Myelinated nerve cell perikaryon in mouse spinal cord

Summary In the thoracic cord (posterior horn region) of a wild mouse, we have observed a small nerve cell soma completely enveloped by a myelin sheath. The number of myelin lamellae varied between 7 and 12. In one place, the existence of an inner mesoperikaryon could also be shown. The significance of this fortuitous finding has not yet been explained.

---

Zusammenfassung Im Thorakalmark (Hinterhornbereich) einer Wildmaus wurde ein kleines Nervenzellperikaryon beobachtet, das vollständig von einer Markscheide umhüllt war. Die Zahl der Markscheidenlamellen variierte zwischen 7 und 12. An einer Stelle konnte ein sogenanntes inneres Mesoperikaryon nachgewiesen werden. Die Bedeutung dieses zufällig erhobenen Befundes ist vorerst noch offen.

     

Presynaptic and postsynaptic organelles of synapses formed in cultures of previously dissociated mouse spinal cord

Summary This study describes some of the ultrastructural features of presynaptic and postsynaptic organelles at synapses developed in cultures of previously dissociated mouse spinal cord cells. Particular attention was paid to the agranular reticulum which is well developed at many presynaptic and postsynaptic sites, either in the form of simple tubules or cisternae, or more complex networks and often closely associated with mitochondria. In addition, the disposition of microtubules at and close to synaptic specializations is described. These and other features of synaptic zones, such as granular vesicles in presynaptic sites, are discussed in relation to cultures developed on feeder layers and synapses in vivo, and in relations to possible degenerative and regenerative events in the cell cultures.     

Ependyma and meninges of the spinal cord of the mouse

Summary In addition to ependymal epithelial cells, numerous tanycytes are found along the entire central canal of the mouse. These tanycytes are arranged in clusters in the cervical, thoracic and lumbar segments of the spinal cord. In the conus medullaris, tanycytes separate and ensheath bundles of myelinated and unmyelinated axons; their processes take part in the formation of the stratum marginale gliae. In the caudal part of the spinal cord, the ventral wall of the central canal is thin and some areas are reduced to a single-cell thickness. In this region, ependymal cells participate directly in the formation of the stratum marginale gliae.The meninges consist of the intima piae, the pia mater, the arachnoid, a subdural neurothelium and the dura mater. The subarachnoid space appears occluded and opens only around the spinal roots. In the vicinity of the spinal ganglia, the dura mater, the subdural neurothelium and the arachnoid form a cellular reticulum.     

Sox11 promotes endogenous neurogenesis and locomotor recovery in mice spinal cord injury

We introduced a lentiviral vector containing the Sox11 gene into injured spinal cords of mice to evaluate the therapeutic potential of Sox11 in spinal cord injury. Sox11 markedly improved locomotor recovery after spinal cord injury and this recovery was accompanied by an up-regulation of Nestin/Doublecortin expression in the injured spinal cord. Sox11 was mainly located in endogenous neural stem cells lining the central canal and in newly-generated neurons in the spinal cord. In addition, Sox 11 significantly induced expressions of BDNF in the spinal cords of LV-Sox11-treated mice. We concluded that Sox11 induced activation of endogenous neural stem cells into neuronal determination and migration within the injured spinal cord. The resultant increase of BDNF at the injured site might form a distinct neurogenic niche which induces a final neuronal differentiation of these neural stem cells. Enhancing Sox11 expression to induce neurogenic differentiation of endogenous neural stem cells after injury may be a promising strategy in restorative therapy after SCI in mammals.     

Functional corticotropin-releasing factor receptors in neonatal rat spinal cord

The present study localized corticotropin-releasing factor (CRF) receptors and studied the actions of CRF in the neonatal rat spinal cord preparation. Lumbar CRF receptors were present in highest concentrations in laminae I and II with progressively lower concentrations in lamina IX and intermediate and central zones respectively. CRF directly and indirectly depolarized lumbar motoneurons in a concentration-related manner and the putative receptor antagonist, alpha helical oCRF(9–41), partially blocked the depolarizing response to CRF. The electrophysiological responses to CRF and the distribution of receptors within the spinal cord suggest that CRF may play a physiological role in regulating spinal cord reflex function.     

