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1 CCMP90-RD-105 (2-2) Neural Mechanisms of Electroacupuncture Effect in the Functional Change of Urinary Bladder in the Rat Electroacupuncture, EA 3Hz 30Hz Hz 10T 20T 40T T Li-4 L6-S1 BL-28 L2-L3 BL-23 BP IVP PANA PENA EUS-EMG PNA 508

2 VT VV RV VD VE 1 VT 2 VE 100% VE 73±7% 3 3Hz,20T BP 109±3.1 % of control % Hz,20T BP 131.7±3.2 % 107.2±2.4 % of control 4 3Hz % Hz 5 VD % mivp % PNA % - Intute of Physiology, College of Medicine National Taiwan University Tsu-Ching Fu Abstract Acupuncture has been used to treat various diseases in China for over 2000 years, and it has been known that mulation of different acupoints could cure the illness of different organs. However, due to the lack of a convincing evidence and a sound scientific basis, it has not been well accepted by the general public, nor possible 509

3 to make much progress in this field. Therefore, the aim of this study is to invegate the effects of electroacupuncture (EA) simulation of major acupoints related to the urinart system on the function of urinary bladder in both anesthetized and conscious animals, to determine the optimal mulation parameters and to clarify its possible neural mechanisms. EA at three acupoints: Hoku (Li-4), P ang-k uang-yu (BL-28, L6-S1) and Shen-yu (BL-23, L2-L3) was conducted with a combination of various frequencies (3, 30, and Hz) with different intensities (10T, 20T and 40T; T is a threshold or minimum current for skeletal muscle contraction) on the wistar rat for 10 min each time. A rhythmic contraction of the urinary bladder was induced by constant infusion of normal saline in the urethane-anesthetized and conscious rats. The blood pressure (BP), intravesical pressure (IVP), pelvic nerve activity (PNA), and external urethra sphincter-electromyogram (EUS-EMG) were recorded simultaneously. The voiding threshold (VT), voided volume (VV), residual volume (RV), voiding duration (VD) and voiding efficiency (VE) were measured, analyzed and compared between those from anesthetized and from conscious rats. The results showed: (1) the VT of the conscious rats were three times bigger than that of anesthetized ones, (2) the VE in conscious rats was 100%, i.e., urine in bladder was totally voided each time, versus 73±7% in anesthetized ones, (3) in anesthetized rats, when Hoku was mulated with 3Hz and 20T, the BP was elevated (109±3.1% of control) and maintained at the peak during mulation; the micturition interval (MI) was prolonged significantly (51.4%) in 5 min following EA started, and recovered gradually after cessation of mulation. On the other hand, at the frequency of 30 Hz and 200 Hz with 20T, the BP was raised to the peak (131.7±3.2 and 107.2± 2.4 % of control respectively) within 1 min, however returned to control level immediately; and the MI was not changed. (4) In conscious rats, MI was significantly prolonged ( %) following EA with 3Hz mulation of either P ang-k uang-yu or Shen-yu, however, at a frequency of 30 Hz or 300Hz mulation, no effect was found; furthermore, (5) 3Hz mulation at 510

4 P ang-k uang-yu could also significantly reduce the VD ( %), lower the mivp ( %) and increased the PNA ( %). Therefore, it is concluded that EA with a low frequency and adequate intensity at an appropriate acupoint can significantly improve the function of urinary bladder by prolongation of MI and increase of VE. The involved neural mechanisms are: (1) activation of somato-sympathetic reflex to increase urine storage by inhibition of detrusor muscle contraction; (2) activation of both spinal and supraspinal micturition centers to make better coordination of the detrusor-sphinctor muscles activities by enhancement and re-modulation of the micturiton reflex at the spinal level. were mulated and the In the anesthetized rats, pelvic efferent nerve activity (PENA) and in the conscious rats, the IVP and pelvic nerve activity (PNA) were recorded simultaneously. to analyze the difference of the micturiton reflex between the anesthetized and the conscious rats. According to the analysis of these parameters before and after EA, we could know the effect of EA at different acupoints on the micturition reflex. rats and the urine were totally expulsive in the conscious rats when the anesthetized rats were only 73±7%. When mulated at Hoku in the anesthetized rats, the BP was raised to a peak and maintained (109±3.1 % of control) during the mulation of 3Hz, 20T and the BP recovered after the mulation. The micturition interval (MI) was prolonged significantly (51.4 %) in 5 minutes after the mulation started. On the other hand, the mulation of 30 and 200Hz, 20T caused the BP raised to a peak (131.7±3.2 and 107.2± 2.4 % of control) in 1 minute after the EA started but fallen down to the baseline quickly and there was no effect on the MI. In the conscious rats, no matter at the P ang-k uang-yu or Shen-yu, the MI was prolonged significantly ( %) after the 3Hz mulation. However, there was no this effect of 30 and 300Hz mulation. Furthermore, 3Hz mulation at the P ang-k uang-yu could also significantly reduced the VD ( %), lower the mivp ( %) and increased the PNA ( %). Therefore our study showed that 3Hz EA mulation at Hoku, P ang-k uang-yu and Shen-yu all could improve the function of 511

