contingent negative variation - Parkinson disease - levodopa
variação contigente negativa - doença de Parkinson - levodopa
The increasing prevalence and incidence of Parkinson’s disease (PD), along with the
increase in the population’s life expectancy, has made it the second most frequent
neurodegenerative disorder. This has a significant socio-economic impact on the quality
of life of PD patients and caregivers due to progressive physical dependence in activities
of daily living, in addition to subsequent cognitive impairment.
Hypomimia, or masked facies, in PD is characterized by reduced automatic and voluntary
facial muscle expressiveness from the early stages, progressing with advancing disease[1] and generating social implications due to the expressionless, apathetic, depressed
and introspective appearance[2]. Among the hypomimia findings, there is a decrease of the frequency of eyelid blink
by different pathways related to the planning and execution of this motor act. This
signal is one component of the extensive bradykinesia presentation.
Bradykinesia results from neuronal damage to pathways related to motor planning[3]. Voids in the dopaminergic and nondopaminergic circuits have been involved in this
pathophysiology, generating an imbalance between inhibition and excitation, deficits
and compensation[4],[5],[6]. Changes in the dopaminergic pathways explain, at least in part, disturbances in
the kinematics of spontaneous eyelid blink[7],[8],[9], and the voluntary one[10] and in the blink reflex habituation of PD[11].
Pharmacological treatment with dopaminergic medications, considered the gold standard[12],[13], results in partial problem-solving of bradykinesia as it improves the speed of
movement[7],[8],[10], but not its rhythm and amplitude[14], although the damage regarding the amplitude is very evident[1].
Bereitschaftspotential (BP), or readiness potential, described by Kornhuber and Deecke
in 1964 corresponds to the negative gradient starting one-and-a-half to two seconds
before the motor potential, seen in electroencephalogram (EEG) retrograde averaging.
This potential is divided into an early component related to planning and a late potential
related to motor execution[15],[16],[17]. This study aimed to evaluate the ocular blink in patients with PD in the off (without levodopa) and on phases (with levodopa) by measuring the BP.
METHODS
Subjects
The study was approved by the University Human Research Ethics Committee (statement
90208). All participants signed an informed consent form.
Ten PD patients (nine males) aged 43 to 78 years were randomly recruited in the Parkinsonian
Patient Care Service (Pro-Parkinson) at the Clinic Hospital of the Federal University
of Pernambuco, Brazil. All patients were diagnosed by a neurologist trained in movement
disorders. Eighteen normal individuals (eight males) aged 17 to 60 years served as
a comparison group. The analysis of this normal data was recently accepted for publication[18].
Procedures
The participants sat comfortably on a chair and were instructed to relax, restrain
moving the muscles of the neck, head or face and fix their gaze on a spot placed two
meters away at eye level, blinking as naturally as possible, once every ten seconds.
There were no auditory or visual clues for the time of blinking and the rhythm was
rehearsed before starting the recordings.
The PD patients who met the inclusion criteria were invited to return the next morning.
The patients were instructed not to take their morning anti-Parkinson’s medication,
before coming to the laboratory. They were also advised to avoid the intake of protein
foods.
The PD patients were initially recorded at least 12 hours after the last anti-Parkinson’s
medication (off phase). Then levodopa/benserazide 100/25 mg was offered and, after 40 minutes, a
second recording section (on phase) was made. The normal controls were recorded only once.
The EEG was recorded from 11 EEG electrodes placed in positions F3, FZ, F4, C3, Cz,
C4, P3, Pz, P4, O1, O2 of the international 10-20 system of electrode placement, referenced
to two electrodes placed on the mastoids and linked together. The vertical electrooculogram
was recorded from two electrodes placed one centimeter above and below the right eye.
The ground electrode was placed on the skin over the right clavicle.
The recordings were made using a Neuron-Spectrum NET polygraph (Neurosoft). The bandpass
of the filters were 0.1 Hz to 35 Hz. Inter-electrode impedances (10 Hz) were kept
below 3kΩ.
Data analysis
Blinks were visually identified as large positive deflections on the vertical electrooculogram
channel and the beginning of the blink potential was marked. The two-second EEG segments
preceding the mark were back-averaged together. Blinks that presented artifacts during
or close to this period were eliminated from the analysis.
The averages were saved in text files and analyzed in a specific program written in
Matlab software. Initially, the waves obtained by the general averaging of each group
of participants were observed. The measurement points were established at -1800 ms
(at the beginning of the BP), -500 ms (at the start of the late component of the BP)
and 0ms (at the onset of the blink potential). To compensate for background noise,
the amplitudes were averaged in a 100 ms segment centered on the latency to be measured.
