Yuji Komaki

Conditions for quantitative evaluation of injured spinal cord by in vivo diffusion tensor imaging and tractography: Preclinical longitudinal study in common marmosets

Conventional magnetic resonance imaging (MRI) can detect hemorrhage, edema, syrinx, and spinal cord atrophy, but not axonal disruption after spinal cord injury (SCI). We previously demonstrated that diffusion tensor tractography (DTT) could depict axonal disruption after hemisection SCI in common marmosets. In the present study, to determine the relationship between DTT results and functional recovery after contusive SCI, we performed longitudinal DTT, behavioral, and histological analyses before and after contusive SCI in common marmosets. By comparing the tract fiber estimate depicted by DTT with neuronal fibers labeled with RT97 and SMI-31, anti-neurofilament antibodies, we determined the optimal fractional anisotropy (FA) threshold for fiber tracking to be 0.40. The ratio of the number of tract fiber estimates at the lesion site to the number before SCI, determined by DTT, was significantly correlated with the functional recovery after SCI. Moreover, comparison of the longitudinal pre- and post-SCI FA and axial diffusivity (λ(‖)) values revealed that they decreased after injury at the sites caudal to the lesion epicenter in the corticospinal tract and rostral to the lesion epicenter in the dorsal column. The FA values, then, showed partial recovery in the dorsal column. FA-value-oriented color DTT was used to represent axonal sparing or regeneration of the different tracts. These findings indicated that DTT analysis might be a versatile non-invasive tool for evaluating the axonal disruption after SCI.

Atlas of the developing brain of the marmoset monkey constructed using magnetic resonance histology

The developmental anatomy of the brain is largely directed by neural-based cues. Despite this knowledge, the developmental trajectory of the primate brain has not yet been fully characterized. To realize this goal, the advance in noninvasive imaging methods and new brain atlases are essential. The common marmoset (Callithrix jacchus), a small New World primate, is widely used in neuroscience research. The recent introduction of transgenic techniques has enabled the marmoset to be used as a genetically modifiable primate model for brain development. Here, a magnetic resonance histology technique involving the use of ultra-high-resolution ex vivo magnetic resonance imaging (MRI) was performed to identify the developmental anatomy of the marmoset brain at different time points from gestational week 8 through to birth. The data allowed the generation of a multidimensional atlas of brain structures at different developmental stages. Furthermore, in utero MRI techniques were developed to noninvasively monitor brain development during the embryonic and fetal stages. The multidimensional atlas and the MRI tools developed herein are anticipated to further our understanding of the developing primate brain.

Diffusion tensor imaging and tractography of the spinal cord: from experimental studies to clinical application.

Diffusion-weighted magnetic resonance imaging provides detailed information about biological structures. In particular, diffusion tensor imaging and diffusion tensor tractography (DTT) are powerful tools for evaluating white matter fibers in the central nervous system. We previously established a reproducible spinal cord injury model in adult common marmosets and showed that DTT could be used to trace the neural tracts in the intact and injured spinal cord of these animals in vivo. Recently, many reports using DTT to analyze the spinal cord area have been published. Based on the findings from our experimental studies, we are now routinely performing DTT of the human spinal cord in the clinic. In this review we outline the basic principles of DTT, and describe the characteristics, limitations, and future uses of DTT to examine the spinal cord.

Parkinson Disease: Diffusion MR Imaging to Detect Nigrostriatal Pathway Loss in a Marmoset Model Treated with 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

PURPOSE To investigate the use of diffusion-tensor imaging (DTI) to detect denervation of the nigrostriatal pathway in a nonhuman primate model of Parkinson disease (PD) after treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MATERIALS AND METHODS This study was approved by the institutional committee for animal experiments. DTI was performed in marmosets (n = 6) by using a 7-T magnetic resonance (MR) imager before and 10 weeks after administration of MPTP. Fixed brains of a normal marmoset and a marmoset model of PD (n = 1) were analyzed by using microscopic tractography. Tyrosine-hydroxylase staining of dopaminergic neurons and three-dimensional histologic analysis also were performed in normal marmosets (n = 2) and a PD marmoset model (n = 2) to validate the course of the nigrostriatal pathway revealed at tractography. Statistical analysis of voxel-based and post hoc region-of-interest analyses of DTI maps was performed by using a paired t test. RESULTS At voxel-based analysis of DTI before and after treatment, MPTP-treated marmoset brains showed significantly increased axial and radial diffusivity in the bilateral nigrostriatal pathway (P < .05, false discovery rate corrected). The largest area of significantly increased diffusivity was an area of axial diffusivity in the right hemispere (177 mm(3)) that corresponded to the location of dopaminergic neurodegeneration at histologic evaluation. Region-of-interest analysis revealed a 27% increase in axial diffusivity in the right hemisphere (1.198 mm(2)/sec ± 0.111 to 1.522 mm(2)/sec ± 0.118; P = .002). Three-dimensional histologic analysis with tyrosine-hydroxylase staining showed the course of the nigrostriatal pathway and degeneration in the PD marmoset model as the absence of a tyrosine-hydroxylase stained region. Microscopic tractography showed that the connection of the substantia nigra to the striatum followed the same course as the nigrostriatal pathway and fewer fiber tracts in the PD marmoset model. CONCLUSION DTI and microscopic tractography showed the loss of fiber structures of the nigrostriatal pathway in the marmoset model of PD. The results of this study provide a potential basis for the use of DTI in the clinical diagnosis of PD.

