Peter K. Stys

Anoxic and ischemic injury of myelinated axons in CNS white matter : from mechanistic concepts to therapeutics

White matter of the brain and spinal cord is susceptible to anoxia and ischemia. Irreversible injury to this tissue can have serious consequences for the overall function of the CNS through disruption of signal transmission. Myelinated axons of the CNS are critically dependent on a continuous supply of energy largely generated through oxidative phosphorylation. Anoxia and ischemia cause rapid energy depletion, failure of the Na+−K+-ATPase, and accumulation of axoplasmic Na+ through noninactivating Na+ channels, with concentrations approaching 100 mmol/L after 60 minutes of anoxia. Coupled with severe K+ depletion that results in large membrane depolarization, high [Na+]i stimulates reverse Na+–Ca2+ exchange and axonal Ca2+ overload. A component of Ca2+ entry occurs directly through Na+ channels. The excessive accumulation of Ca2+ in turn activates various Ca2+-dependent enzymes, such as calpain, phospholipases, and protein kinase C, resulting in irreversible injury. The latter enzyme may be involved in “autoprotection,” triggered by release of endogenous γ-aminobutyric acid and adenosine, by modulation of certain elements responsible for deregulation of ion homeostasis. Glycolytic block, in contrast to anoxia alone, appears to preferentially mobilize internal Ca2+ stores; as control of internal Ca2+ pools is lost, excessive release from this compartment may itself contribute to axonal damage. Reoxygenation paradoxically accelerates injury in many axons, possibly as a result of severe mitochondrial Ca2+ overload leading to a secondary failure of respiration. Although glia are relatively resistant to anoxia, oligodendrocytes and the myelin sheath may be damaged by glutamate released by reverse Na+–glutamate transport. Use-dependent Na+ channel blockers, particularly charged compounds such as QX-314, are highly neuroprotective in vitro, but only agents that exist partially in a neutral form, such as mexiletine and tocainide, are effective after systemic administration, because charged species cannot penetrate the blood–brain barrier easily. These concepts may also apply to other white matter disorders, such as spinal cord injury or diffuse axonal injury in brain trauma. Moreover, whereas many events are unique to white matter injury, a number of steps are common to both gray and white matter anoxia and ischemia. Optimal protection of the CNS as a whole will therefore require combination therapy aimed at unique steps in gray and white matter regions, or intervention at common points in the injury cascades.

Compound action potential of nerve recorded by suction electrode: a theoretical and experimental analysis

Suction electrodes are widely used for recording compound action potentials (CAPs) from peripheral nerves or central tracts. Unfortunately, the recordings obtained with suction electrodes often vary over time, making quantitative measurement of CAP amplitude difficult. We developed an equivalent electrical model which predicts that the magnitude of a recorded potential will be linearly related to the resistance of the electrode with a nerve inserted. Mathematical procedures were developed that allow correction of virtually all variability inherent in this type of recording; this variability may arise from resistance drift, variable stimulus artifact, or potentials generated as a result of the current of injury. The validity of the theoretical analysis was confirmed experimentally using rat optic nerves. The magnitude of the CAP and electrode resistance varied spontaneously by as much as 100% over time, due to changes in electrode resistance and size of the stimulus artifact. Because the CAP was linearly related to resistance, it was therefore best quantified by the slope computed from this relationship. The stimulus artifact, unlike the CAP itself, was shown to be independent of recording electrode resistance and therefore only resulted in a variable offset to the area vs resistance linear relationship; the slope of this relationship was unaffected. In the absence of stimulation, a steady negative DC potential was recorded from the optic nerve, which was greatest immediately after dissection, and was also a linear function of electrode resistance. In contrast to CAP amplitude, the latencies of the component peaks within the CAP were not significantly altered by changes in electrode resistance. The experimental results confirmed the validity of the electrical model and demonstrated that suction electrodes can be a very reliable and quantitative recording method if the signals are properly corrected.

Ultrastructural concomitants of anoxic injury and early post-anoxic recovery in rat optic nerve

To study the effects of anoxia on CNS white matter, we examined the ultrastructure of axons and glial cells in a white matter tract, the rat optic nerve, that was subjected to a standardized anoxic insult in vitro. Previous electrophysiological studies showed that in this model, action potential conduction is rapidly abolished by anoxia, and conduction is restored after reoxygenation in about 30% of axons following a 60-min anoxic period. The present study examined the ultrastructural correlates of anoxic injury and early post-anoxic recovery in this model. Optic nerves examined immediately following 60 min of anoxia displayed numerous large, apparently empty zones located within myelin sheaths adjacent to the axon. The myelin remained compact and retained its periodicity. In some regions, the extracellular space was enlarged. There was mitochondrial swelling with loss of normal cristae. There was also loss of microtubules and, to a smaller degree, of neurofilaments in large-diameter axons. Some nodes of Ranvier in anoxic optic nerves displayed detachment of terminal oligodendroglial loops or retraction of the myelin from the node; the presence of tongue-like processes, extending from nearby cells under the detached myelin loops, suggested a possible role of cell-mediated damage to the paranodal myelin. Bundles of dense astrocyte processes were present, and there was vesicular degeneration of perinodal astrocyte processes. In optic nerves that had been permitted to recover for 60 min in oxygenated Ringers following the anoxic period, empty zones were only rarely observed within myelin sheaths and, when present, were smaller than in optic nerves immediately following 60 min of anoxia. The axoplasm of large fibers continued to show loss of microtubules and neurofilaments, as well as mitochondrial swelling. Myelin appeared normal, and only rare paranodal oligodendroglial processes remained unattached from the axon membrane. These results provide support for the idea that, during anoxia, myelinated axons are damaged with significant injury to cytoskeletal elements, probably due to an influx of calcium. The ultrastructural results, together with our earlier observations on the physiological correlates of anoxia and re-oxygenation, suggest that the development of intramyelinic spaces or damage to paranodes lead to conduction block in the anoxic optic nerve. These results also suggest that repair of these structural abnormalities may provide a morphological basis for the early recovery of conduction that occurs after re-oxygenation.

