Region-Dependent Increase of
Cerebral Blood Flow During
Electrically Induced Contraction of the
Hindlimbs in Rats
Remi Chaney
1
, Philippe Garnier
1
,
2
, Aurore Quirié
1
, Alain Martin
3
, Anne Prigent-Tessier
1
* and
Christine Marie
1
1
INSERM UMR1093-CAPS, Université Bourgogne Franche-Comté, UFR des Sciences de Santé, Dijon, France,
2
Département
Génie Biologique, IUT, Dijon, France,
3
INSERM UMR1093-CAPS, Université Bourgogne Franche-Comté, UFR des Sciences du
Sport, Dijon, France
Elevation of cerebral blood flow (CBF) may contribute to the cerebral benefits of the regular
practice of physical exercise. Surprisingly, while electrically induced contraction of a large
muscular mass is a potential substitute for physical exercise to improve cognition, its effect
on CBF remains to be investigated. Therefore, the present study investigated CBF in the
cortical area representing the hindlimb, the hippocampus and the prefrontal cortex in the
same anesthetized rats subjected to either acute (30 min) or chronic (30 min for 7 days)
electrically induced bilateral hindlimb contraction. While CBF in the cortical area
representing the hindlimb was assessed from both laser doppler flowmetry (LDF
CBF
)
and changes in p-eNOS
Ser1177
levels (p-eNOS
CBF
), CBF was evaluated only from changes
in p-eNOS
Ser1177
levels in the hippocampus and the prefrontal cortex. The contribution of
increased cardiac output and increased neuronal activity to CBF changes were examined.
Stimulation was associated with tachycardia and no change in arterial blood pressure. It
increased LDF
CBF
with a time- and intensity-dependent manner as well as p-eNOS
CBF
in
the area representing the hindlimb. By contrast, p-eNOS
CBF
was unchanged in the two
other regions. The augmentation of LDF
CBF
was partially reduced by atenolol (a ß1
receptor antagonist) and not reproduced by the administration of dobutamine (a ß1
receptor agonist). Levels of c-fos as a marker of neuronal activation selectively increased in
the area representing the hindlimb. In conclusion, electrically induced bilateral hindlimb
contraction selectively increased CBF in the cortical area representing the stimulated
muscles as a result of neuronal hyperactivity and increased cardiac output. The absence of
CBF changes in cognition-related brain regions does not support flow-dependent
neuroplasticity in the pro-cognitive effect of electrically induced contraction of a large
muscular mass.
Keywords: cerebral blood flow (CBF), heart rate (HR), exercise pressor reflex, rat—brain, electrically-induced
muscle contraction
Edited by:
James (Jim) David Cotter,
University of Otago, New Zealand
Reviewed by:
Kazuto Masamoto,
The University of Electro-
Communications, Japan
Kurt Smith,
University of Victoria, Canada
Sae Uchida,
Tokyo Metropolitan Institute of
Gerontology, Japan
*Correspondence:
Anne Prigent-Tessier
Specialty section:
This article was submitted to
Exercise Physiology,
a section of the journal
Frontiers in Physiology
Received: 08 November 2021
Accepted: 04 March 2022
Published: 23 March 2022
Citation:
Chaney R, Garnier P, Quirié A,
Martin A, Prigent-Tessier A and
Marie C (2022) Region-Dependent
Increase of Cerebral Blood Flow During
Electrically Induced Contraction of the
Hindlimbs in Rats.
Front. Physiol. 13:811118.
doi: 10.3389/fphys.2022.811118
Frontiers in Physiology | www.frontiersin.org March 2022 | Volume 13 | Article 8111181
ORIGINAL RESEARCH
published: 23 March 2022
doi: 10.3389/fphys.2022.811118
INTRODUCTION
Induced skeletal muscle contraction (ISMC) has been shown to
improve muscle strength (Duchateau and Hainaut, 1988) and
functional capacity (Maddocks et al., 2016). Over the last few
years, ISMC has broadened its field of action beyond the muscle.
For instance, improvement of motor function by ISMC was
related not only to muscular changes but also to induction of
cortical plasticity (Chipchase et al., 2011). For a few years, chronic
ISMC has been envisaged as a potential substitute for aerobic
physical exercise (EX), i.e., endurance exercise such as walking,
running, cycling to improve brain health including mental health.
To support this, chronic ISMC was reported to increase the
production of myokines involved in neuroplasticity (Sanchis-
Gomar et al., 2019) as well as cerebral levels of brain-derived
neurotrophic factor (BDNF) (Ke et al., 2011; Dalise et al., 2017)a
neurotrophin playing a crucial role in EX-induced synaptic
plasticity, neurogenesis and cognition (Vaynman et al., 2004;
Lu et al., 2008; Erickson et al., 2012). There are also studies that
reported an improvement by chronic ISMC of the negative
psychological state associated with spinal cord injury (Twist
et al., 1992) and memory impairment in Alzheimer’s patients
(Scherder et al., 1995). One unresolved point concerns the
mechanisms underlying improved cognitive abilities in
response to chronic ISMC, while elucidating these mechanisms
is a prerequisite for optimizing protocols of ISMC aimed to
improve cognition.
One mechanism underlying the cognitive benefit afforded by
either chronic ISMC of a large muscular mass or chronic EX such
as running could be the repeated elevation of cerebral blood flow
as a result of increased neuronal activity, hypercapnia and
increased cardiac output (Willie et al., 2014; Smith and
Ainslie, 2017). Each increase of CBF may augment the
delivery of myokines involved in neuroplasticity into the brain.
