The Structure of the Spinal Cord Ependymal Region in Adult Humans Is a Distinctive Trait among Mammals.

In species that regenerate the injured spinal cord, the ependymal region is a source of new cells and a prominent coordinator of regeneration. In mammals, cells at the ependymal region proliferate in normal conditions and react after injury, but in humans, the central canal is lost in the majority of individuals from early childhood. It is replaced by a structure that does not proliferate after damage and is formed by large accumulations of ependymal cells, strong astrogliosis and perivascular pseudo-rosettes. We inform here of two additional mammals that lose the central canal during their lifetime: the Naked Mole-Rat (NMR, Heterocephalus glaber) and the mutant hyh (hydrocephalus with hop gait) mice. The morphological study of their spinal cords shows that the tissue substituting the central canal is not similar to that found in humans. In both NMR and hyh mice, the central canal is replaced by tissue reminiscent of normal lamina X and may include small groups of ependymal cells in the midline, partially resembling specific domains of the former canal. However, no features of the adult human ependymal remnant are found, suggesting that this structure is a specific human trait. In order to shed some more light on the mechanism of human central canal closure, we provide new data suggesting that canal patency is lost by delamination of the ependymal epithelium, in a process that includes apical polarity loss and the expression of signaling mediators involved in epithelial to mesenchymal transitions.

The adult macaque spinal cord central canal zone contains proliferative cells and closely resembles the human.

The persistence of proliferative cells, which could correspond to progenitor populations or potential cells of origin for tumors, has been extensively studied in the adult mammalian forebrain, including human and nonhuman primates. Proliferating cells have been found along the entire ventricular system, including around the central canal, of rodents, but little is known about the primate spinal cord. Here we describe the central canal cellular composition of the Old World primate Macaca fascicularis via scanning and transmission electron microscopy and immunohistochemistry and identify central canal proliferating cells with Ki67 and newly generated cells with bromodeoxyuridine incorporation 3 months after the injection. The central canal is composed of uniciliated, biciliated, and multiciliated ependymal cells, astrocytes, and neurons. Multiciliated ependymal cells show morphological characteristics similar to multiciliated ependymal cells from the lateral ventricles, and uniciliated and biciliated ependymal cells display cilia with large, star-shaped basal bodies, similar to the Ecc cells described for the rodent central canal. Here we show that ependymal cells with one or two cilia, but not multiciliated ependymal cells, proliferate and give rise to new ependymal cells that presumably remain in the macaque central canal. We found that the infant and adult human spinal cord contains ependymal cell types that resemble those present in the macaque. Interestingly, a wide hypocellular layer formed by bundles of intermediate filaments surrounded the central canal both in the monkey and in the human, being more prominent in the stenosed adult human central canal.

Central canal ependymal cells proliferate extensively in response to traumatic spinal cord injury but not demyelinating lesions.

The adult mammalian spinal cord has limited regenerative capacity in settings such as spinal cord injury (SCI) and multiple sclerosis (MS). Recent studies have revealed that ependymal cells lining the central canal possess latent neural stem cell potential, undergoing proliferation and multi-lineage differentiation following experimental SCI. To determine whether reactive ependymal cells are a realistic endogenous cell population to target in order to promote spinal cord repair, we assessed the spatiotemporal dynamics of ependymal cell proliferation for up to 35 days in three models of spinal pathologies: contusion SCI using the Infinite Horizon impactor, focal demyelination by intraspinal injection of lysophosphatidylcholine (LPC), and autoimmune-mediated multi-focal demyelination using the active experimental autoimmune encephalomyelitis (EAE) model of MS. Contusion SCI at the T9-10 thoracic level stimulated a robust, long-lasting and long-distance wave of ependymal proliferation that peaked at 3 days in the lesion segment, 14 days in the rostral segment, and was still detectable at the cervical level, where it peaked at 21 days. This proliferative wave was suppressed distal to the contusion. Unlike SCI, neither chemical- nor autoimmune-mediated demyelination triggered ependymal cell proliferation at any time point, despite the occurrence of demyelination (LPC and EAE), remyelination (LPC) and significant locomotor defects (EAE). Thus, traumatic SCI induces widespread and enduring activation of reactive ependymal cells, identifying them as a robust cell population to target for therapeutic manipulation after contusion; conversely, neither demyelination, remyelination nor autoimmunity appears sufficient to trigger proliferation of quiescent ependymal cells in models of MS-like demyelinating diseases.

Loss of cell adhesion causes hydrocephalus in nonmuscle myosin II-B-ablated and mutated mice.

