Changes in human sensory axonal excitability induced by an ischaemic insult

Clinical Neurophysiology 119 (2008) 2054–2063 www.elsevier.com/locate/clinph

Changes in human sensory axonal excitability induced by an ischaemic insult S.E. Han, Robert A. Boland, Arun V. Krishnan, Steve Vucic, Cindy S.-Y. Lin, Matthew C. Kiernan* Prince of Wales Clinical School & Prince of Wales Medical Research Institute, The University of New South Wales, Barker St., Randwick, NSW 2031, Australia

See Editorial, pages 1945–1946

Abstract Objective: To identify the sensitivity and the patterns of change in sensory excitability that accompany an ischaemic insult. Methods: Sensory excitability studies were undertaken in 10 subjects (mean age 36), and monitored throughout ischaemia and following its release. Ischaemia was induced using a sphygmomanometer inflated to 200 mm/Hg above the elbow. Results: During ischaemia there was reduction in threshold (P < 0.001), associated with a significant increase in refractoriness (106 ± 6.62%; P < 0.001), reduction in superexcitability (30.4 ± 0.42%; P < 0.001), and ‘fanning in’ of threshold electrotonus, all indicative of axonal depolarization. Paraesthesiae were minimal during ischaemia, but became severe on release, at which stage numbness was prominent. Late subexcitability in sensory axons was completely abolished by a relatively shorter period of ischaemia than previously observed in motor axons. Conclusions: The present study has successfully developed a template for changes in sensory axonal excitability parameters that accompany ischaemia, and established their relative sensitivity to an ischaemic change. Further, it is proposed that the inhibition of the Na+/K+ pump, in the setting of increased persistent Na+ currents and abolition of late subexcitability may underlie the development of paraesthesiae during ischaemia. Significance: Changes in axonal excitability induced by ischaemia may serve as a tool to identify and interpret changes in axonal membrane potential recorded in neuropathic patients. Ó 2008 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved. Keywords: Ischaemia; Paraesthesiae; Nerve excitability

1. Introduction Despite the fact that neuropathies typically affect sensory and motor pathways, the clinical manifestation may suggest more predominant involvement of either sensory

Abbreviations: CSAP, compound sensory action potential; Em, membrane potential; Ek, potassium equilibrium potential; I/V, current–voltage; IKs, Nodal slow K+ currents; KCNQ, K – potassium, CN – channel, Q – long QT designation; K+, potassium ion; Na+, sodium ion; SDTC, strength–duration time constant. * Corresponding author. Tel.: +61 2 9382 2422; fax: +61 2 9382 2437. E-mail address: [email protected] (M.C. Kiernan).

or motor axons. Furthermore, pathological investigation may yield no evidence of selective involvement, perhaps suggesting that changes in axonal function, rather than structure, underlie the differences in clinical presentations. In support of such a hypothesis, a number of differences in axonal behaviour between sensory and motor fibres have been identified, related to differences in axonal membrane ion channel function (Bostock and Rothwell, 1997; Kiernan et al., 1996, 2001b, 2004; Lin et al., 2002b; Mogyoros et al., 1997a,b; Vagg et al., 1998). Experimental models of ischaemia have evolved from initial studies that investigated the effects of inflation of a blood pressure cuff around the arm, with the subsequent

1388-2457/$34.00 Ó 2008 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.clinph.2008.04.295

