Accumulation of Fascin⁺ cells during experimental autoimmune neuritis

Male Lewis rats (8–10 weeks old, 200–250 g, Charles River, Sulzfeld, Germany) were housed with equal daily periods of light and darkness, and free access to food and water. All procedures were performed in accordance with the published European Health Guidelines under a protocol approved by the local Administration District Official Committee, and all efforts were made to minimize the number of animals and their suffering. EAN was induced by s.c. Immunization with an emulsion of 100 μg of synthetic neuritogenic P2 peptide/CFA as described [18]. The severity of EAN was scored daily as follows: 0-normal, 1-reduced tonus of tail, 2-limp tail, impaired righting, 3-absent righting, 4-gait ataxia, 5-mild paresis of the hind limbs, 6-moderate paraparesis, 7-severe paraparesis or paraplegia of the hind limbs, 8-tetraparesis, 9-moribund, 10-death. In our present study, a previously reported EAN sciatic nerve library [18], containing tissue at days 7, 11, 13, 15, 17 and 22 (three rats each time point) was used. In addition, tissue from five normal rats was used as normal control.

Rats were deeply anesthetized with ether and perfused intracardially with 4°C, 4% paraformaldehyde (PFA in PBS). Sciatic nerves were quickly removed and post-fixed in 4% PFA overnight at 4°C. Sciatic nerves were cut into two equally long segments. All of these segments were embedded in paraffin, sectioned serially (3 μm) and mounted on silane-covered slides. IHC was performed on 3 μm paraffin-embedded sections using purified polyclonal antibody against Fascin, which is considered DC marker. The specificity of this antibody has been demonstrated by ELISA and Western blotting (product datasheet, Santa Cruz Biotechnology). The specificity of these antibodies has been demonstrated by ELISA and Western blotting (product data sheets, Sigma and Serotec). After dewaxing, sections were boiled (in an 850 W microwave oven) for 15 min in citrate buffer (2.1 g citric acid monohydrate/l, pH 6) (Carl Roth, Karlsruhe, Germany). Endogenous peroxidase was inhibited by 1% H2O2 in pure methanol (Merck, Darm-stadt, Germany) for 15 min. Sections were incubated with 10% normal pig serum (Biochrom, Berlin, Germany) to block non-specific binding of immunoglobulins and then with Fascin antibodies overnight at 4°C. Antibody binding to tissue sections was visualized with biotinylated swine anti-rabbit IgG antibody (1:400; Dako, Hamburg, Germany) and subsequent incubation with a horseradish peroxidase-conjugated streptavidin complex for 30 min (1:100; Dako), followed by development with diaminobenzidine (DAB) substrate (Fluka, Neu-Ulm, Germany). Finally, sections were counterstained with hemalum. As negative controls for immunostaining, the primary antibodies were omitted. By control staining, no immunoreactivity (IR) was detected in EAN sciatic nerves without primary Fascin antibody.

In double-staining experiments, sciatic nerve sections were immunolabeled as described above. Then, they were once more irradiated in a microwave for 15 minutes in citrate buffer and were incubated with 10% normal pig serum (Biochrom). Subsequently, the sections were incubated with the appropriate second primary monoclonal and polyclonal antibodies for 1 h at room temperature. The following antibodies were used: ED1 (1:100; Serotec, Oxford, UK) for activated microglia/macrophages, CD3 (1:50; Serotec) for T-lymphocytes. Consecutively, visualization was achieved by adding secondary antibody at a dilution of 1:400 in tris buffered saline with bovine serum albumin (TBS-BSA) for 30 minutes, and then alkaline phosphatase-conjugated Avidin complex diluted 1:100 in TBS-BSA for another 30 minutes. Finally, immunostaining was developed with Fast Blue BB salt chromogen-substrate solution, but by omission of counterstaining with hemalum.

After immunostaining, sections of each time point were examined by light microscopy for expression of Fascin. To evaluate immunostaining data, the numbers of positively stained cells to areas of sciatic nerve cross-sections were calculated. Briefly, images of sciatic nerve cross-sections were captured under 40 × magnification using Nikon Cool-scope (Nikon, Duesseldorf, Germany) with fixed parameters. Images were analyzed using Metamorph Off-line 7.1 (Molecular Devices, Toronto, Canada). Positively stained cells were counted by two independent observers from randomly chosen areas. For each EAN rat, four cross-sections from root and middle levels of both sides were analyzed. Results were given as arithmetic means of numbers of positively stained cells to areas of sciatic nerve cross-sections and standard errors of means (SEM). Differences in numbers of positively stained cells per unit of area of cross-sections among different time points were analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test (GraphPad 5.0 for Windows, GraphPad Software, San Diego, USA), and data presented as histograms created by GraphPad Prism 5.0 for Windows. Relative abundance of cell populations was presented as the ratio of arithmetic means. Correlation analysis was evaluated by checking Spearman’s correlation coefficient (for nonparametric correlation) (GraphPad Prism 5.0 for windows). For all statistical analyses, significance levels were set at P < 0.05.

Rat EAN model has been established and the time course of neurological scores of our EAN library has been published previously [18]. For this EAN tissue library, the first neurological sign (reduced tonus of tail) was seen at around day 12 (1.3 ± 0.8) and the maximal severity of neurological signs appeared at about 15 days after immunization (5.3 ± 0.3). Thereafter, rats rapidly recovered from EAN and no neurological signs were observed by day 22.

Fascin expression in rat sciatic nerves of EAN rats were studied by IHC. Results are given as means of numbers of Fascin⁺ cell ± ESM per mm², and compared to day 0 (Figure 1A). Following immunization, slight accumulation of Fascin⁺ cells was observed already at Day 7 (Figure 1B) and increases dramatically to Day 11 (Figure 1C). The maximum accumulation of Fascin⁺ cells was observed at Day 15 (Figure 1D), corresponding to the peak of disease severity. Subsequently, a considerable decrease of Fascin⁺ cells was observed and finally returned to normal control level by Day 22 (Figure 1E and F).

In sciatic nerves, the accumulation of Fascin⁺ cells appeared earlier compared with the development of neurological signs. However, the maximum levels of Fasin⁺ cells and clinical scores were found at the same day (day 15), and the significant accumulation of Fascin⁺ cells were observed in accordance with the severity of EAN. Further correlation analysis proved a significant positive correlation of the time course of Fascin⁺ cells accumulation in sciatic nerves with the time course of neurological scores of EAN rats in our observation ( r =0.89; P < 0.05) (Figure 2).

We further characterized the accumulated Fascin⁺ cells by double labeling with monoclonal antibodies directed against activated macrophages (ED1) and T cells (CD3). A double-staining experiment was performed in sciatic nerves of Day 15 EAN rats. Few co-expression of Fascin and CD3 was observed at day 15 (Figure 1G) and few Fascin⁺ cells co-expressed active macrophage marker ED1 (blue) at day 15 (Figure 1H). As DC is the only one that has Fascin in the white blood cells, so we think the infiltrating Fascin⁺ cells around vessels are DCs.