Mesenchymal stem cell conditioned medium increases glial reactivity and decreases neuronal survival in spinal cord slice cultures

Ex vivo spinal cord slice cultures (SCSC) allow study of spinal cord circuitry, maintaining stimuli responses comparable to live animals. Previously, we have shown that mesenchymal stem/stromal cell (MSC) transplantation in vivo reduced inflammation and increased nerve regeneration but MSC survival was short-lived, highlighting that beneficial action may derive from the secretome. Previous in vitro studies of MSC conditioned medium (CM) have also shown increased neuronal growth. In this study, murine SCSC were cultured in canine MSC CM (harvested from the adipose tissue of excised inguinal fat) and cell phenotypes analysed via immunohistochemistry and confocal microscopy. SCSC in MSC CM displayed enhanced viability after propidium iodide staining. GFAP immunoreactivity was significantly increased in SCSC in MSC CM compared to controls, but with no change in proteoglycan (NG2) immunoreactivity. In contrast, culture in MSC CM significantly decreased the prevalence of βIII-tubulin immunoreactive neurites, whilst Ca²⁺ transients per cell were significantly increased. These ex vivo results contradict previous in vitro and in vivo reports of how MSC and their secretome may affect the microenvironment of the spinal cord after injury and highlight the importance of a careful comparison of the different experimental conditions used to assess the potential of cell therapies for the treatment of spinal cord injury.     

Optimized and efficient preparation of astrocyte cultures from rat spinal cord

Astrocytes constitute a major class of glial cells in the CNS, and play crucial roles in physiological functioning, performance and maintenance of the CNS, as well as promotion of neuronal migration and maturation. Astrocytes have also been directly and indirectly implicated in the pathophysiology of various trauma occurrences, development of neurodegenerative diseases and nerve regeneration. To further understand mechanisms by which astrocytes elicit these effects, the first critical step in the study of astrocytes is the preparation of purified astrocytes cultures. Here we describe a simple and convenient procedure for producing rat primary astrocyte cultures of high purity, viability and proliferation. For astrocyte culture, we have optimized the isolation procedures and cultivation conditions including coating substrates, enzyme digestion, seeding density and composition of the culture medium. Using immunofluorescent antibodies against GFAP and OX-42 in combination of Hoechst 33342 fluorescent staining, we found that the purity of the astrocyte cultures was >99%. Astrocytes had high viability as measured by 3-(4, 5-dimethyl-2-yl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay. In addition, flow cytometric analysis was used to measure and observe variations in the cell cycle after 1–2 passages and proliferation of astrocytes was detected with a high percentage of cells stand in S+G₂/M phase. Therefore, the method described here is ideal for experiments, which require highly pure astrocyte cultures.     

Visualization of the NMDA recognition site in rat and mouse spinal cord by [3H]CGS 19755in vitro autoradiography

Summary The possibility to visualize the NMDA recognition site with [³H]CGS 19755in vitro autoradiography was evaluated in rat brain and spinal cord sections and thereafter used to study the distribution of the NMDA recognition site in rat and mouse spinal cord. The [³H]CGS 19755 binding was concentrated to the dorsal horn, in particular to the substantia gelatinosa. Notable binding was also seen in the intermediate area and ventral horn, while some binding was observed in the funiculi. No major differences were seen in [³H]CGS 19755 binding at various levels of the rat or mouse spinal cord, although a more dense binding was seen in the mouse. A similar distribution of the [³H]CGS 19755 specific binding and the NMDA receptor associated ion-channel site, labeled with [³H]TCP, was found in the mouse spinal cord. Taken together, our data indicate that the NMDA recognition site can be visualized in rat and mouse spinal cord byin vitro [³H]CGS 19755 autoradiography.Abbreviations NMDA N-methyl-D-aspartate - CGS 19755 Cis-4-phosphonomethyl-2-piperidine carboxylic acid - D-AP5 D(—)-2-Amino-5-phosphonopentanoic acid - TCP N-(1-2-thienylcyclohexyl)-3,4-piperidine - MK-801 (±)-5-Methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate - AMPA -Amino-3-hydroxy-5-methyl-isoxazolepropionic acid - kainate 2-Carboxy-3-carboxymethyl-4-isopropenyl pyrrolidine - CGP 39653 D,L-(E)-2-amino-4-propyl-5-phosphonopentenoic acid     

