Using non-invasive neuroimaging to enhance the care, well-being and experimental outcomes of laboratory non-human primates (monkeys).

Over the past 10-20 years, neuroscience witnessed an explosion in the use of non-invasive imaging methods, particularly magnetic resonance imaging (MRI), to study brain structure and function. Simultaneously, with access to MRI in many research institutions, MRI has become an indispensable tool for researchers and veterinarians to guide improvements in surgical procedures and implants and thus, experimental as well as clinical outcomes, given that access to MRI also allows for improved diagnosis and monitoring for brain disease. As part of the PRIMEatE Data Exchange, we gathered expert scientists, veterinarians, and clinicians who treat humans, to provide an overview of the use of non-invasive imaging tools, primarily MRI, to enhance experimental and welfare outcomes for laboratory non-human primates engaged in neuroscientific experiments. We aimed to provide guidance for other researchers, scientists and veterinarians in the use of this powerful imaging technology as well as to foster a larger conversation and community of scientists and veterinarians with a shared goal of improving the well-being and experimental outcomes for laboratory animals.

Internalization of Vectorized Liposomes Loaded with Plasmid DNA in C6 Glioma Cells.

We studied internalization of vector nanocarriers loaded with plasmid DNA into C6 glioma cells. For improving selectivity of plasmid delivery, the liposomes were conjugated with monoclonal antibodies to VEGF and its receptor VEGFR2. Flow cytofluorometry and laser scanning confocal microscopy showed more intensive (more than 2-fold) internalization and accumulation of antibody-vectorized liposomes in C6 glioma cells in comparison with the control (liposomes conjugated with non-specific antibodies and non-vectorized liposomes). Using quantitative analysis of fluorescent signal, we showed that cationic immunoliposomes significantly more effective delivered pCop-Green-N plasmid DNA and ensured effective transfection of C6 glioma cells.

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.

Generation of recombinant extracellular fragment of vascular endothelial growth factor receptor 2 and specific monoclonal antibodies to this receptor.

cDNA extracellular Ig-like domains I-III of vascular endothelial growth factor receptor 2 (VEGFR2) was cloned in an expressing vector pET_32a. Western blotting showed immunochemical identity of recombinant VEGFR2I-III produced by prokaryotic expression system to the native receptor. BALB/c mice were immunized with VEGFR2I-III for obtaining specific antibodies to VEGFR2. Hybridomas producing monoclonal antibodies were selected by ELISA, Western blotting, and immunocytochemical assay. Thus, we obtained hybridoma producing monoclonal antibodies to VEGFR2 that selectively interact with both recombinant and native extracellular fragment of the receptor.

Ligand-receptor assay for evaluation of functional activity of human recombinant VEGF and VEGFR-1 extracellular fragment.

cDNA encoding VEGF and Ig-like extracellular domains 2-4 of VEGFR-1 (sFlt-1(2-4)) were cloned into prokaryotic expression vectors pET32a and pQE60. Recombinant proteins were purified (metal affinity chromatography) and renatured. Chemiluminescent study for the interaction of recombinant VEGF and sFlt-1(2-4) showed that biotinylated VEGF specifically binds to the polystyrene-immobilized receptor extracellular fragment. Biotinylated recombinant sFlt-1 interacts with immobilized VEGF. Analysis of the interaction of immobilized recombinant VEGFR-1 and VEGF with C6 glioma cells labeled with CFDA-SE (vital fluorescent dye) showed that recombinant VEGFR-1 also binds to native membrane-associated VEGF. Recombinant VEGF was shown to bind to specific receptors expressed on the surface of C6 glioma cells. Functional activity of these proteins was confirmed by ligand-receptor assay for VEGF and VEGFR-1 (sFlt-1) and quantitative chemiluminescent detection.

Cloning and expression of brain-specific anion transporter BSAT1 and isolation of monoclonal antibodies to its extracellular fragment.

Brain-specific anion transporter BSAT1 extracellular fragment (451-557) cDNA was cloned in a vector for prokaryotic expression, a producer E. coli strain was obtained, and recombinant extracellular fragment BSAT1(451-557)was purified and used for immunization of BALB/c mice. Splenic cells from mice with verified immune response were used for hybridoma generation. Several hybridoma clones producing monoclonal antibodies to BSAT1 extracellular fragment were selected. Antibody specificity was confirmed by ELISA, immunoblotting with recombinant BSAT1(451-557), and immunofluorescent BSAT1 assay on rat brain sections and cultured HEK293 cells. It was demonstrated that the obtained antibodies specifically bind native rat and human BSAT1 and can be used in both fundamental studies of structures forming the blood-brain barrier and development of targeted transport of diagnostic preparations and drugs across the blood-brain barrier.

