TOPOCHEMICAL ASPECTS OF NUCLEIC ACID AND PROTEIN METABOLISM WITHIN THE NEURON-NEUROGLIA UNIT OF THE SPINAL CORD ANTERIOR HORN

Abstract— Using a two-wavelength modification of ultraviolet and visible cytospectro-photometric methods, the content of nucleic acids per cell was determined in neuronal cytoplasm and glial satellite cell-bodies from the spinal cord anterior horns in mice and rats. Mice which had been swimming for 3 and 4 h showed an increase in the content of RNA in the spinal motoneurons with no changes in the neuroglia. Stronger stimulation of the nervous system such as electrical skin irritation (20-40 V, approx. 40 impulses/min) for 5 min resulted in an increase of RNA in the motoneurons of rat spinal cord and a decrease in the surrounding glia. Exhausting actions upon the nervous system (60 min irritation of rat paws by the electrical current, acute clonic convulsions in rats injected with cardiazol (pentamethylenetetrazol, metrazol) or initial free motor activity after 3 weeks of restraint of mice) induced a marked decrease of RNA content throughout the whole neuron-neuroglia unit. After stimulation, return to normal amounts of RNA and protein was more rapid in glia than in neurons. After 1-3 days rest the level of RNA was normal in motoneurons, but a decrease in glial RNA was shown. These trace changes in the glia are believed to reflect an adaptation mechanism in the nervous system at the cellular level. The relationship between neuronal and glial compartments within the neuron-neuroglia unit is discussed; a supporting, homeostatic, secondary role of glial metabolism with respect to adequate reconstruction of neuronal metabolism is outlined.     

Neuroprotective effects of N-acetyl-cysteine and acetyl-L-carnitine after spinal cord injury in adult rats

Following the initial acute stage of spinal cord injury, a cascade of cellular and inflammatory responses will lead to progressive secondary damage of the nerve tissue surrounding the primary injury site. The degeneration is manifested by loss of neurons and glial cells, demyelination and cyst formation. Injury to the mammalian spinal cord results in nearly complete failure of the severed axons to regenerate. We have previously demonstrated that the antioxidants N-acetyl-cysteine (NAC) and acetyl-L-carnitine (ALC) can attenuate retrograde neuronal degeneration after peripheral nerve and ventral root injury. The present study evaluates the effects of NAC and ALC on neuronal survival, axonal sprouting and glial cell reactions after spinal cord injury in adult rats. Tibial motoneurons in the spinal cord were pre-labeled with fluorescent tracer Fast Blue one week before lumbar L5 hemisection. Continuous intrathecal infusion of NAC (2.4 mg/day) or ALC (0.9 mg/day) was initiated immediately after spinal injury using Alzet 2002 osmotic minipumps. Neuroprotective effects of treatment were assessed by counting surviving motoneurons and by using quantitative immunohistochemistry and Western blotting for neuronal and glial cell markers 4 weeks after hemisection. Spinal cord injury induced significant loss of tibial motoneurons in L4-L6 segments. Neuronal degeneration was associated with decreased immunostaining for microtubular-associated protein-2 (MAP2) in dendritic branches, synaptophysin in presynaptic boutons and neurofilaments in nerve fibers. Immunostaining for the astroglial marker GFAP and microglial marker OX42 was increased. Treatment with NAC and ALC rescued approximately half of the motoneurons destined to die. In addition, antioxidants restored MAP2 and synaptophysin immunoreactivity. However, the perineuronal synaptophysin labeling was not recovered. Although both treatments promoted axonal sprouting, there was no effect on reactive astrocytes. In contrast, the microglial reaction was significantly attenuated. The results indicate a therapeutic potential for NAC and ALC in the early treatment of traumatic spinal cord injury.     

Morphologic and cytophotometric indices of the state of the neurons of the spinal cord and spinal cord ganglia following exposure to gravitational stress]

The work presents data on the state of the neurons of the spinal ganglia and the spinal cord of the lumbosacral part in 96 cats subjected to single stresses (10g) and repeated ones (6g). The material was stained with thionin after Nissl. RNA was detected after Einarson. Photometry of the sections was made in MUP.5. Morphological changes were shown to be more pronounced in sensory cells. The method of cytophotometry established the increase by 25% amount of RNA in their cytoplasm after single stresses. Repeated stresses resulted in depletion of RNA. The amount of RNA returned to the initial level within 2 days after single stresses and within 3 days after repeated rotations. The shifts were much less pronounced in motoneurons.     