Atrial natriuretic factor-like immunoreactivity in spinal cord and in primary sensory neurons of spinal and trigeminal ganglia of guinea-pig: correlation with tachykinin immunoreactivity*

Summary Atrial natriuretic factor (ANF) is a cardiac hormone with various functions in body homeostasis. It is also processed in the brain and in the peripheral nervous system where it appears to play a role as a neuromodulator. Little is known about the presence of ANF throughout the spinal cord of the guinea-pig. We therefore examined the distribution of ANF and its possible interrelation with primary sensory afferents in this species. Using enzyme- and fluorescence-immunohistochemistry on deparaffinized sections, ANF-like immunoreactivity was found to be present in nerve fibers in laminae I/II of the spinal cord and in neurons of spinal and trigeminal ganglia. Tachykinins and ANF coexisted in very few fibers of the spinal cord but did not coexist in primary sensory spinal or trigeminal neurons. Our results indicate that spinal ANF-immunoreactive fibers are of dual origin, primary sensory and non-primary sensory. The possibly heterogeneous source of the non-primary sensory ANF, its possible coexistence with other co-transmitters and functional implications are discussed.Preliminary aspects of this study have been reported at the 2nd World Congress of Neuroscience in Budapest (Weihe et al. 1987) and at the Versammlung der Anatomischen Gesellschaft in Zürich (Nohr et al. 1989)     

Nerve growth factor in glia and inflammatory cells of the injured rat spinal cord

Nerve growth factor (NGF) is crucial for the development of sympathetic and small-diameter sensory neurons and for maintenance of their mature phenotype. Its role in generating neuronal pathophysiology is less well understood. After spinal cord injury, central processes of primary afferent fibers sprout into the dorsal horn, contributing to the development of autonomic dysfunctions and pain. NGF may promote these states as it stimulates sprouting of small-diameter afferent fibers and its concentration in the spinal cord increases after cord injury. The cells responsible for this increase must be identified to develop a strategy to prevent the afferent sprouting. Using immunocytochemistry, we identified cells containing NGF in spinal cord sections from intact rats and from rats 1 and 2 weeks after high thoracic cord transection. In intact rats, this neurotrophin was present in a few ramified microglia and in putative Schwann cells in the dorsal root. Within and close to the lesion of cord-injured rats, NGF was in many activated, ramified microglia, in a subset of astrocytes, and in small, round cells that were neither glia nor macrophages. NGF-immunoreactive putative Schwann cells were prevalent throughout the thoracolumbar cord in the dorsal roots and the dorsal root entry zones. Oligodendrocytes were never immunoreactive for this protein. Therapeutic strategies targeting spinal cord cells that produce NGF may prevent primary afferent sprouting and resulting clinical disorders after cord injury.     

