1Department of Medical Physics and Biophysics, University of Nijmegen, NL-6525 EZ Nijmegen; 2Institute of Neurology and 3Department of Child Neurology, University Hospital Nijmegen, NL-6500 HB Nijmegen; and 4SMK Research, NL-6500 GM Nijmegen, The Netherlands
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ABSTRACT |
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van Wezel, B.M.H.,
B.G.M. van
Engelen,
F.J.M. Gabreëls,
A.A.W.M. Gabreëls-Festen, and
J. Duysens.
A Fibers Mediate Cutaneous Reflexes During Human Walking.
J. Neurophysiol. 83: 2980-2986, 2000.
During human gait, transmission of cutaneous reflexes from the foot is
controlled specifically according to the phase of the step cycle. These
reflex responses can be evoked by nonnociceptive stimuli, and therefore
it is thought that the large-myelinated and low-threshold A
afferent
fibers mediate these reflexes. At present, this hypothesis is not yet
verified. To test whether A
fibers are involved the reflex responses
were studied in patients with a sensory polyneuropathy who suffer from
a predominant loss of large-myelinated A
fibers. The sural nerve of
both patients and healthy control subjects was stimulated electrically
at a nonnociceptive intensity during the early and late swing phases while they walked on a treadmill. The responses were studied by recording electromyographic (EMG) activity of the biceps femoris (BF)
and tibialis anterior (TA) of the stimulated leg. In both phases, large
facilitatory responses were observed in the BF of the healthy subjects.
These facilitations were reduced significantly in the BF of the
patients, indicating that A
fibers mediate these reflexes. In TA
similar results were obtained. The absolute response magnitude across
the two phases was significantly smaller for the patients than for the
healthy subjects. The TA responses for the healthy subjects were on
average facilitatory during early swing and suppressive during end
swing. Both facilitations and suppressions were considerably smaller
for the patients, indicating that both types of responses are mediated
by A
fibers. It is concluded that low-threshold A
sensory fibers
mediate these reflexes during human gait. The low threshold and the
precise phase-dependent control of these responses suggest that these
responses are important in the regulation of gait. The loss of such
reflex activity may be related to the gait impairments of these patients.
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INTRODUCTION |
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Somatosensory feedback from muscles, joints, and
skin is essential in the normal execution of human gait. One important
source of afferent input during gait comes from the skin of the foot. A
nonnociceptive electrical stimulus applied to the sural, posterior tibial, or superficial peroneal nerves can elicit both facilitatory and
suppressive reflex responses with latencies of ~50, 80, and 100 ms
(Duysens et al. 1990; Tax et al. 1995
;
Van Wezel et al. 1997
; Yang and Stein
1990
). The reflex responses at a latency of ~80 ms are
observed in a wide variety of muscles in both legs. The amplitude of
these responses is modulated according to the phase of the step cycle
(De Serres et al. 1995
; Duysens et al. 1990
-1992
, 1995
, 1996
; Tax et al. 1995
;
Van Wezel et al. 1997
; Yang and Stein
1990
; Zehr et al. 1997
, 1998b
). This
phase-dependent modulation depends on the muscle (Duysens et al.
1996
; Tax et al. 1995
; Van Wezel et al.
1997
), the leg (Tax et al. 1995
; Van Wezel et al. 1997
), and the location of the stimulus
(Van Wezel et al. 1997
; Zehr et al.
1997
).
The large-myelinated and low-threshold A afferent fibers may be
involved because nonnociceptive stimulation is sufficient to evoke
these reflex responses. However, at present this hypothesis has not yet
been verified. In fact, although the small-myelinated A
fibers
mostly transmit nociceptive information, these also can transmit
nonnociceptive information (Adriaensen et al. 1983
; Handwerker and Kobal 1993
; Millan 1999
).
Moreover, reflex responses after a nonnociceptive stimulus often are
observed in periods of the step cycle when muscles are inactive
(Duysens et al. 1996
; Tax et al. 1995
;
Van Wezel et al. 1997
; Zehr et al. 1997
,
1998b
), a condition that requires A
-fiber activation to
elicit responses under static conditions (Ertekin et al.
