Gender bias in autoimmune diseases is a well-known and hitherto unexplained fact. In particular, MS is more prevalent in females than in males, and this female predominance increases as time goes by. Gender appears to play a critical role also in the progression of MS, suggesting that not only immune reaction, but also remyelination, axonal pathology and neural damage might be gender-dependent . In all cases, the histopathological and molecular mechanisms underlying the inherent differences in male and female MS are still obscure .
In the present study molecular markers for myelin and grey matter that are differentially regulated in male and female rats in the experimental model of the disease have been described. Two areas were investigated: the spinal cord, which is the area with extensive inflammation and demyelination, and the cerebellum, in view of the ataxia symptom in EAE, where inflammatory cellular infiltrates are scare in this disease model . While the clinical profile of the disease differs between sexes being more severe in female than male rats, the inflammatory cellular infiltration in the spinal cord is lower in females, but produces a stronger astroglial reaction than in males. Moreover, disease-induced alteration of several markers is different between the two sexes. In particular, a strong gender-dependent difference in MBP and PDGFaR mRNA expression level in the spinal cord of healthy animals was found, which correspond to a different regulation during the disease.
EAE in male and female Dark Agouti rats
Here we confirm that both male and female Dark Agouti rats are highly susceptible to EAE . While the clinical score profile in the acute phase is similar in males and females, male rats show a more pronounced recovery than female rats. In this EAE model, a massive infiltration by inflammatory cells, and a massive demyelination were observed in the spinal cord, whereas small and localized lesions were spread over the main white tracts, including the cerebellar peduncoli, corpus callosum and optic nerve [15, 16, 26]. The remyelination process starts quite early also in Dark Agouti rats, but it is not yet complete at 40 DPI . In spite of the fact that severity of inflammatory infiltrate in the spinal cord is lower in female compared to male rats, astroglial reaction is more pronounced in female than in male rats. This could be related to humoral rather cellular immune reaction. Overall, the females of all the species used for MS models display stronger immune responses than males [26–28]. This is attributed to cytokines production [29, 30]. For example, cytokine IL-13 is implicated in gender differences in EAE severity in C57BL/6 mice, where the absence of the anti-inflammatory IL-13 entails lower susceptibility to EAE in females vs males or WT females with normal levels of IL-13 .
Gender-dependent differences in white matter during health and inflammatory-demyelinating disease
When comparing the expression level of the genes included in the study in male vs female healthy rats, the most significant result was the 5-fold higher MBP and the 2.5-fold higher PDGFαR expression in males than females in the spinal cord, but not in the cerebellum. This difference is not present at protein level, and, more generally, no significant differences in myelin sheaths in the spinal cord were found between male and female rats. This result might suggest that MBP synthesis could be regulated in a different way at post-transcriptional and translational levels in males and females. In fact, mRNA levels and protein levels do not always correlate [32, 33] and this might be due to the post-transcriptional mechanisms playing and/or to the different in vivo half life of proteins. Gonadal steroid and neurosteroid may elicit effects through non-genomic mechanisms via ERs localized on the plasma membrane, and ligand-independent pathways to activate ERs have been also described . Moreover, the mechanisms controlling the rates of degradation/synthesis for a given mRNA and protein are not homogeneous, even within proteins that have similar functions . Technical limits of the methods for quantifying mRNA and proteins should also be taken into account.
More generally, the issue of gender-related difference in the anatomy of white tracts is complex and controversial. The gender-dependent difference in PDGFαR and MBP mRNA in the spinal cord correlates with data from Cerghet et al. [35, 36] in mice. They found that the density of oligodendrocytes and the content of several myelin proteins in white tracts is higher in males than in females, whereas the lifespan of oligodendrocytes is shorter in females than in males, thus suggesting that myelin turnover is greater in females than in males. It may thus be argued that males have a greater functional reserve than females, whereas females have a greater vulnerability related to higher myelin protein turnover.
We and others already described the variation of MBP protein content, such as different markers for OPCs in male and female rats during EAE [13, 15, 16, 37]. In this study we report that there is a gender-dependent difference in the regulation of PDGFαR and MBP during the experimental disease, involving both genes in the spinal cord, and MBP, only in the cerebellum. This correlates with other reports describing differences in remyelination in old male and female rats  and in cuprizone intoxication model (reversible inflammatory demyelination) .
Neurotrophins and neuronal markers in inflammatory-demyelinating diseases
The EAE model in rodents (and primates) allows neuronal distress/damage to be investigated. We already described how the expression level of the acetylcholine synthesis enzyme choline acetyltransferase mRNA level was transiently reduced in motor neurons in the spinal cord  and in cholinergic neuron of the basal forebrain  during EAE. Here investigation was focused on cerebellum, which is involved in motor symptoms in EAE. In spite of scant inflammation and demyelination, a grey matter atrophy  and a Purkinje cell loss  has been described in the cerebellum. As index of neuronal injury, we investigated the expression level of the mRNAs encoding for the GABA synthesis enzyme γ-aminobutyric acid decarboxylase (GAD) . A transient down-regulation of both GAD65 and GAD67 was observed in male and female EAE rats, thus confirming that the acute phase of the disease is associated with reversible neuron distress.
Due to the neurotrophins role as endogenous neuroprotectors, their expression levels were investigated during EAE, focusing on NGF and BDNF. Previous results from our laboratory have described higher levels of NGF in certain brain areas, like the thalamus and cerebral cortex, but not the spinal cord, in EAE compared to healthy rats, associated to a strong up-regulation of p75- and trkA-like immunoreactivity . This up-regulation diminishes over time and a drop in NGF mRNA expression level was reported in the cerebral cortex at 104 DPI . Here we confirm the up-regulation of NGF and its high-affinity receptor trkA mRNA in the cerebellum in both male and female in the early, inflammatory phase of EAE, while BDNF is down-regulated at the same times. NGF, but also BDNF, modulate inflammation and immune cell function in many diseases [42–45]. Both the high and low affinity NGF receptors are widely expressed in the immune system, thus indicating a potential for responding to this neurotrophin through an autocrine mechanism [43, 45]. NGF increase during EAE may possibly also result in increased neuroprotection , working with the marmoset model, showed that NGF icv administration delayed the onset of clinical EAE, and also prevented the full development of EAE lesions. NGF administration also influences EAE development and progression in rats  by reducing the severity of the disease compared to that in saline treated EAE mice.
The low-affinity receptor p75 is also up-regulated. Notably, the up-regulation of p75 in Purkinje neurons has been described in EAE . The p75 up-regulation in EAE could be related to the Purkinje neurons death and cerebellar atrophy, since p75 can induce autophagy and death in these cells .
Since the original report describing the different content of NGF in the submaxillary gland in male and female mice , many other reports have illustrated sexually dimorphic distribution of NGF in tissues and plasma of different animal species [51–53]. The cerebellum and particularly the Purkinje cells have been recognized as a major source for neurosteroid production , and we reported that neurosteroids are differentially regulated in the cerebellum of control male and female rats, such as during EAE . Our mRNA data indicate that there are no sex differences in the NGF and BDNF levels in cerebellum either in healthy and EAE rats, thus confirming previous reports suggesting that neurotrophin levels do not correlate with estrogen levels in females or with estrogen or testosterone levels in males at this age [55, 56].