Statistical analysis of the BP amplitudes was performed on the C3, C4 and Cz electrodes,
using the STATISTICA version 10 statistical package (StatSoft). The statistical analysis
was performed using analysis of variance (ANOVA) models. A three-way repeated measures
ANOVA model, condition (off and on), time (-1800 ms, -500 ms, 0s) and electrodes (C3, Cz, C4) was used to compare the
PD group during the off phase and the on phase. A mixed-model ANOVA, group (PD and controls) and two repeated-measures, time
(-1800 ms, -500 ms and 0 ms) and electrode (C3, Cz and C4), was used to compare the
PD patients with the normal controls. The Geisser-Greenhouse correction was applied
for violation of sphericity. The Newman-Keuls test was used for post hoc analysis, when necessary. The critical p-value was 0.05.
RESULTS
[Table 1] lists some demographic and clinical characteristics of the studied PD patients.
Table 1
Some characteristics of the studied Parkinson’s disease patients.
Initials
|
Sex
|
Age
|
TD
|
H&Y
|
UPDRS II
|
UPDRS III
|
UPDRS T
|
MMSE
|
AC
|
M
|
43
|
8
|
3
|
9
|
14
|
23
|
29
|
EL
|
M
|
44
|
3
|
1
|
10
|
29
|
39
|
25
|
DA
|
M
|
48
|
6
|
1
|
10
|
15
|
25
|
30
|
SF
|
F
|
52
|
2
|
1
|
10
|
24
|
34
|
26
|
JF
|
M
|
58
|
4
|
1
|
12
|
30
|
42
|
27
|
CE
|
M
|
53
|
5
|
2
|
19
|
90
|
109
|
30
|
MA
|
M
|
61
|
3
|
1
|
11
|
29
|
40
|
30
|
AJ
|
M
|
61
|
5
|
1
|
5
|
20
|
25
|
30
|
EV
|
M
|
68
|
1
|
2
|
8
|
35
|
43
|
24
|
JQ
|
M
|
78
|
4
|
1
|
5
|
7
|
12
|
30
|
M: male; F: female; TD: time of disease; HY: Hoehn and Yahr scale; UPDRS: unified
Parkinson’s disease rating scale; MMSE: mini–mental state examination. Age and TD
in years.
A total of 230 blinks during the off phase and 250 blinks during the on phase were averaged from the 10 PD patients, giving a mean of 23 blinks per patient
during the off phase and 25 blinks per patient during the on phase.
[Figure 1] shows the grand average of the EEG recorded at all electrodes from the PD patients
in the off (dashed line) and on (solid line) phases.
Figure 1 Grand average of the bereitschaftspotential of the Parkinson’s disease patients during
the off phase (dashed line) and on phase (solid line) of medication at all electrode positions.
In both conditions, a negative potential of increasing amplitude was observed starting
around -1,500 ms before the blink onset. This potential had a broad scalp distribution,
but was most evident at the central regions. Around -500ms before the blink potential,
a change in the wave configuration was observed. No obvious differences were seen
between the potentials obtained during the off phase and the on phase.
[Figure 2] shows the analysis of the amplitudes of the potentials at C3, Cz and C4 electrodes
measured -1800 ms, -500 ms and 0 ms before the onset of the blink potential. The waveforms
and measurement times are shown at the top of the figure. Amplitudes were averaged
in a segment of 100ms centered on the latency to be measured (vertical gray shadows).
The means and standard deviations of the amplitudes at each electrode, condition and
time-point are illustrated at the bottom of the figure. [Table 2] shows the ANOVA of the data.
Figure 2 Analysis of the amplitudes of the bereitschaftspotential of the Parkinson’s disease
patients at C3, Cz and C4 electrodes measured -1800, -500 and 0 ms before the onset
of the blink potentials. The waveforms and measurement times are shown on the top
of the figure. Amplitudes were averaged within a segment of 100 ms centered on the
latency to be measured (vertical gray shadows). The means and standard deviations
of the amplitudes at each electrode, condition and time-point are illustrated at the
bottom of the figure.
Table 2
Factorial repeated-measurement analysis of variance of the amplitudes of the bereitschaftspotential
of the Parkinson’s disease patients, with three repeated measurements: condition (off and on medication), time (-1800, -500 and 0ms, relative to the onset of the blink potential)
and electrode (C3, Cz and C4).
