Dysregulation in myelination mediated by persistent neuroinflammation: possible mechanisms in chemotherapy-related cognitive impairment

Abstract

Cognitive impairment is commonly reported as a consequence of chemotherapy and can have considerable impact on everyday life on cancer patients. Thus, it is imperative to have a clear understanding of this phenomenon and the underlying mechanism involved. In the present study we examined the role of neuroinflammation and myelination in chemotherapy-related cognitive impairment. Female Sprague-Dawley rats (12-months old) were used in the study (total n=52, 13rats/group). Rats were randomly assigned to either the chemotherapy or saline control group. The drug combination of cyclophosphamide, methotrexate, and 5-fluorouracil (CMF) was given i.p. once a week for 4weeks. Rats in the control group received normal saline of equal volume. Animals from each group were further randomized to receive either: cyclooxygenase (COX-2) inhibitor, NS-393, to block the inflammatory response or vehicle. NS-398 was given at 10mg/kg i.p. and equal volume of saline (vehicle) was injected i.p. as vehicle. Both NS-398 and vehicle were injected 1h after the first CMF dose and then given daily for 28days then rats were tested in the Y maze. Our data showed that: (1) CMF led to the increase in the levels of inflammatory mediators IL-1β, TNF-α, and COX-2 while levels of the anti-inflammatory cytokine IL-10 decreased; (2) cognitive impairment and neuroinflammation resulting from CMF persisted 4weeks after the treatment ended; and (3) administration of NS-398 attenuated CMF-induced neuroinflammation and effects on myelin and cognitive impairment. These findings suggest the involvement of neuroinflammation in CMF-induced changes in myelin and myelination, and cognitive impairment.

Keywords: COX-2; Corpus callosum; IL-10; IL-1beta; TNF-alpha.

Figure 1. Camera Lucida drawing of the corpus callosum

The region sampled in the study for electron microscopy was the genu.

Figure 2. Expression of Inflammatory markers in the Corpus Callosum

ELISA showed significant increase in IL-1β (A), TNF-α (B), and COX-2 (C) after CMF treatment but administration of COX-2 inhibitors lessened the upregulation of these inflammatory mediators. ELISA also showed that CMF treatment resulted in decrease levels of the anti-inflammatory cytokine IL-10. No significant differences were seen in the saline-treated groups. Representative Western blot is shown on the upper panel (C). Legends: 1 = CMF/NS-398; 2 = CMF/vehicle; 3 = Saline/NS-398; 4 = Saline/vehicle. *p <0.05, **p <0.01.

Figure 3. Behavioral performance in the Y maze

Discrimination ratio in the novelty object test (A) showed no significant differences in all groups. However, CMF rats showed significant impairment in discriminating between the objects presented in the object-in-place test (B) and temporal order memory test (C) compared to the saline group but administration of NS-398 ameliorated the CMF-induced cognitive impairment. However, performance of the CMF-treated rats given NS-398 is still impaired in the objecy-in-place and temporal order memory task when compared to the saline group. No significant differences were seen in the performance of the saline groups. *p <0.05, **p <0.01.

Figure 4. O4 pre-myelinating cells and myelin basic protein (MBP) expression

CMF treatment resulted in decrease O4 (lower left) and MBP (lower right) immunoreactivities in the corpus callosum, but administration of NS-398 diminished the cytotoxic effects of the chemotherapy on O4 and MBP. However, CMF-treated rats that received NS-398 still showed a reduction in both OPCs and MBP compared to the saline-treated rats. Representative photomicrographs of O4 and MBP immunoreactions in the corpus callosum (upper panel). *p <0.05, **p <0.01. Scale bar = 20 μm.

Figure 5. Axon myelination and thickness

CMF treatment resulted in decrease area occupied by myelinated axons (A) and increased area occupied by unmyelinated axons (B) in the genu region of the corpus callosum, but administration of NS-398 diminished the cytotoxic effects of the chemotherapy on myelination. However, CMF-treated rats that received NS-398 still show decreased myelinated axons and increased unmyelinated axons compared to the saline-treated rats. No significant groups differences were seen in the saline-treated animals. Similar pattern of CMF and NS-398 effects were seen when myelin thickness was measured (D). Representative digital electron micrographs (C) showing myelinated axons (arrows) and unmyelinated axons (arrowhead). *p <0.05, **p <0.01. Scale bar = 1 μm.