This study investigated the inconsistencies of demyelination and gliosis occurring within the corpus callosum following standardized cuprizone-induced demyelination. We demonstrate anatomical differences in the extent of demyelination and gliosis within the corpus callosum with the rostral aspects (i.e., genu, and body) being less susceptible to pathological changes than the caudal regions (i.e., isthmus, splenium and dorsal hippocampal commissure). These observations are consistent with results from others (Stidworthy et al., 2003b; Wu et al., 2008
; Xie et al., 2010
). Importantly, to our knowledge this study is the first to demonstrate that the dorsal hippocampal commissure and splenium differ in the myelin repair process upon cuprizone withdrawal.
Histological examination of mice fed cuprizone showed demyelination in the cerebral cortex, and the external capsule as well as the caudate putamen. As in the corpus callosum the demyelination of the cerebellum showed distinct patterning, with the white matter of the vermis mainly spared (; and (Groebe et al., 2009
; Skripuletz et al., 2010
). Of interest, much of the caudate putamen was still noticeably demyelinated after two weeks of recovery, while other structures such as the external capsule, cerebral cortex, and corpus callosum showed substantial repair (not shown). As astrocytes have been shown to attract and induce differentiation of oligodendrocyte progenitor cells in this particular model (Patel et al., 2010
), it is notable that we did not observe as robust astrogliosis in the caudate putamen, whereas microgliosis was a prominent feature. On the other hand, the dorsal hippocampal commissure demonstrated continual astrogliosis two weeks after cuprizone withdrawal but had delayed remyelination when compared to the splenium.
Several different groups have now shown region-specific differences in the demyelination of the corpus callosum resulting from cuprizone intoxication of C57BL/6 mice (Binder et al., 2008
; Stidworthy et al., 2003a
; Wu et al., 2008
; Yang et al., 2009b, a
). In these experiments the degree of demyelination was predicted to be more severe in the caudal aspects of the callosal structure, a finding later confirmed by MRI and diffusion tensor imaging analysis (Stidworthy et al., 2003a
; Wu et al., 2008
; Xie et al., 2010
) and by the current study. The rostral-caudal differences have been indirectly supported by work involving the spatial organization of the repair processes after cuprizone-induced demyelination as well as by examination of the effects of the immunoregulator GAS6 on demyelination (Binder et al., 2008
). Neural progenitor cells injected into the lateral ventricles of cuprizone-demyelinated mice repopulate and repair the superficial aspect of the genu as well as the isthmus and splenium, but not the body of the corpus callosum (Irvine and Blakemore, 2008
). Moreover, in a recent study examining the role of CXCR4 in promoting migration of oligodendrocyte progenitors it was found that its ligand, CXCL12, was more abundantly expressed in the caudal aspect of the corpus callosum compared to the most rostral aspects, suggesting an increased reparative process ongoing in the more caudal corpus callosum (Patel et al., 2010
Our results demonstrate complete demyelination in the caudal regions of the corpus callosum. Importantly, our study provides anatomical parameters for affected callosal regions for use in designing experiments aimed to investigate cuprizone-induced demyelination and the molecular machinery involved in facilitating successful remyelination. These data are particularly relevant when examining coronal sections. Our data strongly suggest that regions caudal to approximately 0.8 mm bregma should be considered when studying remyelination while more rostral regions of the corpus callosum should be avoided as they do not provide adequate separation between groups of mice (). Dual analysis of the dorsal hippocampal commissure and the splenium on sagittal sections may provide valuable insight when examining the effects of treatments on remyelination as these structures appear to repair with a differential kinetics (, ). A recent study using magnetic resonance diffusion tensor imaging techniques to examine remyelination in cuprizone-fed mice demonstrated the importance of the caudal callosum region in identifying factors involved in remyelination (Tobin et al., 2011
). Our findings also suggest that neither the genu nor the body of the corpus callosum are appropriate anatomic locations for the stereotaxic injection of potential therapeutic factors aimed at promoting remyelination, as these locations are not completely demyelinated in the cuprizone model. On the other hand, the callosal body appears to be a more appropriate anatomical area to examine factors that influence demyelination.