Optogenetic activation of CA1 pyramidal neurons at the dorsal and ventral hippocampus evokes distinct brain-wide responses revealed by mouse fMRI.

The dorsal and ventral hippocampal regions (dHP and vHP) are proposed to have distinct functions. Electrophysiological studies have revealed intra-hippocampal variances along the dorsoventral axis. Nevertheless, the extra-hippocampal influences of dHP and vHP activities remain unclear. In this study, we compared the spatial distribution of brain-wide responses upon dHP or vHP activation and further estimate connection strengths between the dHP and the vHP with corresponding extra-hippocampal areas. To achieve this, we first investigated responses of local field potential (LFP) and multi unit activities (MUA) upon light stimulation in the hippocampus of an anesthetized transgenic mouse, whose CA1 pyramidal neurons expressed a step-function opsin variant of channelrhodopsin-2 (ChR2). Optogenetic stimulation increased hippocampal LFP power at theta, gamma, and ultra-fast frequency bands, and augmented MUA, indicating light-induced activation of CA1 pyramidal neurons. Brain-wide responses examined using fMRI revealed that optogenetic activation at the dHP or vHP caused blood oxygenation level-dependent (BOLD) fMRI signals in situ. Although activation at the dHP induced BOLD responses at the vHP, the opposite was not observed. Outside the hippocampal formation, activation at the dHP, but not the vHP, evoked BOLD responses at the retrosplenial cortex (RSP), which is in line with anatomical evidence. In contrast, BOLD responses at the lateral septum (LS) were induced only upon vHP activation, even though both dHP and vHP send axonal fibers to the LS. Our findings suggest that the primary targets of dHP and vHP activation are distinct, which concurs with attributed functions of the dHP and RSP in spatial memory, as well as of the vHP and LS in emotional responses.

Inflammatory cascades mediate synapse elimination in spinal cord compression

BackgroundCervical compressive myelopathy (CCM) is caused by chronic spinal cord compression due to spondylosis, a degenerative disc disease, and ossification of the ligaments. Tip-toe walking Yoshimura (twy) mice are reported to be an ideal animal model for CCM-related neuronal dysfunction, because they develop spontaneous spinal cord compression without any artificial manipulation. Previous histological studies showed that neurons are lost due to apoptosis in CCM, but the mechanism underlying this neurodegeneration was not fully elucidated. The purpose of this study was to investigate the pathophysiology of CCM by evaluating the global gene expression of the compressed spinal cord and comparing the transcriptome analysis with the physical and histological findings in twy mice.MethodsTwenty-week-old twy mice were divided into two groups according to the magnetic resonance imaging (MRI) findings: a severe compression (S) group and a mild compression (M) group. The transcriptome was analyzed by microarray and RT-PCR. The cellular pathophysiology was examined by immunohistological analysis and immuno-electron microscopy. Motor function was assessed by Rotarod treadmill latency and stride-length tests.ResultsSevere cervical calcification caused spinal canal stenosis and low functional capacity in twy mice. The microarray analysis revealed 215 genes that showed significantly different expression levels between the S and the M groups. Pathway analysis revealed that genes expressed at higher levels in the S group were enriched for terms related to the regulation of inflammation in the compressed spinal cord. M1 macrophage-dominant inflammation was present in the S group, and cysteine-rich protein 61 (Cyr61), an inducer of M1 macrophages, was markedly upregulated in these spinal cords. Furthermore, C1q, which initiates the classical complement cascade, was more upregulated in the S group than in the M group. The confocal and electron microscopy observations indicated that classically activated microglia/macrophages had migrated to the compressed spinal cord and eliminated synaptic terminals.ConclusionsWe revealed the detailed pathophysiology of the inflammatory response in an animal model of chronic spinal cord compression. Our findings suggest that complement-mediated synapse elimination is a central mechanism underlying the neurodegeneration in CCM.

Voxel-based morphometry of the marmoset brain: In vivo detection of volume loss in the substantia nigra of the MPTP-treated Parkinson's disease model.

