doi: /brain/awp243 Brain 2009: 132;

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1 doi: /brain/awp243 Brain 2009: 132; BRAIN A JOURNAL OF NEUROLOGY Increasing olfactory bulb volume due to treatment of chronic rhinosinusitis a longitudinal study V. Gudziol, 1 D. Buschhüter, 1 N. Abolmaali, 2 J. Gerber, 3 P. Rombaux 4 and T. Hummel 1 1 Department of Otorhinolaryngology, University of Dresden Medical School, ( Technische Universität Dresden ), Fetscherstrasse 74, Dresden, Germany 2 Radiation Research in Oncology, University of Dresden Medical School, Germany 3 Department of Neuroradiology, University of Dresden Medical School, Germany 4 Department of Otorhinolaryngology, University of Louvain, Brussels, Belgium Correspondence to: Volker Gudziol MD, Smell & Taste Clinic, Department of Otorhinolaryngology, University of Dresden Medical School ( Technische Universität Dresden ), Fetscherstrasse 74, Dresden, Germany volker.gudziol@uniklinikum-dresden.de Differentiation of progenitor cells into neurons in the olfactory bulb depends on olfactory stimulation that can lead to an increase in olfactory bulb volume. In this study, we investigated whether the human olfactory bulb volume increases with increasing olfactory function due to treatment of chronic rhinosinusitis. Nineteen patients with chronic rhinosinusitis were investigated before and after treatment. For comparison, additional measurements were performed in 18 healthy volunteers. Volumetric measurements of the olfactory bulb were based on planimetric manual contouring of magnetic resonance scans. Olfactory function was evaluated separately for each nostril using tests for odour threshold, odour discrimination and odour identification. Measurements were performed on two occasions, 3 months apart. In healthy controls, the olfactory bulb volume did not change significantly between the two measurements. In contrast, the olfactory bulb volume in patients increased significantly from the initial to mm 3 on the left side (P = 0.02) and from to mm 3 on the right side (P50.001). The increase in olfactory bulb volume correlated significantly with an increase in odour thresholds (r = 0.60, P = 0.006, left side; r = 0.49, P = 0.03, right side), but not with changes in odour discrimination or odour identification. Results of this study support the idea that stimulation of olfactory receptor neurons impacts on the cell death in the olfactory bulb, not only in rodents but also in humans. To our knowledge, this is the first longitudinal study that describes an enlargement of the human olfactory bulb due to improvement of peripheral olfactory function. Keywords: olfaction; smell; brain plasticity; sensory system; sinus surgery Introduction Most cells in the central nervous system are born in the embryonic and early post-natal period. The olfactory system differs because in both the sensory epithelium (Graziadei and Metcalf, 1971; Graziadei and DeHan, 1973) and the olfactory bulb, progenitor cells differentiate into neurons well into adulthood. The olfactory bulb the first relay where olfactory information is processed receives progenitor cells via the rostral migration stream from the subventricular zone (Altmann, 1969). The speed of this replacement appears to be dependent on sensory input. Specifically, deprivation from olfactory stimuli leads to a decrease Received June 11, Revised August 10, Accepted August 17, Advance Access publication September 22, 2009 ß The Author (2009). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please journals.permissions@oxfordjournals.org

2 Volume of the olfactory bulb Brain 2009: 132; of olfactory bulb volume by 25% (Meisami, 1976). However, in rats, olfactory bulb size recovers 40 days after normalization of olfactory stimulation (Cummings et al., 1997). In accord with the early investigation in animals, patients with olfactory loss also demonstrate smaller olfactory bulb volume than controls in magnetic resonance imaging studies (Yousem et al., 1999; Mueller et al., 2005b; Rombaux et al., 2006a, b, 2009). Chronic rhinosinusitis is the most frequent cause of olfactory loss in humans. Olfactory disturbances in patients with chronic rhinosinusitis are attributed to sinunasal inflammation that (i) impairs odorant access to the olfactory cleft and (ii) disturbs the function of the olfactory epithelium (Klimek and Eggers, 1997). For example, biopsies from the olfactory epithelium in patients with chronic rhinosinusitis revealed inflammation of the receptor organ (Kern, 2000). Thus, in patients with chronic rhinosinusitis, functional endoscopic sinus surgery and postoperative administration of glucocorticoids are used for the treatment of olfactory function. The aim of the present study was to investigate whether the human olfactory bulb volume increased after short-term treatment of olfactory function. To our knowledge, only one publication has addressed this question, but investigated only a small number of patients (Abolmaali et al., 2008). Our aim, in this study, was to measure olfactory bulb volume and olfactory function