1975
; Rossi et al. 1996
). Hence, at present it
remains unclear whether A
fibers mediate cutaneous reflexes during
human gait.
Therefore the present study investigated whether A fibers contribute
to cutaneous reflexes from the foot during human gait on patients who
suffered from a clinically established sensory polyneuropathy with a
predominant loss of large-myelinated low-threshold A
sensory fibers.
Hence, reduced or absent reflexes during gait of these patients would
indicate that A
fibers are involved. The responses to electrical
stimulation of the sural nerve were studied in the biceps femoris (BF)
and tibialis anterior (TA) of the stimulated leg during walking of
these patients. The BF was chosen because this muscle usually exhibits
large facilitatory responses to sural nerve stimulation during human
gait (Duysens et al. 1996
; Tax et al.
1995
; Van Wezel et al. 1997
) and therefore primarily will provide information about changes in the amplitude of
reflex responses during gait of patients with sensory polyneuropathy. The TA was chosen because stimulation of the sural nerve elicits reflex
responses at ~80 ms in TA, which are on average facilitatory during
early swing, whereas these are suppressive during late swing
(Duysens et al. 1996
; Tax et al. 1995
;
Van Wezel et al. 1997
; Yang and Stein
1990
). In principle, it is possible that these responses are
mediated by fibers with different activation thresholds. In that case,
these two types of responses should be affected differently in patients
with a sensory polyneuropathy. The responses in this muscle therefore
will provide information about potential differences in the excitatory
and inhibitory reflex pathways and their phase-dependent control during
gait of patients with sensory polyneuropathy.
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METHODS |
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The methods in this study were mostly identical to those which
have been described in detail elsewhere (Duysens et al.
1996; Tax et al. 1995
; Van Wezel et al.
1997
). The essentials will be described in the following text,
together with some specific procedures.
Patients and methods
Experiments were performed on a group of 7 patients (5 male, 2 female) with a predominant sensory polyneuropathy (aged between 13 and
80). The selection of patients was based on clinical investigation that
indicated a relatively specific loss of sensory afferents and not motor
efferents and that sensory deficits were restricted largely to the loss
of large-myelinated A and not A
fibers.
In all patients, tactile sensory impairment (vibration/position) was
evident in the feet and lower legs during clinical investigation. Romberg test was impaired in all patients. In none of the patients could a sensory evoked potential be observed during clinical
electrophysiological investigation of the sural nerve, suggesting loss
of large-myelinated A fibers. No abnormal acute pain sensation (pin
prick) was observed in four of the patients, suggesting no severe loss
of small myelinated A
fibers. A predominant loss of large-myelinated
A
afferents, although the small myelinated A
fibers were less
affected (see RESULTS), was evident in three patients for
whom a sural nerve biopsy was available (taken previously for clinical
diagnosis). In six patients, no responses were observed after ankle
taps. In five patients, no responses were observed after patella taps. During clinical examination no signs of weakness were observed in six
patients and weakness only in the feet of one patient, indicating no
severe motor impairments in the muscles that were measured in the
present study (BF and TA). Clinical electrophysiological investigation
on the peroneal nerve confirmed this because in all patients a
motor-evoked potential could be elicited, suggesting no severe loss of motoneurons.
For comparison, 10 normal healthy subjects (aged between 19 and 60) with no known history of neurological or motor disorder participated in this study. In addition, two patients with a predominant motor polyneuropathy (aged 17 and 70) participated to investigate the possibility that any reflex impairments during the walking experiments were due to motor components. All subjects had given informed consent. The experiments were carried out in conformity with the declaration of Helsinki for experiments on humans.
Experimental setup
All subjects were asked to walk on a treadmill while wearing a safety-harness that was fastened to an emergency brake at the ceiling. A second emergency brake was attached close to the subject on the handrail of the treadmill so that the subject could stop the treadmill at any moment. Very thin insole foot-switch systems (designed in collaboration with Algra Fotometaal B. V., Wormerveer, The Netherlands) were used to detect foot contact. Bipolar electromyographic (EMG) activity was recorded in both legs by means of surface electrodes over the long head of the BF and the TA muscles. The EMG signals were (pre-)amplified (by a total factor that was generally in the order of 104-105), high-pass filtered (cutoff frequency at 3 Hz), full-wave rectified and then low-pass filtered (cutoff frequency at 300 Hz). These signals were sampled along with the foot-switch signals and a digital code referring to the stimulus condition. The data were sampled at 500 Hz and stored on hard disk for each trial.