Ablation of nonmuscle myosin (NM) II-B in mice during embryonic development leads to marked enlargement of the cerebral ventricles and destruction of brain tissue, due to hydrocephalus. We have identified a transient mesh-like structure present at the apical border of cells lining the spinal canal of mice during development. This structure, which only contains the II-B isoform of NM, also contains beta-catenin and N-cadherin, consistent with a role in cell adhesion. Ablation of NM II-B or replacement of NM II-B with decreased amounts of a mutant (R709C), motor-impaired NM II-B in mice results in collapse of the mesh-like structure and loss of cell adhesion. This permits the underlying neuroepithelial cells to invade the spinal canal and obstruct cerebral spinal fluid flow. These defects in the CNS of NM II-B-ablated mice seem to be the cause of hydrocephalus. Interestingly, the mesh-like structure and patency of the spinal canal can be restored by increasing expression of the motor-impaired NM II-B, which also rescues hydrocephalus. However, the mutant isoform cannot completely rescue neuronal cell migration. These studies show that the scaffolding properties of NM II-B play an important role in cell adhesion, thereby preventing hydrocephalus during mouse brain development.

Detection of epithelial cell transfer in spinal areas by light microscopy and determining any tissue coring via cell culture during combined spinal-epidural interventions.

BACKGROUND AND OBJECTIVES: Epithelial tissue coring by spinal needles during subarachnoid injections may cause intraspinal epidermal tumors. Previous studies have investigated tissue transfer with different needle types during subarachnoid or epidural injection. This study deals with the transfer of epithelial tissue during combined spinal-epidural (CSE) anesthesia. METHODS: We studied 68 American Society of Anesthesiologists I to III adult patients. CSE anesthesia was induced under aseptic conditions at the L2-3 or L3-4 interspace with patients in the lateral decubitus position. Cerebral spinal fluid, spinal needle stylet, fluid used to flush the interior of the spinal needle, fluid used to wash the exterior of the spinal needle, fluid used to flush the interior of the epidural needle, and fluid used to wash the exterior tip of the epidural needle were examined under light microscopy (n = 30 patients) or incubated in a cell-culture medium (n = 38 patients). Samples were incubated in cell-culture medium alone (n = 13) or in a cell-culture medium for 3 weeks and then in a medium with epidermal growth factor (n = 25). As a positive control, skin tissue samples were taken by punch biopsy from 10 randomly chosen patients who underwent CSE interventions. These samples were incubated in an enriched medium serum. RESULTS: Light microscopy revealed that there was cell transfer in all phases in various rates: samples 1, 2, 3, 4, 5, and 6 contained epithelial cells and debris in ratios of 6.9%, 20.7%, 6.9%, 20.7%, 26.7%, and 33.3%, respectively. Epithelial cell colonization was detected in the cell-culture samples taken from the control group but not in the samples taken from the CSE group. CONCLUSIONS: We could not reproduce the cells or cell debris obtained during the CSE interventions in vivo, which can be explained by a possible structural deformation of cells or the inadequacy of the amount of cells that were transferred.

Recombinant human bone morphogenic protein-2-enhanced anterior spine fusion without bone encroachment into the spinal canal: a histomorphometric study in a thoracoscopically instrumented porcine model.

