A transversely isotropic constitutive model of excised guinea pig spinal cord white matter

Narrowing of the spinal canal generates an amalgamation of stresses within the spinal cord parenchyma. The tissue’s stress state cannot be quantified experimentally; it must be described using computational methods, such as finite element analysis. The objective of this research was to propose a compressible, transversely isotropic constitutive model, an augmentation of the isotropic Mooney–Rivlin hyperelastic strain energy function, to describe the guinea pig spinal cord white matter. Model parameters were derived from a combination of inverse finite element analysis on transverse compression experiments and least squared error analysis applied to quasi-static longitudinal tensile tests. A comparison of the residual errors between the predicted response and the experimental measurements indicated that the transversely isotropic constitutive law that incorporates an offset stretch reduced the error by a factor of four when compared to other commonly used models.     

Axon kinematics change during growth and development

The microkinematic response of axons to mechanical stretch was examined in the developing chick embryo spinal cord during a period of rapid growth and myelination. Spinal cords were isolated at different days of embryonic (E) development post-fertilization (E12, E14, E16, and E18) and stretched 0%, 5%, 10%, 15%, and 20%, respectively. During this period, the spinal cord grew approximately 55% in length, and white matter tracts were myelinated significantly. The spinal cords were fixed with paraformaldehyde at the stretched length, sectioned, stained immunohistochemically for neurofilament proteins, and imaged with epifluorescence microscopy. Axons in unstretched spinal cords were undulated, or tortuous, to varying degrees, and appeared to straighten with stretch. The degree of tortuosity (ratio of the segment's pathlength to its end-to-end length) was quantified in each spinal cord by tracing several hundred randomly selected axons. The change in tortuosity distributions with stretch indicated that axons switched from non-affine, uncoupled behavior at low stretch levels to affine, coupled behavior at high stretch levels, which was consistent with previous reports of axon behavior in the adult guinea pig optic nerve (Bain, Shreiber, and Meaney, J. Biomech. Eng., 125(6), pp. 798-804). A mathematical model previously proposed by Bain et al. was applied to quantify the transition in kinematic behavior. The results indicated that significant percentages of axons demonstrated purely non-affine behavior at each stage, but that this percentage decreased from 64% at E12 to 30% at E18. The decrease correlated negatively to increases in both length and myelination with development, but the change in axon kinematics could not be explained by stretch applied during physical growth of the spinal cord. The relationship between tissue-level and axonal-level deformation changes with development, which can have important implications in the response to physiological forces experienced during growth and trauma.     

Compression behavior of porcine spinal cord white matter

Spinal cord injury often results from a compressive load; however, the compression behavior of spinal cord white matter has not been clearly established. Quantifying the compression behavior is important for advancing our understanding of spinal cord injury mechanics and facilitating the use of finite element models to study injury. The objective of this study was to characterize the unconfined compression behavior of isolated white matter segments and determine the constitutive model which best captured the stress-strain behavior. Spinal cord white matter samples were harvested immediately following sacrifice from juvenile Yorkshire pigs (n=104). The samples were compressed to 40% strain at four strain rates (0.005, 0.05, 0.5, and 5.0/s) and allowed to relax for 60s. The effects of preload, peak strain, sample aspect ratio, and time post mortem on peak stress, and constitutive model parameters were also examined. Strain rate had a significant effect on peak stress (p<0.001). A first-order Ogden model best captured the loading response of spinal cord white matter (R(2)=0.99) and a viscoelastic material model combining a first-order Ogden model with a 3-term Prony series effectively captured the effect of strain rate and the relaxation response. This study showed spinal cord white matter to be less stiff than previously estimated by inverse finite element methods, which will have a significant effect on finite element model predictions of the magnitude and distribution of stresses and strains in the spinal cord. This study is the first to quantify the unconfined compression response of spinal cord white matter.     