Generation of monoclonal antibodies to recombinant vascular endothelial growth factor.

Female BALB/c mice were subcutaneously immunized with recombinant VEGF-164. After 3 immunization cycles, splenic B cells from immunized mouse were fused with immortalized myeloma culture SP2/0-Ag14 cells. Screening of hybrid cells producing anti-VEGF antibodies was performed by ELISA and immunocytochemical analysis on cultured C6 glioma cells. Subsequent cloning yielded hybridoma stably expressing monoclonal anti-VEGF antibodies recognizing recombinant and native VEGF.

Visual adaptation to convexity in macaque area V4.

Aftereffects are perceptual illusions caused by visual adaptation to one or more stimulus attribute, such as orientation, motion, or shape. Neurophysiological studies seeking to understand the basis of visual adaptation have observed firing rate reduction and changes in tuning of stimulus-selective neurons following periods of prolonged visual stimulation. In the domain of shape, recent psychophysical work has shown that adaptation to a convex pattern induces a subsequently seen rectangle to appear slightly concave. In the present study, we investigate the possible contribution of V4 neurons of rhesus monkeys, which are thought to be involved in the coding of convexity, to shape-specific adaptation. Visually responsive neurons were monitored during the brief presentation of simple shapes varying in their convexity level. Each test presentation was preceded by either a blank period or several seconds of adaptation to a convex or concave stimulus, presented in two different sizes. Adaptation consistently shifted the tuning of neurons away from the convex or concave adapter, including shifting response to the neutral rectangle in the direction of the opposite convexity. This repulsive shift resembled the known perceptual distortion associated with adaptation to such stimuli. In addition, adaptation caused a nonspecific response decrease, as well as a specific decrease for repeated stimuli. The latter effects were observed whether or not the adapting and test stimuli matched closely in their size. Taken together, these results provide evidence for shape-specific adaptation of neurons in area V4, which may contribute to the perception of the convexity aftereffect.

Pathology of the olfactory epithelium: smoking and ethanol exposure.

OBJECTIVE: To investigate the effects of tobacco smoke on the olfactory epithelium. Cigarette smoking has been associated with hyposmia; however, the pathophysiology is poorly understood. The sense of smell is mediated by olfactory sensory neurons (OSNs) exposed to the nasal airway, rendering them vulnerable to environmental injury and death. As a consequence, a baseline level of apoptotic OSN death has been demonstrated even in the absence of obvious disease. Dead OSNs are replaced by the mitosis and maturation of progenitors to maintain sufficient numbers of neurons into adult life. Disruption of this balance has been suggested as a common cause for clinical smell loss. This current study will evaluate the effects of tobacco smoke on the olfactory mucosa, with emphasis on changes in the degree of OSN apoptosis. STUDY DESIGN: A rat model was used to assess the olfactory epithelium after exposure to tobacco smoke. METHODS: Rats were exposed to tobacco smoke alone (for 12 weeks), smoke plus dietary ethanol (for the final 5 weeks), or to neither (control). Immunohistochemical analysis of the olfactory epithelium was performed using an antibody to the active form of caspase-3. Positive staining for this form of the caspase-3 enzyme indicates a cell undergoing apoptotic proteolysis. RESULTS: Control rats demonstrated a low baseline level of caspase-3 activity in the olfactory epithelium. In contrast, tobacco smoke exposure triggered a dramatic increase in the degree of OSN apoptosis that affected all stages of the neuronal lineage. CONCLUSIONS: These results support the following hypothesis: smell loss in smokers is triggered by increased OSN death, which eventually overwhelms the regenerative capacity of the epithelium.

Clinical presentation of qualitative olfactory dysfunction.

Many patients with olfactory dysfunction not only experience quantitative reduction of olfactory function, but also suffer from distorted olfactory sensations. This qualitative dysfunction is referred to as parosmia (also called "troposmia") or phantosmia, with the major difference that distorted olfactory sensations are experienced in the presence or absence of an odor, respectively. Our clinical observations corroborate the literature in terms of a general underestimation of the incidence of olfactory distortions. Based on selected cases we try to show that olfactory distortions exhibit a large variance in their clinical appearance. Further, emphasis is placed on the fact that only a detailed and directed history of the patient can provide cues to the correct diagnosis.

