Anticonvulsion Effect of Acupuncture Might Be Related to the Decrease of Neuronal and Inducible Nitric Oxide Synthases

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Ru Yang, M.S., Ph.D. Candidate

Zhi-Nong Huang M.S., Ph.D. Candidate

Jie-Shi Cheng, M.D., Professor of Neurobiology Deputy Director of Institute of Acupuncture Research

(National Laboratory of Medical Neurobiology, Department of Neurobiology, Institute of Acupuncture Research, Shanghai Medical University, Shanghai 200032, P.R. China)

(Received July 2, 1999; Accepted with revisions November 3, 2000)

ABSTRACT:

To measure the levels of hippocampal nitric oxide synthase isoforms in penicillin induced epilepsy and to test the effect of electroacupuncture (EA) on changes of theses levels during epilepsy, we injected penicillin into rat hippocampus to make an epilepsy model and performed electroacupuncture treatment on "Feng Fu" (DU 16) and "Jin Suo" (DU 8) points in Wistar rats. Nitric Oxide synthase (NOS) mRNA levels of rat hippocampus were determined by reverse transcription-polymerase chain reaction (RT-PCR). The neuronal nitric oxide synthase (nNOS) mRNA markedly increased (P<0.01) and inducible nitric oxide synthase (iNOS) mRNA significantly emerged during epilepsy, whereas no significant change in epithelial nitric oxide synthase (eNOS) mRNA was observed. EA inhibited the epilepsy and decreased nNOS (P<0.01) and iNOS (P<0.01) correspondingly but had no effect on the amount of eNOS mRNA. The data suggest that penicillin-induced epilepsy caused an increase in nNOS and iNOS, and the EA anticonvulsant effect might be related to the decrease of these nitric oxide synthases.

KEY WORDS: nitric oxide synthase (NOS), epilepsy, electroacupuncture (EA), hippocampus, RT-PCR.

INTRODUCTION

The occurrence of epilepsy is very high in the human population. The precise pathophysiological mechanisms responsible for epilepsy, however, are incompletely understood. The occurrence of epilepsy is mainly related to the decrease of the GABAergic inhibition and the increase of the glutamatergic excitation. Recent reports suggest that alterations of glutamatergic neurotransmission and of the glutamatergic signal transduction pathway involving calcium, nitric oxide and nitric oxide synthase (NOS) may be implicated. The NOS are a family of isoenzymes catalyzing the oxidation of L-arginine to NO and L-citrulline. NO, a putative neurotransmitter has a variety of physiological actions including the regulation of vascular tone, mediation of immune cell cytotoxicity and neurotransmission. Our laboratory also previously reported the increase of NOS mRNA during kainic acid-induced epilepsy and penicillin-induced epilepsy in the rat hippocampus as determined by Northern Blot technique.

NOS is divided into three types: eNOS, nNOS, iNOS. It remains unclear which NOS isoenzymes are involved in epilepsy. In order to further the research, three types of NOS gene expression were studied in the rat hippocampus in epilepsy and anti-convulsion and appropriate sham-operated control groups.

MATERIALS AND METHODS

Epilepsy model and acupuncture anti-convulsion

Male Wistar rats (n=44, 180-220g) bred by the Experimental Animal Department of Shanghai Medical University were divided into 11 groups: ( 1) unoperated control; ( 2) sham-operated vehicle control; ( 3) EA treated control; ( 4)( 5)( 6)( 7) four epilepsy groups in which the rats were decapitated at 4hrs, 8hrs, 12hrs, 24hrs respectively after penicillin injection; ( 8)( 9)( 10)( 11) four anti-convulsion groups in which the epilepsy rats received EA treatment and were decapitated at 4hrs, 8hrs, 12hrs, 24hrs respectively after penicillin injection. 4 rats were used in each of the eleven groups.

All animals except the unoperated control group were anesthetized with pentobarbital and stereotaxically implanted with a 0.8mm stainless-steel cannula in one side of the hippocampus by P3.0R/L2.0/H3.7 (3.0 mm posterior to bregma, 2.0 mm lateral to the midline, 3.7 mm below the surface of skull) according to George Paxinos Stereotaxic Coordinates. Penicillin (300 units/1.5µl) or artificial cerebrospinal fluid (ACSF, 1.5µl) in ( 2)~( 11) groups was microinjected into one side of the hippocampus through the cannula after three days. EA was added to "Jin Suo" [Non-Ascii Text], DU.8), "Feng Fu" ([Non-Ascii Text], DU. 16) points at 100 Hz, 6 mA for 60 min to induce anti-convulsion models.(Fig1)

GLO:1I5/01SEP00:138N1.JPG DIAGRAM: Fig 1. Schematic diagram showing the electroacupuncture gl

The hippocampi were dissected rapidly on ice and homogenized immediately or kept frozen at -80 degrees C until use.

