Normal Human Spinal Cord Research Articles

Zonally Magnified Oblique Multislice and Non-Zonally Magnified Oblique Multislice DWI of the Cervical Spinal Cord

The zonally magnified oblique multislice EPI (ZOOM-EPI) diffusion-weighted sequence has been visually shown to provide superior MR diffusion image quality compared with the full-FOV single-shot EPI sequence (non-ZOOM-EPI) in the adult cervical spinal cord. The purpose of this study was to examine the diffusion tensor imaging indices in the normal human cervical spinal cord between ZOOMED and non-ZOOMED DTI acquisitions and determine whether DTI values are comparable between direct and indirect age-matched groups. Fifty-four subjects 23-58 years of age (9 direct age-matched and 45 indirect age-matched) were scanned using a 1.5T scanner. Diffusion tensor indices including fractional anisotropy, mean diffusivity, axial diffusivity, and radial diffusivity were generated from the DTI dataset. These DTI values were calculated for both ZOOM and non-ZOOM acquisitions and compared at each intervertebral disc level. The variability of the DTI values for ZOOM and non-ZOOM sequences was measured using a coefficient of variation within direct and indirect age-matched controls. The mean diffusivity, axial diffusivity, and radial diffusivity values obtained along the cervical spinal cord in the age-matched controls showed a significant decrease using the ZOOM sequence (P = .05, P = .002, and P < .001). Mean fractional anisotropy showed a significant increase (P = .04) using the ZOOM sequence. The indirect age-matched controls showed a statistically significant increase in fractional anisotropy (P = .03) and a decrease in mean diffusivity (P = .002), axial diffusivity (P < .001), and radial diffusivity (P = .002) using the ZOOM sequence. Less variability has been shown in DTI using the ZOOM sequence compared with the non-ZOOM sequence in both direct and indirect age groups. The ZOOM sequence exhibited higher SNR (SNRZOOM = 22.84 ± 7.59) compared with the non-ZOOM sequence (SNRnon-ZOOM = 19.7 ± 7.05). However, when we used a 2-tailed t test assuming unequal variances, the ZOOM sequence did not demonstrate a statistically significant increase. ZOOM DTI acquisition methods provide superior image quality and precision over non-ZOOM techniques and are recommended over conventional full-FOV single-shot EPI DTI for clinical applications in cervical spinal cord imaging.

Angiogenin loss‐of‐function mutations in amyotrophic lateral sclerosis

Heterozygous missense mutations in the coding region of angiogenin (ANG), an angiogenic ribonuclease, have been reported in amyotrophic lateral sclerosis (ALS) patients. However, the role of ANG in motor neuron physiology and the functional consequences of these mutations are unknown. We searched for new mutations and sought to define the functional consequences of these mutations. We sequenced the coding region of ANG in an independent cohort of North American ALS patients. Identified ANG mutations were then characterized using functional assays of angiogenesis, ribonucleolysis, and nuclear translocation. We also examined expression of ANG in normal human fetal and adult spinal cords. We identified four mutations in the coding region of ANG from 298 ALS patients. Three of these mutations are present in the mature protein. Among the four mutations, P(-4)S, S28N, and P112L are novel, and K17I has been reported previously. Functional assays show that these ANG mutations result in complete loss of function. The mutant ANG proteins are unable to induce angiogenesis because of a deficiency in ribonuclease activity, nuclear translocation, or both. As a correlate, we demonstrate strong ANG expression in both endothelial cells and motor neurons of normal human spinal cords from the developing fetus and adult. We provide the first evidence that ANG mutations, identified in ALS patients, are associated with functional loss of ANG activity. Moreover, strong ANG expression, in normal human fetal and adult spinal cord neurons and endothelial cells, confirms the plausibility of ANG dysfunction being relevant to the pathogenesis of ALS.