STUDY DESIGN: A thoracoscopically assisted 5-level anterior spinal fusion and instrumentation model analyzing new bone formation when using recombinant human bone morphogenic protein-2 (rhBMP-2) with a collagen hydroxyapatite-tricalcium phosphate (HA/TCP) composite sponge carrier. OBJECTIVE: To determine whether new bone formation extends beyond the posterior confines of the vertebral body encroaching into the spinal canal when rhBMP-2 is used to enhance anterior fusion. SUMMARY OF BACKGROUND DATA: A possible concern regarding the use of rhBMP-2 to enhance spinal fusion is the risk of unwanted bone formation leading to inadvertent fusion of adjacent levels or compression of neural elements. The safety of rhBMP-2 in one spinal application does not ensure similar results in other applications. Therefore, the expanded use of rhBMP-2 should occur only after carefully monitored preclinical and clinical studies for each new application. METHODS: Eighteen pigs underwent thoracoscopically-assisted instrumentation and fusion of 5 contiguous levels (T5-T10) and randomly assigned to 4 treatment groups: group 1 (n = 6): rh-BMP-2 on a HA/TCP-collagen sponge (Medtronic Sofamor Danek, Memphis, TN); group 2 (n = 4): iliac crest autograft; group 3 (n = 4): empty; group 4 (n = 4): HA/TCP-collagen sponge (Medtronic Sofamor Danek) only. In groups 1 and 4, the HA/TCP collagen sponge was morselized into small granules and pushed through a bone delivery funnel for implantation into the disc. At 4 months after surgery, spines were sectioned longitudinally through the midsagittal plane and underwent undecalcified processing. Bone formation extending beyond the margins of the original discectomy and the confines of vertebral body were evaluated histomorphometrically at each operative level. RESULTS: Recombinant human bone morphogenic protein-2 on a HA/TCP-collagen sponge induced significant new bone formation extending anterior to the confines of the vertebral body compared with the other treatment groups (P < 0.05). In addition, rhBMP-2 on a HA/TCP-collagen sponge induced significant new bone formation extending posterior to the original margins of the discectomy (P < 0.05). However, there was no new bone formation beyond the confines of the posterior vertebral body. The total bone volume in the rhBMP-2-HA/TCP-collagen sponge group was significantly greater compared with all other groups in both the discectomy fusion area and beyond the discectomy area (P < 0.05). CONCLUSIONS: Recombinant human bone morphogenic protein-2 on a HA/TCP-collagen sponge enhanced anterior spinal fusion and induced significant new bone formation extending beyond the margins of the original discectomy and anterior vertebral body, most likely secondary to migration of some morselized carrier fragments from the disc space. However, the new bone formation did not extend beyond the posterior confines of the vertebral body to encroach into the spinal canal because of the intact posterior anulus and/or posterior longitudinal ligament.

Immunocytochemical localization of zinc transporter 3 in the ependyma of the mouse spinal cord.

We report, for the first time, the light microscopical and ultrastructural appearance of ZnT3-immunoreactivities in the ependymal cells of the central canal of the mouse spinal cord. Light microscopy revealed the presence of ZnT3-immunoreactive (Ir) ependymal cells in 1 microm thick epon sections stained by the ABC method. The ZnT3-Ir cells were observed at all levels of the spinal cord, but were a little more numerous in lumbosacral segments than in cervicothoracic segments. The ZnT3-Ir cells had large, ovoid nuclei with abundant cytoplasm, and protruded into the lumen of the central canal. Our ultrastructural findings suggest that the ZnT3-Ir ependymal cells possess secretory activity directed towards the central canal. We propose that they may play a role in the trans-ependymal mechanism responsible for zinc homeostasis between cerebrospinal fluid and the central area of the gray matter.

Neuronal progenitor-like cells expressing polysialylated neural cell adhesion molecule are present on the ventricular surface of the adult rat brain and spinal cord.

In the adult rodent brain, it is now well established that neurons are continuously generated from proliferating neuronal progenitor cells located in the subventricular zone of the lateral ventricle (SVZ) and the dentate gyrus of the hippocampus. Recently, it has been shown that neurons can also be generated in vitro from various regions of the adult brain and spinal cord ventricular neuroaxis. As the highly polysialylated neural cell adhesion molecule (PSA-NCAM) has been shown to be specifically expressed by neuronal progenitor cells of the SVZ and the hippocampus, the present study was designed to determine whether cells expressing this molecule could be detected in the vicinity of the ventricular system of the adult rat brain and spinal cord. After double or triple immunostaining for different neuronal and glial markers, confocal microscopy was used to examine the surface of the ventricular neuroaxis in either 40- to 50-microm-thick transverse vibratome sections cut through different brain regions, or in 200- to 300-microm-thick tissue slices including the intact surface of the brain ventricles or of the spinal cord central canal. In untreated rats, PSA-NCAM, microtubule associated protein 2 (MAP2) and class III-beta-tubulin were found to be associated with a number of neuron-like cells located on the surface of the third and fourth ventricles and of the spinal cord central canal. The proliferation of the PSA-NCAM-immunoreactive (IR) neuron-like cells detected on the surface of the third and fourth ventricles was not affected by injection of epidermal growth factor (EGF) or basic fibroblast growth factor (bFGF) into these ventricles, but was stimulated by the combined injection of EGF + bFGF. These data indicate that cells exhibiting features of neuronal progenitors are present on the ependymal surface of the adult rat brain and spinal cord ventricular axis.

Anatomy of soft tissues of the spinal canal.

