Glutamate is the most abundant excitatory neurotransmitter in the mature brain. Astroglia contribute to ~10-20% of total glutamate in brain which is highly regulated in the brain¹. Sub-acute administration of neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in mice causes selective dopaminergic cell death in the substantia nigra and striatum leading to inflammation and astrogliosis². ¹³C NMR studies have been utilized to study the astroglial activity in the brain but they are limited by the low sensitivity, hence less spatial resolution. Recently, GluCEST MRI has been applied in animals as well as humans to map small changes in regional cerebral glutamate homeostasis^3,4,5. The objective of the current study is to map the changes in regional glutamate levels in the brain of mice treated with MPTP using GluCEST MRI and determine the correlation with motor function loss measured from neurobehavioral tests. These findings suggest a role for GluCEST as a biomarker of neuroinflammation or glial function derangements.

Male C57BL6 mice were treated with MPTP/Saline for 7 days. Loss of motor function due to MPTP administration was assessed by performing the four limb endurance test. For MR studies, mice were anesthetized with Isoflurane/O₂ (1-2%, 1 L/hr) and positioned in a 9.4T Horizontal bore (Agilent Technologies Inc., USA) spectrometer. GluCEST imaging of striatum and thalamus was performed using a custom-programmed RF spoiled gradient echo readout pulse sequence in 2 mm slices with a frequency selective continuous wave saturation preparation pulse. CEST images were collected using a 1 second saturation pulse at peak B₁ of 5.9 μT for the frequencies ±2.5-3.5 ppm from water resonance with step size of 0.25 ppm. The B₀ inhomogeneity corrected images at 3 ppm (M_+3ppm) and -3 ppm (M_−3ppm) were used for computing the percent GluCEST contrast, which is equal to 100×[(M_−3ppm − M_+3ppm)/M_−3ppm]. ROIs were manually segmented from T2-weighted images. Fixed mouse brain sections were used for immunohistochmeical analysis. Student’s t-test was used to determine the significance of difference between the two groups.Sub-acute exposure to MPTP in mice led to a significant loss in motor function measured by the four limb hanging test. GluCEST contrast maps clearly show a higher contrast in the striatum of MPTP treated mice as compared to control group (Control 23.3±1.5 %, MPTP 26.3±1.1 %; p=0.00005) (Fig. 1 A,B,C). GFAP staining in the brain slices containing striatum show a drastic elevation of in the astroglial reactivity (~350% higher than control) in the region (Fig 1 H,I). The neuronal count and vesicular glutamate transporter-1 was not found to be changed in the striatum pointing toward glial proliferation as the cause of elevated glutamate. Interestingly, GluCEST contrast was also found to be elevated in the motor cortex of mice treated with MPTP significantly (Control 23.9±1.3 %, MPTP 26.3±1.5 %; p=0.0003) (Fig 1 D,E). Both GluCEST contrast and GFAP reactivity were found to be unchanged in the thalamus region of the brain (Fig 1 F,G,J,K). GluCEST contrast elevation in the striatum and motor cortex was also found to be negatively associated with the motor function of the MPTP treated mice (Fig 2 A,B). This implies that with higher glutamate in striatum and motor cortex leads to lower motor control in mice. GluCEST MRI demonstrates elevated glutamate with high spatial resolution and can potentially provide a biomarker for the detection of neuroinflammation and regional perturbation in the glial function in human brain at early disease stages. Additionally, GluCEST MRI may be applied to assess the efficacy of novel therapeutic interventions and treatment modalities for PD and related neurodegenerative disorders in humans.