High Levels of Ascorbic Acid, Not Glutathione, in the CNS of Anoxia-Tolerant Reptiles Contrasted with Levels in Anoxia-Intolerant Species

Abstract: Ascorbic acid and glutathione (GSH) are antioxidants and free radical scavengers that provide the first line of defense against oxidative damage in the CNS. Using HPLC with electrochemical detection, we determined tissue contents of these antioxidants in brain and spinal cord in species with varying abilities to tolerate anoxia, including anoxia-tolerant pond and box turtles, moderately tolerant garter snakes, anoxia-intolerant clawed frogs (Xenopus laevis), and intolerant Long-Evans hooded rats. These data were compared with ascorbate and GSH levels in selected regions of guinea pig CNS, human cortex, and values from the literature. Ascorbate levels in turtles were typically 100% higher than those in rat. Cortex, olfactory bulb, and dorsal ventricular ridge had the highest content in turtle, 5–6 µmol g^?1 of tissue wet weight, which was twice that in rat cortex (2.82 ± 0.05 µmol g^?1) and threefold greater than in guinea pig cortex (1.71 ± 0.03 µmol g^?1). Regionally distinct levels (2–4 µmol g^?1) were found in turtle cerebellum, optic lobe, brainstem, and spinal cord, with a decreasing anterior-to-posterior gradient. Ascorbate was lowest in white matter (optic nerve) in each species. Snake cortex and brainstem had significantly higher ascorbate levels than in rat or guinea pig, although other regions had comparable or lower levels. Frog ascorbate was generally in an intermediate range between that in rat and guinea pig. In contrast to ascorbate, GSH levels in anoxia-tolerant turtles, 2–3 µmol g^?1 of tissue wet weight, were similar to those in mammalian or amphibian brain, with no consistent pattern associated with anoxia tolerance. GSH levels in pond turtle CNS were significantly higher (by 10–20%) than in rat for several regions but were generally lower than in guinea pig or frog. GSH in box turtle and snake CNS were the same or lower than in rat or guinea pig. The distribution GSH in the CNS also had a decreasing anterior-to-posterior gradient but with less variability than ascorbate; levels were similar in optic nerve, brainstem, and spinal cord. The paradoxically high levels of ascorbate in turtle brain, which has a lower rate of oxidative metabolism than mammalian, suggest that ascorbate is an essential cerebral antioxidant. High levels may have evolved to protect cells from oxidative damage when aerobic metabolism resumes after a hypoxic dive.     

The dynamics of axolemmal disruption in guinea pig spinal cord following compression

Membrane damage has been postulated as a critical factor in mediating axonal degeneration in brain and spinal cord trauma. Despite compelling evidence of membrane disruption as a result of physical insults in both in vivo and in vitro studies, the dynamics of such damage over the time post injury in in vivo studies has not been well documented. Using a well-characterized in vivo guinea pig spinal cord compression model and horseradish peroxidase exclusion assay, we have documented significant membrane disruption at 1 hr, 3 days, and 7 days following injury. Furthermore, the membrane damage was found to spread laterally 10 mm beyond the center of original compression site in both rostral and caudal directions. A second-degree polynomial fit of the measured data predicts a bilateral spread of approximately 20–21 mm of membrane disruption from the epicenter of injury over a period of about 20 days. Thus, this study shows that membrane damage exists days, and possibly weeks, after spinal cord trauma in live guinea pigs. This provides the evidence necessary to investigate the role of membrane damage in triggering axonal deterioration in the future. Furthermore, this study has also revealed a long therapeutical window for membrane repair and functional enhancement following traumatic injury in the central nervous system.     

Spontaneous and Experimental Neuritis and the Distribution of the Myelin Protein P2 in the Nervous System

The P2 contents of nervous tissues from the human, rabbit, guinea pig, and Lewis rat were measured by radioimmunoassay. The ventral spinal roots contained more P2 than any other tissue. Human dorsal roots and peripheral nerves contained 41-65% of the amount in human ventral roots. Human olfactory and optic nerves and brain contained 1.1-2.7%, spinal cord, 2.8%, cranial nerve VIII, 11%, and cerebral grey matter, 0%. The relative amounts in the rabbit nervous system were similar except that the spinal cord contained 20% of the amount in the ventral roots. Qualitative estimates in the guinea pig showed that the spinal roots and peripheral nerves contained more P2 than the spinal cord, and that none was present in the brain. In the Lewis rat, P2 could be detected in the spinal roots and peripheral nerves but not in the CNS. The distribution of P2 in the human nervous system parallels the incidence and severity of lesions in acute polyradiculoneuritis. It also explains the absence of any lesions in the CNS when experimental allergic neuritis is induced in the Lewis rat.     

