Mechanisms of axonal dysfunction after spinal cord injury: with an emphasis on the role of voltage-gated potassium channels.

Dysfunction of surviving axons which traverse the site of spinal cord injury (SCI) appears to contribute to posttraumatic neurological deficits, though the underlying mechanisms remain unclear. Although demyelination of injured but surviving axons following trauma appear to be a major contributor of axonal conduction deficits, altered activity of ion channels may also play an important role. It has been theorized that exposure of K+ channels as a result of demyelination would result in a reduced safety factor of action potential propagation across the demyelinated region of the axon. This theory and electrophysiological studies using K+ channel blockers on animal nerve preparations prompted the investigation of 4-aminopyridine (4-AP), a blocker of rapidly activating voltage-gated K+ channels, as a therapeutic agent in both multiple sclerosis and spinal cord injured patients. Several preliminary clinical trials have already demonstrated therapeutic benefit of 4-AP in both multiple sclerosis and spinal cord injured patients. In this review, we shall give a comprehensive summary of the mechanisms of axonal dysfunction following SCI and how axonal dysfunction may have resulted due to specific pathological changes following trauma including the ultrastructural and molecular changes that occur to myelinated axons. The pathology of spinal cord injury is very complex and many different mechanisms may contribute to axonal conduction deficits and the associated sensory and motor loss.

Changes in axonal physiology and morphology after chronic compressive injury of the rat thoracic spinal cord.

The spinal cord is rarely transected after spinal cord injury. Dysfunction of surviving axons, which traverse the site of spinal cord injury, appears to contribute to post-traumatic neurological deficits, although the underlying mechanisms remain unclear. The subpial rim frequently contains thinly myelinated axons which appear to conduct signals abnormally, although it is uncertain whether this truly reflects maladaptive alterations in conduction properties of injured axons during the chronic phase of spinal cord injury or whether this is merely the result of the selective survival of a subpopulation of axons. In the present study, we examined the changes in axonal conduction properties after chronic clip compression injury of the rat thoracic spinal cord, using the sucrose gap technique and quantitatively examined changes in the morphological and ultrastructural features of injured axonal fibers in order to clarify these issues. Chronically injured dorsal columns had a markedly reduced compound action potential amplitude (8.3% of control) and exhibited significantly reduced excitability. Other dysfunctional conduction properties of injured axons included a slower population conduction velocity, a longer refractory period and a greater degree of high-frequency conduction block at 200 Hz. Light microscopic and ultrastructural analysis showed numerous axons with abnormally thin myelin sheaths as well as unmyelinated axons in the injured spinal cord. The ventral column showed a reduced median axonal diameter and the lateral and dorsal columns showed increased median diameters, with evidence of abnormally large swollen axons. Plots of axonal diameter versus myelination ratio showed that post-injury, dorsal column axons of all diameters had thinner myelin sheaths. Noninjured dorsal column axons had a median myelination ratio (1.56) which was within the optimal range (1.43-1.67) for axonal conduction, whereas injured dorsal column axons had a median myelination ratio (1.33) below the optimal value. These data suggest that maladaptive alterations occur postinjury to myelin sheath thickness which reduce the efficiency of axonal signal transmission.In conclusion, chronically injured dorsal column axons show physiological evidence of dysfunction and morphological changes in axonal diameter and reduced myelination ratio. These maladaptive alterations to injured axons, including decrease in myelin thickness and the appearance of axonal swellings, contribute to the decreased excitability of chronically injured axons. These results further clarify the mechanisms underlying neurological dysfunction after chronic neurotrauma and have significant implications regarding approaches to augment neural repair and regeneration.

Point mutant mice with hypersensitive alpha 4 nicotinic receptors show dopaminergic deficits and increased anxiety.

