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Altered pH in early Alzheimer’s disease detected by creatine chemical exchange saturation transfer magnetic resonance imaging
Lin Chen1,2, Peter C.M. van Zijl1,2, Zhiliang Wei1,2, Hanzhang Lu1,2, Wenzhen Duan3, Philip C. Wang4,5, Tong Li4,5, and Jiadi Xu1,2
1Department of Radiology and Radiological Science, Johns Hopkins University, Baltimore, MD, United States, 2F.M. Kirby Research Center for Functional Brain Imaging, Kennedy Krieger Research Institute, Baltimore, MD, United States, 3Department of Psychiatry and Behavioral Sciences, Johns Hopkins University, Baltimore, MD, United States, 4Department of Pathology, Johns Hopkins University, Baltimore, MD, United States, 5Department of Neuroscience, Johns Hopkins University, Baltimore, MD, United States

Synopsis

We demonstrate the feasibility of creatine chemical exchange saturation transfer (CrCEST) MRI in detecting altered pH in Alzheimer’s disease (AD) mouse brain. In two early-stage AD models, namely Tau and APP mice, CrCEST contrast in the brain was significantly reduced compared to that of WT mice at 6-7 months (P<0.007). From MRS experiments, the brain creatine concentration between WT and AD mice was the same within error, which indicates that the reduced CrCEST contrast in the AD brain can be contributed mainly to pH reduction. Immunohistochemical analysis showed neuroinflammation in the APP mice, a potential factor for causing pH reduction.

Purpose

Previous studies have shown that abnormal cerebral pH plays an important role in the aggregation of Alzheimer’s disease associated proteins (1-3). Some studies also implicate neuroinflammation as a cause of the cerebral pH reduction involved in developing AD(4-6). Hence, in vivo assessment of cerebral pH could be a useful biomarker for the differentiation between AD and healthy controls (7-9). Currently, there are limited methods for noninvasively detecting pH in vivo. Previous studies demonstrate that the exchange of protons between water and guanidinium protons from creatine is highly sensitive to pH change(10-13). Hence we aim to apply creatine chemical exchange saturation transfer (CrCEST) as a sensitive method to detect cerebral pH changes in two AD mouse models, the amyloid precursor protein/presenilin-1 (APP) model (14) and the tau mouse model (Tau4RDK) (15).

Methods

Thirty female mice (age 6-7 months) were employed for the study, of which ten were Tau4RDK (Tau) (15), ten APPswe:PS1DE9 (APP), and another ten age-matched C57BL/6J (WT). All MRI experiments were performed on a horizontal bore 11.7 T Bruker Biospec system equipped with a 72mm volume resonator and 2×2 phased array coil. A double-tuned 31P/1H coil was employed for the collection of in vivo 31P MRS. All CEST experiments were performed using continuous-wave CEST (cwCEST). MR images were acquired using a Turbo Spin Echo (TSE) sequence with TE = 18 ms, TR=5 s, TSE factor = 20, slice thickness =1.5 mm and a matrix size of 64×64. The saturation field strength (B1) and length for CrCEST were 2 μT and 1 s, respectively (16,17). The extraction and quantification of the CEST signal were achieved using polynomial and Lorentzian line-shape fitting (PLOF) as detailed previously (11,16-19). The concentration of total Cr (tCr=Cr+PCr) was determined by 1H MRS, which was performed with a voxel of 3×3×3 mm3 using a stimulated echo acquisition mode (STEAM) sequence (TE = 3 ms, TM = 10 ms, TR = 2.5 s, NA = 256). The PCr concentration was determined by in vivo 31P MRS experiments, which were performed using a single pulse sequence (TR = 2 s, NA = 512, bandwidth = 50 ppm, acquisition time = 100 ms). Antibodies specific to human Ab and phosphorylated S422 of tau were used to monitor plaque and tangle formation, respectively. Neuroinflammation in the AD brain was determined by examining reactive astrocytes and microglia using polyclonal antibodies against GFAP and IBA1, respectively.

