Anoxia & Ischemic Injury Of Myelinated CNS Axons

Dorsal Column Axons of the rat Spinal Cord are partially protected from Anoxic injury following blockade of voltage-sensitive Na⁺ channels and the Na⁺/ Ca²⁺ exchanger.

To examine the potential contribution of voltage-gated Ca²⁺ channels to Anoxic injury of Spinal Cord Axons, we studied Axonal Conduction in rat dorsal columns in vitro following a 60-min period of Anoxia.

Glass MicroElectrodes were used to record field potentials from the Dorsal Columns following distal local surface stimulation.

Perfusion solutions containing blockers of voltage-gated Ca²⁺ channels were introduced 60 min prior to onset of Anoxia and continued until 10 min after ReOxygenation.

Pharmacological blocking agents which are relatively selective for L- (Verapamil, Diltiazem, Nifedipine) and (N-omega-ConoToxin GVIA) type Calcium channels were significantly protective against Anoxia-induced loss of conduction, as was non-specific block using divalent Cations.

Other Ca²⁺ channel blockers (Neomycin and omega-ConoToxin MVIIC) that affect multiple Ca²⁺ channel types were also NeuroProtective.

Ni2⁺, which preferentially blocks R-type Ca²⁺ channels more than T-type channels, was also protective in a dose-dependent manner.

These data suggest that the influx of Ca²⁺, through L-, N- and possibly R-type voltage-gated Ca²⁺ channels, participates in the PathoPhysiology of the Ca²⁺-mediated injury of Spinal Cord Axons that is triggered by Anoxia.

Copyright 1999 Elsevier Science B.V.

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 the synthesis of ATP by Phosphorylation of ADP.

For which energy is obtained by electron transport and which takes place in the Mitochondria during aerobic respiration (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⁺ 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 Gamma-AminoButyric Acid (GABA) 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.

Electron probe x-ray microanalysis was used to measure water content and concentrations (mmol/kg dry weight) of elements (Na, P, S, Cl, K, Ca, and Mg) in Myelinated Axons and Glial Cells of rat Optic Nerve exposed to in vitro Anoxia and ReOxygenation.

In response to Anoxia, large, medium, and small diameter fibers exhibited an early (5 min) and progressive loss of Na⁺ and K⁺ regulation which culminated (60 min) in severe depletion of respective TransMembrane gradients.

As Axoplasmic Na⁺ levels increased during Anoxic exposure, a parallel rise in Ca²⁺ content was noted.

For all Axons, mean water content decreased progressively during the initial 10 min of Anoxia and then returned toward normal values as Anoxia continued.

Analyzes of Mitochondrial areas revealed a similar pattern of elemental disruption except that Ca²⁺ concentrations rose more rapidly during Anoxia.

Following 60 min of PostAnoxia ReOxygenation, the majority of larger fibers displayed little evidence of recovery, whereas a subpopulation of Small Axons exhibited a trend toward restoration of normal elemental composition.

Glial Cells and Myelin were only modestly affected by Anoxia and subsequent ReOxygenation.

Thus, Anoxic injury of CNS Axons is associated with characteristic changes in Axoplasmic distributions of Na⁺, K⁺, and Ca²⁺.

The magnitude and temporal patterns of elemental Na⁺ and Ca⁺ disruption are consistent with reversal of Na⁺-Ca²⁺ exchange and subsequent Ca²⁺ entry (Stys et al., 1992).

During ReOxygenation, elemental deregulation continues for most CNS fibers, although a subpopulation of Small Axons appears to be capable of recovery.

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:

1. Mitochondrial swelling
2. Dissolution of Cristae
3. SubMyelinic vacuoles
4. PeriNodal Oligodendrocyte-Axon loop detachment
5. Severe CytoSkeletal damage
6. MicroTubule and NeuroFilament loss in Axoplasma

To further examine the coupling between Na⁺ influx and Ca²⁺-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 UultraStructure.

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.

White Matter of the mammalian CNS suffers irreversible injury when subjected to Anoxia/Ischemia.

