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.