Changes in plasma calcitonin gene-related peptide and serum neuron specific enolase in rats with acute cerebral ischemia after low-frequency electrical stimulation with different waveforms and intensities＊☆

Department of Rehabilitation Medicine, Key Laboratory of Rehabilitation Medicine of Sichuan Province, West China Hospital, Sichuan University, Chengdu 610041, Sichuan Province, China

lNTRODUCTlON

Calcitonin gene-related peptide (CGRP), a product of the calcitonin gene, is a new type of endogenous neural peptide.CGRP is widely distributed in the brain, and acts on neurons in the amygdala, hippocampus and hypothalamus as well as other regions[1].CGRP can induce vasodilationviaCGRP receptors in human cerebral arteries of various diameter and other large arteries[2-3].Following ischemia, CGRP can improve cerebral blood flow, decrease cerebral edema, and promote the survival of ischemic neurons[4].CGRP also significantly decreases infarction volume[5]and reduces tissue damage in brain tissue[6].Measurement of changes in CGRP levels could be helpful in determining the severity of brain ischemia and evaluating the efficacy of the interventions.

Neuron specific enolase (NSE) is a dimerized intracellular enzyme involved in glucose metabolism that is presents in neuronal cell bodies of the central or peripheral nervous system.After cerebral infarction or injury neurons release NSE is released into the cerebrospinal fluid and passes through the blood-brain barrier to the plasma, resulting in elevated serum NSE.A higher concentration of serum NSE is associated with more severe brain damage[7-8].Therefore, serum NSE is considered a biological indicator of neuronal damage[8-9]that can give useful information about short- and long-term neurological outcome[10].Several studies have used measurements of serum NSE to determine the effectiveness of the interventions or whether the intervention would lead to neuronal injury[11].

Starting in the 1960s, technological advances have widened the field of electrical stimulation (ES) to include various applications in physical medicine and rehabilitation.Primarily, ES was used in muscular training programs to improve skeletal muscle force in individuals with muscular wasting or atrophy.Recently the influence of low-frequency ES has been described in various clinical conditions such as seizures,migraine, Alzheimer’s disease, traumatic brain injury and neuropathic pain[12-13].However, its effect on CGRP and NSE after cerebral ischemia remains poorly understood.

The waveform and intensity are the important parameters which may influence the effect of ES[14].In a broader attempt to provide some basis for clinical research and practice, we hypothesized that low-frequency ES with different waveforms and intensities would have effects on the levels of plasma CGRP and serum NSE and the volume of brain infarction in rats with acute cerebral ischemia.

RESULTS

Quantitative analysis of experimental animals

A total of 48 healthy, male, Sprague-Dawley rats were included in the experiment.These experimental rats were equally and randomly assigned to six groups:sham-surgery; model [middle cerebral artery occlusion(MCAO)]; ES I (MCAO and ES treatment with a square wave at 2-4 mA and 3-8 Hz; ES II (MCAO and ES treatment with a square wave at 4-6 mA and 3-8 Hz); ES III (MCAO and ES treatment with a triangular wave at 2-4 mA and 2-6 Hz); ES IV (MCAO and ES treatment with a triangular wave at 4-6 mA and 2-6 Hz).ES treatment was conducted at 24 hours following cerebral ischemia (supplementary Figure 1 online).All the rats were included in the final analysis.

Effect of ES at different waveforms and intensities on the plasma CGRP and serum NSE content in rats with acute cerebral ischemia

The plasma CGRP and the serum NSE content was determined with radioimmunology at 5 days post-ES.The plasma CGRP decreased and the serum NSE increased in the MCAO model group compared with the sham group (P＜0.01).The plasma CGRP significantly increased and serum NSE significantly decreased in ES I, ES II, ES III and ES IV groups compared with the model group (P＜0.05 orP＜0.01), and there were no significant differences between the four ES groups(Table 1).

Table 1 Plasma CGRP and serum NSE content in each group

Effect of ES at different waveforms and intensities on the volume of brain infarction in rats with acute cerebral ischemia

Image-Pro Plus software analysis showed that the infarction volume increased in the model group compared with sham-surgery group at 5 days post-ES (P＜0.01), significantly decreased in ES I, ES II, ES III and ES IV groups compared with the model group(P＜0.01), and there were no significant differences between the four ES groups (P＞ 0.05; Figure 1).

Figure 1 The infarction volume in each group.aP＜0.01,vs.sham-surgery group; bP＜0.01, vs.model group.Data are expressed as mean±SD of 8 rats in each group.A one-way analysis of variance was performed to test values separately and the LSD-ttest was used to compare the values among groups.ES: Electrical stimulation.