Spinal cord evoked potentials and edema in the pathophysiology of rat spinal cord injury

Summary The possibility that nitric oxide is somehow involved in the early bioelectrical disturbances following spinal cord injury in relation to the later pathophysiology of the spinal cord was examined in a rat model of spinal cord trauma. A focal trauma to the rat spinal cord was produced by an incision of the right dorsal horn of the T 10–11 segments under urethane anaesthesia. The spinal cord evoked potentials (SCEP) were recorded using epidural electrodes placed over the T9 and T12 segments of the cord following supramaximal stimulation of the right tibial and sural nerves in the hind leg. Trauma to the spinal cord significantly attenuated the SCEP amplitude (about 60%) immediately after injury which persisted up to 1h. However, a significant increase in SCEP latency was seen at the end of 5h after trauma. These spinal cord segments exhibited profound upregulation of neuronal nitric oxide synthase (NOS) immunoreactivity, and the development of edema and cell injury. Pretreatment with a serotonin synthesis inhibitor drug p-chlorophenylalanine (p-CPA) or an anxiolytic drug diazepam significantly attenuated the decrease in SCEP amplitude, upregulation of NOS, edema and cell injury. On the other hand, no significant reduction in SCEP amplitude, NOS immunolabelling, edema or cell changes were seen after injury in rats pretreated with L-NAME. These observations suggest that nitric oxide is somehow involved in the early disturbances of SCEP and contribute to the later pathophysiology of spinal cord injury.     

Perchloric acid-soluble protein regulates cell proliferation and differentiation in the spinal cord of chick embryos

The role of perchloric acid-soluble protein (PSP) was investigated in chick embryos. Fluorescently labeled anti-chick liver (CL)-PSP IgG was injected into the yolk sac in ovo at embryonic day 3, and became localized in neuroepithelial cells. Within 12 h, morphological changes were observed in 37.5% of anti-CL-PSP IgG-injected embryos, and the neuroepithelial cells formed a wavy line. No significant changes were observed in embryos injected with non-immune IgG or PBS. Increased expression of PCNA and decreased expression of neuronal class III beta-tubulin were observed in the spinal cord after anti-CL-PSP IgG injection. These results suggest that PSP controls the proliferation and differentiation of neuroepithelial cells in chick embryos.     

Guanosine reduces apoptosis and inflammation associated with restoration of function in rats with acute spinal cord injury

Spinal cord injury results in progressive waves of secondary injuries, cascades of noxious pathological mechanisms that substantially exacerbate the primary injury and the resultant permanent functional deficits. Secondary injuries are associated with inflammation, excessive cytokine release, and cell apoptosis. The purine nucleoside guanosine has significant trophic effects and is neuroprotective, antiapoptotic in vitro, and stimulates nerve regeneration. Therefore, we determined whether systemic administration of guanosine could protect rats from some of the secondary effects of spinal cord injury, thereby reducing neurological deficits. Systemic administration of guanosine (8 mg/kg per day, i.p.) for 14 consecutive days, starting 4 h after moderate spinal cord injury in rats, significantly improved not only motor and sensory functions, but also recovery of bladder function. These improvements were associated with reduction in the inflammatory response to injury, reduction of apoptotic cell death, increased sparing of axons, and preservation of myelin. Our data indicate that the therapeutic action of guanosine probably results from reducing inflammation resulting in the protection of axons, oligodendrocytes, and neurons and from inhibiting apoptotic cell death. These data raise the intriguing possibility that guanosine may also be able to reduce secondary pathological events and thus improve functional outcome after traumatic spinal cord injury in humans.     

Dll1 and Dll4 function sequentially in the retina and pV2 domain of the spinal cord to regulate neurogenesis and create cell diversity