Non-synaptic mechanisms of Ca2+-mediated injury in CNS white matter

Clinical deficits after injury to the CNS are due, in large part, to dysfunction of white matter (myelinated fiber tracts), including descending and ascending tracts in the spinal cord. A crucial set of questions, in the search for strategies that will preserve or restore function after CNS injury, centers on the pathophysiology of, and mechanisms underlying recovery of conduction in, CNS white matter. These questions are relevant both to spinal cord injury, and to brain infarction, which frequently affects white matter.

Protection of the axonal cytoskeleton in anoxic optic nerve by decreased extracellular calcium

Since CNS white matter tracts contain axons, oligodendrocytes and astrocytes but not synapses, it is likely that anoxic injury of white matter is mediated by cellular mechanisms that do not involve synapses. In order to test the hypothesis, that anoxic injury of white matter is mediated by an influx of Ca2+ into the intracellular compartment of axons, we compared the ultrastructure of axons in rat optic nerve exposed to 60 min of anoxia in artificial cerebrospinal fluid (aCSF) containing normal (2 mM) Ca2+, and in aCSF containing zero-Ca2+ together with 5 mM EGTA. Optic nerves fixed at the end of 60 min of anoxia in 2 mM Ca2+ exhibit extensive ultrastructural alterations including disruption of microtubules and neurofilaments within the axonal cytoskeleton, development of membranous profiles and empty spaces between the axon and the ensheathing myelin, and swelling of mitochondria with loss of cristae. Bathing the nerves in zero-Ca2+ aCSF during anoxia protected the axons from cytoskeletal changes; after 60 min of anoxia, optic nerve axons retained normal-appearing microtubules and neurofilaments. Membranous profiles were rare, and empty spaces between axons and myelin did not develop in anoxic optic nerves bathed in zero-Ca2+ aCSF. Disorganization of cristae in axonal mitochondria was observed in anoxic optic nerves even when Ca2+ was omitted from the medium. Because Ca(2+)-mediated injury is known to disrupt the axonal cytoskeleton, these results support the hypothesis that anoxia triggers an abnormal influx of Ca2+ into myelinated axons in CNS white matter.

Anoxic injury of rat optic nerve: ultrastructural evidence for coupling between Na+ influx and Ca2+-mediated injury in myelinated CNS axons

Physiological studies in the anoxic rat optic nerve indicate that irreversible loss of function, measured by the compound action potential, is due to depolarization and run-down of the transmembrane Na+ gradient which triggers Ca2+ entry through reverse Na(+)-Ca2+ exchange. EM studies in the anoxic optic nerve have demonstrated characteristic changes, including mitochondrial swelling and dissolution of cristae, submyelinic vacuoles, detachment of perinodal oligodendrocyte-axon loops, and severe cytoskeletal damage with loss of microtubules and neurofilaments within the axoplasm. To further examine the coupling between Na+ influx and Ca(2+)-mediated injury in myelinated axons within anoxic white matter, we have examined the ultrastructural effects of tetrodotoxin (TTX), in the anoxic optic nerve. Optic nerves, maintained in an interface brain slice chamber, were exposed to a 60-min period of anoxia. TTX (1 microM) was introduced 10 min before the onset of anoxia. Nerves were examined at the end of the anoxic period, or after 80 min in 1 microM TTX for normoxic controls. Under normoxic conditions, optic nerve axons exposed to TTX exhibited a normal ultrastructure. In optic nerves exposed to TTX studied at the end of a 60-min period of anoxia, mitochondria showed swelling and loss of cristae, and terminal oligodendroglial loops were detached from the nodal axon membrane. Cytoskeletal architecture was preserved in anoxic optic nerve axons treated with TTX, and axonal microtubules and neurofilaments maintained their continuity. Submyelinic empty spaces were not present. Perinodal astrocyte processes often appeared to be replaced by cellular remnants containing multiple membranous profiles; clusters of shrunken astrocytic processes were present between myelinated axons.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of polyvalent cations and dihydropyridine calcium channel blockers on recovery of CNS white matter from anoxia

