This work was supported by USPHS grants MH-091460 (E.N.), MH-084018 (A.S.), MH-094268 Silvo O. Conte center (A.S.), MH-069853 (A.S.), MH-085226 (A.S.), MH-088753 (A.S.), MH-092443 (A.S.), and MH-096208 (K.I.), grants from DANA (E.N.), Stanley (A.S.), RUSK (A.S.), S-R foundations (A.S.), NARSAD (A.S. and K.I.), and Maryland Stem Cell Research Fund (A.S. and K.I.).
We sincerely appreciate the efforts and contributions of Pearl Kim, Maria Papapavlou, Nao Gamo, Youjin Chung, Yukiko Lema and Mark Christie towards coordination of the biopsy process.

NOTE: All research volunteers in this study were administered informed consent documents approved by the Institutional Review Board of Howard University and Johns Hopkins University. Only participants who consented by signing the informed consent documents were enrolled into the study. The study base for this analysis consisted of: 20 subjects (12 BD and 10 controls; 30% males) and mean age (s.d.) of 38.2 (14.1) years. 
NOTE: Spray all surfaces with RNase Zap to remove RNase contamination from glass and plastic surf...

<ol>
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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene-expression+differences+in+peripheral+blood+between+lithium+responders+and+non-responders+in+the+Lithium+Treatment-Moderate+dose+Use+Study+(LiTMUS).">Gene-expression differences in peripheral blood between lithium responders and non-responders in the Lithium Treatment-Moderate dose Use Study (LiTMUS).</a> Pharmacogenomics J. 14 (2), 182-191 (2013).</li><li>Horiuchi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+cells+via+nasal+biopsy+reflect+the+developing+brain+in+gene+expression+profiles:+utility+and+limitation+of+the+surrogate+tissues+in+research+for+brain+disorders.">Olfactory cells via nasal biopsy reflect the developing brain in gene expression profiles: utility and limitation of the surrogate tissues in research for brain disorders.</a> Neurosci Res. 77 (4), 247-250 (2013).</li><li>Dryer, L., Graziadei, P. 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G., Takaki, M., Lin, S., Sawa, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurodevelopmental+involvement+in+schizophrenia:+the+olfactory+epithelium+as+an+alternative+model+for+research.">Neurodevelopmental involvement in schizophrenia: the olfactory epithelium as an alternative model for research.</a> J Neurochem. 102 (3), 587-594 (2007).</li><li>Sawa, A., Cascella, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+olfactory+system+for+clinical+and+basic+psychiatry:+a+promising+entry+point+to+the+mystery+of+brain+mechanism+and+biomarker+identification+in+schizophrenia.">Peripheral olfactory system for clinical and basic psychiatry: a promising entry point to the mystery of brain mechanism and biomarker identification in schizophrenia.</a> Am J Psychiatry. 166 (2), 137-139 (2009).</li><li>Mor, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-382+expression+is+elevated+in+the+olfactory+neuroepithelium+of+schizophrenia+patients.">MicroRNA-382 expression is elevated in the olfactory neuroepithelium of schizophrenia patients.</a> Neurobiol Dis. 55, 1-10 (2013).</li><li>Kano, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+profiling+of+multiple+histone+methylations+in+olfactory+cells:+further+implications+for+cellular+susceptibility+to+oxidative+stress+in+schizophrenia.">Genome-wide profiling of multiple histone methylations in olfactory cells: further implications for cellular susceptibility to oxidative stress in schizophrenia.</a> Mol Psychiatry. 18 (7), 740-742 (2013).</li><li>Toritsuka, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deficits+in+microRNA-mediated+Cxcr4/Cxcl12+signaling+in+neurodevelopmental+deficits+in+a+22q11+deletion+syndrome+mouse+model.">Deficits in microRNA-mediated Cxcr4/Cxcl12 signaling in neurodevelopmental deficits in a 22q11 deletion syndrome mouse model.</a> Proc Natl Acad Sci U S A. 110 (43), 17552-17557 (2013).</li><li>Evgrafov, O. 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A new platform for obtaining enriched olfactory neuronal layers by combining nasal biopsy and LCM is presented and has been validated in this study. This technique can have widespread implications. It can be applied towards biomarker studies, including those for treatment response, and drug discovery efforts for other neuropsychiatric conditions, making a broader impact in the field.
To obtain high quality neurons amenable to downstream applications, a few critical and troubleshooting steps mu...