In addition, increased blood flow in the cerebral microvasculature
may stimulate NO production as a consequence of
phosphorylation of endothelial NO synthase (eNOS) at serine
1177 by shear stress (Dimmeler et al., 1999) while NO secreted by
the endothelium of cerebral capillaries plays a key role in
neurogenesis and neuroplasticity (Chen et al., 2005; Hopper
and Garthwaite, 2006). Surprisingly, the effect ISMC of a large
muscular mass on regional CBF was never investigated. This
effect cannot be extrapolated from studies reporting CBF
elevation during acute EX. Indeed, the pattern of neuronal
activation differs between voluntary and involuntary
contraction (Wegrzyk et al., 2017) and whether neuronal
activation occurs in cognition-related brain regions during
stimulation as observed during acute EX is not known. In
addition, while many studies reported a contribution of
cardiac output to CBF (Meng et al., 2015), an independent
relationship between cardiac output and CBF was recently
questioned (Castle-Kirszbaum et al., 2021).
In this context, the present study aimed to investigate the effect
of ISMC of a large muscular mass on regional CBF. For this
purpose, CBF was measured at the microvascular level directly by
laser doppler flowmetry (LDF
CBF
) and indirectly by changes in
tissue p-eNOS
Ser1177
levels (p-eNOS
CBF
) in anesthetized rats
subjected to induced bilateral hindlimb contraction or in
unstimulated rats. Selective blockade of ß1 cardiac receptors
by atenolol, their activation by dobutamine as well as
modulation of stimulation intensity (2.5 vs. 5-fold the motor
threshold) were used as strategies to assess the contribution of
increased cardiac output to CBF changes. Changes in neuronal
activity were assessed from the measurement of c-fos levels. CBF
and c-fos levels were measured in the area representing the
hindlimb and in two cognition-related brain regions (the
hippocampus and the prefrontal cortex). Electrically induced
bilateral hindlimb contraction was performed according to an
acute stimulation protocol (stimulation for 30 min under
ketamine/xylazine or chloral hydrate anesthesia) since an
interaction between anesthesia and the effect of ISMC on
blood pressure (BP) was previously suspected by Ishide et al.
(2002) or to a subchronic stimulation protocol (stimulation of
30 min a day for 7 consecutive days under isoflurane anesthesia).
MATERIALS AND METHODS
Animals and Drugs
Animals. Experiments were conducted according to the French
Department of Agriculture guidelines (license 21-CAE-102),
approved by local ethics committee (Ethic committee of
animal experiment, Dijon, aggregation number 105) and
conformed to ARRIVE guidelines. They were carried out on
88 male Wistar rats (7 to 8 week-old) purchased from Janvier
Labs (Le Genest Saint Isle, France). Rats were housed under a
12 h/12 h light/dark cycle and allowed free access to food
and water.
Anesthetic agents. In experiments for which rats were placed
on a stereotaxic frame, volatile anesthesia could not be used.
Therefore, electrically induced contraction was first induced in
rats anesthetized with ketamine (Virbac, Carros, France)/xylazine
(Bayer, Leverkuse, Germany) that is the recommended no volatile
anesthesia in rodents. The administration of a mixture (0.115 ml/
100 g, i.p) of ketamine (75 mg/kg) and xylazine (8 mg/kg) was
preceded (15 min) by the administration of buprenorphine
(0.05 mg/kg, s.c, Buprécare, Axience, Pantin, France). An
additional injection of ketamine (35 mg/kg) was administrated
as indicated by loss of withdrawal reflex to pinching of the
hindpaw and/or spontaneous increases in heart rate (HR).
Then, anesthesia was induced by chloral hydrate. This
anesthetic agent has the reputation to not depress the
cardiovascular system even though it is not recommended
because of its depressive effect on ventilation when used at the
dose required for surgical anesthesia, its irritating effect
(peritonitis) when chronically administered by i.p route, its
cancerogenic effect when administered chronically. However,
in the present study, respiratory depression and toxicity of
chloral hydrate could not occur since rats were placed under
assisted ventilation and euthanized less than 100 min after
induction of anesthesia, respectively. Anesthesia by chloral
hydrate consisted in the administration of a 4% chloral
hydrate (Sigma-Aldrich, Saint-Quentin Fallavier, France)
solution (saline). This solution was administered under a
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Chaney et al. Hemodynamics During Induced Hindlimbs Contraction
volume of 10 ml/kg (i.p) to induce anesthesia and then
(30–40 min later) under a volume of 5 ml/kg (i.v) to maintain
deep anesthesia. Pentobarbital anesthesia (60 mg/kg) was used to
explore the effect of dobutamine on CBF. For experiments not
requiring to place rats on a stereotaxic frame, anesthesia was
induced by 4% iso flurane in a clear induction chamber and then
maintained with 2% isoflurane in air in a standard rat nose mask.
Advantage of gaseous anesthesia is that it allows a rapid
awakening as compared to anesthesia with chloral hydrate,
thus limiting potential interaction between CBF and
anesthesia. Body temperature of anesthetized rats was
maintained around 37
°
C with a heating pad.
Drugs. Atenolol (Sigma-Aldrich, Saint-Quentin Fallavier,
France), a water-soluble and specific ß1 receptor competitive
antagonist was administered at 10 mg/kg (1 ml/kg, i.v).
Dobutamine (Sigma, D0676), a water-soluble specificß1
receptor agonist was infused at 10 μg/kg/min (5.5 μL/min/
100 g for 30 min, i.v). Dosages of atenolol and dobutamine
were determined from dosages used in patients to treat
tachycardia and low cardiac output hypoperfusion states.
