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Laser-assisted microdissection.

Oberbeck D - Alcohol Res Health (2008)

Bottom Line: The challenge then becomes how to isolate small numbers of cells from a specific brain region without including unwanted cells.One approach to solving this problem is to use a technology known as laser-assisted microdissection (LMD).This article reviews some of the principles of LMD and its use in alcohol research.

View Article: PubMed Central - PubMed

Affiliation: Department of Behavioral Neuroscience, Oregon Health & Science University, and in the Research Service, Portland VA Medical Center, both in Portland, Oregon.

ABSTRACT
When analyzing alcohol's effects on the brain, researchers often want to look at small clusters of cells that can be studied in isolation from the surrounding brain tissue rather than at the entire brain or larger brain areas. This implies that relatively small numbers of cells have to be retrieved from the brain and studied in culture or subjected to biochemical analyses. The challenge then becomes how to isolate small numbers of cells from a specific brain region without including unwanted cells. One approach to solving this problem is to use a technology known as laser-assisted microdissection (LMD). This article reviews some of the principles of LMD and its use in alcohol research.

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Representative photomicrographs showing a section of the mouse brain before and after laser microdissection of two subnuclei of the amygdala. A) Thionin-stained section of brain tissue viewed at 4x magnification before dissection. B) Computer graphic overlay (light grey circles) on photograph showing regions to be dissected. C) Section after microdissection; the nuclei have been removed. D) The same brain region at lower magnification (1.25x), showing the surrounding neuronal architecture. E) Microdissected region as seen isolated in a microcentrifuge tube.NOTE: BMA = basomedial nucleus of the amygdala; CeA = central nucleus of the amygdala.
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f8-arh-31-3-249: Representative photomicrographs showing a section of the mouse brain before and after laser microdissection of two subnuclei of the amygdala. A) Thionin-stained section of brain tissue viewed at 4x magnification before dissection. B) Computer graphic overlay (light grey circles) on photograph showing regions to be dissected. C) Section after microdissection; the nuclei have been removed. D) The same brain region at lower magnification (1.25x), showing the surrounding neuronal architecture. E) Microdissected region as seen isolated in a microcentrifuge tube.NOTE: BMA = basomedial nucleus of the amygdala; CeA = central nucleus of the amygdala.

Mentions: As LMD becomes more widely used, laboratories should adopt some common quality-control procedures to ensure that indeed the desired cells have been isolated and no contamination with other cells occurs. For example, when trying to analyze cells from individual nuclei in the brain (e.g., subregions of the amygdala), researchers could measure the relative expression of three calcium-binding proteins called parvalbumin, calretinin, and calbindin. These proteins are produced in all brain regions, but their relative amounts differ among different regions or even nuclei. For example, if an experimental design requires dissecting (in the mouse brain) the central nucleus of the amygdala and the basomedial amygdala, the dissection of these two nuclei is relatively simple from an LMD perspective (see figure 8). However, subtle changes in dissection technique could lead to some substantial differences in the actual nature of the tissue acquired and presumably the results that are obtained. Therefore, as the experiment proceeds over time, with samples acquired from different animals, it will be critical to know that the dissection procedures have remained constant. At this point, biochemical expression analyses such as RT-PCR can be used to measure the expression of the calcium-binding proteins in each sample obtained. The expression of parvalbumin is relatively low in both the central and the basomedial nuclei of the amygdala; the expression of calretinin and calbindin, however, shows some variation between these nuclei. Moreover, the central nucleus can be further divided into three main divisions (lateral, medial, and capsular), which also show differential expression of the three genes. Thus, determining the relative expression of the three genes helps researchers determine exactly whether they have isolated the correct cells. With such quality-control strategies in place, LMD can help researchers gain valuable information about the functions and properties of very specific brain regions as well as about the effects that alcohol and other drugs have on these cells.


Laser-assisted microdissection.