Variable
|
SS
|
df
|
MS
|
F
|
p
|
Intercept
|
11.706
|
1
|
11.706
|
12.145
|
0.007
|
Error
|
8.675
|
9
|
0.964
|
|
|
Condition
|
0.212
|
1
|
0.212
|
0.284
|
0.607
|
Error
|
6.716
|
9
|
0.746
|
|
|
Time
|
9.552
|
2
|
4.776
|
7.072
|
0.005*
|
Error
|
12.157
|
18
|
0.676
|
|
|
Electrode
|
0.063
|
2
|
0.032
|
0.099
|
0.906
|
Error
|
5.730
|
18
|
0.318
|
|
|
Condition × time
|
0.260
|
2
|
0.130
|
0.275
|
0.762
|
Error
|
8.494
|
18
|
0.472
|
|
|
Condition × electrode
|
0.021
|
2
|
0.010
|
0.207
|
0.815
|
Error
|
0.8900
|
18
|
0.049
|
|
|
Time × electrode
|
0.064
|
4
|
0.016
|
0.176
|
0.950
|
Error
|
3.293
|
36
|
0.092
|
|
|
Condition × time × electrode
|
0.018
|
4
|
0.005
|
0.063
|
0.992
|
Error
|
2.534
|
36
|
0.070
|
|
|
Newman-Keuls test of the main effect TIME
|
-1800 ms × 0 ms
|
0.005*
|
-1800 ms × -500 ms
|
0.024*
|
-500 ms × 0 ms
|
0.233
|
SS: Sum of squares; Df: Degrees of freedon; MS: mean squares; F: ANOVA Statistic *
P<0.05
The ANOVA showed a significant effect for time. No interactions were significant.
The post hoc analysis (Newman-Keuls) showed that the amplitudes at -500ms (onset of the late component)
and 0ms (end of the BP) were both significantly larger than the amplitude at -1800
ms (just before the onset of the early BP component). The amplitudes at -500 and 0ms
were not different. This is consistent with the presence of a BP preceding the voluntary
blink of PD patients, during both off and on phases. No significant differences between the amplitudes during the off phase and the on phase were observed.
A total of 900 blinks were averaged from the 18 normal controls, giving a mean of
50 blinks per individual. As there were no significant differences between the measured
amplitudes of the BP during off and on phases, all 580 blinks from the 10 PD patients were averaged, giving a mean of 58
blinks per participant.
[Figure 3] shows the analysis of the amplitudes of the potentials at C3, Cz and C4 measured
-1800, -500 and 0 ms before the onset of the blink potentials from the PD patients
(solid line) and the normal controls (dashed line). The waveforms and measurement
times are shown at the top of the figure. The means and standard deviations of the
amplitudes at each electrode, condition and time-point are illustrated at the bottom
of the figure. Different scales were used for the PD patients (right side) and for
the control group (left side). [Table 3] shows the ANOVA of the data.
Figure 3 Analysis of the amplitudes of the bereitschaftspotential of the Parkinson’s disease
patients (grand average of all recordings during the off phase and the on phase) and the normal controls at C3, Cz and C4 electrodes measured -1800, -500 and
0 ms before the onset of the blink potentials. The waveforms and measurement times
are shown on the top of the figure. Amplitudes were averaged within a segment of 100ms
centered on the latency to be measured (vertical gray shadows). The means and standard
deviations of the amplitudes at each electrode, condition and time-point are illustrated
at the bottom of the figure. Different scales were used for the PD patients (right
side) and the normal controls (left side).
Table 3
Factorial mixed-model analysis of variance of the amplitudes of the bereitschaftspotential
of the Parkinson’s disease patients and normal controls: Group (PD patients and normal
controls) and two repeated measures: time (-1800, -500 e 0 ms, relative to the onset
of the blink potential) and electrode (C3, Cz and C4).
Variable
|
SS
|
Df
|
MS
|
F
|
p*
|
Intercept
|
227.770
|
1
|
227.770
|
10.379
|
0.004*
|
Group
|
128.687
|
1
|
128.687
|
5.864
|
0.024*
|
Error
|
504.716
|
23
|
21.944
|
|
|
Time
|
134.349
|
2
|
67.175
|
7.847
|
0.001*
|
Time × group
|
74.475
|
2
|
37.238
|
4.350
|
0.019*
|
Error
|
393.785
|
46
|
8.561
|
|
|
Electrode
|
9.394
|
2
|
4.697
|
2.296
|
0.112*
|
Electrode × group
|
9.731
|
2
|
4.866
|
2.379
|
0.104*
|
Error
|
94.087
|
46
|
2.045
|
|
|
Time × electrode
|
2.860
|
4
|
0.715
|
1.244
|
0.298*
|
Time × electrode × group
|
2.665
|
4
|
0.666
|
1.159
|
0.334*
|
Error
|
52.878
|
92
|
0.5748
|
|
|
Newman-Keuls test of the interaction time × group
|
Parkinson × Control at -1800 ms
|
0.902
|
Parkinson × Control at -500 ms
|
0.033*
|
Parkinson × Control at 0 ms
|
0.012*
|
SS: Sum of squares; Df: Degrees of freedon; MS: mean squares; F: ANOVA Statistic*
P<0.05
The ANOVA showed a significant interaction between the time and the group. The post hoc analysis showed that the amplitudes at -500 ms and 0ms were both significantly larger
in the control group than in the PD group. At -1800 ms the amplitudes were not different
between groups. Therefore, the amplitude of the BP of PD patients is significantly
smaller than the BP of normal controls at all analyzed electrode positions.