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development of post-ischaemic paraesthesiae in the digits and palm (Lewis et al., 1931). It is now well understood that an ischaemic insult to human peripheral nerve inhibits energy-dependent processes, particularly the electrogenic Na+/K+ pump. This process induces depolarization of sensory and motor axons, producing first an increase, followed by a reduction in nerve excitability (Bostock et al., 1991a,b, 1994; Kiernan et al., 1999; Lin et al., 2001, 2002a,b). A previous study investigated the effects of ischaemia on multiple parameters of excitability for motor axons and defined the relative sensitivity of excitability parameters to changes in membrane potential induced by polarizing currents (Kiernan and Bostock, 2000). That study established a template which has been subsequently employed to interpret the pathophysiology of different neuropathic processes (Kanai et al., 2006; Kiernan et al., 2002a, 2001a, 2005, 2001b, 2002b; Krishnan et al., 2005a,b, 2006, 2002; Nodera et al., 2004; Sung et al., 2004; Z’Graggen et al., 2006). Importantly, that study also established that the effects of ischaemia could be distinguished from pure changes in membrane potential alone, as induced by the application of polarizing currents. To expand the earlier studies limited to motor axons, the present study was undertaken to establish the patterns of change of sensory excitability induced by ischaemia. The study also investigated changes in sensory nerve excitability parameters in relation to the development of neuropathic symptoms, both ‘‘positive” (paraesthesiae) and ‘‘negative” (numbness), to explore whether peripheral changes in excitability were the critical factors underlying symptom generation. 2. Methods Two series of experiments were carried out on 10 normal subjects (aged 24–44 years; mean 36 years; 7 male 3 female). Studies were approved by the South East Sydney Area Health Service Human Research Ethics Committee (Eastern Section) and the Committee on Experimental Procedure Involving Human subjects, University of New South Wales. Informed consent was provided by each subject and studies were undertaken in accordance with the Declaration of Helsinki. The median nerve was stimulated electrically at the wrist (Fig. 1A) through non-polarisable surface electrodes (Unilect Long-Term, Unomedical Ltd., Great Britain). Stimulation was delivered by a purpose built isolated linear bipolar constant current stimulator (maximal output ±50 mA). Reference electrodes were placed 10 cm proximal to the cathode over the lateral radius. Compound sensory action potentials (CSAP) were recorded from digit II using ring electrodes. The response was amplified (Medelec Ltd., Woking, Surrey, England), digitized with an analogue/digital board (DT2812, Data Translation Inc., Marlboro, Mass., USA.) and imported to a personal computer. Stimulation and recording protocols were determined by QTRAC (version 5.2a, copyright Institute of Neurology, London). Skin temperature was taken at the site of stimu-

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Median nerve stimulation site

Recording site

200mmHg

Ch1

Ch2

Ch3

Supramaximal stimulus

Unconditioned stimulus (0.2 ms) to produce CSAP 30% of Ch1

Unconditioned stimulus (1 ms) to produce identical CSAP to Ch2

Ch4

Refractoriness 2.5 ms

Ch5

Superexcitability 7 ms

Fig. 1. (A) Experimental setup. Excitability studies were undertaken by stimulating the median nerve at the wrist and recording compound sensory action potential (CSAP) from the second digit. During ischaemia, the sphygmomanometer cuff was inflated to 200 mmHg and secured with tape. (B) Configuration of stimulus channels. Vertical arrows in channel 2 to channel 5 indicate threshold tracking of test stimulus aiming to elicit 30% of maximal response measured in channel 1.

lation and monitored throughout each study and was maintained above 32 °C (mean temperature 32.8 °C). Ischaemia was applied using a sphygmomanometer cuff wrapped around the right upper arm with the pressure level maintained above 200 mmHg. This pressure was sufficient to produce ischaemia, as each subject’s systolic blood pressure was below 150 mm/Hg. In the first series of studies, nerve excitability parameters were monitored continuously. Different channels in QTRAC were used to record nerve excitability parameters (Fig. 1B) as follows: channel 1 delivered supramaximal stimulus of 0.2 ms duration and monitored the maximal compound sensory action potential (CSAP) response; channels 2 and 3 were used to track 30% of the maximal response using short (0.2 ms) and long (1 ms) duration stimuli; channel 4 monitored refractoriness, using a supramaximal conditioning stimulus 2.5 ms before the test stimulus; channel 5 measured superexcitability, using a test stimulus delivered 7 ms after a supramaximal conditioning stimulus. The ratio between stimuli from channels 2 and 3 was used to calculate the strength–duration time constant