in patients with chronic rhinosinusitis and in healthy controls, the day before and 3 months after surgery. Subjects and Methods The longitudinal, prospective study was performed according to the Declaration of Helsinki on Biomedical Research Involving Human Subjects. Written consent was obtained by each participant. It was approved by the Ethics Committee of the Faculty of Medicine Carl Gustav Carus of the Technische Universität Dresden, Germany. Patients Nineteen patients, with chronic rhinosinusitis with polyps, who were scheduled for functional endoscopic sinus surgery in the Department of Otorhinolaryngology of the University of Dresden Medical School, were investigated. Patients age was between 36 and 73 years [ years (mean standard deviation)]. Nine female and 10 male patients were investigated. Patients were recruited from October 2007 to May Surgery was performed when two or more of the following symptoms persisted over a period of 43 months, despite appropriate medical treatment: nasal obstruction, increased retronasal dripping of mucous, facial or cranial pain, or facial pressure and smell disorder. Postoperative care included nasal tamponade for 2 days to avoid bleeding. On the second postoperative day, patients started using the intranasal topical oil [contents: I.E. Retinol, oil of peanut, orange and citron, butylhydroxyanisol (E320), butylhydroxytoluol (E321)] and decongestant spray (xylometazoline) when contraindications were excluded. The postoperative situs was cleaned from crust and mucous by suction under endoscopic control. In all cases, appropriate wound healing was achieved. From the fifth postoperative day, patients used brine irrigation of the nose. Nasal spray containing mometasone furoate monohydrate was applied twice daily from the 10 th postoperative day. Healthy controls Eighteen volunteers without history and endoscopic signs of chronic rhinosinusitis were investigated on two occasions. Healthy controls ages were between 20 and 54 years ( years). Ten females and eight males were investigated. Subjects younger than 18 years and those with a history of acute rhinitis, within at least 4 weeks before the first examination and within the follow-up period, were excluded. Healthy controls were investigated between September 2007 and February Olfactory testing On two different occasions, olfactory function was evaluated separately for each nostril. In patients, the first examination was performed the day before surgery and the second on the day of the follow-up examination. The side which was tested first was randomly selected. For assessment of olfactory function, pen-like odour dispensing devices ( Sniffin Sticks ) were employed (Hummel et al., 1997, 2007). This kit comprised of three subtests of olfactory function; namely tests for phenyl ethyl alcohol odour threshold, odour discrimination and odour identification. For a more comprehensive analysis of the results, data from the three tests: phenyl ethyl alcohol odour threshold (T), odour discrimination (D) and odour identification (I) were summed up to a composite score, the so-called TDI-score. Measurement of the olfactory bulb volume All scans were performed with a 1.5-T magnetic resonance imaging scanner (Magnetom Sonata; Siemens, Erlangen, Germany) using an eight-channel head coil. Volumes of the right and left olfactory bulb were determined using the MRI scans of the brain and a standardized protocol for olfactory bulb volume analysis. The protocol included a T 2 -weighted fast spin-echo 2D-sequence in the coronal plane covering the anterior and middle segments of the base of the skull [repetition time (TR) = 4.8 s, echo time (TE) = 152 ms, slice thickness 2 mm, gap 0.4 mm, matrix , number of slices 30, averages 4, resolution mm). Volumetric measurements of the right and left olfactory bulb were performed independently by two observers, blinded to the group and the olfactory test results, by manual segmentation of the coronal slices through the olfactory bulbs using the AMIRA 3D visualization and modelling system (Visage Imaging, Carlsbad, USA). As suggested by Yousem et al. (1998), the sudden change in the diameter at the beginning of the olfactory tract was used as the proximal demarcation of the olfactory bulb. In summary, olfactory bulb volumes were calculated by planimetric manual contouring (surface in square millimetres) and all surfaces were added and multiplied by two because of the 2 mm slice thickness to obtain a volume in cubic millimetres. The accuracy and reliability of olfactory bulb volume measurements has been demonstrated previously (Yousem et al., 1997; Mueller et al., 2005a). The method used provides intraclass coefficients of correlation, for repeated measurements by a single observer, greater than 0.92 and intraclass coefficients of correlation, for measurements across observers, greater than Comparing the measured volume with the true volume of investigated phantoms revealed a mean measurement error of 7.3%. Hereafter, data from a single measurement by the most experienced observer (N.A.) were used.

