Discussion
This study serves as a preliminary investigation into the astrocytic
response following BBB rupture from shunt insertion and shows a
significant increase in cellular attachment when exposed to blood
(P <0.0001). Rupture of the BBB causes and increase in
VZ permeability similar to other chronic indwelling devices within the
brain (28). Using this 3D printed in vitromodel, we were able to mimic breaking the BBB and subsequent
astrogliosis. In a study done by Castaneyra-Ruiz, it was described that
the optimal concentration of blood was 3% over a 48-hour time period
with higher concentrations, 4% blood, showing significant cell death.
This decrease in cells prevented any observations, at higher
concentrations, past the 3-hour time point. Since our experiment ran for
two weeks, a significantly longer time period, we opted to decrease our
blood concentration to 1.5% in the media to prevent excessive cell
death and allow for a sufficient number of cells to survive for
analysis. Certainly, concentration and time-point dependencies will
exist, which will be studied in future work. Additionally, the
incorporation of a bioreactor-based flow system at physiologic flow and
shear rates may augment the possibility for dynamic changes in blood
protein adsorption.
Replication of industrial catheters using a leather punch created a
rough surface similar to that of industrial made catheters. In previous
work, it was shown that these imperfections cause an increase in
cellular adhesion (29). The addition of this rough
surface may perhaps give rise to an increased opportunity for increased
blood protein adsorption and/or astrocyte attachment. Silicone catheters
have been known to have relatively hydrophobic properties allowing for
an increase in cellular adsorption to the surface(30). Along with the addition of holes creating a
rough surface, the hydrophobic characteristic creates a device prone to
more cellular attachment.
Choosing to limit the investigation to astrocytes alone was due to the
known reactivity after exposure to blood along with its prevalent role
in general shunt obstruction (24),(31),(32).
Certainly, the acute- and chronic- response to the shunt catheter is
dynamic, multi-factorial, and dependent on environmental conditions.
Included in this response, we must consider that the ependymal layer
that makes up the ventricular wall breaks down when exposed to blood
allowing the astrocytes to migrate into the ventricular space. Catheter
contact with the ventricular wall also increases astrocyte density close
to the interface between the wall and the shunt
catheter(33). Astrogliosis will occur when the cells
are exposed to blood following shunt insertion and subsequent breaking
of the BBB (20). In future work, we plan run a similar
experiment utilizing ependymal cells due to their importance in allowing
the activation of astrocytes due to VZ breakdown (14).
Astrocytic response is likely to be due to the bodies intuitive need to
repair itself following a trauma (19). Data presented
here indicates that the activation of astrocytes, due to blood,
increases proliferation of astrocytes following their exposure due to VZ
disruption causing the migration to the shunt surface(19). Cell exposure to blood products to represent
breaking of the BBB was done and showed an increase of cellular
attachment to the surface of the catheter around the holes(34)–(36). When comparing the average total cell
count for each sample type the DAPI stain, for cell nuclei, is 94.7±44.5
for the control and 392.0±317 for the blood exposed samples (Figure 4).
After running the Mann-Whitney test the p-value was <0.0001,
which indicates a significant difference between the two cell counts.
From this we can determine that the blood had a negative effect on the
cells causing an increase in catheter obstruction.
Investigation into the astrocytic response was analyzed by staining with
GFAP stains to visualize the astrocytes on the catheter. Comparisons
between the control and blood exposed samples show a significant
difference in GFAP expression (P <0.0001). These data
suggest that there is a potential effect from blood proteins, when added
into the media, enhances cell spread and/or cell reactivity. Astrocyte
activation occurs in response to stimuli in an effort to repair the
brain via proliferation and extension of astrocyte processes
[37,38]. Perhaps increased concentration and type of protein
adsorbed to the polydimethylsiloxane shunt surface enhances the
receptor-integrin interplay and increases cell attachment, not just in
cell number, but in cell affinity to the surface (Figure 5). Increased
GFAP expression after whole blood exposure may also be indicative of
enhanced neuroinflammation, cytoskeletal changes, and/or increased cell
communication. Since GFAP stains within the cytoplasm, it allows for a
single astrocyte to be stained multiple times. The software takes into
account intensity and signal diameter when analyzing the cell count, but
still can be added to the total count. This could be a methodological
explanation for the higher expression in comparison to DAPI. When
comparing the cell counts of the control and blood exposed samples of
the DAPI and GFAP stains, they were analyzed separately to account for
the antibody’s expression behaviors.
Cellular attachment to the surface of the shunt showed an insignificant
difference of DAPI and GFAP expression along the side of the catheter
compared to holes in direct contact to cells for the control samples
(P >0.05). The difference in DAPI signal of the blood
exposed sample was also insignificant as well, but GFAP expression
showed a significantly higher count around the row of holes along the
side of the catheter (P <0.05). One possible reason for
an insignificant difference is due to the removal of the catheter from
the culture. There could have been a potential ripping of cells from the
surface of the catheter upon extraction of the shunt. A significant
difference in GFAP expression of the blood exposed sample may further
indicate that a morphological change occurs following activation of
astrocytes.
Blood within the chamber contained no cells that would be positively
stained with GFAP, indicating that the expression is limited only to
astrocytes. The use of DAPI to stain the DNA within the nucleus is not
affected by the addition of blood due to the small amount of blood used
along with the majority of the components of blood not containing a
nucleus. Small amounts of cells within the blood that could have been
pick up by the DAPI stain were filtered out by setting a specific
diameter, such that the software only counts the astrocytic nuclei.