The electrical stimulus (a train of 5 rectangular pulses of 1-ms duration given at 200 Hz) was applied by a custom-made constant-current stimulator through a bipolar stimulation electrode (with poles of 0.5 cm and an interpole distance of 2.0 cm) that usually was positioned over the left sural nerve (approximately halfway between the lateral malleolus and the Achilles tendon), firmly pressed with elastic straps. In one patient, the electrode was positioned over the right sural nerve because his left sural nerve was taken for a biopsy for clinical diagnosis. For each subject, the exact position of the stimulation electrode was determined so that an electrical stimulus evoked a clear sensation along the innervation area of the sural nerve (lateral side of the foot).
Stimulus stability
Both voltage and current of the stimulus appeared to be quite
constant for the two phases. The maximum deviation from the mean of all
stimuli was <2.5% for the voltage and <1.5% for the current.
However, this does not rule out the possibility that the nerve itself
might receive a variable input due to changes in the ankle positions
during the course of a step cycle. In previous publications, it was
discussed that this possibility did not play a large role
(Duysens et al. 1995, 1996
). Furthermore the stability was also verified in one subject by measuring the compound sensory action potentials of the sural nerve for several ankle positions under
stationary conditions (Van Wezel et al. 1997
). In
addition, the responses of the ipsilateral tibialis anterior muscle in
this study agree with those of other groups (see RESULTS),
including those that have controlled for the stimulation by using mixed nerve stimulation and monitoring of the M waves (De Serres et al. 1995
; Yang and Stein 1990
; Zehr et
al. 1997
). Hence there are no indications that there is a large
phase-dependent variation of the stimulus delivered to the nerve.
Experimental protocol
Before each experimental run, during quiet standing, the
perception threshold (PT) was determined by gradually increasing (to
above PT) and decreasing (to below PT) the stimulus amplitude. During
the experiment, the electrical stimuli were delivered to the sural
nerve during the early and end swing phases at an intensity of two PT.
During the same phases, control values (i.e., no stimulus) of the EMG
also were measured. Every stimulus condition was presented 10 times
in an experimental run. All trials occurred in random order. The
successive stimulus conditions were separated by a random interval in
the range of 3.5-6.5 s. Hence, two stimuli always were separated by at
least two step cycles without a stimulus. At the end of each
experimental run the perception threshold was determined once more. It
was slightly lowered (8% on average) as compared with the measurement
taken immediately before the experimental run.
Data analysis
The overall effect after nerve stimulation was obtained by
averaging the 10 trials of each stimulus condition and subsequently by
subtracting the average EMG control trials from the corresponding average EMG stimulus trials ("pure" responses or
"subtractions"). To quantify these responses, the average EMG value
was calculated within a time window around the responses
(Duysens et al. 1991, 1996
; Tax et al.
1995
; Van Wezel et al. 1997
; Yang and
Stein 1990
). When a muscle showed little or no responses, no
adequate window could be set. In that case, an average window was used,
calculated from the time windows used to measure responses in (in order
of priority) other muscles or the average of other subjects (cf. Tax et al. 1995
).
For each trial, the EMG was averaged within the applicable window. Then the average and standard error were calculated for corresponding stimulus conditions (n = 10 trials per condition). The resulting data underwent an amplitude normalization with respect to the maximum control value in the step cycle so that a proper intersubject comparison could be made. The statistical significance of the observed reflex responses was tested using the Wilcoxon signed-rank test (significance level P < 0.05).
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RESULTS |
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Unperturbed treadmill walking
Gait on the treadmill of the patients with sensory polyneuropathy
clearly was impaired. During a short walking period, the patients were
asked to determine the speed at which they walked most comfortably.