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using Weiss’s formula (Bostock, 1983; Mogyoros et al., 1996; Weiss, 1901). Each excitability measurement was observed at 1 Hz and the automated program rotated sequentially through the five channels. Measurements were continuously taken during a 4 min control period at the beginning of each experiment, and subsequently during 21 min of applied ischaemia, and for 30 min of recovery following the release of ischaemia. Subjects were seated in a chair and it was ensured that the subject’s initial posture was comfortable and that they remained still throughout the experiment. In a second series of experiments, which was carried out on a separate occasion (at least 3 weeks after the completion of the first series of studies), an automated sensory nerve excitability protocol (TRONDXS) was employed to measure multiple nerve excitability parameters (Kiernan et al., 2000; Kiernan et al., 2001b). Stimulus-response curves were generated separately for current pulses of 0.1 and 0.5 ms durations, by increasing the stimuli in 6% steps until maximal CSAPs were reached. CSAP was measured from negative to positive peak after the baseline subtraction. Threshold electrotonus measured changes in threshold (the current required to generate the target response) induced by a prolonged subthreshold current, which caused electrotonic changes of membrane potential of the nodal and internodal axolemma. The changes in membrane potential were induced by 100 ms polarizing currents, set to +40% (depolarizing) and 40% (hyperpolarizing) of the control threshold current (current evoking 40% of maximal CSAP). The resultant changes in threshold were measured using a test stimulus of 0.5 ms duration at various time intervals before, during and after subthreshold polarizing currents. A current-threshold relationship (I/V), an indicator of the rectifying properties of nodal and internodal axolemma, was then obtained by tracking changes of 0.5 ms test stimuli at the end of 200 ms subthreshold polarizing currents which were varied from +50% to 100% of the control threshold in 10% steps. Finally, the recovery cycle of excitability was recorded in each subject. The recovery cycle consisted of the absolute and relative refractory periods, superexcitability and late subexcitability, and was recorded by tracking the changes in threshold of 0.5 ms duration test stimuli as the conditioning–test intervals increased from 2 to 200 ms. A complete set of the above automated protocol was acquired during baseline, and after 5 min of ischaemia. Three series of recordings were then sequentially acquired during the post-ischaemic period. In total, the experimental duration again comprised a baseline period (15 min), followed by an application of 21 min of upper limb ischaemia, and the 45 min of recovery, with the five sequential TRONDXS protocols lasting 10–15 min each. Throughout both series of experimental protocols, subjects were asked to rate the intensities of numbness and paraesthesiae every 30 s using a 0–10 visual analogue scale,

with 0 reflecting no symptoms and 10 being unbearable paraesthesiae and severe numbness (Levine et al., 1993). 2.1. Analysis All results are expressed as mean ± standard error of the mean. Threshold and peak CSAP results were normalised to the mean of the pre-ischaemic values. Paired t-tests were used for single comparisons of excitability parameters with a significance level of P < 0.05 considered significant. Comparison of changes in late subexcitability in sensory axons from the present study was undertaken using data obtained from healthy motor axons, published previously (Kiernan and Bostock, 2000). Motor studies were undertaken on 4 healthy subjects (aged 24–54 years). The methodology used to record the recovery cycle of excitability of motor axons in the previous study (Kiernan and Bostock, 2000) was similar to that from the present study, the only difference being that a 1 ms duration test stimuli was utilized for motor axons, compared to 0.5 ms for sensory axons. As in the present series, the previous study also measured excitability before, during (at 5 min) and after the release of ischaemia. 3. Results A complete sequence of recordings was obtained from all 10 subjects, with an experimental duration of 58.3 ± 1.1 min in the first series, and 79.7 ± 1.2 min in the second. Prior to the application of the ischaemic insult, excitability parameters were recorded for cutaneous afferents at rest and established a mean refractoriness of 14.9 ± 3.1% and superexcitability of 15.3 ± 1.5%, both within previously established normative values (Kiernan et al., 2001b). In addition, the CSAP amplitude of 24.8 ± 3.4 lV and the latency of 2.6 ± 0.1 ms, were well within previously published control values for these conventional nerve conduction parameters (Burke et al., 1974), confirming normal peripheral nerve function in this cohort of clinical subjects. 3.1. Threshold changes during ischaemia Threshold decreased in all subjects during ischaemia, consistent with ischaemic depolarization due to inhibition of the electrogenic Na+/K+ pump. Threshold for the 0.2 ms-duration stimulus decreased by 25.8 ± 0.94% (P < 0.001) compared with 33.0 ± 1.19% (P < 0.001) for 1 ms-duration stimulus (Fig. 2A). This threshold decrease reached a peak after 9.5 min of ischaemia. These changes in threshold were associated with a mean reduction of 25.1 ± 4.94% (P < 0.005) in CSAP amplitude during ischaemia (Fig. 2B), and one subject developed conduction block (CSAP reduction 54.4%). These reductions in threshold and peak amplitude were associated with an increase in

