The effect of A1 and A2 reactive astrocyte expression on hydrocephalus shunt failure

Abstract

Background: The composition of tissue obstructing neuroprosthetic devices is largely composed of inflammatory cells with a significant astrocyte component. In a first-of-its-kind study, we profile the astrocyte phenotypes present on hydrocephalus shunts.

Methods: qPCR and RNA in-situ hybridization were used to quantify pro-inflammatory (A1) and anti-inflammatory (A2) reactive astrocyte phenotypes by analyzing C3 and EMP1 genes, respectively. Additionally, CSF cytokine levels were quantified using ELISA. In an in vitro model of astrocyte growth on shunts, different cytokines were used to prevent the activation of resting astrocytes into the A1 and A2 phenotypes. Obstructed and non-obstructed shunts were characterized based on the degree of actual tissue blockage on the shunt surface instead of clinical diagnosis.

Results: The results showed a heterogeneous population of A1 and A2 reactive astrocytes on the shunts with obstructed shunts having a significantly higher proportion of A2 astrocytes compared to non-obstructed shunts. In addition, the pro-A2 cytokine IL-6 inducing proliferation of astrocytes was found at higher concentrations among CSF from obstructed samples. Consequently, in the in vitro model of astrocyte growth on shunts, cytokine neutralizing antibodies were used to prevent activation of resting astrocytes into the A1 and A2 phenotypes which resulted in a significant reduction in both A1 and A2 growth.

Conclusions: Therefore, targeting cytokines involved with astrocyte A1 and A2 activation is a promising intervention aimed to prevent shunt obstruction.

Keywords: A1 and A2 reactive astrocyte phenotype; Glial Scar; Hydrocephalus; Neuroprosthetic device failure; Targeted drug delivery.

Fig. 1

Expression of C3, EMP1 astrocyte activation genes assessed by qPCR on obstructed and non-obstructed shunts. A Comparing the expression of the A2-specific gene EMP1 and the A1-specific gene C3 to the housekeeper gene hRPLP0 in every single patient for obstructed and non-obstructed shunts. Statistical analysis for fold changes was carried out using two-way ANOVA; *p < 0.05. B Representative images for obstructed and non-obstructed shunts collected from patients. Obstructed and non-obstructed shunts were characterized based on the degree of actual tissue blockage on the shunt surface instead of clinical diagnose

Fig. 2

Expression of C3, EMP1 astrocyte activation genes assessed by RNAscope fluorescent in situ hybridization on obstructed and non-obstructed shunts. A SLC1A3 was used as an astrocyte marker to confirm that C3 and EMP1 signal represented the A1 and A2 astrocytes specifically. Data for obstructed and non-obstructed shunts are shown. For normalization, the C3 and EMP1 signals were dividing by SLC1A3 signals. Statistical analysis was carried out using two-way ANOVA; *p < 0.05. B Representative RNAscope fluorescent in situ hybridization images for obstructed and non-obstructed astrocyte gene C3 (red) and EMP1(yellow) showing colocalization with the astrocyte marker SLC1A3 (green). Separated channels for C3 and EMP1 for each condition are also presented (scale bar = 500 μm)

Fig. 3

Cerebrospinal fluid cytokine concentrations for obstructed and non-obstructed shunts. Analytes include C3, C1q, and IL-1α (A1 astrocyte markers), IL-1β and IL-6 (A2 astrocyte markers), TNF-α, IL-8 and IL-10. For normalization, the concentration of each cytokine is divided by the total protein concentration for each group. Statistical analysis was carried out using Mann Whitney U test; p > 0.05 (n = 10 per group, mean ± SEM)

Fig. 4

Targeted therapies that inhibit cell activation to reduce adhesion on the shunt surface. A A1 reactive astrocytes treated with neutralizing antibodies to TNF-α, IL-1α, and anti-inflammatory cytokine TGF-β. B A2 reactive astrocytes treated with neutralizing antibodies to TNF-α, IL-1β, and IL-6. Statistical analysis was carried out using two-tailed unpaired Student’s t-test; *p < 0.05 (n = 3 per group, mean ± SEM). Cell count covering each well of a 24-well plate with a surface area of 1.9 cm² is measured