Position paper on olfactory dysfunction.

Background: Olfactory dysfunction is an increasingly recognised condition, associated with reduced quality of life and major health outcomes such as neurodegeneration and death. However, translational research in this field is limited by heterogeneity in methodological approach, including definitions of impairment, improvement and appropriate assessment techniques. Accordingly, effective treatments for smell loss are limited. In an effort to encourage high quality and comparable work in this field, among others, we propose the following ideas and recommendations. Whilst the full set of recommendations are outlined in the main document, points include the following: • Patients with suspected olfactory loss should undergo a full examination of the head and neck, including rigid nasal endoscopy with small diameter endoscopes. • Subjective olfactory assessment should not be undertaken in isolation, given its poor reliability. • Psychophysical assessment tools used in clinical and research settings should include reliable and validated tests of odour threshold, and/or one of odour identification or discrimination. • Comprehensive chemosensory assessment should include gustatory screening. • Smell training can be helpful in patients with olfactory loss of several aetiologies. Conclusions: We hope the current manuscript will encourage clinicians and researchers to adopt a common language, and in so doing, increase the methodological quality, consistency and generalisability of work in this field.

Position paper on olfactory dysfunction.

BACKGROUND: Olfactory dysfunction is an increasingly recognised condition, associated with reduced quality of life and major health outcomes such as neurodegeneration and death. However, translational research in this field is limited by heterogeneity in methodological approach, including definitions of impairment, improvement and appropriate assessment techniques. Accordingly, effective treatments for smell loss are limited. In an effort to encourage high quality and comparable work in this field, among others, we propose the following ideas and recommendations. Whilst the full set of recommendations are outlined in the main document, points include the following: - Patients with suspected olfactory loss should undergo a full examination of the head and neck, including rigid nasal endoscopy with small diameter endoscopes. - Subjective olfactory assessment should not be undertaken in isolation, given its poor reliability. - Psychophysical assessment tools used in clinical and research settings should include reliable and validated tests of odour threshold, and/or one of odour identification or discrimination. - Comprehensive chemosensory assessment should include gustatory screening. - Smell training can be helpful in patients with olfactory loss of several aetiologies. CONCLUSIONS: We hope the current manuscript will encourage clinicians and researchers to adopt a common language, and in so doing, increase the methodological quality, consistency and generalisability of work in this field.

Extraskeletal osteosarcoma: a very rare case report of primary tumor of the colon-rectum and review of the literature.

Extraskeletal osteosarcoma (ESOS) is a malignant mesenchymal tumor in which neoplastic cells produce bone osteoid in variable amounts. An 81-year-old woman presented with severe abdominal pain, tenesmus, constipation and rectal bleeding. The digital rectal exploration showed a large lesion of hard consistency, occupying the lumen, with the presence of splinters that "pierced" the endoscopist's glove. Endoscopic examination and CTscan revealed an ulcerative exophytic neoplasia of the lower rectum in which multiple calcified areas were found. The lesion showed no bone involvement. An abdominal perineal resection sec Miles was performed. The histological examination revealed a highly cellular mesenchymal lesion, with spindle and epithelioid cells with moderate nuclear pleomorphism. The calcified component consisted of widespread osteoid deposition. The immunohistochemical investigations of neoplastic cells showed strong positivity for vimentin and osteonectin. The definitive histological diagnosis of primary extraskeletal osteosarcoma arising from the colon-rectum was made. To our knowledge, only one previous case of colonic osteosarcoma was published in the literature in 2001, reported by Shimazu and other authors. The extreme rarity of the tumor at this location, also confirmed by morphological and immunohistochemical data, prompted us to present this case report and to review the literature.

Pulmonary delivery of peptide YY for food intake suppression and reduced body weight gain in rats.

