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

Summary

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.

Abstract

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.

Introduction

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 respon....

Protocol

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 surfaces.

1. Biopsy Procedure

1. Spray topical lidocaine (xylocaine) liquid 4% and oxymetazoline HCl 0.05% in the nas....

Results

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,.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Tissue Preparation
Tissue-Tek Cryomold Molds	Sakura Finetek	4557
Tissue-Tek O.C.T. Compound	Sakura Finetek	4583
Cryosectioning
Membrane Slide 1.0 PEN (D)	Carl Zeiss Microscopy	415190-9041-000
Rnase Zap	Ambion	AM9780
DEPC Treated Water	Quality Biological	351-068-131
Microdissection
Microscope: PALM Series MicroLaser System	Carl Zeiss Microscopy		Model: Axiovert 200M Software: Robo v3.2
No. 5 Dumont Microdissction Forceps	Roboz	RS-49085
RNA Extraction
RNAqueous Micro Kit	Ambion	AM1931
cDNA Synthesis
SuperScript III First Strand Synthesis Kit	Invitrogen	18080-051
OMP qPCR
SYBR GreenER qPCR SuperMix	Invitrogen	11760-500
Taqman qPCR
TaqMan Expression Assay Probes	Applied Biosystems	Various
TaqMan Gene Expression Master Mix	Applied Biosystems	4369016