This preferred walking speed was generally slower for the patients than
for the healthy subjects. On average, the most comfortable belt speed
for the patients was 2.4 ± 0.6 (SD) km/h, whereas healthy
subjects usually are studied at 4 km/h (De Serres et al.
1995; Duysens et al. 1990
, 1996
; Van
Wezel et al. 1997
; Yang and Stein 1990
;
Zehr et al. 1997
, 1998b
). To exclude possible influence
of walking speed on the results, the healthy subjects, used as controls
in this study, also were investigated at 2.5 km/h.
When possible, patients were asked to walk without any support devices. However, four of the seven patients needed support from the side bars of the treadmill. When they were asked to release the side bars, they usually staggered for a few steps and then grasped the side bars again to prevent falling. The patients who walked without support tended to have a shorter average step-cycle duration (1,159 ± 63 ms across patients, n = 3) than the healthy subjects (1,327 ± 122 ms across subjects, n = 10), whereas the patients who did walk with support of the side bars tended to have a longer average step-cycle duration (1,568 ± 50 ms across patients, n = 4). The variability of the step-cycle duration was on average almost four times larger for the patients who walked without support (SD 8.1% of the average step cycle) than for the healthy subjects (SD 2.1% of the average step cycle). The patients who walked with support also had an increased variability of the step-cycle duration (SD 3.5% of the average step cycle).
Reflex responses to sural nerve stimulation
The sural nerve was stimulated at an intensity of two times perception threshold (2 PT). At this intensity both patients and healthy subjects felt a tactile, nonnociceptive irradiation to the lateral side of the foot, which is the innervation area of the sural nerve. During gait the stimulus was applied during the early and late swing phases. To obtain the average net effect after stimulation of the sural nerve, for both stimulus and control (i.e., no stimulus) conditions, the 10 trials of the two stimulus phases were averaged and the average control trials were subsequently subtracted from the corresponding average stimulus trials (Fig. 1A).
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Figure 1B shows the subtracted responses of the BF for a healthy subject, a patient with a predominant motor polyneuropathy, and a patient with a predominant sensory polyneuropathy. The stimuli were presented during the end swing period, a phase in which the BF was active during unperturbed gait in all subjects. In the healthy subject a large facilitatory reflex response is observed at a latency of ~80-90 ms (Fig. 1B, top). When the same experiment was performed on two patients with predominant motor polyneuropathy, the responses were similar to those observed in the healthy subject (example in Fig. 1B, middle). In contrast, the responses in a patient with a predominant sensory polyneuropathy were much smaller or even absent (Fig. 1B, bottom).
To quantify the responses a time window was set around the responses, in which the mean EMG value is calculated. On average for all subjects, this time window started at 85 ± 7 ms and ended at 112 ± 10 ms. Subsequently, for all stimulus conditions (i.e., for early- and late-swing phases and for both control and stimulus conditions), both the average and the standard error were calculated for these window-averaged trials (n = 10 trials per stimulus condition). The data of the individual subjects were normalized to the maximum control EMG value in the step cycle (see METHODS). The resulting values were subsequently averaged for both patients and healthy subjects (Fig. 2).
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Consistent with previous results (Duysens et al. 1996;
Tax et al. 1995
; Van Wezel et al. 1997
),
large responses were observed in BF for the healthy subjects, with no
statistically significant phase-dependent effect in the early and late
swing phases. For the patient group, the responses in BF were
considerably smaller and on average no significant responses were
observed (Fig. 2). The difference between healthy subjects and patients
was statistically significant (Wilcoxon rank sum test:
P < 0.01).
Similar results were obtained for the responses in the TA muscle. For
the healthy subjects, the well-known "reflex reversal" (Duysens et al. 1990; Tax et al. 1995
;
Van Wezel et al. 1997
; Yang and Stein
1990
) was observed from facilitatory responses during early
swing to suppressive responses during late swing. This modulation
tended to be similar in the patients with sensory polyneuropathy,
although the responses were smaller. Both the facilitatory responses
during early swing and the suppressive responses during late swing were
smaller for the patients than for the healthy subjects (Fig. 2). For
the absolute response magnitude across the two phases, the difference
between healthy subjects and patients was statistically significant
(Wilcoxon rank sum test: P < 0.01).