Knock-in mice were generated that harbored a leucine-to-serine mutation in the alpha4 nicotinic receptor near the gate in the channel pore. Mice with intact expression of this hypersensitive receptor display dominant neonatal lethality. These mice have a severe deficit of dopaminergic neurons in the substantia nigra, possibly because the hypersensitive receptors are continuously activated by normal extracellular choline concentrations. A strain that retains the neo selection cassette in an intron has reduced expression of the hypersensitive receptor and is viable and fertile. The viable mice display increased anxiety, poor motor learning, excessive ambulation that is eliminated by very low levels of nicotine, and a reduction of nigrostriatal dopaminergic function upon aging. These knock-in mice provide useful insights into the pathophysiology of sustained nicotinic receptor activation and may provide a model for Parkinson's disease.

Role of L- and N-type calcium channels in the pathophysiology of traumatic spinal cord white matter injury.

Recent work has suggested a potential role for voltage-gated Ca(2+) channels in the pathophysiology of anoxic central nervous system white matter injury. To examine the relevance of these findings to neurotrauma, we conducted electrophysiological studies with inorganic Ca(2+) channels blockers and L- and N-subtype-specific calcium channel antagonists in an in vitro model of spinal cord injury. Confocal immunohistochemistry was used to examine for localization of L- and N-type calcium channels in spinal cord white matter tracts. A 30-mm length of dorsal column was isolated from the spinal cord of adult rats, pinned in an in vitro recording chamber and injured with a modified clip (2g closing force) for 15s. The functional integrity of the dorsal column was monitored electrophysiologically by quantitatively measuring the compound action potential at two points with glass microelectrodes. The compound action potential decreased to 71.4+/-2.0% of control (P<0. 05) after spinal cord injury. Removal of extracellular Ca(2+) promoted significantly greater recovery of compound action potential amplitude (86.3+/-7.6% of control; P< 0.05) after injury. Partial blockade of voltage-gated Ca(2+) channels with cobalt (20 microM) or cadmium (200 microM) conferred improvement in compound action potential amplitude. Application of the L-type Ca(2+) channel blockers diltiazem (50 microM) or verapamil (90 microM), and the N-type antagonist omega-conotoxin GVIA (1 microM), significantly enhanced the recovery of compound action potential amplitude postinjury. Co-application of the L-type antagonist diltiazem with the N-type blocker omega-conotoxin GVIA showed significantly greater (P<0.05) improvement in compound action potential amplitude than application of either drug alone. Confocal immunohistochemistry with double labelling for glial fibrillary acidic protein, GalC and NF200 demonstrated L- and N-type Ca(2+) channels on astrocytes and oligodendrocytes, but not axons, in spinal cord white matter. In conclusion, the injurious effects of Ca(2+) in traumatic central nervous system white matter injury appear to be partially mediated by voltage-gated Ca(2+) channels. The presence of L- and N-type Ca(2+) channels on periaxonal astrocytes and oligodendrocytes suggests a role for these cells in post-traumatic axonal conduction failure.

Abnormal axonal physiology is associated with altered expression and distribution of Kv1.1 and Kv1.2 K+ channels after chronic spinal cord injury.

Dysfunction of surviving axons which traverse the site of spinal cord injury (SCI) has been linked to altered sensitivity to the K+ channel blocker 4-aminopyridine (4-AP) and appears to contribute to post-traumatic neurological deficits although the underlying mechanisms remain unclear. In this study, sucrose gap electrophysiology in isolated dorsal column strips, Western blotting and confocal immunofluorescence microscopy were used to identify the K+ channels associated with axonal dysfunction after chronic (6-8 weeks postinjury) clip compresssion SCI of the thoracic cord at T7 in rats. The K+ channel blockers 4-AP (200 microM, 1 mM and 10 mM) and alpha-dendrotoxin (alpha-DTX, 500 nM) resulted in a significant relative increase in the amplitude and area of compound action potentials (CAP) recorded from chronically injured dorsal column axons in comparison with control noninjured preparations. In contrast, TEA (10 mM) and CsCl (2 mM) had similar effects on injured and control spinal cord axons. Western blotting and quantitative immunofluorescence microscopy showed increased expression of Kv1.1 and Kv1.2 K+ channel proteins on spinal cord axons following injury. In addition, Kv1.1 and Kv1.2 showed a dispersed staining pattern along injured axons in contrast to a paired juxtaparanodal localization in uninjured spinal cord axons. Furthermore, labelled alpha-DTX colocalized with Kv1.1 and Kv1.2 along axons. These findings suggest a novel mechanism of axonal dysfunction after SCI whereby an increased 4-AP- and alpha-DTX-sensitive K+ conductance, mediated in part by increased Kv1.1 and Kv1.2 K+ channel expression, contributes to abnormal axonal physiology in surviving axons.