Normalised Threshold

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1.2 1.1 1 0.9 0.2ms 1ms

0.8 0.7 0.6

Normalised CSAP

1.2 1.1 1 0.9 0.8 0.7 0.6

Latency (ms)

3.4 3.2 3 2.8 2.6 2.4 0

5

10

15

20

25

30

35

40

45

50

Elapsed time (min) Fig. 2. Threshold and peak response (n = 10) before, during, and after 21 min of upper arm ischaemia (bold horizontal bar indicates period of ischaemia). (A) Normalised threshold for stimuli of 0.2 ms duration (filled circles) and 1 ms duration (open square). Thresholds were normalised to pre-ischaemic baseline values. (B) Normalised peak compound sensory action potential (CSAP) amplitude following supramaximal stimulation (0.2 ms duration). Peak CSAP amplitude was normalised to pre-ischaemic baseline values. (C) Mean changes in latency of the compound sensory action potential during and after ischaemia.

latency from 2.6 ± 0.1 ms to 3.13 ± 0.1 ms (P < 0.001; Fig. 2C). 3.2. Changes in sensory excitability measures during ischaemia The reduction in peak response CSAP and the increase in latency were paralleled by an increase in the refractoriness, reflecting greater inactivation of voltage-gated transient Na+ channels, as occurs with membrane depolarization (refractoriness increased from 14.9 ± 3.08% to 120 ± 7.85%; P < 0.001; Fig. 3A). Strength–duration time constant (SDTC) is a measure of the rate at which the threshold current for a target potential decreases as stimulus duration increases, and is dependent on persistent Na+ conductances. [While local factors including nerve geometry may influence the absolute value, SDTC remains a relatively sensitive parameter to monitor longitudinal changes in nerve excitability (Kiernan and Bostock, 2000; Mogyoros et al., 1997a)]. During ischaemia, there was an

overall increase of 36.6 ± 4.71% in SDTC (Fig. 3B, P < 0.001) reflecting up-regulation of persistent Na+ conductances. Superexcitability decreased during ischaemia, consistent with ischaemic depolarization (Fig. 3C). In fact, superexcitability completely disappeared during ischaemia, to be overwhelmed by refractoriness. The peak reduction of superexcitability during ischaemia was 30.4 ± 0.42% (P < 0.001). 3.3. Post-ischaemic changes in excitability Following the release of ischaemia, a rebound increase in the activity of the axonal Na+/K+ pump resulted in the development of axonal hyperpolarization. Threshold for 0.2 ms-duration stimulus increased by 25.3 ± 3.36% (from the pre-ischaemic value; P < 0.05) and by 29.1 ± 3.32% (P < 0.05) for 1 ms-duration stimulus (Fig. 2A). These maximal increases were reached within 3 min of the release of ischaemia in every individual. This

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Refractoriness (*100%)

A

Normalised SDTC

B

1.4 1.2 1 0.8 0.6 0.4 0.2 0 1.4 1.3 1.2 1.1 1 0.9 0.8

Superexcitability (*100%)

C 0.2 0.1 0 -0.1 -0.2 -0.3 0

5

10

15

20 25 30 35 Elapsed time (min)

40

45

50

Fig. 3. Excitability parameters recorded from median nerve (n = 10) before, during, and after 21 min of upper arm ischaemia (bold horizontal bar indicates period of ischaemia). (A) Refractoriness expressed as the percentage change in threshold at a conditioning-test interval of 2.5 ms. (B) Strength duration time constant (SDTC), calculated from test durations of 0.2 and 1 ms duration. (C) Superexcitability expressed as the percentage change in threshold at a conditioning-test interval of 7 ms.