Bipolar disorder (BD) is a severe neuropsychiatric disorder, characterized by pathological changes in mood, drive and cognition1. Lithium used for treatment of BD has been shown to alter steady state mRNA levels of a large number of genes in animal studies2, but it remains unknown if any of these molecules is associated with clinical response in humans2. Understanding the mechanism of lithium response would require investigating lithium-induced molecular changes in neuronal tissues. Unfortunately, it is not practical to obtain brain biopsies of BD patients pre- and post-lithium therapy to identify molecular signatures of lithium response. Post-mortem brain tissues have been used to study biomarkers in BD, however, they cannot be used to assess molecular markers associated with dynamic changes in emotions, cognition and drive; and the validity of retrospectively ascertained lithium treatment response can be problematic4. Lymphocytes and other blood cells could be useful, but molecular changes in blood cells may not reflect neuronal alterations3-5. Cerebrospinal fluid may be insufficient to obtain information on intracellular molecules that may reflect disease-associated and medication-reversible intrinsic changes.
The olfactory epithelium (OE) is a unique part of the central nervous system (CNS), embryologically-related to limbic structures6; and it is easily accessible through nasal biopsies. It consists of glial-like supporting (i.e., sustentacular) cells, basal proliferating cells, and olfactory receptor neurons at different stages of development7-9. Therefore, OE provides a unique opportunity to accessibly study dynamic changes in the CNS of patients with neuropsychiatric diseases7. Studies are demonstrating the utility of the OE as a surrogate tissue for investigating disease associated events that reflect those occurring in brain neurons8,9. For example, studies have utilized OE to investigate molecular profiles associated with psychiatric conditions10-14. Olfactory system also serves to identify clinical endophenoytpes such as smell deficits that are associated with negative symptoms of schizophrenia15. Additionally, neurodevelopmental processes continue in the OE throughout life, providing a useful avenue to model the underlying pathophysiology of psychiatric conditions8,9.
However, a drawback to the use of this tissue is the substantial contamination of olfactory biopsies with non-neuronal cells16. For instance, total RNA used for gene expression studies in previous OE studies contained RNA extracted from the entire nasal biopsied tissue including RNA from non-neuronal cells17. Therefore, previous approaches have been limited by the quality of cells. To overcome this issue, a novel approach to obtain enriched neuronal cell populations by combining nasal biopsies with laser-capture microdissection (LCM) has been developed18.
LCM is a technique that allows for the selective isolation of cells using UV laser cutting combined with infra-red laser19-21. Combining LCM with OE approach minimizes substantial contamination of OE by non-neuronal cells, thereby enhancing enrichment of neuronal cells18. Moreover, the neuronal layer can be distinguished from the submucosa layer under a microscope, thereby eliminating the need for staining. Neuronal cell types can further be distinguished from other cell populations using primary antibodies which are expressed by the cell type of interest7. Therefore, this procedure establishes an easier method for the enrichment of almost purely neuronal cell population that can be used for gene expression studies, immunohistochemistry and other morphologic investigations.
This study aims to establish an experimental platform to investigate molecular changes in olfactory neurons associated with disease states and treatment response. To address this, a small set of non-smoking patients who met the DSM-IV diagnostic criteria for BD based on the Diagnostic Interview for Genetic Studies (DIG)22 was recruited to undergo two nasal biopsies: one biopsy pre-treatment with lithium and the second biopsy, after 6 weeks of daily oral lithium therapy. Furthermore, eligible BD patients must be: symptomatic for depression, based on scoring &#8805;10 out of 60 in the clinician administered Montgomery-Asberg Rating Scale (MADRS)23; symptomatic for hypomania or mania, based on a score &#8805;10 out of 56 on the clinician administered Young-Mania Rating Scale (YMRS)24; or &#8805;10 on both MADRS and YMRS. Rater-Inter-Rater coefficient of agreement between clinicians for both scales is &#62;0.96. After biopsies, neurons were enriched from OE by LCM. Following additional quality control measures, to ensure extraction of high quality RNA from the tissue and neuronal enrichment, Real Time RT PCR was conducted to investigate pre- and post-treatment expression levels of genes of interest. The ensuing sections contain a description of the validation of this approach, highlighting the optimization of the protocol and the strategies that were applied for trouble-shooting the protocol.