30 cycles of 1-min ramp to 94 degrees C, 1-min ramp to 63 degrees C and 2-min ramp to 72 degrees C, with a subsequent extension of 10min at 72 degrees C; eNOS: 5-min ramp to 94 degrees C followed by 3 cycles of 1-min ramp to 94 degrees C, 1-min ramp to 52 degrees C and 1-min ramp to 72 degrees C, and then followed by 32 cycles of 1-min ramp to 94 degrees C, 1-min ramp to 56 degrees C and 1-min ramp to 72 degrees C, with a subsequent extension of 10min at 72 degrees C. Negative (distilled water) controls were included in all PCRs.

After PCR amplification, 20 µl of the total 50-µl reaction mixture was electrophoresed through a 2% agarose gel containing ethidium bromide. The gel was photographed by UV transillumination.

RESULTS

1. RT-PCR amplification of total hippocampal RNA using the primers for rat nNOS yielded a 472 bp product which was expressed higher in epilepsy rats compared to controls and the increase of nNOS showed enhancement within 24hrs after penicillin injection. Meanwhile the PCR product was expressed lower in EA anti-epilepsy rats compared to epilepsy ones at the same time point, nNOS mRNA levels increased markedly in hippocampi during epilepsy to 210%, 255%, 347% and 431% of control levels respectively at 4hrs, 8hrs, 12hrs and 24hrs after epilepsy. The increased levels were decreased by EA to 56%, 67%, 64% and 55% of epilepsy levels of the corresponding time-point.
2. RT-PCR amplification of total hippocampal RNA using the primers for rat iNOS yielded a 222 bp product which almost could not be observed in control rats but was expressed significantly in epilepsy rats compared to controls. The amount of iNOS in epilepsy rats showed an increase within 24hrs after penicillin injection, whereas the content of the PCR product was decreased in EA anti-epilepsy rats compared to epilepsy ones at the same time point respectively. The induction levels of iNOS mRNA in hippocampus during epilepsy were decreased by EA to 51%, 60%, 67% and 64% of epilepsy levels of the corresponding time-point.
3. RT-PCR amplification of total hippocampal RNA using the primers for rat eNOS yielded a 214 bp product which was expressed markedly in control rats, epilepsy rats and EA anti-epilepsy rats. And the levels of eNOS among all eleven groups had no significant changes.

No NOS bands were observed if the RNA was not added to the reaction or if the RT step was omitted. (Fig 2)

GLO:1I5/01SEP00:141N1.JPG GRAPH: FIG2. The transcription on nNOS and INOS gl

DISCUSSION

In present study, we found that penicillin-induced epileptic-form seizure activity increased nNOS mRNA level and triggered iNOS expression in hippocampus whereas has no effect on the amount of eNOS. Electroacupuncture not only inhibited epileptic-form seizure activity in EEG and behavior but also decreased the rise of nNOS and iNOS level. Meanwhile, the levels of nNOS mRNA and iNOS mRNA were enhanced more and more gradually at 4hrs, 8hrs, 12hrs, 24hrs time-points during epilepsy and EA decreased the level rise respectively at the corresponding time-points. The results suggested: 1, Both nNOS and iNOS involved in epilepsy and the involvement lasted a long-term process of epilepsy. 2, The EA anti-convulsion effect may be related to decrease change of nNOS and iNOS level. 3, No evidence was shown whether or not eNOS took part in epilepsy and anti-epilepsy.

mRNA measurement by RT-PCR

Total RNA was extracted from the hippocampal tissue by the method of Chomczynski( 1) using TRIzol Reagent. The integrity of the RNA was determined on denaturing formaldehyde gels. Three types of NOS mRNA were reversedly transcribed and PCR-amplified in a two-step reaction. Briefly, first strand cDNA synthesis was performed with 2µg RNA, 10mM Tris-HCl (pH9.0 @ 25 degrees C), 50mM KC1, 0.1% Triton X-100, 5mM MgC1[ 2], lmM dNTPs, 40U of RNasin, 15U of AMV RTase and 0.5µg dT 15 in a total reaction volume of 20µl according to manufacture instructions. The reaction was incubated for 45min at 42 degrees C, heated for 5min at 99 degrees C and immediately cooled on ice for 5min.

