Abstract
Neuromuscular electrical stimulation
(NMES) is commonly used for producing repetitive active muscular contraction, which
is believed that one of the outcomes is to promote plastic reorganisation after
stroke. Previous microdialysis studies showed that motor activity triggered the
increase in the levels of several amino acids/ neurotransmitters such as
glutamate. Massive release of glutamate during cerebral ischemia is the main
pathway leading to neuronal death. The objective of this study is to
investigate the effect of NMES on the levels of neurotransmitters released by
the hippocampal neurons after ischemic damage. The levels were monitored by
microdialysis for two weeks. Basal percentage of glutamate was found to be
increased after NMES intervention and then dropped gradually. This trend
persisted throughout a week (Day 1 to Day 7 after stroke), whereas the levels
remained steady in control group. The increase in glutamate may lead to
additional excitotoxic damage in hippocampus following the intervention. Taurine
level was increased after NMES on Day 1, but this pattern did not persisted
afterwards. Its basal concentration kept increasing
throughout 2 weeks while glutamate basal concentration exhibited an opposite
trend. Taurine may act as an inhibitor to the
excitotoxicity by excessive glutamate.
1.
INTRODUCTION
Stroke is a leading cause of serious, long-term
disabilities, which include loss of motor, sensory and cognitive functions.
Approximately forty percent of stroke sufferers experience moderate to severe
impairments who requiring special care. An effective rehabilitation strategy
promotes early motor recovery and thus allows the people after stroke to
reintegrate into the society as soon as possible. Neuromuscular electrical
stimulation (NMES), which generates repetitive active muscle contraction [1],
has been used as one of the post-stroke rehabilitation strategies. Liberson et al pioneered to use electrical
stimulation to improve the gait of hemiplegic people. It assists a weak or
paralyzed movement. Advanced techniques for brain imaging revealed the altered
post-stroke activation patterns of specific brain structures, which may be an
implication of plastic reorganization [2].
Bland et al’s study showed the increased level
of glutamate, taurine and asparate in hippocampus triggered by motor activity
in rats. It was suggested that these neurotransmitters may play a role in
recovery of motor function after brain injury [3]. Following cerebral ischemia,
the excessive amounts of glutamate might activate a delayed excitotoxic neuronal
injury. This excitotoxic cellular damage leads to a persistant impairment of
protein synthesis in hippocampal neurons [4]. Thus, it is still unclear that
whether NMES would exacerbate the brain damage or promote recovery following
cerebral ischemia. This study aimed at investigating the effect of NMES on the
levels of neurotransmitters (glutamate and taurine) released by the hippocampal
neurons after ischemic damage. This might provide more basic understandings on
the motor recovery mechanisms by neuromuscular electrical stimulation after
stroke.
2. METHODS
The protocols for the animal experiment were performed
according to the Procedures for the Care of Laboratory Animals and Codes of
Ethics of the
Animals from ES group (n=4) were stimulated for two
consecutive weeks (twice a day, 30 minutes/section, 5 days/week). They were
stimulated with biphasic stimulation. Frequency
of the stimulation was 20Hz and the pulse width was set to 200μsec. The
stimulation was delivered by a clinical stimulator. Current was determined by
increasing the current of the stimulator gradually until contraction of TA was
observed, which was enough to produce a dorsiflexion of ankle joint. The rats
with implanted electrodes were allowed to move freely on the treadmill while
electrical stimulation was applied on the left TA muscle (see Figure 1).
Stimulator treadmill Wires
connecting stimulator to the implanted wires
Figure 1. Rat with implanted cannula and electrodes ran on the treadmill while neuromuscular electrical stimulation was applying by the clinical stimulator.
In vivo samplings
using microdialysis were carried out in all rats 2 days before inducing stroke,
day 1, 2, 4, 7 and 14 after stroke. The microdialysis probe (BAS MD-2204) was
inserted through the cannula. The probes were connected to a microinfusion pump
(BAS Inc.) perfused with artificial cerebrospinal fluid at a constant flow rate
of 2μL min-1. The
microdialysis samples were collected every 15 minutes in vials. The total
collection time was about 5.5 hours. Glutamate and taurine were assayed by High Performance Liquid Chromatography (HPLC) with
fluorescence detection, using the precolumn derivatisation method with
ortho-phthaldialdehyde (OPA) and an automatic system from Shimadzu Instruments for
HPLC (
3. RESULTS
Data are given as means ± SEM. Significant level was not analysed due to
small sample size. Figure 2 shows the level of hippocampal glutamate (in basal percentage)
changed with time and day. Glutamate was increased after ES intervention and
then gradually dropped in the course of time (min) in both Day 1 and 7. The
levels of glutamate in control group kept steady with time (min) in both days.
Figure 2. Effect of 30min of NMES on hippocampal glutamate levels in
ES (solid lines) and Control (broken lines) animals on Day 1 (cross) and Day 7
(triangle). Data are expressed as means ± SEM. *Rats in control group received no intervention during ES duration.
As shown in Figure 3, the basal % level of
taurine was increased after ES intervention and dropped back to basal level on
Day 1, but no increase on Day 7. Similar to the glutamate levels, hippocampal
taurine in control group remained at steady levels with time on both Day 1 and
Day 7. According to Figure 4, basal concentration of glutamate in both groups
was decreasing, whereas taurine exhibited an increasing trend throughout the 2
weeks.
Figure 3. Effect of 30min of NMES on hippocampal Taurine levels in ES (solid
lines) and Control (broken lines) animals on Day 1 (cross) and Day 7
(triangle). Data are expressed as means ± SEM. *Rats in control group received no intervention during ES duration
Figure 4. Basal concentration of Glutamate and Taurine in hippocampus of rats in control and ES groups before stroke and at different days after stroke. Data are presented as means ± SEM.
4. DISCUSSION AND CONCLUSIONS
In Risedal et al’s study, cortical infarct volume
was found to be larger in the early training group compared with the control
group. It was suggested that in the presence of excitatory and toxic substances
from ischemic tissue, an additional release of glutamate induced by motor activity
may worsen the injury in the early postischemic stage [6]. In our study, there
is a trend of increase in glutamate levels in ES group after NMES intervention
throughout a week. It may imply additional excitotoxic damage triggered by the
intervention after stroke for one week.
Some studies showed
that taurine may regulate glutamate level and thus attenuate neuronal injury
during ischemia [7]. Although taurine was found to be increased only on Day 1 in
ES group after stroke, its basal concentration kept increasing throughout 2
weeks (Figure 4). While taurine was increasing, glutamate level exhibited an
opposite trend. Probably, there are some interactions between the
actions of glutamate and taurine. Taurine may inhibit the excitotoxicity caused
by excessive release of glutamate.
Further evidences
such as indexes measuring the extent of brain damage (infarct volume, deficit
scores, etc.) are necessary to explain the actual effect of glutamate induced
by NMES on the hippocampal neuronal death. In clinical studies, functional
improvement was observed in people using NMES in chronic stage of stroke, while
this study showed early NMES intervention (started one day after stroke) may
induce different effects. Future study will be focused on investigating whether
NMES intervention prescribed at different stages of stroke would result in
different neurochemical effects on the ischemic brain.
References
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Acknowledgements
The authors would like to acknowledge the colleagues at ZJU and PolyU for critical assistance and materials. The investigations were supported by the PolyU research grant and the scheme of Joint Supervision of PhD students with ZJU.