A method to immunolabel rodent spinal cord neurons and glia for molecular study in specific laser microdissected cells involved in neurodegenerative disorders

Neuron-glia interaction is involved in physiological function of neurons, however, recent evidences have suggested glial cells as participants in neurotoxic and neurotrophic mechanisms of neurodegenerative/neuroregenerative processes. Laser microdissection offers a unique opportunity to study molecular regulation in specific immunolabeled cell types. However, an adequate protocol to allow morphological and molecular analysis of rodent spinal cord astrocyte, microglia and motoneurons remains a big challenge. In this paper we present a quick method to immunolabel those cells in flash frozen sections to be used in molecular biology analyses after laser microdissection and pressure catapulting.     

Neuroprotective Effects of Estradiol on Motoneurons in a Model of Rat Spinal Cord Embryonic Explants

Amyotrophic lateral sclerosis (ALS) is an adult-onset degenerative disorder characterized by motoneuron death. Clinical and experimental studies in animal models of ALS have found gender differences in the incidence and onset of disease, suggesting that female hormones may play a beneficial role. Cumulative evidence indicates that 17β-estradiol (17βE2) has a neuroprotective role in the central nervous system. We have previously developed a new culture system by using rat spinal cord embryonic explants in which motoneurons have the singularity of migrating outside the spinal cord, growing as a monolayer in the presence of glial cells. In this study, we have validated this new culture system as a useful model for studying neuroprotection by estrogens on spinal cord motoneurons. We show for the first time that spinal cord motoneurons express classical estrogen receptors and that 17βE2 activates, specifically in these cells, the Akt anti-apoptotic signaling pathway and two of their downstream effectors: GSK-3β and Bcl-2. To further validate our system, we demonstrated neuroprotective effects of 17βE2 on spinal cord motoneurons when exposed to the proinflammatory cytokines TNF-α and IFN-γ. These effects of 17βE2 were fully reverted in the presence of the estrogen receptor antagonist ICI 182,780. Our new culture model and the results presented here may provide the basis for further studies on the effects of estrogens, and selective estrogen receptor modulators, on spinal cord motoneurons in the context of ALS or other motoneuron diseases.     

Gallocyanin-Chrome Alum: I. Technique and Specificity

Certain technical aspects of gallocyanin-chrome alum were examined relative to its supposed specificity for nucleic acids. Five different lake formulae were prepared using four different batches of gallocyanin. Spectrophotometric curves were made of each lake and of each dye in a simple water solution. Paraffin sections 6-8 μ thick of spinal cords from albino rats and from cats fixed in CaCl₂-formalin or plain formalin were stained 10 min to 48 hr with gallocyanin lakes made with chrome alum, ferric alum, strontium chloride and copper nitrate. Similar sections were treated with ribonuclease or perochloric acid and stained in the same manner. The spectrophotometric data indicates considerable variation in dye content between different batches and different lakes. Chrome alum was the best of the 4 mordants and a 12-15 hr staining time with Einarson's 1932 preparation was optimal. Neither perchloric acid nor ribonuclease destroyed cytoplasmic basophilia as revealed by gallocyaninchrome alum. Staining was more intense after CaCl₂-formalin fixation than after plain formalin. Variation of the dye content in the different batches of dyes, the poorly understood role of boiling in preparing the lakes, and the inability of ribonuclease or perchloric acid to destroy cytoplasmic basophilia indicates that we are not dealing with a histochemically specific reagent for nucleic acid, but only a desirable nuclear stain.     

Evaluation of Avulsion-Induced Neuropathology in Rat Spinal Cords with 18F-FDG Micro-PET/CT

Brachial plexus root avulsion (BPRA) leads to dramatic motoneuron death and glial reactions in the corresponding spinal segments at the late stage of injury. To protect spinal motoneurons, assessment of the affected spinal segments should be done at an earlier stage of the injury. In this study, we employed ¹⁸F-FDG small-animal PET/CT to assess the severity of BPRA-induced cervical spinal cord injuries. Adult Sprague-Dawley rats were randomly treated and divided into three groups: Av+NS (brachial plexus root avulsion (Av) treated with normal saline), Av+GM1 (treated with monosialoganglioside), and control. At time points of 3 day (d), 1 week (w), 2 w, 4 w and 8 w post-injury, ¹⁸F-FDG micro-PET/CT scans and neuropathology assessments of the injured spinal roots, as well as the spinal cord, were performed. The outcomes of the different treatments were compared. The results showed that BPRA induced local bleeding and typical Wallerian degeneration of the avulsed roots accompanied by ¹⁸F-FDG accumulations at the ipsilateral cervical intervertebral foramen. BPRA-induced astrocyte reactions and overexpression of neuronal nitric oxide synthase in the motoneurons correlated with higher ¹⁸F-FDG uptake in the ipsilateral cervical spinal cord during the first 2 w post-injury. The GM1 treatment reduced BPRA-induced astrocyte reactions and inhibited the de novo nNOS expressions in spinal motoneurons. The GM1 treatment also protected spinal motoneurons from avulsion within the first 4 w post-injury. The data from this study suggest that ¹⁸F-FDG PET/CT could be used to assess the severity of BPRA-induced primary and secondary injuries in the spinal cord. Furthermore, GM1 is an effective drug for reducing primary and secondary spinal cord injuries following BPRA.     

