CJA
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Résumé de cet Article
Right arrow Full Text (PDF)
Right arrow Submit a scholarly reply
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nishiyama, T.
Right arrow Articles by Yamaguchi, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nishiyama, T.
Right arrow Articles by Yamaguchi, T.
Canadian Journal of Anesthesia 48:288-294 (2001)
© Canadian Anesthesiologists' Society, 2001
Regional Anesthesia and Pain

Synergistic analgesic effects of intrathecal midazolam and NMDA or AMPA receptor antagonists in rats

Tomoki Nishiyama, MD PhD*,{dagger}, Laszlo Gyermek, MD PhD{dagger}, Chingmuh Lee, MD{dagger}, Sachiko Kawasaki-Yatsugi, PhD{ddagger} and Tokio Yamaguchi, PhD{ddagger}

* From the Department of Surgical Center, Institute of Medical Science, University of Tokyo, Tokyo, Japan, the Department of Anesthesiology,
{dagger} Harbor-University of California, Los Angeles Medical Center, Torrance, California, USA, and the Institute for Drug Discovery Research,
{ddagger} Yamanouchi Pharmaceutical Company, Ibaraki, Japan.

Address correspondence to: Dr. Tomoki Nishiyama, 3-2-6-603, Kawaguchi, Kawaguchi-shi, Saitama, 332-0015, Japan. Phone: 81-3-5449-5356 (Dept.); Fax. 81-3-5449-5356 (Dept.); E-mail: nishiyam{at}ims.u-tokyo.ac.jp


   Abstract
TOP
 Abstract
Materials and methods
 Results
 Discussion
 References
 
Purpose: To investigate the interaction of midazolam and N-methyl-D-aspartate (NMDA) receptor or -amino-3- hydroxy-5-methyl isoxazole-4-propionic acid (AMPA) receptor antagonist on the effects of persistent inflammatory nociceptive activation.

Methods: Male Sprague-Dawley rats were implanted with lumbar intrathecal catheters and were tested for their responses to subcutaneous formalin injection into the hindpaw. Saline, midazolam (1 to 100 µg), AP-5 (1 to 30 µg), a NMDA receptor antagonist, or YM872 (0.3 to 30 µg), an AMPA receptor antagonist was injected intrathecally 10 min before formalin injection. The combinations of midazolam and AP-5 or YM872 in a constant dose ratio based on the 50% effective dose (ED50) were also tested and were analysed with an isobologram.

Results: Dose-dependent effects were observed with midazolam (ED50 was 1.34 µg and 1.21 µg in phase 1 and 2 of the formalin test, respectively), AP-5 (7.64 µg and 1.4 µg) and YM872 (0.24 µg and 0.21 µg). Synergistic effects in both phases were obtained when combining midazolam with AP-5 or YM872. The ED50 of midazolam decreased to 0.012 µg (phase 1) and 0.27 µg (phase 2) with AP-5 and to 0.09 µg (phase 1) and 0.35 µg (phase 2) with YM872 (P < 0.01).

Conclusions: These results suggest a functional coupling of benzodiazepine—aminobutyric acid (GABA)A receptor with NMDA and AMPA receptors in acute and persistent inflammatory nociceptive mechanisms in the spinal cord.

IN the spinal cord, both the excitatory neurotransmitter, glutamate1 and the inhibitory neurotransmitter, - aminobutyric acid (GABA)2 are involved in nociceptive mechanisms. The N-methyl-D-aspartate (NMDA) receptors are thought to mediate persistent nociceptive activation,1 while the -amino-3-hydroxy-5-methyl isoxazole-4- propionic acid (AMPA) receptors are considered to have a role in acute nociception.3 The AMPA receptors also mediate facilitated state of pain processing.4 Both GABAA and GABAB receptors also have roles in spinally mediated nociception.5 The benzodiazepine binding site is present on the GABAA receptor (Cl– channel) in the spinal cord6 and benzodiazepines have spinally mediated antinociceptive effects.7