Decreased Uptake and Release of D-Aspartate in the Guinea Pig Spinal Cord After Dorsal Root Section

This study attempts to determine if L-glutamate and/or L-aspartate may be transmitters of dorsal sensory neurons. The uptake and the electrically evoked release of D-[3H]aspartate, a putative marker for L-glutamate and L-aspartate, were measured in the cervical enlargement (segments C4-T1) of the guinea pig spinal cord before and after cutting dorsal roots C5-T1 on the right side. The uptake and the release of gamma-aminobutyric acid (GABA) also were measured as indices of the integrity of GABAergic neurons in the spinal cord. The cervical enlargement was excised and divided into left and right halves, then into dorsal and ventral quadrants. Quadrants from unlesioned animals took up D-aspartate and GABA, achieving concentrations in the tissues which were 14-25 times that in the medium. Subsequently, electrical stimulation evoked a Ca2+-dependent release of D-aspartate and of GABA. The uptake and release of D-aspartate and GABA were similar in tissues taken from intact and sham-operated animals. However, dorsal rhizotomy, without damage to dorsal radicular or spinal blood vessels, depressed the uptake (by 22-29%) and the release (by 50%) of D-aspartate only in quadrants ipsilateral to the lesion. The uptake and the release of GABA were unchanged. In transverse sections of the cervical enlargement, stained to reveal degenerating fibers, by far the heaviest loss of axons occurred in the cuneate fasciculus and in the gray matter ipsilateral to the cut dorsal roots. These findings suggest that the synaptic endings of dorsal sensory neurons probably mediate the uptake and the release of D-aspartate and, therefore, may use L-glutamate or L-aspartate as a transmitter. When spinal blood vessels were damaged during dorsal rhizotomy, the deficits in D-aspartate uptake and release were larger than those in the absence of vascular damage and were accompanied by deficits in GABA uptake and release. These findings imply that vascular damage results in the loss of intraspinal neurons, some of which probably mediate the uptake and release of D-aspartate and, therefore, may use L-glutamate and/or L-aspartate as a transmitter.     

Changes within maturing neurons limit axonal regeneration in the developing spinal cord

Embryonic birds and mammals display a remarkable ability to regenerate axons after spinal injury, but then lose this ability during a discrete developmental transition. To explain this transition, previous research has emphasized the emergence of myelin and other inhibitory factors in the environment of the spinal cord. However, research in other CNS tracts suggests an important role for neuron-intrinsic limitations to axon regeneration. Here we re-examine this issue quantitatively in the hindbrain-spinal projection of the embryonic chick. Using heterochronic cocultures we show that maturation of the spinal cord environment causes a 55% reduction in axon regeneration, while maturation of hindbrain neurons causes a 90% reduction. We further show that young neurons transplanted in vivo into older spinal cord can regenerate axons into myelinated white matter, while older axons regenerate poorly and have reduced growth cone motility on a variety of growth-permissive ligands in vitro, including laminin, L1, and N-cadherin. Finally, we use video analysis of living growth cones to directly document an age-dependent decline in the motility of brainstem axons. These data show that developmental changes in both the spinal cord environment and in brainstem neurons can reduce regeneration, but that the effect of the environment is only partial, while changes in neurons by themselves cause a nearly complete reduction in regeneration. We conclude that maturational events within neurons are a primary cause for the failure of axon regeneration in the spinal cord.     

A micromechanical procedure for modelling the anisotropic mechanical properties of brain white matter

This paper proposes a micromechanics algorithm utilising the finite element method (FEM) for the analysis of heterogeneous matter. The characterisation procedure takes the material properties of the constituents, axons and extracellular matrix (ECM) as input data. The material properties of both the axons and the matrix are assumed to have linear viscoelastic behaviour with a perfect bonding between them. The results of the modelling have been validated with experimental data with material white input from brainstem by considering the morphology of brainstem in which most axons are oriented in longitudinal direction in the form of a uniaxial fibrous composite material. The method is then employed to examine the undulations of axons within different subregions of white matter and to study the impact due to axon/matrix volume fractions. For such purposes, different unit cells composed of wavy geometries and with various volume factions have been exposed to the six possible loading scenarios. The results will clearly demonstrate the undulation and axon volume fraction impacts. In this respect, undulation affects the material stiffness heavily in the axon longitudinal direction, whereas the axons' volume fraction has a much greater impact on the mechanical properties of the white matter in general. Also the results show that the created stresses and strains in the axons and matrix under loading will be impacted by undulation change. With increase in undulation the matrix suffers higher stresses when subjected to tension, whereas axons suffer higher stresses in shear. The axons always exhibit higher stresses whereas the matrix exhibits higher strains. The evaluated time-dependent local stress and strain concentrations within a repeating unit cell of the material model are indicative of the mechanical behaviour of the white tissue under different loading scenarios.     