Electrical Stimulation
Circular (7 mm) electrodes (Contrôle Graphique Medical, Brie-
Compte-Robert, France) were connected to an electrical
stimulator (DS7AH, Digitimer, Hertfordshire,
United Kingdom) controlled by TIDA software (Tida, Heka
Elektronik, Lambrecht/Pfalz, Germany) to trigger stimulation.
After abdomen and back shaving in anesthetized rats, the anode
was placed on the abdomen and the cathode on lumbar (L6)
nerve roots to induce the simultaneous contraction of the two
hindlimbs. Electrical stimulation was induced with a rectangular
biphasic current of 100 Hz frequency, 200 µs of pulse duration
with an alternating of 6 s ON (contraction) and 3 s OFF (rest).
The current intensity was set to 2.5- or 5- fold the motor
threshold (MT) i.e., the smallest stimulation intensity to
induce the contraction of both hindlimbs (7 mA). The
intensity was continuously increased (up to 70 mA) to
maintain the target torque output.
Two protocols of stimulation were used. According to the
acute stimulation protocol, ventilated rats anesthetized with
ketamine/xylazine or chloral hydrate were subjected to a
30 min-long stimulation period. End-tidal carbon dioxide
(CO
2
) was continuously recorded with a capnograph
(CapnoScan, Kent Scientific, United States) and maintained at
35 mmHg by adjusting the ventilation rate, thus allowing us to
eliminate the effect of hypercapnia on CBF. According to the
chronic stimulation protocol, a daily (30 min) stimulation was
repeated during 7 consecutive days in rats anesthetized with
isoflurane and not placed under assisted ventilation. The rationale
for this protocol is that a treadmill activity 30 min a day during 7
consecutive days was reported by our laboratory to increase levels
of both c-fos and p-eNOS
Ser1177
in the sensorimotor cortex, the
hippocampus and the prefrontal cortex and to reduce memory
deficit induced by scopolamine (Banoujaafar et al., 2014; Cefis
et al., 2019; Pedard et al., 2019). The parameters of stimulation
did not differ between the acute and chronic protocols of
stimulation.
Methods to Investigate Cerebral Blood Flow
Changes in CBF during stimulation were assessed at the
microvascular level as interrogation of the cerebral
microvasculature provides a more accurate assessment of
actual tissue perfusion than macrovascular hemodynamic
measurements. Thus, CBF was directly assessed from laser
doppler flowmetry (LDF
CBF
) and indirectly from changes in
tissue p-eNOS
Ser1177
levels (p-eNOS
CBF
). LDF method
measures red blood cells velocity in the cerebral
microcirculation while changes in cerebral p-eNOS
Ser1177
levels
reflect changes in shear stress in vessels of the cerebral
microcirculation mainly the capillaries. Supporting changes in
p-eNOS
Ser1177
levels as a reliable marker of changes in CBF at the
microvascular level, cerebral peNOS
Ser1177
levels decreased in
response to interruption of the carotid circulation (Banoujaafar
et al., 2016) and increased in response to EX in the sensory-motor
cortex, the prefrontal cortex and the hippocampus (Cefis et al.,
2019; Pedard et al., 2019). Notably, even though eNOS
phosphorylation is induced by both flow-and receptor-
dependent mechanisms, an increase in microvasculature flow
obligatorily translates into the induction of eNOS
phosphorylation at serine 1177.
Laser Doppler Flowmetry
LDF
CBF
was expressed as arbitrary tissue perfusion units
(TPU) and continuously recorded before, during and after
acute stimulation using a probe (diameter of 1.2 mm)
connected to a laser Doppler flowmetry apparatus (BLF21,
Transonics Systems, NY, United States). After anesthesia (with
chloral hydrate or pentobarbital) and heparin administration
(50UI/100g,i.v,Lovenox,Aventis,Strasbourg,France),
arterial BP and heart rate (HR) were recorded from a
catheter inserted into a common carotid artery, the caudal
artery or a femoral artery using a BP monitor (Easy Graf,
GOULD, United States). Rats were then ventilated (Harvard
Apparatus, Fircroft, Eddenbridge, United Kingdom) with
room air through an endotracheal tube and placed on a
stereotaxic f rame (Model 900, Kopf Instruments, Tujunga,
CA, United States). The tip of the probe was placed
perpendicularly to the cortical surface and centered on the
area representing the hindlimb according to the following
stereotaxic coordinates: AP = − 1.8 mm, L = 2.8 mm from
bregma as reference (Atlas of Paxinos and Wats on). F or this
purpose, a circular area of 5 mm diameter overlying the
cortical hindlimb representation was thinned using a dental
drill until a translucent cranial plate remained. Then, this plate
was removed before positioning the probe on the dura mater.
Placement of the probe on area with large vessels was avoided.
Rats with a mean arterial BP (MABP) below 60 mmHg were
excluded for further experiments. LDF
CBF
was recorded in rats
stimulated at low (2.5x MT) or high intensity (5x MT), in rats
with or without treatment with atenolol as well as in
unstimulated rats receiving dobutamine.
Western Blotting Analysis
The cortical area representing t he hindlimb, t he hippocampus
and the pref rontal cortex were collect ed 20 m in aft er ce ssation
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Chaney et al. Hemodynamics During Induced Hindlimbs Contraction
of stimulation in rats subjected to the acute stimulation
protocol, 2 4 h after the last session of stimulation in rats
subjected to the sub-chronic sti mulation proto col and at
equivalent times in corresponding unstimulated sham rats.