Protein concentration in the neuron--neuroglia system of the anterior horns of the spinal cord in rats following exposure to the tranquilizer diazepam against a background of anticipation stress

Anticipation stress was induced in 16 day-old male rats by placing the animals daily for 7 days into individual cells for 45 min. In the end of each 45 min session, an electric stimulation of paws of the animals was done for 2 min. It was shown by visible cytospectrophotometry of amido black-stained spinal cord sections that the anticipation stress for 7 days resulted in an accumulation of the nuclear and cytoplasmic total proteins in the motoneurons of spinal cord anterior horns, with no changes in the body (in fact, in the nuclei) of the glial cells adjacent to the neurons. Intraperitoneal injection of the tranquilizer diazepam (10 mg per kg) 40 min. before the beginning of the last anticipation stress session gave rise to the return to the normal of the protein content per cell in the motoneuron nucleus and cytoplasm while inducing an increase in the quantity of neuroglia cell protein. Differences in the protein metabolism between the neurons and the neuroglia are discussed.     

Expression of nerve growth factor receptor mRNA is developmentally regulated and increased after axotomy in rat spinal cord motoneurons

In situ hybridization histochemistry and RNA blot analysis were used to study expression of nerve growth factor receptor (NGF-R) mRNA in rat spinal cord motoneurons. The results show that NGF-R mRNA is expressed at high levels in rat spinal cord motoneurons at the time of naturally occurring cell death. This expression is sustained, but reduced, during synapse formation and is subsequently greatly reduced in the adult spinal cord. A unilateral crush lesion of the sciatic nerve resulted in an 8-fold increase in NGF-R mRNA in adult rat spinal cord motoneurons 3 days after lesion, compared with the nonlesioned side. NGF-R mRNA induction was even more pronounced 7 and 14 days after lesion, reaching levels 12 times higher than those on the nonlesioned side. However, 6 weeks after lesion, when the motor function of the leg was largely restored, NGF-R expression had decreased to levels similar to those on the contralateral side. We therefore suggest that NGF-R mediates a trophic or axonal guidance function for developing and regenerating spinal cord motoneurons.     

Progesterone neuroprotection in spinal cord trauma involves up-regulation of brain-derived neurotrophic factor in motoneurons

Progesterone (PROG) provides neuroprotection to the injured central and peripheral nervous system. These effects may be due to regulation of myelin synthesis in glial cells and also to direct actions on neuronal function. Both types of cells express classical intracellular PROG receptors (PR), while neurons additionally express the PROG membrane-binding site called 25-Dx. In motoneurons from rats with spinal cord injury (SCI), PROG restores to normal the deficient levels of choline acetyl-transferase and of alpha3 subunit Na,K-ATPase mRNA, while levels of the growth associated protein GAP-43 mRNA are further stimulated. Recent studies suggest that neurotrophins are possible mediators of hormone action, and in agreement with this assumption, PROG treatment of rats with SCI increases the expression of brain-derived neurotrophic factor (BDNF) at both the mRNA and protein levels in ventral horn motoneurons. In situ hybridization (ISH) has shown that SCI reduces BDNF mRNA levels by 50% in spinal motoneurons, while PROG administration to injured rats (4mg/kg/day during 3 days, s.c.) elicits a three-fold increase in grain density. In addition to enhancement of mRNA levels, PROG increases BDNF immunoreactivity in perikaryon and cell processes of motoneurons of the lesioned spinal cord, and also prevents the lesion-induced chromatolytic degeneration of spinal cord motoneurons as determined by Nissl staining. Our findings strongly indicate that motoneurons of the spinal cord are targets of PROG, as confirmed by the expression of PR and the regulation of molecular parameters. PROG enhancement of endogenous neuronal BDNF could provide a trophic environment within the lesioned spinal cord and might be part of the PROG activated-pathways to provide neuroprotection. Thus, PROG treatment constitutes a new approach to sustain neuronal function after injury.     