Cortical neurite outgrowth and growth cone behaviors reveal developmentally regulated cues in spinal cord membranes.

Corticospinal axon outgrowth in vivo and the ability to sprout or regenerate after injury decline with age. This developmental decline in growth potential has been correlated with an increase in inhibitory myelin-associated proteins in older spinal cord. However, previous results have shown that sprouting of corticospinal fibers after contralateral lesions begins to diminish prior to myelination, suggesting that a decrease in growth promoting and/or an increase in inhibitory molecules in spinal gray matter may also regulate corticospinal axon outgrowth. To address this possibility, we carried out in vitro experiments to measure neurite outgrowth from explants of 1-day-old hamster forelimb sensorimotor cortex that were plated onto membrane carpets or membrane stripe assays prepared from white or gray matter of 1-to 22-day-old cervical spinal cord. On uniform carpets and in the stripe assays cortical neurites grew robustly on young but not older membranes from both white and gray matter. Mixtures of membranes from 1- and 15-day spinal cord inhibited neurite outgrowth, suggesting that the presence of inhibitory molecules in the 15-day cord overwhelmed permissive or growth promoting molecules in membranes from 1-day cord. Video microscopic observations of growth cone behaviors on membrane stripe assays transferred to glass coverslips supported this view. Cortical growth cones repeatedly collapsed at borders between permissive substrates (laminin or young membrane stripes) and nonpermissive substrates (older membrane stripes). Growth cones either turned away from the older membranes or reduced their growth rates. These results suggest that molecules in both the gray and white matter of the developing spinal cord can inhibit cortical neurite outgrowth.     

Finite Element Study of the Mechanical Response in Spinal Cord during the Thoracolumbar Burst Fracture

Background

The mechanical response of the spinal cord during burst fracture was seldom quantitatively addressed and only few studies look into the internal strain of the white and grey matters within the spinal cord during thoracolumbar burst fracture (TLBF). The aim of the study is to investigate the mechanical response of the spinal cord during TLBF and correlate the percent canal compromise (PCC) with the strain in the spinal cord.

Methodology/Principal Findings

A three-dimensional (3D) finite element (FE) model of human T12-L1 spinal cord with visco-elastic property was generated based on the transverse sections images of spinal cord, and the model was validated against published literatures under static uniaxial tension and compression. With the validated model, a TLBF simulation was performed to compute the mechanical strain in the spinal cord with the PCC. Linear regressions between PCC and strain in the spinal cord show that at the initial stage, with the PCC at 20%, and 45%, the corresponding mechanical strains in ventral grey, dorsal grey, ventral white, dorsal white matters were 0.06, 0.04, 0.12, 0.06, and increased to 0.14, 0.12, 0.23, and 0.13, respectively. At the recoiled stage, when the PCC was decreased from 45% to 20%, the corresponding strains were reduced to 0.03, 0.02, 0.04 and 0.03. The strain was correlated well with PCC.

Conclusions/Significance

The simulation shows that the strain in the spinal cord correlated well with the PCC, and the mechanical strains in the ventral regions are higher than those in the dorsal regions of spinal cord tissue during burst fracture, suggesting that the ventral regions of the spinal cord may susceptible to injury than the dorsal regions.     

