J Neurol (2012) 259:921–928
DOI 10.1007/s00415-011-6279-3
ORIGINAL COMMUNICATION
Microcirculation response to local cooling in patients
with Huntington’s disease
Ziva Melik • Jan Kobal • Ksenija Cankar •
Martin Strucl
Received: 5 May 2011 / Revised: 3 October 2011 / Accepted: 4 October 2011 / Published online: 20 October 2011
Ó Springer-Verlag 2011
Abstract Altered autonomic nervous system (ANS)
functioning in early stages of Huntington’s disease (HD)
has been suggested, presumably due to distorted high-order
autonomic control. ANS functioning in the early stages of
HD was further investigated. Laser-Doppler (LD) flux in
the skin of the fingertips, heart rate (HR), HR variability,
systolic and diastolic blood pressure were measured during
rest and during a 6 min cooling of one hand at 15°C. Data
of 15 presymptomatic gene mutation carriers (PHD), 15
early symptomatic HD patients (EHD), and two groups of
15 age- and sex-matched controls were compared. The area
under the low frequency (LF) and high frequency (HF)
bands of the HR variability spectrum were calculated. An
augmented reduction of cutaneous LD flux was found in
response to the direct cooling in the PHD group (37.5 ±
8.5% of resting value) compared to the PHD controls
(67.27 ± 8.4%) (p \ 0.05). In addition, the PHD group
had higher (LF/(LF ? HF) index of primary sympathetic
modulation of the HR at rest (53.6 ± 3.3) compared to the
EHD patients (39.7 ± 4.2) (p \ 0.05). In the EHD group, a
significantly smaller change of HR during cooling
(100.26 ± 1.2%) was found compared to the EHD controls
(95.9 ± 1.0%) (p \ 0.05). The results are in line with the
hypothesis that ANS dysfunction occurs even in PHD
subjects. Further, they support the hypothesis that dys-
function of the high-order autonomic centres are involved
in HD.
Z. Melik (&)
K. Cankar
M. Strucl
Medical Faculty, Institute of Physiology,
University of Ljubljana, Zaloska 4, 1000 Ljubljana, Slovenia
e-mail: ziva.melik@mf.uni-lj.si
J. Kobal
Department of Neurology, University Medical Centre Ljubljana,
Zaloska 2, 1000 Ljubljana, Slovenia
Keywords Huntington’s disease
Central autonomic
network
Heart rate variability
Microcirculation