Calcitonin gene-related peptide immunoreactivity in chronic human spinal cord injury

Histopathological study of the human spinal cord. International Collaboration on Repair Discoveries, Vancouver, BC, Canada. In animals, primary dorsal root afferent fibers, which are immunoreactive for calcitonin gene-related peptide (CGRP), sprout following spinal cord injury (SCI) into deeper laminas of the dorsal horn below the level of injury. It has been suggested that this aberrant sprouting plays a role in altering cardiovascular control after SCI and could be responsible for life-threatening episodes of autonomic dysreflexia (AD). To observe the changes of CGRP distribution after SCI and compare the differences between normal and injured human spinal cord. Upper thoracic segments from individuals with chronic cervical SCI (n=4) and individuals with intact spinal cords (n=5) were processed immunocytochemically to identify CGRP fibers and histologically to identify the severity of degeneration. Semiquantitative analysis showed a significant increase in CGRP immunoreactivity in the dorsal horns of individuals with chronic SCI (P<0.001). Furthermore, one of the SCI individuals in this study displaying significant CGRP sprouting had well documented episodes of AD. Our observations suggest that SCI in humans results in significant sprouting of CGRP fibers. This aberrant sprouting of sensory fibers could contribute to the abnormal cardiovascular control and pain commonly observed following chronic human SCI.

Differential microglial regulation in the human spinal cord under normal and pathological conditions

As the primary intrinsic immune effector cells of the central nervous system, microglia are involved in virtually all pathological processes of the brain and spinal cord including inflammatory, neurodegenerative, traumatic, neoplastic and vascular diseases. Despite this important role, there is a lack of data concerning microglial distribution and protein expression in the human spinal cord. In this study, we immunohistochemically investigated 10 normal human spinal cords to establish reference data and compared these results with 15 pathological human spinal cords deriving from distinct pathologies. Each spinal cord was evaluated at eight different levels for three white and two grey matter areas for both constitutive (MHC-II, CD68, IL-16, AIF-1, LCA, CD4) and reactive (MRP-8, MRP-14) microglial antigens. Whereas previous studies revealed significant regional differences in microglial distribution and protein expression in human brain, normal spinal cord displayed a uniform expression pattern, reaching levels of up to 17% MHC-II positive cells of the total cell population. This datum formed the basis for the further evaluation of microglia expression levels in pathological spinal cords, where levels of up to 45% positive cells were observed. Our results represent important reference values for future neuropathological diagnostic and therapeutical approaches in spinal cord pathologies.

Expression of neurotrimin in the normal and injured adult human spinal cord

Neurotrimin (Ntm) is a member of the family of neural cell adhesion molecules. Its expression pattern suggests that Ntm promotes axonal fasciculation, guides nerve fibers to specific targets and stabilizes synapses as it accumulates coincident with synaptogenesis. Strong labeling of Ntm was observed in motor and sensory areas of the postnatal rat cortex. It is not known whether Ntm is present in adult human spinal cord (SC). In the present study, a monoclonal antibody specific for Ntm (1B1), is applied to the first study of the expression of Ntm in normal and injured adult human SC. (1) To investigate the expression pattern of Ntm in adult normal human SC, and (2) to observe the changes of Ntm expression after SC injury and compare the differences between normal and injured adult human SC. Human SC tissue was obtained from necropsies of patients with (n=5) and without (n=4) SC injury. The 1B1 Ntm monoclonal antibody was used for immunohistochemical staining on paraffin embedded sections with an ABC kit. (1) In total, 12 slides were analyzed for each group from both cervical and thoracic levels. Motor neurons and Clarke's neurons and glial-like cells were mild to moderately positive in all uninjured SC specimens. (2) In injured SC, no staining was observed in the injury epicenter between two and three levels proximally and distally, but was detected five levels away. (3) In patients older than 67 years of age, Ntm-positive inclusions were present in the white matter of the SC with or without injury. (4) Some meningeal cells were strongly Ntm-positive, especially in the uninjured human SC. Ntm is expressed by motor and Clarke's neurons and glial cells in uninjured human SC. The downregulation of Ntm in the injured SC suggests that its expression is regulated by afferent input.