Recurrent axon collaterals of lumbar motor neurons in turtles

A combined morphophysiological study was made of connections between motoneurons on the superfused isolated lumbar spinal cord of Testudo horsfieldi. Postsynaptic potentials of motoneurons, followed by antidromic stimulations of ventral root filaments (VR-PSPs), were recorded intracellularly. Depolarizing VP-PSPs had short latencies (1.0-1.5 mc) and amplitudes in the range of 0.3-3.0 mV. At the constant stimulus intensity, the fluctuations of amplitudes were recorded. In some motoneurons, hyperpolarizing VP-PSRs with the latencies 2.5-3.0 mc were observed. A possible structural basis of VR-PSPs was studied by the horseradish peroxidase (HRP) method. After HRP application on thin ventral root filaments the retrograde staining of motoneurons revealed recurrent axon collaterals of labeled motoneurons. Three-dimensional computer reconstructions showed one to three collaterals given off by motoneuron axons. There were up to 19 points of branching in a single collateral. In some cases the full length of collateral trees reached 4.0 mm. The collateral branches had up to 72 "en passant" and terminal axon swellings. The swellings (presumed contacting boutons) were distributed in the ventral and intermedial gray matter and in the ventromedial while matter and revealed on motoneurons and inerneurons. These data suggest the participation of the motor axon collaterals in the motoneuron--motoneuron communication in the turtle spinal cord whereas only dendro-dendritic contacts had been discussed earlier.     

Cortical neurite outgrowth and growth cone behaviors reveal developmentally regulated cues in spinal cord membranes

Corticospinal axon outgrowth in vivo and the ability to sprout or regenerate after injury decline with age. This developmental decline in growth potential has been correlated with an increase in inhibitory myelin‐associated proteins in older spinal cord. However, previous results have shown that sprouting of corticospinal fibers after contralateral lesions begins to diminish prior to myelination, suggesting that a decrease in growth promoting and/or an increase in inhibitory molecules in spinal gray matter may also regulate corticospinal axon outgrowth. To address this possibility, we carried out in vitro experiments to measure neurite outgrowth from explants of 1‐day‐old hamster forelimb sensorimotor cortex that were plated onto membrane carpets or membrane stripe assays prepared from white or gray matter of 1‐to 22‐day‐old cervical spinal cord. On uniform carpets and in the stripe assays cortical neurites grew robustly on young but not older membranes from both white and gray matter. Mixtures of membranes from 1‐ and 15‐day spinal cord inhibited neurite outgrowth, suggesting that the presence of inhibitory molecules in the 15‐day cord overwhelmed permissive or growth promoting molecules in membranes from 1‐day cord. Video microscopic observations of growth cone behaviors on membrane stripe assays transferred to glass coverslips supported this view. Cortical growth cones repeatedly collapsed at borders between permissive substrates (laminin or young membrane stripes) and nonpermissive substrates (older membrane stripes). Growth cones either turned away from the older membranes or reduced their growth rates. These results suggest that molecules in both the gray and white matter of the developing spinal cord can inhibit cortical neurite outgrowth. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 393–406, 1999     

Mechanisms of uniformity of yield strains for trabecular bone

Variations in yield strains for trabecular bone within a specific anatomic site are only a small fraction of the substantial variations that exist for elastic modulus and strength, and yet the source of this uniformity is not known. Our goal was to investigate the underlying mechanisms by using high-resolution, materially nonlinear finite element models of 12 human femoral neck trabecular bone specimens. The finite element models, used to obtain apparent yield strains in both tension and compression, assumed that the tissue-level yield strains were the same across all specimens. Comparison of the model predictions with the experimental data therefore enabled us to isolate the combined roles of volume fraction and architecture from the role of tissue material properties. Results indicated that, for both tensile and compressive loading, natural variations in volume fraction and architecture produced a negligible coefficient of variation (less than 3%) in apparent yield strains. Analysis of tissue-level strains showed that while bending of individual trabeculae played only a minor role in the apparent elastic behavior, the combined effects of this bending and tissue-level strength asymmetry produced apparent-level failure strains in compression that were 14% lower than those at the tissue level. By contrast, tissue and apparent-level yield strains were equivalent for tensile loading. We conclude that the uniformity of apparent yield strains is primarily the result of the highly oriented architecture that minimizes bending. Most of the variation that does occur is the result of the non-uniformity of the tissue-level yield strains.     