BACKGROUND AND OBJECTIVES: Important issues regarding the spread of solutions in the epidural space and the anatomy of the site of action of spinal and epidural injections are unresolved. However, the detailed anatomy of the spinal canal has been incompletely determined. We therefore examined the microscopic anatomy of the spinal canal soft tissues, including relationships to the canal walls. METHODS: Whole mounts were prepared of decalcified vertebral columns with undisturbed contents from three adult humans. Similar material was prepared from a macaque and baboon immediately on death to control for artifact of tissue change after death. Other tissues examined included nerve root and proximal spinal nerve complex and dorsal epidural fat obtained during surgery. Slides were examined by light microscopy at magnifications of 10-40x. RESULTS: There is no fibrous tissue in the epidural space. The epidural fat is composed of uniform cells enclosed in a fine membrane. The dorsal fat is only attached to the canal wall in the dorsal midline and is often tenuously attached to the dura. The dura is joined to the canal wall only ventrally at the discs. Veins are evident predominantly in the ventral epidural space. Nerve roots are composed of multiple fascicles which disperse as they approach the dorsal root ganglion. An envelope of arachnoid encloses the roots near the site of exit from the dura. CONCLUSIONS: These features of the fat explain its semifluid consistency. Lack of substantial attachments to the dura facilitate movement of the dura relative to the canal wall and allow distribution of injected solution. Fibrous barriers are an unlikely explanation for asymmetric epidural anesthesia, but the midline fat could impede solution spread. Details of nerve-root structure and their envelope of pia-arachnoid membrane may be relevant to anesthetic action.

Ependymal development, proliferation, and functions: a review.

A survey of the literature shows that proliferation of ependyma occurs largely during the embryonic and early postnatal periods of development in most species. Differentiation of these cells proceeds along particular regional and temporal gradients as does the expression of various cytoskeletal (vimentin, cytokeratins, glial fibrillary acidic protein) and secretory proteins (S-100). Turnover declines significantly postnatally, and only low levels of residual activity persist into adulthood under normal conditions. Although the reported response of ependyma to injury is somewhat equivocal, only limited regenerative capacity appears to exist and to varying degrees in different regions of the neuraxis. Proliferation has been most often observed in response to spinal cord injury. Indeed, the ependyma plays a significant role in the initiation and maintenance of the regenerative processes in the spinal cord of inframammalian vertebrates. In the human, however, ependyma appears never to regenerate at any age nor re-express cytoskeletal proteins characteristic of immature cells. The functions of ependyma including tanycytes, a specialized form of ependymal cell that persists into adulthood within circumscribed regions of the nervous system, are still largely speculative. Fetal unlike mature ependyma is believed to be secretory and is believed to play a role in neurogenesis, neuronal differentiation/axonal guidance, transport, and support. In the adult brain, mature ependyma is not merely an inert lining but may regulate the transport of ions, small molecules, and water between the cerebrospinal fluid and neuropil and serve an important barrier function that protects neural tissue from potentially harmful substances by mechanisms that are still incompletely understood.

[Presence of epithelial cells in the medullary canal after spinal anesthesia]. / Presenza di cellule epiteliali nel canale midollare dopo anestesia spinale.

BACKGROUND AND AIM: Several authors have focused on a causal link between the onset of neurological complications after lumbar injections and the fact that epithelial cells may be drawn into the vertebral canal during these procedures. Complications may arise both early (cephalea, septic and aseptic meningitis) and late (epidermoid tumours). The authors aimed to evaluate whether skin fragments which are carried down by the needle during subarachnoid anesthesia may even be present in the epidural or subarachnoid space three days later and may therefore justify the onset of the above neurological syndromes. METHODS: Five adult cats under narcosis underwent subarachnoid anesthesia using disposable 22G Quincke type needles. Between 0.7 and 1 ml isobaric bupivacaine at 0.50% was injected. The presence of the motor block of the lower limbs was ascertained once the effects of general anesthesia wore off. On the third day, again under general anesthesia, cardio-respiratory arrest was provoked by intravenous injection. Samples of meninges were collected in the injection area. After fixation in a phosphate glutaraldehyde buffer, dehydration in acetone, dehydration by critical point and gold metalisation, the samples were examined using SEM. RESULTS: No epidermal cells were found on the surface of the meninges. On the other hand, a squamous epithelial cell was observed which drained inside a sectioned epidural vessel towards the systemic circulation. CONCLUSIONS: This study confirms the possibility that, after subarachnoid anesthesia using 22G Quincke needles, skin fragments may enter the spinal canal. The permanence or otherwise of the epithelial fragments on the third day depends on the size of the fragment drawn down and the efficacy of the drainage system which removes isolated epithelial cells. This phenomenon may justify the self-limiting character of cephalea and meningisms which, even if not treated, regress in a few days, as well as the scarce development of epidermoid tumours.

NADPH-diaphorase and cytosolic urea cycle enzymes in the rat spinal cord.