5 the urinary bladder by significantly prolong the MI and increased the VE. These effects may come from the somato-sympathetic reflex and the inhibition of the pontine micturition center. Keywords electroacupuncture, bladder, nerve activity, EMG urinary bladder neck urethra external urethral sphincter, EUS parasympathetic nervous system pelvic nerve sympathetic nervous system hypogastric nerve somatic nervous system pudendal nerve Kuru, 1965 de Groat & Booth, 1980 de Groat, 1995, 1997 Lumbar Sacral Jänig & Mclachlan, 1987 Jänig & Koltzenburg, 1990 Ness & Gebhart, 1990 pelvic afferent nerve L6-S1 dorsal root ganglia pelvic preganglionic efferent nerve L6-S1 Applebaum et al., 1980 Nadelhaft & Booth, 1984 Jancsó & Maggi, 1987 Ness & Gebhart, 1990 Vera & Nadelhaft, 1992 hypogastric afferent nerve T13-L3 hypogastric efferent nerve L1-L3 Aldskogius et al., 1986 Jänig & Mclachlan, 1987 Ness & Gebhart, 1990 Vera & 512

6 Nadelhaft, 1992 L6-S2 McKenna & Nadelhaft, 1986 Pacheco et al., 1989 de Groat et al., 1998 detrusor Kuru, 1965 de Groat et al., 1980 de Groat, 1995 pontine micturition center, PMC Barrington s nucleus Kuru, 1965 de Groat & Booth, 1980 de Groat et al., 1995 Mallory et al., 1989 intravesical pressure, IVP external urethral sphincter-electromyogram, EUS-EMG high-frequency contraction, 7-9Hz high-frequency oscillation, HFO voiding duration, VD Mersdorf et al., 1993 Chien et al, 2000 Maggi 1986 bladder dome d-tubocurarine duration of micturition contraction Kruse

7 pancuronium bromide Chien 2000 Kruse 1993 T9-T11 burng activity tonic EUS contraction 15±2 % 54±4 % Steers & de Groat, detrusor-urethral sphincter dyssynergia Kuru, 1965 Mallory et al.,1989 myelinated A unmyelinated C fiber Sengupta & Gebhart, 1994 de Groat, 1995 early reflex late reflex T8 spinal reflex supraspinal reflex A Mallory et al., 1989 C fiber Maggi 1984, 1986c C fiber capsaicin-sensitive C fiber 514

8 C fiber de Groat et al., 1990 Cheng et al., 1993, 1995 Barrington 1921 dorsolateral pontine tegmentum DLPT Kuru, 1965 Blok et al., 1995 de Groat et al., 1998 L5-S1 laterodorsal tegmental nucleus LDT L6-S1 Loewy et al., 1979 Ding et al., 1997 de Groat et al., 1998 Bolk & Holstege, 2000 dorsolateral pons locus coeruleus LC nucleus tegmentalis lateralis NTL LC LDT LC LDT LDT LDT LDT L6-S1 LDT Elam et al., 1986 Lumb & Morrison, 1987 Noto et al., 1989, 1991 urethane

9 3 Maggi & Meli, 1986b, Matsuura & Dowine, 2000 Van Der Meer et al., 1975, Matsuura & Dowine, 2000 acute experiment chronic experiment Maggi 1984a Yoshiyama 1994 MK-801 NMDA non-competitive NMDA receptor agonist MK-801 Mk-801 NMDA Maggi & Meli, 1986a, Yoshiyama et al., 1994 Cragg & Stephenson,

10 genetically well-defined species Nadelhaft & Booth, 1984, Mersdorf et al., Yaksh PE-50 PE-100 Yaksh Morikawa et al., 1989 Yoshiyama % 15% acupuncture Gunn, et al, 1976 Wu, 1992 Yao,