increase in threshold was accompanied by recovery in CSAP amplitude and a reduction in latency, both approaching their pre-ischaemic values. Associated with these changes in threshold, amplitude and latency, refractoriness declined to 23.6 ± 4.97% (P < 0.001) within 4 min of the release of ischaemia, and ultimately completely recovered to pre-ischaemic levels after 21 min of recovery. Superexcitability returned, on average 2.1 ± 0.4 min after the release of ischaemia. Later, there was a maximal post-ischaemic increase in superexcitability reaching 25.9 ± 2.5% (P < 0.001), providing further evidence for post-ischaemic axonal hyperpolarization. 3.4. Changes in multiple excitability measures using an automated protocol To better clarify the nature of the excitability changes induced by ischaemia, a second series of readings was undertaken that measured multiple parameters using an

automated protocol. Quantitatively similar (almost identical) changes were recorded in threshold, refractoriness and superexcitability to those obtained in the first series of ischaemia studies. Overall, it was evident that during ischaemia, marked and distinctive changes occurred in all measures of axonal excitability – changes consistent with axonal depolarization (Fig. 4). The slope of the currentthreshold relationship reflecting the rectifying properties of the axon, both nodal and internodal, became steeper, indicating reduced activation of inward rectification (Fig. 4A). There was a ‘‘fanning in” of threshold electrotonus curves due to changes in activation of nodal slow K+ channels and inactivation of Na+ channels. And finally, recording of the recovery cycle demonstrated a significant increase in the refractoriness at 2.5 ms (from 14.4 ± 4.06% at rest to 197.8 ± 17.2% during ischaemia; P < 0.001) as membrane depolarization inactivated transient Na+ channels at the expense of superexcitability (Fig. 4B). Subexcitability also substantially decreased from 9.44 ± 2.05% to 2.85 ± 0.63% (P < 0.005). Taken together,

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Threshold change (%)

Current (% threshold)

100 0

PI

C

I

I

0

C PI

-100 -500 0 Threshold reduction (%)

10 100 Interstimulus interval (ms)

Fig. 4. The effects of ischaemia on nerve excitability parameters, n = 10 (C, control; I, 5 min ischaemia; PI, post ischaemic recovery phase). (A) Current– threshold relationship. (B) Recovery cycle.

these changes were those expected from axonal depolarization (Kiernan and Bostock, 2000). In turn, this axonal depolarization triggered voltage-dependent regenerative Na+ conductances, leading to spontaneous activity as indicated by mild short-lasting paraesthesiae in subjects (see following section). In contrast, the release of ischaemia reversed the changes in excitability described above, such that the axon became hyperpolarized: the stimulus-response curve shifted to the right; the current-threshold relationship became less steep (Fig. 4A); threshold electrotonus curves ‘fanned out”; and the relative refractory period almost disappeared to be replaced by enormous superexcitability (Fig. 4B). This post-ischaemic axonal hyperpolarization is induced by rebound overactivity of the Na+/K+ pump attempting to normalise the ionic gradients that have developed during the period of ischaemia when the pump was inhibited.

mum (mean value of 0.8/10) and 7 subjects reported no paraesthesiae towards the end of ischaemic period. Upon the release of ischaemia, paraesthesiae sharply increased to a peak level of 8.4/10. Perhaps somewhat paradoxically, paraesthesiae became most intense when axons became hyperpolarized with the release of ischaemia. In contrast to paraesthesiae, numbness increased steadily throughout the ischaemic phase, with the pattern of increase in numbness closely matching the pattern of change in refractoriness (Figs. 3A and 5). Numbness reached a peak of 7.3/10 by the end of ischaemia (Fig. 5). The time taken for numbness to resolve after the release of ischaemia varied between individuals, and the overall variation, ranging from 1 to 24 min across subjects, was larger than that observed for paraesthesiae. 3.6. Sensitivity of excitability parameters to ischaemia

3.5. Symptom generation during & after ischaemia Paraesthesiae experienced during the period of ischaemia were mild (during the first 4 min mean peak of 4.1/ 10; Fig. 5), and likely reflect alterations in persistent Na+ conductances, as reflected by the increase in SDTC (Fig. 3B). Paraesthesiae subsequently declined to a mini-

In order to investigate the relative sensitivity of excitability to ischaemia, changes in each excitability parameters were plotted against changes in threshold, taken to reflect membrane potential (Kiernan and Bostock, 2000). Linear regression analysis confirmed that changes in excitability parameters during and after ischaemia were closely

Sensation Scale (0-10)

10 8

numbness

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paresthesia

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Fig. 5. Sensory symptomatic rating (0–10) of 10 subjects before, during and after 21 min ischaemia (bold horizontal bar indicates period of ischaemia) for numbness (filled circles) and paraesthesiae (open triangles).

