Laser capture microdissection and analysis of amplified antisense RNA from distinct cell populations of the young and aged rat brain: effect of traumatic brain injury on hippocampal gene expression

Shimamura, Megumi; Garcia, Jeanna M.; Prough, Donald S.; Hellmich, Helen L.

doi:10.1016/j.molbrainres.2003.11.015

Cited by 47 publications

(43 citation statements)

References 61 publications

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“…Previous investigations demonstrated changes in gene expression after traumatic injury (Dash et al, 2004;Hayes et al, 1995;Hellmich et al, 2005Hellmich et al, ,2008Shimamura et al, 2004Shimamura et al, ,2005. Mechanical injury (Morrison et al, 2000) or other types of trauma, including ischemia or seizures, can lead to disruption of calcium homeostasis that can produce alterations in gene expression (Bazan et al, 1995;West et al, 2001).…”

Section: Discussionmentioning

confidence: 99%

“…The use of LCM methods permitted the examination of the effects of TBI on Cx gene expression specifically in endothelial or smooth muscle cells, while RPA allowed for absolute, quantitative measures of mRNA levels (Shimamura et al, 2004). Vascular smooth muscle cells contain all four of the vascular Cxs.…”

Section: Discussionmentioning

confidence: 99%

“…Cells were obtained from cerebral blood vessels using LCM (Hellmich et al, 2007;Shimamura et al, 2004). Frozen 10-lm sections were collected on uncoated RNase-free slides (Fisher Scientific, Pittsburgh, PA), and kept at -20°C until staining.…”

Section: Laser Capture Microdissectionmentioning

confidence: 99%

“…Probes were cloned using reverse transcription-polymerase chain reaction (RT-PCR) as previously described (Shimamura et al, 2004 P-UTP. The sizes of the protected fragments were 200, 199, 246, and 297 base pairs, respectively, and the sequences of the primers and probes are listed in Table 1.…”

Section: Laser Capture Microdissectionmentioning

confidence: 99%

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Cerebrovascular Connexin Expression: Effects of Traumatic Brain Injury

Avila

Sell

Hawkins

et al. 2011

Journal of Neurotrauma

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Traumatic brain injury (TBI) results in dysfunction of the cerebrovasculature. Gap junctions coordinate vasomotor responses and evidence suggests that they are involved in cerebrovascular dysfunction after TBI. Gap junctions are comprised of connexin proteins (Cxs), of which Cx37, Cx40, Cx43, and Cx45 are expressed in vascular tissue. This study tests the hypothesis that TBI alters Cx mRNA and protein expression in cerebral vascular smooth muscle and endothelial cells. Anesthetized (1.5% isoflurane) male Sprague-Dawley rats received sham or fluid-percussion TBI. Two, 6, and 24 h after, cerebral arteries were harvested, fresh-frozen for RNA isolation, or homogenized for Western blot analysis. Cerebral vascular endothelial and smooth muscle cells were selected from frozen sections using laser capture microdissection. RNA was quantified by ribonuclease protection assay. The mRNA for all four Cx genes showed greater expression in the smooth muscle layer compared to the endothelial layer. Smooth muscle Cx43 mRNA expression was reduced 2 h and endothelial Cx45 mRNA expression was reduced 24 h after injury. Western blot analysis revealed that Cx40 protein expression increased, while Cx45 protein expression decreased 24 h after injury. These studies revealed significant changes in the mRNA and protein expression of specific vascular Cxs after TBI. This is the first demonstration of cell type-related differential expression of Cx mRNA in cerebral arteries, and is a first step in evaluating the effects of TBI on gap junction communication in the cerebrovasculature.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Laser Capture Microdissectionmentioning

confidence: 99%

Section: Laser Capture Microdissectionmentioning

confidence: 99%

See 2 more Smart Citations

Cerebrovascular Connexin Expression: Effects of Traumatic Brain Injury

Avila

Sell

Hawkins

et al. 2011

Journal of Neurotrauma

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“…the rats with 1.5-2.0% isoflurane in oxygen: air (70:30) and prepare them for parasagittal fluid-percussion injury as previously described. 8,10 4. Sacrifice rats at the appropriate time point after injury depending on the experimental design.…”

Section: Introductionmentioning

confidence: 99%

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

Boone

Sell

Hellmich

2013

JoVE

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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.

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Neurotrauma investigation through spatial omics guided by mass spectrometry imaging: Target identification and clinical applications

Mallah

Zibara

Kerbaj

et al. 2021

Mass Spectrometry Reviews

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Traumatic brain injury (TBI) represents one of the major public health concerns worldwide due to the increase in TBI incidence as a result of injuries from daily life accidents such as sports and motor vehicle transportation as well as militaryrelated practices. This type of central nervous system trauma is known to predispose patients to several neurological disorders such as Parkinson's disease, Alzheimer's disease, chronic trauamatic encephalopathy, and age-related Dementia. Recently, several proteomic and lipidomic platforms have been applied on different TBI studies to investigate TBI-related mechanisms that have broadened our understanding of its distinct neuropathological complications. In this study, we provide an updated comprehensive overview of the current knowledge and novel perspectives of the spatially resolved microproteomics and microlipidomics approaches guided by mass spectrometry imaging used in TBI studies and its applications in the neurotrauma field. In this regard, we will discuss the use of the spatially resolved microproteomics and assess the different microproteomic sampling methods such as laser capture microdissection, parafilm assisted microdissection, and liquid microjunction extraction as accurate and precise techniques in the field of neuroproteomics. Additionally, we will highlight lipid profiling applications and their prospective potentials in characterizing molecular processes involved in the field of TBI. Specifically, we will discuss the phospholipid metabolism acting as a precursor for proinflammatory molecules such as eicosanoids. Finally, we will survey the current state of spatial neuroproteomics and microproteomics applications and present the various studies highlighting their findings in these fields.

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Laser capture microdissection and analysis of amplified antisense RNA from distinct cell populations of the young and aged rat brain: effect of traumatic brain injury on hippocampal gene expression

Cited by 47 publications

References 61 publications

Cerebrovascular Connexin Expression: Effects of Traumatic Brain Injury

Cerebrovascular Connexin Expression: Effects of Traumatic Brain Injury

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

Neurotrauma investigation through spatial omics guided by mass spectrometry imaging: Target identification and clinical applications

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