DlSCUSSlON

When ES was applied to the rats’ necks, it stimulated the skin, fascia, muscles, vascular, nerves, and other soft tissues.The ES could be seen as neuromuscular stimulation when it stimulated muscles and nerves.The nociceptive receptors from skeletal muscle are located in the fascia and blood vessels[15], and neuromuscular stimulation can act as a proprioceptive stimulation[16].Many previous studies have shown that ES treatment can increase CGRP.A review summarized that antidromic activation of group IV somatic afferent nerves produced CGRP and had a vasodilative affect on skeletal muscle blood flow[17].Kurosawaet al[18]showed that the increase in meningeal blood flow following ES treatment on the dura mater is mediated by the release of CGRP and speculated that the dural afferent and sympathetic and parasympathetic efferent nerve fibers contributed to this response.Olivar’s study[19]suggested that electrically evoked neurogenic vasodilation in rabbit basilar artery has a large component resulting from the release of sensory neuropeptides, in particular CGRP.Furthermore, ES-induced muscle contraction could produce CGRP mRNA and increase CGRP[20].The effect of ES and the consequent liberation of CGRP from perivascular sensory nerve fibers cannot be inhibited by CGRP scavengers[21].Therefore, we suggest that in our study when low-frequency ES is applied to the rats’ neck complex actions on sensory and motor afferents lead to the release of CGRP, thus decreasing the infarction volume of the brain.

A study showed that peripheral ES could increase brain blood flow in ischemic or normal rats[22].As a result of the increasing blood flow in the brain, the collateral circulation of the ischemic side increased, the ischemic penumbra was rescued and neuronal damage was reduced;leading to a decrease in NSE release.A large number of studies have shown that oral and pharyngeal stimulation can promote functional reorganization of the cerebral cortex[23-24].Neuromuscular stimulation of pharyngeal muscles can re-establish a swallowing pattern[25].We speculated that the movement induced by the low-frequency ES might stimulate the reorganization of the brain and affect the levels of plasma CGRP and serum NSE in rats with cerebral ischemia.

In addition, when ES was applied to the neck of the rats,the vagus nerve in the neck could be stimulated.Ayet al[12]conducted ES on the cervical part of the right vagus nerve on the rats with cerebral ischemia and found that the volume of the ischemic lesion was smaller and the function scores were better in the experimental animals compared with control animals.This evidence suggests that stimulation of the soft tissues in the neck including the underlying vagus nerve may contribute to the change in the plasma CGRP, serum NSE and infarction volume.The present study showed that low-frequency ES with either square- or triangular-wave forms and low or high intensity could increase the plasma CGRP, decrease the serum NSE and decrease the infarction volume of the brain in rats with cerebral ischemia.Interestingly, there was no significant difference among different wave forms or intensities.

MATERlALS AND METHODS

Design

A randomized, controlled, animal experiment.

Time and setting

The experiment was performed at the Neurobiological Laboratory of Sichuan University, China from October 2008 to March 2009.

Materials

A total of 48 healthy, male, specific pathogen free Sprague-Dawley rats, aged 2-3 months and weighing 180 ±10 g, were provided by the Laboratory Animal Center of Sichuan University, China (certification No.SCXK2008-10).The rats were housed under controlled temperature 20±2°C and lighting conditions (07: 00 to 19: 00), with food and water made availablead libitumthroughout the experiments.The experimental procedures were performed in accordance with theGuidance Suggestions for the Care and Use of Laboratory Animals,formulated by the Ministry of Science and Technology of China[26].

Methods

Establishment of acute cerebral ischemia model

A focal cerebral ischemia model was established in all groups except the sham-surgery group by occlusion of the right middle cerebral artery using the modified thread embolism method[27].The rats were anesthetized by abdominal injection of 10% chloral hydrate (0.3 mL/ 100 g).The proximal parts of the right common carotid artery and internal carotid artery, as well as the approaching bifurcation of the right external carotid artery, were ligated.Next, a standardized occlusion was established by advancing fishing thread exactly 17 mm into the internal carotid artery, from the origin of the external carotid artery, in each rat.Rectal temperature was maintained at 37.0-37.5°C during the procedure.The incision was sutured and the animals were kept warm until they recovered from anesthesia.In the sham-surgery group an incision was made in the neck skin but no vascular occlusion was performed.

The model was considered successful if the rat regained consciousness and exhibited one of following symptoms following surgery[27]: (1) flexion of wrist and elbow, and internal rotation of the shoulder on the forelimb contralateral to the injured hemisphere; (2) lack of strength of the left forelimb, and/or falling down towards the left side while walking; (3) crawling or circling towards the left side and restlessness.Rats were excluded if they presented the following symptoms: (1)adduction without extension on the forepaw opposite to the infarction hemisphere; (2) subarachnoid hemorrhage observed during dissection; (3) death prior to the appointed time points.