Expression of metabotropic glutamate receptor mRNAs in the human spinal cord: implications for selective vulnerability of spinal motor neurons in amyotrophic lateral sclerosis

To elucidate the relevance of metabotropic glutamate receptors (mGluRs) to the selective vulnerability of motor neurons in the spinal cord in patients with amyotrophic lateral sclerosis (ALS), we investigated the distribution of mRNAs coding mGluR1-5 in the normal human spinal cord. The mRNAs for mGluR1, 4 and 5 were observed in the spinal gray matter, whereas mGluR2 mRNA was absent in the spinal cord and mGluR3 mRNA was displayed only on glial cells in the white matter. Signals for mGluR1 and mGluR5 were enriched in the dorsal horn, while mGluR4 mRNA was abundant in the ventral horn. Since agonists to group I mGluRs (mGluR 1 and 5) have been demonstrated to have neuroprotective effects on spinal motor neurons, less expression of mRNAs coding mGluR1 and mGluR5 in the ventral horn than in the dorsal horn may be implicated in the selective susceptibility of spinal motor neurons in ALS.

Immunolocalization of NAIP in the human brain and spinal cord.

The neuronal apoptosis inhibitory protein (NAIP) is known to have anti-apoptotic functions, and its gene is often mutated in severe cases of spinal muscular atrophy (SMA), a disease characterized by motor neuron degeneration. In this study, we examined the distribution of the endogenous NAIP protein in normal human spinal cord and brain tissue by using a polyclonal antibody against NAIP. Immunohistochemical staining demonstrated that NAIP is strongly expressed in anterior horn and motor cortex neurons of normal brains, and it is not altered in the remaining motor neurons of patients with amyotrophic lateral sclerosis (ALS). NAIP is also located in human fetal neurons and in adult choroid plexus cells. These results suggest that the anti apoptotic molecule NAIP may be important in motor neurons, but it specifically does not appear to be altered in ALS.

Location of the phrenic nucleus in the human spinal cord.

Eight normal human spinal cords were studied. Spinal segments were identified and embedded in paraffin wax. Serial cross sections were cut at 25 microm and stained by cresyl violet. Motor columns were reconstructed adapting Elliott's (1942) methods. Motor columns were classified into the medial and lateral divisions and were numbered sequentially from medial to lateral at the level of C1. In the cervical cord, 8 motor columns were traced. Column 1, corresponding to the medial column, presented 3 subdivisions designated as 1a, 1b and 1c with ventral, dorsal and lateral positions respectively. Columns 1a and 1b extended throughout the cervical region while 1c was confined to 3rd, 4th and 5th cervical segments. At the level of C3, 1c was a discrete column situated lateral to 1a and 1b but at C4 and C5 it became displaced medially close to the medial margin of the ventral horn. In cross section, it presented smaller medial and large lateral part. With the help of clinical and developmental evidence an attempt was made to correlate column 1c with the phrenic nucleus.

A study of motoneuron groups and motor columns of the human spinal cord.

Eight normal human spinal cords were studied for motoneuron (Mn) groups and columns. Spinal segments (C1 to Coc.) were identified and embedded in paraffin wax. Serial cross sections were cut at 25 microm and stained by cresyl violet. Cross-sectional profiles of the spinal cord were traced for each segmental level and the outlines of the various Mn groups superimposed. These charts (maps) were used to examine intra and intersegmental changes in the relative positions of the columns. An attempt was made to provide topographical picture of Mn groups of individual segments. In the cervical region neuronal groups were more numerous but smaller and less distinct, while in the lumbosacral region they were fewer, larger and at many levels better circumscribed. The average number of Mn groups at any segmental level was 3-4 and never exceeded 5. C4, C5, C6, C7, L4, L5 and S1 contained numerous Mn groups. Maximum intrasegmental changes were noted at C3, C4, C7, T1, and S2, while at C5, C6, all thoracic, L1 L2 and L3, the pattern was constant throughout the segment. Eleven motor columns were traced in the human spinal cord. Column 1 belonged to the medial division and columns 2-11 to the lateral division of the ventral grey horn. Columns 1 and 2 were the most extensive as they were traceable from the lower medulla to S3 segment. Columns 3-8 were confined to cervical segments (including T1), while columns 9-11 were traced in lumbosacral segments. In general, motor columns followed a definite mode for their appearance and disappearance. Many of them showed rotation from a dorsal to a ventromedial direction.

