Fibrinogen contributes to myelin deficit and cognitive impairment in aged mice after anesthesia and surgery

Abstract

Perioperative neurocognitive disorder (PND) is a common complication of anesthesia and surgery, which is more prevalent in elderly patients. Fibrinogen is known to contribute to the pathophysiology of neurodegenerative disorders. This study investigated whether fibrinogen induces myelin deficit and cognitive impairment in aged mice after anesthesia and surgery. Here, abdominal surgery was performed on 17-month-old C57BL/6 mice to establish a PND model. Following anesthesia and surgery, cognitive function and exploratory locomotion of mice were assessed using behavioral tests. We used in vivo two-photon brain microscopy to track the perivascular accumulation of blood-derived fibrinogen in the central nervous system (CNS). Immunostaining, electron microscopy (EM), and western blotting were used to measure myelin sheath density and oligodendrocyte alterations, and inflammatory markers were determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). In the current study, we found that fibrinogen deposited in the CNS after blood-brain barrier (BBB) disruption, induces oligodendrocyte loss, myelin deficits and causes behavioral abnormalities in PND model. Fibrinogen depletion could reverse myelin deficits and cognitive function which induced by anesthesia and surgery. In summary, our data support that fibrinogen is a key determinant in the early pathogenesis of PND.

Keywords: Perioperative neurocognitive disorder; blood-brain barrier; fibrinogen; myelination; oligodendrocyte.

Figure 1.

Fibrinogen extravasation following BBB disruption in aged mice after anesthesia and surgery. (a) Longitudinal in vivo two-photon brain microscopy imaging of fibrinogen (Alexa594-labeled fibrinogen, red) in the cortex of 15-month-old mice. Images were acquired in the same cortical area before anesthesia and surgery and 0, 6, 24, 48 h after anesthesia and surgery, n = 3 per group. Scale bar: 425.10 μm. (b–c) Lectin-positive endothelial profiles (green) and extravascular fibrinogen deposits (red) in the hippocampus (b) and cortex (c) of mice, n = 6 per group. (d) Quantification of fibrinogen deposits in the hippocampus and cortex of mice. (e) mRNA levels of Claudin-5 and Occludin at 24 hours after anesthesia and surgery in hippocampus and cortex, n = 3–5 per group and (f) blots show Claudin-5 and Occludin of mice in the hippocampus, n = 3–5 per group. (g) The relative protein expression levels of Claudin-5 and Occludin were normalized to β-actin and (h) mRNA levels of IL-1β and TNF-α at 24 hours after anesthesia and surgery in hippocampus and cortex. Data are presented as mean ± standard deviation (SD). All experiments were repeated three times.

Figure 2.

Myelin deficits and loss of oligodendrocytes after anesthesia and surgery in aged mice. (a–b) Representative images of MBP+ (red) area in the hippocampus (a) and cortex (b), n = 6 per group. Scale bar represents 100 μm. (c) Quantification of MBP intensity in the hippocampus and cortex of mice. (d) mRNA levels of MBP in the hippocampus, n = 3–5 per group. (e) Blots show MBP of mice in the hippocampus, n = 3–5 per group. (f) The MBP expression level was normalized to β-actin. (g–i) Electron micrographs and quantification of myelinated axons in the corpus callosum of mice, n = 3 per group. (j–k) Confocal images of Olig2, CC1 and DAPI in the corpus callosum (j) and cortex (k) of mice, n = 6 per group. Scale bar represents 50 µm and (l) quantification showing the percent of CC1+ cells among Olig2 cells in corpus callosum and cortex. Data are presented as mean ± standard deviation (SD). All experiments were repeated three times.

Figure 3.

Fibrinogen depletion mitigates myelin deficits after anesthesia and surgery in aged mice. (a) Study design and endpoints. (b) Plasma fibrinogen levels in mice, n = 4 per group. (c–d) Lectin-positive endothelial profiles (green) and extravascular fibrinogen deposits (red) in the hippocampus (c) and cortex (d) of mice, n = 6 per group and (e) quantification of fibrinogen deposits in the hippocampus and cortex of mice. Data are presented as mean ± standard deviation (SD). All experiments were repeated three times.

Figure 4.

Fibrinogen depletion mitigates myelin deficits after anesthesia and surgery in aged mice. (a) Blots show MBP of mice in the hippocampus, n = 3–5 per group. (b) The MBP expression level was normalized to β-actin. (c) mRNA levels of MBP in the hippocampus, n = 3–5 per group. (d–e) Representative images of MBP+ (red) area in the hippocampus (d) and cortex (e), n = 6 per group. Scale bar represents 100 μm. (f) Quantification of MBP intensity in the hippocampus and cortex of mice and (g–i) electron micrographs and quantification of myelinated axons in the corpus callosum of mice, n = 3 per group. Data are presented as mean ± standard deviation (SD). All experiments were repeated three times.

Figure 5.

Fibrinogen depletion mitigates myelin deficits after anesthesia and surgery in aged mice. (a–b) Confocal images of Olig2, CC1 and DAPI in the corpus callosum (a) and cortex (b) of mice, n = 6 per group. Scale bar represents 50 µm and (c) quantification showing the percent of CC1+ cells among Olig2 cells in corpus callosum and cortex. Data are presented as mean ± standard deviation (SD). All experiments were repeated three times.

Figure 6.

Fibrinogen depletion rescues cognitive decline after anesthesia and surgery in aged mice. (a) Representative movement traces of open field, n = 8–10 per group. (b) Total distance in the open field test. (c) Novel object recognition index of mice, n = 8–10 per group. (d) Representative movement traces on day 5 of water maze, n = 8–10 per group. (e–g) Water maze test revealed the latency to platform in the acquisition phase (e), their number of crossing platform quadrant (f), time in target sector (g). Data are presented as mean ± standard deviation (SD).

Figure 7.

Working model for the role of fibrinogen in CNS myelin deficits in PND. Fibrinogen enters the CNS after BBB disruption and has pleiotropic effects on suppressing of oligodendrocyte maturation and myelin deficits.