PET Imaging On Neuro-injury Repair And Neural Functional Changes

Ischemic stroke is one of the leading and life-threatening diseases in modern society.Existing conventional therapeutic strategies(including interventional procedures,surgery and rehabilitation training)could partly improve the clinical symptoms of ischemic stroke、but could not replace the lost brain parenchymal cells.Recently,stem cell-based therapy has provided hope for enhancing tissue repair and functional recovery after ischemic stroke.Among various stem cell types,neural stem cells(NSCs)have shown great potential in cell replacement and circuitry repair of the infarct area,since they can directly differentiate into neurons,glial cells and other brain parenchymal cells.In addition,structural damage of the brain parenchyma in cerebral ischemia often leads to many mental disorders such as anxiety and depression.Positron emission tomography(PET),as one of the most advanced modern molecular imaging technology,can reflect the functional and metabolic changes at cellular or molecular levels,which breaks through the limitations of anatomical structure imaging.Since PET molecular imaging can detect brain functional changes sensitively,which could provide an effective research approach for further understanding the mechanism of mental disorders such as anxiety and depression.In this study,PET imaging was used to evaluate the dynamic metabolic changes after NSCs transplantation in a rat mode of focal cerebral ischemia.The role of NSCs in reconstructing the damaged neural circuits was investigated by using patch clamp and virus tracing technique.Our results showed that NSCs could not only promote the metabolic recovery of focal infarct,but also establish the neural circuit connections with the ventral thalamus of rat brain.To evaluate the potential role of dorsal periaqueductal gray(dPAG)in the PD pathogenesis,we established a rat panic anxiety-like model by precisely stimulating the dPAG with optogenetic approach,and further studied the neurofunctional changes in the related brain areas during panic attacks by using PET molecular imaging.In conclusion,PET molecular imaging is an effective method to non-invasively monitor the neuro-injury repair and brain function changes,and is helpful for evaluating therapeutic strategies and studying mechanisms of disease.Part 1:18F-FDG PET Imaging of Metabolic Changes after Neural Stem Cells Transplantation in a Rat Model of Cerebral Ischemia Objective:The aim of this study is to non-invasively and sensitively monitor the metabolic recovery of ischemic infarct after NSCs transplantation with the use of 18F-FDG(2-[18F]-fluoro-2-deoxy-D-glucose)PET imaging.Immunofluorescence,patch clamp and viral-based tracing technique were used to evaluate the neural circuitry damage and repair.Methods:Focal cortical infarct in rats was established by using the photothrombotic method.Magnetic resonance imaging(MRI)was used to identify the exact location and size of the infarct,which provided a precise guidance for transplantation of cortical-fated NSCs derived from human induced pluripotent stem cells(iPSCs)to the adjacent cortex of infarct.Then,PET imaging was utilized to monitor the metabolic recovery of infarcted cortex after NSCs transplantation.Meanwhile,in vivo BLI was used to evaluate the survival,proliferation and distribution of NSCs.Moreover,we used immunofluorescence and patch clamp to evaluate the differentiation and maturation of NSCs after transplantation.Viral-based anterograde monosynaptic tracing technique was used to confirm the establishment of direct synaptic connections between progeny cells of NSCs and ventral thalamus of rat brain.Results:A rat model of focal cerebral ischemia(confined to unilateral somatosensory cortex)was successfully established by using the photothrombotic method.18F-FDG PET imaging showed that the glucose metabolism began to improve at the 1st week after NSCs transplantation(L/H=0.80),and the maximum improvement was achieved by the 4th week(L/H=0.90).BLI results showed that the number of NSCs increased gradually with the time of transplantation.At 2 months after transplantation,immunofluorescence and patch clamp results confirmed that NSCs successfully differentiated into all layers of neurons of normal brain cortex,and the progeny neurons obtained fairly mature electrophysiological characteristics.Moreover,virus-based anterograde monosynaptic tracing result demonstrated that the grafted neurons receive direct synaptic inputs from ventral thalamus of rat brain.Conclusion:Human iPSC-derived cortex-fated NSCs have a strong capability of proliferation and survival,and could promote the metabolic recovery of focal infarct(somatosensory cortex).NSCs could differentiate into six layers of neurons of the normal cerebral cortex,and then replace the cells in the necrotic cortex.NSCs exhibit highly mature electrophysiological characteristics and can establish circuit connections with the ventral thalamus of rat brain,thus repairing danaged neural circuitry to a certain extent.Part 2:18F-FDG PET Imaging on Neurofunctional Changes after Optogenetic Stimulation in a Rat Model of Panic AnxietyObjective:Panic disorder(PD)is one of the clinical common mental disorder which impairs the quality of patients life,but its pathophysiology is still not well understood.This study aimed to map the neurofunctional changes after optogenetic stimulation to the dPAG by using the 18F-FDG PET imaging.Methods:In this study,rats were randomly divided into two groups:the experimental rats were injected with AAV2/9-CaMKIIα-ChR2-mCherry(ChR2+group,n=9),and the control rats were injected with AAV2/9-CaMKIIα-mCherry(ChR2-group,n=9).Three weeks after the AAV virus injection,each group underwent the 18F-FDG PET scans twice(baseline and post-optogenetic stimulation)on the microPET R4 scanner.Accurate optogenetic stimulation to dPAG was conducted to induce panic anxiety-like behaviors.18F-FDG PET imaging was performed to evaluate the neurofunctional changes before and after optogenetie stimulation.The expression of c-Fos protein in dPAG was confirmed by the immunofluorescent staining-Results:We observed obvious panic anxiety-like behaviors,such as galloping,jumping and rotation in the ChR2+ rats after optogenetic stimulation to the excitatory neurons of dPAG.In the ChR2 group,optogenetic stimulation PET images demonstrated significantly increased 18F-FDG accumulation in the dPAG,euneiform nucleus,cerebellar lobule,cingulate cortex,alveus of the hippocampus,primary visual cortex,septohypothalamic nucleus and retrosplenial granular cortex(P<0.001 in each comparison);but decreased in the basal ganglia,frontal cortex,forceps minor corpus callosum,primary somatosensory cortex,primary motor cortex,secondary visual cortex and dorsal lateral geniculate nucleus(P<0.001 in each comparison)compared to the baseline.However,these widespread changes in 18F-FDG accumulation were not observed in ChR2-control.The immunofluorescent results confirmed that optogenetic stimulation resulted in a significantly increased c-Fos protein in dPAG of ChR2+experimental group(P<0.001).Conclusion:Our results suggested that the dPAG plays a key role in the genesis of panic attacks.18F-FDG PET could identify the brain regions involved in the panic attacks.18F-FDG PET combined with optogenetics might be a new paradigm for precise evaluating the role of specific brain regions in brain diseases.