During the experiments, four of the seven patients with a sensory polyneuropathy needed support from the side bars of the treadmill. This did not play a large role because three patients did not use support from the side bars, and in these three patients, the amplitude of the reflex responses also were reduced significantly. Furthermore the two patients with a motor neuropathy also used the side bar for support. These two patients exhibited reflexes that were normal compared with the healthy subjects.
In three patients, a sural nerve biopsy that was taken for clinical
purposes showed a predominant loss of large-myelinated A fibers.
Normally, the histogram of myelinated fiber density versus fiber
diameter in the sural nerve is bimodal: one group of thick fibers (A
fibers; low-activation threshold) is clustering around a diameter of 9 µm; another group of thin fibers (A
fibers; higher threshold) has
a peak of approximately 3-4 µm with an overlap between both groups
(Fig. 3,
). In these three patients,
there was a significant difference of 50% in the remaining amount of A
as compared with A
fibers (Fig. 3,
). The density of large diameter fibers (range 6-14 µm) was on average 10 ± 7% of the normal density. In contrast, the density of small diameter fibers (range 1-6 µm) was 77 ± 13% of the normal density. The
reflexes in BF of these three patients were on average 9 ± 4% of
the magnitude of the reflexes of the healthy subjects (Fig.
4).
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DISCUSSION |
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A fibers mediate cutaneous reflexes during walking
This study showed that reflexes at a latency of ~80 ms evoked by
nonnociceptive stimulation of the sural nerve were significantly smaller for patients with a sensory polyneuropathy than for healthy subjects during gait. Clinical investigation of these patients revealed
severe sensory deficits for tactile perception (vibration and
position), suggesting functional loss of ascending A fibers. In
contrast, acute pain perception (pin prick) was less affected, indicating a higher degree of functionally intact ascending A
fibers. This fitted well with both sural nerve clinical
electrophysiology and morphometry indicating a selective loss of
large-myelinated A
fibers. A sural nerve biopsy, which was available
for three patients, showed a distinct difference between the loss of
large-myelinated A
fibers (reduction to 10%) and the loss of small
myelinated A
fibers (reduction to 77%). A large decrease in the
amplitude of the reflexes in BF of these three patients was observed
(reduction to 9%). Hence the present results provide direct
experimental evidence that A
fibers mediate reflexes at ~80 ms
during human gait.
This is compatible with other studies that indicated that
nonnociceptive stimulation is sufficient under static conditions in
active muscles (Aniss et al. 1992; Burke et al.
1991
; Kukulka 1994
; Meinck et al.
1981
; Rossi et al. 1996
). An average conduction velocity of ~45 m/s was reported for the reflex afferents involved, suggesting contribution from A
fibers (Rossi et al.
1996
). In contrast, nociceptive stimuli are usually necessary
to evoke reflex responses in inactive muscles under stationary
conditions (Ertekin et al. 1975
; Rossi et al.
1996
). For these reflexes, A
fibers are believed widely to
be involved because of the close relation between the amplitude of
these responses and subjective pain score (Chan and Dallaire
1989
; Willer 1977
; Willer et al.
1984
) and because of the observed conduction velocity of the
reflex afferents involved (~10-25 m/s, Ertekin et al.
1975
; ~27 m/s, Rossi et al. 1996
).
Studies that used the spatial facilitation technique suggested that
nociceptive A and nonnociceptive A
fibers converge on common
interneurons (Behrends et al. 1983
; Rossi et al.
1996
). Hence it is not unlikely that at least part of the
pathways is common for the nonnociceptive and the nociceptive reflexes.
On the other hand, during gait, there is evidence that the two types of
input, nonnociceptive and nociceptive, use largely different reflex
pathways. After nonnociceptive stimulation of the sural nerve during
the midswing phase, facilitatory reflex responses were observed in TA,
which induced dorsiflexion movements of the ankle (Duysens et
al. 1992
). In contrast, a nociceptive stimulus to the same
nerve elicited suppressive reflex responses with the same latency
accompanied by ankle plantar flexion (Duysens et al.
