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Effect of Ninjin-yoei-to (Rensheng-Yangrong-Tang) on olfactory behavior after olfactory nerve transection.

Abstract

Ninjin-yoei-to (NYT), a Japanese traditional medicine, is used to treat athrepasia due to surgery, anorexia, cold constitution, and anemia. There are reports of the effects of NYT on the nervous system; however, there have been no behavioral studies of the effect of NYT on olfactory function. The olfactory system undergoes continuous replacement of sensory neurons. Morphologic and behavioral studies have shown that the olfactory system recovers after bilateral olfactory nerve transection (BNX). However, in the humans, olfactory function does not always recover. In this study, we examined the effect of oral NYT on behavioral recovery after BNX. Fourteen mice were subjected to BNX. The regular diet was mixed with 2% NYT (NYT diet). Mice were separated into two groups; seven mice were fed the regular diet (control group), and seven mice were fed the NYT diet (NYT group). NYT was administered beginning 7 days prior to BNX and continuing for 35 days after BNX. Mice in both groups had free access to food and water. Olfactory function was evaluated by testing each mouse's ability to avoid cotton balls treated with acetic acid. After BNX, mice lost their ability to avoid cotton balls treated with acetic acid. In the control group, the time for behavioral recovery after BNX was 28 days. In the NYT group, the time for behavioral recovery after BNX was 21 days. NYT hastened behavioral recovery after BNX. NYT may have therapeutic benefits for patients with olfactory disorders.

[c] 2007 Elsevier GmbH. All rights reserved.

Keywords: Olfactory; Behavior; Olfactory nerve transection; Ninjin-yoei-to; Traditional medicine; Regeneration

Introduction

Many researchers reported that the olfactory system undergoes continuous replacement of sensory neurons. The olfactory system recovers after bilateral olfactory nerve transection (BNX). BNX leads to degeneration of olfactory receptor neurons in the olfactory epithelium. Globose basal cells differentiate into mature olfactory receptor neurons sending axons to the olfactory bulb. There is functional reconnection of the new receptor neurons with second odor cells in the olfactory bulb, and the olfactory system is restored. Harding and Wright (1979) reported that the time for recovery of olfactory function after olfactory nerve transection was 23-25 days in mice. However, in the humans, olfactory function does not always recover.

It has been reported that the members of neurotrophin family play an important role in regeneration of the olfactory system. Martin et al. (2002) reported that nerve growth factor (NGF) binds to olfactory axons and facilitates their elongation in vitro. Brain-derived neurotrophic factor (BDNF) promotes survival of neurons cultured from the olfactory nerve (Buckland and Cunningham, 1998, 1999; Holcomb et al., 1995). Isoyama et al. (2004) reported that BDNF suppressed degeneration of bulbectomized olfactory epithelium and Briscoe et al. (2006) reported that BDNF increased the axon length of axons on cultured olfactory neurons.

Ninjin-yoei-to (NYT), a Japanese traditional medicine, is used to treat patients with athrepsia due to surgery, anorexia, cold constitution, and anemia. NYT has many pharmacologic actions, including various actions on the nervous system. Cui et al. (2004) reported that Rehmanniae radix, a component of NYT, could improve learning and memory in rats, and its mechanism may be related to increased expression of hippocampal NGF. Yabe et al. (2003) reported that NYT increased NGF secretion by cultured rat astrocytes. NYT may have therapeutic benefits for patients with olfactory disorders by increasing neurotrophin secretion.

Although the physiologic and morphologic effects of NYT and its components have been reported, little is known about the effect of NYT on olfactory nerve function. Behavioral analysis is important for the study of sensory nerve recovery. In the present study, we examined the effect of oral administration of NYT on the olfactory-mediated behavior of mice subjected to bilateral olfactory nerve transection.

Materials and methods

Materials

Thirty male ICR (Japan SLC, Hamamatsu, Japan) mice (6 weeks old) were used in this study. All animals weighed 28-33 g. Mice were housed in groups of 3 or 4 mice per cage in a room maintained at 24[degree]C on a 12-h light/dark cycle. All experimental protocols were reviewed by the Committee for Ethics on Animal Experiments of Yamaguchi University School of Medicine. All experiments were carried out in accordance with these guidelines and Federal Law No. 105 and Notification No. 6 of the Japanese government.

Experiment 1: the efficacy of our method for studying olfactory-mediated behavior

Sixteen mice were used to evaluate the efficacy of our method for studying olfactory-related behavior. Mice had free access to regular diet and water during all phases of the experiment.

Surgery

Eight of the 16 mice were subjected to bilateral olfactory nerve transection (BNX group), and the remaining 8 mice underwent a sham surgery (sham group). Animals in the BNX group were anesthetized by intraperitoneal injection of sodium pentobarbital (80mg/kg). A skin incision was made at the midline over the anterior skull. The frontal bone was removed, the bilateral olfactory bulbs were exposed, and the cribriform plate was identified. The dura covering the dorsal surface of the bilateral olfactory bulbs was cut and retracted. A pick was inserted between the olfactory bulbs and the cribriform plate, and a needle was run across the base of the olfactory bulbs, resulting in transection of all olfactory nerve fibers where they emerged from the cribriform plate and penetrated the bulbs. The skin incision was then sutured. Animals in the sham group underwent the same surgical procedure but without transection. Animals were observed carefully until fully recovered from anesthesia and were returned to their home cages. Animals were allowed free access to food and water before and after surgery.

Experiment 2: the effect of NYT on olfactory function

Fourteen mice were used to examine the effect of NYT on olfactory behavior during recovery from olfactory nerve transection.

Surgery

Fourteen mice underwent surgery. All mice were subjected to BNX as deseribed in Experiment 1.

Drugs

NYT is a herbal supplement comprised of Rehmanniae radix (4.0 g, root of Rehmannia glutinosa), Angelicae redix (4.0g, root of Angelica acutiloba), Poria (4.0g, fungus of Poria cocos), Atractylodes rhizoma (4.0 g, root of Atractylodes japonica), Ginseng radix (3.0 g, root of Panax ginseng), Cinnamomi cortex (2.5 g, bark of Cinnamomum cassia), Polygalae radix (2.0 g, root of Polygala tenuifolia), Paeoniae radix (2.0 g, root of Paeonia lactiflora), Aurantii nobilis pericarpium (2.0 g, peel of Citrus unshiu), Astragali radix (1.5 g, root of Astragalus mongholicus), Glycyrrhizae radix (1.0 g, root of Glycyrrhiza uralensis), and Schisandrae fructus (1.0 g, fruit of Schisandra chinensis) (Table 1). 3-Dimensional HPLC analysis was carried out to known broad chemical profile of NYT (Fig. 1). NYT was supplied by Tsumura Co. (Tokyo, Japan) as dried powder. The regular chow, CE-2 (Clea, Tokyo, Japan), was mixed with 2% NYT (NYT diet). The 14 mice were separated into two groups; seven mice were fed the regular diet (control group), and 7 mice were fed the NYT diet (NYT group). Mice in both groups had free access to food and water. NYT was administered beginning 7 days prior to surgery and continuing for 35 days after surgery. The amount of chow consumed per mouse was approximately 3.4 g/kg/day.

[FIGURE 1 OMITTED]
Table 1
Rehmanniae Rehmannia 4.0 Catalpol
 g

Radix glutinosa [beta]-Sitosterol Mannitol
 D-glucose D-galactose D-fructose
 Sucrose Arginine

Angelicae Angelica 4.0 Ligustilide
 g

Radix acutiloba n-Butylidenephthalide Palmitic
 acid Scopoletin Falcarinol
 Vitamin [B.sub.12]

Atractylodis Atractylodes 4.0 Atractylon
 g

Rhizoma japonica Atractylenolide I
 Diacetyl-atractylodiol (6E,
 12E)-Tetradecadiene-8 10-Diyne-l
 3-Diol diacetate Atractan A, B,
 C

Poria Poria cocos 4.0 Pachymic acid Eburicoic acid
 g Dehydroeburicoic acid
 3[beta]-0-acetyltumulosic acid
 3[beta]-O-acetyldehydrotumulosic
 acid Pachymaran Ergosterol

Ginseng Panax 3.0g Ginsenoside Ro, Ra~Rn

Radix ginseng Glucoginsenoside-Rf Panaxynol
 (falcarinol) Dihidropanaxacol
 [beta]-Sitosterol
 [beta]-Sitosterol glucoside
 D-glucose Maltose Trisaccharide
 A, B, C ATP

Cinnamomi Cinnamomum 2.5 Cinnamic aldehyde
 g

Cortex cassia Cinnamic acid Cinncassiol A-E
 Cinnzeylanol Cinnamoside
 D-glucose Procyanidin B-2
 Cinnamtannin I Melilotic acid

Polygalae Polygala 2.0 Tenuifoliside A-D
 g

Radix tenuifolia Onjisaponin A-G Onjixanthone I-II
 Sinapic acid

 Polygalitol

Paeoniae Paeonia 2.0 Paeoniflorin
 g

Radix lactiflora Oxypaeoniflorin
 Benzoylpaeoniflorin Albiflorin
 Penta-galloylglucose Paeonol
 Sucrose

Aurantii Citrus 2.0 D-limonene
 unshiu g

Nobilis

Pericarpium Hesperidin Nobiletin Synephrine

Astragali Astragalus 1.5g Formononetin

Radix mongholicus 3'-Hydroxyformononetin
 4'-Dihydroxy-5 6'
 -Dimethoxyisoflavone
 Astragaloside I-VIII Soyasaponin
 1 [gamma]-Aminobutyric acid
 Linoleic acid Linolenic acid
 [beta]Sitosterol

Glycyrrhizae Glycyrrhiza l.Og Glycyrrhizin

Radix uralensis Glabric acid Liquiritin apioside
 Liquiritin Licoricone
 Formononetin

Schisandrae Schisandra l.Og Schizandrin A-D

Fructus chinensis (gomisin A-D) Gomisin A-D
 Deoxyschizandrin Citral

[beta]-Chamigrene
Citric acid


Olfactory behavior analysis

For both Experiments 1 and 2 olfactory function was evaluated by testing each mouse's ability to avoid cotton balls treated with acetic acid placed in a corner of a square chamber. Experiments were conducted between 20:00h and 21:00h in a dark room isolated from external noise. A light reflector (1.5 cm diameter) was attached with adhesive to the back of each mouse. Experiments were performed in a dark, soundproof chamber (30 cm x 30 cm) equipped with a ventilation system. A cotton ball (1.5 cm) was placed in each of the four corners of the chamber. Three milliliters of a 40% acetic acid solution was added to one of the cotton balls. The position of the cotton ball treated with acetic acid was changed with every trial. Mice were placed into the chamber and were allowed to move freely about the chamber (Fig. 2). Behavior of the mice was recorded with an infrared digital video camera (Sony, Tokyo, Japan) for 3 min. Videos were analyzed with NIH Image software on a personal computer (Apple Computer, Cupertino, CA). Movement was translated onto positional coordinates. The chamber was divided into four areas (acetic acid area, opposite area, side area 1, and side area 2) (Fig. 2). The positional coordinates of each mouse at each time point were calculated (Fig. 3), and the mean time spent in each area was calculated such as in Fig. 4. In Experiment 1, experiments were conducted once per day on the day before surgery and on postoperative days 1, 7, 14, 21, 28, 35, 42, 56, and 70. In Experiment 2, experiments were conducted once per day on the day before surgery and on postoperative days 1, 7, 14, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, and 35.

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

Statistical analysis

The mean times spent in the acetic acid and opposite areas were compared by Mann-Whitney U test with Stat View 5.0 (SAS Institute, Cary, NC). A result was considered significant if p<0.05.

Results

Experiment 1: the efficacy of our method for studying olfactory-mediated behavior

In the sham group, the mean time spent in the acetic acid area was significantly less than that in the opposite area throughout the observation period (p < 0.01, Fig. 5). In the BNX group, before surgery, the mean time spent in the acetic acid area was significantly less than that in the opposite area (p< 0.01). After bilateral olfactory nerve transection, mice lost their ability to avoid cotton balls treated with acetic acid. There was no significant difference between the mean time spent in the acetic acid area and that spent in the opposite area up to 21 days after surgery. By 28 days after surgery, the mean time spent in the acetic acid area was again significantly less than that in the opposite area (p<0.01, Fig. 6).

[FIGURE 5 OMITTED]

[FIGURE 6 OMITTED]

3.2. Experiment 2: the effect of NYT on olfactory function

In the control group, before surgery, the mean time spent in the acetic acid area was significantly less than that in the opposite area (p<0.01). After bilateral olfactory nerve transection, mice lost their ability to avoid cotton balls treated with acetic acid, and there was no significant difference between the mean time spent in the acetic acid area and that spent in the opposite area up to 27 days after surgery. At 28 days after surgery, the mean time spent in the acetic acid area was again significantly less than that in the opposite area (p<0.05, Fig. 7).

[FIGURE 7 OMITTED]

In the NYT group, before surgery, the mean time spent in the acetic acid area was significantly less than that in the opposite area (p<0.01). After bilateral olfactory nerve transection, mice lost their ability to avoid cotton balls treated with acetic acid. There was no significant difference between the mean time spent in the acetic acid area and that spent in the opposite area up to 20 days after surgery. At 21 days after surgery, the mean time spent in the acetic acid area was again significantly less than that in the opposite area, (p<0.01, Fig. 8). Food intake per day or body weight did not differ significantly between the NYT group and control group during the experiments.

[FIGURE 8 OMITTED]

Discussion

Experiment 1: the efficacy of our method for studying olfactory-mediated behavior

Various behavioral methods for analysis of olfactory function have been reported. However, there are disadvantages to these conventional methods. Ducray et al. (2002) evaluated olfactory function in mice by testing the ability to discriminate between two odorants in a T-maze test. Their test requires a large T-shaped apparatus, and several trials are necessary to evaluate olfactory function. Edwards et al. (1972) and Yee and colleagues also tested olfactory function in mice and rats with a buried food task (Yee and Costanzo, 1995; Yee and Wysocki, 2001). In such tests, animals are fasted before the test, and this may cause undue stress on the animals. Other groups have used a foot-shock freezing paradigm (Herzog and Otto, 2002; Otto et al., 1997), which may be more stressful than a buried food paradigm. These conventional methods are complex and can require large amounts of time, and some tests require special equipment and preconditioning (Darling and Slotnick, 1994; Herzog and Otto, 2002). In the present study, we established a simple and reproducible method based on avoidance of acetic acid to evaluate olfactory function in mice. We found that all mice avoided the acetic acid on the first trial and that the mean time spent in the acetic acid area was significantly less than that in the opposite area, indicating that preconditioning is not necessary. In addition, each trial requires only 3 min. Stressors are not present, and all mice are allowed free access to food and water during the study period.

One possible issue with our method is potential stimulation of the trigeminal never by acetic acid, which may influence behavior in a manner independent of olfaction. BNX mice are impaired only with respect to olfactory function. Olfactory nerve transection damage mature olfactory receptor neurons without affecting other cells within the olfactory epithelium (Graziadei and Graziadei, 1979). If the avoidance behavior were due to trigeminal nerve stimulation, BNX mice would have continued to avoid the acetic acid area. However, BNX mice spent similar amounts of time in all areas from 1 to 21 days after surgery, indicating that trigeminal nerve stimulation was not involved. Sham mice avoided acetic acid throughout the experiments, suggesting that mice do not adapt to acetic acid odor stimulation.

After surgery, BNX mice lost the ability to avoid acetic acid. There was no significant difference between the mean time spent in the acetic acid area and that spent in the opposite area 21 days after surgery. Twentyeight days after surgery, the mean time spent by BNX mice in the acetic acid area was again significantly less than that in the opposite area, indicating that olfactory function had recovered. In our method, the time for recovery of olfactory function after olfactory nerve transection was 28 days. This is consistent with previous reports of 23-25 days for recovery of olfactory function in mice after olfactory nerve transection (Harding and Wright, 1979). Our method allows simple, reproducible evaluation of olfactory-mediated behavior in mice.

Experiment 2: the effect of NYT on olfactory function

We examined the effect of NYT on olfactory function during recovery after olfactory nerve transection. Oral administration of NYT had no effect on food intake and body weight of mice. In the control group, at 28 days after surgery, the mean time spent in the acetic acid area was again significantly less than that in the opposite area, indicating that the time for recovery of olfactory function after olfactory nerve transection was 28 days. In the NYT group, the time for recovery of olfactory function after olfactory nerve transection was 21 days. These findings suggest that NYT hastens recovery of olfactory function after olfactory nerve transection.

NYT and its components have various effects on the nervous system. Ginseng radix increases the survival rate of nerve cell cultured from cerebral cortex neurons and promotes production of NGF in brain (Himi et al., 1989; Salim et al., 1997). Ginsenoside Rbl isolated from Ginseng radix potentiates NGF-induced neurite outgrowth of cultured chick embryonic dorsal root ganglia (Nishiyama et al., 1994). Catalpol, which is from Rehmanniae radix, has neuroprotective effect. Liu et al. (2006) reported that BDNF levels were up-regulated in the hippocampus of catalpol-treated rats after 10 days of treatment. Polygalae radix promotes production of NGF and BDNF in brain (Yabe et al., 1997). Song et al. (2001) reported that NYT increases NGF levels in olfactory bulb.

Members of the neurotrophin family play an important role in the olfactory system. NGF in olfactory bulb plays an important role in maintenance and development, and hastens regeneration in the olfactory system. NGF is produced in the olfactory bulb (Ebendal, 1992), and transported to the olfactory epithelium (OE) (Miwa et al., 1998). NGF receptor (NGFR) is expressed in the mouse OE after olfactory nerve transection, although NGFR does not appear to be expressed in the OE of adult control mice (Miwa et al., 1993). NGF present in the OE may modulate turnover of neurons (Aiba et al., 1993). Yasuno et al. (2000) reported that NGF may protect against degenerative changes in olfactory receptor neurons following axotomy. Martin et al. (2002) reported that NGF binds to olfactory axons and facilitates their elongation in vitro.

BDNF mRNA and protein are expressed by cells in the olfactory bulb (Deckner et al., 1993; Guthrie and Gall, 1991). BDNF contributes to trophic regulation of olfactory neuron pathway (Buckland and Cunningham, 1998). Isoyama et al. (2004) reported that BDNF suppresses degenerative changes in bulbectomized OE. Briscoe et al. (2006) reported that BDNF increases the axon length of cultured olfactory neurons. It is possible that NYT hastens regeneration of olfactory receptor neurons by increasing expression of NGF and BDNF in olfactory bulb.

In conclusion, we report that NYT speeds recovery of olfactory-mediated behavior after olfactory nerve transection in mice. NYT may have therapeutic benefits in patients with olfactory disorders.

Acknowledgements

This work was supported by a grant from the Japanese Ministry of Education, Culture, Sports, Science and Technology (17659534).

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* Corresponding author.

E-mail address: hiro-shi@yamaguchi-u.ac.jp (H. Yamashita).

0944-7113/$-see front matter (c) 2007 Elsevier GmbH. All rights reserved. doi:10.10.16/j.phymed.2007.08.006

Aigo Yamasaki (a), Kazuma Sugahara (a), Tsuyoshi Takemoto (a), Takuo Ikeda (b), Hiroshi Yamashita (a),*

(a) Department of Otolarynology, Yamaguchi University Graduate School of Medicine, Yumaguchi, Japan

(b) Department of Otolaryngology, Tsustumigaura Children's Medical Center, Yamaguchi, Japan

Received 29 March 2007; accepted 8 August 2007
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Author:Yamasaki, Aigo; Sugahara, Kazuma; Takemoto, Tsuyoshi; Ikeda, Takuo; Yamashita, Hiroshi
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9JAPA
Date:May 1, 2008
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