Resistance of isolated mammalian spinal cord white matter to oxygen-glucose deprivation

We found that isolated guinea pigspinal cord white matter is resistant to acute oxygen-glucosedeprivation. Sixty minutes of oxygen-glucose deprivation resulted in a60% reduction of compound action potential (CAP) conductance, andthere was a near complete recovery after 60 min reperfusion.Corresponding horseradish peroxidase-exclusion assay showed littleaxonal membrane damage. To further deprive the axons of metabolicsubstrate, we added 2 mM sodium cyanide or 2 mM sodium azide, bothmitochondrial suppressors, to the ischemic medium, whichcompletely abolished CAP and resulted in a 15 to ~30% recoverypostreperfusion. Both compounds preferentially reduced the conductanceof large diameter axons. We suggest the residual ATP in ourischemic model can protect anatomic integrity and physiological functioning of spinal axons following ischemic insult. Thisfurther suggests that oxygen-glucose deprivation alone cannot be solely responsible for short-term functional and anatomic damage. The damagingeffects of ischemia in vivo may be mediated by factors originating from the gray matter of the cord or other systemic factors;both were largely eliminated in our in vitro white matter preparation.

     

Coherent anti-stokes Raman scattering imaging of axonal myelin in live spinal tissues

We present a vibrational imaging study of axonal myelin under physiological conditions by laser-scanning coherent anti-Stokes Raman scattering (CARS) microscopy. We use spinal cord white matter strips that are isolated from guinea pigs and kept alive in oxygen bubbled Krebs' solution. Both forward- and epi-detected CARS are used to probe the parallel axons in the spinal tissue with a high vibrational contrast. With the CARS signal from CH₂ vibration, we have measured the ordering degree and the spectral profile of myelin lipids. Via comparison with the ordering degrees of lipids in myelin figures formed of controlled lipid composition, we show that the majority of the myelin membrane is in the liquid ordered phase. By measuring the myelin thickness and axon diameter, the value of g ratio is determined to be 0.68 with forward- and 0.63 with epi-detected CARS. Detailed structures of the node of Ranvier and Schmidt-Lanterman incisure are resolved. We have also visualized the ordering of water molecules between adjacent bilayers inside the myelin. Our observations provide new insights into myelin organization, complementary to the knowledge from light and electron microscopy studies of fixed and dehydrated tissues. In addition, we have demonstrated simultaneous CARS imaging of myelin and two-photon excitation fluorescence imaging of intra- and extraaxonal Ca²⁺. The current work opens up a new approach to the study of spinal cord injury and demyelinating diseases.     

Development, neurochemical properties, and axonal projections of a population of last-order premotor interneurons in the white matter of the chick lumbosacral spinal cord

There is general agreement that last-order premotor interneurons-a set of neurons that integrate activities generated by the spinal motor apparatus, sensory information and volleys arising from higher motor centres, and transmit the integrated signals to motoneurons through monosynaptic contacts-play crucial roles in the initiation and maintenance of spinal motor activities. Here, we demonstrate the development, neurochemical properties, and axonal projections of a unique group of last-order premotor interneurons within the ventrolateral aspect of the lateral funiculus of the chick lumbosacral spinal cord. Neurons expressing immunoreactivity for neuron-specific enolase were first detected in the ventrolateral white matter at embryonic day 9 (E9). The numbers of immunoreactive neurons were significantly increased at E10-E12, while most of them were gradually concentrated in small segmentally arranged nuclei (referred to as major nuclei of Hofmann) protruding from the white matter in a necklace like fashion dorsal to the ventral roots. The major nuclei of Hofmann became more prominent at E12-E16, but substantial numbers of cells were still located within the ventrolateral white matter (referred to as minor nucleus of Hofmann). The distribution of immunoreactive neurons achieved by E16 was maintained during later developmental stages and was also characteristic of adult animals. After injection of Phaseolus vulgaris-leucoagglutinin unilaterally into the minor nucleus of Hofmann, labeled fibres were detected in the ventrolateral white matter ipsilateral to the injection site. Ascending and descending fibres were revealed throughout the entire rostro-caudal length of the lumbosacral spinal cord. Axon terminals were predominantly found within the lateral motor column and the ventral regions of lamina VII ipsilateral to the injection site. Several axon varicosities made close appositions with somata and dendrites of motoneurons, which were identified as synaptic contacts in a consecutive electron microscopic study. With the postembedding immunogold method, 21 of 97 labeled terminals investigated were immunoreactive for glycine and 2 of them showed immunoreactivity for gamma-aminobutyric acid (GABA). The axon trajectories of neurons within the minor nucleus of Hofmann suggest that some of these cells might represent a population of last-order premotor interneurons. J. Exp. Zool. 286:157-172, 2000.     