11 Kline et al., 1978 Sato et al., 1992 somatic nervous system autonomic nervous system - somatosympathetic reflex Kline et al., 1978 Ernst & Lee, 1985 Sato & Schmidt, 1987 Lin et al., pressor response depressor response Sato & Schmidt, 1973, 1987 depressor response group group group A fiber group C fiber Sato & Schmidt, 1973, 1987 Sato et al., 1980, 1981 Yao et al., 1982 Kimura et al., 1995 Lovick et al., 1995 Ohsawa et al., 1995 Kiyono et al., 1996 Knardahl et al., 1998 Sato 1975 rubbing pinching Hz group group Sato Hz 518

12 Morrison Hz electroacupuncture, EA Terui et al., 1981 Chen et al., 1996 Lin 1998 Li-4 3Hz 30Hz Hz Li-4 blood pressure, BP pelvic afferent nerve activity, PANA pelvic efferent nerve activity, PENA Lin 1998 L6-S1 BL-28 L2-L3 BL-23 pelvic nerve activity, PNA 3Hz 519

13 Wistar Rat urethane 1.2gm/kg:2/3i.p.,1/3s.c PE mm heparin, 50I.U. PE-50 PE PE 3. bladder dome PE-50 PE T Infors CH-4130, U.S.A

14 1mm 5mm 8mm 5mm l 2mm 6. Li-4 0.5cm Trueline Instruments INC., U.S.A. 5mm 5mm Grass S88, U.S.A. Grass SIU5B, U.S.A. Grass CCU1A, U.S.A. T 10T 20T 40T 3Hz 30Hz 200Hz paraffin oil paraffin pool 521

15 Dow Corning, cat.no. silac tubing , ID=0.76 mm, OD=1.65 mm, U.S.A. sodium pentobarbital, 35mg/kg, i.p % autoclave Stainless Steel Guide ID=2.15 mm, OD=2.76 mm, Small Parts INC., U.S.A. PE mm 1A mm PE silac tubing # B silac tubing Stainless Steel Guide ID=2.15 mm, OD=2.76 mm, Small Parts INC., U.S.A. Stainless Steel Guide silac tubing silac tubing transducer 3. Teflon-coated Stainless Steel Wire OD=125 um, A-M System INC., U.S.A Male Pin Connector 1C silac tubing # , ID=0.31 mm, OD=0.64 mm 522

16 10mm Teflon-coated Stainless Steel Wire 15 silac tubing # , ID=0.51 mm, OD=0.94 mm Teflon-coated Stainless Steel Wire silac tubing # , 6mm 2.5mm Teflon Coating 1C 1mm Stainless Steel Guide ID=4.43 mm, OD=5.13 mm, Small Parts INC., U.S.A. Male Pin Connector 6mm silac tubing # Male Pin Connector Male Pin Connector Teflon-coated Stainless Steel Wire paraffin oil paraffin oil Male Pin Connector 8-10mm silac tubing paraffin oil silac tubing paraffin oil silac tubing silac tubing # , ID=1.57 mm, OD=2.41 mm silac tubing silac tubing 523

17 Statham Transducer, P23D, U.S.A. Grass, 79D, D.C., Polygraph, U.S.A Grass P511 A.C. Preamplifier, U.S.A. 1 Gould Oscilloscope 1604, U.K. 2 Grass AM8 Audio Monitor, U.S.A. 3 Neuro-Corder Model DR-890, U.S.A. Vedio Tape Recorder 2200s, Japan 4 World Precision Instrument 121 Window Discriminator, U.S.A. Gould Recorder 2200s, U.S.A. 4. EUS-EMG PANA PENA 4 PNA 18 MI mivp max IVP 524

18 0.12ml/min voiding threshold, VT 0.12ml/min 0.2ml/min 2ml ml/min VT VT 2ml mivp mivp mivp 40mmHg VE ml/min IVP EUS-EMG PANA PENA voided volume, VV residual volume, RV VT ethrane L6 S1 L2 L3 13mm Trueline Instruments INC, U.S.A. 5-10mm 5-10mm 525

19 ml/min 3Hz 30Hz 300Hz BP micturition interval, MI mivp VD VT VV RV PNA MI VD mivp VT VV RV VT VV PNA 526

20 PNA t-test p 0.05 VT VT mivp mivp VE VE 70% 100% 1. 3Hz 10T BP MI mivp 20T BP 107.1±2.1% of control 109.9±3.1 % of control 109±3.4 % of control 40T 527