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Table 1 Sensitivity of various excitability parameters to 10% threshold reduction during ischaemic depolarization Y intercept (a) Rheobase (mA) S-R slope Peak CSAP (uV) SDTC (ms) Rest I/V slope Min I/V slope Refractoriness (%) Superexcitability (%) Subexcitability (%) TEd (10–20 ms) TEh (10–20 ms) TEd (90–100 ms) TEh (90–100 ms)

Slope (b)

1.12 1.70 6.36 0.71 3.69 1.44 348.82 79.77 2.14 25.57 39.21 8.51 4.85

0.78 0.17 7.50 0.07 0.43 0.18 48.07 14.64 1.68 4.35 1.31 5.69 9.43

Sensitivity(10%) [(Y10

Yr)/Yr]

0.13 0.04 0.12 0.14 0.30 0.44 2.05 0.52 0.12 0.06 0.03 0.08 0.09

Correlation coefficient (r)

Significance (P)

0.95 0.45 0.35 0.72 0.57 0.65 0.57 0.54 0.36 0.44 0.12 0.61 0.40

0.001 0.045 0.129 0.001 0.008 0.002 0.018 0.017 0.121 0.053 0.61 0.004 0.082

In the equation Y = a + bX,X represents threshold as an indirect marker of resting membrane potential; Y represents each excitability parameter. Yr represents the baseline value of the excitability parameter, while Y10 represents each excitability value when threshold is reduced by 10%. Each correlation coefficient was estimated from two values (pre and during ischaemia) for ten subjects. SDTC, strength–duration time constant; S-R, stimulus response; I/V, current-threshold relationship; TEh, late hyperpolarizing threshold electrotonus; TEd, depolarizing threshold electrotonus.

linked with alterations in membrane potential (Table 1), as indirectly related by current threshold. Comparison of the effects of ischaemia on different excitability parameters indicated that refractoriness was the most sensitive parameter, where the sensitivity indicates fractional percentage changes in each excitability parameter relative to their resting values per 10% reduction of threshold. 3.7. Changes in late subexcitability for sensory and motor axons during ischaemia Comparison of the late subexcitable phase of sensory and motor axons at rest, confirmed that sensory axons had less late subexcitability than motor axons (Fig. 6A), consistent with the previous studies (Kiernan and Bostock, 2000; Kiernan et al., 1996). Furthermore, after 5 min of ischaemia, late subexcitability was completely abolished in sensory axons, whereas in motor axons the reduction was relatively minor (Fig. 6B).

The present study has successfully demonstrated the changes that occur in multiple parameters of axonal excitability of cutaneous afferents during an ischaemic insult. The results have confirmed that these changes were consistent with axonal membrane depolarization during ischaemia, and hyperpolarization after the release of an ischaemic insult. Taken in total, changes in sensory axonal excitability as documented in the present study, and their relative sensitivity to ischaemic change, may be usefully incorporated as a template for the interpretation of changes in axonal resting membrane potential recorded from patients with focal neuropathies. 4.1. Excitability as an index of ischaemic change In the present study, the excitability parameter which showed the highest sensitivity to ischaemia, and thereby

100

Motor Sensory Late subexcitability

0

10 100 Interstimulus interval (ms)

Threshold change (%)

Threshold change (%)

100

4. Discussion

Motor Sensory Late subexcitability

0

10 100 Interstimulus interval (ms)

Fig. 6. Comparison of recovery cycles of excitability measured from sensory and motor axons during ischaemia: (A) before ischaemia and (B) after 5 min of ischaemia in sensory (open circles) and motor axons. Data for motor ischaemia are re-plotted from the original study (Kiernan and Bostock, 2000). It can be observed that compared to control measures (A) late subexcitability is completely abolished in sensory axons, whilst remaining relatively preserved in motor axons, despite clear changes in refractoriness and superexcitability.