Depolarizing agent-induced cyclic AMP accumulation in isolated rat spinal cord

The stimulation of cyclic adenosine 3',5'-monophosphate (cyclic AMP) accumulation by the depolarizing agents K+, ouabain and veratridine, was studied in rat and guinea pig spinal cord tissue slices. Significantly increased accumulation of cyclic AMP was produced by each of the agents in a concentration-dependent manner. Veratridine and ouabain were equipotent (EC50 = 5 x 10(-5)M) and approximately 500 fold more potent than K+ (EC50 = 10(-2)M). Depolarizing agent-induced cyclic AMP accumulation in slices from guinea pig spinal cord was approximately double the response in rat spinal cord. Maximum stimulation occurred within 2.5 min of incubation with these agents and lasted for at least 30 min. Regional studies demonstrated that the maximal accumulation of cyclic AMP occurred to a greater degree in tissue slices from the dorsal section of spinal cord from both rat and guinea pig. Whereas the ouabain and veratridine stimulatory responses are completely dependent on extracellular Ca++, the K+ response is only partially dependent. Stimulation due to ouabain and veratridine is dependent, and K+ is independent, of release of neurohumoral substances such as norepinephrine or adenosine from spinal neurons. These experiments indicate the possible modulatory role of depolarization-linked events in regulating the spinal cord cyclic AMP system.     

Pathological changes in the white matter after spinal contusion injury in the rat

It has been shown previously that after spinal cord injury, the loss of grey matter is relatively faster than loss of white matter suggesting interventions to save white matter tracts offer better therapeutic possibilities. Loss of white matter in and around the injury site is believed to be the main underlying cause for the subsequent loss of neurological functions. In this study we used a series of techniques, including estimations of the number of axons with pathology, immunohistochemistry and mapping of distribution of pathological axons, to better understand the temporal and spatial pathological events in white matter following contusion injury to the rat spinal cord. There was an initial rapid loss of axons with no detectable further loss beyond 1 week after injury. Immunoreactivity for CNPase indicated that changes to oligodendrocytes are rapid, extending to several millimetres away from injury site and preceding much of the axonal loss, giving early prediction of the final volume of white matter that survived. It seems that in juvenile rats the myelination of axons in white matter tracts continues for some time, which has an important bearing on interpretation of our, and previous, studies. The amount of myelin debris and axon pathology progressively decreased with time but could still be observed at 10 weeks after injury, especially at more distant rostral and caudal levels from the injury site. This study provides new methods to assess injuries to spinal cord and indicates that early interventions are needed for the successful sparing of white matter tracts following injury.     

D-Aspartate Uptake and Release in the Guinea Pig Spinal Cord After Partial Ablation of the Cerebral Cortex

This study attempts to determine if L-glutamate and L-aspartate may be transmitters of the guinea pig corticospinal tract. Unilateral ablations were made of the frontal and parietal neocortex which destroyed most of the motor and somatosensory areas in the right cerebral hemisphere. In lesioned animals, transverse sections of the cervical enlargement of the spinal cord (segments C6--T1) were stained to reveal degenerating fibers. Degeneration of axons first appeared 4 days after surgery, reached a maximum on the seventh day, and began to wane by the ninth day. The most prominent loss of axons appeared deep in the dorsal funiculus and in laminae IV-IX of the gray matter contralateral to the cortical lesion. Ipsilaterally, there was very sparse degeneration of fibers in the dorsal and ventral funiculi and in the spinal gray matter. The uptake and release of D-[3H]aspartate, a putative nonmetabolizable marker for L-glutamate and L-aspartate, were measured in dissected quadrants of the cervical enlargement taken from intact and lesioned animals. The uptake and the electrically evoked, Ca2+-dependent release of D-[3H]aspartate were depressed by 29-35% at 4 and 7 days after surgery, but only in tissue that was contralateral to the cortical ablation. The lesion had no effect on the uptake and release of exogenous gamma-[14C]aminobutyric acid, which were measured as indices of the postlesion integrity of neurons in the spinal gray matter. These findings suggest that the synaptic endings of corticospinal fibers probably mediate the uptake and release of D-[3H]aspartate and, therefore, may use L-glutamate and/or L-aspartate as a transmitter.