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.

     

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.     

The location of spinothalamic axons within spinal cord white matter in cat and squirrel monkey

The locations of spinothalamic (STT) fibers in the spinal cord white matter have been identified in cat and squirrel monkey by light-microscopic visualization of labeled fibers following multiple thalamic injections of wheatgerm agglutinin conjugated to horseradish peroxidase. Thalamic injections were combined with either a constricting dural tie or an intraspinal injection of colchicine to facilitate axonal labeling at more rostral spinal levels. In the cat, the ventral-to-dorsal distribution of labeled STT fibers was bimodal. In the ventrolateral white matter, labeled axons were coarse in nature and were primarily concentrated peripherally. In the dorsolateral white matter, labeled STT axons consisted of fine-caliber fibers concentrated in the ventral portion of the dorsolateral funiculus and were equally distributed throughout the medial and lateral white matter. In the squirrel monkey, the distribution of STT fibers was unimodal, extending from the ventral surface of the spinal white matter to the ventralmost portion of the dorsolateral funiculus. As in the cat, however, the ventrally located axons were large and coarse and were primarily located in the peripheral white matter, whereas the dorsalmost STT fibers were of fine caliber and were distributed equally in the medial and lateral white matter.     

Spread of a neurotropic coronavirus to spinal cord white matter via neurons and astrocytes.

Mouse hepatitis virus strain JHM (MHV-JHM) causes a chronic encephalomyelitis in susceptible mice, with histological evidence of demyelination in the spinal cord. After intranasal inoculation, virus spreads retrogradely to several brain structures along neuroanatomic projections to the main olfactory bulb. In the absence of experimental intervention, mice become moribund before the spinal cord is infected. In this study, infusions of anti-MHV neutralizing monoclonal antibodies were administered to protect mice from the MHV-JHM-induced acute encephalitis and to allow survival until virus spread to the spinal cord. Under these conditions, virus was observed to enter specific layers (primarily laminae V to VII) in the gray matter of the upper spinal cord, consistent with transneuronal spread. While the brain structures which are the sources for virus spread to the spinal cord cannot be determined with certainty, the ventral reticular nucleus is likely to be important since it is consistently and extensively labeled in all mice and receives projections from subsequently infected areas of the spinal cord. After initial entry into the gray matter, virus rapidly spread to the white matter of the spinal cord. During the early stages of this process, extensive infection of astrocytes was noted, suggesting that cell-to-cell spread via these glial cells is an important part of this process. Reports from other laboratories using cultured cells strongly suggested that astrocytes serve as important regulators of oligodendrocyte function and, by extrapolation, have a major role in vivo in the processes of both demyelination and remyelination. Thus, our results not only outline the probable pathway used by MHV-JHM to infect the white matter of the spinal cord but also, with the assumption that infection of astrocytes leads to subsequent dysfunction, raise the possibility that infection of these cells contributes to the demyelinating process.     

Soluble and bound acid protease activity of myelin from bovine cerebral white matter and spinal cord

Isolated myelin of bovine spinal cord was found to degrade exogenous myelin basic protein (MBP) at pH 4.4. Electrophoretic peptide patterns were consistent with limited proteolysis of MBP. Some of the proteolytic activity was soluble at increased ionic strength, some remained bound, withstanding extraction at 37°C for up to 12 hr. While being measurable with exogenous MBP, bound protease degraded neither bound MBP nor any other major intrinsic myelin protein. Both soluble and bound protease activity was completely inhibited by pepstatin A. The patterns of limited proteolysis of MBP they produced were identical. Myelin of cerebral white matter also exhibited soluble and bound acid protease activity which was likewise inhibited by pepstatin A. Protease activity of spinal cord and cerebral myelin is therefore suggested to be due to a cathepsin D-like endopeptidase, present in a loosely and tightly bound form. Both forms increased by 50 to 80% in activity when myelin was isolated from mixtures of white and cortical gray matter. While increased soluble activity of myelin is consistent with binding of cathepsin D of lysosomal origin during the isolation of myelin the tightly bound form might point to a principal mechanism through which exogenous proteins may become attached to the myelin sheath in vivo.     

Correlations between tissue-level stresses and strains and cellular damage within the guinea pig spinal cord white matter

Strain magnitude, strain rate, axon location, axon size, and the local tissue stress state have been proposed as the mechanisms governing primary cellular damage within the spinal cord parenchyma during slow compression injury. However, the mechanism of axon injury has yet to be fully elucidated. The objective of this study was to correlate cellular damage within the guinea pig spinal cord white matter, quantified by a horseradish peroxidase (HRP) exclusion test, with tissue-level stresses and strains using a combined experimental and computational approach. Force-deformation curves were acquired by transversely compressing strips of guinea pig spinal cord white matter at a quasi-static rate. Hyperelastic material parameters, derived from a Mooney-Rivlin constitutive law, were varied within a nonlinear, plane strain finite element model of the white matter strips until the computational force-deformation curve converged to the experimental results. In addition, white matter strips were subjected to nominal compression levels of 25%, 50%, 70%, and 90% to assess axonal damage by quantifying HRP uptake. HRP uptake density increased with tissue depth and with increased nominal compression. Using linear and nonlinear regression analyses, the strongest correlations with HRP uptake density were found for groups of tissue-level stresses and groups of log-transformed tissue-level strains.     

