Chronic pain affects 15% of the world’s population and poses a major burden on patients’ lives due to insufficient treatment options.¹ Classically used opiates lack long-term efficiency and the development of novel treatments has been largely unsuccessful due to poor translatability of preclinical findings. Using mouse models of chronic pain, we studied functional and neurochemical changes in the brain longitudinally in an effort to try to reveal specific imaging readouts for evaluation of novel treatments.Mouse models of chronic pain from bone cancer were prepared by intramedullar injection of EO771 (tebu-bio, 940001-A) and 4T1 (ATCC, CRL-2539) breast cancer cells into the tibia of female C57BL/6 and Balb/cJRj mice (n=12 per group). Functional and neurochemical changes in the brain were assessed longitudinally using a Bruker Biospec 94/30 small animal MR system with a four-element receive only cryogenic phased array coil (Bruker BioSpin AG) with a linearly polarized room temperature volume resonator for transmission. Resting-state functional MRI (rs-fMRI) data was acquired using a gradient-echo echo-planar imaging (GE-EPI) sequence: TR=1000ms, TE=12ms, flip angle 60°. Rs-fMRI data was analyzed using independent component analysis (ICA) followed by linear mixed model analysis. Neurochemical profiles were assessed in the right ventral hippocampus by proton magnetic resonance spectroscopy (¹H-MRS) using a stimulated echo (STEAM) sequence: TR=2500ms, TM=10ms, 500 averages, 20min 50sec acquisition time in a 3µl volume. Voxel specific shimming was performed using field mapping and water signal was suppressed using VAPOR pulses. ¹H MRS data were analyzed using LCModel, metabolites were quantified as relative concentrations to the creatine/phosphocreatine pool. Animals were anesthetized using a combination of s.c. administered medetomidine hydrochloride (0.05 mg/kg as a bolus and 0.1 mg/kg/h for maintenance), intubated and ventilated with low dose isoflurane (0.5%) in a 20% O2/80% air mixture at a rate of 80breaths/min. For immobilization pancuronium bromide was administered s.c. as a bolus at a dose of 0.5mg/kg. Behavioral readouts of pain were performed using an electrical von Frey aesthesiometer (IITC Life Science) assessing the paw withdrawal threshold (PWT) in grams.Pain induced alterations in functional networks: Prominent significant functional changes were detected in C57BL/6 mouse brain networks comprising limbic structures like the hippocampus, cingulate cortex, amygdala and striatal regions. Additionally, cortical networks comprising the contralateral thalamus, motor cortex and areas of the parietal cortex were found to be significantly altered compared to sham mice. All of these altered cortical and limbic networks showed increased correlation values by 20% on average in tumor-bearing animals. The specificity of these findings has been validated in a cross-model study using Balb/cJRj mice injected with 4T1 cells, although fewer cortical networks appeared affected in this strain. Neurochemical alterations in hippocampus: We focused on hippocampus as this structure showed consistent changes in functional connectivity in pain models. Single voxel ¹H-MRS yielded significantly decreased levels of myo-inositol (-30.7%), glutamate (-7.5%) and glycerylophosphorylcholine (-51.7%) in tumor-bearing animals. Glutamine levels were found to be significantly increased (32.6%). The time course of these changes matched connectivity changes in the brain as well as behavioral measurements of pain except for glutamine.Limbic structures such as the hippocampus, striatum, cingulate cortex and amygdala are emerging as primarily affected areas in the development of a chronic pain state. The specificity of the presented results has been validated in a cross-model validation study where directionality and time course of functional changes was found to match previous results. Moreover, these findings mirror results from a large clinical study performing rs-fMRI on patients suffering from chronic back pain as well as preclinical findings indicating deficits in hippocampus-dependent memory extinction tasks in rodents.² Neurochemical profiling in the ventral hippocampus revealed decreased glutamate levels, which have been ascribed to neurodegeneration and mitochondrial stress.³ This hypothesis is supported by decreased myo-inositol and glycerylophosphorylcholine levels indicating decreased cell proliferation and neurogenesis, which has been reported previously in rats suffering from chronic neuropathic pain after a spared nerve injury (SNI).⁴ This indicates that these inherently translatable functional and metabolic readouts might be characteristic for the development of a chronic pain state.We characterized functional connectivity changes and neurochemical profiles in a mouse brain during development of a chronic pain state. The findings were found to be stable across different mouse strains and reflect changes observed in clinical studies with chronic back pain patients very closely. Given the translatability, these readouts could potentially be used to evaluate novel treatments more specifically than commonly used behavioral readouts of pain, which are known to be rather unspecific. In an ongoing study we try to modulate these readouts using analgesic treatments.