Potentiation of ischemia-related behavioral altera-t i o n s by electro-acupuncture in gerbils

Leonardo Restivoa,b

Silvia Middeia

Luo Mingfuc

Rosaria Reggiod

Enrica Passinoa,b

Rita Formisanoa

Martine Ammassari-Teulea,b

a IRCCS S. Lucia Foundation, Rome, Italyb CNR Institute of Neuroscience, Laboratory of Psychobi-ology and Psychopharmacology, Rome, Italyc Department of Neurobiology, Institute of Acupuncture,China Academy of Traditional Chinese Medicine, Beijing,C h i n ad Department of Pharmacology, Italian Superior Instituteof Health, Rome, Italy

Reprint requests to: Dr L. Restivo, CNR Institute of Neuroscience, Laboratory of Psychobiology and Psychopharmacology,IRCCS S. Lucia Foundation, Via Ardeatina, 306 - 00179 Rome - ItalyE-mail: l.restivo@hsantalucia.it

Accepted for publication: February 3, 2004

Summary

Gerbils subjected to global ischemia or sham-ischemiareceived electro-acupuncture (EA) or sham EA atpoints 26 Du (Renzhong) and 8 Du (Junsuo). All ani-mals were then tested for motor activity in an openfield, and for spontaneous alternation in a T maze. Re-sults show that EA alone did not affect any behavioralparameter. Ischemia alone increased motor activitywithout significantly interfering with spontaneous al-ternation. EA in ischemic gerbils potentiated the in-crease of motor activity and elicited a decrease inspontaneous alternation. Thus, our data show an inter -action between global ischemia and EA applied at spe-cific acupoints which, however, consists of a potentia-tion rather than an alleviation of the behavioral alter-ations consecutive to the ischemic insult.

KEY WORDS: electro-acupuncture, functional recovery, global is -

chemia, motor activity, spontaneous alternation.

Introduction

Global transitory ischemia can produce permanent neu-rological alterations in animals as in man. In rodents,and particularly in gerbils, several areas are remarkably

sensitive to ischemia with even brief occlusion of thecarotid arteries being found to induce extensive cellloss in the CA1 hippocampal subfield, the dorsolateralstriatum, the lateral septum, and layer 5 of the somato-sensory cortex (1-3) that is apparent as soon as oneday after the ischemic episode (4). As a consequenceof this targeted neuronal damage, gerbils subjected toglobal ischemia show sensory motor deficits togetherwith a number of behavioral alterations such as in-creased locomotor activity (4,5) and poor performancein hippocampal-dependent memory tasks (6-9).There is evidence that electro-acupuncture (EA) ap-plied in various body points, identified by Chinese tradi-tional medicine, can exert a neuroprotective effect oreven enhance recovery of neurobiological alterationsfollowing experimental ischemia. For example, in therat, EA applied at the late stage of the ischemic insulthas been shown to restore the amplitude of somatosen-sory evoked potentials (10), the cortical levels of nitricoxide (NO) and endothelin (11), and the extracellularcontent of glutamate, aspartate and taurine in the stria-tum (12). EA has also been found to stimulate mecha-nisms involved in neuroprotection, such as the expres-sion of basic fibroblast growth factor in astrocytes (13).However, to the best of our knowledge, no study has,until now, been conducted to examine whether EA isable to alleviate ischemia-related behavioral alterations.To investigate this possibility, gerbils with ischemia pro-duced by bilateral occlusion of carotid arteries or sham-ischemia were subjected to EA or sham EA and thentested for locomotor activity in an open field (experi-ment 1) and for spontaneous alternation in a T maze(experiment 2).