The relations between different receptors involved in spinal nociceptive mechanisms are studied primarily with opioid receptors. Morphine, a µ-opioid receptor agonist, possesses a synergistic antinociceptive interaction with {alpha}2 adrenergic agonist,8 midazolam,9 or AMPA receptor antagonist.3 However, it did not show synergy with NMDA receptor antagonist or NMDA glycine site antagonist.3 The GABA and glutamate receptors may operate in concert to regulate nociceptive signals.10 There are few studies that address the relation between GABA receptors and glutamate receptors in spinal nociceptive mechanisms. We have reported that midazolam, a benzodiazepine-GABAA receptor complex agonist, exhibited synergistic analgesia for thermally induced acute nociception both with NMDA and AMPA receptor antagonists.11 However, there are no reports of the interaction between these receptors in other types of nociceptive mechanisms. The purpose of the present study was to investigate the relationships of the benzodiazepine-GABAA receptor complex and the NMDA receptor or the AMPA receptor in the mechanisms of persistent inflammatory nociceptive activation in the formalin test of intrathecally catheterized rat model.


   Materials and methods
TOP
 Abstract
 Materials and methods
Results
 Discussion
 References
 
Animal preparations
The protocol was approved by the Research and Education Institute of Harbor - UCLA Medical Center. Male Sprague-Dawley rats (280–300 g; BK Universal, Fremont, CA, USA) were implanted with chronic lumbar intrathecal catheters under halothane (2%) anesthesia according to the previously described method.12 Briefly, an 8.5-cm polyethylene (PE-10; Clay Adams, Parsippany, NJ, USA) catheter was advanced caudally through an incision in the atlanto-occipital membrane, to the thoracolumbar level of the spinal cord. The external part of the catheter was tunnelled subcutaneously to exit on the top of the skull and plugged with a 28G stainless steel wire. The position of the catheter was checked by the aspiration of cerebrospinal fluid at the implantation and was directly verified after sacrificing the rat. Only rats with normal motor function and behaviour five days after surgery were used. In total, 176 rats were used.

Drugs and administration
Midazolam (a benzodiazepine-GABAA receptor agonist; Sigma, St. Louis, MO, USA) 1, 3, 10, 30, and 100 µg, and AP-5 (2-amino-5-phosphonovaleic acid, a NMDA receptor antagonist; Sigma, St. Louis, MO, USA) 1, 3, 10, and 30 µg were dissolved in 10 µL saline. YM872 {[2,3-Dioxo-7-(1H-imidazol-1-yl)-6-nitro-1,2,3,4-tetrahydro-1-quinoxalinyl] acetic acid}, an AMPA receptor antagonist (Yamanouchi Pharmaceutical Co. Ltd., Tsukuba, Japan) 10 mg was dissolved in 0.97 mL distilled water with 30 µL, 1N NaOH added to adjust pH to 7.3–7.5. Solutions of 0.3 (0.86), 1 (2.86), 3 (8.59), 10 (28.63), or 30 (85.89) µg (nMol) per 10 µL were made using normal saline. Normal saline, 10 µL, was used in the control group. After intrathecal drug or saline injection, the catheter was flushed with an injection of 10 µL normal saline to clear the dead space of the catheter (7 ± 0.4 µL, mean ± standard error). Micro injector syringes were used for all injections. In each dose group, eight randomly selected rats were used.

Nociceptive test
Rats were kept in an open Plexiglas chamber with a diameter of 40 cm for 30 min before drug injection. Ten minutes after the intrathecal administration of the agent, 50 µL formalin 5% were injected subcutaneously into the dorsal surface of the right hindpaw with a 30 G needle. Immediately after injection, the rat was placed in an open Plexiglas chamber and observed for 60 min. Quantification of pain behaviour was made by counting the incidence of spontaneous flinches/shaking of the injected paw at 1–2 min, 5–6 min and at 5-min intervals during 10- to 60-min periods after formalin injection. The animals were then sacrificed with an overdose of halothane. As previously described,13 two distinct phases were observed after formalin injection: phase 1, during 0–6 min after injection, and phase 2, beginning about 10 min after injection.

Experimental paradigm
The first series of experiments were performed to determine the dose-dependency of the antinociceptive effects of intrathecally administered midazolam, AP-5, and YM872 on the formalin test.