A 50µl PCR reaction mixture was prepared to amplify a 472-bp fragment of nNOS cDNA consisting of base pairs 4113-4584( 2), a 222-bp fragment of iNOS cDNA consisting of base pairs 1723-1944( 3)( 4), and a 214-bp fragment of eNOS cDNA consisting of base pairs 244-457( 5)

The primers used for the nNOS sequence were based on the rat sequence and were 5'-CTTCC GAAGC TTCTG GCAAC AGCGA CAATT-3' (sense) and 5'-GGACT CAGAT CTAAG GCGGT TGGTC ACTTC-3' (antisense). The primers used for the iNOS sequence were based on the rat sequence and were 5'-GCATG GAACA GTATA AGGA AACA-3' (sense) and 5'-GTTTC TGGTC GATGT CATGA GCAA-3' (antisense). The primers used for the eNOS sequence were based on the human sequence and were 5'-CAAGT TCCCT CGTGT GAAGA ACTG-3' (sense) and 5'-TAAAG GTCTT CTTCC TGGTG ATGCC-3' (antisense). Beta-Actin cDNA was amplified using the sense and anti-sense primers, with respective sequences, 5' to 3' CTA TTG GCA ACG AGC GGT TC and CTT ACJG AGT GGG GGT GGC TT These primers yielded a 577 bp PCR product. Amplification efficiency was determined in a kinetic study to ensure that all the experiments were performed within the exponential phase of amplification where PCR products remains proportional to the number of cycles.

The PCR mixture consisted of the RT product, 10mM Tris-HCl (pH9.0 @ 25 degrees C), 50mM KCI, 0.1% Triton X-100, 2mM MgC12, 0.2mM dNTPs, lµM 5' and 3' primers, and 0.03 unit/µl Taq DNA polymerase. Amplification was performed as follows: nNOS: 4-min ramp to 94 degrees C followed by 35 cycles of 30-second ramp to 94 degrees C, 30-second ramp to 55 degrees C and 1-min ramp to 72 degrees C, with a subsequent extension of 10min at 72 degrees C; iNOS: 6-min ramp to 94 degrees C followed by

Our data are in general agreement with previous studies in our laboratory ( 6)( 7)( 8) and other laboratories ( 9)( 10) in which both nNOS mRNA and nNOS protein were determined to increase and the enhancement of two matters reached a peak at 24hrs after epilepsy by Northern Blot and immunohistochemistry. Our results are also consistent with other studies in our laboratory in which the 7-nitroindazole (a NOS inhibitor) preinjected into lateral ventricle of brain before penicillin injection into hippocampus reduced the severity of seizure that was measured by EEG power spectrum. Moreover, data from the present study showed that epilepsy caused induction of iNOS and EA anti-epilepsy decreased the content of iNOS at the level of gene expression.

The role of endogenous nitric oxide, which was produced by the catalysis of NOS in epileptic-form seizure, is unclear. For when lots of studies suggest that inhibitors of NOS are proconvulsant on kainic acid-, pilocarpine- or bicuculline-evoked seizures, other works indicated the opposite on NMDA-, kainic acid- or pentylenetetrazole-induced epileptic activity ( 10). From early studies by Mollace( 11) to recent reports by Proctor( 12), it was found that nitric oxide and the nitric oxide precursor L-arginine enhance seizure-triggering activity in response to a subconvulsive dose of NMDA, suggesting that NO is a pro-convulsant mediator. In contrast, data from Theard( 13) and other laboratories showed when NOS is acutely inhibited, the duration of bicuculline-induced seizures was doubled. Many evidences implicated that lots of factors governed pro- and anti-convulsant effect of NOS inhibitors including difference in inhibitors, difference in models of seizure, difference in doses of drug and even difference in injected regions. However, although the evidence for the role of NO in this disease is controversial, it is no doubt that NO may play an important role in the emergence and development of epilepsy. In our present study, we tried to determine which ones of three isoforms of NOS related to epilepsy from one point of view in penicillin-induced seizure model.

CONCLUSIONS

In conclusion, our results, together with some literature data, indicate that nitric oxide may be regarded as a proconvulsant substance in penicillin-induced seizures. The increase of nNOS and iNOS, which contributes to an increased release of NO, may be related to epileptogenesis. Anticonvulsion effect of acupuncture may result from the decrease in the transcription of nNOS and iNOS mRNA, while no change of eNOS transcription was observed in our experiment. The differential regulation of the three isoforms of the NOS gene by excessive neuronal activity provides further evidence for a complex role of NO during epilepsy.

ACKNOWLEDGEMENT

The study is supported by the National Natural Science Foundation of China. (No. 39570886)
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By Ru Yang; Zhi-Nong Huang and Jie-Shi Cheng

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