Materials and methods

Forty male three-month-old gerbils purchased from theCharles River rearing center (Calco, CO, Italy) wereused. They were housed in groups of 4 in a room with a12h:12h light-dark cycle (lights on: 07.00-19.00) andfood and water ad libitum. At the beginning of the ex-periments, they were approximately three months oldand their weights ranged from 60 to 75 g. Surgery wasperformed under isofluorane anesthesia (3% during theinduction phase and 1% during the maintenancephase) delivered in a mixture of 30% O2 and 70% NO2

by means of a Fluotec 3 vaporizer. The animals wereplaced in supine position and a midline incision of thecutaneous tissue was performed to expose the carotidarteries. Global ischemia (ISCH) was performed byclamping the carotid arteries with microaneurismal clipsfor ten minutes, while the procedure for sham ischemiasimply omitted the occlusion step. EA was administeredat the same time as ischemia or sham ischemia in the

Functional Neurology 2004; 19(1): 19-23 19

anesthetized animals. That is, the needles were insert-ed in the muscle at the intended acupoints (see dia-grams published elsewhere showing the location ofacupoints Du 26 (14) and Du 8 (15) in rodents) fiveminutes before the incision of the cutaneous tissue,then left in situ during surgery and for an additional pe-riod of 20 minutes. EA consisted of a total 30-minuteelectrostimulation (7 Hz, 5 mA). On each of the follow-ing seven days, the animals, non anesthetized, wererestrained in a plastic cage and subjected to EA, againfor 30 minutes. The acupoints used were points Du 26(Renzhong) and Du 8 (Junsuo). Traditionally, stimula-tion of point Du 26 (Renzhong) is used to enhance arte-rial blood pressure and heart rate in infarcted subjects(16), whereas stimulation of point Du 8 (Junsuo), fre-quently associated with stimulation of Du 16 (Feng Fu),has been reported to exert a neuroprotective effect no-tably by reducing the level of inducible NO synthase inischemic animals (17). Sham EA was performed simplyby restraining the animals in the cage used for deliver-ing EA. Accordingly, the following groups were includedin the experimental design: ISCH+EA, ISCH+sham EA,Sham ISCH+EA, Sham ISCH+Sham EA (controlgroup). In addition, to avoid possible confounds result-ing from aspecific factors, an additional group of is-chemic subjects receiving EA in two non acupoints lo-cated in the same area was considered (ISCH+pseudoEA). All groups had 8 subjects.Behavioral testing started 24 hours after the last EAstimulation and was carried out during a single day.Five animals (one per condition) were subjected to be-havioral testing on the same day. Experiments wereconducted in compliance with the European Council di-rective (86/609/EEC) on the use and care of laboratoryanimals. Animals were first tested for motor activity inan open field (Auto Track System, Columbus Instru-ments, Columbus Ohio, USA), then for spontaneousalternation in a T maze. The open field consisted of asquare cage (perimeter 40×4 cm) with a transparentfloor. Infrared light sources situated on two oppositesides of the cage at two different heights (5 cm and 15cm) from the floor allowed recording of various behav-ioral parameters, such as distance traveled, time spentresting, and number of rearing episodes. These behav-iors were quantified through an interface (Auto Track)connected to a PC. The dependent variable collectedwas the distance (in cm) travelled in a period of fiveminutes. The spontaneous alternation phenomenon isthe natural tendency of animals to explore alternatelythe right arm and the left arm of a T maze across suc-cessive runs, even in the absence of any reinforcingevent (18,19). At the beginning of testing, animalswere placed at the entrance of the starting arm (length:40 cm) and allowed to explore the maze until theyreached the end of the right (length: 25 cm) or the left(length: 25 cm) arm. Upon completion of every run,they were manually returned to the entrance of thestarting arm. Each animal performed one trial consist-ing of nine consecutive runs. Four of the inter-run inter-vals lasted 5 seconds (short intervals) and the otherfour lasted 60 seconds (long intervals). The short andthe long intervals were randomly distributed throughoutthe trial. The sequence of arm choices allowed us tocalculate the alternation rate in each interval (short orlong) condition.

Upon completion of the experiments, the gerbils weresacrificed by an overdose of chloral hydrate. Thebrains were fixed in formalin (10% solution) and cut incoronal sections of 40 µm using a cryostat. The sec-tions were stained with cresyl violet according to theNissl method.