AIMS: Peptide YY (PYY) is an endogenous anorectic gut-secreted peptide that has been shown to suppress appetite in animals and humans, when given by injection. This study tested if needle-free pulmonary delivery of PYY enables food intake suppression and reduced body weight gain in rats. The PYY pharmacokinetics and effects on brain neuropeptide levels were also examined. METHODS: Rats received single or once-daily 7-day pulmonary administration of saline or PYYs. Food intake and body weight gain were monitored to study the effects of different doses (0.08-0.90 mg/kg) of PYY3-36, PYY1-36 and PYY13-36. Plasma PYY pharmacokinetics were determined via enzyme-linked immunosorbent assay. Changes in orexigenic neuropeptide Y (NPY) and c-Fos protein levels in the hypothalamus arcuate nucleus (ARC) were measured by immunofluorescence microscopy. RESULTS: PYY3-36 caused dose-dependent and 4- to 6-h food intake suppression following pulmonary delivery. At 0.80 mg/kg, the effect was significant with 35.1 ± 5.7 and 19.7 ± 4.2% suppression at 4 and 6 h, respectively. Repeated administration for 7 days reduced cumulative body weight gain by 39.4 ± 11.0%. PYY1-36, but not PYY13-36, was equipotent to PYY3-36 in food intake suppression. The plasma PYY concentration reached its peak at 10 min following pulmonary delivery with 12-14% of bioavailability. Increased c-Fos and reduced NPY expressions were observed in the hypothalamus ARC, consistent with the magnitude of food intake suppression by each of the PYYs. CONCLUSIONS: Pulmonary delivery of PYY enabled significant 4- to 6-h food intake suppression via 12-14% of lung absorption and hypothalamic ARC interaction, leading to reduced body weight gain in rats.

Impact of olfactory impairment on quality of life and disability.

OBJECTIVE: To determine whether olfactory loss affects patients' quality of life or level of disability. DESIGN: Retrospective survey using questionnaire data and clinic database review. SETTING: Two university medical center smell and taste clinics. PATIENTS: A total of 1407 patients were tested for smell and taste disturbances from 1984 through 1998. Surveys were mailed to 1093 patients who had abnormal test scores; 420 (38.4%) returned completed surveys. Patients were grouped by self-rated ability to smell as "impaired" (those reporting persisting deficits) or "improved" (those reporting no smell problem when surveyed). MAIN OUTCOME MEASURES: Response frequencies were compared between the 2 groups for questions regarding ability to perform common activities of daily living and quality-of-life issues. RESULTS: Mean (+/-SD) number of activities of daily living affected by olfactory loss was 4.70 +/- 3.56 for the impaired group and 0.61 +/- 1.58 for the improved group (P < .001). Among specific activities, the most common cited impairments were ability to detect spoiled food (impaired vs improved groups, 75% vs 12%; P < .001), gas leaks (61% vs 8%; P < .001), or smoke (50% vs 1%; P < .001); eating (53% vs 12%; P < .001); and cooking (49% vs 12%; P < .001). Differences in quality-of-life issues were reported primarily in the areas of safety and eating. Overall satisfaction with life was reported by 87% of the improved group but only 50% of the impaired group (P < .001). CONCLUSIONS: Patients reporting persistent olfactory impairment after previously documented olfactory loss indicate a higher level of disability and lower quality of life than those with perceived resolution of olfactory compromise.

Rhinotopy is disrupted during the re-innervation of the olfactory bulb that follows transection of the olfactory nerve.

Re-innervation of the olfactory bulb was investigated after transection of the olfactory nerve using monoclonal antibody RB-8 to assess whether rhinotopy of the primary olfactory projection is restored. In normal animals RB-8 heavily stains the axons, and their terminals, that project from the ventrolateral olfactory epithelium onto glomeruli of the ventrolateral bulb (termed RB-8(+)). In contrast, axons from dorsomedial epithelium are unlabeled (RB-8(-)) and normally terminate in the dorsomedial bulb. Sprague-Dawley rats underwent unilateral olfactory nerve transection and survived for 6 weeks prior to perfusion, sectioning and immunostaining with RB-8. Nerve lesion does not shift the position of the boundary between RB-8(+) and RB-8(-) regions of the epithelium. However, following transection and bulb re-innervation, the distribution of RB-8(+) and RB-8(-) axons is markedly abnormal. First, in all 10 experimental animals RB-8(-) axons displace RB-8(+) axons from anterior glomeruli. Furthermore, the usual target of the RB-8(-) fibers, i.e. the dorsomedial bulb at more posterior levels of the bulb, remains denervated, judging by the lack of staining with antibodies that label axons derived from all epithelial zones. Finally, RB-8(+) fibers invade foreign territory in the dorsolateral bulb on the lesioned side in some cases. The shifts in terminal territory in the bulb after transection contrast with the restoration of the normal zonal patterning of the projection after recovery from methyl bromide lesion, but is consistent with reports of mistargeting by a receptor-defined subset of neurons after transection.

Immunocytochemical study of G(i)2alpha and G(o)alpha on the epithelium surface of the rat vomeronasal organ.