3 3098 Brain 2009: 132; V. Gudziol et al. Statistical analyses Data were investigated using Statistical Package for the Social Sciences (SPSS) 16.0 for Windows TM. t-tests for paired samples were used to compare results from different test sessions. An -level of less than 0.05 was regarded as significant. Correlations according to Pearson were computed between olfactory bulb volume and the subtests for olfactory function. For analyses of a possible impact of age and gender on the test results, analyses of variance for repeated measures (rm-anova) were performed. The within-subject factors were olfactory bulb volume (first or second test). Age and gender were used as between-subject factors. Results The interval between the two tests of olfactory function and olfactory bulb volume was 3 months in both patients and healthy controls. In healthy controls the TDI-score was (mean standard error) on the left side and on the right side in the first test. In the second test, TDI-score was and for the left and right side, respectively. t-test for paired samples did not show a significant change (P = 0.71) for the left side but a significant increase of the TDI-score on the right side was observed (P = 0.03). In patients, the TDI-score increased significantly (P50.001) from to on the left side and from to (P50.001) on the right side. In healthy controls, the olfactory bulb volume (Table 1) was mm 3 on the left side and mm 3 on the right side, in the first test. In the second test, the olfactory bulb volume was mm 3 on the left side and mm 3 on the right side. The volume was not significantly different, on either side, between the two measurements (P = 0.052, left side; P = 0.33, right side). In contrast, olfactory bulb volume in patients (Table 1) increased significantly (P = 0.02) from the initial to mm 3 on the left side and from to mm 3 on the right side (P50.001) (Fig. 1). The patient s age and gender did not show a significant impact on the change in olfactory bulb volume (P = 0.27 and 0.33 for age and gender, respectively). Specific analyses of the three subtests of olfactory function in patients revealed that the change of thresholds correlated significantly (r = 0.60, P = 0.006, left side; r = 0.49, P = 0.03, right side) with the change of olfactory bulb volume. However, changes in odour discrimination and identification did not show a significant correlation with the change in olfactory bulb volume on either side. Discussion The present study provided the following major results: (i) in healthy subjects, olfactory bulb size was constant over a period of 3 months; (ii) medical and surgical treatment of chronic rhinosinusitis led to an increase in olfactory function which was, after 3 months, accompanied by an increase in the left and right olfactory bulb volume by 9.4 and 18.9%, respectively; and (iii) changes in odour threshold correlated with the change in olfactory bulb size. It has been previously demonstrated, in animals, that olfactory bulb size increases when olfactory deprivation is eliminated (Cummings et al., 1997). Thus, it was important to investigate whether improvement of olfactory function in humans, due to Figure 1 Box plots of olfactory bulb volume in healthy controls at two test occasions (1 st and 2 nd test) and in patients before treatment (1 st test) and 3 months afterwards (2 nd test). The boundary of the boxes closest to zero indicates the 25 th percentile, a line within the box marks the median and the boundary of the box farthest from zero indicates the 75 th percentile. Whiskers above and below the box indicate the 90 th and 10 th percentiles. The horizontal brackets indicate the P-values for comparison of paired samples. When the P-value was 0.05 or bigger it is indicated as non-significant (n.s.). Table 1 Olfactory bulb volumes in cubic millimetres (mean standard error) of healthy controls and patients, at two test occasions Subject and side 1 st observation 2 nd observation Difference Significance, P-value Healthy controls Left side Right side Patients Left side Right side P50.001

4 Volume of the olfactory bulb Brain 2009: 132; treatment of chronic rhinosinusitis, can also lead to an enlargement of the olfactory bulb. A constant olfactory bulb volume in a healthy population was required to evaluate the expected changes of the olfactory bulb volume in patients with improvement of olfactory function, 3 months after surgery. The 3 months postoperative interval was chosen because it was demonstrated that, at this point, olfactory function was best in patients who had undergone functional endoscopic sinus surgery for chronic rhinosinusitis (Blomqvist et al., 2001). In the healthy subjects, olfactory function and olfactory bulb volumes were in the range of subjective normosmic individuals (Hummel et al., 2007; Buschhuter et al., 2008). Olfactory bulb volume did not show major changes on either side, although there was a small but significant increase of 1.3 points in the TDI-score, on the right side. An increase of 1.3 points in the TDI-score would; however, not be noticed as a subjective improvement of olfactory function by the vast majority of subjects (Gudziol et al., 2006), and most likely reflects learning processes found in response to repetitive testing (Hummel et al., 1997). Patients initially demonstrated olfactory loss as indicated by the average TDI-scores of 20.0 and 18.6 for the left and right side, respectively. Olfactory function improved significantly due to treatment of chronic rhinosinusitis. Three months after surgery, patients scored an average of 7.2 and 8.4 points higher for the left and the right side, respectively, which indicates a significant gain