Once collected, the structures were immediately weighed and
frozen at -80
°
C. Le vels of p-eNOS
Ser1177
as an indirect marker of
CBF (p-eNOS
CBF
) and of c-fos as a marker of neuronal
activation were measured by Western blotting analysis using
Stain-Free imaging technology (Biorad). Briefly, equal amounts
of protein were loaded on sodium dodecyl
sulfate–polyacrylamide (SDS-PAGE) TGX Stain-Free
FastCast gel electrophoresis (TGX Stain-Free FastCast
Acrylamide Kit, 7.5%, 1610181, Bio-Rad) and
electrophoretically transferred to polyvinylidene difluoride
(PVDF) membranes using Turbo Transblot technology
(1704150, Biorad). After blocking non-specific binding sites
for 1 h at room temperature (RT), m embran es were in cubated
overnight at 4
°
C with primary antibody directed against
p-eNOS
Ser1177
(1/1000, mouse monoclonal, 612,383, BD
Biosciences) or c-fos (1/3,000, rabbit polyclonal, GTX129846,
GeneTex). Membranes were then incubated (1 h, RT) with
secondary antibody conjugated with horseradish peroxidase
(1/25000, anti-mouse: 115-035-166, anti-rabbit: 111-035-144,
Jackson ImmunoResearch). Membranes were then placed in
Chemidoc imagin g syste ms. A st ain-free i mage of t he blot was
captured to control the total protein loading and normalize
data. Protein-antibody complexes were visualized using the
enhanced chemiluminescence Western blotting detection
system (ECL 2, 1151-7371, Fisher Scientific). Band densities
were analysed with ImageLab software (Bio-Rad) and
standardized on total protein. Gels were run in duplicate.
The appropriate amounts of total proteins to be analysed
were previously determined from concentration (increasing
amounts of proteins)/response (optical density of the band)
curves.
Experimental Design
Four sets of experiments were conducted. The first set aimed to
assess the peripheral cardiovascular effect (HR and BP
recorded from a catheter inserted in a common carotid
artery) of acute stimulation in rats anesthetiz ed with either
ketamine/xylazine (n = 12) or chloral hydrate (n =6).The
second set of experiments investigated the effect of acute
stimulation on CBF in the area representing the hindlimb,
the hippocampus and the prefrontal cortex in rats anesthetized
with chloral hydrate (n = 31). In t he same stimulated rats,
LDF
CBF
was measured in t he area representing the hindlim b
while p-e NOS
CBF
was measured in the three brain regions of
interest. HR and BP were recorded fr om a catheter in serted
either in a common c arotid artery (n =13)orinthecaudal
artery (n = 12) in order to exclude a potential interaction
between CBF and unilateral carotid occlusion. The third set of
experiments investig ated the con tribution of cardiac output to
the CBF response to acute stimulation (n = 29). It consisted in
the rec ording of HR, BP (recorded from a common caro tid
artery, a caudal artery, or a femoral artery) an d LDF
CBF
in the
area representing the hindlimb either in stimulated rats
treated with a β-blocker (n = 12), in rats stimulated at a
lower intensity (n = 5) than that used in the previous
experimentsorinunstimulatedratstreatedwith
dobutamine (n =12).Theaimofthefourthsetof
experiments (n =10)wastoinvestigatetheeffectsof
subchronic stimulation on levels of c-fos and p-eNOS
Ser1177
in the three regions of interest in isoflurane-anesthetized rats
(5 stimulated rats and 5 sham rats).
Statistical Analysis
Values are expressed as mean ± standard deviation (SD). The
normality of the values was carried out by a Shapiro-Wilk test. To
study the effect of stimulation on HR, MABP and CBF over time,
a one-way or two-way ANOVA (time x group) was performed
with time 0 or −10 min as reference time (control values). p values
were subjected to a Bonferroni correction. Differences in
p-eNOS
Ser1177
and c-fos levels between groups of rats were
assessed using unpaired t-test. Statistical significance was set at
the 5% level.
RESULTS
Preliminary Experiments to Indirectly
Assess the Effect of Stimulation on Cardiac
Output
Increased cardiac output was reported to contribute to the
elevation of flow in the cerebral arteries that occurs during
acute EX involving a large body mass (Seifertetal.,2009).
Therefore, to investigate whether cardiac output increased
during our protocol of stimulation, we measured the
response of BP and HR to acute stimulation protocol
(intensity at 5-fold motor threshold). Experim ents were first
conducted in ventilated rats anesthetized with ketamine/
xylazine (n = 12), BP and HR being recorded from a
catheter inserted into the right common carotid. Among 12
ketamine/xylazine-anesthetized rats, half died before the end
of stimulation likely as a result of cardiovascular arrest. In the
surviving rats, stimulation induced a time-dependent increase
in HR but did not change BP (Figure 1). Tach ycardia at 30 min
of stimulation r eached + 25% of the pre-stimulation values and
the HR rapidly recovered pre-stimulation values after stopping
the stimulation. From these data, increased cardiac output was
expected to occur during stimulation. However, the substantial
mortality (50%) observed in rats anesthetized with ketamine/
xylazine in combination with the suspi cion of inter acti on
between anesthesia and the effect of ISMC on BP (Ishide
et al., 2002) led us to conduct the same experiments in
artificially ventilated rats anesthetized with choral hydrate.
As observed under ketamine/xylazine anesthesia, stimulation
at 5-fold motor threshold induced a time-dependent elevation
of HR without associated changes in BP (Figure 1)inchloral
hydrate-anesthetized rats (n = 6). The superimposable
response of HR and BP to stimulation between rats
anesthetized with ketamine/xylazine and those anesthetized
with chloral hydrate indicated that stress (adrenalin
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Chaney et al. Hemodynamics During Induced Hindlimbs Contraction
secretion)- if occurs during stimulation- did not differ
between the two protocols of anesthesia. Furthermore,
unlike ketamine/xylazine, chloral hydrate did not induce
animal death. Therefore, chloral hydrate was used in the
further experiments on rats subjected to acute stimulation
protocol.
