Gender effect on neurodegeneration and myelin markers in an animal model for multiple sclerosis

Abstract

Background: Multiple sclerosis (MS) varies considerably in its incidence and progression in females and males. In spite of clinical evidence, relatively few studies have explored molecular mechanisms possibly involved in gender-related differences. The present study describes possible cellular- and molecular-involved markers which are differentially regulated in male and female rats and result in gender-dependent EAE evolution and progression. Attention was focused on markers of myelination (MBP and PDGFαR) and neuronal distress and/or damage (GABA synthesis enzymes, GAD65 and GAD67, NGF, BDNF and related receptors), in two CNS areas, i.e. spinal cord and cerebellum, which are respectively severely and mildly affected by inflammation and demyelination. Tissues were sampled during acute, relapse/remission and chronic phases and results were analysed by two-way ANOVA.

Results: 1. A strong gender-dependent difference in myelin (MBP) and myelin precursor (PDGFαR) marker mRNA expression levels is observed in control animals in the spinal cord, but not in the cerebellum. This is the only gender-dependent difference in the expression level of the indicated markers in healthy animals; 2. both PDGFαR and MBP mRNAs in the spinal cord and MBP in the cerebellum are down-regulated during EAE in gender-dependent manner; 3. in the cerebellum, the expression profile of neuron-associated markers (GAD65, GAD67) is characterized by a substantial down-regulation during the inflammatory phase of the disease, which does not differ between male and female rats (two-way ANOVA); 4. there is an up-regulation of NGF, trkA and p75 mRNA expression in the early phases of the disease (14 and 21 days post-immunization), which is not different between male and female.

Conclusions: It is reported herein that the regulation of markers involved in demyelination and neuroprotection processes occurring during EAE, a well-established MS animal model, is gender- and time-dependent. These findings might contribute to gender- and phase disease-based therapy strategies.

Figure 1

Clinical score evolution (A) and body weight variations (B) in experimental animals (control female: white triangle, control male: white circle, EAE female: black triangle, EAE male: black circle). Significant differences were observed in the clinical disability score between male and female animals (two-way ANOVA, gender effect, **p = 0.0028, F(1,649) = 9.027; days post-immunization (DPI) effect, *** p < 0.0001, F(27,649) = 54.32.

Figure 2

Histopathology of the spinal cord in control and EAE animals was performed by E&E (A-D), GFAP-immunostaining for astroglial reaction (E-H) and FluoroMyelin staining for white tractd and myelin sheaths (K-M and related inserts). Micrograph in A illustrates a spinal cord hemi-sections in control, male rat; micrograph in B a spinal cord hemi-sections showing the extensive inflammatory infiltrates in EAE, as detailed in high power micrographs in C and D. Arrows indicate the intraparenchimal infiltrates asterisk in C a perivascular infiltrate. Image analysis indicates that there is a difference in the extension of inflammation infiltrate in female vs male EAE animals (N, Student's t test, p = 0.00249). Micrographs from E to H illustrate the astroglial reaction in EAE animals (F: female; H: male) with control animals (E: female; G: male). Image analysis indicates that astroglial reaction is stronger in female than in male EAE animals (O, one way ANOVA and Tukey multiple comparison test, **p < 0.01; ***p < 0.001). Micrographs from K to M illustrate white matter in EAE (I: female; M: male) compared with control animals (K: female; L: male). The color inserts refer to high power magnification, to shown the myelin sheath morphology. The original image has been processed using a deconvolution procedure. Image analysis indicates that demyelination is not different in female and male EAE animals (one way ANOVA and Tukey multiple comparison test, **p < 0.01; ***p < 0.001). Graph bars: M, 100 μm; color insert 10 μm.