To investigate the interaction between midazolam and AP-5 or YM872, an isobolographic analysis14 was used. The method is based on comparisons of the dose ratios that are determined to be equieffective. First, the respective 50% effective dose (ED50) values were determined from the dose-response data of each agent alone. Subsequently, dose-response data were obtained by coadministration of the two drugs in a constant dose ratio based on the ED50 values of the phase 2 of the single agents; i.e., combinations of each 1/2 ED50, 1/4 ED50, 1/8 ED50, or 1/16 ED50 doses. From the dose-response data of the combined drugs, the ED50 value of the mixture was calculated (calculated ED50 values). The theoretical additive values were the 1/2 ED50s of the single agents. Eight rats were used for each dose combination.

Data analysis and statistics
The ED50 values were calculated using the % maximum possible effect (% MPE) by a computer program made in the Anesthesiology Laboratory of University of California, San Diego.3,11 The % MPE was the % against the value in the control group.

To describe the magnitude of interaction between the agents, a total fractional dose value was calculated as follows: [(ED50 dose of drug 1 in combination) / (ED50 value for drug 1 alone)] + [(ED50 dose of drug 2 in combination) / (ED50 value for drug 2 alone)]. The values were normalised by assigning the ED50 values of the agents given alone as 1. Values near to 1 suggest an additive interaction, values >1 imply an antagonistic interaction, while values <1 indicate a synergistic interaction.

Student's t test was used to compare the calculated ED50 values with the theoretical additive values. A P value <0.05 was considered statistically significant.


   Results
TOP
 Abstract
 Materials and methods
 Results
Discussion
 References
 
Analgesic effects of each agent
Midazolam, AP-5 and YM872 dose-dependently decreased the number of flinches in both phase 1 and phase 2 (time course was shown in the Figures 1A, 1B, and 1CGo, and dose response was shown in the Figures 2A and 2BGo). The ED50 values are shown in the TableGo




View larger version (53K):
[in this window]
[in a new window]
  		FIGURE 1 Time course of the effects of intrathecal midazolam (A), AP-5 (NMDA receptor antagonist, B), YM872 (AMPA receptor antagonist, C), AP-5 + midazolam (D), and YM872 + midazolam (E) on the formalin test. Each point represents mean ± SEM of eight animals. AP: AP-5, Mid: midazolam, YM: YM872. The numbers after symbols, AP, Mid and YM are the doses expressed in µg.

 



View larger version (29K):
[in this window]
[in a new window]
  		FIGURE 2 Dose response curves of the effects of intrathecal midazolam, AP-5 (NMDA receptor antagonist), YM872 (AMPA receptor antagonist) (A, B), AP-5 + midazolam, and YM872 + midazolam (C, D) on phase 1 and 2 of the formalin test. Each point represents mean ± SEM of eight animals. AP: AP-5, Mid: midazolam, YM: YM872.

 

View this table:
[in this window]
[in a new window]
  		TABLE ED50 (50% effective dose) values (µg)
 
Analgesic interaction between midazolam and AP-5 or YM872
Intrathecal administration of the combination of midazolam and AP-5 produced dose-dependent analgesic effects on both phase 1 and phase 2 of the formalin test (time course was shown in the Figure 1DGo, and dose response was shown in the Figures 2C and 2DGo). The ED50 values for midazolam and AP-5 decreased in the combination compared with each single agent (TableGo). Total fractional dose values were 0.01 in phase 1 and 0.45 in phase 2. Isobolographs are shown in the Figure (Figures 3A, 3BGo).




View larger version (37K):
[in this window]
[in a new window]
  		FIGURE 3 Isobolograms for the intrathecal interaction of AP-5 or YM872 and midazolam in both phase 1 and 2 of the formalin test. Horizontal and vertical bars indicate 95% confidence interval. The oblique lines between the x axis and y axis are the theoretical additive lines. The points in the middle of this line are the theoretical additive points calculated from separate ED50 values. The experimental points lie far below the additive line , indicating very marked significant synergism (P <0.01 in phase 1 (A) and P=0.038 in phase 2 (B) of AP-5 + midazolam, P <0.01 in phase 1 (C) and P=0.029 in phase 2 (D) of YM872 + midazolam).