<table border="0" cellpadding="0" cellspacing="0" width="600">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Reagent</td>
			<td style="width:143px;">Manufacturuer&#160;</td>
			<td style="width:175px;">Manufacturer Catalog #</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue Preparation</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue-Tek Cryomold Molds</td>
			<td>Sakura Finetek</td>
			<td>4557</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue-Tek O.C.T Compound</td>
			<td>Sakura Finetek</td>
			<td>4583</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cryosectioning</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Membrane Slide 1.0 PEN (D)</td>
			<td>Carl Zeiss Microscopy</td>
			<td>415190-9041-000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rnase Zap</td>
			<td>Ambion</td>
			<td>AM9780</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DEPC Treated Water</td>
			<td>Quality Biological</td>
			<td>351-068-131</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microdissection</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope: PALM Series MicroLaser System</td>
			<td>Carl Zeiss Microscopy</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Model: Axiovert 200M</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Software: Robo v3.2</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">No.5 Dumont Microdissction Forceps</td>
			<td>Roboz</td>
			<td>RS-49085</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNA Extraction</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNAqueous Micro Kit</td>
			<td>Ambion</td>
			<td>AM1931</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">cDNA Synthesis</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SuperScript III First Strand Synthesis Kit</td>
			<td>Invitrogen&#160;</td>
			<td>18080-051</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">OMP qPCR</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SYBR GreenER qPCR SuperMix</td>
			<td>Invitrogen&#160;</td>
			<td>11760-500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Taqman qPCR</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan Expression Assay Probes</td>
			<td>Applied Biosystems</td>
			<td>Various</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TaqMan Gene Expression Master Mix</td>
			<td>Applied Biosystems</td>
			<td>4369016</td>
		</tr>
	</tbody>
</table>

olfactory neurons obtained through nasal biopsy combined with laser-capture microdissection: a potential approach to study treatment response in mental disorders

Bipolar disorder (BD) is a severe neuropsychiatric disorder with poorly understood pathophysiology and typically treated with the mood stabilizer, lithium carbonate. Animal studies as well as human genetic studies indicate that lithium affects molecular targets that are involved in neuronal growth, survival and maturation, and notably molecules involved in Wnt signaling. Given the ethical challenge to obtaining brain biopsies for investigating dynamic molecular changes associated with lithium-response in the central nervous system (CNS), one may consider the use of neurons obtained from olfactory tissues to achieve this goal.The olfactory epithelium contains olfactory receptor neurons at different stages of development and glial-like supporting cells. This provides a unique opportunity to study dynamic changes in the CNS of patients with neuropsychiatric diseases, using olfactory tissue safely obtained from nasal biopsies. To overcome the drawback posed by substantial contamination of biopsied olfactory tissue with non-neuronal cells, a novel approach to obtain enriched neuronal cell populations was developed by combining nasal biopsies with laser-capture microdissection. In this study, a system for investigating treatment-associated dynamic molecular changes in neuronal tissue was developed and validated, using a small pilot sample of BD patients recruited for the study of the molecular mechanisms of lithium treatment response.

The protocol and troubleshooting strategy in the study of molecular signatures were successfully optimized. RNA quality and neuronal enrichment criteria for samples to be used in further downstream Real Time-PCR analysis were standardized. RIN and RNA concentration were examined as confounding factors to detected expression levels. Based on the analysis of several samples with RINs ranging from 1 - 10, it was determined that a minimum RIN of ~3.0 and above is sufficient for downstream analysis in this study. As a result,...

Watch this Scientific Journal Video about Olfactory Neurons Obtained through Nasal Biopsy Combined with Laser-Capture Microdissection: A Potential Approach to Study Treatment Response in Mental Disord

Olfactory Neurons Obtained through Nasal Biopsy Combined with Laser-Capture Microdissection: A Potential Approach to Study Treatment Response in Mental Disorders

In this study, a novel platform to investigate intraneuronal molecular signatures of treatment response in bipolar disorder (BD) was developed and validated. Olfactory epithelium from BD patients was obtained through nasal biopsies. Then laser-capture microdissection was combined with Real Time RT PCR to investigate the molecular signature of lithium response in BD.

Bipolar disorder (BD) is a severe neuropsychiatric disorder with poorly understood pathophysiology and typically treated with the mood stabilizer, lithium ...

olfactory-neurons-obtained-through-nasal-biopsy-combined-with-laser

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Neuroscience

9.7K visualizações.  Johns Hopkins University.  Neste estudo, um romance plataforma para investigar assinaturas moleculares intraneuronal de resposta ao tratamento do transtorno bipolar (TB) foi desenvolvido e validado. O epitélio olfatório de pacientes com TB foi obtido através de biópsias nasais. Em seguida, captura a laser microdissection foi combinado com Tempo real RT PCR para investigar a assinatura molecular de resposta de lítio em BD. 

Vídeo: Olfactory Neurons Obtained through Nasal Biopsy Combined with Laser-Capture Microdissection: A Potential Approach to Study Treatment Response in Mental Disorders