Purpose

About 60 % of the human body is composed of water, which is essential for life. In the brain, water plays a vital role. Water kinetics of the human body are regulated by aquaporins, particularly by aquaporin-4 (AQP4) in the brain. Dysfunctions of AQP4 lead to brain diseases, such as neuromyelitis optica [1], brain edema [2], Alzheimer’s disease [3], and vascular dementia [4]. Brain edema may be cellular or vasogenic. Geoffrey T et al. [2] reported water intoxication-induced cellular edema and its decreased degree in AQP4-knockout (AQP4-KO) mice. Water kinetics could elucidate the pathophysiological mechanism of brain diseases, although still unclear.
Magnetic resonance imaging (MRI) is a non-invasive and high-resolution imaging modality for the brain in both research and clinical settings. Recently, ¹⁷O-labeled water was developed as a new contrast agent owing to its advantages of safety and free diffusion [5,6]. Its application can be direct or indirect. The direct method requires specific hardware to detect MRI signals from the nucleus of ¹⁷O but has the disadvantage of high cost. Indirect MRI is used to detect ¹⁷O-induced proton signals because the current clinical MRI machines can be diverting.
In this study, we performed T2-weighted indirect MRI with ¹⁷O-labeled water, which is the latest imaging modality, to evaluate the water kinetics of the brain of the intoxication model, or AQP4-KO, mice and elucidate the role of AQP4.

Materials and Methods

All animal care and experimental procedures were approved by the Institutional Animal Care and Use Committee and were performed in accordance with the institutional guidelines for animal experiments. Eleven AQP4-KO and eight wild-type (WT) control mice (C57BL6) were scanned with 7T MRI (Bruker BioSpin MRI GmbH) using the Rapid Acquisition with Relaxation Enhancement (RARE) sequence. The scanning conditions were: repetition time, 5000 ms; echo time, 88.09 ms; RARE factor, 64; field of view, 19.2 mm; matrix, 128 × 128; slice thickness, 1.0 mm; number of slices, 9; number of excitation, 1; single scan time, 5 s; number of dynamic phases, 540; total scan time, 45 min. The animals were sedated with isoflurane via inhalation. 5 min after the scanning started, 10% ¹⁷O-labeled distilled water (0.1 mL/g) with desmopressin (0.4 µg/kg) was administered intraperitoneally.
Postprocessing of the imaging data was performed using in-house software (Perfusion Mismatch Analyzer: PMA) [7]. ROIs were manually placed in the basal ganglia, cerebral parenchyma, midbrain (two parts), lateral ventricle (two parts) and 3rd ventricle (Fig. 1). Signals in ROIs were converted to ¹⁷O concentrations. The acquired ¹⁷O concentrations versus time curves were averaged every 2 min and processed data were compared between AQP4-KO and WT mice using the independent t-test. P < 0.05 represented a statistically significant difference.

Results

Fig. 2 shows the time-curves of ¹⁷O concentrations of AQP4-KO and WT mice. After intraperitoneal administration of ¹⁷O-labeled water, concentrations began increasing rapidly, peaked, gradually decreased, and reached a steady state. The ¹⁷O concentrations in five representative phases were analyzed with the independent t-test between AQP4-KO and WT mice (Fig. 3).
In (a) basal ganglia, (b) cerebral parenchyma and (c) midbrain, AQP4-KO showed slightly higher concentrations in latter phases than WT did. In both (d) lateral ventricle 1 and (f) 3rd ventricle, similar curve trends with (a), (b) and (c) were seen, but standard deviations of them were much larger. In (e) lateral ventricle 2, curve trend was opposite and standard deviations were much larger as well.

Discussion

In this study, the water kinetics of the brain of intoxication model mice was compared using indirect MRI with ¹⁷O-labeled water between AQP4-KO and WT mice. There were significant differences in the steady-state phases of ROIs in the brain parenchyma.
The intraperitoneally administered ¹⁷O-labeled water entered the abdominal capillaries and reached the brain vessels via systemic circulation. In the brain capillaries, some water molecules passed the vascular endothelial cell gap and entered the paravascular space. Subsequently, the water molecules crossed AQP4 in the blood-brain barrier formed by the astrocyte end-feet.
There was accumulation of ¹⁷O-labeled water molecules in the ROIs of the brain parenchyma. A previous study on intoxication model mice [2] reported that brain edema decreased in AQP4-KO mice, implying that water did not reach the brain parenchyma and that water might accumulate in spaces other than the intravascular and parenchymal spaces. Previous studies reported expansion of the extracellular space in AQP4-KO mice [8] and increase perivascular space with less expression of AQP4 in the vascular dementia model mice [3]. This suggests that water might accumulate in the paravascular space.
However, the results for the ventricles are the limitation of this study. Variations in ¹⁷O concentrations were large on 2D MRI, as 1.0-mm slice thickness could cause a partial volume effect. This study revealed sufficient temporal resolution. Higher spatial resolution and lower temporal resolutions are required for precise measurements.

Conclusion

The water kinetics of the brain of AQP4-KO mice on indirect MRI with ¹⁷O-labeled water shows that the deficiency of AQP4 might lead to water accumulation in the paravascular space. The latest imaging modality should be further developed to elucidate the pathophysiological mechanism of brain diseases.

Acknowledgements

We would like to thank Editage (www.editage.com) for English language editing.

References

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