 
The combination of midazolam and YM872 also induced dose-dependent decreases of flinches in both phase 1 and phase 2 of the formalin test (time course was shown in the Figure 1EGo, and dose response was shown in the Figures 2C and 2DGo). The ED50 values for midazolam and YM872 decreased in the combination compared with each single agent (TableGo). Total fractional dose values were 0.07 in phase 1 and 0.57 in phase 2. Isobolographs are shown in the Figure (Figures 3C, 3DGo).


   Discussion
TOP
 Abstract
 Materials and methods
 Results
 Discussion
References
 
Intrathecally administered midazolam (benzodiazepine-GABAA receptor agonist), AP-5 (NMDA receptor antagonist) and YM872 (AMPA receptor antagonist) produce dose-dependent decreases in the number of flinches in both phase 1 and phase 2 of the formalin test in rats. Midazolam showed synergistic suppressive effects with AP-5 and YM872 on the number of flinches of both phase 1 and phase 2. The synergism of midazolam was more pronounced with AP-5 than with YM872 in phase 1 of the formalin test.

The GABAA receptors are proposed to exist at the primary afferent terminal in the spinal cord; and the GABAergic system plays an important role in the presynaptic inhibition of primary afferents.13 Benzodiazepine receptors are concerned with pain transmission in the dorsal horn of the spinal cord.7 Benzodiazepine receptor agonists increase the intrinsic efficacy of GABA at the GABAA receptor coupling with benzodiazepine receptor in the spinal cord6 by increasing Cl– conductance.5 Midazolam, a benzodiazepine derivative, has spinally mediated analgesic7,11and anesthetic16 effects in animal experiments. In the present study, we could show analgesic effects of intrathecally administered midazolam on both formalin induced acute nociception and persistent nociceptive activation. Goodchild et al. reported that intrathecal midazolam caused spinally mediated antinociception in rats by a mechanism involving opioid receptor activation.17 We did not use any antagonists for GABA or opioid receptors in the present study. Therefore, further studies are necessary to verify the detailed mechanism of the analgesic action of midazolam in the spinal cord.

Glutamates also exist in primary afferents and in interneurons.18 The NMDA receptors are involved in persistent nociceptive input of deep dorsal horn cells.19 Accordingly, NMDA receptor antagonists are effective in phase 2 of the formalin test1 rather than on acute nociception.3 This is consistent with our present results in which the ED50 value of AP-5 was smaller in phase 2 than in phase 1 of the formalin test.

The AMPA receptors are localized in superficial laminae of the dorsal horn pre- and postsynaptically.20 These receptors alter acute afferent evoked excitation. Intrathecal application of AMPA receptor antagonists are commonly thought to decrease the responses to acute noxious stimulus.11,21 However, on the formalin test of the animals, Hunter et al. reported that the AMPA receptor antagonist inhibited only phase 1, but not phase 2 responses.13 In contrast, Simmons et al. reported that the AMPA receptor antagonist reduced phase 2 response but not phase 1.4 In our present study, YM872 inhibited both phase 1 and phase 2 responses. The discrepancies in these results might be due to the different compounds used: NBQX used by Hunter and Singh,13 YM872 used in our present study, and other compounds in the study of Simmons et al.4 have different affinities to the AMPA and kainate subtypes of ionotropic glutamate receptors.4,22 Further experiment to compare the affinity of these compounds to different subtypes in the same study is necessary to discuss the discrepancies of the analgesic effect furthermore.

As we already reported,11 midazolam and NMDA receptor antagonist or AMPA receptor antagonist had synergistic antinociception for acute thermal stimulation. Similarly in the present study, midazolam was very markedly synergistic with NMDA receptor antagonist and AMPA receptor antagonist in suppressing phase 1 response of the formalin test. Curiously, AP-5, which is usually less effective for acute pain, enhanced the effect of midazolam in phase 1 of the formalin test more than YM872. The phase 1 response is due to the direct stimulation of nociceptors by formalin or tissue damage and is thought to be an acute pain reaction.23 In addition, in the phase 2 response of the formalin test, midazolam had synergistic antinociception with NMDA and AMPA receptor antagonists in the present study. The phase 2 response is due to subsequent inflammation after formalin injection and central sensitization related to C fibre activity.24 Therefore, in the spinal cord, benzodiazepine-GABAA receptor and NMDA or AMPA receptor might have some functional coupling in both acute and persistent inflammatory nociception.