Regulation of clusterin expression following spinal cord injury

We have investigated the localization and regulation of a putative extracellular chaperone, clusterin, in the rat spinal cord after lesion. In control animals, clusterin is expressed in motoneurons, in meningeal and ependymal cells, and in astrocytes mainly located beneath the pial surface. Beginning at day 2 after hemisection at segmental level C6, clusterin levels increase in GFAP-positive astrocytes within the lesioned segment. Three weeks after trauma, clusterin mRNA and protein are elevated in neurons close to the lesion site and in glial elements within scar tissue and within degenerating fiber tracts rostral and caudal to the lesion. This study provides evidence for a role of clusterin in the subacute and late phase of spinal cord injury.     

Embryonic stem cell-derived L1 overexpressing neural aggregates enhance recovery after spinal cord injury in mice

An obstacle to early stem cell transplantation into the acutely injured spinal cord is poor survival of transplanted cells. Transplantation of embryonic stem cells as substrate adherent embryonic stem cell-derived neural aggregates (SENAs) consisting mainly of neurons and radial glial cells has been shown to enhance survival of grafted cells in the injured mouse brain. In the attempt to promote the beneficial function of these SENAs, murine embryonic stem cells constitutively overexpressing the neural cell adhesion molecule L1 which favors axonal growth and survival of grafted and imperiled cells in the inhibitory environment of the adult mammalian central nervous system were differentiated into SENAs and transplanted into the spinal cord three days after compression lesion. Mice transplanted with L1 overexpressing SENAs showed improved locomotor function when compared to mice injected with wild-type SENAs. L1 overexpressing SENAs showed an increased number of surviving cells, enhanced neuronal differentiation and reduced glial differentiation after transplantation when compared to SENAs not engineered to overexpress L1. Furthermore, L1 overexpressing SENAs rescued imperiled host motoneurons and parvalbumin-positive interneurons and increased numbers of catecholaminergic nerve fibers distal to the lesion. In addition to encouraging the use of embryonic stem cells for early therapy after spinal cord injury L1 overexpression in the microenvironment of the lesioned spinal cord is a novel finding in its functions that would make it more attractive for pre-clinical studies in spinal cord regeneration and most likely other diseases of the nervous system.     

Binding Patterns of Lectins with GalNAc specificity in the mouse dorsal root ganglia and spinal cord

To localize membrane glycoconjugates in neurons of the mouse spinal cord and dorsal root ganglia (DRG), cryostat sections of newborn (P0), 7 day-old (P7), P14, P21 and P31 animals were stained with ten FITC-conjugated plant lectins, the majority of them recognizing N-acetyl-D-galactosamine (GalNAc) terminal sugar residues. In the dorsal root ganglia of P0 animals, the different lectins showed distinct patterns of labeling in either cells of the nervous system, including neurons, or other structures such as nerves or blood vessels. Moreover, some of these lectins showed important changes in their pattern of labeling during postnatal development. This was especially relevant for lectins that label a subpopulation of small-sized cells that have been previously identified as the nociceptive cells of the DRG. Enzymatic digestion of sections with neuraminidase removes sialic acid from the carbohydrate chains of glycoconjugates thus exposing novel sugar residues. When this treatment was applied to DRG sections from postnatal animals the pattern of lectin staining was either changed or eliminated and heterogeneous subsets of glycoconjugates normally masked by this sugar were exposed. In the spinal cord of PO animals, none of the lectins labeled cells in the central gray matter. However, after the enzymatic digestion of sections with neuraminidase, spinal cord motoneurons and some other cells were labeled by two of the lectins suggesting that GalNAc residues present in these cells are normally masked by terminal sialic acid. Altogether, these results show important changes in the temporal and spatial expression of glycoconjugates that may be relevant for the postnatal development of the CNS and PNS of mice.     

Developmental changes in unique cell surface antigens of chick embryo spinal motoneurons and ganglion cells

