Periventricular leukomalacia is the leading cause of cerebral palsy and currently no effective treatment exists. Ischemia–reperfusion and inflammatory processes are considered the underlying culprits for the cascade of injury that eventually leads to abnormal myelination and axonal injury. A mouse model for PVL can be helpful for further understanding of pathologic mechanisms involved in the molecular cascade leading to white matter injury and serves as a testbed for the evaluation of experimental therapeutics. In this study, we present an ischemia model of PVL, induced by performing unilateral carotid artery ligation at a very early postnatal age.
The first important finding in our study is that unilateral carotid artery ligation when performed on P5 in CD-1 mouse pups leads to quite selective injury to the white matter in most of the animals. Our group and others have reported unilateral ischemia combined with hypoxia in neonatal mice, which typically leads to unilateral striatal, hippocampal, and cortical injuries (Johnston et al, 2005
). In all these studies, hypoxia–ischemia was induced in older mice typically between P7 and P12 and typically adds a period of hypoxia, leading to more severe injury. Our group has shown that C57Bl/6 and C3HeB/FeJ mice were much less vulnerable to seizures and brain injury after unilateral carotid ligation than were CD-1 mice at P12 (Comi et al, 2009
). Differences in vascular anatomy and other genetic strain-related differences are likely to contribute to different vulnerability. Furthermore, Back et al (1998)
have shown age-selective vulnerability of OPCs, which along with data regarding the development of glutamate receptors in the cortex (Johnston, 2009
) was the basis for selection of P5 as a ligation time point for our white matter injury model.
The majority of apoptotic cells that we found during the acute phase in our study were in the white matter, and had a morphology similar to oligodendrocyte progenitors or immature oligodendrocytes and many coexpressed aCasp3 and PDGFRα
, leading to the hypothesis that these cells are selectively targeted by ischemia or the downstream excito-oxidative cascade known to be triggered by ischemia, and ultimately the apoptosis of these oligodendrocyte lineage cells leads to dysmyelination. We also found evidence of axonal injury in the corpus callosum in our model, similar to recent reports from autopsy cases of PVL (Billiards et al, 2008
). Therefore, an alternative hypothesis can be offered here: The process of final differentiation and myelin synthesis of OPCs is orchestrated by a complex interaction between axons and these cells (Bozzali and Wrabetz, 2004
). These immature cells are known to express a high level of glutamate receptors that are excitatory and respond to stimuli received from axons (Bergles et al, 2010
). Altered signaling between axons and oligodendrocyte progenitors can activate an apoptotic pathway in these progenitors. Hence, we postulate that ischemia or related downstream effects will primarily compromise axons, which then leads to abnormal signaling to oligodendrocyte progenitors, resulting in their apoptosis. This phenomenon could explain the bilateral dysmyelination observed in our study, even though arterial ligation was unilateral. A third hypothesis would be that ischemia and related downstream effects lead to a differentiation arrest or alternative differentiation of OPCs into astrocytes. In line with this theory, we found a striking increase in the proportional target area of GFAP-stained sections, suggesting increased astrocytic cell body size, or increased number of astrocytes, or both. To answer this question, further studies using transgenic mice with cell-specific inducible reporter genes would be necessary, which could allow the detection of the fate of these cells after ischemia by inducing the reporter gene at the time of injury. As our model uses mice, we plan to conduct these studies in the future.
The second important finding in our study is that the MTR maps can nicely detect the severity of white matter injury in a quantitative manner in the living mouse. Although postmortem histology can be used as the ultimate outcome, it does not allow the assessment of evolving changes of function within an individual. In terms of functional testing of rodents, it is believed that neurologic abnormalities are very subtle in mouse models of perinatal brain injury and that these models usually do not exhibit prominent motor impairment, as seen in patients with PVL-associated cerebral palsy (Johnston et al, 2005
). Ment et al
have shown locomotor hyperactivity in the chronic sublethal hypoxia mouse model, and others have shown abnormal open-field testing after neonatal hypoxia–ischemia in mice (Weiss et al, 2004
). Rather than using these limited functional studies, which often require animal adaptation to a new environment and are time consuming, one could use the in vivo
imaging studies we report here as surrogate markers for disease progression and outcome. However, one weakness in our study is the lack of functional assessment in our model. We plan to conduct these studies in the future and correlate the findings with the MRI results.
Interestingly, there were bilateral reductions in MTR, MBP density, and in the number of mature oligodendrocytes (CC1+), but the reduction observed in corpus callosum volume was limited to the ligated side. Abnormal SMI32 staining at P60, combined with normal PanNF staining, is consistent with persistent axonopathy without axonal loss. We speculate that although there is equal bilateral reduction of myelin content in the corpus callosum, pathologic changes in axons originating in the ligated hemisphere may result in smaller caliber axons, and a thinner corpus callosum on the ligated side. Similarly, others have shown that in cuprizone-induced demyelination, the loss of oligodendrocytes is associated with SMI32 staining of axons and a reduction in axon caliber (Lindner et al, 2009
; Mason et al, 2001
). The thinning of axons would not affect the MT-MTR signal, because the main molecules that are believed to affect MT are the large lipid moieties of myelin. We also note that the lack of axonal loss is not consistent with reported human autopsy cases in which there is loss of axons and neurons (Billiards et al, 2008
). A number of hypotheses can be speculated here. (1) Our injury model may be milder than the reported human autopsy cases, because these cases are likely to reflect the more severe end of PVL; (2) It is possible that there is a greater degree of axonal regeneration in our rodent model than in human infants with PVL. Future electron microscopy experiments at different time points after ischemia are required to further delineate the axonal pathology in this model.
A third and perhaps most important finding in our study is the striking degree of correlation between the in vivo
MT-MRI and the postmortem myelin density. Magnetization transfer imaging has been widely used in multiple sclerosis and other demyelinating conditions as a sensitive tool to detect demyelination (Fatemi et al, 2005
; Horsfield, 2005
; McGowan et al, 2000
). Here, we find that in vivo
MTR is an excellent predictor of myelin density. A strong correlation has been shown between MT and the severity of demyelination in cuprizone-induced white matter injury (Zaaraoui et al, 2008
) and in inflammatory-induced demyelination models (Gareau et al, 2000
); however, these other injury models show quite severe demyelination and inflammation. Our model shows a mild-moderate degree of dysmyelination, and although inflammation may have an important role shortly after ischemia, at the time MRI was conducted (P60), there was no evidence of edema or microglial activation. Therefore, we conclude that the MTR decrease is likely a direct correlate of myelin content. One limitation of our study is the fact that precise coregistration between in vivo
imaging and postmortem histology is extremely difficult to achieve. However, one would expect that limitations of coregistration should reduce the amount of correlation, rather than artificially increase it.
In summary, we report an ischemia-induced neonatal mouse model of PVL in which we find selective vulnerability of the white matter and histologic abnormalities that resemble autopsy reports in infants with PVL. We further validate in vivo MRI techniques as quantitative monitoring tools for assessment of injury severity and predictors of histopathology. This model has an advantage over previously reported mouse models for PVL, because the injury is induced at a single time point postnatally, hence opening a window for acute interventions. This model, therefore, combined with the techniques presented can serve as a feasible testbed for evaluation of experimental therapeutics.