Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury

Abstract

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that is critical for learning, memory and executive function.1,2 Hence our studies focus on gene expression analysis of specific neuronal populations in distinct subregions of the hippocampus. The technique of laser capture microdissection (LCM), introduced in 1996 by et al.,3 has allowed for significant advances in gene expression analysis of single cells and enriched populations of cells from heterogeneous tissues such as the mammalian brain that contains thousands of functional cell types. 4 We use LCM and a well established rat model of traumatic brain injury (TBI) to investigate the molecular mechanisms that underlie the pathogenesis of TBI. Following fluidpercussion TBI, brains are removed at pre-determined times post-injury, immediately frozen on dry ice, and prepared for sectioning in a cryostat. The rat brains can be embedded in OCT and sectioned immediately, or stored several months at -80 °C before sectioning for laser capture microdissection. Additionally, we use LCM to study the effects of TBI on circadian rhythms. For this, we capture neurons from the suprachiasmatic nuclei that contain the master clock of the mammalian brain. Here, we demonstrate the use of LCM to obtain single identified neurons (injured and degenerating, Fluoro-Jade-positive, or uninjured, Fluoro-Jade-negative) and enriched populations of hippocampal neurons for subsequent gene expression analysis by real time PCR and/or whole-genome microarrays. These LCM-enabled studies have revealed that the selective vulnerability of anatomically distinct regions of the rat hippocampus are reflected in the different gene expression profiles of different populations of neurons obtained by LCM from these distinct regions. The results from our single-cell studies, where we compare the transcriptional profiles of dying and adjacent surviving hippocampal neurons, suggest the existence of a cell survival rheostat that regulates cell death and survival after TBI.

Gene expression profiling of the astrocyte transcriptome in multiple sclerosis normal appearing white matter reveals a neuroprotective role

“…An increasing number of studies in a range of neurological diseases have used this approach to identify disease-relevant, cell-specific gene expression changes (Boone et al, 2013, Chu et al, 2009, Kumar et al, 2013, Pietersen et al, 2009, Simpson et al, 2011, Baker et al, 2015. To date, only two studies have employed LCM in the investigation of human MS cases: one identifying gene expression changes in total extracts from NAWM, peri-lesional and lesional regions compared to control WM (Mycko et al, 2012) and the other identifying transcriptomic changes in the vascular compartment in MS (Cunnea et al, 2010); with neither study specifically assessing changes in the astrocyte transcriptome.…”

Enrichment of single neurons and defined brain regions from human brain tissue samples for subsequent proteome analysis

“…The literature contains numerous staining protocols for different stains in the context of LMD processing. Common stains include hematoxylin and eosin (De Souza et al 2004;Dos Santos et al 2007), toluidine blue (Sridharan and Shankar 2012;Kulkarni et al 2013;Kirana et al 2009;Lawrie et al 2001;Mouledous et al 2002) and cresyl violet (Boone et al 2013;Aaltonen et al 2011). Immunohistochemical staining is also popular for LMD processing because it can distinguish single cell types (e.g.…”

“…Neurons are not easily detached from their parenchyma, are highly branched and interact in networks. Recently, laser microdissection (LMD) has emerged as a promising alternative for isolating neurons for genomics and transcriptomics (Boone et al 2013;Kumar et al 2013;Majer et al 2012;Moulédous et al 2003;Friedrich et al 2012;Decarlo et al 2011;Simunovic et al 2009;Cantuti-Castelvetri et al 2007;Elstner et al 2011). For example, Simunovic et al (2009) made a gene expression profile of dopamine neurons isolated by LMD for a comparison study of idiopathic PD and control subjects.…”

Abordagem para análise proteômica de neurônios contendo neuromelanina na substância negra, isolados por microdissecção a laser

“…Este fato é particularmente relevante para o tecido encefálico, que consiste em vários tipos celulares morfologicamente e/ou neuroquimicamente distintos cercados por vários tipos de células, como por exemplo, células gliais (oligodendrócitos, microglia e astrócitos). Portanto, a capacidade de isolar populações de células específicas, como por exemplo, neurônios surgiu recentemente como uma alternativa promissora (Banks et al, 1999;Craven e Banks, 2001;Moulédous et al, 2003;Caudle et al, 2008;Boone et al, 2013;Kumar et al, 2013) e unido a técnicas moleculares, pode acentuadamente melhorar a compreensão de diversos processos biológicos (Kummari et al, 2015).…”

“…No entanto, a especificidade do processo de coloração e o seu efeito sobre a amostra, devem ser considerados (Standaert, 2005 (Aaltonen et al, 2011;Boone et al, 2013). A técnica de imunohistoquímica também é utilizada para realizar a MCL (Fend et al, 1999) e permite distinguir tipos de células individuais, como neurônios (Pietersen et al, 2009).…”