Results and Discussion

Typical CrCEST maps, which are represented by the true apparent relaxation rates of Cr ( on AD and WT mouse brains, are shown in Figs. 1d-f together with T1 maps (Figs. 1g-i) and T2 maps (Figs. 1k-m). To compare the results among different types of mice, regional , T1 and T2 values were extracted and summarized in Fig. 2. Three regions of interest (ROIs), namely cortex (cx), thalamus (th), and corpus callosum (cc), were chosen (Fig. 1a). Significant difference was observed between WT and APP values in all three regions (P<0.001). The values for APP mice were slightly lower than those for Tau mice in the cortex (Tau: 0.104±0.014 s-1; APP: 0.088±0.014 s-1; P=0.04). No significant difference was observed for the regional T1 values among the three types of mice. The T2 values were similar between WT and Tau mice, while the T2 values were slightly higher in the APP mice, particularly in the thalamus (WT: 35.56±0.60 ms; Tau: 35.29±0.59 ms; and APP: 36.34±0.90 ms), though this did not reach statistical significance (P=0.08).
To exclude potential contributions from creatine concentration variations to the reduced CrCEST contrast observed in APP and Tau mouse brains, the PCr and total Cr (tCr = PCr + Cr) concentrations were measured using 31P and 1H MRS, respectively (Fig. 3). The MRS showed that the concentrations of PCr and Cr in AD and WT mouse brain were not significantly different. This result suggests that altered pH is the major contributor to the reduced CEST contrast observed in AD mouse brain.
Typical immunohistochemical staining results in the cortex of WT, APP and Tau mouse brains are demonstrated in Fig. 4. At the age when altered PH was detected, the plaques are already abundant in APP mice (Fig. 4a), while Tau tangles are less obvious (Fig. 4b). Hypertrophic GFAP positive astrocytes were observed in the cortical regions of APP mice but not Tau mice (Fig. 4c). Similar to the astrocyte results, the IBA1 histological staining results indicated activation of microglia in the APP mice, while weaker microglial activation was observed in the Tau mice. Inflammation is a widely demonstrated pathological feature in the AD human brain characterized by an accumulation of activated microglia and astrocytes around plaques and tau tangles(20,21). Hence, neuroinflammation could be a major cause of the intracellular pH reduction in both APP and Tau mice (5,6) as confirmed by the current immunohistochemical analysis.

Conclusion

Young AD mice have reduced intracellular pH compared to WT mice, which preceded the tangle and plaque formation. pH has the potential to be a biomarker for early diagnosis of AD.

Acknowledgements

This work was supported by NIH: P41EB015909, R01HL149742, R03NS109664, R21NS118079, R21AG065794, P50AG05146 and DOD W81XWH-18-1-0797

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Figures

Figure 1: Typical S0 (a, b, c), CrCEST (d, e, f), T1 (g, h, i) and T2 maps (k,l,m) for WT (a, d, g, k), Tau (b, e, h, l), and APP (c, f, i, m) mice. The CrCEST maps (RCr) of Tau and APP mice showed clear reduction compared to that of WT mice, while the T1 and T2 maps were closely resembled among the three types of mice. The typical ROIs used to extract regional values are indicated in (a).

Figure 2. The CrCEST values (RCr) represented by Cr apparent relaxation rate for cortex (a ), thalamus (b), and corpus callosum (c) regions in WT (green square), Tau (red circle) and APP (blue diamond) mice. The T1 values for cortex (d), thalamus (e), and corpus callosum regions (f) in WT (green square), Tau (red circle) and APP (blue diamond) mice. The T2 values for cortex (g), thalamus (h), and corpus callosum regions (i) in WT (green square), Tau (red circle) and APP (blue diamond) mice.

Figure 3. Typical in vivo proton MRS spectra for (a) WT, (b) Tau and (c) APP mouse brains. (d) The tCr concentrations were extracted from the proton spectra through fitting with LCModel. There were no clear tCr concentration differences between the three types of mice. The averaged 31P MRS spectra (n=4) of (e) WT, (f) Tau, and (g) APP mouse brains. The coil coverage is identical to Fig. 1g.

Figure 4. Brain sections of WT, APP and Tau mice stained by antibodies specific to Ab (6E10) (a), phosphorylated tau: Tau-pS422 (b), reactive astrocytes (GFAP) (c), and microglia (IBA1) (d). Scale bars are 200 mm in (a-c) and 100 mm in (d).

Proc. Intl. Soc. Mag. Reson. Med. 29 (2021)
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