Effect of Induced Contraction on LDF
CBF
in
the Area Representing the Hindlimb
In the preceding experiment, BP was recorded from a catheter
inserted into the common carotid artery due to the ability to
easily insert a catheter into this large vessel and of the obtention
ofalargepulsatilepressurefromwhichHRcanbeestimatedwithout
any difficulty. However, vascular catheterization resulted in a
permanent occlusion of the vessel that may interact with the
response of CBF to stimulation. Indeed, acute common carotid
artery occlusion was reported to reduce basal global CBF (De Ley
et al., 1985). Therefore, the effect of the acute stimulation protocol on
local CBF was investigated in rats in which HR and BP were recorded
from either the left common carotid artery (carotid series) or the
caudal artery (caudal series). The probe was placed on the right
cortical area representing the hindlimb. This region and the two
cognition-related brain regions were then collected for further
determinations of p-eNOS
Ser1177
levels. Notably, CBF was not
measured in six rats (on 31) as their MABP dropped below
60 mmHg during LDF measure. As shown below, no statistical
difference was observed between the two series indicating that
unilateral carotid occlusion did not interact with the response of
LDF
CBF
to electrically induced contraction.
In unstimulated sham rats of both series (n = 5 rats each), a slight
(less than 7% of control values) and unsignificant decrease in HR,
MABP and CBF were observed over time (not shown). By contrast,
induced contraction (5x MT) evoked a time-dependent elevation in
LDF
CBF
and HR that rapidly returned to pre-stimulation values after
cessation of stimulation (Figure 2). Thus, at 30 min of stimulation
FIGURE 1 | Effects of induced contraction on heart rate (HR) and blood pressure (BP). HR and mean arterial blood pressure (MABP) were recorded before, during
(0–30 min) and after electrically induced contraction of hindlimbs (5x MT) in rats anesthetized either with ketamine/xylazine or chloral hydrate. Values are expressed as
means ± SD, n = number of rats. * different from pre-stimulation values at p < 0.01 and no difference between anesthetic agents after two-way (time x group) ANOVA and
Bonferroni correction.
FIGURE 2 | Effects of induced contraction on LDF
CBF
in the area representing the hindlimb LDF
CBF
in the cortical area representing the hindlimb and HR were
measured before, during (0–30 min) and after electrically induced contraction of hindlimbs (5x MT). HR was calculated from a BP trace that was recorded from the
common carotid artery (carotid series) or the caudal artery (caudal series) in rats anesthetized with chloral hydrate. Values are expressed as means ± SD, n = num ber of
rats. * significantly different from pre-stimulation values at p < 0.01 and no difference between series after two-way (time x group) ANOVA and Bonferroni correction.
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Chaney et al. Hemodynamics During Induced Hindlimbs Contraction
and as compared to pre-stimulation values, CBF was significa ntly
increased by 84% in rats of the carotid series (n =8)and65%inratsof
the caudal series (n =7)(Figure 2A, with no significant difference
between the two series). Moreover, as previously observed (see results
in 3.1) stimulation increased HR (+ 25% at 30 min of stimulation) in
both series without differences between the series (Figure 2B)anddid
not change MABP (not shown). No statistical difference in HR was
observed between carotid and caudal series.
Effect of Induced Contraction on
p-eNOS
CBF
in the Area Representing the
Hindlimb, the Hippocampus and the
Prefrontal Cortex
Regarding the experimental difficultytosimultaneouslymeasure
CBF in different brain regions with LDF and the lack of validation of
LDF method to explore subcortical CBF, changes of p-eNOS
Ser1177
expression were used as an indirect marker of changes in CBF. Levels
of p-eNOS
Ser1177
were measured in the regions of interest in rats in
which LDF
CBF
was recorded (see paragraph 3.2). The brains were
collected 20 min after cessation of stimulation (at 5x MT) in
stimulated rats or at the equivalent time in unstimulated sham
rats. The results are summarized in Figure 3. Supplemental Figure
S1 shows full membranes as well as loading controls (stain-free)
from which graphs of Figure 3 were constructed. In accordance with
elevation of LDF
CBF
in the area representing the hindlimb
(Figure 2), this region exhibited increased p-eNOS
Ser1177
levels in
response to stimulation. Thus, as shown in Figure 3A, the rise in
p-eNOS
Ser1177
expression in the area representing the hindlimb
reached +63% in the carotid series and +69% in the caudal series
(p < 0.001), as compared to values obtained in unstimulated sham
rats. By contrast, p-eNOS
Ser1177
levels in the prefrontal cortex
(Figure 3B) and the hippocampus (Figure 3C) did not differ
between stimulated and unstimulated sham rats suggesting that
CBF did not increase in these cognition-related regions.
Contribution of Cardiac Output to LDF
CBF
The contribution of cardiac output to the elevation of LDF
CBF
in
the area representing the hindlimb in response to stimulation was
first investigated by the measurement of LDF
CBF
in stimulated
rats treated with the β-blocker atenolol. Atenolol was
administrated 10 min before the onset of stimulation at 5 x
MT, HR and BP being recorded either from the left common
carotid (carotid series, n = 6) or the caudal artery (caudal series,
n = 6) with the LDF probe placed on the right area representing
the hindlimb. Results are summarized in Figure 4. Atenolol fully
prevented the chronotropic effect of stimulation either in the
carotid series or in the caudal series (Figure 4A, note the
bradycardia induced by atenolol before induction of
stimulation). By contrast, stimulation still induced a time-
dependent increase in LDF
CBF
(Figure 4B) but the increase
was significantly lower than that observed in untreated rats
(Figure 4C). Thus, CBF values at 30 min of stimulation were
26.8 and 27.6 TPU in the carotid and caudal series, respectively
(i.e., 131 and 135% of control values). BP remained unchanged
before and after stimulation in atenolol-treated rats (not shown).