However, the mechanisms of Anoxic injury in Central Myelinated Tracts are not well understood.

Although White Matter injury depends on the presence of ExtraCellular Ca²⁺, the mode of entry of Ca²⁺ into cells has not been fully characterized.

We studied the mechanisms of Anoxic injury using the in vitro rat Optic Nerve, a representative Central White Matter Tract.

Functional integrity of the Nerves was monitored ElectroPhysiologically by quantitatively measuring the area under the compound Action Potential, which recovered to 33.5 +/- 9.3% of control after a standard 60 min Anoxic insult.

Reducing Na⁺ influx through voltage-gated Na⁺ channels during Anoxia, by applying Na⁺ channel blockers (TTX, SaxiToxin) substantially improved recovery.

TTX was protective even at concentrations that had little effect on the control compound Action Potential.

Conversely, increasing Na⁺ channel permeability during Anoxia with Veratridine resulted in greater injury.

Manipulating the transmembrane Na⁺ gradient at various times before or during Anoxia greatly affected the degree of resulting injury.

Applying zero-Na⁺ solution (Choline or Li⁺ substituted) before Anoxia, significantly improved recovery; paradoxically, the same solution applied after the start of Anoxia resulted in more injury than control.

Thus, Ionic conditions that favored reversal of the normal transmembrane Na⁺ gradient during Anoxia promoted injury.

Suggesting that Ca²⁺ loading might occur via reverse operation of the Na⁺- Ca²⁺ exchanger.

Na⁺- Ca²⁺ exchanger blockers (Bepridil, Benzamil, DichloroBenzamil) significantly protected the Optic Nerve from Anoxic injury.

Together, these results suggest the following sequence of events leading to Anoxic injury in the rat Optic Nerve:

Anoxia causes rapid depletion of ATP and membrane depolarization leading to Na⁺ influx through incompletely inactivated Na⁺ channels.

The resulting rise in the IntraCellular Na⁺, coupled with membrane depolarization, causes damaging levels of Ca²⁺ to be admitted into the IntraCellular compartment, through, reverse operation of the Na⁺- Ca²⁺ exchanger.

These observations emphasize that differences in the PathoPhysiology of Gray and White Matter Anoxic injury are likely to necessitate multiple strategies for optimal CNS protection.

Axonal degeneration can be an important cause of permanent disability in Neurological Disorders in which inflammation is prominent, including Multiple Sclerosis and Guillain-Barre Syndrome.

The mechanisms responsible for the degeneration remain unclear, but it is likely that Axons succumb to factors produced at the site of inflammation, such as Nitric Oxide (NO).

We previously have shown that Axons exposed to NO in vivo can undergo degeneration, especially if the Axons are electrically active during NO exposure.

The Axons may degenerate because NO can inhibit Mitochondrial respiration, leading to IntraAxonal accumulation of Na²⁺ and Ca²⁺ Ions.

Here, we show that Axons can be protected from NO-mediated damage using low concentrations of Na²⁺ Channel Blockers, or an inhibitor of Na⁺/Ca²⁺ exchange.

Our findings suggest a new strategy for Axonal protection in an inflammatory environment, which may be effective in preventing the accumulation of permanent disability in patients with NeuroInflammatory Disorders.

Objective
To evaluate markers of Axonal Damage in CSF and Serum of patients with different subtypes of MS in relation to measures of disease progression on MRI.

Methods
In 51 patients with MS (21 Relapsing/Remitting, 20 Secondary/Progressive, 10 Primary/Progressive), levels of heavy and light NeuroFilaments (NfH and NfL) and AntiBodies to NeuroFilaments (Anti-NfL and -NfH) as well as the total ImmunoGlobulin G (IgG) were analyzed.

MRI analysis included T₂ HyperIntense, T₁ HypoIntense, and Gadolinium enhancing lesions and markers of Cerebral Atrophy (Ventricular and Parenchymal Fractions).