The monoclonal antibody technique was used to investigate neuronal heterogeneity and its developmental changes in the chick embryo trunk especially at the thoracic level. We report here four monoclonal antibodies (called SC 1, SC 2, SC 3, and SC 4) that bound to cell surface antigens. These antigens appeared to be proteins or glycoproteins because of their susceptibility to trypsin. In the spinal cord, antibody SC 3 stained all cells, but antibody SC 1 specifically stained motoneurons and ventral epithelial cells. The staining of motoneurons by antibody SC 1 was transient. It appeared at early stages (stage 16-17; Hamburger and Hamilton), but decreased markedly in intensity at older stages (stage 30-31). Antibody SC 2 did not stain cells in the spinal cord. It stained only neurons in the dorsal root and sympathetic ganglia. Antibody SC 4 stained only cells derived from the neural crest at the early stages (stage 16-20). At later stages, it stained a wider population of cells, including sensory neurons, Schwann cells, and cells in the central nervous system. In the dorsal root ganglion, antibodies SC 1 and SC 2 stained only neuronal cells whereas antibodies SC 3 and SC 4 stained both neuronal and glial cells. The dorsal root ganglionic antigens recognized by these antibodies were not expressed concurrently but appeared in a developmental sequence. Staining with antibodies SC 3 and SC 4 appeared first, then SC 1, and finally SC 2. Among these four antigens, the antigens common to both neuronal and glial cells appeared earlier than the neuron specific antigens. Thus, our monoclonal antibodies revealed heterogeneities in cell surface neuronal molecules and their transient and sequential appearance during embryonic development.     

Morphofunctional characteristics of the rat distal spinal cord after its complete experimental transection with subsequent animal treadmill training

Motor activity of rats was studied after experimental complete transection of the spinal cord at lower thoracic level. Treadmill training 1 day after the surgery was shown to lead to the appearance of movements in hindlimbs and restoration of the body weight support function. According to our data, the key moment in initiation of locomotor movements is stimulation of foot. Morphoimmunohistochemical investigation of the lumbar enlargement (study of proliferating cell nuclear protein, synaptophysin, and glial fibrillary acidic protein immunohistochemistry) revealed a rearrangement of motoneurons, interneurons, and the afferent chain in the distal part of the transected spinal cord. In the trained animals, there was observed the normal structure of motoneurons and the appearance of aggregates of the synaptophysin-immunoreactive structures lost after the surgery.     

Growth of the axons in organotypic culture of the spinal cord

Peculiarities of the axons growth in the culture of 14-day old chick embryo spinal cord after 24, 48 hr, 3, 5 and 7 days in the Maximov's chamber were observed. For the stimulation of axon growth the spinal cord was cultivated simultaneously with the explants of the muscle tissue and in the medium after the addition of supernatant of the somatic muscle. It has been demonstrated that the growth of the axons stimulated with the muscle explants or muscle supernatant takes place through the growth cones, while in the absence of growth stimulation effect glial cells can take part in the axons growth. It is supposed that the glial cells are capable of playing the role of the cells, which direct axons growth in the absence of influence of specific target factor.     

AMINO ACID AND SUBSTANCE P CONTENTS IN SPINAL CORD OF CATS WITH EXPERIMENTAL HIND-LIMB RIGIDITY PRODUCED BY OCCLUSION OF SPINAL CORD BLOOD SUPPLY

Abstract— Experimental hind-limb rigidity of spinal origin was produced in cats by temporary occlusion of thoracic aorta and internal mammary arteries. In the lumbar segments (L6- S1) of these rigid cats, the monosynaptic reflex recorded from ventral roots was enhanced whereas the polysynaptic reflexes as well as the dorsal root reflexes were almost abolished. On morphological examination of the lumbar spinal cord, the number of interneurons was greatly reduced, whereas the small sized cells, presumably glial cells, were increased by about two times. Ventral horn motoneurons were also reduced. The lumbar spinal cords of the rigid cats were analysed for amino acid and substance P contents. Four major amino acids, aspartate, glutamate, glycine and GABA, were definitely reduced in both grey and white matter except that the glutamate level in the dorsal white was within the normal range. Content and distribution pattern of substance P were not altered in the lumbar cord of the rigid cats. These results are consistent with the notions that GABA occurs in the dorsal horn interneurons subserving primary afferent depolarisation, and that substance P is concentrated in primary afferent fibre terminals. The implications of the decrease of aspartate, glutamate and glycine in the spinal cord of rigid cats are discussed.     

Characterization of a Heat-Shock Process for Reduction of the Nucleic Acid Content of Candida utilis

A process for reducing the nucleic acid content of Candida utilis NRRL Y900 has been developed. The optimal process consists of heating the cells suspended in spent medium initially at pH 4.0 for various times at three different temperatures. Initially a heat-shock at 68 C for 1 to 3 sec is performed followed by incubation for 1 hr at 45 to 50 C and for a 2nd hr at 52 to 55 C. The distribution of degradation products has been characterized. Initially 90% of the nucleic acids were in a polymerized form (extractable by hot perchloric acid). After 30 min, much of this material was hydrolyzed but remained within the cell (extractable by cold perchloric acid). After 2 hr, most of the hydrolysis products leaked into the surrounding medium with only a small amount of low-molecular-weight material remaining within the membrane. Predominantly 3'-mononucleotides accumulated within the cell and eventually leaked from the cell.