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to changes in membrane potential, was refractoriness with a calculated sensitivity of 2.05 (i.e. 205% increase per 10% threshold drop). The next most sensitive parameter to ischaemic depolarization was superexcitability with a sensitivity of 52%. Resting I/V slope and minimum I/V slope also showed relatively high sensitivities (30% and 44% increase per 10% threshold drop). Rheobase, on the other hand, could be regarded as the most accurate single parameter for detecting small changes in membrane potential during ischaemia, given its strong correlation with ischaemic depolarization (r = 0.95). The comparative sensitivities of different excitability parameters to ischaemia shown in Table 1 are similar to the voltage-dependencies reported previously for human cutaneous afferents (Burke et al., 1998). In particular, refractoriness and superexcitability were very sensitive to changes induced by depolarizing afferents, as also established by the present study for depolarization induced by ischaemia. Although, as a single excitability parameter, rheobase could serve accurately as an index of ischaemic membrane depolarization in sensory axons, small changes in membrane potential (e.g. < 10% of normal) may be harder to identify because of the relatively lower sensitivity of this parameter. Therefore, a better index of ischaemic membrane depolarization would include additional excitability parameters that demonstrated higher sensitivity to ischaemic depolarization such as refractoriness, resting and minimum I/V slopes. The value of the multiple excitability protocol used in the present study is that information from parameters that depend on membrane potential and nodal Na+ channels (e.g. refractoriness) can be collected with information from parameters that depend more on membrane potential and internodal K+ channels (e.g. resting I/V slope). As observed in a previous study of motor axons, the best index of axonal membrane potential that can be obtained noninvasively is perhaps likely to be a combination of different excitability parameters. 4.2. Sensitivity of late subexcitability to ischaemia Ischaemia is associated with complex changes in axonal excitability. Previous studies of human motor axons demonstrated that late subexcitability disappeared during ischaemia but not with applied membrane depolarization (Kiernan and Bostock, 2000). It was suggested that nodal slow K+ currents (IKs), which are largely responsible for late subexcitability, are determined by the electrochemical gradient of K+, and reflect a balance between the membrane potential (Em) and the potassium equilibrium potential (Ek). With ischaemic depolarization, extracellular K+ accumulates (Bostock et al., 1991b; Bostock and Grafe, 1985), and ultimately late subexcitability disappeared in motor axons with prolonged ischaemia (Kiernan and Bostock, 2000). A more detailed explanation of ischaemia-induced excitability changes may also invoke a contri-

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bution from KCNQ2 (a potassium channel proposed to mediate the slow nodal potassium current, IKs) particularly in relation to the development of symptoms reflecting hyperexcitability such as paraesthesiae (Schwarz et al., 2006). In sensory axons from the present series, subexcitability subsided following a much shorter duration of ischaemia when compared to motor axons, and was abolished at 5 min whereas in the previous study on motor axons such observation was only made after at least 15 min of ischaemia (Fig. 6); (Kiernan and Bostock, 2000). These differences in late subexcitability between sensory and motor axons may in turn influence symptom generation. It is known for instance that human sensory axons express a greater proportion of non-inactivating or persistent Na+ conductance when compared to motor axons (Bostock and Rothwell, 1997). This persistent Na+ conductance appears to be active at resting membrane potential, producing a persistent inward movement of Na+ ions. Activation of this conductance decreases with hyperpolarization but increases further with depolarization (Bostock and Rothwell, 1997). Therefore, the inward leak of Na+ ions present at resting membrane potential would increase as it produced further depolarization, a regenerative process likely to result in impulse initiation. That this does not occur indicates that there must be some counteractive process at work. One suggestion is that this counteraction is provided by the electrogenic Na+/K+ pump, supported by the finding that inhibition of the pump results in axonal depolarization (Kaji and Sumner, 1989). This implies that the Na+/K+ pump provides a hyperpolarizing influence at resting membrane potential. Given the duration of ischaemia used in the present series of studies, it would seem unlikely that the Na+/Ca+ exchanger would contribute to these processes (Steffensen et al., 1997; Tatsumi and Katayama, 1995). Consequently, it would be expected that were the Na+/K+ pump to be inhibited in some way, then sensory axons would be more likely to develop spontaneous activity than motor axons, given the greater expression of persistent Na+ channels in sensory axons. Furthermore, the abolition of late subexcitability after relatively brief periods of ischaemia will only serve to promote the generation of spontaneous activity, particularly that which occurs in bursts. 4.3. Clinical implications Clinically, the mechanisms of spontaneous activity that underlie symptoms such as paraesthesiae are important because they may reflect the mechanisms operating in many disease processes. Many nerve lesions produce focal compression and ischaemia, or as is the case in diabetic neuropathy, have generalized ischaemic changes that may be accompanied by reduction in late subexcitability (Krishnan and Kiernan, 2005). Inhibition of the Na+/K+ pump, in the setting of increased persistent Na+ conductances and abolition of late subexcitability as documented by the present study, is likely to underlie the greater tendency

