In the present work, we explored the effects of CX3CR1 or CCR2 deficiency on normal and inflammatory states of the CC WM, informed by how absence of CX3CR1 or CCR2 modifies the infiltration of circulating immune cells into the CC and influences the microglial activation. Furthermore, we examined whether the absence of either receptor would alter the neurophysiological properties of the CC WM tract in healthy and LPS treated littermates. As mentioned in introduction, we intended to clarify which chemokine and receptor would dominate the infiltration of circulating immune cells into the CC following systemic LPS. We applied immunostaining of CD45 plus MPO to identify the infiltrating immune cells due to 1) CD45 is a leukocyte common marker that anchors on the majority of circulating immune cells [30, 31], and has been frequently used to discriminate infiltrating and resident macrophages [37, 38]; 2) murine monocytes express most CD45 isoforms [31], and 3) MPO labels all neutrophils and is used to distinguish monocytes from neutrophils [39]. The result of this research was contrary to our hypothesis that was based on a line of previous studies [24, 25, 40]. It is well-documented in the existing literatures that CCR2 equipped circulating monocytes are inflammatory and are predominantly recruited by inflamed tissue [24, 25, 40]; though, a few studies implicated CX3CR1high myeloid cells as inflammatory mediators equivalent to the CCR2high monocytes [41, 42]. Accordingly, the number of infiltrating circulating immune cells would be significantly elevated in the CC of CCR2+/+CX3CR1-/- mice following systemic LPS if the CCR2 equipped monocytes are predominantly recruited. But instead, the number of infiltrating cells in the CC of CX3CR1+/+CCR2-/- mice was nearly significantly increased following systemic LPS, which is similar to the result from WT mice that normally express both receptors (see Fig. 5A left and middle).
Meanwhile, both CX3CR1 and CCR2 mediated microglia activation have been described in a number of previous studies. For instance, the CX3CL1-CX3CR1 pathway was documented to be engaged in detrimental microglia activation in the ischemic brain [28, 43], epilepsy, and some neurodegenerative diseases [26, 44]. While, the CCL2-CCR2 axis was shown to control microglia activation in a traumatic brain injury [45], in an intracerebral hemorrhage model [27] and in circumstances of systemic LPS [46]. Hence, we explored the effect of either CX3CR1 or CCR2 deficiency on microglia activation in the CC WM following systemic LPS. It appears that the CX3CR1 plays a more significant role than CCR2 does to activate microglia in the inflamed CC, since our data showed that Iba-1 positive proportional area in the CC was significantly increased in WT and CX3CR1+/+CCR2-/- mice (see Fig. 5C left and middle) after LPS challenge, but not increased in CCR2+/+CX3CR1-/-mice (5C right). In the normal condition without endotoxemia, the CC microglial proportional area values in either receptor deleted mice was evidently lower, reached significance in CCR2+/+CX3CR1-/- mice comparing to the value in WT mice (see Fig. 5B left). This fact suggests that lack of CX3CR1 may interrupt microglial migration or proliferation before the CC is inflamed or possibly during the brain development. Nevertheless, the CCR2 may also facilitate migration, proliferation and activation of the microglia, as our data exhibited that 1) the CC microglial proportional area in both CX3CR1+/+CCR2-/- and CCR2+/+CX3CR1-/- mice was lower than that in WT mice (see Fig. 5B left); 2) the microglial proportional area in the absence of either receptor was significantly less expanded following systemic LPS, comparing to the conspicuous enlargement of proportional area in LPS treated WT mice (see Fig. 5B right).
Then, we intended to elucidate whether and how the inflammatory states, as described above, would disturb the action potential conduction through the CC WM tract. We firstly examined whether deficiency of CX3CR1 or CCR2 would affect the CC WM tract function without LPS challenge, since this data was not found through a literature search. Generally, it looked like absence of these receptors impacted the myelinated axon bundle more prominently than it impacted unmyelinated fibers (see Figs. 6 and 7). Meanwhile, the overall CC axon responsiveness, indexed by I/O curves, in either CX3CR1 or CCR2 deficiency mice was not significantly different from that in the WT mice (see Fig. 6A, B; Fig. 7A, B). The myelinated fibers’ CAP recorded from normal CCR2+/+CX3CR1-/- mice was significantly lower at 0.40 and 0.45 mA stimulation intensity compared to the CAP recorded from the CC of naïve WT mice (see Fig. 6B). In inflammatory states, the CC neurophysiological property of both myelinated and unmyelinated fibers exhibited an observable difference in the CX3CR1+/+CCR2-/- mice comparing to the normal ones (see Fig. 6C, and 7C). In these littermates, the overall down-shifting of both N1 and N2 I/O curves was not significant indicated by ANOVA analysis of AUC; whereas the decline of CAP by LPS challenge was significant at 0.40–0.50 mA stimulation points in the N1 component of CAP (Fig. 6C), and at 0.5 mA in the N2 component of CAP (Fig. 7C). These kinds of neurophysiological variations in reaction to systemic LPS, or to neuroinflammation, were not identified in CCR2+/+CX3CR1-/- mice (as shown in Fig. 6D and 7D).