Catalytic nitric oxide synthase activity in the white and gray matter regions of the spinal cord of rabbits

The latest research reveals that nitric oxide as a gas messenger may diffuse into the surrounding extracellular fluid and act locally upon neighboring target cells. However, several observations raise the possibility that nitric oxide may also be released at a greater distance from the neuronal cell body. The catalytic nitric oxide synthase (cNOS) activity was therefore studied in the cervicothoracic and lumbosacral segments of the spinal cord of rabbits, including the white matter of dorsal columns (DC), lateral columns (LC) and ventral columns (VC), as well as the gray matter of dorsal horns (DH), intermediate zone (IZ) and ventral horns (VH). Lower cNOS activity was found in the white matter of both cervicothoracic (47%) and lumbosacral (30%) regions, whereas that detected in the gray matter of the lumbosacral part of the spinal cord was considerably higher (70%). Enzyme activity varied from 43.4 to 77.2 dpm/microg protein in the cervicothoracic segments of the gray matter in the descending order: DH>VH>IZ. Similar cNOS activity was found in the white matter of the cervicothoracic segments (42.1-62.8 dpm/microg protein). When the activity of cNOS was compared in the lumbosacral segments, the highest enzyme activity was found in DH of the gray matter (198.7 dpm/microg protein) and the lowest cNOS in DC (45.8 dpm/microg protein) of the white matter. It was concluded that the white matter of the spinal cord contains similar cNOS activity in comparison to the gray matter.     

Inelastic behavior in repeated shearing of bovine white matter

Understanding the brain's response to multiple loadings requires knowledge of how straining changes the mechanical response of brain tissue. We studied the inelastic behavior of bovine white matter and found that when this tissue is stretched beyond a critical strain threshold, its reloading stiffness drops. An upper bound for this strain threshold was characterized, and was found to be strain rate dependent at low strain rates and strain rate independent at higher strain rates. Results suggest that permanent changes to tissue mechanics can occur at strains below those believed to cause physiological disruption or rupture of axons. Such behavior is characteristic of disentanglement in fibrous-networked solids, in which strain-induced mechanical changes may result from fiber realignment rather than fiber breakage.     

Effect of spinal cord compression on cyclic 3',5'-guanosine monophosphate in the white matter columns of rabbit

Changes in the level of cyclic 3',5'-guanosine monophosphate (cGMP) were studied one day after a surgically induced spinal cord constriction performed at the Th7 segment level in the dorsal, lateral and ventral white matter columns and in the non-compartmentalized white matter of Th5-Th6 segments, i.e., above the site of the spinal cord constriction and in Th8-Th9 segments, located below the spinal cord constriction. The midthoracic spinal cord constriction caused a significant decrease in the level of cGMP in the ventral column of Th5-Th6 segments and a significant increase in the lateral column of Th8-Th9 segments. The level of cGMP in the dorsal column, located either rostrally or caudally to the site of the spinal cord injury, remained unchanged. In addition, no significant changes in the level of cGMP were found in the non-compartmentalized white matter of Th5-Th6 and Th8-Th9 segments in response to constriction of the Th7 segment.     

Progressive white matter pathology in the spinal cord of transgenic mice expressing mutant (P301L) human tau

Transgenic mice expressing mutant (P301L) tau develop paresis, neurofibrillary tangles and neuronal loss in spinal motor neurons beginning at 4 to 6 months of age. Astrocytes and oligodendrocytes acquire filamentous tau inclusions at later ages. Here we report pathology in the spinal white matter of these animals. Progressive white matter pathology, detected as early as 2 months of age, was most marked in lateral and anterior columns, with sparing of posterior columns until late in the disease. Early changes in Luxol fast blue/periodic acid Schiff (LFB/PAS) and toluidine blue stained sections were vacuolation of myelin followed by accumulation of myelin figures within previous axonal tubes and finally influx of PAS-positive macrophages. Myelin debris and vacuoles were found in macrophages. At the ultrastructural level, myelinated axons showed extensive vacuolation of myelin sheaths formed by splitting of myelin lamellae at the intra-period line, while axons were atrophic and contained densely packed neurofilaments. Other axons were lost completely, resulting in collapse and phagocytosis of myelin sheaths. Also present were spheroids derived from swollen axons with thin myelin sheaths containing neurofilaments, tau filaments and degenerating organelles. Many oligodendrocytes had membrane-bound cytoplasmic bodies composed of tightly stacked lamellae capped by dense material. The vacuolar myelopathy in this model to some extent resembles that reported in acquired immune deficiency syndrome and vitamin B12 deficiency. The progressive axonal pathology is most consistent with a dying-back process caused by abnormal accumulation of tau in upstream neurons, while vacuolar myelinopathy may be a secondary manifestation of neuroinflammation.     

Depolarization-induced Ca2+ release in ischemic spinal cord white matter involves L-type Ca2+ channel activation of ryanodine receptors

The mechanisms of Ca(2+) release from intracellular stores in CNS white matter remain undefined. In rat dorsal columns, electrophysiological recordings showed that in vitro ischemia caused severe injury, which persisted after removal of extracellular Ca(2+); Ca(2+) imaging confirmed that an axoplasmic Ca(2+) rise persisted in Ca(2+)-free perfusate. However, depletion of Ca(2+) stores or reduction of ischemic depolarization (low Na(+), TTX) were protective, but only in Ca(2+)-free bath. Ryanodine or blockers of L-type Ca(2+) channel voltage sensors (nimodipine, diltiazem, but not Cd(2+)) were also protective in zero Ca(2+), but their effects were not additive with ryanodine. Immunoprecipitation revealed an association between L-type Ca(2+) channels and RyRs, and immunohistochemistry confirmed colocalization of Ca(2+) channels and RyR clusters on axons. Similar to "excitation-contraction coupling" in skeletal muscle, these results indicate a functional coupling whereby depolarization sensed by L-type Ca(2+) channels activates RyRs, thus releasing damaging amounts of Ca(2+) under pathological conditions in white matter.