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Turner et al. LPS preconditioning neuroprotective

because it undergoes robust biochemical changes GFAP expression in the cortex differed significantly
following impact-acceleration TBI. [19] The software between experimental groups (F 3,32 = 57.92; P <
selected random 75-µm counting frames with 0.001) [Figure 3]. LPS preconditioning significantly
a depth of 6 µm, and the object of interest was reduced GFAP levels after TBI according to one-
marked by an investigator blinded to treatment. The way ANOVA and post-hoc Tukey’s test (q = 8.70,
region of interest volume was previously identified, P < 0.001). No difference was observed between
and the number of cells marked by the observer was sham-injured and LPS-treated animals (q = 2.89, P
quantified. > 0.05), indicating that peripheral administration of
LPS did not activate astrocytes.
Statistical analysis
Data were analyzed using GraphPad Prism version LPS preconditioning reduces M1 microglia
4.0. One-way ANOVA with post-hoc Tukey’s test activation after TBI
was used to compare histological findings across To investigate the effect of LPS preconditioning on
control and various experimental groups. Repeated classically activated microglia, CD68 expression
measures two-way ANOVA was used to analyze was quantified by stereology. Figure 4 shows a
the total activity data. An overlap coefficient of significant difference in CD68 expression between
0.6 or greater indicated strong co-localization, a experimental groups (F 3,32 = 28.22; P < 0.001).
coefficient between 0.4 and 0.6 indicated medium The presence of M1 microglia (CD68 expression)
co-localization, and a coefficient < 0.4 indicated was significantly reduced after TBI following LPS
weak co-localization. A P value of < 0.05 was preconditioning compared with no LPS pretreatment
considered statistically significant for all studies. according to one-way ANOVA with post-hoc Tukey’s
test (q = 9.77, P < 0.001). Importantly, no significant
RESULTS differences in CD68 expression were observed
between injured animals with LPS preconditioning
LPS induces transient acute sickness and sham-injured animals (q = 2.121, P > 0.05).
behavior These findings were consistent with IBA-1 staining
LPS injection induces systemic sickness in rodents for undifferentiated microglia, which showed a
as evidenced by an acute reduction in activity. [10,20] qualitative reduction in LPS pre-conditioned animals
Figure 1 shows a significant reduction in total activity [Figure 5].
after LPS administration compared with saline-
treated animals based on time (F 3,66 = 8.14, P < LPS preconditioning reduces OSMR
0.001), treatment (F 1,66 = 18.67, P < 0.001), and the expression in astrocytes after TBI
time treatment interaction (F 3,66 = 22.92, P < 0.001) One of the primary mechanisms for regulating
using two-way repeated measures ANOVA. Total astrocyte activation is neuropoietic cytokine signaling
[21]
activity was reduced by approximately 71% and through the gp130 receptor-signaling complex. TBI
59% at 2 and 4 h post-injection, respectively. This associated with significant vascular injury upregulates
was resolved within 24 h, indicating that sickness members of the neuropoietic cytokine family, including
behavior was acute.

LPS preconditioning reduces neuronal
degeneration and glial activation following
TBI
Previous studies have demonstrated a
neuroprotective effect of LPS preconditioning
following controlled cortical impact injury with
large vascular insult. Figure 2 shows a significant
[2]
difference in cortical FJB expression between
experimental groups (F 3,32 = 59.79; P < 0.001). LPS
preconditioning significantly reduced FJB levels
following TBI according to one-way ANOVA with
Tukey’s post-hoc test (q = 8.50, P < 0.001). No
difference was observed between sham-injured and
LPS-treated animals (q = 0.13, P > 0.05), indicating Figure 1: Total locomotor activity after LPS injection by number of
beam breaks at 2, 4, 24, and 48 h after injection. Acute sickness
that peripheral administration of LPS did not induce was present at 2 and 4 h but resolved by 24 h. ***P < 0.001. LPS:
neurodegeneration. lipopolysaccharide
Neuroimmunology and Neuroinflammation ¦ Volume 4 ¦ January 20, 2017 9