In combining with our morphological data, we think that this difference was due to the CX3CR1 being expressed and activated, as we have observed more circulating immune cells being recruited into the CC following systemic LPS if the CX3CR1 is normally expressed (see Fig. 5A). Furthermore, the presence of the CX3CR1 would also facilitate microglia activation after systemic LPS challenge, but if CX3CR1 was knocked out with only the CCR2 equipped, the increment of microglia proportional area by LPS treatment would not become significance (Fig. 5C). Correspondingly, these inflammatory state variations would probably impact the neurophysiological property of the CC WM tract (see Fig. 6C and 7C). A line of previous published data inferred that pro-inflammatory cytokines, released by infiltrating immune cells and/or activated microglia, play a significant role to compromise brain function and/or basic integrities [44, 47–49]. For examples, it was documented that tumor necrosis factor-alpha (TNF-α), interleukin-1 beta and interleukin-6 would affect neuronal excitability and induce epilepsy [44, 48], or participate in the modulation of synaptic plasticity and long-term potentiation during neuroinflammatory states [6, 49]. These pro-inflammatory cytokines would also interrupt integrity of the blood-brain barrier, which subsequently increases influx of neurotoxic elements into the parenchyma from the systemic circulation [44, 47]. Direct effects of pro-inflammatory cytokines on axonal conduction or integrity of WM tract has been less explored; nonetheless, several studies have revealed that TNF-α and interferon-gamma facilitated myelin antigens or cuprizone triggered demyelination [50, 51].
More significantly, Zhang and colleagues has demonstrated the direct contacts of microglial pseudopodia upon Ranvier’s node, paranodal myelin sheath, and node-like clusters of sodium channels by means of confocal and electronic microscopy [52]. In light of this finding and other previous studies, these direct contacts and/or close apposition of microglial pseudopodia upon Ranvier’s node, paranodal myelin sheath, and node-like sodium channel clusters might play a role in monitoring and modulating potassium and calcium ion concentration around the node [52–55]. It is well-known that nodes of Ranvier and myelin sheath are key structures for generation of saltatory conduction of action potentials [56], and electrolytic homeostasis surrounding the node or node-like cluster maintains appropriate excitability of the node and sustains saltatory conduction [56, 57]. Under inflammatory conditions, stretching of microglial pseudopodia would be dramatically distorted, constituting a striking morphological change from delicate ramified resting microglia to hypertrophic and hyper-ramified activated microglia [58] (see Fig. 4). Consequently, the contacts of microglial pseudopodia on the node and/or paranode would be dramatically altered, which might disturb microglia monitory function and break the balance of potassium and calcium surrounding the node. This could result in interruption of action potential conduction through the CC axonal bundles, especially the conduction through myelinated nerve fibers. In the optic nerve, the degree of fiber bundle CAP declines had been verified to be significantly correlated with the extent of nerve fiber injuries identified by diffusion tensor imaging and immunohistochemistry staining [59]. Taking these data into account, it is rational to conclude that the existence or absence of CX3CR1 on microglia influence WM tract neurophysiological properties, and potentially lead a step closer to associating the WM tract disruption with cognitive problems [10].
In summary, in the present work, we have demonstrated: 1) the CX3CR1 is more significantly dominant in the infiltration of circulating immune cells into the CC WM following systemic LPS, without excluding the similar function of the CCR2; 2) the CX3XR1 plays more significant role than the CCR2 does to mediate activation of microglia in the CC WM in circumstances of endotoxemia; 3) absence of either CX3CR1 or CCR2 reduces the density of microglia in the CC of normal mice, with a significant reduction in the CC of CX3CR1 deficient ones; 4) endotoxemia induces a downshift of N1 I/O curve plotted with CAP recorded from the CC of CX3CR1+/+CCR2-/-mice at an approximately significant level, and a slight downshift of N2 I/O curve as well, which is not found in CCR2+/+ CX3CR1-/- mice; 5) a potential cause for more detrimental effects of systemic LPS on myelinated nerve fibers is discussed, which is probably due to LPS treatment changed interacting states between microglial pseudopodia and Ranvier’s node or node like sodium channel clusters; 6) the association of CX3CR1 equipped microglia with the CC WM tract malfunction, and with the potential risk of cognitive problems is proposed.