To investigate in detail the distribution of G protein subtypes G(i)2alpha and G(o)alpha along the surface of the vomeronasal epithelium, we used double labeling immunocytochemical methods and electron microscopy. We examined the immunoreactivity of these surface structures with antibodies against G(i)2alpha and G(o)alpha. G(i)2alpha- and G(o)alpha-positive cells were observed at the epithelial surface and were evenly distributed. Electron microscopy revealed that strong immunoreactivities to both antibodies were observed on the microvilli and knob-like surface structures of receptor cells. No immunoreactivity was found on the microvilli or surface membranes of supporting cells. This expression pattern is similar to that reported for putative pheromone receptors. These data confirm that there are two distinct classes of vomeronasal receptor cells expressed at the surface of the epithelium. These two classes of receptors correspond to the same G(i)2alpha- and G(o)alpha-positive cells distributed in cell body layers of the epithelium and in the axon terminals in the accessory olfactory bulb.

Olfactory epithelium grafts in the cerebral cortex: an immunohistochemical analysis.

OBJECTIVE: To develop an alternative model for studying the regenerative capacity of olfactory neurons. STUDY DESIGN: An immunohistochemical analysis of mouse olfactory epithelium transplanted to the cerebral cortex. METHODS: Strips of olfactory epithelium removed from donor mice at postnatal day 5 to day 20 were inserted into the parietal cortex of adult mice. Recipient animals were allowed to survive for 25 to 120 days and then perfused with 4% paraformaldehyde 1 hour after bromodeoxyuridine injection. The brains were processed, and frozen sections were obtained. Sections through transplant tissue were analyzed using immunohistochemistry and compared with normal olfactory epithelium. RESULTS: Graft survival approached 85% with mature olfactory neurons detected in 35% of the transplants stained for olfactory marker protein. Transplant epithelium resembled normal olfactory epithelium containing mature olfactory neurons and axon bundles. CONCLUSIONS: Studies of olfactory neuron regeneration have been limited by the inability to produce cultures with long-term viability. Olfactory epithelial grafts to the cerebral cortex provide an alternative approach to the study of olfactory neuron regeneration.

Surface changes in the rat vomeronasal epithelium during degeneration and regeneration of sensory receptor cells.

To investigate cell turnover in the vomeronasal epithelium we used electron microscopy to obtain quantitative measurements of changes observed at the surface of the sensory epithelium. Receptor cell degeneration was induced by sensory nerve transection and animals were examined at postoperative recovery times of 2, 4, 6, 10, 15, 35 and 60 days. We measured the number and density of receptor and supporting cells, and membrane length at the surface of the sensory epithelium. The number of receptor cells rapidly decreased during the degeneration period, reaching a minimum at 6 days. After 15 days of recovery the number and density of receptor cells returned to control levels. The surface membrane length for regenerated receptor cells was similar to that of controls, however the morphological appearance was characteristic of immature cells. In contrast to the receptor cells, the number and density of supporting cells did not change during degeneration and regeneration. However, there was a significant increase in the length of supporting cell-surface membranes. These results suggest that during receptor cell degeneration, supporting cell membranes compensate for the loss of receptor cells by expanding their surface membrane length to help to maintain the continuity of the epithelial surface. Thus, an important role of vomeronasal supporting cells may be to maintain the structural integrity of the epithelium during turnover of the receptor cell population.

Rewiring the olfactory bulb: changes in odor maps following recovery from nerve transection.

Recent studies have shown that axons from olfactory receptor subtypes converge onto glomeruli in fixed positions within the olfactory bulb. Different receptor subtypes project to different glomeruli, forming a spatial distribution of odor information or 'odor maps'. Olfactory receptor neurons are continuously replaced throughout the life span of an animal, yet they preserve this highly localized mapping of receptor subtypes. In this study we used a transgenic mouse (P2-IRES-tau-lacZ) to map axons from a single receptor subtype (P2 receptors) in order to determine if regenerating axons were able to re-establish the P2 receptor map following nerve transection. Results confirm that P2 receptor axons retain their capacity to grow back to the olfactory bulb and converge onto glomeruli following nerve transection. However, the location and number of convergence sites was significantly altered compared to the control map. This change in the spatial distribution of axons alters the topography of odor mapping and has important implications for the processing of olfactory information. Findings from this study may explain why animals recovering from nerve injury require odor training before odor discrimination is restored. Future studies of olfactory receptor mapping could prove helpful in planning strategies to rewire connections in the brain and to restore function following injury or neurological disease.

Morphological and histochemical changes in the regenerating vomeronasal epithelium.