The relationship between agricultural environments and olfactory dysfunction.

OBJECTIVES: To examine the olfactory ability of farmers and identify exposures in agricultural environments associated with olfactory loss. STUDY DESIGN: Cross-sectional. METHODS: 405 individuals (214 males and 191 females) completed a questionnaire assessing agricultural exposures, health status, and olfactory history. Participants then completed a scratch-and-sniff 12-item odor identification test. Data analysis was conducted using the method of least squares to fit general linear models. Equality of outcome measures (the number of correctly identified odorants) between groups was evaluated using the Wilcoxon rank sum test. Differences were considered to be significant at the 0.05 level. RESULTS: Approximately 80% of participants reported active participation in farm work. Farmers correctly identified an average of 9.3 of 12 odorants, compared to 10.1 of 12 correctly identified by non-farmers, a difference that was not statistically significant (p = 0.2). Participants reporting sneezing after handling soybeans, wheat, or oats scored significantly lower than those without symptoms. There were suggestive associations between olfactory ability and exposure to anhydrous ammonia, history of wheezing and asthma, and a history of flu-like illness after farm work. CONCLUSIONS: Results indicate that, in general, farming is not associated with olfactory loss. However, we identified certain groups of farmers with inflammatory-type reactions who appeared to be susceptible to olfactory loss. The association between inflammatory reactions or conditions and olfactory loss is a novel finding.

Dynamic assessment of paranasal sinus ventilation using xenon-enhanced computed tomography.

Disease of the paranasal sinuses is a common and costly condition. Evaluation of the efficacy of either medical or surgical methods of treatment is limited by the lack of quantitative methods to characterize sinus ventilation, which may be an important determinant of the baseline physiological state of the sinuses. Xenon-enhanced computed tomography (Xe-CT) measurement of sinus ventilation provides a noninvasive method of quantifying maxillary sinus ventilation using the nonradioactive, radiodense gas Xe as a tracer. Study subjects breathed a mixture of Xe gas and oxygen through a close-fitting nasal mask during serial CT imaging of a single radiographic plane through the maxillary sinuses--a generally well-tolerated protocol. Analysis of the sinus density-time curves allowed calculation of first-order exponential time constants from which specific ventilation rates could be determined for individual sinuses. Previously developed data analysis techniques were used to assess the statistical significance of the data and determine confidence intervals, allowing examination of the effects of noise in the data, and to demonstrate areas for further study protocol refinement. We conclude that Xe-CT measurement of sinus ventilation is a potentially valuable noninvasive technique for the diagnostic imaging of the human maxillary sinus.

Effect of nasal dilators on nasal structures, sniffing strategies, and olfactory ability.

This paper describes the effects that nasal dilators have on olfactory ability. Experimental results demonstrate that nasal dilators increase odorant identification, lower odorant threshold, and increase perceptual odorant intensity. In other experiments, magnetic resonance imaging (MRI) data demonstrates that the size of the nasal cavity especially around the region of the nasal valve is increased when nasal dilators are worn. Additionally, pneumotachograph data demonstrates that during a sniff, the peak flow, maximum flow rate, volume, and duration are all increased when nasal dilators are worn. Taken together, the increase in olfactory ability can most easily be explained by an increase in both the amount and the proportion of inspired odorant molecules that are directed to the olfactory mucosa and are, therefore, available for odorant perception.

Prototype-referenced shape encoding revealed by high-level aftereffects.

We used high-level configural aftereffects induced by adaptation to realistic faces to investigate visual representations underlying complex pattern perception. We found that exposure to an individual face for a few seconds generated a significant and precise bias in the subsequent perception of face identity. In the context of a computationally derived 'face space,' adaptation specifically shifted perception along a trajectory passing through the adapting and average faces, selectively facilitating recognition of a test face lying on this trajectory and impairing recognition of other faces. The results suggest that the encoding of faces and other complex patterns draws upon contrastive neural mechanisms that reference the central tendency of the stimulus category.

Mutation in the gene responsible for cystic fibrosis and predisposition to chronic rhinosinusitis in the general population.

