Fig 1.
Sepsis survivor mice do not show a deficit in contextual fear conditioning or trace fear conditioning 50 days after CLP, but do demonstrate impaired extinction of conditioned fear.
A: In a contextual fear conditioning paradigm, mice were placed in a defined context for 180 s (white bar) at rest. A tone was played for 30 s (gray bar), followed immediately by a foot shock (star). In the trace fear conditioning paradigm, a 30 s delay is introduced after the tone and before the administration of the foot shock. B: In contextual fear conditioning, there is no difference between the rate at which sepsis survivor and control mice associated the freezing response with the context. C: After three days of training, there was no difference in the response of post-CLP and control mice to the tone alone in a novel context. D: There was no difference among post-CLP and control mice in the freezing response to the conditioned context. E-G. In the trace fear conditioning paradigm, there were also no differences among groups in the acquisition of the freezing response, response to tone, or response to context. H. Post-CLP mice demonstrate increased freezing in the conditioned context compared to sham-operated and unoperated mice during repeated exposures without foot shock, indicated a deficit in extinction learning.
Fig 2.
CLP does not result in significant changes to the structure of the hippocampus.
There is no difference in the morphology of the hippocampus in 50 days after CLP (C) compared to 50 days after sham operation (B) or in age matched unoperated controls (A). There is no difference in the volume of the ventral stratum pyramidale of the cornu ammonis (D). The lack of change in hippocampal volume 50 days after CLP is concordant with the absence of FluoroJade staining, which necrotic or apoptotic cell death, 14 days after CLP (E). Positive FluoroJade staining in the hippocampus after status epilepticus is shown for comparison (F). Golgi staining was used to determine synaptic density on pyramidal cell dendrites of the dentate gyrus (G). There was no measurable difference among sham operated and post-CLP mice (H).
Fig 3.
IgG and C1q deposition in the brain after CLP.
Immunofluorescent staining for total mouse IgG does not reveal any tissue deposition in unoperated mice (A). In sham operated mice, there are sparse, focal areas of IgG immunoreactivity (B). After CLP, large areas of IgG immunoreactivity are present in a perivascular pattern (C). There is increased immunoreactivity for complement component C1q in the dentate gyrus in after CLP (arrows, E) compared to unoperated control mice (D, F).
Fig 4.
CLP results in long-lasting myeloid inflammation in the brain.
A: 14 days after operation, an increased proportion of CD11b+/ CD45hi cells are observed in the brains of post-CLP mice compared to sham controls. B: CD11b+/CD45hi cells are divided into Ly6G+/Ly6Cmid and Ly6G-/Ly6Chi subsets, corresponding to PMN and monocytes (left). Monocytes also express CCR2, while PMN do not (middle). Neither subset of CD11b+/CD45hi cells express CX3CR1 (right, dashed line indicates isotype control intensity). C: CD11b+/CD45mid cells (left, dotted circle) express the microglial markers CX3CR1 and CD64 (middle) and do not express CCR2 (right). D: Five days after CLP (filled squares) or sham operation (open circles), both groups demonstrate a small increase in PMN (left) and monocytes (right) compared to unoperated controls (gray line). The proportion of PMN and monocytes increase at 14 days in post-CLP mice, however, while decreasing in sham operated controls. By 50 days after CLP, the proportion of PMN and monocytes decreases nearly to baseline.
Fig 5.
CLP results in patchy perivascular infiltration by monocytes.
Unoperated mice do not demonstrate any CCR2 immunoreactivity in the brain (A). 14 days after sham operation, sham operated mice demonstrate sparse perivascular CCR2 immunoreactivity (B). CLP operated mice, however, demonstrate multiple cortical and subcortical dense patches of CCR2 immunoreactivity (C, white arrows). Highly immunoreactive cells include both elongated perivascular morphologies (E), extravasating cells (F), and punctate perivascular cells (G), all consistent with monocytes. Notable perivascular immunoreactivity is present in isotype stained tissue, likely reflecting cross-reactivity with nonspecific IgG deposition (E, inset).
Fig 6.
Gene expression measured from whole brain homogenates.
Bars indicate differences with p<0.05 when corrected by the Bonferroni criterion for multiple comparisons.
Fig 7.
CNS myeloid inflammation is independent of peripheral monocytosis and CCR2 signaling.
There was no change in the proportion of brain monocytes in CCR2-/- mice after CLP (A). In contrast, CCR2 deficiency markedly decreases blood monocytosis (B). CCR2 deficiency has no effect on CNS (C) or blood (D) neutrophilia, as PMN do not express CCR2.
Fig 8.
Microglia after CLP demonstrate distinct transcriptional differences, despite absence of morphological signs of activation.
There is no difference in intensity of Iba1 immunoreactivity in somatosensory cortex among unoperated (A), sham operated (B) and post-CLP mice (C) 14 days after operation (E). There is also no difference in Iba1 immunoreactivity of microglia in regions of somatosensory cortex with evidence of vasculopathy (IgG deposition, D) compared to regions without IgG immunoreactivity (C, F). Despite the absence of activated amoeboid morphology, microglia in 14 day post-CLP mice demonstrate a distinct transcriptional profile compared to both sham operated and unoperated controls (G).