The goal of the present study was twofold: (1). to show proof-of-principle for adopting DTI methods to study the impact of obstructive HCP on the brain in a rodent model; and (2) to evaluate the tissue integrity in the hydrocephalic brain using DTI and correlate it with cytopathology. Our results demonstrate that DTI can successfully be used to quantify the underlying structural abnormality as reflected in the changes of the regional anisotropic diffusion properties in both GM and WM; even on a sub-millimeter scale in rat brain. In addition, these results show that the DTI findings have a positive correlation to the histopathological changes in HCP rats. Thus we establish that DTI parameters may be used as a surrogate marker to evaluate cerebral tissue integrity during the development of HCP, further supporting its use in translational research studies in the clinical setting.
As summarized by Beaulieu et al
], anisotropic diffusion properties are strongly influenced by the micro-structural components within the brain. The deviation of diffusion indices based on DTI from the normal range is believed to be an indication of the integrity, or the lack thereof, in various structural components, such as the myelin sheath or axonal membrane. Neuronal degeneration is often reported to be a reflection of decreased FA accompanied by an increased MD [36
]. Conversely, increased FA accompanied by decreased radial diffusivity and increased axial diffusivity is often regarded as an indication of compression of tissue, as seen in space-occupying lesions, such as non-invasive tumors [38
]. In clinical DTI studies of pediatric hydrocephalus, different patterns of change in DTI indices have been described in specific WM regions [18
]. For example, infants with HCP have been reported to demonstrate abnormally low FA in the corpus callosum and abnormally high FA in the internal capsule [20
]. Similarly, older patients (>7 yrs) also showed analogous patterns of diffusion [18
]. We speculate that multiple injury mechanisms may coexist in the HCP patient. The clinical manifestation of injury in HCP may depend on various factors such as the location of the structure under study, and the intrinsic tissue resistance, as well as the timing and duration of HCP during critical time points of CNS development. Among the four WM regions of interest examined in the present study (Tables and ), the EC in most HCP rats was unidentifiable using DTI maps. This can be considered an indication of severe tissue damage. Even though anatomically identifiable during the preparation for immunohistochemical staining, partial volume effect prevented us from making meaningful measurements in this region in any of the HCP rats. In the CC and the IC, significant decreases in FA and increases in MD values were found in rats with HCP. The FX showed similar DTI trends even though the difference did not reach statistical significance. These group differences based on data averaged over time (between P7 and P12) can be found as early as P9 when the comparison was made for individual post natal days. This may serve as preliminary evidence of the early impact of HCP as reflected from the DTI measurement. As described earlier, a decrease in FA usually indicates degenerative damage in the myelin or axonal membrane [6
], a change that is regarded as less reversible than a mere physical compression which often presents as an increase in FA [38
]. Combining the early timing and the potential nature of the changes, our results support early management in the treatment of HCP. In the clinical setting it would translate to having a pre-operative MRI demonstrating a significant reduction in FA values from our standard ROIs [20
DTI is seldom used to quantify the anisotropy in GM structures. However, relatively high anisotropy has been reported previously in the neonatal brain of both experimental models and clinical studies [39
]. In the normal individual, the elevated FA is followed by a rapid decrease until a plateau is reached in adulthood. This change in anisotropy is attributed to the developmental change in neuronal proliferation/migration, apoptosis and axonal pruning, and synaptogenesis occurring during the postnatal period [39
]. In the present study, DTI was used to determine the anisotropic diffusion properties in three GM structures: the CX, CPu, and HC. When comparing our data to the FA values at a corresponding age reported by Bockhorst et al
], our FA measurements in normal rats were similar in the CX but higher than previously reported in the HC and CPu. In the present study, HCP rats had a significantly reduced FA (from 0.25 to 0.17) in the CX, indicating that the impact of HCP not only affects the adjacent periventricular WM and the subcortical WM, but also the GM structures that are further peripherally located within the cranial vault.
It should be stressed that the DTI measurement is an objective but indirect reflection of the underlying micro-structural integrity or pathological condition under study. As a non-invasive imaging biomarker, DTI results need to be examined and correlated to the histopathological data which is regarded as the gold standard for studying the injury mechanisms at the cellular level. In this study, we examined GFAP immunoreactivity to characterize the temporal-spatial changes of reactive gliosis following HCP. Our results from GFAP stains showed that a strong glial reaction was seen in the HCP rats in the IC, EC, and FX at both P11 and P22/P23. Astrocytosis was also present at P11 in the CC. The correlation analysis of DTI with our GFAP ranking showed a moderate to strong correlation in the CC at P11. Likewise, the microglial reaction (Iba-1) also demonstrated a moderate to strong correlation with the DTI measurements in the CC at P11. LFB staining was very weak at P11 in control animals; this result is expected given that these observations occurred only three days after the initiation of myelin maturation (P7) [39
]. Consequently, the paucity of LFB staining in hydrocephalic brains at P11 was difficult to interpret. At P22/P23, even though the reduction of myelination was, as expected, statistically significant in IC in HCP rats (Table ), only a moderate correlation (without statistical significance) was found between the LFB staining in IC at P22/P23 and the DTI measurement at P7-P9 (Table ).
In the present study, astrocytes and microglial cells demonstrated an early and continuous response to CNS injury in untreated HCP. The correlations between DTI and those cellular changes reveal initial evidence for the potential of DTI to serve as an imaging biomarker in studying the progression of HCP.
The results presented in this study add new important value to the field of advanced imaging in experimental HCP. However, caution should be exercised in the interpretation of the results due to the overall small sample size. Our data do not allow for a more comprehensive analysis to evaluate the causal effect or the predicting power of DTI. Moreover, it is possible that partial volume effects may have artificially increased the differences in diffusivity measurements between the groups. Within a 1.5 mm thick slice as used in the study, the structures examined may have changed diffusion direction. The impact of this potential confounding factor will be reduced by increasing the imaging resolution using 3 D fast DTI imaging sequence, e.g., EPI or RARE based DTI sequence. With regard to the animal model, the current study will benefit if the progression of ventricular enlargement can be controlled and managed both in time and in the degree of severity. We have begun experiments to address these shortcomings. In addition, monitoring the longitudinal changes between the pre- and post-shunting conditions will further increase our understanding of the underlying injury mechanisms as well as the course for recovery.