Local cooling
Laser-Doppler flow
Introduction
Huntington’s disease (HD) is a neurodegenerative disorder
leading to the progressive death of neurons in various brain
regions. There is a triad of movement, behavioural, and
cognitive disorders. There are also some other clinical
features associated with the disease. Symptoms suggestive
of autonomic nervous system (ANS) dysfunction have been
reported. Patients with HD experience significantly more
gastrointestinal, urinary, cardiovascular and sexual prob-
lems [1]. Accordingly, clinical testing has revealed hypo-
function of the ANS in mid and advanced HD patients
[2–4]. Moreover, there are signs of ANS dysfunction even
in early symptomatic HD patients (EHD) and presymp-
tomatic HD gene mutation carriers (PHD). A recent study
[5] found a significantly attenuated heart rate response
during a cognitive stress test (mental arithmetic) and an
exaggerated response during a sensory stressor with aver-
sive characteristics (cold pressure test) in EHD and in PHD
individuals. The neural basis for the progression of the
autonomic nervous system dysfuction is largely unknown
[6]. The evidence of cerebral cortical and hypothalamic
pathology [7–11] in the early stages of HD might suggest
distorted activity in cortical and subcortical structures that
are involved in the higher order central autonomic network
(CAN) [12–14]. Recent research has suggested that the
cortical parts of CAN are involved very early in PHD
subjects [15]. There are some interesting reports of hypo-
thalamic involvement at the beginning of the clinical dis-
ease in EHD patients [10, 11, 16–19] that could imply the
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successive involvement of the higher order parts of CAN
during progress of the disease. We, therefore, hypothesized
that the changes in the cardiovascular ANS functioning
could be detected as early as in the presymptomatic phase
of the disease due to the successive involvement of the
higher order parts of CAN. The common cardiovascular
tests like the Valsalva manoeuvre, deep breathing and
orthostatic test did not confirm the differences between
PHD and EHD subjects [5, 20]. In the present study we
introduced new tests that could show more subtle differ-
ences in ANS function in PHD and EHD subjects. For this
purpose skin microcirculation was examined with laser-
Doppler flowmetry [21, 22] during the local cooling of one
hand, along with cardiovascular autonomic testing. The
differences between PHD and EHD individuals were
examined with regard to their microcirculatory responses
during local cooling that triggers ANS partially through the
thermoregulation centre in the hypothalamus [23–26].
Skin microcirculation in humans is under the complex
control of neural reflexes and local factors [27–30]. At the
cooling site (direct response of skin vessels) cold decreases
the amount of norepinephrine released from nerve-endings
[31], reduces the enzymatic breakdown of norepinephrine
within the vessel wall [32], decreases smooth muscle
contractility, diminishes the affinity of alpha1 adrenocep-
tors for norepinephrine [33–35], and stimulates the mobi-
lization of alpha2c adrenoceptors from the Golgi apparatus
to the vascular smooth muscle plasma membrane [36–38].
In contrast, at the site remote from the cooling (indirect
response) the response mediated by the sympathetic vaso-
motor reflexes prevails [39].
The skin is a suitable site for the evaluation of micro-
vascular dysfunction due to the ease and harmlessness of
access [27]. Moderate local cooling of the skin was used to
avoid pain as individual cold pain perception has important
influence on the parameters of cardiovascular reactivity
[40]. Furthermore, lower temperatures induce stronger
sympathetic stimulation which results in maximum
response in all groups of subjects and the disappearance of
eventual differences between PHD and EHD subjects.
Subjects and methods
Subjects
The study enrolled 15 PHD and 15 EHD subjects. HD gene
mutation was confirmed by an expanded number of CAG
triplets [41]. Predictive testing in PHD subjects was per-
formed according to the IHA/WFN HD committee guide-
lines [42]. PHD subjects were treated according to IHA/
WFN proposals during the genetic consultation. They were
enrolled to study at least 6 months after the presymptomatic
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J Neurol (2012) 259:921–928
diagnostic process and when suggested by the consulting
clinical psychologist.
Participants were evaluated clinically not more than a
week before the study entry. The exclusion criteria were
strict: they included diseases and/or conditions that may
influence ANS function; medical disorders like cardiac
insufficiency, arrhythmia, arterial hypertension requiring
treatment, renal and/or liver disease, obstructive pulmonary
disease requiring treatment with bronchodilators, endocrine
disorders (e.g. diabetes mellitus, thyroid dysfunction). All
the PHD subjects were free from regular drug abuse. We
tolerated oral contraceptives or occasional intake of non-
steroidal anti-inflammatory drugs. In EHD patients we
tolerated all of the above mentioned plus short acting
selective serotonin reuptake inhibitors (SSRI). None of the
patients were taking drugs with known major and/or long
term effect on ANS [43]. Five of our 15 EHD patients were
taking low dose of escitalopram (up to 10 mg daily). As
SSRI inhibitors might have a mild short term anticholin-
ergic effects, a 48 h wash out period was considered before
testing, as recommended [44]. None of the patients pre-
sented signs of neurological disorders other than HD nor
did they suffer from peripheral neuropathy or psychiatric
disease such as schizophrenia, major depressive and/or
anxiety disorder as defined by ‘‘Diagnostic and statistical
manual of mental disorders - fourth edition’’ criteria.
Because cardiovascular parameters highly depend on
age and sex, two distinct age- and sex-matched healthy
control groups for the PHD and EHD group were selected.
Each patient or premanifest subject had a control subject of
the same age and sex. Control subjects were healthy,
without acute or chronic disease and without regular drug
therapy. All the PHD/EHD as the controls were students or
employees, originated from similar social environments,
and lived a similar life style. We calculated body mass
index (BMI) from obtained subjects’ data. There was no
statistically significant difference in BMI index values
among groups of subjects. Table 1 present the basic data of
the study sample.
The study was approved by the national medical ethics
committee, and written informed consent was obtained
from each subject.
Methods
Clinical evaluation
Before entering the study, participants were examined by a
neurologist experienced in HD and a neuropsychiatrist to
rule out neurological and/or psychiatric disorders aside
from HD. The PHD subjects were carefully observed
clinically to detect motor symptoms that may precede the
obvious signs of extrapyramidal dysfunction. Clinical
J Neurol (2012) 259:921–928 923