Attempts to identify herpes simplex virus DNA in normal and diseased human peripheral nerve.

PCR analysis of DNA extracted from 31 sections of formalin-fixed sural nerve biopsies did not reveal herpes simplex virus (HSV) DNA. Unlike the presence of HSV DNA sequences in normal human brain, spinal cord, and ganglia, HSV DNA is not present in normal or diseased human distal peripheral nerve.

Kinesin and cytoplasmic dynein in spinal spheroids with motor neuron disease.

Kinesin and cytoplasmic dynein are two major molecular motors responsible for fast axonal transport. As visualized by immunohistochemistry with monoclonal antibodies, both motors were found to be distributed throughout the cell bodies, dendrites and axons of motor neurons in normal human spinal cords. Large axonal swellings, spheroids, in the spinal cords of patients with motor neuron disease showed massive accumulation of kinesin co-localized with highly phosphorylated neurofilaments. Of 114 spheroids in five spinal cords, 87% were stained heavily with the three anti-kinesin antibodies used in this study. Cytoplasmic dynein was scarce or absent in most of the spheroids. These findings suggest that kinesin selectively accumulates in the spheroids of motor neuron axons, causing disturbance of the machinery for anterograde fast axonal transport in motor neuron disease.

Distribution of B-50(GAP-43) mRNA and protein in the normal adult human spinal cord.

B-50(GAP-43) is a phosphoprotein mainly found in the nervous system which plays a major role in neurite growth during development and regeneration as well as in synaptic remodelling. In the mature intact central nervous system, intense B-50 immunoreactivity (B-50-IR) can still be detected in regions which maintain residual capacity for structural re-organization. B-50 expression has been studied extensively in laboratory animals; however, its distribution and regulation in the human spinal cord is largely unknown. As a first step to analyze lesion-induced structural alterations, we investigated the distribution of B-50 protein and mRNA in the normal adult human spinal cord and dorsal root ganglia. Intense B-50-IR was localized to the superficial laminae of the dorsal horn at all segmental levels, the intermediolateral nucleus at thoracic levels and Onuf's nucleus at sacral levels. Scattered neurons, particularly in the ventral horn of lumbar and sacral segmental levels (and occasionally also in Clarke's nucleus) displayed intense B-50-IR in close apposition to the perikaryal and proximal dendritic surfaces. Nonradioactive in situ hybridization indicated that B-50 mRNA could also be detected in neurons of the ventral horn and also in the intermediolateral nucleus. The distribution of B-50 mRNA and protein in the normal human spinal cord shows a marked similarity to that reported in experimental animals, including the selective labelling of Onuf's nucleus. However, the strong B-50-IR on the surface of some large anterior horn motor neurons has not been observed in other mammals. This finding might reflect a particular state of readiness for synaptic plasticity.

Immunocytochemical study of the distribution of the free radical scavenging enzymes CU/ZN superoxide dismutase (SOD1); MN superoxide dismutase (MN SOD) and catalase in the normal human spinal cord and in motor neuron disease