Oberbeck D - Alcohol Res Health (2008)

Representative photomicrographs showing a section of the mouse brain before and after laser microdissection of two subnuclei of the amygdala. A) Thionin-stained section of brain tissue viewed at 4x magnification before dissection. B) Computer graphic overlay (light grey circles) on photograph showing regions to be dissected. C) Section after microdissection; the nuclei have been removed. D) The same brain region at lower magnification (1.25x), showing the surrounding neuronal architecture. E) Microdissected region as seen isolated in a microcentrifuge tube.NOTE: BMA = basomedial nucleus of the amygdala; CeA = central nucleus of the amygdala.
© Copyright Policy - public-domain
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3860489&req=5

f8-arh-31-3-249: Representative photomicrographs showing a section of the mouse brain before and after laser microdissection of two subnuclei of the amygdala. A) Thionin-stained section of brain tissue viewed at 4x magnification before dissection. B) Computer graphic overlay (light grey circles) on photograph showing regions to be dissected. C) Section after microdissection; the nuclei have been removed. D) The same brain region at lower magnification (1.25x), showing the surrounding neuronal architecture. E) Microdissected region as seen isolated in a microcentrifuge tube.NOTE: BMA = basomedial nucleus of the amygdala; CeA = central nucleus of the amygdala.
Mentions: As LMD becomes more widely used, laboratories should adopt some common quality-control procedures to ensure that indeed the desired cells have been isolated and no contamination with other cells occurs. For example, when trying to analyze cells from individual nuclei in the brain (e.g., subregions of the amygdala), researchers could measure the relative expression of three calcium-binding proteins called parvalbumin, calretinin, and calbindin. These proteins are produced in all brain regions, but their relative amounts differ among different regions or even nuclei. For example, if an experimental design requires dissecting (in the mouse brain) the central nucleus of the amygdala and the basomedial amygdala, the dissection of these two nuclei is relatively simple from an LMD perspective (see figure 8). However, subtle changes in dissection technique could lead to some substantial differences in the actual nature of the tissue acquired and presumably the results that are obtained. Therefore, as the experiment proceeds over time, with samples acquired from different animals, it will be critical to know that the dissection procedures have remained constant. At this point, biochemical expression analyses such as RT-PCR can be used to measure the expression of the calcium-binding proteins in each sample obtained. The expression of parvalbumin is relatively low in both the central and the basomedial nuclei of the amygdala; the expression of calretinin and calbindin, however, shows some variation between these nuclei. Moreover, the central nucleus can be further divided into three main divisions (lateral, medial, and capsular), which also show differential expression of the three genes. Thus, determining the relative expression of the three genes helps researchers determine exactly whether they have isolated the correct cells. With such quality-control strategies in place, LMD can help researchers gain valuable information about the functions and properties of very specific brain regions as well as about the effects that alcohol and other drugs have on these cells.

Bottom Line: The challenge then becomes how to isolate small numbers of cells from a specific brain region without including unwanted cells.One approach to solving this problem is to use a technology known as laser-assisted microdissection (LMD).This article reviews some of the principles of LMD and its use in alcohol research.

View Article: PubMed Central - PubMed

Affiliation: Department of Behavioral Neuroscience, Oregon Health & Science University, and in the Research Service, Portland VA Medical Center, both in Portland, Oregon.

ABSTRACT
When analyzing alcohol's effects on the brain, researchers often want to look at small clusters of cells that can be studied in isolation from the surrounding brain tissue rather than at the entire brain or larger brain areas. This implies that relatively small numbers of cells have to be retrieved from the brain and studied in culture or subjected to biochemical analyses. The challenge then becomes how to isolate small numbers of cells from a specific brain region without including unwanted cells. One approach to solving this problem is to use a technology known as laser-assisted microdissection (LMD). This article reviews some of the principles of LMD and its use in alcohol research.

Show MeSH
Related in: MedlinePlus