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for subjects to experience paraesthesiae (rather than fasciculation) during ischaemia, a manoeuvre that affects pump function. Similarly, biophysical differences may also contribute to the greater incidence of paraesthesiae in peripheral nerve disorders, rather than fasciculation or myokymia. In terms of the unexpected sensitivity of late subexcitability in sensory axons during ischaemia when compared to motor axons, it remains possible that ischaemia results in greater extracellular K+ accumulation in sensory axons in the periaxonal space. Alternatively, the present study again raises the possibility that sensory axons may have a greater dependence on Na+/K+ pump activity than motor axons (Kiernan et al., 2004; Lin et al., 2002b). Such dependence may underlie the difference in susceptibility to extracellular K+ accumulation in sensory axons, and hence the more prominent changes recorded in late subexcitability. Biophysical differences between sensory and motor axons, particularly the relatively greater reduction in late subexcitability in sensory axons during ischaemia, may also predispose sensory axons to the development of repetitive or continuous impulse generation. Acknowledgements The support of the Prince of Wales Clinical School Postgraduate Research Scholarship, the National Health and Medical Research Council of Australia (Project grant 400938) and Medical Advances without Animals Doctoral Research Scholarship is gratefully acknowledged. S.H. was awarded the Tow Prize of the Coast Medical Association for the presentation of these studies. The authors thank Professor Hugh Bostock for useful discussions. References Bostock H. The strength–duration relationship for excitation of myelinated nerve: computed dependence on membrane parameters. J Physiol 1983;341:59–74. Bostock H, Baker M, Grafe P, Reid G. Changes in excitability and accommodation of human motor axons following brief periods of ischaemia. J Physiol 1991a;441:513–35. Bostock H, Baker M, Reid G. Changes in excitability of human motor axons underlying post-ischaemic fasciculations: evidence for two stable states. J Physiol 1991b;441:537–57. Bostock H, Burke D, Hales JP. Differences in behaviour of sensory and motor axons following release of ischaemia. Brain 1994;117:225–34. Bostock H, Grafe P. Activity-dependent excitability changes in normal and demyelinated rat spinal root axons. J Physiol 1985;365:239–57. Bostock H, Rothwell JC. Latent addition in motor and sensory fibres of human peripheral nerve. J Physiol 1997;498:277–94. Burke D, Mogyoros I, Vagg R, Kiernan MC. Quantitative description of the voltage dependence of axonal excitability in human cutaneous afferents. Brain 1998;121:1975–83. Burke D, Skuse NF, Lethlean AK. Sensory conduction of the sural nerve in polyneuropathy. J Neurol Neurosurg Psychiatry 1974;37:647–52. Kaji R, Sumner AJ. Ouabain reverses conduction disturbances in single demyelinated nerve fibers. Neurology 1989;39:1364–8. Kanai K, Kuwabara S, Misawa S, Tamura N, Ogawara K, Nakata M, et al. Altered axonal excitability properties in amyotrophic lateral

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