Low-frequency ES treatment

Preliminary experiments determined that a current between 2 mA and 6 mA was sufficient to produce electrical stimulation while remaining within the rats’tolerance levels.At 24 hours of ischemia, rats in groups ES I–IV received low-frequency ES using a pair of bipolar electrodes on the cervical muscle groups.The rats were fixed in place by the rat holder without anesthesia.The rats in ES I were treated with a square wave at 2-4 mA and 3-8 Hz; rats in ES II were treated with a square wave at 4-6 mA and 3-8 Hz; rats in ES III were treated with a triangular wave at 2-4 mA and 2- 6 Hz; and rats in ES IV were treated with a triangular wave at 4-6 mA and 2-6 Hz.Figure 2 shows the oscillograph of the square wave and triangular wave.

ES was applied for 10 minutes, twice per day and maintained for 5 days.A low-frequency electrical stimulator was purchased from Sichuan University (Chengdu, Sichuan, China).The sham-surgery group and MCAO model groups were fixed in the same way for 10 minutes,twice per day, without current stimulation.

Figure 2 Oscillograph of the electrical stimulation.

Determination of plasma CGRP in femoral artery

Rats in all groups were decapitated on day 7 after MCAO(5 days after ES).Two blood samples of 2 mL each were taken from the femoral artery.EDTA 30 μL and aprotinin 40 μL were added to the first blood sample, which was centrifuged at 3 000 r/min for 10 minutes at 4°C and stored at -20°C for the test of plasma CGRP content.The CGRP test was a double antibody radio-immunoassay performed as previously described[1]:100 μL aliquots of sample and standard rat CGRP were mixed with 100 μL purified rabbit anti-human CGRP polyclonal antiserum and incubated for 48 hours at 4°C.Using the 100 μL125I-chloramine T method[1], labeled CGRP (human recombinant-125I-CGRP) was added(6 000 counts per min/100 μL) and the solution was incubated for an additional 24 hours.Free and antibody-bound CGRP were separated with 50 μL goat anti-rabbit solid phase second antibody coated cellulose suspension.Samples were left at room temperature for 30 minutes.The reaction was blocked with 1 mL distilled H2O.Samples were then centrifuged at 3 000×gfor 20 minutes at 4°C and the supernatants were decanted.Pellets were counted in a gamma counter (Wallac, Turku, Finland) for 3 minutes.The count value was input into a computer and processed with the professional RIA application software (Wallac), choosing the four-parameter Logistic Model to compute the concentration of the substance.The detection limit of the CGRP assay was 3.9 pmol/L and the intra- and inter-assay coefficients of variation were 5% and 14%, respectively.CGRP kits were provided by Beijing Puer Weiye Biotechnology Limited Company, China.

Determination of serum NSE content in femoral artery

Rats in all groups were decapitated on day 7 after MCAO(5 days after ES).Two blood samples of 2 mL each were taken from the femoral artery.The second sample was centrifuged at 3 000 r/min for 10 minutes at 4°C for the test of serum NSE content.All samples were sealed in EP tubes and preserved at -20°C.The NSE was tested by a double antibody radio-immunoassay[28].NSE kits were provided from Beijing Puer Weiye Biotechnology Limited Company, China.

Determination of infarction volume in brains

Brains were sectioned into 2-mm-thick coronal slices with the aid of a brain matrix on day 7 after MCAO (5 days after ES).Slices were placed in the vital dye 2, 3,5-triphenyltetrazolium chloride (2%; Sigma, St Louis, MO,USA) at 37°C in the dark for 30 minutes and then stored in phosphate-buffered 4% paraformaldehyde overnight prior to analysis.The area of damaged parenchyma(unstained tissue) was measured on the posterior surface of each slice using Image-Pro Plus software (Media Cybernetics, Maryland, USA).Whole brain infarction volume was calculated as the sum of the infarct area per slice multiplied by the slice thickness.Both the surgeon and image analyzer operator were blinded to the treatment given to each animal.

Statistical analysis

All data values are presented as mean±SD.A one-way analysis of variance was performed to test values separately and the LSD-ttest was used to compare the values among groups.Statistical analysis data were analyzed by SPSS 13.0 software (SPSS, Chicago, IL, USA) andP＜0.05 was considered statistically significant.

Author contributions:Qiang Gao participated in the study design, statistical analysis, and revision of the manuscript.Yonghong Yang and Shasha Li carried out the experiments and drafted the manuscript.Jing He was responsible for study design and data analysis.Chengqi He participated in the study design and wrote the manuscript.

Conflicts of interest:None declared.

Funding:This project received financial support from the National High-Tech R&D Program of China (863 Program; Independent development and clinical therapeutic evaluation research on dysphagia therapeutic apparatus for dysphagic post-stroke patients), No.2007AA022Z482.

Ethical approval:The project gained full ethical approval from the Ethics Committee of Sichuan University in China.

Supplementary information:Supplementary data associated with this article can be found, in the online version, by visiting www.nrronline.org, and entering Vol.6, No.28, 2011 item after selecting the “NRR Current Issue” button on the page.

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