Intrauterine growth retardation has been linked to the development of cerebral palsy in children who were born preterm or term. Using a model of uteroplacental insufficiency that is associated with fetal growth retardation, oxidative stress and mitochondrial dysfunction (21
), we found oligodendrocyte and myelin maturation defects in addition to motor deficits; features commonly found in affected children (36
). While the pathogenesis of cerebral palsy in children who were formerly IUGR is complex and poorly understood, our data provide a mechanistic link between oxidative stress and increased BMP4 expression, which inhibits oligodendrocyte maturation and myelination. BMP4 was upregulated during the period of delayed myelination and returned to near normal levels as the rats matured and myelination increased. However, despite near normalization of myelination, mild motor deficits persisted, indicating that defects in myelination induced during fetal life can have life-long consequences.
The IUGR rat model demonstrated typical features of white matter injury, including a myelination defect and astrogliosis. At P14, the midpoint for myelin development in the rat, the myelination abnormality was characterized by a decrease in the number of oligodendrocytes and myelinating axons and a reduction in myelin protein expression. Myelin protein levels were similarly reduced at P21, indicating that the deficiencies in oligodendrocytes and myelin persisted through the end of the myelination period. Astrogliosis was also present at P14. The combination of lack of mature oligodendrocytes and astrogliosis is common in animal models of white matter disease as well as the brains of babies with white matter injury (1
Despite the paucity of oligodendrocytes in the IUGR animals compared to sham, the number of NG2-positive or PDGFRα-positive OPCs did not differ between the IUGR and sham animals. Thus, precursor number was not affected by the injury and precursors remain in the white matter and may retain their potential to mature and myelinate. One might expect OPCs to accumulate in these animals because many of them are unable to differentiate. However, this is not the case and is comparable to genetic models in which a paucity of mature oligodendrocytes during development does not reflect a change in the number of precursors (12
). It is unclear if these oligodendrocyte precursors give rise to normally functioning oligodendrocytes. Studies of PWMI in humans as well as experimental animals have shown reduced arborization of processes in oligodendrocytes labeling with mature markers. One study linked this finding to NMDA receptors located on the processes of oligodendrocytes that detach and disintegrate in an oxygen glucose deprivation model due to excitotoxicity (6
). This mechanism may play a role in our model as well; indeed, the oligodendrocyte shown in has truncated process growth. Our finding that motor deficits persist in IUGR adults also suggests that these precursors give rise to abnormally functioning oligodendrocytes.
Surprisingly, we did not see significant cell death by TUNEL or caspase staining at P14. The small foci of TUNEL-positive cells in the subventricular zone are considered normal and did not differ between IUGR and sham rats. This suggests that cell death at P14 cannot account for the lack of mature oligodendrocytes. Nevertheless, it is possible that cell death occurred much earlier and was cleared by P14 or is cleared at such a high rate that it cannot be appropriately captured by assaying a single time point.
Previous work has implicated oxidative stress as the primary mechanism for white matter injury (4
). Our IUGR model features oxidative stress, BMP upregulation, and a lack of oligodendrocytes. To demonstrate that these are linked, we treated OPCs in vitro with agents that cause oxidative stress by 2 different mechanisms and demonstrated that oxidative stress executes this function by upregulating BMP signaling, which in turn inhibits OPC differentiation leading to impaired myelination. We have previously shown that oxidant treatment aborts oligodendrocyte differentiation by downregulating transcription factors and nuclear proteins needed for differentiation such as Olig 2 and Sox 10 while upregulating those that inhibit differentiation such as Ids 2 and 4 and acetylated histone H3 and H4 (7
). BMP is known to inhibit oligodendrocyte differentiation through the regulation of key transcription factors and nuclear proteins mentioned above (46
). Our present data showing the differentiation and myelination defects can be prevented by blocking BMP signaling even in the presence of oxidative stress validate our model.
Evidence that BMP signaling is an important modulator of oligodendrocyte maturation and myelination is well established as is a role for this signaling pathway in white matter diseases. The function of BMP in oligodendrocyte development has been shown by our laboratory and others to arrest oligodendrocyte precursor maturation, inhibit myelin protein expression and increase astrogliogenesis (9
). Furthermore, it has been established that levels of BMP decrease around the time of birth, thus permitting OPC differentiation (9
). A number of studies have shown increased expression of BMPs, especially BMP4, in spinal cord injury or demyelinating disease in the adult (13
). BMP upregulation has recently been demonstrated in 2 neonatal white matter injury models as well (19
). Abrogation of BMP signaling has been shown to improve myelination, decrease astrogliosis, and increase functional recovery (16
It is not yet clear which cell types are making the BMP. Studies, including our own, using demyelinating models, have implicated astrocytes (13
), macrophages (15
), neurons (52
) and oligodendrocytes (15
). Because BMP4 is a secreted and diffusible protein, identification of the cell of origin is difficult and it is possible that BMPs are made by multiple cell types. While knowing the cell of origin is of inherent interest, it is not necessary for the consideration of therapy to rescue myelination during IUGR or other conditions in which BMP inhibits myelination. Although our in vitro data indicates that BMP directly affects oligodendrocytes, representing a possible cause of demyelination, we cannot rule out the possibility that other cell types, such as astrocytes, are affected by BMP upregulation and indirectly inhibit myelination. This will be the subject of future research.
Despite the paucity of oligodendrocytes and lack of myelin at P14 and P21, myelin in mature IUGR animals appeared essentially normal by IHC, cell counts, and myelin protein quantification. GFAP expression also returned to normal levels. Thus, OPCs in the IUGR animals are eventually able to overcome the differentiation block and this coincides with decreased BMP4 expression. In contrast, a study of IUGR in which uterine artery ligation was performed much earlier in gestation showed a decrease in the thickness of the corpus callosum in the motor cortex at P60 (54
). However, this model results in a significant decrease in overall brain size and thus represents a very severe global phenotype (55
Of particular importance and relevance to human IUGR and cerebral palsy is the persistence of motor and coordination disturbances found in the adult IUGR animals, even though the myelination differences have resolved. These data suggest a developmental timing requirement for oligodendrocyte differentiation and myelination, which, if delayed, results in persistent defects that are also seen in children who were born growth retarded (36
). However, the behavioral differences in the adult rats were more statistically significant in the females. There are several possible reasons for this. First, multiple behavioral studies have shown that there are often differences between genders in behavioral testing, particularly motor performance and anxiety, with females performing better (i.e. more activity) in open field and other motor tasks (56
). The reason for these differences not well established but is likely due to the effects of sex hormones on the brain (58
). Because the normal females perform better than normal males, differences due to IUGR will be more pronounced in females, as our represented in our data. Second, in premature infants, there is substantial literature about sex differences in outcomes (59
). Our behavioral data could be related to the mechanisms underlying these differences, which are not well understood.
In summary, we have provided a mechanism for the increase in BMP4 during demyelinating injury. Oxidative stress is a common and significant component of IUGR, as well as other perinatal injuries causing demyelination and adult demyelinating disease such as MS (4
). Our previous work demonstrates that oxidative stress in vitro inhibits differentiation by altering the cellular and molecular program of maturation (7
). We now show that BMP is upregulated by oxidative stress and oxidants cannot inhibit oligodendrocyte differentiation when BMP signaling is abrogated. This is the first demonstration that oxidative stress operates through BMP in the nervous system in general, and in oligodendrocytes in particular, and provides a mechanism to explain BMP upregulation in demyelinating diseases.