Signalling mediated by Notch receptors is known to have multiple functions during vertebrate neural development, regulating processes like progenitor differentiation and cell type diversification. Various Notch ligands are expressed in the developing nervous system and their activities might contribute to this multiplicity of functions. Here, we show that two Delta-like genes, Dll1 and Dll4, are sequentially expressed in differentiating neurons of the embryonic mouse retina and spinal cord's pV2 domain, with Dll1 starting to be expressed before Dll4. Analysis of Dll1 mutants reveals this gene is necessary and sufficient to maintain a pool of progenitors in the embryonic neuroepithelium. Accordingly, in the spinal cord domains where Dll1 is the only expressed Notch ligand, its inactivation leads to an increased rate of neurogenesis and premature differentiation of neural progenitors. In contrast, in the pV2 domain and retina where Dll1 is co-expressed with Dll4, progenitors are not exhausted and cell diversity is maintained. Together, our results support a model where Dll1 and Dll4 are part of a unique genetic circuitry that regulates subsequent steps of neurogenesis in the retina and pV2 domain: while Dll1 serves to prevent the untimely differentiation of neural progenitors, Dll4 might function to generate diversity within the population of differentiating neurons.     

Growth factor binding and bioactivity in human kidney epithelial cell cultures

Summary Insulinlike growth factors (IGF) and epidermal growth factor (EGF) are produced in renal tissue, as are specific receptors for these hormones. To evaluate the significance of these observations to regulation of renal tubular cell proliferation, we have examined the interaction of IGF and EGF with cultured human proximal tubular epithelial cells (HPT). HPT cells showed specific binding of IGF-1, insulin, and EGF. IGF-1 binding was inhibited by antibody to the type 1 IGF receptor (α-IR3). Insulin receptors and type 1 IGF receptors were identified by bifunctional cross-linking. IGF-1, insulin, and EGF stimulated [³H]thymidine incorporation by 77, 73, and 87%, respectively. Haft maximal stimulation by IGF-1, insulin, and EGF was produced with 4×10⁻⁹ M, 2.5×10⁻⁸ M, and 8×10⁻¹⁰ M concentrations of these hormones. α-IR3 inhibited stimulation of thymidine incorporation by IGF-1 and insulin but had no effect in EGF-stimulated thymidine incorporation. EGF and high concentrations of insulin both stimulated cell proliferation by 83 and 79%, respectively. These data are consistent with regulation of tubular epithelial proliferation by IGF-1, insulin, and EGF and suggest that the mitogenic activity of both insulin and IGF-1 is mediated by the type 1 IGF receptor. Supported by grants CA37887 and DK32889 from the National Institutes of Health, Bethesda, MD, and by a Medical University of South Carolina institutional grant.     

Pretreatment with calpain inhibitor CEP-4143 inhibits calpain I activation and cytoskeletal degradation, improves neurological function, and enhances axonal survival after traumatic spinal cord injury

The pathophysiology of traumatic spinal cord injury (SCI) involves abnormal activation of the neutral cysteine protease calpain I (EC 3.4.22.17). In the present study we examined the effect of the calpain inhibitor CEP-4143 on cytoskeletal protection and neurological recovery after SCI in adult rats. Microinjection of 50 mM CEP-4143 into the T7 vertebral segment 10 min before a 35-g clip compression injury resulted in inhibition of calpain activation at 2 and 4 h postinjury, as determined by western blotting for calpain I-mediated spectrin degradation, and significantly attenuated the degradation of dephosphorylated NF200 neurofilament protein at 4 and 8 h postinjury. To examine the in vivo chronic neuroprotective effects of CEP-4143, animals underwent microinjection with saline or 50 mM CEP-4143 10 min before injury, followed by weekly blinded behavioral assessments for 6 weeks. Animals receiving CEP-4143 treatment showed significant improvement over saline-treated controls on the Basso Beattie Bresnahan locomotor rating scale (p < 0.02) and inclined plane test (p < 0.05). Counts of neurons in the red nucleus retrogradely labeled by fluoro-gold after introduction distal to the injury site were significantly higher in CEP-4143-treated animals. Finally, morphometric assessment of the injury site by computer-assisted image analysis revealed significant tissue preservation in CEP-4143-treated animals. We conclude that the calpain antagonist CEP-4143 exhibits biochemical, behavioral, and anatomical neuroprotection following traumatic SCI.     