Results

Histology

Figure 1 shows the neural tissue in a control (shamISCH+sham EA) gerbil (A) and in gerbils subjected toglobal ischemia and EA. In general, ischemia producedextensive bilateral loss of pyramidal cells in the CA1hippocampal subfield (C), although some animals ex-hibited only unilateral damage (B). The proportion of bi-lateral and unilateral damage was similar in all thegroups of gerbils subjected to ischemia. Light mi-croscopy examination of the neural tissue did not revealany difference between ischemic groups receiving EA,sham EA or pseudo EA.

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L. Restivo et al.

F i g u r e 1 - Representative coronal sections stained with cresyl vio-let showing cell loss in the CA1 subfield of the hippocampus ingerbils subjected to global ischemia and electro-acupuncture atpoints Du 26 (Renzhong) and Du 8 (Junsuo) (top) and in gerbilssubjected to ischemia and sham electro-acupuncture (middle) atthe same acupoints.No cell loss is evident in gerbils subjected tosham ischemia and sham electro-acupuncture (bottom).

Behavior

Motor activity scores (Fig. 2) were analyzed by a one-way ANOVA (group factor, five levels). Alternationscores (Fig. 3) were analyzed by a two-way ANOVAwith group (five levels) and delay (two levels) as mainfactors. Simple effects and pair comparisons (NewmanKeuls ’test) were calculated where necessary.The ANOVA performed on the motor activity data re-vealed a significant effect of the group factor [F(1,4) =7.20, p<.001] indicating that the distance traveled variedaccording to the treatment received. Subsequent paircomparisons revealed that motor activity was significant-ly higher in the ischemic gerbils subjected to EA than inthe four other groups. Ischemia alone enhanced locomo-tor activity (p<.05) relative to the control group. Interest-ingly, the scores of gerbils subjected to ischemia fol-lowed either by sham EA or pseudo EA (that is, EA ei-ther not administered or administered in a non acupoint),were not statistically different (p=.9) thus ruling out anaspecific effect of the electrical stimulation on motor ac-tivity scores. Finally, EA was not found to increase motoractivity in non ischemic gerbils. It is therefore apparentthat EA delivered at the acupoints considered potentiatesthe increase of motor activity induced by ischemia, butdoes not affect motor activity in non ischemic gerbils.The ANOVA performed on the spontaneous alternationdata indicated a significant group x delay interaction [F(4,29)=3.89, p<.02]. Alternation scores in the short (5 s)delay condition were generally high in all the groups ex-cept ISCH+EA, that is, a significant effect of the “groupfactor” was found for the 5 s delay [F (4,29)=3.55,p<.02] with pair comparisons indicating that gerbils sub-jected to both ischemia and EA alternated significantlyless than the other groups (p<.05 for all pair compar-isons). This observation might suggest that the combi-nation of the two treatments produces a disruptive effect

on working memory that does not emerge followingeach single treatment. However, this hypothesis of aworking memory deficit in the ISCH+EA group was chal-lenged by the consideration that these gerbils, whichshowed enhanced motor activity, could simply be im-paired in controlling their behavior at the choice point ofthe T maze. In fact, two observations are consistent withthe latter assumption. First, no main effect of the “groupfactor” was found in the longer delay condition [F (4,29)=0.48, p=.74]. That is, all gerbils were now dis-playing similar alternation scores, whereas a possibleworking memory deficit in the ISCH+EA group shouldhave been more pronounced in the long delay condition.Second, there was no significant main effect of the de-lay factor, [F(1,29)=0.7, p=.4]. Thus, a longer delay in-terposed between runs did not significantly decreasespontaneous alternation in any group but just restoredthe alternation scores of the ISCH+EA group to a levelsimilar to that recorded in the other groups.

Functional Neurology 2004; 19(1): 19-23 21

Electro-acupunture in ischemic gerbils

F i g u r e 2 - Mean distance (cm) traveled (+ S.E.) during a fiveminute session in the open field by gerbils subjected to global is-chemia or sham ischemia, and electro-acupuncture or sham orpseudo electro-acupuncture delivered at points Du 26 (Ren-zhong) and Du 8 (Junsuo). § different from all other groups (p<.05). * p<.05.