Interest in free radical-mediated mechanisms of motor neuron injury has arisen following the discovery of point mutations in the CU/ZN superoxide dismutase (SOD1) gene in a proportion of cases of familial motor neuron disease (MND). Evidence is emerging which indicates that oxidative stress may contribute to the pathophysiology of sporadic MND. Free radical scavenging enzymes form a major component of the anti-oxidant defense system. The aim of this study was to compare the distribution and density of immunoreactivity to CU/ZN SOD, Mn SOD and catalase in the spinal cord of sporadic MND cases ( n = 10) compared to normal controls ( n = 8). There was abundant expression of CU/ZN SOD, Mn SOD and catalase in spinal motor neurons, suggesting important roles for these enzymes in neuroprotective pathways in this cell group. In MND cases, there was no evidence in surviving motor neurons of a consistent alteration in the protein expression of any of these enzymes. There was evidence of increased expression of these enzymes in glial cells present in the ventral and intermediate grey matter and degenerating descending motor pathways of the spinal cord in MND cases. The changes observed were more marked in the cervical compared to lumbar spinal segments. Further investigation is required to determine whether these findings represent a compensatory response to a pathophysiological process involving oxidative stress.

Morphologic Features of the Normal Human Cadaveric Spinal Cord

The cross-sectional area and diameter of the normal cadaveric spinal cord at each segmental level were measured, and the morphologic features were presented. To provide accurate anatomic descriptions and morphometric data of the human spinal cord. There is a large individual variation in human spinal cord size, and no authorized standard of measurement has been established. There have been few detailed descriptions of the normal morphologic features of the spinal cord. The authors measured the cross-sectional area and diameter of the spinal cord at each segment from C2 and S3 in 12 cadaveric specimens, and the morphologic features of each segment were described. The relative ratio of the cross-sectional area of each segment to that of the C3 segment was similar in all the specimens examined despite a large individual variation in absolute cord size. Each segment had distinct qualitative and quantitative morphologic features. The normal cross-sectional area of the spinal cord at any segment in an individual is calculable from measurements of a given single normal segment. This value appears to be an appropriate and practical standard of measurement of the normal morphologic features of the spinal cord.

Internalization of IgG in motoneurons of patients with ALS

The hypothesis that abnormal antibodies may be involved in the pathogenesis of ALS has been supported in part by IgG's being present within motoneurons of ALS patients more frequently than in motoneurons of controls. IgG, as well as other serum proteins, is also present in motoneurons of normal human and animal spinal cords. We attempted to determine whether the IgG found in motoneurons of ALS patients was localized by an immune-specific or nonspecific process. To address this question, we used immunocytochemistry to evaluate the presence and relative density of different serum proteins in spinal cords from nine patients with ALS. Both IgG and alpha 2-macroglobulin (alpha 2Mac) were present in motoneurons in all nine cases. More important, there was a close concordance between the IgG and alpha 2Mac immunolabeling of motoneurons. The presence of a nonimmune plasma protein--alpha 2Mac--in a similar distribution to IgG and with a similar intensity implies that the internalization of these proteins in motoneurons of patients with ALS is best explained by a nonselective mechanism of endocytosis of extracellular fluid.

Distribution of secretoneurin-like immunoreactivity in comparison with that of substance P in the human spinal cord

Secretoneurin (SN), a neuropeptide of 33 amino acids, was determined in comparison with substance P (SP) by immunocytochemistry in normal human spinal cord. The density of secretoneurin-like immunoreactivity (SN-IR) was high in the superficial dorsal horn and in the lateral column of autonomic arcs. The ventral horn displayed low to moderate density of SN-IR and prominently outlined motoneurons. The congruent distribution of SN and SP to the termination of primary afferents may indicate that SN is involved in modulation of pain.

Stress-response (heat-shock) protein 90 expression in tumors of the central nervous system: an immunohistochemical study.

This retrospective study deals with the expression of stress-response (heat-shock) protein 90 (srp 90) in a series of 148 human brain tumors. Immunohistochemical procedures were employed; cells of the human breast cancer line MCF7 exposed to hyperosmolar stress served as positive controls. Deposits of reaction products were found in the cytoplasm and they displayed a granular pattern. srp 90 was detected in 14/31 meningiomas and 5/10 breast cancer metastases to the brain. The protein was also present in 6/13 glioblastomas and 7/18 astrocytomas. In addition, a positive reaction was found in 2/10 medulloblastomas, 2/14 primitive neuroectodermal tumors, 1/11 pituitary tumor, 2/21 schwannomas and 2/11 lung tumor metastases; however, oligodendrogliomas and primary malignant lymphomas were not stained. The srp 90 was detected in Western blots of meningioma tissue homogenates. No significant immunohistochemical reaction was seen with sections of normal human cerebra, brain stem, cerebella, pituitary glands and spinal cords. These results document the expression of srp 90 by a variety of primary and metastatic intracranial tumors.