Nitric oxide synthase (NOS), argininosuccinate synthetase (ASS), and argininosuccinate lyase (ASL) compose a cyclic pathway to form nitric oxide (NO). These enzymes, however, are localized differentially in most regions of the brain. To find out whether NOS, ASS, and ASL are colocalized in neurons of the spinal cord, we examined the distribution of these enzymes by using a double-labeling procedure combining fluorescent immunohistochemistry with an assay for reduced nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d). Results indicate that neurons in the dorsal horn, the intermediolateral nucleus, and the central canal region were NADPH-d active (+) and NOS-, ASS-, and ASL-like immunoreactive (-LI). In laminae II and III of the dorsal horn, some NADPH-d (+) neurons were ASL-LI (8-30%) but only a few were ASS-LI (0.5-7%). In the nucleus intermediolateralis, a large portion of NADPH-d (+) neurons were ASL-LI (30-60%), whereas only a small portion of NADPH-d (+) neurons were ASS-LI (10-20%). In the central canal region, some NADPH-d (+) neurons were ASL-LI (15-40%), and a few NADPH-d (+) neurons were ASS-LI (3-16%). Thus, the results suggest that, in the nucleus intermediolateralis and the central canal region, NOS, ASS, and ASL are colocalized and form a cyclic pathway to produce NO, whereas, in the dorsal horn, these enzymes are more characteristically localized in different neurons, which may transport the substrates intercellularly.

Effects of nitric oxide availability on responses of spinal wide dynamic range neurons to excitatory amino acids.

The role of nitric oxide (NO) in responses of spinal dorsal horn neurons to excitatory amino acids and to cutaneous mechanical stimuli was examined. Extracellular recordings were made from wide dynamic range neurons excited with iontophoretically applied excitatory amino acid agonists, N-methyl-D-aspartate (NMDA) and (R,S)-alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) or kainic acid. Nitric oxide availability was decreased by iontrophoretic application of NO synthase inhibitors, N omega-nitro-L-arginine methyl ester (L-NAME) or L-N5-(1-iminoethyl)ornithine (L-NIO), or elevated by the NO donating compound, S-nitroso-N-penicillamine (SNAP). When cells were excited with successive application of NMDA and non-NMDA excitatory amino acid receptor agonists, application of NO synthase inhibitors led to a decrease in responses to NMDA in 60% of neurons. In more than a third of the cells tested, inhibition of NO synthase caused reciprocal changes in responses to glutamate receptor agonists: NMDA-evoked responses were significantly decreased whereas responses to the non-NMDA receptor agonists (AMPA or kainic acid) were increased. Application of the NO donating compound, S-nitroso-N-penicillamine, revealed an opposite tendency, increasing responses to NMDA in more than half of the neurons tested. In approximately 40% of the cells, reciprocal changes in responses to excitatory amino acid receptor agonists of NMDA versus non-NMDA types were observed after application of S-nitroso-N-penicillamine, such that the increase in NMDA responses was accompanied by decreases in the responses to kainic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

The ventriculus terminalis and filum terminale of the human spinal cord.

Serial sections of the conus medullaris and the filum terminale of 23 randomly selected human spinal cords were studied by light and electron microscopy, and following immunoperoxidase staining for glial fibrillary acidic protein (GFAP), vimentin, neuron-specific enolase (NSE), amyloid beta protein, and S-100 protein. The intradural portion of the filum contains bundles of GFAP-positive glial fibers, scattered silver- and NSE-positive neurons, segments of peripheral nerve, blood vessels, fibrous connective tissue, and fat. Glial cell clusters varying from five to 100 cell layers thick at times constitute the bulk of the filum. The periependymal glial cells possess moderate amounts of eosinophilic cytoplasm and relatively uniform round to ovoid nuclei containing evenly distributed chromatin. They are distributed diffusely with no specific pattern of organization, although some of them showed a tendency to form acinar structures. A minority of the glial cells showed GFAP immunoreactivity, and some were immunoreactive for vimentin. Electron microscopy demonstrated the presence of periependymal cells showing cilia, microvilli, and the formation of intercellular junctional complexes, as well as cells containing bundles of glial filaments within the cytoplasm. Degenerated NSE-positive neurons and degenerated neurites resembling neuritic plaques were also demonstrated. However, immunoperoxidase staining for amyloid beta protein was negative in these structures. Thus, the filum terminale is endowed with an abundance of glial cells and neurons and is not simply a fibrovascular tag. Periependymal glial cells in the filum terminale should not be mistaken for neoplasm. The presence of neuropil with profuse astroglial and neuronal components within the filum terminale suggests a possible functional role for these structures.