Receptor cell degeneration and regeneration within the vomeronasal organ (VNO) of the rat was studied using both electron microscopy and histochemical methods. Electron microscopy was employed to examine the morphological changes along the surface of the sensory epithelium, and histochemical markers were used to monitor the changes in the epithelial cell layers. Transection of the vomeronasal nerves induced selective degeneration of the receptor cells, and within six days, a significant decrease in the number of receptor cells was observed. During the subsequent stage of receptor cell regeneration, cilia and bud-like structures characteristic of a developing sensory epithelium were seen. By day 15, thin microvilli covering the surface of the receptor cells reappeared in the sensory epithelium. The neural cell adhesion molecule (NCAM) and two vomeronasal system-specific lectins; 1) Bandeiraea simplicifolia lectin (BSL-I) and 2) Vicia villosa agglutinin (VVA) were used as the histochemical markers. NCAM immunoreactivity on the surface of the epithelium was observed to be decreased significantly six days after nerve transection, and was restored during receptor cell regeneration (day 15). The reactivity of the two lectins, BSL-I and VVA, was decreased slightly during degeneration, but was still detectable at the time of maximum receptor cell degeneration (day 6). Lectin reactivity was restored to control levels by day 15. These findings suggest that (1) NCAM is a useful marker for vomeronasal receptor cells and that the vomeronasal system-specific lectins may bind to both receptor and supporting cells and (2) degeneration of vomeronasal receptor cells occurs during the first week (day 6) following nerve transection and the receptor cell population begins to recover within 15 days. The morphological changes observed during receptor cell regeneration suggest that the stages of VNO receptor cell regeneration are similar to those observed during development.

Continual neurogenesis of vomeronasal neurons in vitro.

We developed a culture system of vomeronasal neurons in which continuous degeneration and regeneration of axon bundles were observed. Partially dissociated vomeronasal cells from rat embryonic day 15 were grown in culture and formed a miniature vomeronasal-like epithelium. We called these structures vomeronasal pockets. They contained both vomeronasal neurons and supporting cells. They formed a spherical structure with a central cavity where microvilli protruded from supporting cells. Mature vomeronasal neurons with well-developed microvilli were not observed in the vomeronasal pocket. The time period between degeneration of axon bundles and the next was about 2 weeks. When vomeronasal pockets were incubated with 5 microgram/mL aphidicolin, an inhibitor of cell division, regeneration of axon bundles was not observed after degeneration. These results suggest that vomeronasal neurons in culture undergo continuous regeneration but do not fully mature. In this culture system, vomeronasal pockets survived for over 1 year.

Changes in odor quality discrimination following recovery from olfactory nerve transection.

Following recovery from olfactory nerve transection, animals regain their ability to discriminate between odors. Odor discrimination is restored after new neurons establish connections with the olfactory bulb. However, it is not known if the new connections alter odor quality perception. To address this question, 20 adult hamsters were first trained to discriminate between cinnamon and strawberry odors. After reaching criterion (> or = 90% correct response), half of the animals received a bilateral nerve transection (BTX) and half a surgical sham procedure. Animals were not tested again until day 40, a point in recovery when connections are re-established with the bulb. When BTX animals were tested without food reinforcement, they could not perform the odor discrimination task. Sham animals, however, could discriminate, demonstrating that the behavioral response had not been extinguished during the 40 day period. When reinforcement was resumed, BTX animals were able to discriminate between cinnamon and strawberry after four test sessions. In addition, their ability to discriminate between these two familiar odors was no different than that of BTX and sham animals tested with two novel odors, baby powder and coffee. These findings suggest that, after recovery from nerve transection, there are alterations in sensory perception and that restoration of odor quality discrimination requires that the animal must again learn to associate individual odor sensations with a behavioral response.

Replacement of receptor cells in the hamster vomeronasal epithelium after nerve transection.

Chemoreceptor cells in the vomeronasal and olfactory epithelium are replaced following experimentally induced degeneration. This study analyzes quantitatively the time course and degree of vomeronasal receptor cell replacement. Unilateral transection of the vomeronasal nerves in adult hamster was used to induce a retrograde degeneration of receptor cells in the vomeronasal organ. Histological measurement of both number of receptor cells and epithelial thickness were made for recovery times from 0 to 60 days. After nerve transection, there was a gradual degeneration of receptor cells, the number decreasing to 50% of control by day 2 and 16% by day 6. During days 7-15 maximum receptor cell replacement was observed. Cell number increased rapidly and reached a peak on day 15. At recovery times of 40-60 days, cell number returned to the control level. Epithelial thickness, however, decreased to 60-70% during the degeneration period (days 4-6) and did not return to control levels. After 40-60 days epithelial thickness remained at 70% of control. These results demonstrate that vomeronasal receptor cells are replaced following degeneration, but epithelial thickness does not return to control levels. These findings suggest that the number of replacement cells is not limited by the reduced thickness of the epithelium, and that recovery mechanisms may function to restore an optimum number of receptor cells.