in subjective olfactory function for the majority of patients (Gudziol et al., 2006). On comparing the preoperative size of the olfactory bulb with normative data from Buschhueter et al. (2008), nine olfactory bulbs (23.7%) of the patients with chronic rhinosinusitis were smaller than the 10 th percentile of olfactory bulb volumes in healthy subjects. The olfactory loss leading to a significant decrease in olfactory bulb volume, within months, indicates on a structural level that olfactory dysfunction was already present in the majority of the patients before the first examination (Haehner et al., 2008). In the present study, olfaction improved within 3 months of surgery. Within this interval, olfactory bulb volume increased significantly on both sides. In animal studies, it was found that the reduction in olfactory bulb size was due to a decrease in the size of existing mitral cells and a decrease in the number of tufted, granule and glia cells (Brunjes, 1994). Additionally, olfactory deprivation led to increased death of progenitor cells in the subventricular zone and the rostral migration stream, resulting in fewer progenitor cells that are able to differentiate in the olfactory bulb (Mandairon et al., 2003). On the other hand, increasing olfactory stimulation leads to an increased number of progenitor cells in the olfactory bulb and to decreased cell mortality (Rochefort et al., 2002). The finding, in the present study, of increasing olfactory bulb volume with increasing olfactory function supports the hypothesis that changes in olfactory function regulate cell death, differentiation and the number of progenitor cells, not only in rodents but also in humans. Analysis of the three subtests of olfactory function revealed a significant correlation between the change of odour threshold perception and the change in olfactory bulb volume, which has been reported previously (Haehner et al., 2008). In contrast, changes in suprathreshold olfactory functions (odour discrimination and odour identification) did not correlate with the change in olfactory bulb volume. Odour discrimination and odour identification can be regarded as functions which require higher order processing, while odour thresholds reflect primarily peripheral olfactory function (Jones-Gotman and Zatorre, 1988a, b; Hornung et al., 1998). This result supports the assumption that ingrowing olfactory receptor neuron axons regulate the size of the olfactory bulb (Stout and Graziadei, 1980). Figure 2 Scatterplot of the change of olfactory bulb volume and the change of odour threshold in patients separately for each side. Positive values on the axes indicate an increase of olfactory sensitivity or an enlargement of the olfactory bulb at the follow up examination. Significant correlation was found for the right side (r = 0.49, P = 0.03) and the left side (r = 0.60, P = 0.006).

5 3100 Brain 2009: 132; V. Gudziol et al. Even though the patients and controls were matched for gender, the healthy controls were younger than the patients. Olfactory bulb volume shrinks and olfactory function decreases in subjects older than 50 years (Doty et al., 1984; Yousem et al., 1998). Since olfactory bulb plasticity is regulated by peripheral olfactory stimuli in animals, one could hypothesize that the ability to increase olfactory bulb volume is reduced in older patients (Rochefort et al., 2002). In contrast to this hypothesis, a significant increase in olfactory bulb volume was identified in the investigated patients, with an average age of 53 years. In fact, in the present study, the patient s age had no impact on the change of olfactory bulb volume. It is thought that a study with older subjects could reveal whether the plasticity of the olfactory bulb decreases in individuals older than 50 years. Olfactory bulb volume was not adjusted to overall brain volume, normalized for head volume, for three reasons: (i) in healthy subjects older than 65 years, the overall brain volume decreases by 0.45% (standard deviation = 0.53%) per year (Fotenos et al., 2005). Thus, it appeared to be problematic to adjust a potentially variable structure with a structure which would exhibit a more uniform decrease in volume; (ii) adjustment of a very small volume (olfactory bulb) to a relatively large structure (whole brain volume) would increase noisiness of the data set and (iii) in this longitudinal study, the change of the olfactory bulb volume was investigated in relation to an intra-individual standard. Conclusion Treatment of patients with chronic rhinosinusitis is accompanied by increased olfactory function and increased left and right-sided olfactory bulb volumes, by 9.4% and 18.9%, respectively, within 3 months of onset of therapy. The results of the present study support the hypothesis that olfactory stimulation of olfactory receptor neurons impacts on the cell death in the olfactory bulb, the rostral migration stream and the differentiation of progenitor cells from the subventricular zone in the olfactory bulb, not only in rodents, but also in humans. To our knowledge, this is the first study that describes an enlargement of the human olfactory bulb due to an increase of peripheral olfactory sensory function. Acknowledgements We would like to thank Heike Hoffmann and Franziska Seltmann for their help in obtaining some of the data. Supplementary material Supplementary material is available at Brain online. References Abolmaali N, Gudziol V, Hummel T. Pathology of the olfactory nerve. Neuroimaging Clin N Am 2008; 18: , preceding x. Altmann J. Autoradiographic and histological studies of postnatal neurogenesis. IV. 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