Serial recording of somatosensory and myoelectric motor evoked potentials: role in assessing functional recovery after graded spinal cord injury in the rat.

Accurate functional outcome measures are essential in assessing therapeutic interventions after experimental spinal cord injury (SCI). We examined the hypothesis that serial recording of somatosensory (SSEP) and myoelectric motor evoked potentials (mMEPs) would provide complementary information to standard methods of behavioral analysis in a rat model of SCI and would allow objective discrimination of functional recovery in sensory and motor tracts. Clip compression injury of varying severity (sham, 23 g, 34 g, 56 g) and transections were performed at T1 in adult rats. SSEPs were recorded from the right sensorimotor cortex (SMC) after stimulation of the contralateral hind paw; mMEPs were recorded from the paraspinal, quadriceps, and the tibialis anterior muscles after anodal stimulation of the SMC. The inclined plane and Tarlov techniques were used to assess clinical neurological function. All outcome measures were assessed weekly prior to and up to 6 weeks following injury. Changes in clinical neurological function as assessed by the inclined plane and Tarlov methods varied with increasing injury severity (R = -0.72 and R = -0.73, respectively). SSEP latency was strongly correlated with injury severity (R = 0.92) and with clinical behavioral scores (R = -0.93 for inclined plane). The tibialis anterior mMEP correlated significantly, though weakly, with changes in inclined plane (R = 0.49) and Tarlov scores (R = 0.41). Although the mMEPs were sensitive to the presence of SCI, these recordings did not discriminate between severities of injury. We conclude that serial recording of SSEPs but not myoelectric MEPs correlates closely with the extent and temporal course of clinical neurological recovery after graded SCI in the rat.

A new model of acute compressive spinal cord injury in vitro.

A novel in vitro method of spinal cord injury was developed to facilitate the study of cellular and molecular mechanisms underlying neural trauma. A 3-cm length of thoracic spinal cord was removed from the adult Wistar rat. A strip of dorsal column and its associated dorsal horn gray matter was excised and pinned in an in vitro recording chamber where it was constantly perfused with oxygenated Ringer's solution at either 25 degrees C or 33 degrees C. Injury was performed by compressing the dorsal column segment in vitro with a modified aneurysm clip (closing force 2.0 g) for 15 s. Microelectrode and sucrose gap recordings were generated to characterize the physiological effects of compressive injury. Longitudinal thin sections of control and injured dorsal column segments were examined by electron microscopy. At 25 degrees C, injured axons were characterized by a significant reduction in amplitude of the compound action potential (CAP) to 76.9 +/- 2.4% (P < 0.0005) and an increase in response latency to 112.5 +/- 2.5% (P <0.005). At 33 degrees C, the effects of injury on the CAP amplitude were accentuated (P< 0.0001). With the K+ channel blocker, 4-AP (1 mM), there was broadening of the CAP of injured axons and a delay in repolarization of the axonal resting membrane potential, suggesting myelin disruption with exposure of paranodal K+ channels. Ultrastructurally, injured dorsal column segments showed considerable axonal and myelin pathology including splaying of the myelin sheath and vesicular degeneration.

Changes in pharmacological sensitivity of the spinal cord to potassium channel blockers following acute spinal cord injury.