21 BP 106.8±1.5 % of control 6A 20T MI ±12.7 % of control 40T 139.6±10.9 % of control 123.6±6.4 % of control 5A 7A 2. 30Hz 20T 40T BP 131.7±3.2 % of control 141±4.5 % of control 5B 6B MI 10T MI 20T 116.9±3.3 % of control 40T 117.3±11.8 % of control 5B 7B Hz20T BP 107.2±2.4 % of control 5C 10 BP 102.3±0.0 % of control BP 40T BP 103.5±1.2 % of control BP 6C MI 20T 115.0±4.8 % of control 40T MI 5C 7C mivp 8 EUS-EMG PANA PENA 9 3Hz,20T 528

22 mA 2mA 1. L6-S Hz MI ±5.6 % of control ±7.5 % of control 10 14A 15A mivp ±3.6 % of control ±3.5 % of control ±3.3 % of control 10 16A 17A VD 77.6±4.8 % of control ±1.9 % of control ±4.7 % of control A 20A PNA ±2.2 % of control 30 PNA ±2.5 % of control PNA 21 27A 28A Hz MI 87±4.7 % of control A 15B mivp VD 12 16A 17B 19A 20B PNA PNA 10 PNA 74.5±8.2 % of control ±11.9 % of coontrol 22 27A 28B 529

23 Hz MI 84±5 % of control A 15C mivp 94.1±2.8 % of control ±3.6 % of control 40 16A 17C VD ±10 % of control 19A 20C PNA PNA 20 PNA 76±11.4 % of control 40 PNA 86.5±3.6 % of control 23 27A 28C Hz 2. L2-L Hz MI 76.1±6.4 % of control ±3.3 % of control ±4.8 % of control 11 14B 15A mivp 16B 17A VD 87.3±8 % of control ±5 % of control 19B 20A 24 PNA PNA ±16.6 % of control PNA 24 27B 28A Hz MI mivp 530

24 12 14B 15B 16B 17B VD 86.8±9.1 % of control 19B 20B PNA ±8.1 % of control 25 27B 28B Hz MI 82.4±7 % of control 13 14B 15C mivp 84.1±4.9 % of control B 17C VD ±4.1 % of control ±4.9 % of control 19B 20C 26 PNA PNA 79±5.8 % of control B 28C BP MI BP MI MI mivp VD PNA MI mivp VD PNA 29 MI mivp VD PNA MI mivp VD PNA

25 Yaksh 1986 PE silac tubing chloroform PE PE PE silac tubing chloroform PE PE PE PE 5-10mm silac tubing silac tubing silac tubing Yaksh VT 0.2ml VT 1ml Morikawa 1989 VT VT 1.5ml VT 2ml 3ml Yaksh Morikawa VT VT VT Yaksh Morikawa 532

26 Yaksh Morikawa Male Pin Connector paraffin oil silac tubing paraffin oil silac tubing Cheng 1995 isovolumetric contraction outlet obstruction n=2 20mmHg 44% 533

27 stress Pert et al., 1981 Lin T Lin 5T T T Sato Schmidt T 10T group 20T group - Sato et al., 1975 Sato et al., 1980, 1981 Sato et al., 1992 Lin et al., 1998 group group group group Sato et al., 1980 Sato & Schmidt,

28 L6-S1 MI mivp VD PNA MI MI VT - Noto 1989 Kruse 1990 PMC MI mivp VT PMC VT MI PNA PNA Sato 1992 MI MI Araki 1981, 1984 Sato somato-hormonal secretion effect catecholamine hormonal effect VD mivp VD mivp - 535

29 VD mivp Kruse et al., 1993 Yoshiyama et al., 1999 VD mivp VE L2-L3 MI - MI PMC mivp VD PNA Knardahl et al., 1998 Terui Hz group group Lin 1998 pressor response negative feedback pressor response PMC opioid 536

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39 1 A B C 546

40 547 2

41 3 548

42 4 EUS-EMG PANA PENA IVP 549

43 n=4 (n=3) (n=7) (n=6) (n=3) (n=6) VT (ml) mivp mmhg VV (ml) RV (ml) VE (%) VT voiding threshold mivp max intravesical pressure VV voided volume RV residual volume VE voiding efficiency VV/VT 100 ( p<0.05) 550

44 5 A 3Hz,20T B 30Hz,20T C 200Hz,20T (B) 551

45 BP(% of control) T(n=3) (A) 3Hz 20T(n=6-7) 40T(n=4-5) BP(% of control) BP(% of control) Time(min) (B) 30Hz Time(min) (C) 200Hz Time(min) 6 Li-4 BP A 3Hz,10T 20T B C Hz p<