Results
For the total group, correlations were found between the Anti-NfL index and the Parenchymal Fraction (PF) (r = -0.51, p < 0.001), T₂ lesion load (r = 0.41, p < 0.05), Ventricular Fraction (r = 0.37, p < 0.05), and T₁ lesion load (r = 0.37, p < 0.05).

For the Anti-NfH index, a correlation was found with the PF (r = -0.39, p < 0.05). No correlations were found between the IgG index and MRI measures.

Conclusions
Intrathecal production of Anti-NfL AntiBodies may serve as a marker of tissue damage, particularly Axonal Loss, in MS.

Inflammatory DeMyelinating Neuropathies such as Guillain-Barre Syndrome (GBS) and its animal model, Experimental Autoimmune Neuritis (EAN), are typically acute monophasic diseases of the PNS.

That can leave affected individuals with permanent disability due primarily to Axonal degeneration. The mechanisms underlying the degeneration are not understood.

But, we have previously shown in vitro and in vivo that Axons can degenerate when exposed to the inflammatory mediator Nitric Oxide.

And, that Axons can be protected by application of the Sodium Channel-blocking agent, Flecainide.

Here we examine whether Flecainide administration can similarly reduce Axonal Degeneration in the periphery in animals with EAN.

EAN was induced in Lewis rats (n = 116, in three independent trials), and rats received either Flecainide (Flec) (30 mg/kg/day) or vehicle (Veh) from the onset of disease expression.

Flecainide administration significantly reduced the mean (SD) scores for Neurological deficit at both the peak of disease (Flec: 5.7 (2.7), Veh: 8.0 (3.6), P < 0.001) and at the termination of the trials 25-29 days post-inoculation (Flec: 2.2 (2.4), Veh: 4.2 (4.2), P < 0.001).

Histological examination of the Tibial Nerve of EAN animals revealed that Flecainide provided significant protection against Axonal Degeneration.

So that 80.0% of the normal number of Axons survived in Flecainide-treated rats compared with 62.8% in vehicle-treated rats (P < 0.01).

These findings may indicate a novel avenue for Axonal protection in GBS and other Inflammatory DeMyelinating Neuropathies.

We studied the magnitude and route(s) of Ca2⁺ flux from extra- to IntraCellular compartments during Anoxia in adult Rat Optic Nerve (RON), a Central White Matter Tract, using Ca2⁺ sensitive MicroElectrodes to monitor ExtraCellular [Ca2⁺] ([Ca2⁺]o).

One hour of Anoxia caused a rapid loss of the stimulus-evoked Compound Action Potential (CAP), which partially recovered following Re-Oxygenation, indicating that irreversible injury had occurred.

After an initial increase caused by ExtraCellular Space shrinkage, Anoxia produced a sustained decrease of 0.42 mM (29%) in [Ca2⁺]o.

We quantified the [Ca2⁺]o decrease as the area below baseline [Ca2⁺]o during Anoxia and used this as a qualitative index of suspected Ca2⁺ influx.

The degree of RON injury was predicted by the amount of Ca2⁺ leaving the ExtraCellular Space.

Bepridil, 0 Na⁺ artificial CerebroSpinal Fluid or TetrodoToxin reduced suspected Ca2⁺ influx during Anoxia implicating reversal of the Na⁺/Ca2⁺ exchanger as a route of Ca2⁺ influx.

Diltiazem reduced suspected Ca2⁺ influx during Anoxia, suggesting that Ca2+ influx via L-type Ca2⁺ Channels is a route of toxic Ca2⁺ influx into Axons during Anoxia. ImmunoCytoChemical staining was used to demonstrate and localize high-threshold Ca2+ Channels.

Only alpha1(C) and alpha1(D) subunits were detected, indicating that only L-type Ca2⁺ Channels were present. Double labeling with anti-Neurofilament Antibodies or Anti-Glial Fibrillary Acidic Protein AntiBodies localized L-type Ca2+ Channels to Axons and Astrocytes.

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.

And, 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 Ca2⁺-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.

Central Nervous System Myelin is a specialized structure produced by Oligodendrocytes that ensheaths Axons, allowing rapid and efficient saltatory conduction of Action Potentials.