Then, the cardiac response to stimulation was modulated by
changing intensity of stimulation. Thus, LDF
CBF
in the area
representing the hindlimb, HR and BP (recorded from
common carotid artery only) were measured before, during,
and after stimulation at 2.5x MT in chloral hydrate
anesthetized rats (n = 5) and compared to those obtained after
stimulation at high (5x MT) intensity . As shown in Figure 5,
stimulation at low intensity increased HR only by 10% (NS) and
CBF by 44% (p = 0.0251) at 30 min of stimulation as compared to
pre-stimulation values. These changes were significantly lower
than those evoked by high-intensity stimulation, at least from 20
to 30 min of stimulation but significantly higher (p = 0.0317) than
those observed in corresponding sham rats (not shown). Of note,
MABP remained to pre-stimulation values during stimulation at
low intensity (not shown).
FIGURE 3 | Effect of induced contraction on p-eNOS
CBF
levels of p-eNOS
Ser1177
were measured in the sensorimotor cortex (A), prefrontal cortex (B) and
hippocampus (C) 20 min after cessation of a 30 min electrically induced contraction of hindlimbs (5x MT) in rats with catheterization of the common carotid artery (carotid
series) or the caudal artery (caudal series). These rats are the same than those used to measure LDF
CBF
. Values are expressed as means ± SD, n = number of rats. ***
significantly different from sham rat values after t-test at p < 0.001.
Frontiers in Physiology | www.frontiersin.org March 2022 | Volume 13 | Article 8111186
Chaney et al. Hemodynamics During Induced Hindlimbs Contraction
Lastly, we investigated the effect of dobutamine (an agonist of
β1-receptor able to increase cardiac output but unable to alter
neuronal activity as a result to its incapacity to cross the blood-
brain barrier) in unstimulated rats. LDF
CBF
was continuously
recorded in the cortical area representing the hindlimb, BP and
HR being measured from a catheter inserted into a femoral artery
in rats anesthetized with pentobarbital. As shown in Figure 6 and
as compared to saline, dobutamine increased HR by 18% (+80
beats/min) after 30 min of perfusion (Figure 6A), while it
changed neither LDF
CBF
(Figure 6B) nor MABP (not shown).
Cerebral Effects of Subchronic Stimulation
Subchronic stimulation was used to investigate the effect of
stimulation of c-fos as a marker of neuronal activation. Levels
of c-fos and p-eNOS
Ser1177
were measured in the three regions of
interest that were collected 24 h after the last session of
stimulation. In fact, c-fos was not investigated in rats
subjected to the acute stimulation protocol because -in these
rats- the brain regions were collected 20 min after cessation of
stimulation, a time too short to reveal potential c-fos protein
upregulation. As shown in Figure 7 and as compared to
unstimulated sham rats, induced contraction increased c-fos
and p-eNOS
Ser1177
levels in the cortical area representing the
hindlimb (Figure 7A) with no change in the prefrontal cortex
(Figure 7B) and the hippocampus (Figure 7C). Supplemental
Figure S2 shows full membranes as well as loading controls
(stain-free) from which graphs of Figure 7 were constructed.
Importantly, the selective increase in p-eNOS
Ser1177
in the area
representing the hindlimb suggests that hypercapnia did not
occur during stimulation. Consistently, spontaneous
hyperventilation was noticed during each period of stimulation.
DISCUSSION
The main results provided by the present study are that 1)
stimulation increased LDF
CBF
and p-eNOS
Ser1177
levels in the
area representing the stimulated muscles but did not change
p-eNOS
Ser1177
levels in the prefrontal cortex and the
hippocampus, 2) LDF
CBF
elevation was dependent on the
intensity of stimulation, blunted by atenolol administration
and not reproduced by dobutamine administration in
unstimulated rats, 3) stimulation resulted in a selective
augmentation in c-fos levels in the area representing the
stimulated muscles.
In opposition to the plethora of studies on the cardiovascular
(CV) effects of physical activity, the CV effects of ISMC are poorly
documented. Nevertheless, tachycardia was consistently reported
during induced contraction (Crayton et al., 1979; Ishide et al.,
2002). More controversial and for still hypothetical reasons
(Ishide et al., 2002) is the effect of ISMC on arterial BP (an
augmentation, a decrease or no effect), while BP increases during
physical activity as a consequence of increased cardiac output. In
the present study, tachycardia increased by 25% after 30 min of
stimulation while BP remained to pre-stimulation values
irrespective of the anesthetic agent (ketamine/xylazine or
chloral hydrate). Of note, tachycardia without an associated
change in BP was previously reported during bilateral sciatic
nerve stimulation in anesthetized rats (Ishide et al., 2002).
