Neuroapoptosis is present in the perilesioned area of AL animals.

<p>Fluoro-Jade B and Hoechst staining of brain sections from AL animals 2, 7, 14, 28 days after injury. Numerous Fluoro-Jade B/Hoechst positive cells (merged) were observed in the AL group on the 2^nd day following injury (arrows); these cells were not detected at later time points. Images are representative of brain sections at the site of the lesion (n = 3 animals per experimental group); c – physiological control; 2, 7, 14, 28 – days following injury; blood vessels (arrowheads); magnification 40×.</p

CR treatment suppresses neuroapoptosis in the perilesioned area.

<p>Fluoro-Jade B and Hoechst staining of brain sections from CR animals 2, 7, 14, 28 – days after injury. Fluoro-Jade B/Hoechst positive cells were not detected in the CR group at any time point. Images are representative of brain sections at the site of the lesion (n = 3 animals per experimental group); c – physiological control; 2, 7, 14, 28 – days after the injury; blood vessels (arrowheads); magnification 40×.</p

Description of the qualitative scoring system for microglia activation state.

<p>Description of the qualitative scoring system for microglia activation state.</p

CR suppresses activation of microglia in the perilesioned area when applied prior to cortical stab injury.

<p>Brain sections from AL and CR animals 2, 7, 14, 28 days after injury were stained for the Iba-1 marker of microglial cells. Activated microglial cells displaying highly amoeboid and round-shaped morphology (Fig. 1a) were observed only in the AL group on the 2^nd day following injury (Fig. 1A). A number of moderately activated microglial cells (Fig. 1b, c) were observed in AL animals on the 7^th and 14^th day following injury (Fig. 1B, C). In the CR group microglial cells retained a mainly ramified structure throughout the recovery period (Fig. 1 E–H). Images are representative of 3 animals per experimental group; asterisk – site of the lesion. Scale bars: 50 µm and 25 µm for 20× and 40× magnification, respectively.</p

Number of microglial cells.

<p>Number of Iba-1 positive cells classified according to their morphology in the perilesioned area 2,7,14 and 28 days following SCI in AL and CR animals. Data are expressed as means ± S.E.M. (n = 3 animals per experimental group).</p>*<p>p<0.05 vs. AL control;</p>#<p>p<0.05 vs. CR control;</p>$<p>p<0.05 AL vs. CR;</p>&<p>p<0.05 vs. other time points of AL.</p

CR modulates microglial morphology in the perilesioned area.

<p>Quantification of each morphological type of microglial cell (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037215#pone-0037215-t002" target="_blank">Table 2</a>) is depicted as the percentage distribution per AL and CR groups as a function of time following injury. Percentage of both <i>intense</i> and <i>moderate</i> microglial cells was much higher in the AL group compared to the CR group at all time points. By far the greatest percentage of <i>intense</i> microglial cells was observed in the AL group on the 2^nd day following injury (83%). In the CR group the percentage of activated microglia (both <i>intense</i> and <i>moderate</i>) did not exceed 15% of the microglia population. c – physiological control; 2, 7, 14, 28 – days following injury.</p

CR treatment inhibits the induction of soluble TNF-α and active caspase-3.

<p>Western blot analysis of the temporal expression of soluble TNF-α (A) and active caspase-3 (B) revealed the induction of both proteins only in the ipsilateral cortex of AL animals on the 2^nd day following injury. Expression of both proteins was not detected in the CR group. Actin bands are the loading control. Representative immunoblots (n = 5 animals per experimental group) are presented; c – physiological control; 2, 7, 14, 28 – days following injury.</p