicrotome equipment.     

Three-dimensional evaluation of the spinal local neural network revealed by the high-voltage electron microscopy: a double immunohistochemical study

Three-dimensional (3-D) analysis of anatomical ultrastructures is important in biological research. However, 3-D image analysis on exact serial sets of ultra-thin sections from biological specimens is very difficult to achieve, and limited information can be obtained by 3-D reconstruction from these sections due to the small area that can be reconstructed. On the other hand, the high-penetration power of electrons by an ultra-high accelerating voltage enables thick sections of biological specimens to be examined. High-voltage electron microscopy (HVEM) is particularly useful for 3-D analysis of the central nervous system because considerably thick sections can be observed at the ultrastructure level. Here, we applied HVEM tomography assisted by light microscopy to a study of the 3-D chemical neuroanatomy of the rat lower spinal cord annotated by double-labeling immunohistochemistry. This powerful methodology is useful for studying molecular and/or chemical neuroanatomy at the 3-D ultrastructural level.     

In vivo imaging of the mouse spinal cord using two-photon microscopy

In vivo imaging using two-photon microscopy in mice that have been genetically engineered to express fluorescent proteins in specific cell types has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task. We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.     

Photobiomodulation by diffusing optical fiber on spinal cord: A feasibility study in piglet model

Previous studies on spinal cord injury (SCI) have confirmed that percutaneous photobiomodulation (PBM) therapy can ameliorate immunoinflammatory responses at sites of injury, accelerate nerve regeneration, suppress glial scar formation and promote the subsequent recovery of locomotor function. The current study was performed to evaluate a large‐animal model employing implanted optical fibers to accurately irradiate targeted spinal segments. The method's feasibility and irradiation parameters that do not cause phototoxic reaction were determined, and the methodology of irradiating the spinal cord with near‐infrared light was investigated in detail. A diffusing optical fiber was implanted above the T9 spinal cord of Bama miniature pigs and used to transfer near‐infrared light (810 nm) onto the spinal cord surface. After daily irradiation with 200, 300, 500 or 1000 mW for 14 days, both sides of the irradiated area of the spinal cord were assessed for temperature changes. The condition of the spinal cord and the position of optical fiber were investigated by magnetic resonance imaging (MRI), and different parameters indicating temperature increases or phototoxicity were measured on the normal spinal cord surface due to light irradiation (ie, heat shock responses, inflammatory reactions and neuronal apoptosis), and the animals' lower‐limb neurological function and gait were assessed during the irradiation process. The implanted device was stable inside the freely moving animals, and light energy could be directly projected onto the spinal cord surface. The screening of different irradiation parameters preliminary showed that direct irradiation onto the spinal cord surface at 200 and 300 mW did not significantly increase the temperature, stress responses, inflammatory reactions and neural apoptosis, whereas irradiation at 500 mW slightly increased these parameters, and irradiation at 1000 mW induced a significant temperature increase, heat shock, inflammation and apoptosis responses. HE staining of spinal cord tissue sections did not reveal any significant structural changes of the tissues compared to the control group, and the neurological function and gait of all irradiated animals were normal. In this study, we established an in‐vivo optical fiber implantation method, which might be safe and stable and could be used to directly project light energy onto the spinal cord surface. This study might provide a new perspective for clinical applications of PBM in acute SCI.