Abstract
Background: Current methods for murine brain microvasculature isolation requires the pooling of brain cortices while disregarding the rest of the CNS, making the analysis of single individuals non feasible. New method: Efficient isolation of brain microvessels requires the elimination of meninges, vessels of high caliber vessels and choroid plexus, commonly done by rolling the over filter paper, but can't be done on other CNS regions. We overcome this hurdle by using a double-pronged pick, as well as elution and filtration through cell strainers after centrifugation. Results: We were able to develop a region-specific murine CNS microvessels isolation, that allows for the comparison of the neurovascular unit from these regions both within the same individual and between multiple individuals and/or treatment groups without pooling. Additionally, we were able to adapt this method to macaque CNS tissue. Comparison with existing method(s): Although similar to a previously published method that requires no enzymatic dissociation and no ultracentrifugation, it does differ in its ability to isolate from a single experimental animal and from non-cortical tissues. However, it relies heavily on the researcher dissecting skills and careful elution and filtration of re-suspended samples. Conclusions: CNS region-specific microvessels comparison can inform of molecular and/or cellular differences that would otherwise be obscured by excluding non-cortical tissue. Additionally, it allows for the unmasking of variations between individuals that remained hidden when pooling of multiple samples is the norm. Lastly, isolation of region-specific microvessels for non-human primate CNS allows for more translationally relevant studies of the BBB.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 17-33 |
| Number of pages | 17 |
| Journal | Journal of Neuroscience Methods |
| Volume | 318 |
| DOIs | |
| State | Published - Apr 15 2019 |
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Keywords
- Astrocytic end-feet
- Blood-brain barrier
- Endothelial cell
- Junctional proteins
- Microvasculature
- Neurovascular unit
- Pericyte
ASJC Scopus subject areas
- Neuroscience(all)
Cite this
Straightforward method for singularized and region-specific CNS microvessels isolation. / Dayton, Jacquelyn Rose; Franke, Marissa Cindy; Yuan, Yinyu; Cruz-Orengo, Lillian.
In: Journal of Neuroscience Methods, Vol. 318, 15.04.2019, p. 17-33.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Straightforward method for singularized and region-specific CNS microvessels isolation
AU - Dayton, Jacquelyn Rose
AU - Franke, Marissa Cindy
AU - Yuan, Yinyu
AU - Cruz-Orengo, Lillian
PY - 2019/4/15
Y1 - 2019/4/15
N2 - Background: Current methods for murine brain microvasculature isolation requires the pooling of brain cortices while disregarding the rest of the CNS, making the analysis of single individuals non feasible. New method: Efficient isolation of brain microvessels requires the elimination of meninges, vessels of high caliber vessels and choroid plexus, commonly done by rolling the over filter paper, but can't be done on other CNS regions. We overcome this hurdle by using a double-pronged pick, as well as elution and filtration through cell strainers after centrifugation. Results: We were able to develop a region-specific murine CNS microvessels isolation, that allows for the comparison of the neurovascular unit from these regions both within the same individual and between multiple individuals and/or treatment groups without pooling. Additionally, we were able to adapt this method to macaque CNS tissue. Comparison with existing method(s): Although similar to a previously published method that requires no enzymatic dissociation and no ultracentrifugation, it does differ in its ability to isolate from a single experimental animal and from non-cortical tissues. However, it relies heavily on the researcher dissecting skills and careful elution and filtration of re-suspended samples. Conclusions: CNS region-specific microvessels comparison can inform of molecular and/or cellular differences that would otherwise be obscured by excluding non-cortical tissue. Additionally, it allows for the unmasking of variations between individuals that remained hidden when pooling of multiple samples is the norm. Lastly, isolation of region-specific microvessels for non-human primate CNS allows for more translationally relevant studies of the BBB.
AB - Background: Current methods for murine brain microvasculature isolation requires the pooling of brain cortices while disregarding the rest of the CNS, making the analysis of single individuals non feasible. New method: Efficient isolation of brain microvessels requires the elimination of meninges, vessels of high caliber vessels and choroid plexus, commonly done by rolling the over filter paper, but can't be done on other CNS regions. We overcome this hurdle by using a double-pronged pick, as well as elution and filtration through cell strainers after centrifugation. Results: We were able to develop a region-specific murine CNS microvessels isolation, that allows for the comparison of the neurovascular unit from these regions both within the same individual and between multiple individuals and/or treatment groups without pooling. Additionally, we were able to adapt this method to macaque CNS tissue. Comparison with existing method(s): Although similar to a previously published method that requires no enzymatic dissociation and no ultracentrifugation, it does differ in its ability to isolate from a single experimental animal and from non-cortical tissues. However, it relies heavily on the researcher dissecting skills and careful elution and filtration of re-suspended samples. Conclusions: CNS region-specific microvessels comparison can inform of molecular and/or cellular differences that would otherwise be obscured by excluding non-cortical tissue. Additionally, it allows for the unmasking of variations between individuals that remained hidden when pooling of multiple samples is the norm. Lastly, isolation of region-specific microvessels for non-human primate CNS allows for more translationally relevant studies of the BBB.
KW - Astrocytic end-feet
KW - Blood-brain barrier
KW - Endothelial cell
KW - Junctional proteins
KW - Microvasculature
KW - Neurovascular unit
KW - Pericyte
UR - http://www.scopus.com/inward/record.url?scp=85062220235&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85062220235&partnerID=8YFLogxK
U2 - 10.1016/j.jneumeth.2019.02.007
DO - 10.1016/j.jneumeth.2019.02.007
M3 - Article
C2 - 30797797
AN - SCOPUS:85062220235
VL - 318
SP - 17
EP - 33
JO - Journal of Neuroscience Methods
JF - Journal of Neuroscience Methods
SN - 0165-0270
ER -