F i g u r e 3 - Percentage of spontaneous alternation in the T mazefollowing 5 s (top) or 60 s (bottom) inter-run intervals in gerbilssubjected to global ischemia or sham ischemia, and electro-acupuncture or sham or pseudo electro-acupuncture delivered atpoints Du 26 (Renzhong) and Du 8 (Junsuo).

Discussion

Taken together, these data allow the following pointsto be made. Ischemia alone increased motor activitywhereas EA alone did not. The first observation isconsistent with data from the literature showing thatglobal ischemia, because of the hippocampal damageproduced by the carotid occlusion, increases motoractivity as other unilateral (20) or bilateral hippocam-pal lesions do (21). The second observation, on theother hand, seems to conflict with previous data show-ing an increase in motor activity following EA in the rat(22). In this latter experiment, however, EA was ap-plied at a different acupoint (Bai Hui point) and wasfound specifically to activate central monoamine neu-rotransmission. Thus, the main result of the presentexperiments is that EA administered at the 26 Du(Renzhong) and 8 Du (Junsuo) acupoints does not re-duce but potentiates the increase of motor activity pro-duced by ischemia, even though examination of theneural tissue does not reveal a different extent of cellloss in ischemic gerbils subjected to EA vs those re-ceiving sham EA. Clearly, the absence of increasedmorphological damage in ischemic gerbils subjectedto EA could be explained by the fact that gerbils weresacrificed shortly after behavioral testing thus prevent-ing long-term effects of EA-related functional brainmodifications – such as NO synthase synthesis – onbrain morphology from being observed.The spontaneous alternation data also show that ger-bils subjected to both ischemia and EA poorly alternateunder the short delay condition. This finding, however,which might point to an impairment of working memoryin the ISCH+EA group, is likely to depend on the highlevel of motor activity recorded in this group. Indeed,the absence of a significant effect of ischemia alone onspontaneous alternation is intriguing. However, it isworth noting that spontaneous alternation deficits fol-lowing ischemia have often been observed in gerbils,both tested in more complex alternation tasks [e.g., re-inforced alternation, longer inter-run intervals, (23)] andsubjected to repeated occlusion episodes (24).A possible explanation of the negative effect of EA onthe behavior of ischemic gerbils relates to the physio-logical effects induced by stimulation of the 26 Du(Renzhong) acupoint. That is, stimulation of 26 Du(Renzhong) alone is traditionally used to enhance heartrate and blood pressure in infarcted patients. In the fieldof experimental ischemia, there is evidence that oxida-tive stress related to NO synthesis reaches a peak dur-ing reperfusion that, however, is remarkably diminishedby treatments reducing cerebral blood flow (25). It couldbe, therefore, that electrostimulation at point Du 26(Renzhong), via its effects on blood pressure, acceler-ates reperfusion injury by increasing cerebral blood flowthat, in turn, might enhance the levels of NO synthaseand NO end-products. Clearly, this possibility wouldhave to be more carefully examined by monitoring cere-bral blood flow following application of EA at the Du 26(Renzhong) acupoint. Thus, in contrast with evidenceshowing neuroprotection through a reduction of NOsynthase when EA was applied at both Du 16 (FengFu) and Du 8 (Junsuo) in ischemic gerbils (17), the pre-sent findings suggest that electrostimulation of both Du26 (Renzhong) and Du 8 (Junsuo) enhances rather

than prevents ischemia-related behavioral alterations.The possibility that EA can negatively influence func-tional recovery following ischemia should be takencarefully into account when considering the potentialtherapeutic applications of acupuncture.

Acknowledgements

This experiment was supported by a grant from the Ital-ian Ministry of Health (Project “Unconventional Thera-pies”, Art. 502, Fasc. 99/S). L.M. was a visiting scientistat the Paracelso Institute, Rome, Italy, supported by theabove grant.

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Electro-acupunture in ischemic gerbils