Tachycardia during stimulation related to activation of bulbar
sympathetic centers and subsequent increased sympathetic
outflow as evidenced from the complete prevention of
tachycardia by atenolol. Notably, while sympathetic activation
observed during voluntary contraction is driven by the central
command (feed-forward mechanism originating from higher
brain centers) (Williamson et al., 2006), the exercise pressor
reflex (a feed-back mechanism originating from skeletal
muscle) and the arterial barore flex (a negative feed-back
FIGURE 4 | Cardiovascular effect of induced contraction in atenolol-
treated rats. Atenolol (10 mg/kg, i.v.) was administrated 10 min before (time
-10) bilateral hindlimb contraction at 5x motor threshold. (A) HR was
calculated from BP trace that was recorded from the common carotid
artery (carotid series) or the caudal artery (caudal series), (B) LDF
CBF
was
measured in the cortical area representing the hindlimb, (C) Difference in CBF
expressed as % of cont rol values at 30 min of stimulation in rats treated with
atenolol (full squares) vs. untreated rats (empty squares). Values are
expressed as means ± SD, n = number of rats. * significantly different from
pre-stimulation values at p < 0.01 and no difference between series after two-
way (time x group) ANOVA and Bonferroni correction.
Frontiers in Physiology | www.frontiersin.org March 2022 | Volume 13 | Article 8111187
Chaney et al. Hemodynamics During Induced Hindlimbs Contraction
mechanism originating from the carotid sinus and aortic arch)
(Fisher et al., 2015), it is driven only by the exercise pressor reflex
during our protocol of involuntary contraction since the central
command is absent during stimulated contraction and BP did not
change during stimulation. Thus, only the activation of sensory
fibers originating from muscles may be responsible for the
sympathetic activation observed during stimulation. According
to our protocol, activation of these fibers was due not only to
muscle contraction but also to electrical stimulation of the dorsal
roots (that contain sensory fibers).
To the best of our knowledge, our study is the first to
investigate the effect of involuntary contraction involving a
large muscular mass on regional CBF. CBF was directly and
indirectly investigated by LDF measures (LDF
CBF
) and changes in
p-eNOS
Ser1177
levels (p-eNOS
CBF
), respectively. Our results
showed a region-dependent increase in CBF during electrically
induced bilateral hindlimb contraction. More precisely, LDF
CBF
and p-eNOS
CBF
increased in the area representing the hindlimb,
while p-eNOS
CBF
did not change in cognition-related brain
regions in rats subjected to acute stimulation. The mechanisms
that control blood supply to the brain are complex and multiple.
However, both local factor i.e neuronal activity and systemic
factor i.e BP, arterial partial pressure of CO
2
and cardiac output
are involved (Willie et al., 2014). In the present study, LDF
CBF
in
the cortical area representing the hindlimb related neither to
hypercapnia nor change in BP as end-tidal CO
2
pressure was
maintained at 35 mmHg during stimulation by increasing the
ventilation rate and BP remained to pre-stimulation values
during stimulation. By contrast, our results on atenolol and
stimulation intensity support the contribution of increased
cardiac output to LDF
CBF
elevation. However, despite the
elevation of cardiac output evoked by stimulation, p-eNOS
CBF
was not increased in all the regions examined indicating that
increased cardiac output cannot by itself induce CBF elevation.
Consistently, dobutamine failed to reproduce the effect of
stimulation on LDF
CBF
. Of note, pharmacologically induced
cardiac output and MABP elevation by dobutamine was
previously reported to not change global CBF in monkeys
FIGURE 5 | Influence of stimulation intensity of induced contraction-associated cardiovascular effects. HR and LDF
CBF
in the cortical area representing the hindlimb
were recorded before, during (0–30 min) and after electrically induced contraction of hindlimbs at 2.5 or 5x motor threshold. Values are expressed as means ± SD, n =
number of rats. * significantly different from pre-stimulation values at p < 0.01,
#
significant difference between low and high intensity after two-way (time x group) ANOVA
and Bonferroni correction.
FIGURE 6 | Cardiovascular effects of dobutamine. Dobutamine (10 μg/kg/min, 5.5 μl/min/100 g for 30 min, i.v) or saline was perfused to unstimulated rats. (A) HR
was calculated from BP trace that was recorded from the femoral artery, (B) LDF
CBF
was measured in the cortical area representing the hindlimb. Values are expressed
as means ± SD, n = number of rats. * significantly different from control values after one-way ANOVA and Bonferroni correction at p < 0.05.
Frontiers in Physiology | www.frontiersin.org March 2022 | Volume 13 | Article 8111188
Chaney et al. Hemodynamics During Induced Hindlimbs Contraction
(Bandres et al., 1992) while this strategy was recently reported to
increase blood flow in the external carotid artery and reduce flow
in the internal carotid artery in human beings (Ogoh et al., 2017).
Such redistribution of cardiac output to extra-cranial structures
likely emphasizes the importance of preventing brain
overperfusion. In other words, increased cardiac output
observed during stimulation cannot increase CBF in regions
where neuronal activation is absent. This is confirmed by our
results on c-fos that revealed a selective increase in neuronal
activity in the area representing the hindlimb after chronic
stimulation and pointed the importance of the voluntary
nature of the contraction to expect increased neuronal activity
in the cognition-related brain regions. The absence of c-fos
elevation in the cognition-related brain regions is in line with
a recent study showing no change in hippocampal c-fos in mice
subjected to bilateral hindlimb contraction twice a week during
7 weeks (Gardner et al., 2020). Our results showing that increased
levels of c-fos coexisted with increased levels p-eNOS
Ser1177
confirm that it is neuronal activation that drives CBF elevation
at the microvascular level during stimulation and argue for a
contribution of increased cardiac output to CBF elevation only in
the region where increased neuronal activity is present. Notably,
EX (treadmill activity, 30 min a day for 7 consecutive days) was
reported by our laboratory to induce c-fos upregulation and
phosphorylation of eNOS at serine 1177 not only in the
sensorimotor cortex but also in the hippocampus and the
prefrontal cortex (Banoujaafar et al., 2014; Cefis et al., 2019;
Pedard et al., 2019). These data are in line with studies that
reported increased CBF (Nishijima et al., 2012) and neuronal
activation (Nguyen et al., 2004) in cognition-related brain regions
during EX. Such difference between induced and voluntary
contraction indicates that muscle contraction alone cannot
fully reproduce the effect of EX on cognition-related brain
regions. Moreover, increased neuronal activity in the area
representing the hindlimbs is likely lower during stimulation
than during EX at least in rats. Indeed, assuming that motor-
sensory overlap is important for the hindlimb representation in
rats (Hummelsheim and Wiesendanger, 1985), neuronal
activation in this region involves both the voluntary motor
command and activation of sensory fibers originating from
active muscles during EX as expected from the projection on
the somatosensitive cortex of proprioceptive information
(Landgren and Silfvenius, 1969; Wiesendanger and Miles,
1982) but only the latter during stimulation. Supporting this,
changes in p-eNOS
Ser1177
and c-fos levels in the area representing
the hindlimb were higher after EX than after subchronic
stimulation. Of note, it is unlikely that anesthesia was a
confounding factor in the interpretation of our results since
neither chloral hydrate nor isoflurane reduced basal CBF as
compared to conscious rats (Suzuki et al., 2021).
