The interaction between cells in the central nervous system and immune cells are key in neuroinflammation, and play a role in the survival of neurons during neuropathology. Flow cytometry is a technique that enables the separation of cells from a mixed population.
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By using a digestion protocol, researchers were able to optimize the yield of these cells which could then be analyzed by flow cytometry and allow for both neural cells and leukocytes.
How do you study neuroimmune cells?
Studies have shown that microglia and astrocytes in the brain alongside leukocytes from the bloodstream are key cells in neuroinflammation, and are a factor in the survival of neurons in certain neuropathologies. These range from in vitro studies involving the use of cell lines, as well as using primary glial or astrocyte cultures.
While this does provide information on the function of these cells in certain situations, this does not provide information on how these cells interact with each other and with other cells (such as leukocytes) that enter the central nervous system.
With an in vivo approach, it would be ideal to isolate cell populations from the brain, this is not easily achieved. This is due to the fact that central nervous system tissue needs to be made into a single cell suspension.
Can you study a multiple sclerosis disease model using flow cytometry?
Another challenge that Legroux et al. wanted to address was identifying neural cells and leukocytes at the same time. To achieve this, the authors investigated various methods of preparing murine central nervous system tissue before analyzing the resulting suspension by flow cytometry. For this, they utilized a disease model of multiple sclerosis, experimental encephalomyelitis, in mice.
The experimental encephalomyelitis was induced in mice, and then the central nervous system tissue was isolated using a variety of “digestion mixes”; the authors found that the most effective mix included enzymes such as collagenase and DNase. The digestion was carried out for 15 minutes at 37°C.
After the digestion, the central nervous system suspension was placed in a separation mixture made of Percoll™; this is colloidal silica coated with polyvinylpyrrolidone. This was either a gradient of varying concentration, or a single concentration. The combined central nervous system suspension and separation mixture was then centrifuged for 10 mins without using brakes.
This allowed the myelin and debris from the central nervous system suspension to be separated from the cells. The authors found that using the single concentration of the separation mixture resulted in more cells being recovered from the central nervous system suspension.
Finally, the authors used fluorescently labeled antibodies to identify specific cell types within the central nervous system suspension.
Previous studies had revealed that there is an influx of leukocytes during experimental encephalomyelitis, therefore the authors wanted to see if the above flow cytometry protocol could be used to study differences in the cell populations found in mice with experimental encephalomyelitis compared to mice without the disease.
The protocol revealed that there was an increase in leukocytes in mice with experimental encephalomyelitis, therefore this protocol was able to successfully recover neural cells and leukocytes and reveal differences in the numbers found.
Are there differences in adult and neonatal mice?
Calvo et al. used a similar protocol to Legroux et al. to separate the cellular components of the murine central nervous system. Here, they utilized non-enzymatic dissociation or an enzymatic digestion mix containing DNase, papain and dispase, alongside the same separation mixture.
Interestingly, the authors found that the digestion process had different efficiency depending on the age of the mouse; papain was more efficient for digesting neonatal central nervous system tissue, while non-enzymatic dissociation and dispase was more effective at digesting adult central nervous system tissue.
Using flow cytometry to investigate neuroinflammation after brain injury
It is known that after a traumatic brain injury, neurons can still be damaged by a process known as “chronic/secondary degeneration”. This is a poorly understood process, with consequences for cognitive function. While studies have shown that neuroinflammatory processes occur at the time of traumatic brain injury, it is unknown whether this contributes to chronic degeneration.
Ertürk and co. set out to investigate whether neuroinflammation played a role in chronic degeneration by using a mouse model of traumatic brain injury. As a part of this study, the authors used flow cytometry to analyze the influx of leukocytes after the traumatic brain injury.
The preparation of the central nervous system tissue was similar to the processes used in the studies described above; briefly, enzymatic and non-enzymatic methods were used to disrupt the central nervous system tissue, then this was placed in the same separation mixture. This study used a concentration gradient of the separation mixture rather than a single concentration. The resulting suspension was then labeled with antibodies.
When the cell suspension was analyzed, the authors found elevated levels of lymphocytes 7 days after the traumatic brain injury. This was also seen 4 months after the traumatic brain injury. When analyzed further, it was revealed that the initial lymphocyte population had increased T-helper cells and natural killer cells when compared to the lymphocyte population at 4 months.
Legroux, L. et al. (2015) An optimized method to process mouse CNS to simultaneously analyze neural cells and leukocytes by flow cytometry. Journal of Neuroscience Methods https://doi.org/10.1016/j.jneumeth.2015.03.021
Calvo, B. et al. (2020) Dissociation of neonatal and adult mice brain for simultaneous analysis of microglia, astrocytes and infiltrating lymphocytes by flow cytometry. IBRO Reports https://doi.org/10.1016/j.ibror.2019.12.004
Ertürk, A. et al. (2016) Interfering with the Chronic Immune Response Rescues Chronic Degeneration After Traumatic Brain Injury. The Journal of Neuroscience https://doi.org/10.1523/JNEUROSCI.1898-15.2016