Many disorders promote damage to and eventual loss of the Myelin Sheath, which often results in significant Neurological morbidity.

However, little is known about the fundamental mechanisms that initiate Myelin damage, with the assumption being that its fate follows that of the parent Oligodendrocyte.

Here we show that NMDA (N-Methyl-D-Aspartate) Glutamate Receptors mediate Ca2⁺ accumulation in Central Myelin in response to chemical Ischemia in vitro.

Using two-photon microscopy, we imaged fluorescence of the Ca2⁺ indicator X-rhod-1 loaded into Oligodendrocytes and the cytoplasmic compartment of the Myelin Sheath in adult rat Optic Nerves.

The AMPA (alpha-Amino-3-Hydroxy-5-Methyl-4-Isoxazole Propionic Acid)/kainate Receptor antagonist NBQX completely blocked the Ischemic Ca2⁺ increase in Oligodendroglial cell bodies, but only modestly reduced the Ca2⁺ increase in Myelin.

In contrast, the Ca2⁺ increase in Myelin was abolished by broad-spectrum NMDA Receptor antagonists (MK-801, 7-ChloroKynurenic Acid, d-AP5), but not by more selective blockers of NR2A and NR2B subunit-containing Receptors (NVP-AAM077 and Ifenprodil).

In vitro Ischemia causes ultrastructural damage to both Axon cylinders and Myelin. NMDA Receptor antagonism greatly reduced the damage to Myelin.

NR1, NR2 and NR3 subunits were detected in Myelin by ImmunoHistoChemistry and ImmunoPrecipitation, indicating that all necessary subunits are present for the formation of functional NMDA Receptors.

Our data show that the mature Myelin Sheath can respond independently to injurious stimuli.

Given that Axons are known to release Glutamate, our finding that the Ca2⁺ increase was mediated in large part by activation of Myelinic NMDA Receptors suggests a new mechanism of Axo-Myelinic signalling.

Such a mechanism may represent a potentially important therapeutic target in disorders in which DeMyelination is a prominent feature, such as Multiple Sclerosis, NeuroTrauma, infections (for example, HIV Encephalomyelopathy) and aspects of Ischemic Brain injury.

Electron probe X-ray microanalysis was used to measure water content and concentrations of elements (i.e. Na, K, Cl and Ca) in selected morphological compartments of rat Optic Nerve Myelinated Axons.

TransAxolemmal movements of Na⁺ and Ca2⁺ were modified experimentally and corresponding effects on Axon element and water compositions were determined under control conditions and following in vitro Anoxic challenge.

Also characterized were effects of modified Ion transport on Axon responses to PostAnoxia reoxygenation.

Blockade of Na⁺ entry by Tetrodotoxin (1 microM) or zero Na⁺/Li⁺-substituted perfusion reduced Anoxic increases in Axonal Na and Ca concentrations.

Incubation with zero-Ca2⁺/EGTA perfusate prevented Axoplasmic and Mitochondrial Ca accumulation during Anoxia but did not affect Na increases or K losses in these compartments.

Inhibition of Na⁺-Ca2⁺ exchange with Bepridil (30 microM) selectively prevented increases in Intra-Axonal Ca, whereas neither Nifedipine (5 microM) nor Nimodipine (5 microM) influenced the effects of Anoxia on Axonal Na, K or Ca

X-ray microanalysis also showed that prevention of Na and Ca influx during Anoxia obtunded (blunted) severe elemental deregulation normally associated with reoxygenation.

Results of the present study suggest that during Anoxia, Na⁺ enters Axons mainly through voltage-gated Na⁺ channels and that subsequent increases in Axoplasmic Na⁺ are functionally coupled to Extra-Axonal Ca2⁺ import.

Na⁺i-dependent, Ca2⁺o entry is consistent with reverse operation of the Axolemmal Na⁺-Ca⁺⁺ exchanger and we suggest this route represents a primary mechanism of Ca2⁺ influx.

Our findings also implicate a minor route of Ca2⁺ entry directly through Na⁺ channels.