Remyelination capacity and the extent of axonal pathology following cuprizone withdrawal vary between the splenium and dorsal hippocampal commissure within individual mice. While the mechanism(s) underlying the regional differences is unknown, it is interesting to note that impaired remyelination and increased axonal pathology were concurrent with persistent gliosis in the dorsal hippocampal commissure despite that there appeared to be more NG2+
progenitors when compared with the splenium. Reactive astrocytes are capable of producing a multitude of factors including trophic factors and cytokines that can either promote or hinder remyelination (Williams et al., 2007
). Thus, future experiments designed to elucidate physiological differences between reactive astrocytes in the splenium versus the dorsal hippocampal commissure may provide insight into the differences in remyelination processes between these two regions.
The differential sensitivity to cuprizone-induced demyelination along the corpus callosum may have a few implications. As the largest compact white-matter structure in the brain, the corpus callosum is anatomically divided into five separate regions, from rostral to caudal, the lamina rostralis, the genu, the body, the isthmus, and the splenium (Raybaud, 2010
). The corpus callosum can also be subdivided into two physiologically separate regions. While the genu and body are responsible for the commissural transfer of myelinated axons originating from the prefrontal cortex, premotor cortex, primary motor cortex, and supplemental motor area, the splenium and dorsal hippocampal commissure are involved in the transfer of sensory information from the posterior parietal cortex temporal lobe and occipital lobe (Raybaud, 2010
). The isthmus, which is located at the junction of the fornix and callosal body between the septum pellucidum and hippocampal commissure, contains fibers from the motor strip, somato-sensory strip and the primary auditory cortex (Raybaud, 2010
). As such, it can be predicted that mice undergoing cuprizone-induced demyelination might experience more difficulty with sensory modalities rather than motor function. This is consistent with the fact that cuprizone mice do not have obvious clinical signs (Ludwin, 1978
). However, this interpretation must be made with caution as we and others have demonstrated demyelination in other structures, including the caudate putamen and the cerebellum (Groebe et al., 2009
; Pott et al., 2009
; Skripuletz et al., 2010
), which may in part contribute to the decreased performance of cuprizone mice in the rotarod test as observed by several groups.
Toxin induced neuropathies have been known for a long time to also differentially affect specific regions of the brain. For example, isonicotinic acid hydrazide given to Peking ducks caused severe demyelination of the cerebellar medulla, white matter folia and deep cerebellar nuclei, while the medullary layer of occipital lobe, tectothalamic tract and cerebellar peduncles are less affected (Carlton and Kreutzberg, 1966
). Additionally, hexachlorophene toxicosis and triethyltin can cause specific demyelination pattering and gliosis with similarities to that of described for cuprizone (Poppenga et al., 1990
). In humans, inhalation of heroin vapor, industrial solvents, methanol misuse, alcohol abuse and nutritional deficiency can cause distinct patterns of demyelination with characteristic appearance upon imaging (Smith and Smirniotopoulos; Smith and Smirniotopoulos, 2010). The corpus callosum is suspected to be affected in almost all MS patients (Gean-Marton et al., 1991
). Unlike the regional distribution following cuprizone intoxication, diffusion tensor image mapping studies of MS patients have, however, demonstrated the callosal body and isthmus as being most affected areas while the genu and splenium the least (Ge et al., 2004
; Lou et al., 2009
; Ozturk et al.). Therefore, while it is peculiar that the isthmus, splenium and dorsal hippocampal commissure are more susceptible to cuprizone-induced demyelination when compared to the genu and body, this is not necessarily surprising as different demyelinating diseases with different etiologies show distinctive pathological patterning.
In conclusion, we have demonstrated rostral-caudal differences in demyelination and gliosis within the corpus callosum structure following cuprizone intoxication. We have mapped the location for gliosis and demyelination of the corpus callosum to approximately 0.5 mm caudal to bregma, and have demonstrated substantial differences in remyelination of these regions following cuprizone withdrawal. Together, these data provide guidance for the design of studies aimed at uncovering the mechanisms involved in demyelination and remyelination using this animal model.