In this investigation we studied changes in the pharmacological sensitivity of dorsal column white matter to a variety of K+ channel blockers, including 4-aminopyridine (4-AP), following acute spinal cord injury (SCI) in vitro using a modified aneurysm clip. Compound action potentials (CAPs) were recorded extracellularly with microelectrodes and by the sucrose gap recording technique. With acute trauma, injured axons showed significantly enhanced sensitivity to 4-AP in comparison to uninjured controls as early as 10 min following injury. Microelectrode derived field potential recordings showed a significantly greater increase in a delayed positive component (P2) of the CAP at both 1 and 5 mM 4-AP in injured as compared to noninjured axons. Sucrose gap recordings showed an increase in CAP area and amplitude of injured axons with 1 mM 4-AP at 22 degrees C. The relative improvement in CAP area and amplitude with 4-AP was even more pronounced (P < 0.05) at higher temperatures (37 degrees C). As shown by sucrose gap, 4-AP also caused a delay in repolarization of the CAP and depolarization of the resting membrane potential of acutely injured axons. TEA (0.1 mM and 10 mM), when infused alone and with CsCl (10 mM), produced similar effects on injured and intact axons. In conclusion, the results of this study show an altered sensitivity of the spinal cord to 4-AP following acute SCI. In contrast, TEA and CsCl exhibit no difference in their effects on low frequency axonal conduction between injured and noninjured axons. The data suggest that acute traumatic myelin disruption following SCI causes axonal dysfunction partly due to abnormal activation of 4-AP-sensitive 'fast' K+ channels.

Assessment of axonal dysfunction in an in vitro model of acute compressive injury to adult rat spinal cord axons.

An in vitro model of spinal cord injury was developed to study the pathophysiology of posttraumatic axonal dysfunction. A 25 mm length of thoracic spinal cord was removed from the adult male rat (n = 27). A dorsal column segment was isolated and pinned in a recording chamber and superfused with oxygenated (95% O2/5% CO2) Ringer. The cord was stimulated with a bipolar electrode, while two point responses were recorded extracellularly. Injury was accomplished by compression with a modified aneurysm clip which applied a 2 g force for 15 s. With injury the compound action potential (CAP) amplitude decreased to 53.7 +/- 5.4% (P < 0.001), while the latency increased to 115.6 +/- 3.1% (P < 0.0025) of control values. The absolute refractory period increased with injury from 1.7 +/- 0.1 ms to 2.1 +/- 0.1 ms (P < 0.05). The infusion of 5 mM 4-aminopyridine (4-AP), a blocker of voltage-sensitive 'fast' K channels confined to internodal regions, resulted in broadening of the CAP of injured axons to 114.9 +/- 3.1% of control (P < 0.05). Ultrastructural analysis of the injured dorsal column segments revealed marked axonal and myelin pathology, including considerable myelin disruption. In conclusion, we have developed and characterized an in vitro model of mammalian spinal cord injury which simulates many of the features of in vivo trauma. Injured axons display characteristic changes in physiological function including a shift in refractory period and high frequency conduction failure. The ultrastructural data and response of injured axons to 4-AP suggest that myelin disruption with exposure of 'fast' K+ channels contributes to posttraumatic axonal dysfunction.

EEG rhythms of the sensorimotor region during hand movements.

The aim of this study was to determine what motor behaviors or conditions were associated with an increased occurrence of beta activity in the sensorimotor region of human subjects. EEG recordings were obtained from 8 electrodes symmetrically arranged around C3, with 3 cm interelectrode spacing. The electrode montage allowed calculation of the Laplacian operator at two positions, C3r and C3c, overlying the hand area of the motor cortex and of the somatosensory cortex, respectively. A variety of tasks involving right-hand movements of different levels of complexity, attention and preparation were performed. The corresponding EEG power spectra were subsequently computed for frequencies between 7 and 50 Hz. Repetitive hand movements alone (either drawing circles or writing one's signature) did not result in significantly increased beta activity in the sensorimotor region compared to relaxed conditions. However, both motor preparations and focused attention, whether movements were performed or not, were associated with an increase of high frequency beta activity (30-50 Hz) in the sensorimotor region. Therefore, the facilitatory effect of attention and motor preparation and not the functional activation of the sensorimotor cortex by hand movements was associated with an increase in synchronized fast beta activity.