Benzodiazepines increase the Cl– conductance of GABAA on the primary afferents hyperpolarizing the afferent,6 therefore, reducing the release of glutamate in the spinal cord.25 The glutamate receptor antagonists inhibit the binding of glutamate post-synaptically reducing the depolarization of the postsynaptic cell. Reduction of glutamate release and binding would decrease transmission and, therefore, pain perception. It is also reported that a GABAA receptor agonist inhibited the behavioural effects of NMDA, quisqualic acid and kainic acid.10 Thus, GABAA receptors might have some functional coupling with glutamate receptors in the spinal cord with regard to nociceptive transmission.

Regarding side effects, we did not investigate any behavioural effects in this study. However, in our previous study,11 the combinations of midazolam with AP-5 or YM872 decreased side effects compared to each single agent. Therefore, we also expected decreased side effects in the same combinations in the present study.

In conclusion, intrathecal coadministration of midazolam (a benzodiazepine-GABAA receptor agonist) with AP-5 (a NMDA receptor antagonist) or midazolam with YM872 (an AMPA receptor antagonist) produced synergistic analgesia on formalin induced acute nociception and persistent nociceptive activation. These results suggest a functional coupling of benzodiazepine-GABAA receptor with NMDA and AMPA receptors in various nociceptive mechanisms in the spinal cord. These synergisms can reduce dose requirements (increase therapeutic index) in consequence can reduce toxicity when projected to possible clinical application.


   Acknowledgments
 
We thank to Dr. Ang Ji, Dr. Young-moon Cho, and Nguyen B. Nguyen, Department of Anesthesiology, Harbor / University of California, Los Angeles, for their assistance.

Accepted for publication November 18, 2000.


   References
TOP
 Abstract
 Materials and methods
 Results
 Discussion
 References
 
1 Näsström J, Karlsson U, Post C. Antinociceptive actions of different classes of excitatory amino acid receptor antagonists in mice. Eur J Pharmacol 1992; 212: 21–9.[Medline]

2 Buckett WR. Induction of analgesia and morphine potentiation by irreversible inhibitors of GABA- transaminase. Br J Pharmacol 1979; 68: 129–30.

3 Nishiyama T, Yaksh TL, Weber E. Effects of intrathecal NMDA and non-NMDA antagonists on acute thermal nociception and their interaction with morphine. Anesthesiology 1998; 89: 715–22.[Medline]

4 Simmons RMA, Li DL, Hoo KH, Deverill M, Ornstein PL, Iyengar S. Kainate GluR5 receptor subtype mediates the nociceptive response to formalin in the rat. Neuropharmacology 1998; 37: 25–36.[Medline]

5 Sawynok J. GABAergic mechanisms of analgesia: an update. Pharmacol Biochem Behav 1987; 26: 463–74.[Medline]

6 Unnerstall JR, Kuhar MJ, Niehoff DL, Palacios JM. Benzodiazepine receptors are coupled to a subpopulation of -aminobutyric acid (GABA) receptors: evidence from a quantitative autoradiographic study. J Pharmacol Exp Ther 1981; 218: 797–804.[Abstract/Free Full Text]

7 Goodchild CS, Serrao JM. Intrathecal midazolam in the rat: evidence for spinally-mediated analgesia. Br J Anaesth 1987; 59: 1563–70.[Abstract/Free Full Text]

8 Monasky MS, Zinsmeister AR, Stevens CW, Yaksh TL. Interaction of intrathecal morphine and ST-91 on antinociception in the rat: dose-response analysis, antagonism and clearance. J Pharmacol Exp Ther 1990; 254: 383–92.[Abstract/Free Full Text]

9 Yanez A, Sabbe MB, Stevens CW, Yaksh TL. Interaction of midazolam and morphine in the spinal cord of the rat. Neuropharmacology 1990; 29: 359–64.[Medline]