Movement dysfunction in Parkinsons disease (PD) is caused by the degeneration of dopaminergic (DA) neurons in the substantia nigra (SN). Here, we established a method for voxel-based morphometry (VBM) and automatic tissue segmentation of the marmoset monkey brain using a 7-T animal scanner and applied the method to assess DA degeneration in a PD model, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated animals, with tyrosine-hydroxylase staining. The most significant decreases of local tissue volume were detected in the bilateral SN of MPTP-treated marmoset brains (-53.0% in right and -46.5% in left) and corresponded with the location of DA neurodegeneration found in histology (-65.4% in right). In addition to the SN, the decreases were also confirmed in the locus coeruleus, and lateral hypothalamus. VBM using 7-T MRI was effective in detecting volume loss in the SN of the PD-model marmoset. This study provides a potential basis for the application of VBM with ultra-high field MRI in the clinical diagnosis of PD. The developed method may also offer value in automatic whole-brain evaluation of structural changes for the marmoset monkey.

Connectomics: comprehensive approaches for whole-brain mapping

The aim of connectomics analysis is to understand whole-brain neural connections. This is accomplished using new biotechnologies. Here, we provide an overview of the recent progress in connectomics analysis. The entire neural network of an organism was revealed for the first time in the nematode. Caenorhabditis elegans (C. elegans) have an advantage of their limited number of neurons and their transparency, allowing the neural network to be visualized using light and electron microscopes (EMs). It is practically impossible to adopt the same approach for mammals because of the large number of neural cells and the opacity of the central nervous system. A variety of new technologies are being developed to perform computer-assisted high-throughput image acquisition and analysis to obtain whole-brain maps for higher species, including mammals. Diffusion tensor magnetic resonance imaging and tractography and three-dimensional imaging with the EM are examples of novel approaches to connectomics. These new technologies will soon be applied not only to Drosophila, C. elegans and rodent research, but also to comprehensive connectomics analysis in a wide range of species including humans and primates. In the near future, results from connectomics analysis will reveal the neural circuitry of the whole brain and enhance our understanding of the human mind and neuropsychiatric diseases.

Physiological effects of a habituation procedure for functional MRI in awake mice using a cryogenic radiofrequency probe

BACKGROUND Functional magnetic resonance imaging (fMRI) in mice is typically performed under anesthesia due to difficulties in holding the head of awake mice stably with a conventional three-point fixation method that uses a tooth-bar and earplugs. Although some studies have succeeded in fMRI in awake mice by attaching a head-post on the skull, this cannot be applied to fMRI using a high signal-to-noise ratio (SNR) cryogenic MRI-detector, CryoProbe, because it covers the head of a mouse closely. NEW METHOD We developed head-fixation implements for awake mice that are applicable to fMRI using CryoProbe. RESULTS A head-bar was surgically attached to the skull of a mouse that was then habituated to a mock fMRI-environment, two hours/day for eight days with physiological examinations of body-weight, fecal weight, electromyogram (EMG), and electrocardiogram. EMG power decreased with just one day of habituation, whereas heart rate decreased after at least seven days of habituation. Estimated head motions of awake mice during fMRI were significantly smaller than a voxel size. Unexpectedly, temporal SNR of fMRI signals for awake mice was higher than that for anesthetized mice held by a conventional method. Functional connectivity in the brain of both anesthetized and awake mice showed bilateral and unilateral networks. COMPARISON WITH EXISTING METHOD(S): fMRI using CryoProbe had been performed on anesthetized mice previously. Our method does not use anesthetics during habituation or fMRI. CONCLUSION Our method would be beneficial for translational research using fMRI in mice and humans because human fMRI is typically performed without anesthetics.

In vivo microscopic voxel-based morphometry with a brain template to characterize strain-specific structures in the mouse brain

Hundreds of inbred mouse strains are established for use in a broad spectrum of basic research fields, including genetics, neuroscience, immunology, and cancer. Inbred mice exhibit identical intra-strain genetics and divergent inter-strain phenotypes. The cognitive and behavioral divergences must be controlled by the variances of structure and function of their brains; however, the underlying morphological features of strain-to-strain difference remain obscure. Here, in vivo microscopic magnetic resonance imaging was optimized to image the mouse brains by using an isotropic resolution of 80 μm. Next, in vivo templates were created from the data from four major inbred mouse strains (C57Bl/6, BALB/cBy, C3H/He, and DBA/2). A strain-mixed brain template was also created, and the template was then employed to establish automatic voxel-based morphometry (VBM) for the mouse brain. The VBM assessment revealed strain-specific brain morphologies concerning the gray matter volume of the four strains, with a smaller volume in the primary visual cortex for the C3H/He strain, and a smaller volume in the primary auditory cortex and field CA1 of the hippocampus for the DBA/2 strain. These findings would contribute to the basis of for understanding morphological phenotype of the inbred mouse strain and may indicate a relationship between brain morphology and strain-specific cognition and behavior.