Electrophysiological characterization of the olfactory bulb during recovery from sensory deafferentation.

Axonal transection results in degeneration of the olfactory sensory neurons (OSNs). Replacement OSNs reinnervate the olfactory bulb. To document reinnervation, lateral OSN fibers were stimulated while bulb negative evoked potentials (NEPs) were recorded. For control adult hamsters and at 20, 30, and 120 days after transection, lateral fibers were connected to the lateral more than the medial bulb. The percentage of lateral bulb positions with NEPs was similar to control at 30 and 120 days, but NEP amplitude did not reach control level.

Restoration of olfactory mediated behavior after olfactory bulb deafferentation.

Transection of olfactory nerve fibers leads to deafferentation of olfactory bulbs and a loss of olfactory mediated behavior. Nerve transection studies have shown that during recovery, olfactory nerve fibers can reestablish connections with the olfactory bulbs. Two groups of experimental animals were studied to determine if olfactory mediated behavior returns after recovery. One group (n = 18) received bilateral olfactory nerve transection (BTX), while the second group (n = 4) received a sham surgical procedure. Performance on odor detection and discrimination tasks was measured during recovery periods ranging from 1-120 days. Return of olfactory mediated behavior was first observed 19 days after nerve transection. Performance levels improved with recovery time and by day 40 animals returned to criterion level (> or = 90% correct response). Sham animals maintained a criterion level of performance throughout the recovery period. Horseradish peroxidase (HRP) was used to trace reconnection of olfactory nerve fibers. The absence of HRP label in the bulbs of animals examined one day after BTX, verified the completeness of the nerve transection procedure. After 10 days of recovery, a few HRP labeled axons were observed and the amount of HRP in the bulb increased with recovery time. The results of this study demonstrate that olfactory receptor axons can reestablish functional connections with the deafferented olfactory bulb and these connections are sufficient to restore olfactory mediated behavior.

Regeneration of olfactory sensory neurons and reconnection in the aging hamster central nervous system.

Olfactory neuron recovery and axon growth was studied in 12-24 month old hamsters after unilateral olfactory nerve transection. At recovery times ranging from 4 to 126 days olfactory nerve regeneration and axon reconnection to the olfactory bulb were examined by anterograde horseradish peroxidase (HRP) neurohistochemistry and electron microscopy. Nerve transection produced immediate retrograde neuron degeneration and there was no HRP reaction product in the bulb at 4 days post transection. By day 35, centrally growing olfactory axons had reached the bulb. Axons formed glomeruli smaller than those in the unoperated control bulb and they were not always confined to the glomerular layer of the bulb. Some animals showed robust fiber growth with axon fascicles penetrating the different layers of the bulb and forming ectopic glomeruli along their path. Second order bulb neurons contained wheat germ agglutinin HRP reaction product, indicating that transneuronal transport had occurred. Electron microscopy confirmed transneuronal transport between olfactory axons and second order bulbar neurons. These results show that the capacity for olfactory neuron recovery and reconnection persists in the hamster well into old age.

Is nestin a marker for chemosensory precursor cells?

The vomeronasal and olfactory systems are unique in that their chemosensory neurons undergo continuous neurogenesis after development. Immunoreactivity to nestin, a neuronal precursor marker protein, was investigated in the developing rat vomeronasal organ to determine its potential as a cell marker. From postnatal day 1 (P1) to P22, the distribution of nestin positive cells became progressively restricted to the area adjacent to the basement membrane. By P29, the vomeronasal organ reached structural maturity and only a few nestin positive cells were observed. Results suggest that nestin may be a useful marker for neuronal precursor cells in studies of neurogenesis and development of chemosensory systems.

Morphology of olfactory epithelium in humans and other vertebrates.

Human olfactory epithelium is similar in organization and cell morphology to that of most vertebrate species. The epithelium has a pseudostratified columnar organization and consists of olfactory neurons, supporting and basal cells. Near the mucosal surface there are also microvillar cells. These cells have neuron-like features and may be chemoreceptors. Human olfactory epithelium is not a uniform sensory sheet. Patches of non-sensory tissue often appear in what was thought to be a purely olfactory region. The significance of these patches has not been determined, but they could reflect exposure to environment agents or changes that occur during the normal aging process. In order to better understand the human olfactory system, further knowledge of the normal structure is necessary. This review addresses the morphology of the human olfactory epithelium and the remarkable plasticity of the vertebrate olfactory system.