Limitations of the study. A limitation of the present study is
that CBF changes in cognition-related brain regions were
indirectly assessed from changes in p-eNOS
ser1177
.This method
is less accurate than LDF
CBF
method for two reasons. The first
reason is that it requires to compare p-eNOS
Ser1177
levels in a
group of sham unstimulated rats vs. a group of stimulated rats,
while by contrast LDF
CBF
is measured before and after
stimulation in the same rats. However, as shown in
supplemental figures, the interindividual variability in
p-eNOS
Ser1177
levels in unstimulated rats is low. The second
reason is that p-eNOS
Ser1177
levels were measured by Western
blotting analysis that is a semi quantitative method. Another
limitation of the present study is the lack of demonstration that
increased CBF in the area representing the hindlimb was
FIGURE 7 | Effect of subchronic stimulation on p-eNOS
Ser1177
and c-fos brain levels. Levels of c-fos and p-eNOS
Ser1177
were measured in the area representing
the hindlimb (A), prefrontal cortex (B) and hippocampus (C) 24 h after the last session of a 30 min electrically induced contraction of hindlimbs (5x MT) repeated during 7
consecutive days in rats anesthetized with iso flurane and in sham unstimulated. Values are expressed as means ± SD, n = number of rats. * (p < 0.05) and ** (p < 0.01)
significantly different from sham rat values after t-test.
Frontiers in Physiology | www.frontiersin.org March 2022 | Volume 13 | Article 8111189
Chaney et al. Hemodynamics During Induced Hindlimbs Contraction
effectively driven by increased neuronal activity even though
increased CBF was observed only in regions with increased
neuronal activity. Nevertheless, the possibility that increased
CBF in the area representing the stimulated muscles involved
the activation of the intracerebral vasodilating cholinergic nerve
fibers originating from the basal forebrain (Sato et al., 2001)
cannot be excluded. However, evidence that activation of the
basal forebrain observed during mastication muscle activity was
induced by the cerebral command from the motor cortex,
independently of feedback from contracting muscles
(Hotta et al., 2020) does not argue for the
occurrence of basal forebrain activation during hindlimb
stimulation.
We concluded that stimulated contraction of a large muscle
mass increases CBF in the area representing the stimulated
muscles but not in cognition-related regions and that this
selective CBF increase involves an increase in both neuronal
activity and cardiac output. The absence of CBF changes in
cognition-related brain regions does not support the
involvement of flow-dependent neuroplasticity (cerebral
hemodynamics) in the pro-cognitive effect of electrically
induced contraction. From a physiological point of view, our
results provide evidence that local CBF does not increase in
response to increased cardiac output alone (without changes in
BP and arterial pCO
2
) at least in regions where metabolic needs
are not increased. Importantly, during the review processing of
the present study was published a work that explored the
effect of electrical myostimulation on large muscle mass on
CBF in normal human beings using a color-coded ultrasound
system (Ando et al., 2021). The authors reported increased
flow in the internal carotid artery (+12%), but no change in
the vertebral artery. Regarding the a ssociation observed
between CBF and pCO
2
changes for the carotid, but not
the vertebral circulation, the d ifferential effect of
stimulation between the two circulations was related to the
difference in the cerebrovascular response to hypercapnia.
However, the mechanisms underlying increased flow in the
internal carotid artery were not investigated. Our results
support the involvement of an increase in both neuronal
activity and cardiac output.
DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in
the article/Supplementary Material, further inquiries can be
directed to the corresponding author.
ETHICS STATEMENT
The animal study was reviewed and approved by Ethic committee
of animal experiment, Dijon, aggregation number 105.
AUTHOR CONTRIBUTIONS
AT, CM, AM and PG contributed to the study design. RC
performed the research. RC and CM conducted the statistical
analysis and interpretation. RC, CM and AT prepared the
manuscript. All authors contributed to the critical revision of
the manuscript.
FUNDING
The authors thanks Hayat Banoujaafar for their technical support
and the Foundation of Medical Research for their financial
support (file number: PBR202006012208).
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at:
https://www.frontiersin.org/articles/10.3389/fphys.2022 .811118/
full#supplementary-material
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Conflict of Interest: The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a
potential conflict of interest.
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Frontiers in Physiology | www.frontiersin.org March 2022 | Volume 13 | Article 81111811
Chaney et al. Hemodynamics During Induced Hindlimbs Contraction