Figure 3

PDGFαR and MBP mRNAs expression level in the spinal cord. mRNA levels were studied by real-time PCR. Results are expressed as relative gene expression, from data obtained by using the equation 2^-ΔΔC_T . Results are presented as mean ± SEM. Eight animals were included in each group (male and female) at each time point. Panels A and B refers to the gender effect in control groups, panels C and D refers to the disease effect. PDGFαR mRNA expression level was higher in control males than in control females (A. Student's t test *p = 0.0175), and MBP mRNA in control males was 6 times higher than in control females (B. Student's t test ***p < 0.0004). C: Relative expression of PDGFαR mRNA temporal profile in EAE animals at 14, 21 and 40 DPI, in females (white triangle) and males (black circle), compared with respective/own sex control group. The statistical analysis (two-way ANOVA) indicates a gender effect (*** p < 0.0001, F(1,50) = 18.08) and a disease effect (* p = 0.0259, F(3,50) = 3.358; Bonferroni post-test: control, * p < 0.05). D: Relative expression of MBP mRNA in EAE at the different disease phases compared with control sex group. The statistical analysis (two-way ANOVA) indicates a gender effect (*** p < 0.0001, F(1,46) = 51.51) and a disease effect (*** p = 0.0004, F(3,46) = 7.454; Bonferroni post-test: control, ** p < 0.01; 14 DPI, ** p < 0.01; 21 DPI, *** p < 0.001; 40 DPI, * p < 0.05).

Figure 4

Spinal cord MBP protein level in control rats. The levels of MBP protein, isoforms 21.5, 18.5 and 17.5 KDa were studied using Western-blot. The graphs in A, B and C show the results of densitometric analysis after normalizing MBP with GAPDH (38 KDa). Results are expressed as mean R.O.D. (relative optical density) values ± SEM. D: representative blot with the bands corresponding to female and male GAPDH (38 KDa) and MBP 21.5 18.5 and 17.5 KDa isoforms. No differences were found between male and female control rats for the investigated MBP isoforms.

Figure 5

PDGFαR and MBP mRNA expression level in the cerebellum. mRNA levels were studied by real-time PCR. Results are expressed as relative gene expression, from data obtained by using the equation 2^-ΔΔC_T . Results are presented as mean ± SEM. Eight animals were included in each group (male and female) at each time point. Panels A and B refers to the gender effect in control groups, panels C and D refers to the disease effect. No differences were observed in PDGFαR and MBP mRNA expression in female and male healthy controls (A, B), such as in PDGFαR mRNA expression in female (white triangle) and male (black circle) EAE animals at different DPI (C). On the contrary, statistical analysis (two-way ANOVA) shows that relative MBP mRNA expression in EAE animals in different in male and female rats (** p = 0.0028, F(1,52) = 9.831) and at different time points (*** p < 0.0001, F(3,52) = 25.14; Bonferroni post-test: 40 DPI, ** p < 0.01).

Figure 6

GAD65 and GAD67 mRNAs expression level in the cerebellum. mRNA levels were studied by real-time PCR. Results are expressed as relative gene expression, from data obtained by using the equation 2^-ΔΔC_T . Results are presented as mean ± SEM. Eight animals were included in each group (male and female) at each time point. Panels A and B refers to the gender effect in control groups, panels C and D refers to the disease effect. No differences were observed in GAD65 and GAD67 mRNA expression in female and male healthy controls (A, B). Statistical analysis (two-way ANOVA) shows that relative GAD65 and GAD67 mRNA expression in EAE animals is similar in male (white triangle) and female (black circle) rats (p = 0.3393 and p = 0.2009, respectively), whereas both mRNAs differ at different time points (disease effect: GAD65 **p = 0.0023, F(3,52) = 5.528; GAD67 *p = 0.0171, F(3,52) = 3.708).

Figure 7

NGF, BDNF, p75 and trkA mRNAs expression level in the cerebellum. mRNA levels were studied by real-time PCR. Results are expressed as relative gene expression, from data obtained by using the equation 2^-ΔΔC_T . Results are presented as mean ± SEM. Eight animals were included in each group (male and female) at each time point. Panels A to D refers to the gender effect in control groups, panels E and H refer to the disease effect. No differences female and male healthy controls were observed in this group of transcripts (A, B). Statistical analysis (two-way ANOVA) shows that mRNA expression in EAE animals is similar in male (black circle) and female (white triangle) rats for all transcripts, whereas they differ at different time points (NGF: *p = 0.0253, F(3,53) = 3.365; p75: *** p < 0.0001 F(3,52) = 9.878; trkA: ** p = 0.0029, F(3,52) = 5.299).