Using LPS to induce microglia activation ex vivo, we have previously demonstrated that conditioned microglia activation in rat CC resulted in white matter tract malfunction as illustrated by a reduction of CAP magnitude and an impairment of fast axon transport reflected by accumulation of β-APP . The extent of microglia activation was correlated to the alterations of CAP and fast axon transport of β-APP. Based on our previous experimental results  and the findings by others [31, 32], we further investigated and compared, using an in vivo semi-quantification test, the extent of white matter tract abnormalities with the states of microglia activation, focusing on the CC region. Our results showed that microglia activation induced by systemic administration of LPS produced white matter tract malfunction in the CC and minocycline, a semisynthetic tetracycline derivative, attenuated LPS-induced microglia activation and resultant white matter abnormalities in the CC.
Increasing evidence indicate that neuroinflammation provoked by microglia activation plays an important role in the pathogenesis of neurological disorders [6–12]. Using LPS to mimic an etiological factor to induce microglial activation in many neurological disorders is widely used in animal studies [33–35]. It has been shown that LPS at a dose of 1 mg/kg/day through i.p. delivery was not possible to impair blood brain barrier and enter the brain freely [33, 34]. In contrast, three consecutive deliveries were able to effectively activate microglia and fully activation was observed at the third day after systemic delivery [31, 32]. Using a three-day delivery protocol we did observe microglia activation and functional impairment in the CC. Our results were in a fully agreement with those observations made by others [31, 32].
In a recent study, the authors reported that systemic delivery of LPS for 3 days resulted in a significant malfunction of hippocampus and the CC nerve fibers with robust microglia activation in both brain regions . Three days post LPS delivery, hippocampal function largely recovered with a reduced microglia activation, while the CC nerve fibers malfunction exacerbated . Their results were in consistent with a prior report that activation of microglia was detected 8–24 h after systemic LPS delivery and explicit morphological change was visualized at the 3rd day following 1 or 2.5 mg/kg LPS injection . Noteworthy, comparison of microglia activation levels in the CC with that in the hippocampus at that time point showed a significant higher in the CC than that in hippocampus .
In the present work, we adopted a similar procedure to evoke microglia activation as employed by other researchers [31, 32] and examined potential alterations on white matter integrity and function in the CC. We observed that systemic administration of LPS induced microglia activation as determined by labeling Iba-1 positive microglial cells and the morphological changes as measured and assessed using a proportional area comparison [36, 37]. LPS-induced microglia activation was supported by elevated expression levels of inflammatory markers, such as iNOS and TNF-α. The observed changes associated with LPS-stimulated microglia activation include: 1) an interruption of the CC fibers linear integrity detected by diffusion tensor magnetic resonance imaging (DT-MRI), which can map movements of water molecules and image linear integrity of the white matter tract ; 2) a reduction on magnitude of electrical evoked axonal CAPs, implying for a functional change of the white matter tracts [17, 31]; 3) an accumulation of β-APP (which travels along axons through fast axon transportation), a well-accepted marker for white matter tracts injury [39, 40]. These alterations were partially rescued or significantly reversed by administration of minocycline. Besides, we analyzed the expression levels of myelin basic protein (MBP) and neurofilament (NF) by immunostaining and found no significant change on MBP and slightly reduction of NF, suggesting that degeneration or demyelination of nerve fibers was not ensued in the time course adopted in this study.
How systemically administered LPS enters the brain is not fully understood. A study using radiocarbon labeled LPS to examine how the LPS passes blood brain barrier (BBB) demonstrated that binding to LPS receptors on endothelium membrane is probably the predominant pathway through which the LPS enters the brain parenchyma . The LPS might bind to endothelial CD14 (cluster of differentiation 14) and/or toll-like receptors (TLR) 4 and 2 to cross the BBB, as expression levels of endothelial CD14 mRNAs were up-regulated shortly after LPS injection and overexpression of TLR mRNAs lasted for a longer time . Epithelial cells of the choroid plexus also express CD14 and their mRNAs in the epithelium were significantly elevated for much longer time than in BBB endothelium following system LPS treatment . Moreover, significant increment of pro-inflammatory immune cells crossing choroid plexus into the cerebrospinal fluid (CSF) is observed after system LPS [42, 43]. These reports imply the first delivery of LPS might ensue a little amount of LPS into the brain parenchyma. This implication was supported by experimental results that the concentration of LPS in the brain parenchyma was only 0.025% of the circulating LPS following a single injection of doses from 0.1 to 5 mg/kg . In addition, the first cytokine released into the circulation after stimulation of the immune system with LPS was TNF-α , suggesting that TNF-α might be the first pro-inflammatory cytokine entering the brain from the circulation following systemic LPS. However, a study in rats using isotope labeled TNF-α to monitor the rate of its crossing the BBB in both physiological condition and systemic LPS unveiled no significant change in up-taking rate for isotope labeled TNF-α between 4 and 24 h after administration (i.p.) of LPS . TNF receptors 1 and 2 on BBB endothelium transport TNF-α crossing BBB whenever the BBB is intact [46, 47] and their mRNA expression levels in the endothelium were up-regulated following LPS treatment but their receptor proteins exhibited no significant increase . This paradox may explain why no significant change in up-taking rate for isotope labeled TNF-α was detected after administration (i.p.) of LPS .
In light of aforementioned studies, we assume that: 1) the first LPS injection might prime resident microglia through limited amount of LPS and TNF-α entrance into the brain from circulation. The microglia in the CC immediately adjacent to the ventricles might be activated in an earlier time fashion, considering the pro-inflammatory monocytes, granulocytes and lymphocytes in the CSF are significantly increased [42, 43]. 2) the second injection of LPS might activate most of microglia and the third delivery further challenged microglia, leading to production of significant amount of pro-inflammatory cytokines including TNF-α, as reflected by our western blot results. 3) microglia were eventually agitated to become neurotoxic because up-regulated iNOS expression, as revealed by our western blot results, is a sign for detrimental microglia activation [48, 49]. In this case, the observed malfunction of the white matter tract was most likely due to activated microglia since our proportional area measurement of Iba-1 immunoreactivity  displayed a positive correlation between microglia activation and severity of white matter malfunction. The direct adverse effect of LPS and/or circulating TNF-α on white matter tract, if any, could be ignorable.
It has been shown that systemic administration of LPS and over activated microglia affected both grey matter in the hippocampus and white matter in the CC, with prolonged adverse effect on the CC . The mechanism underlying such a difference is still unclear. Based on our recent findings that microglia pseudopodia directly contact node of Ranvier or node like sodium channel clusters on the CC nerve fibers, it might be possible that activation of microglia changed the contacting ratio and pattern between pseudopodia and node like structures . In addition, large CC area abutting to CSF in the ventricles may facilitate interaction between pro-inflammatory substances in the CSF and the microglia on the CC border [42, 43]. Nevertheless, the present in vivo studies demonstrated that activation of microglia had a noxious effect in the CC. Such effect might be ensued as a significant increment of iNOS, an established sign for detrimental microglia activation [48, 49]. In parallel, malfunction of the CC nerve fibers was reflected by interruption of fiber bundle linear integrity as detected by DT-MIR, reduction of CC nerve fibers CAP magnitude and accumulation (decelerated transportation) of β-APP through axons in the CC.