Table 1 Basic details of the study groups (mean values ± SE)

PHD (N = 15) EHD (N = 15) PHD control (N = 15) EHD control (N = 15) Statistical test

Age (years) 34.0 ± 1.9 39.9 ± 2.4 33.9 ± 2.2 40.0 ± 2.3 Dunnett’s test
Age range (years) 24–50 23–55 24–52 26–54
BMI index 23.8 ± 0.7 23.3 ± 0.7 24.1 ± 0.6 24.0 ± 0.7 Dunnett’s test
Male 9 (60.0%) 7 (46.7%) 9 (60.0%) 7 (46.7%) v2 test
Female 6 (40.0%) 8 (53.3%) 6 (40.0%) 8 (53.3%)
CAG 44.3 ± 0.9 44.7 ± 0.9 Not defined Not defined Dunnett’s test
CAG range 40–51 40–51 Not defined Not defined
UHDRS 1.0 ± 0.4 14.9 ± 1.7a 0b 0c Dunnett’s test
UHDRS range 0–4 6–25 0 0
a
Statistical significant difference between PHD and EHD (p \ 0.05)
b
Statistical significant difference between PHD and PHD controls (p \ 0.05)
c
Statistical significant difference between EHD and EHD controls (p \ 0.05)

status of PHD/EHD individuals was evaluated by the
Unified Huntington’s Disease Rating Scale (UHDRS) [45]
motor score. No subject receiving more than 4 points was
enrolled to PHD group and patients with 5–25 points were
enrolled to EHD group. Physical examination, resting
electrocardiogram (ECG) and complete routine laboratory
tests were performed in all cases.
ANS experimental protocol
Testing was performed in the morning after abstaining
from smoking, alcohol, and caffeine for at least 8 h. The
experiment took place in a quiet and comfortable atmo-
sphere with a room temperature of 23–25°C after 15 min of
acclimatization. Tests were performed in a supine position.
The patients were asked not to perform major voluntary
movements during the measurements in order to avoid
movement artefacts.
Continuous finger arterial blood pressure was measured
by 2300 Finapres monitor (Ohmeda, USA), providing
values of systolic (SBP), diastolic (DBP) and mean arterial
pressure.
The surface electrocardiogram was monitored through
the standard lead II using a conventional ECG apparatus to
determine the average R–R interval at rest and during the
cooling period.
Laser-Doppler (LD) flux was measured with two Peri-
flux P4001 Master/4002 Satellite LD monitors (Perimed,
Sweden). The flux was calculated as the product of con-
centration of moving blood cells and velocity of moving
blood cells. The principle governing measurement of skin
perfusion has been described elsewhere [46].
LD flux, blood pressure and ECG were measured for
6 min of rest and during the 6 min of local cooling of one
hand. The LD probes (PF401) were attached to the pulp of
the index finger of both hands. Cooling was achieved with
flexible cold packs (Comfort Pack, 3 M USA,
200 9 300 mm) at 15°C; the hand was placed between two
packs.
Heart rate variability analysis
Individual R–R intervals were used for spectral analysis of
the 5 min recordings at rest. The Autoregressive Transform
method was employed. Results are expressed in Power
Spectral Density, the squared amplitude calculated for each
frequency. The area under the power spectrum curves of
the high frequency band (HF 0.15–0.4 Hz) and the low
frequency band (LF 0.04–0.15 Hz) was determined, the
former being an indicator of parasympathetic nervous
system activity [47–49] and the latter being particularly
sensitive to cardiac sympathetic nerve activity. The coef-
ficients LF/HF (sympathovagal balance), LF/(LF ? HF)
(primary sympathetic modulation of the heart rate) [50],
and baroreflex sensitivity (BRS) during rest were calcu-
lated. BRS was determined by the sequence method, based
on the computer identification in the time domain of
spontaneously occurring sequences of four or more con-
secutive beats characterized by either a progressive rise in
SBP and lengthening in R–R interval or by a progressive
decrease in SBP and shortening in R–R interval [51, 52].
Nevrokard software was used for the analysis of the
gathered data (Medistar, Slovenia).
Statistical analysis
In the resting precooling condition, the average of 6 min
LD flux, blood pressures and heart rate (HR) were calcu-
lated and used for the statistics. During cooling, we cal-
culated average values of all parameters for each 2 min
intervals of 6 min cooling period. For HRV analysis, 5 min
recording of R–R intervals were used during resting period.