10 Aanonsen LM, Wilcox GL. Muscimol, -aminobutyric acidA receptors and excitatory amino acids in the mouse spinal cord. J Pharmacol Exp Ther 1989; 248: 1034–8.[Abstract/Free Full Text]

11 Nishiyama T, Gyermek L, Lee C, Kawasaki-Yatsugi S, Yamaguchi T. Analgesic interaction between intrathecal midazolam and glutamate receptor antagonists on thermal-induced pain in rats. Anesthesiology 1999; 91: 531–7.[Medline]

12 Yaksh TL, Rudy TA. Chronic catheterization of the spinal subarachnoid space. Physiol Behav 1976; 17: 1031–6.[Medline]

13 Hunter JC, Singh L. Role of excitatory amino acid receptors in the mediation of the nociceptive response to formalin in the rat. Neurosci Lett 1994; 174: 217–21.[Medline]

14 Tallarida RJ, Porreca F, Cowan A. Statistical analysis of drug-drug and site-site interactions with isobolograms. Life Sci 1989; 45: 947–61.[Medline]

15 Sluka KA, Willis WD, Westlund KN. Inflammation-induced release of excitatory amino acids is prevented by spinal administration of a GABAA but not by a GABAB receptor antagonist in rats. J Pharmacol Exp Ther 1994; 271: 76–82.[Abstract/Free Full Text]

16 Bahar M, Cohen ML, Grinshpon Y, Chanimov M. Spinal anaesthesia with midazolam in the rat. Can J Anaesth 1997; 44: 208–15.[Abstract/Free Full Text]

17 Goodchild CS, Guo Z, Musgreave A, Gent JP. Antinociception by intrathecal midazolam involves endogenous neurotransmitters acting at spinal cord delta opioid receptors. Br J Anaesth 1996; 77: 758 –63.[Abstract/Free Full Text]

18 Battaglia G, Rustioni A. Coexistence of glutamate and substance P in dorsal root ganglion neurons of the rat and monkey. J Comp Neurol 1988; 277: 302–12.[Medline]

19 Dickenson AH, Sullivan AF. Differential effects of excitatory amino acid antagonists on dorsal horn nociceptive neurones in the rat. Brain Res 1990; 506: 31–9.[Medline]

20 Furuyama T, Kiyama H, Sato K, et al. Region-specific expression of subunits of ionotropic glutamate receptors (AMPA-type, KA-type and NMDA receptors) in the rat spinal cord with special reference to nociception. Mol Brain Res 1993; 18: 141–51.[Medline]

21 Dougherty PM, Palecek J, Paleckova V, Sorkin LS, Willis WD. The role of NMDA and non-NMDA excitatory amino acid receptors in the excitation of primate spinothalamic tract neurons by mechanical, chemical, thermal, and electrical stimuli. J Neurosci 1992; 12: 3025–41.[Abstract]

22 Kohara A, Okada M, Tsutsumi R, et al. In-vitro characterization of YM872, a selective, potent and highly water-soluble -amino-3-hydroxy-5-methylisoxazole-4-propionate receptor antagonist. J Pharm Pharmacol 1998; 50: 795–801.[Medline]

23 Tjølsen A, Berge O-G, Hunskaar S, Rosland JH, Hole K. The formalin test: an evaluation of the method. Pain 1992; 51: 5–17.[Medline]

24 Li J, Simone DA, Larson AA. Windup leads to characteristics of central sensitization. Pain 1999; 79: 75–82.[Medline]

25 Haefely WE. Benzodiazepines. Int Anesthesiol Clin 1988; 26: 262–72.[Medline]




This article has been cited by other articles:


Home page
 Anesth. Analg.Home page
T. Nishiyama and K. Hanaoka
Midazolam Can Potentiate the Analgesic Effects of Intrathecal Bupivacaine on Thermal- or Inflammatory-Induced Pain
Anesth. Analg., May 1, 2003; 96(5): 1386 - 1391.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Résumé de cet Article
Right arrow Full Text (PDF)
Right arrow Submit a scholarly reply
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nishiyama, T.
Right arrow Articles by Yamaguchi, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nishiyama, T.
Right arrow Articles by Yamaguchi, T.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS