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Chemical Senses Advance Access originally published online on June 4, 2008
Chemical Senses 2008 33(7):633-637; doi:10.1093/chemse/bjn030
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© The Author 2008. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Odor Detection Ability and Thallium-201 Transport in the Olfactory Nerve of Traumatic Olfactory-Impaired Mice

Hideaki Shiga1, Yayoi Kinoshita1, Kohshin Washiyama2, Daisuke Ogawa2, Ryohei Amano2, Kyoko Hirota1, Toshiaki Tsukatani1, Mitsuru Furukawa1 and Takaki Miwa1

1 Department of Otorhinolaryngology, Graduate School of Medical Science, Kanazawa University, 13-1 Takaramachi, Kanazawa, Ishikawa 920, Japan 2 Department of Forefront Medical Technology, Graduate School of Medical Science, Kanazawa University, 13-1 Takaramachi, Kanazawa, Ishikawa 920, Japan

Correspondence to be sent to: Hideaki Shiga, Department of Otorhinolaryngology, Graduate School of Medical Science, Kanazawa University, 13-1 Takaramachi, Kanazawa, Ishikawa 920, Japan. e-mail: shigah{at}med.kanazawa-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Funding
 Acknowledgements
 References
 
Although olfactory nerve damage is a contributing factor in the diagnosis of posttraumatic olfactory loss, at present, there are no methods to directly assess injury to these nerves. We have shown that following olfactory nerve injury in mice, thallium-201 (201Tl) transport from the nasal cavity to the olfactory bulb decreases. To determine if olfactory function after nerve injury could be assessed with nasal administration of 201Tl, we measured the correlation between odor detection ability (ODA) and the rate of transport of 201Tl in olfactory nerves. Both ODA and 201Tl transport were measured after bilateral olfactory nerve transection for a 4-week period. Cycloheximide solution was used for ODA against tap water. 201Tl transport was measured as the ratio of radioactivity in the nasal cavity and olfactory bulb with gamma spectrometry. There was a significant correlation between ODA and the rate of 201Tl transport in the olfactory nerve. These findings suggest that olfactory function after nerve injury can be objectively evaluated with the nasal administration of 201Tl.

Key words: odor detection, posttraumatic olfactory impairment, thallium-201


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 Acknowledgements
 References
 
Injuries to the olfactory bulb and tract (88% of patients), subfrontal region (60%), and temporal lobe (32%) have been shown using magnetic resonance imaging (MRI) in patients with posttraumatic olfactory dysfunction; however, this did not correlate well with olfactory test scores (Yousem et al. 1996Go), and it is difficult to visualize damage to olfactory nerve fibers with MRI (Fujii et al. 2002Go). It would be useful to use objective measurements to assess damage to olfactory nerve fibers and also to predict the course of traumatic olfactory impairment.

Functional MRI (fMRI) signals by response to odor stimuli in the piriform cortex, amygdala, and orbitofrontal cortex have been shown in humans (Zatorre et al. 1992Go; Zald and Pardo 1997Go; Sobel et al. 1998Go; Gottfried et al. 2002Go); however, the possibility of diagnosing a lesion in olfactory nerve fibers with fMRI is unconfirmed. Furthermore, MRI and fMRI are not available for individuals who have metal in their bodies. Other neuroimaging techniques therefore need to be developed to diagnose patients with traumatic olfactory impairment.

We have previously established a simple and reliable method to examine olfactory function in rodents by the cycloheximide solution avoidance behavior (Miwa 1989Go; Moriizumi et al. 1994Go; Fukushima et al. 2002Go; Miwa et al. 2004Go). This is based on the ability to detect the odor of cycloheximide solution.

Thallium-201 (201Tl) is transported in the olfactory nerve of mice when administered intranasally (Kanayama et al. 2005Go). The transport of 201Tl in the olfactory nerve is decreased following transection of the olfactory nerve fibers (Kinoshita et al. 2008Go). 201Tl has been widely used by systemic administration in isotope imaging for clinical diagnosis (Iida and Eberl 1998Go; Higuchi et al. 2005Go).

The purpose of this study was to determine whether olfactory impairment due to head injury can be diagnosed based on the rate at which 201Tl is transported in olfactory nerve fibers after nasal administration of 201Tl. Our results show a positive correlation between odor detection ability (ODA) and the 201Tl transport rate in the olfactory nerve of mice.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 Acknowledgements
 References
 
Materials

Fifteen Male ICR mice, 8 weeks of age (Japan SLC, Shizuoka, Japan), were divided into 5 groups by survival day after surgery (2 days, 7 days, 14 days, and 28 days) and sham operation. Mice were housed in a 22 °C air-conditioned room with a 12:12 h light:dark cycle and freely provided food (Charles River Laboratories Japan, Inc., Yokohama, Japan) and water. The Kanazawa University Animal Experiment Committee approved all animal experimental procedures in advance (No. 26142).

Odor detection ability

Cycloheximide has a peculiar odor and unpleasant taste. After being deprived of water for 48 h, mice were trained to avoid the cycloheximide solution. The cycloheximide solution avoidance behavior was examined by testing the ability to distinguish a 0.01% cycloheximide solution from tap water. The positions of the cycloheximide solution bottle and tap water bottle were randomized according to a uniform random number (the cycloheximide bottle on the right side of the cage if an odd number; the water bottle on the right if an even number). Each mouse was examined twice before nerve transection for conditioning to the odor of cycloheximide. Ten trials were repeated each time.

Bilateral olfactory nerve transection

The olfactory nerve fibers in mice were transected according to a method previously described (Kinoshita et al. 2008Go). We exposed both the right olfactory bulb (OBR) and the left olfactory bulb, cutting the frontal bones of mice under anesthesia (ether inhalation; intraperitoneal administration of pentobarbital sodium, 0.03 mg/g). The olfactory nerve fibers were carefully transected bilaterally with a Teflon knife avoiding damage to olfactory bulbs (bilateral olfactory nerve transection, BNTX). The skin incision was closed using a nylon suture. Sham-operated mice had their olfactory bulbs exposed, but the olfactory nerves were not transected.

ODA test and 201Tl experiments

Mice were assessed for both ODA and the measurement of 201Tl transport at days 2 (N = 3), 7 (N = 3), 14 (N = 3), and 28 (N = 3) following nerve transection (BNTX olfactory-impaired mice). The ODA was 60% or less in all BNTX mice at 2 days after nerve transection. Control mice (N = 3) were observed 28 days after the sham operation. 201TlCl saline solution (78 MBq/ml) was obtained from Nihon Medi-Physics (Kobe, Japan), and 10 µl was carefully instilled into the right nostril of each mouse while avoiding sneeze with a microinjection pipette under anesthesia (ether inhalation). The mice were sacrificed under ether anesthesia 3 h later. Tissue samples were obtained from the right nasal turbinate and the OBR.

The radioactivity of the samples was measured with gamma spectrometry using the Auto Well Gamma System (model ARC-380; Aloka, Tokyo, Japan) after weight measurement. The uptake percentage (percent dose) of the isotope in each sample was calculated as a percentage of the radioactivity of each sample per radioactivity of 1% of 10-µl 201TlCl solution. The 201Tl uptake percentage per weight (percent dose per gram) was calculated as a percentage of the uptake percentage of each sample per wet weight.

The transport of 201Tl by olfactory nerve fibers was calculated as a percentage of the uptake percentage per weight (percent dose per gram) of the isotope in the OBR divided by the isotope uptake percentage per weight (percent dose per gram) in the right nasal turbinate.

Autoradiography

The mice were given 201TlCl solution (10 µl) for autoradiography into their nasal cavity. Three hours later, they were sacrificed under ether anesthesia and the head was dissected. After removal of the skin and muscle, the head was embedded in Tissue-Tek OCT Compound (Sakura Finetechnical, Tokyo, Japan) and frozen in liquid nitrogen. The frozen heads were sectioned at 50 µm. Coronal sections were obtained and dried. The sections were adhered to an isotope imaging plate (FUJIFILM, Tokyo, Japan) and then assessed with a Bio-imaging Analyzer (FUJIFILM). Three mice from each group (control, 2-day BNTX, and 28-day BNTX) were assessed.

Statistical analysis

The Kruskal–Wallis test was applied to compare ODA in multiple groups. The Spearman correlation was applied to compare ODA and the transport of 201Tl in olfactory nerve fibers. A statistical comparison of median values was performed using the Mann–Whitney U test. All P values were 2 tailed (Prism 5, GraphPad, San Diego, CA). A P value <0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 Acknowledgements
 References
 
Odor detection ability

To determine the change of olfactory ability after the transection of olfactory nerve, ODA was assessed based on the cycloheximide solution avoidance behavior. ODA improved with time after recovery from nerve transection (Figure 1; N = 3 for each group; Kruskal–Wallis test, *P < 0.03).


Figure 1
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  		Figure 1 ODA after transection of olfactory nerve fibers in 2-day, 7-day, 14-day, and 28-day BNTX mice and controls. ODA improved with time after transection (N = 3 for each group; Kruskal–Wallis test, *P < 0.03). Bars indicate medians.

 
Correlation between ODA and the transport rate of 201Tl in the olfactory nerve

To determine the transport rate of 201Tl between the nasal cavity and the olfactory bulb, we divided the uptake percentage per weight (percent dose per gram) in the OBR by the uptake percentage per weight (percent dose per gram) in the right nasal turbinate. The transport rate of 201Tl in olfactory nerve fibers was significantly correlated with ODA in 2-day, 7-day, 14-day, and 28-day bilateral olfactory nerve transection (BNTX) mice and the control (Figure 2; N = 15, Spearman correlation, ***P < 0.0001). The transport rate of 201Tl between the nasal cavity and the olfactory bulb following nasal administration may reflect the level of damage to olfactory nerve fibers in mice.


Figure 2
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  		Figure 2 Correlation between ODA and the transport rate of 201Tl in the olfactory nerve in 2-day, 7-day, 14-day, and 28-day BNTX mice and controls. The transport rate of 201Tl in olfactory nerve fibers was significantly correlated with ODA (N = 15; Spearman correlation, ***P < 0.0001).

 
201Tl transport in olfactory nerve in the early and late posttransection groups

The transport rate of 201Tl in olfactory nerve fibers was significantly higher in 14-day and 28-day BNTX mice (late post-nerve transection group) than in the 2-day and 7-day BNTX mice (early post-nerve transection group) (Figure 3; N = 6 for each group; Mann–Whitney U test, **P < 0.005; early group: minimum, 1.312; median, 6.269; maximum, 13.08; 25th percentile, 3.183; 75th percentile, 10.27; late group: minimum 12.89; median, 14.8; maximum, 28.63; 25th percentile, 13.28; 75th percentile, 28.38). The transport rate of 201Tl in the olfactory nerve improved with time after nerve transection.


Figure 3
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  		Figure 3 The significant difference between the early posttransection group (2-day and 7-day BNTX mice) and late posttransection group (14-day and 28-day BNTX mice) in the transport rate of 201TI to the OBR from the right nasal cavity. The transport of 201Tl in olfactory nerve fibers (201Tl uptake percentage per weight in OBR/201Tl uptake percentage per weight in the right nasal turbinate) was significantly higher in the late group than in the early group (N = 6 for each group; Mann–Whitney U test, **P < 0.005). Bars indicate medians.

 
201Tl uptake images with autoradiography

To show the relationship between 201Tl uptake images in the olfactory bulb and the extent to which olfactory nerve fibers were damaged, we compared 201Tl uptake images of mouse heads among control, 2-day BNTX mice, and 28-day BNTX mice using autoradiography. 201Tl uptake in the OBR 2 days after nerve transection and 201Tl administered into the right nasal cavity were significantly lower than the increased uptake in 28-day BNTX mice (Figure 4). The regeneration of olfactory nerve fibers could be assessed using this 201Tl-based imaging method.


Figure 4
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  		Figure 4 Representative isotope images of coronal sections of the head 3 h after the unilateral nasal administration of 201TICl solution (control, 2-day, and 28-day BNTX mice). Circles outline the olfactory bulb.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 Acknowledgements
 References
 
We demonstrated a significant correlation between ODA (the cycloheximide solution avoidance behavior) and the 201Tl transport rate in the olfactory nerves of mice with olfactory nerve transection and controls. The regeneration and recovery of olfactory nerve fibers may be related to the transport rate of thallium from the nasal cavity to the olfactory bulb. Furthermore, our results show that the transport rate of 201Tl in the olfactory nerve improved with time after nerve transection, and the regeneration of olfactory nerve fibers could be determined using 201Tl-based imaging method. Our 201Tl-based imaging method may be useful for the analysis of the connections between nasal cavity and olfactory bulb in olfactory-impaired animal models.

Meredith and O'Connell reported horseradish peroxidase (HRP) uptake from the nasal cavity to the brain in mammals (Meredith and O'Connell 1988Go). HRP reaction was assessed by histochemistry in the previous report. 201Tl uptake in samples can be counted by gamma spectrometry; therefore, thallium may be more simple and quantitative than HRP. Furthermore, if 201Tl uptake in the olfactory bulb and nasal cavity could be visualized with a gamma camera in live animals, follow-up would be available in vivo without sacrifice after nerve transection.

Current olfactory function tests (the University of Pennsylvania Smell Identification Test [Doty et al. 1984Go], the Connecticut Chemosensory Clinical Research Center Test [Cain et al. 1988Go], and Sniffin sticks [Hummel et al. 1997Go; Kobal et al. 2000Go]) depend on the patient's voluntary response to odor. So far, we have no method to visualize the connection between olfactory epithelium and the olfactory bulb in the clinic.

201Tl is routinely used intravenously in nuclear medicine. If we could adapt a nasal 201Tl administration technique to a patient with head injuries resulting in anosmia, the decrease of odor-evoked signals in fMRI due to disconnection of olfactory nerve fibers could be validated when damage to olfactory nerve fibers is assessed with fMRI. We are planning to test the new olfactory nerve imaging technique with thallium on humans after approval from the ethics committee of Kanazawa University Hospital.

It was shown that the inhalation of manganese-54 (54Mn) results in extensive uptake of 54Mn by the olfactory bulb in rats (Brenneman et al. 2000Go). The detection of damage to the olfactory system with fMRI in live animals administered manganese into the nasal cavity has been reported (Drobyshevsky et al. 2006Go); however, high doses of manganese inhalation are known to be associated with neurotoxicity in both animals and humans (Aschner et al. 2007Go). Therefore, 201Tl scintigraphy with nasal administration of 201Tl may be more safety than imaging with 54Mn administration. The effects of the nasal 201Tl administration technique on olfactory function are under investigation in animals.

Recovery of chemosensory deficits occurs up to 12–18 months after a traumatic event (Reiter et al. 2004Go). In humans, the recovery rate in patients with traumatic olfactory impairment is less than 30% (Fujii et al. 2002Go). Patients with intact olfactory nerve fibers may be well selected by means of a new isotope imaging technique for the long-term treatment of olfactory dysfunction. 201Tl scintigraphy with nasal administration of 201Tl may be also useful for the analysis of treatment efficacy with new medicines for patients with posttraumatic olfactory impairment.

In conclusion, damaged olfactory nerve fibers could be assessed in an animal model of olfactory impairment through the nasal administration of 201Tl. The uptake of 201Tl in the olfactory bulb correlated with the ODA of the mice.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
Acknowledgements
 References
 
Kanazawa University (No.1817211 to H.S., No.1816220 to K.W.); Grant-in-Aid for Scientic Research from the Ministry of Education, Science and Culture of Japan (C18591860 to T.M.).


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 Acknowledgements
References
 
We are grateful to Division of Radioisotopes, Kanazawa University Hospital for the technical support. We are also grateful to Professor Hiroyuki Nakamura (Kanazawa University) for his advice on the statistical analysis. We thank Professor Richard Costanzo (Virginia Commonwealth University) for his comments on the manuscript.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 Acknowledgements
 References
 
Aschner M, Guilarte TR, Schneider JS, Zheng W. Manganese: recent advances in understanding its transport and neurotoxicity. Toxicol Appl Pharmacol (2007) 221:131–147.[CrossRef][Web of Science][Medline]

Brenneman KA, Wong BA, Buccellato MA, Costa ER, Gross EA, Dorman DC. Direct olfactory transport of inhaled manganese ((54)MnCl(2)) to the rat brain: toxicokinetic investigations in a unilateral nasal occlusion model. Toxicol Appl Pharmacol (2000) 169:238–248.[CrossRef][Web of Science][Medline]

Cain WS, Gent JF, Goodspeed RB, Leonard G. Evaluation of olfactory dysfunction in the Connecticut Chemosensory Clinical Resarch Center (CCCRC). Laryngoscope (1988) 98:83–88.[Web of Science][Medline]

Doty RL, Shaman P, Kimmelman CP, Dann MS. University of Pennsylvania smell identification test: a rapid quantitative olfactory function test for the clinic. Laryngoscope (1984) 94:176–178.[Web of Science][Medline]

Drobyshevsky A, Robinson AM, Derrick M, Wyrwicz AM, Ji X, Englof I, Tan S. Sensory deficits and olfactory system injury detected by novel application of MEMRI in newborn rabbit after antenatal hypoxia-ischemia. Neuroimage (2006) 32:1106–1112.[CrossRef][Web of Science][Medline]

Fujii M, Fukazawa K, Takayasu S, Sakagami M. Olfactory dysfunction in patients with head trauma. Auris Nasus Larynx (2002) 29:35–40.[CrossRef][Web of Science][Medline]

Fukushima N, Oikawa S, Yokouchi K, Kawagishi K, Moriizumi T. The minimum number of neurons in the central olfactory pathway in relation to its function: a retrograde fiber tracing study. Chem Senses (2002) 27:1–6.[Abstract/Free Full Text]

Gottfried JA, Deichmann R, Winston JS, Dolan RJ. Functional heterogeneity in human olfactory cortex: an event-related functional magnetic resonance imaging study. J Neurosci (2002) 22:10819–10828.[Abstract/Free Full Text]

Higuchi T, Taki J, Sumiya H, Kinuya S, Nakajima K, Namura M, Tonami N. Characterization of cartilaginous tumors with 201Tl scintigraphy. Ann Nucl Med (2005) 19:95–99.[Web of Science][Medline]

Hummel T, Sekinger B, Wolf S, Pauli E, Kobal G. ‘Sniffin’ sticks': olfactory performance assessed by the combined testing of odor identification, odor discrimination and olfactory threshold. Chem Senses (1997) 22:39–52.[Abstract/Free Full Text]

Iida H, Eberl S. Quantitative assessment of regional myocardial blood flow with thallium-201 and SPECT. J Nucl Cardiol (1998) 5:313–331.[CrossRef][Web of Science][Medline]

Kanayama Y, Enomoto S, Irie T, Amano R. Axonal transport of rubidium and thallium in the olfactory nerve of mice. Nucl Med Biol (2005) 32:505–512.[CrossRef][Web of Science][Medline]

Kinoshita Y, Shiga H, Washiyama K, Ogawa D, Amano R, Ito M, Tsukatani T, Furukawa M, Miwa T. Thallium transport and the evaluation of olfactory nerve connectivity between the nasal cavity and olfactory bulb. Chem Senses (2008) 33:73–78.[Abstract/Free Full Text]

Kobal G, Klimek L, Wolfensberger M, Gudziol H, Temmel A, Owen CM, Seeber H, Pauli E, Hummel T. Multicenter investigation of 1,036 subjects using a standardized method for the assessment of olfactory function combining tests of odor identification, odor discrimination, and olfactory thresholds. Eur Arch Otorhinolaryngol (2000) 257:205–211.[CrossRef][Medline]

Meredith M, O'Connell RJ. HRP uptake by olfactory and vomeronasal receptor neurons: use as an indicator of incomplete lesions and relevance for non-volatile chemoreception. Chem Senses (1988) 13:487–515.[Abstract/Free Full Text]

Miwa T. Study of behavioral and histological change in mice following olfactory nerve section. J Juzen Med Soc (1989) 98:391–414.

Miwa T, Watanabe A, Mitsumoto Y, Furukawa M, Fukushima N, Moriizumi T. Olfactory impairment and Parkinson's disease-like symptoms observed in the common marmoset following administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Acta Otolaryngol (2004) 553(Suppl):80–84.

Moriizumi T, Tsukatani T, Sakashita H, Miwa T. Olfactory disturbance induced by deafferentation of serotonergic fibers in the olfactory bulb. Neuroscience (1994) 61:733–738.[CrossRef][Web of Science][Medline]

Reiter ER, DiNardo LJ, Costanzo RM. Effects of head injury on olfaction and taste. Otolaryngol Clin North Am (2004) 37:1167–1184.[CrossRef][Web of Science][Medline]

Sobel N, Prabhakaran V, Desmond JE, Glover GH, Goode RL, Sullivan EV, Gabrieli JD. Sniffing and smelling: separate subsystems in the human olfactory cortex. Nature (1998) 392:282–286.[CrossRef][Web of Science][Medline]

Yousem DM, Geckle RJ, Bilker WB, McKeown DA, Doty RL. Posttraumatic olfactory dysfunction: MR and clinical evaluation. AJNR Am J Neuroradiol (1996) 17:1171–1179.[Abstract]

Zald DH, Pardo JV. Emotion, olfaction, and the human amygdala: amygdala activation during aversive olfactory stimulation. Proc Natl Acad Sci USA (1997) 94:4119–4124.[Abstract/Free Full Text]

Zatorre RJ, Jones-Gotman M, Evans AC, Meyer E. Functional localization and lateralization of human olfactory cortex. Nature (1992) 360:339–340.[CrossRef][Web of Science][Medline]

Accepted 9 May 2008


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