How does the motor relearning program improve neurological function of brain ischemia monkeys?

The motor relearning program can significantly improve various functional disturbance induced by ischemic cerebrovascular diseases. However, its mechanism of action remains poorly understood. In injured brain tissues, glial fibrillary acidic protein and neurofilament protein changes can reflect the condition of injured neurons and astrocytes, while vascular endothelial growth factor and basic fibroblast growth factor changes can indicate angiogenesis. In the present study, we induced ischemic brain injury in the rhesus macaque by electrocoagulation of the M1 segment of the right middle cerebral artery. The motor relearning program was conducted for 60 days from the third day after model establishment. Immunohistochemistry and single-photon emission CT showed that the numbers of glial fibrillary acidic protein-, neurofilament protein-, vascular endothelial growth factor- and basic fibroblast growth factor-positive cells were significantly increased in the infarcted side compared with the contralateral hemisphere following the motor relearning program. Moreover, cerebral blood flow in the infarcted side was significantly improved. The clinical rating scale for stroke was used to assess neurological function changes in the rhesus macaque following the motor relearning program. Results showed that motor function was improved, and problems with consciousness, self-care ability and balance function were significantly ameliorated. These findings indicate that the motor relearning program significantly promoted neuronal regeneration, repair and angiogenesis in the surroundings of the infarcted hemisphere, and improve neurological function in the rhesus macaque following brain ischemia.

Generation of nonhuman primate retinitis pigmentosa model by in situ knockout of RHO in rhesus macaque retina

Retinitis pigmentosa(RP)is a form of inherited retinal degenerative diseases that ultimately involves the macula,which is present in primates but not in the rodents.Therefore,creating nonhuman primate(NHP)models of RP is of critical importance to study its mechanism of pathogenesis and to evaluate potential therapeutic options in the future.Here we applied adeno-associated virus(AAV)-delivered CRISPR/SaCas9 technology to knockout the RHO gene in the retinae of the adult rhesus macaque(Macaca mulatta)to investigate the hypothesis whether non-germline mutation of the RHO gene is sufficient to recapitulate RP.Through a series of studies,we were able to demonstrate successful somatic editing of the RHO gene and reduced RHO protein expression.More importantly,the mutant macaque retinae displayed clinical RP phenotypes,including photoreceptor degeneration,retinal thinning,abnormal rod subcellular structures,and reduced photoresponse.Therefore,we suggest somatic editing of the RHO gene is able to phenocopy RP,and the reduced time span in generating NHP mutant accelerates RP research and expands the utility of NHP model for human disease study.

Co-editing PINK1 and DJ-1 Genes Via Adeno-Associated Virus-Delivered CRISPR/Cas9 System in Adult Monkey Brain Elicits Classical Parkinsonian Phenotype

Whether direct manipulation of Parkinson’s disease(PD)risk genes in the adult monkey brain can elicit a Parkinsonian phenotype remains an unsolved issue.Here,we used an adeno-associated virus serotype 9(AAV9)-delivered CRISPR/Cas9 system to directly co-edit PINK1 and DJ-1 genes in the substantia nigras(SNs)of two monkey groups:an old group and a middle-aged group.After the operation,the old group exhibited all the classic PD symptoms,including bradykinesia,tremor,and postural instability,accompanied by key pathological hallmarks of PD,such as severe nigral dopaminergic neuron loss(>64%)and evidentα-synuclein pathology in the gene-edited SN.In contrast,the phenotype of their middle-aged counterparts,which also showed clear PD symptoms and pathological hallmarks,were less severe.In addition to the higher final total PD scores and more severe pathological changes,the old group were also more susceptible to gene editing by showing a faster process of PD progression.These results suggested that both genetic and aging factors played important roles in the development of PD in the monkeys.Taken together,this system can effectively develop a large number of genetically-edited PD monkeys in a short time(6–10 months),and thus provides a practical transgenic monkey model for future PD studies.

Continuous subcellular resolution three-dimensional imaging on intact macaque brain

To decipher the organizational logic of complex brain circuits,it is important to chart long-distance pathways while preserving micron-level accuracy of local network.However,mapping the neuronal projections with individual-axon resolution in the large and complex primate brain is still challenging.Herein,we describe a highly efficient pipeline for three-dimensional mapping of the entire macaque brain with subcellular resolution.The pipeline includes a novel poly-N-acryloyl glycinamide(PNAGA)-based embedding method for long-term structure and fluorescence preservation,high-resolution and rapid whole-brain optical imaging,and image post-processing.The cytoarchitectonic information of the entire macaque brain was acquired with a voxel size of 0.32μm×0.32μm×10μm,showing its anatomical structure with cell distribution,density,and shape.Furthermore,thanks to viral labeling,individual long-distance projection axons from the frontal cortex were for the first time reconstructed across the entire brain hemisphere with a voxel size of 0.65μm×0.65μm×3μm.Our results show that individual cortical axons originating from the prefrontal cortex simultaneously target multiple brain regions,including the visual cortex,striatum,thalamus,and midbrain.This pipeline provides an efficient method for cellular and circuitry investigation of the whole macaque brain with individual-axon resolution,and can shed light on brain function and disorders.

Feature-reduction and semi-simulated data in functional connectivity-based cortical parcellation

Recently, restingstate functional magnetic resonance imaging has been used to parcellate the brain into functionally distinct regions based on the information available in functional connectivity maps. However, brain voxels are not independent units and adjacent voxels are always highly correlated, so functional connectivity maps contain redundant information, which not only impairs the computational efficiency during clustering, but also reduces the accuracy of clustering results. The aim of this study was to propose featurereduction approaches to reduce the redundancy and to develop semisimulated data with defined ground truth to evaluate these approaches. We proposed a featurereduction approach based on the Affinity Propagation Algorithm （APA） and compared it with the classic feature reduction approach based on Principal Component Analysis （PCA）. We tested the two approaches to the parcellation of both semisimulated and real seed regions using the Kmeans algorithm and designed two experiments to evaluate their noise resistance. We found that all functional connectivitymaps （with/without feature reduction） provided correct information for the parcellation of the semi simulated seed region and the computational efficiency was greatly improved by both feature reduction approaches. Meanwhile, the APAbased featurereduction approach outperformed the PCA based approach in noiseresistance. The results suggested that functional connectivity maps can provide correct information for cortical parcellation, and featurereduction does not significantly change the information. Considering the improvement in computational efficiency and the noiseresistance, featurereduction of functional connectivity maps before cortical parcellation is both feasible and necessary.