The effects of anoxic injury on the functional integrity of mammalian central white matter were studied electrophysiologically using the rat optic nerve model. Previous studies on this model have shown that extracellular Ca2+ is critical to the production of irreversible anoxic injury, and suggest that during anoxia Ca2+ crosses the membrane to enter the intracellular compartment. We attempted to elucidate the mechanism by which this damaging Ca2+ influx occurs. The inorganic Ca2+ channel blockers Mn2+ (1 mM), Co2+ (1 mM) or La3+ (0.1 mM) had no effect on recovery of the area under the compound action potential after a standard 60 min period of anoxia; only Mg2+ (10 mM) significantly improved recovery (54.9 +/- 8.9% vs. 28.7 +/- 10.1%, P less than 0.005). Treatment with organic Ca2+ channel blockers of the dihydropyridine class, nifedipine (1-10 microM) or nimodipine (1-40 microM), also had no effect on recovery from anoxia. We conclude that Ca2+ influx during anoxia does not occur via conventional Ca2+ channels sensitive to polyvalent cations or dihydropyridines.

Reverse Operation of the Na+ -Ca2+ Exchanger Mediates Ca 2+ Influx during Anoxia in Mammalian CNS White Mattera

Central white matter (WM) tracts in the mammalian central nervous system (CNS), such as subcortical pathways and spinal cord tracts that are critical to the functional integrity of the CNS, suffer irreversible injury after anoxia/ischemia. Anoxia-induced cell death appears to be caused by sustained increases in intracellular CaZf. In gray matter, Ca2+ influx into the cytoplasm during anoxia is thought to occur via NMDAreceptor-gated The mechanisms of anoxic injury in CNS WM are less well understood; specifically, the critical step by which extracellular Ca2+ enters the cytoplasmic compartment is not known. We have studied this question using the in vitro rat optic nerve, a representative CNS WM tract. Our results indicate that a large part of the damaging CaZ+ influx that occurs during anoxia in WM is mediated by the Na+-Ca2+ exchanger, forced to operate in the reverse mode due to Na+ influx via voltage-gated Naf channels.

Effects of Temperature on Evoked Electrical Activity and Anoxic Injury in CNS White Matter

Temperature is known to influence the extent of anoxic/ischemic injury in gray matter of the brain. We tested the hypothesis that small changes in temperature during anoxic exposure could affect the degree of functional injury seen in white matter, using the isolated rat optic nerve, a typical CNS white matter tract (Foster et al., 1982). Functional recovery after anoxia was monitored by quantitative assessment of the compound action potential (CAP) area. Small changes in ambient temperature, within a range of 32 to 42°C, mildly affected the CAP of the optic nerve under normoxic conditions. Reducing the temperature to <37°C caused a reversible increase in the CAP area and in the latencies of all three CAP peaks; increasing the temperature to >37°C had opposite effects. Functional recovery of white matter following 60 min of anoxia was strongly influenced by temperature during the period of anoxia. The average recovery of the CAP, relative to control, after 60 min of anoxia administered at 37°C was 35.4 ± 7%; when the temperature was lowered by 2.5°C (i.e., to 34.5°C) for the period of anoxic exposure, the extent of functional recovery improved to 64.6 ± 15% (p < 0.00001). Lowering the temperature to 32°C during anoxic exposure for 60 min resulted in even greater functional recovery (100.5 ± 14% of the control CAP area). Conversely, if temperature was increased to >37°C during anoxia, the functional outcome worsened, e.g., CAP recovery at 42°C was 8.5 ± 7% (p < 0.00001). Hypothermia (i.e., 32°C) for 30 min immediately following anoxia at 37°C did not improve the functional outcome. Many processes within the brain are temperature sensitive, including O2 consumption, and it is not clear which of these is most relevant to the observed effects of temperature on recovery of white matter from anoxic injury. Unlike the situation in gray matter, the temperature dependency of anoxic injury cannot be related to reduced release of excitotoxins like glutamate, because neurotransmitters play no role in the pathophysiology of anoxic damage in white matter (Ransom et al., 1990a). It is more likely that temperature affects the rate of ion transport by the Na+–Ca2+ exchanger, the transporter responsible for intracellular Ca2+ loading during anoxia in white matter, and/or the rate of some destructive intracellular enzymatic mechanism(s) activated by pathological increases in intracellular Ca2+.

Ischemic White Matter Damage

Publisher Summary This chapter discusses the deleterious events triggered to induce anoxic or ischemic damage in mammalian white matter. The highly specialized architecture of myelinated fibers renders them prone to functional disruption when any of the critical components are deranged. A variety of axonal disorders is characterized by irreversible compromise of conduction through central tracts, resulting in varying degrees of clinical disability, which depends on the severity of damage and location of the affected pathways. Common examples include acute stroke, hypoxic or ischemic white-matter injury that can result in periventricular leukomalacia and cause cerebral palsy, and more chronic states such as vascular dementia from long-standing microangiopathic pathology, trauma, and a variety of demyelinating disorders such as multiple sclerosis. Together, these disorders represent a huge personal and socioeconomic burden on western populations. It is noted that a key to devising successful therapies for these disorders lies in a thorough understanding of the fundamental mechanisms of ischemic CNS injury.