CONTEXT: Chronic rhinosinusitis (CRS) is a common condition in the US general population, yet little is known about its underlying molecular cause. Chronic rhinosinusitis is a consistent feature of the autosomal recessive disorder cystic fibrosis (CF). OBJECTIVE: To determine whether mutations in the cystic fibrosis transmembrane regulator (CFTR) gene, which is responsible for CF, predispose to CRS. DESIGN: Case-control study conducted from 1996 to 1999 in which the DNA of CRS patients and controls was typed for 16 mutations that account for 85% of CF alleles in the general population. Chronic rhinosinusitis patients with 1 CF mutation were evaluated for a CF diagnosis by sweat chloride testing, nasal potential difference measurement, and DNA analysis for additional mutations. SETTING: Otolaryngology-head and neck clinic of a US teaching hospital. PARTICIPANTS: One hundred forty-seven consecutive adult white patients who met stringent diagnostic criteria for CRS and 123 CRS-free white control volunteers of similar age range, geographic region, and socioeconomic status. MAIN OUTCOME MEASURES: Presence of CF mutations by DNA analysis among CRS patients vs controls. RESULTS: Eleven CRS patients were found to have a CF mutation (DeltaF508, n = 9; G542X, n = 1; and N1303K, n = 1). Diagnostic testing excluded CF in 10 of these patients and led to CF diagnosis in 1. Excluding this patient from the analyses, the proportion of CRS patients who were found to have a CF mutation (7%) was significantly higher than in the control group (n = 2 [2%]; P =.04, both having DeltaF508 mutations). Furthermore, 9 of the 10 CF carriers had the polymorphism M470V, and M470V homozygotes were overrepresented in the remaining 136 CRS patients (P =.03). CONCLUSION: These data indicate that mutations in the gene responsible for CF may be associated with the development of CRS in the general population. JAMA. 2000;284:1814-1819.

Anterior distribution of human olfactory epithelium.

OBJECTIVES/HYPOTHESIS: To functionally investigate the distribution of the olfactory epithelium in humans by means of the electro-olfactogram (EOG) and anatomically located biopsy specimens. STUDY DESIGN: Prospective, nonrandomized, investigational. METHODS: Supra-threshold EOG recordings were made on 12 healthy, trained volunteers (6 women, 6 men; age range, 21-48 y). Vanillin was used as the stimulus, since it exclusively excites olfactory receptor neurons. The EOG was recorded with tubular electrodes that were placed using thin-fiber endoscopic guidance. Biopsy specimens were obtained of anterosuperior nasal cavity mucosa in the same regions as the positive EOGs in 15 smell-tested patients (7 women, 8 men; age range, 22-60 y) during routine nasal and sinus surgery. This biopsied tissue was histologically processed and stained for olfactory and neural proteins. RESULTS: Viable responses to EOG testing were obtained in 7 of 12 subjects. In these seven subjects it was possible to identify nine sites above or below the anterior middle turbinate insertion where EOGs were obtained. The biopsy results showed mature olfactory receptor neurons in this same area. CONCLUSIONS: Human olfactory epithelium appears to be distributed more anteriorly than previously assumed.

Sinus mucosal IL-8 gene expression in chronic rhinosinusitis.

BACKGROUND: Epithelial hyperplasia and mucosal infiltration of leukocytes are common features of chronic rhinosinusitis. The epithelium can produce chemoattractant cytokines that may contribute to leukocyte infiltration in rhinosinusitis. OBJECTIVE: We sought to determine whether mucosal IL-8 gene expression is increased in chronic rhinosinusitis and to relate IL-8 gene expression to disease severity. METHODS: We used both a noncompetitive and a quantitative, competitive reverse transcription-polymerase chain reaction to examine IL-8 gene expression in samples of sinus mucosal tissue obtained during surgery from 22 patients with chronic rhinosinusitis and 9 normal control subjects. IL-8 gene expression was related to disease severity assessed by sinus computed tomography (CT) scores and to symptom scores assessed by means of a questionnaire. RESULTS: Sinus mucosal IL-8 gene expression was not detected in any of the control subjects but was present in 12 of 22 (55%) patients with rhinosinusitis. Sinus CT scores and symptom scores were both significantly higher in patients with positive mucosal IL-8 gene expression than in subjects with no detectable IL-8 gene expression. Positive IL-8 gene expression was not predicted by history of prior surgery nor by atopic or asthmatic status. In 9 subjects with positive IL-8 gene expression, levels of mRNA expression, assessed by competitive reverse transcription-polymerase chain reaction, correlated significantly (rho = 0.72, P <.05) with sinus CT scores. CONCLUSION: Sinus mucosal expression of the gene for IL-8 is increased in patients with chronic rhinosinusitis, and the level of IL-8 gene expression correlates with disease severity.