46 MI(% of control) (A) 3Hz 10T(n=3-4) 20T(n=6-7) 40T(n=4-5) Time(min) MI(% of control) (B) 30Hz Time(min) MI(% of control) (C) 200Hz Time(min) 7 MI A 10T 3Hz 20T B C 30Hz 200Hz 20T 5 30Hz40T 200Hz20T p<

47 mivp(% of control) mivp(% of control) mivp(% of control) (A) 3Hz 10T(n=3-4) 20T(n=6-7) 40T(n=4-5) (B) 30Hz Time(min) (C) 200Hz Time(min) Time(min) 8 mivp 554

48 9 3Hz,20T IVP EUS-EMG PANA PENA 555

49 10 3Hz L6-S1 B A A

50 11 3Hz L2-L

51 12 30Hz 558

52 13 300Hz L6-S

53 MI(% of control) (A) L6-S1 3Hz(n=8-10) 30Hz(n=9-10) 300Hz(n=8-9) Time(min) MI(% of control) (B) L2-L Time(min) 14 3Hz A L6-S1 B L2-L Hz p<

54 MI(% of control) MI(% of control) MI(% of control) (A) 3Hz L6-S1(n=8-9) L2-L3(n=8-10) (B) 30Hz Time(min) (C) 300Hz Time(min) Time(min) 15 A 3Hz 20 B 30Hz C 300Hz p<

55 110 (A) L6-S1 3Hz(n=7-9) 30Hz(n=7-9) 300Hz(n=7-9) mivp(% of control) Time(min) mivp(% of control) (B) L2-L Time(min) 16 A 3Hz Hz 30 B 300Hz p<

56 mivp(% of control) mivp(% of control) mivp(% fo control) (A) 3Hz L6-S1(n=7) L2-L3(n=8-9) Time(min) (B) 30Hz Time(min) (C) 300Hz Time(min) 17 A 3Hz B 30Hz C 300Hz 30 p<

57 18 3Hz A B VD PNA IVP C 20 PNA IVP 564

58 120 (A) L6-S1 3Hz(n=5-6) 30Hz(n=5) 300Hz(n=5-6) VD(% of control) VD(% of control) Time(min) (B) L2-L Time(min) 19 VD A 3Hz B 3 300Hz 10 30Hz p<

59 VD(% of control) VD(% of control) VD(% of control) (A) 3Hz L6-S1(n=5) L2-L3(n=6) Time(min) (B) 30Hz (C) 300Hz Time(min) Time(min) 20 A 3Hz 10 B 30Hz C 300Hz 10 p<

60 21 3Hz

61 22 30Hz

62 23 300Hz

63 24 3Hz 570

64 25 30Hz

65 26 300Hz

66 PNA(% of control) (A) L6-S1 3Hz(n=5) 30Hz(n=4) 300Hz(n=4-5) PNA(% of control) Time(min) (B) L2-L Time(min) 27 PNA A 3Hz Hz 300Hz 40 B 300Hz 30 p<

67 PNA(% of control) PNA(% of control) PNA(% of control) (A) 3Hz L6-S1(n=4-5) L2-L3(n=4-5) (B) 30Hz Time(min) (C) 300Hz Time(min) Time(min) 28 A 3Hz 20 B 30Hz C 300Hz p<

68 % of control % of control % o f control % o f control (A ) M I 3Hz(n=8) 30Hz(n=9) 300Hz(n=9) (B ) m IVP Time(min) 3 H z ( n = 7 ) 3 0 H z ( n = 7 ) H z ( n = 7 ) (C) VD Tim e (m 3 H z ( n = 5 ) 3 0 H z ( n = 5 ) H z ( n = 6 ) (D) PNA in) T im e (m in) 3Hz(n=5) 30Hz(n=4) 300Hz(n=4) Time(min) 29 3Hz A B C D Hz 30Hz 300Hz 40 p<

69 % o f control % o f c o n t r o l (A ) M I 3Hz(n=10) 30Hz(n=10) 300Hz(n=8) (B ) m IV P Tim e (m H z ( n = 9 ) 3 0 H z ( n = 9 ) H z ( n = 8 ) Tim e (m i n ) in) (% o f control) H z ( n = 6 ) (C ) V D 3 0 H z ( n = 5 ) H z ( n = 6 ) T im e (m i n ) (% of control) (D) PNA 3Hz(n=5) 30Hz(n=4) 300Hz(n=5) T im e (m in) 30 A 3Hz 300Hz B 300Hz C 3 300Hz 10 D 300Hz 30 p<