Inhibitory Smads differentially regulate cell fate specification and axon dynamics in the dorsal spinal cord

The roof plate resident BMPs have sequential functions in the developing spinal cord, establishing cell fate and orienting axonal trajectories. These activities are, however, restricted to the dI1–dI3 neurons in the most dorsal region of the spinal cord. What limits the extent of the action of the BMPs to these neurons? To address this question, we have examined both the distribution of the inhibitory Smads (I-Smads), Smad6 and Smad7 in the spinal cord and the consequence of ectopically expressing the I-Smads in chicken embryos. Our studies suggest that the I-Smads function in vivo to restrict the action of BMP signaling in the dorsal spinal cord. Moreover, the I-Smads have distinct roles in regulating the diverse activities of the BMPs. Thus, the ectopic expression of Smad7 suppresses the dI1 and dI3 neural fates and concomitantly increases the number of dI4–dI6 spinal neurons. In contrast, Smad6 most potently functions to block dI1 axon outgrowth. Taken together, these experiments suggest that the I-Smads have distinct roles in spatially limiting the response of cells to BMP signaling.     

Growth and differentiation of murine cartilage cells in vitro followng a short-term exposure to triamcinolone acetonide

Summary Measurements of ³H-thymidine incorporation, quantitative autoradiography and morphometry were used to evaluate cell behavior during the recovery of mandibular condylar cartilage cultures following short-term exposure to a corticosteroid hormone in vitro. Apical segments of mandibular condyles of newborn mice were initially incubated in the presence of the hormone triamcinolone acetonide (10^-6 M) for 24 h and were thereafter cultured for additional 6 days in hormone-free medium. The present results indicated that the treatment led to a decrease in the rate of incorporation of ³H-thymidine, a feature that lasted for 48 h following the removal of the hormone. Quantitative ³H-thymidine autoradiography of explants that were labeled in the presence of the hormone further substantiated the initial suppressive effect of the hormone on cellular proliferation, a feature that was followed by a recovery. Differences were noted in the pattern of distribution of labeled cells: in control explants, labeled cells progressively moved from the chondroprogenitor compartment into the differentiated portion of the cartilage; in hormone-treated explants, ³H-thymidine labeled cells were confined to the progenitor layer up to 5 days after the treatment and only then appeared in the chondrocytic compartment. The hormone's adverse effect upon differentiation was manifested by both morphology, and by causing a significant increase in the size of the progenitor layer (up to 50.5% on 4th post-treatment day) along with a 70.5% reduction in the size of chondroblastic layer. We conclude that a short-term exposure to a glucocorticoid hormone in vitro interferes with proliferation of chondroprogenitor cells and their subsequent differentiative pathway. While the proliferative activity was restored within 48 h, the hormone's effect on differentiation lasted for a considerably longer period of time.     

Distinct expression of midkine and pleiotrophin in the spinal cord and placental tissues during early mouse development

Midkine and pleiotrophin comprise a family of heparin-binding growth factors, and are expressed in overlapping tissues during the mid- to late-gestation periods of mouse development. Their distinct expression during early mouse development, as revealed by in situ hybridization, was reported. Midkine was expressed in the embryonic ectoderm from as early as embryonic day (E5.5). In the neural tube midkine was expressed specifically in the neuroepithelium, that is, in the whole area of the neural tube at E9.5, and in the ventricular zone from E10.5-13.5. At E15.5, when the neuroepithelium disappeared, midkine concomitantly became undetectable. In contrast, pleiotrophin expression started exclusively in the neural plate at E8.5, and in the lateral plate of the neural tube at E9.5. It then became restricted to a dorsal ventricular zone from E11.5-13.5, and finally to the central gray neurons at E15.5. Moreover, pleiotrophin was expressed in the ventral horns. Among placental tissues, midkine was detected in the chorion, the fetal component of the placenta, whereas pleiotrophin was found in the decidua basalis, the maternal component of the placenta. The distinct expression of midkine and pleiotrophin suggests their differential role in early development.