3H] d-aspartate binding sites in the normal human spinal cord and changes in motor neuron disease: a quantitative autoradiographic study

The distribution and density of glutamate transporter sites was determined in human cervical and lumbar spinal cord, by quantitative autoradiography using [ 3H] d-aspartate. In the normal human spinal cord ( n = 8) there was specific binding of [ 3H] d-aspartate throughout the spinal grey matter, with the highest levels observed in the substantia gelatinosa and central grey matter. In the ventral horns, particularly at the L5 level, focal hot spots of binding were observed in a distribution corresponding to that of lower motor neuron somata. Comparison of motor neuron disease (MND) cases ( n = 12) with normal controls showed a reduction in the density of [ 3H] d-aspartate binding in the intermediate grey matter and the substantia gelatinosa of the lumbar cord. These changes were more marked in the amyotrophic lateral sclerosis (ALS) compared to the progressive muscular atrophy (PMA) subgroup, and may be due to loss of glutamatergic terminals of the corticospinal tract. The changes observed in the cervical cord were milder and did not reach statistical significance. No differences were found between [ 3H] d-aspartate binding in the spinal cords of the normal controls and a neurological disease control group ( n = 6), suggesting that the changes observed in MND are disease specific. These findings provide further evidence in support of a disturbance of glutamatergic neurotransmission in MND.

A quantitative autoradiographic study of [ 3H]kainate binding sites in the normal human spinal cord, brainstem and motor cortex

The quantitative autoradiographic distribution of the kainate subtype of non-NMDA receptor in the normal human motor cortex, brainstem and spinal cord has been investigated using [ 3H]kainate. In the motor cortex specific [ 3H]kainate binding was present in all cortical laminae with the highest density in laminae and II and the upper part of III and lower densities in the middle and deep laminae. In the premotor cortex a band of high density was found in laminae V and VI as well as in the superficial laminae. In the normal brainstem kainate receptors had a heterogeneous distribution. Brainstem motor nuclei which tend to be affected in motor neuron disease (MND) had very low densities of binding sites, whereas the oculomotor nucleus had a higher density. Specific [ 3H]kainate binding was found throughout the spinal grey matter, the greatest density being found in the substantia gelatinosa and much lower densities in the rest of the grey matter including the ventral horns. Excitotoxicity at non-NMDA receptors has been implicated in the pathogenesis of MND. This study shows that the motor neuron groups vulnerable in MND express a low density of [ 3H]kainate binding sites and suggests that the density of kainate receptors does not account for selective vulnerability in this disorder.

Calcitonin gene-related peptide (CGRP)-like immunoreactivity in motoneurons of the human spinal cord following injury.

Changes that occur in the localization of calcitonin gene-related peptide-like-immunoreactivity (CGRP-LI) in motoneurons, following injury to the human spinal cord, were examined. CGRP-LI above and below the level of injury was compared to normal human spinal cord. Vibratome sections were cut and processed for immunostaining using the avidin-biotin immunoperoxidase method. Whereas motoneurons above the lesion contained CGRP-LI, below the lesion the CGRP-LI was reduced. In 3 spinal cords from patients who had survived between 1 and 8 years after injury, CGRP-LI in motoneurons of lumbar spinal cord was absent or considerably diminished. In the spinal cord from one patient who had died several years after injury, there was a unilateral augmentation of CGRP immunostaining. The results suggest that CGRP levels in motoneurons are affected by spinal cord injury in humans and that CGRP levels in motoneurons may be regulated either by supraspinal afferent inputs or by muscle activity.