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All values obtained during cooling were compared to the
resting values before the cooling by a one way ANOVA on
repeated measurements (Dunnett’s test).
We performed a one-way ANOVA (Dunnett’s test) to
test the differences between both groups of patients and the
groups of control subjects in the relative changes of LD
flux, arterial blood pressure and HR during cooling.
All results are expressed in mean values and standard
errors of means (SE) with the significance criterion
p \ 0.05.
Results
All data had normal distribution. The resting values of
SBP, DBP, HR, and LD flux are shown in Table 2. The
PHD group exhibited a significantly higher DBP compared
to the EHD group (Dunnett’s test, p \ 0.05). The resting
LD flux was nearly the same in all four groups (one way
ANOVA). In the EHD group the HF power was signifi-
cantly higher, thus their LF/HF and LF/(LF ? HF) coef-
ficients were significantly lower than those of the PHD
group (Dunnett’s test, p \ 0.05). The BRS of the EHD and
the PHD subjects did not differ. See Table 3.
No patient or healthy control subject perceived cold
packs as painful. In response to direct cooling (direct
response) LD flux decreased during the entire cooling
period for all four test groups (Fig. 1a) (Dunnett’s test,
p \ 0.05). The LD flux decrease in the PHD group was
significantly greater compared to their control. There was
no difference in the direct response between the EHD
patients and their controls or between the EHD and PHD
groups except during the last 2 min of cooling (paired
t test, p \ 0.05).
In the contralateral hand (indirect response) the LD flux
decreased in the PHD and EHD groups during the entire
cooling period (Dunnett’s test, p \ 0.05). However, in the
PHD and EHD control groups LD flux increased after a
significant fall during first 2 min of cooling (Fig. 1b)
(Dunnett’s test, p \ 0.05). There was no significant dif-
ference in the indirect response among the groups.
SBP increased significantly during cooling in all groups
(Dunnett’s test, p \ 0.05) (Fig. 2a). In contrast, DBP
increased in the EHD patients and in both control groups
(Fig. 2b) but not in the PHD subjects.
During first 2 min of cooling significantly lower SBP
and DBP was found in the PHD subjects compared to their
controls (paired t test, p \ 0.05). The difference between
the PHD and EHD patients and that between the EHD
patients and their controls was not significant.
While the PHD subjects and the PHD and EHD controls
demonstrated a significant decrease in HR during the entire
cooling period (Fig. 2c) (Dunnett’s test, p \ 0.05), the
EHD patients did not, resulting in a significant difference
between the EHD patients and their controls during the last
4 min of cooling (paired t test, p \ 0.05).
Discussion
The main findings of this study are: (1) the PHD group
responded to direct cooling with a significantly greater
reduction of cutaneous LD flux than their controls; (2) the
PHD subjects responded to cooling with smaller changes of
SBP and DBP than their controls; (3) PHD individuals at
rest and during cooling had higher SBP, DBP and HR,
although not always significantly; (4) at rest the PHD group
had a significantly greater LF and lower HF component of

