Effects of acute and repeated exposure to lipopolysaccharide on cytokine and corticosterone production during remyelination

Abstract

Chronic exposure to the copper-chelating agent, cuprizone (CPZ), is an increasingly popular model for producing demyelination. More importantly, cessation of cuprizone exposure allows for full remyelination, which represents a window of opportunity for determining the influence of environmental factors on regenerative processes. In the present study, CPZ-treated animals were assessed for functional status of systemic and central cytokine responsiveness to LPS, as well as assessment for signs of body weight changes. Exposure of male C57BL/6J mice to 5 weeks of 0.2% CPZ in the diet was optimal in producing demyelination and microglial activation, as measured by myelin basic protein, CD11b, and CD45 immunohistochemistry. Acute challenge with LPS at the end of 5 weeks CPZ treatment did not alter IL-1beta, IL-6, nor TNFalpha responses in the spleen and corpus callosum. Similarly, repeated exposure to LPS during the remyelination phase (CPZ removal) did not influence these measures to LPS. Plasma corticosterone was unaffected following acute challenge of CPZ-pretreated animals, but after repeated LPS treatment, there was a significant augmentation of the corticosterone response in CPZ-pretreated mice. Interestingly, the basal concentration of IL-1beta in the corpus callosum of CPZ treated animals was significantly increased, which was in keeping with the increase in activated microglial cells. In conclusion, the cuprizone model of demyelination and remyelination does not appear to influence the systemic nor central IL-1, IL-6, and TNF responses to acute nor repeated LPS. This opens up the possibility for studying the contribution of systemic inflammatory processes on remyelination after cessation of CPZ treatment.

Fig. 1

Summary of experimental design. Animals were given 0.2% cuprizone (CPZ) or control feed for 5 weeks to induce demyelination. After that point, animals were either sacrificed after an acute injection of LPS or saline (Expt 1), or allowed to remyelinate for a two week period throughout which they were given injections of LPS or saline every three days (Expt 2). Arrows represent days of sacrifice, which was 2 hrs after injection.

Fig. 2

Immunohistochemistry for Myelin Basic Protein (MBP). (a). Representative overview panel of the corpus callosum (CC) at 4X magnification. The large arrow points to the region of the CC assessed in this study. (b–g). High magnification (20X) insets of the CC. (b) Figure 2b shows extensive MBP immunoreactivity, as indicated by the brown staining in control animals not fed CPZ. (c) After three weeks on a CPZ-containing diet, MBP staining is still evident, but is dramatically diminished after five weeks on the CPZ diet (see panel d). However, once CPZ was removed from the diet, MBP staining in 5-week fed animals reappeared (see panel f), being comparable to the control. (e and g) isotype controls for nonspecific immunoreactivity; note the darker staining of the green counterstain for the 5 wk CPZ treated isotype control. This is due to hypercellularity of the CC during demyelination, and probably due to microglial/macrophage accumulation at the site of injury (eg., Figures 4 and 5).

Fig. 3

(a) Percent of baseline weight. Animals that received LPS showed a decline in the percent of baseline body weight throughout the experiment compared to their starting weight at day 0 (N = 6/group). Arrows indicate day of LPS treatment. (b) Percent of body weight measured at the time of each LPS or Saline injection. The CPZ-fed and control animals that received LPS continued to display a reduction in weight after each injection (N = 6/group). Error bars represent standard error of the mean.

Fig. 4

Representative images of immunohistochemistry for CD11b. (a) Representative overview panel of the CC at 4X magnification. The large arrow shows area of detail where numbers of heavily stained CD11b⁺ cells were observed and quantified (see Figure 6). The panels show CD 11b immunoreactive cells (arrows) in the CC of animals fed CPZ for 5 weeks (b) or taken off CPZ at the end of 5 weeks feeding and perfused after one (c) or two (d) weeks. (e) Representative immunohistochemistry conducted on brains from mice fed a diet that did not contain CPZ. (f and g) Isotype controls for nonspecific immunoreactivity.

Fig. 5

Representative images of immunohistochemistry for CD45. See Figure 4 for details.

Fig. 6

Numbers of CD11b⁺ and CD45⁺ cells in the corpus callosum. Area of detail where counts were obtained corresponds to that shown in panel A of Figures 4 and 5. N = 3–4/group; *p < 0.05. Error bars represent standard error of the mean.

Fig. 7

Mean splenic TNFα, IL-1β, and IL-6 concentrations (+/− SE) after acute (a, c, and e) or chronic (b, d, f) LPS and Saline injections. N= 10–14/group for acute LPS Expt.; N= 9–12/group for chronic Expt. See Materials and Methods, as well as Figure 1, for details. *p < 0.05.

Fig. 8

Mean plasma IL-1β concentration (+/− SE) after acute (a) or chronic (b) LPS and Saline injections. Acute LPS Expt (N= 6–18/group); Chronic LPS Expt (N = 9–12/group). *p < 0.05

Fig 9

Mean concentration (+/− SE) of IL-1β and IL-6 in the corpus callosum of mice after acute (a) or chronic (b) LPS and Saline injections. For further details see Fig. 8 *p < 0.05

Fig. 10

Mean plasma corticosterone concentration after (a) acute, and (b) chronic LPS and Saline injections. (N = 9–12/group for all LPS groups; N= 4–9 for all saline groups). *p < 0.05