MRNA assessed by realtime PCR

Figure 7.1: Real-time mRNA for various cytokines/CD200 in the brain of groups of young/aged BL/6 mice before and after injection of LPS (5 p,g/mouse) ip. Data show mean ± SD for five mice per group. All mRNAs are expressed relative to a composite control for three housekeeping genes, GAPDH, RpL13A, ribosomal protein L13a, and CypA, cyclophlinA (Lee, Manuel, and Gorczynski 2006).

It is clear from these data that under basal conditions aged mice express less CD200 in the CNS, and comparatively more of the inflammatory cytokines IL-1P, TNF-a, IL-6, and TGFP than their young counterparts, as might be predicted from the anti-inflammatory regulatory role postulated for CD200. Interestingly, following peripheral LPS administration, dramatic increases in expression of CD200 and the aforementioned cytokines was observed for young mice (ranging from 4- to 10-fold increases), but not in the aged animals. Both the altered basal levels of cytokines, and the altered response to LPS stimulation, is consistent with other data reported in the literature, and may help explain in part the disrupted sleep patterns in elderly animals. Equally striking however, were the effects observed when we repeated the same studies in groups of mice previously treated chronically (4 weekly injections) with CLP (Fig. 7.2).

Comparison of expression of various mRNAs in brain of young (12wk) or aged (84wk) BL/6 mice following im injections with 100^g CLP (x4wk) and 2d after 5^g/mouse LPS (ip)

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IZZlYoung-PBS

IZZlYoung-PBS

Figure 7.2. Real time mRNA with brain tissue of groups of young/aged BL/6 mice before and after injection of LPS (5 p,g/mouse) ip (see also Fig. 7.1). Unlike the data in Fig. 7.1, all mice were pretreated for 4 weeks with either saline (PBS) ip or CLP (100 p,g/mouse im). LPS was given 7 days after the last CLP injection.

TNIfr Il-1 p IL-6 CD200 TGFp mRNA assessed by real-time PCR

Figure 7.2. Real time mRNA with brain tissue of groups of young/aged BL/6 mice before and after injection of LPS (5 p,g/mouse) ip (see also Fig. 7.1). Unlike the data in Fig. 7.1, all mice were pretreated for 4 weeks with either saline (PBS) ip or CLP (100 p,g/mouse im). LPS was given 7 days after the last CLP injection.

In this case, CLP produced little change in the LPS stimulated levels of CD200 or cytokine mRNAs in young mice when compared with PBS injected control young mice. However, a clear effect was seen when CLP was given to aged mice in comparison to PBS controls. Following CLP injection the response (to LPS) was restored such that in these animals LPS trigger significantly more marked increases in CD200 and the inflammatory cytokines shown above compared with controls. One model to explain these data suggests that CD200 is an important regulator of inflammation in the CNS, and thus the decline in expression of CD200 with age results in increased endogenous inflammatory cytokine production, with resultant sleep disruption (including fragmented sleep). CLP injections, by reversing the decline in CD200 expression, simultaneously restores the pattern of endogenous cytokine production to that more typical of younger animals, and similarly (we predict) restores normal sleep physiology. This hypothesis implies a potentially important effect for peripheral CLP injections on both sleep and CNS inflammation.

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