Table 2 Resting values of systolic (SBP), diastolic blood pressure (DBP), heart rate (HR) and laser-Doppler (LD) flux (mean values ± SE)
PHD EHD PHD control EHD control

SBP (mmHg) 125.6 ± 5.1 114.8 ± 4.0 117.2 ± 3.2 116.2 ± 3.8
DBP (mmHg) 80.5 ± 3.7 71.3 ± 1.8a 73.2 ± 2.3 71.5 ± 2.5
HR (beats/min) 74.9 ± 3.3 67.8 ± 2.7 68.3 ± 2.3 68.3 ± 2.3
LD flux (PU) 236.1 ± 39 218.8 ± 28 243.3 ± 24 227.0 ± 21
a
Statistically significant difference between PHD subjects (N = 15) and EHD patients (N = 15) at p \ 0.05

Table 3 Resting heart rate variability values and values of baroreflex sensitivity (mean values ± SE)
PHD EHD PHD control EHD control

HF power (N.U.) 38.1 ± 3.6 59.2 ± 5.0a 50.7 ± 5.4 50.9 ± 4.7
LF/(LF ? HF) 53.6 ± 3.3 39.7 ± 4.2a 45.4 ± 3.9 43.9 ± 4.1
a
LF/HF 2.70 ± 0.3 1.31 ± 0.4 2.31 ± 0.6 2.12 ± 0.5
BRS 17.3 ± 2.5 14.5 ± 1.8 21.6 ± 4.4 16.4 ± 2.8
a
Statistically significant difference between PHD subjects (N = 15) and EHD patients (N = 15) at p \ 0.05

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Fig. 1 Relative changes in
laser-Doppler (LD) flux
(percentage of resting values)
measured on cooling hand
(a) and on the contralateral hand
(b) in PHD subjects, EHD
patients, and their controls
(asterisk indicates statistically
significant difference between
PHD and EHD at p \ 0.05;
dagger indicates statistically
significant difference between
PHD and their control at
p \ 0.05)
HRV than the EHD group; (5) the EHD patients responded
to cooling with a significantly smaller change in HR than
their controls.
The physiological effects of local cooling have been
established elsewhere [27–39]. It is generally known that
the response to moderate local cooling is normally char-
acterised by a decrease in skin blood flow, an increase of
blood pressure, and a fall in HR. The decrease of skin
blood flow is observable everywhere but is most pro-
nounced at the site of cooling [53].
In the present study the augmented direct response to
local cooling in the PHD group may result from local or
central factors. Altered interactions of mutant huntingtin
with its associated partners could contribute to affected
exocytotic processes that are involved in the translocation
of alpha2c receptors that participate in the direct micro-
vascular response to cold exposure [36–38, 54, 55]. This
would result in attenuated rather than augmented response
to cooling and could not explain our results.
An alternative explanation would be the central ANS
modification of the microvascular response. Preganglionic
sympathetic and parasympathetic neurons have been
proved to be under the control of CAN [12]. The insular,
medial, and other regions of the prefrontal cortex are
shown to be involved in high-order autonomic control [12].
Consequently, the emotional mental state of the PHD
925
subjects with possible anxious reaction could have also
contributed to the higher sympathetic activity in spite of
the previous psychological treatment and attenuated heart
rate response to mental stress [5]. This mechanism could be
at least partially challenged by the fact that, during cooling
PHD subjects respond with lesser increase of SBP and DBP
than controls which suggests that PHD subjects were not
more anxious as healthy controls.
Another possible explanation of the obtained results
would be progressive degeneration of various parts of the
CNS, from the cortex through the hypothalamus to the
brainstem [7, 8, 56, 57] during HD. After initial neuronal
loss, cerebral cortical activity decreases, hypothalamic
nuclei become disinhibited and sympathetic activity might
rise. Later, the disease also involves hypothalamic nuclei and
sympathetic activity might decrease. Similar findings were
observed by Feigin et al. 2007 [6], who found in PHD ele-
vated thalamic metabolism that fell to subnormal levels in
those subjects who developed symptoms. Ghilardi et al. 2008
[58] also found improved explicit learning in PHD when
attentional demands decrease. In the present study, higher LF
component of HRV was obtained in the PHD group, sug-
gesting predominantly higher sympathetic activity and this is
the most plausible reason for higher SBP, DBP and HR.
However, changes in our present study are too subtle to prove
the disinhibition theory of ANS dysfunction.
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Fig. 2 Relative changes in