1992
). This indicates entirely different reflexes and
behavioral reactions after nonnociceptive and nociceptive stimulation
during human walking. Combined with the present results, this suggests that during gait the presently described reflex responses would be
mediated solely by A
fibers and not by A
fibers, and therefore a
purely nocifensive role for these reflexes during gait in terms of a
flexion reflex can be excluded.
The phase-dependent reflex reversal in TA from facilitation in early
swing to suppression during end swing is in agreement with previous
studies (Duysens et al. 1996; Tax et al.
1995
; Van Wezel et al. 1997
). In the patients,
both the facilitations and the suppressions were reduced indicating
that both types of responses are mediated by A
fibers. This is
important because the TA reflex reversal often is used as one of the
characteristic features of the phase-dependent modulation of cutaneous
reflexes (De Serres et al. 1995
; Duysens et al.
1992
, 1996
; Jones and Yang 1994
; Tax et
al. 1995
; Van Wezel et al. 1997
; Yang and
Stein 1990
).
Functional considerations
Just as cutaneous afferents are known to play a role in the
control of posture (Do et al. 1990; Thoumie and
Do 1996
; Wu and Chang 1997
), A
fibers may
provide low-threshold sensory information during gait. For example,
during the stance phase of walking, activity from low-threshold
cutaneous mechanoreceptors (Sinkjaer et al. 1994
) could
provide information about phase transitions [as was observed in cats
(Duysens 1977
)] or irregularities on the ground surface. Furthermore
when the foot strikes an object during the swing phase, the so-called
stumbling reaction can be evoked in humans (Eng et al.
1994
; Schillings et al. 1996
, 1999
). Low-threshold cutaneous reflexes could participate in these corrective responses, as has been proposed previously (cats: Drew and
Rossignol 1987
; Forssberg 1979
; humans:
Zehr et al. 1997
).
If such information is processed through reflexes such as described in
the present study, then one would expect abnormalities in the walking
patterns if the reflexes are abnormal. A relation between abnormal
cutaneous reflexes from the foot and impaired gait was observed in
patients with other forms of neurological disorders, such as patients
suffering from an incomplete spinal-cord injury (Jones and Yang
1994) or a stroke (Zehr et al. 1998a
). Because
descending control clearly is affected in those patients, these studies
could point to supraspinal or even transcortical involvement in these
reflexes (see also Christensen et al. 1999
; Nielsen et al. 1997
). Nevertheless in those patients,
the causal relation between abnormal reflexes and abnormal gait is hard
to establish because the neural pathways that are affected are not precisely known. The present study, in contrast, describes a population of patients in which the primary cause of abnormal reflexes during gait
is the neurological disorder itself (loss of large-myelinated afferent
fibers). This (partial) deafferentation may be comparable to studies on
cats which show that deafferentation of the limbs cause abnormalities
in the gait patterns (Giuliani and Smith 1987
; Goldberger 1977
, 1988a
,b
; Grillner and Zangger
1984
; Rasmussen et al. 1986
; Wetzel et
al. 1976
). Hence, the combination of gait impairments such as
described in the present study (and by Lajoie et al.
1996
) and the loss of reflex activity mediated by low-threshold A
fibers would support the possibility that the loss of sensory input, in part channeled and processed through cutaneous reflex pathways, is one of the contributing factors causing abnormalities in
human walking.
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ACKNOWLEDGMENTS |
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The authors thank I. Gommans for participating in the experiments, A. M. Schillings for critically reading the manuscript, G. Windau for software development, and A. M. van Dreumel and J.W.C. Kleijnen for technical assistance.
This work was supported by the Prinses Beatrix Fonds in The Netherlands.
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FOOTNOTES |
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Address for reprint requests: B.M.H. Van Wezel, Dept. of Medical Physics and Biophysics, University of Nijmegen, Geert Grooteplein 21, 6525 EZ Nijmegen, The Netherlands.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 26 July 1999; accepted in final form 10 February 2000.
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REFERENCES |
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