DISCUSSION
This study demonstrated that there is a BP preceding voluntary blinks in PD patients.
This BP does not differ significantly during the off phase and the on phase of medication. To our knowledge, there have been no studies on BP preceding
blinks in PD patients. In a previous recently-accepted study[18], we reported the presence of a BP preceding voluntary (but not spontaneous blinks)
in normal participants.
The BP of DP patients had a broad scalp distribution, most clearly identified at the
central regions, similar to the BP of normal controls and to the BP preceding other
movements in PD and normal controls[19],[20],[21].
The amplitude of the BP of the PD patients was much smaller than the BP of the control
group, even though the PD patients were at a relatively mild stage of the disease.
At its largest negativity (just preceding the blink) the mean amplitudes of the BP
of the PD patients and the normal controls were respectively 0.6 μV and 3.3 μV. This
suggests a significant dysfunction of the motor neural networks, even in patients
with few symptoms. This finding is consistent with previous descriptions of reduced
amplitude of BP preceding other movements in PD patients, even in the early stages[12],[22],[23],[24],[25],[26]. It is also in accordance with the early clinical finding of paucity of facial expression
and reduced blink rate in PD patients, suggesting that neural systems related to motor
planning, especially those related to facial expression and blinks, are affected early
in PD[1].
Most PD patients were at stage 1 of the H&Y scale and showed low scores on the motor
and daily living activities of the Unified Parkinson’s Disease Rating Scale. Although
the disease has a progressive symptomatology, it is known that damage to neuronal
pathways begins 8–17 years before the appearance of the first symptoms, with different
progression characteristics depending on the compensatory mechanisms and the age of
onset. Therefore, even patients who are diagnosed early may present with significant
neural dysfunction.
Although most studies have reported smaller BP amplitudes in PD patients compared
to normal controls, some studies have reported no differences[21],[24],[26] or even larger amplitudes[27],[28]. The reasons for these discrepancies are not clear.
In our study, the dopaminergic drug did not significantly affect the BP. A larger
amplitude during the on phase has been described in BP preceding the movement of the fingers[22],[23]. The absence of an influence by levodopa on the BP preceding the eye blink may result
from an earlier, and more severe, dysfunction of the dopaminergic networks involved
in the planning of eye blinks, than finger movements.
Although levodopa is considered the gold standard drug for PD therapy, it does not
appear to act on all the mechanisms that cause bradykinesia[14]. Nondopaminergic pathways play a role in the clinical improvement of motor planning
and BP amplitude in PD patients, as observed after unilateral posteroventral pallidotomy[28] and after neurofeedback techniques[25]. These studies corroborate the presence and importance of nondopaminergic or dopaminergic
pathways resistant to modulation mechanisms of the motor planning in PD[6].
The amplitudes at C3, Cz and C4 were not significantly different in PD patients. In
our previous study with normal controls[18], we found larger amplitudes at C3 and C4 in relation with Cz. This was also observed
by Yamamoto[29], but not Shimizu and Okiyama[20], in their study with saccadic movements. A lateral component of the BP source complex,
perhaps the face motor cortex, is less active in PD patients than in normal subjects.
A source analysis study may shed some light on this issue.
The main objective of this study was to determine if a BP preceding voluntary blinks
could be demonstrated in PD patients. We believe this was accomplished. The potentials
we obtained had very low amplitudes in comparison with the potentials obtained from
normal participants. Although the normal participants we used were not perfectly age-matched
with the PD patients, we believe the differences in amplitudes are obvious. Furthermore,
it has been shown that there is no significant change in BP amplitudes with increasing
age, within certain limits[30].
In conclusion, this is the first study demonstrating a BP preceding eyelid blinks
in PD patients. The BP of the PD patients had much smaller amplitudes than the BP
of normal subjects, even though the PD patients were in the early stages of the disease.
These potentials could be used in the future as early diagnostic and evolutionary
markers of Parkinson’s disease. Further investigations should be conducted. Currently
considered the gold standard drug in Parkinson’s disease, levodopa did not obviously
modify the BP preceding blinks, suggesting that the resistant nondopaminergic or dopaminergic
accessory pathways related to motor planning should be investigated in search of more
effective medications.