systolic pressure (a), diastolic
pressure (b), and heart rate
(c) (percentage of resting
values) measured during local
cooling in PHD, EHD and
control groups (dagger indicates
statistically significant
difference between PHD
subjects and their control at
p \ 0.05; double dagger
indicates statistically significant
difference between EHD
patients and their control at
p \ 0.05)

In advanced HD patients clinical testing of autonomic
function has revealed a hypofunction of the parasympa-
thetic and sympathetic parts of the ANS [2, 3]. In addition,
autonomic dysfunction in mid-stage patients has been found
[4]. Nevertheless, only a few studies on ANS function in
presymptomatic and early symptomatic HD patients have
been performed. Sympathetic predominance was found in
presymptomatic and mildly and moderately affected HD
patients [20], but another study [5] found no difference in
LF and HF values between control, PHD and EHD group.
Reasons for the discrepancies might be subtle ANS chan-
ges, different clinical methods of evaluation, differences in
sample size and individual differences in disease onset and
progression. However, it does seem noteworthy that the HF
and LF components of HRV of both control groups in the
present study were between the values of the PHD and EHD
groups. We assume that measured values of the HF and LF
components of HRV vary with age and the stage of the
disease and at one point match those of healthy controls.
The results of the present study are in line with recent
observations suggesting that brain degeneration in HD
extends beyond the striatum to involve nucleus caudatus,
putamen, nucleus accumbens, pallidum and cortical
regions in the presymptomatic and early stages of the
disease [8–11, 17, 55, 59, 60]. The results are also con-
sistent with the concept of CAN, suggesting an intimate
connection of the cortical parts of CAN to each other and to
the hypothalamus [12].
Conclusions about underlying mechanisms cannot be
made on the basis of this study alone, which is limited by a

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lack of information about the functional-structural changes
in CNS over the course of the disease. Further studies are
necessary to confirm the ANS activity pattern.
Conclusions
The results confirm the hypothesis that ANS dysfunction
appears in subjects with a HD gene mutation even before
the motor symptoms of the disease manifest. The results
support the possibility that a dysfunction of high-order
autonomic centres is involved in HD.
The present study appears to be the first to evaluate
microcirculation in patients with HD. The study demon-
strates altered functioning of microcirculation in response
to local cooling, especially in the group of PHD subjects.
The use of such a harmless test would then seem appro-
priate for further evaluation of ANS dynamics in PHD.
Further cross sectional and longitudinal studies are sug-
gested in combination with structural/functional studies.
Acknowledgments The study was supported by Ministry of Higher
Education, Science and Technology (Grant No.: PO-510-381),
Slovenia.
Conflict of interest None.
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