Keigo Hikishima

Visualization of peripheral nerve degeneration and regeneration: monitoring with diffusion tensor tractography.

We applied diffusion tensor tractography (DTT), a recently developed MRI technique that reveals the microstructures of tissues based on its ability to monitor the random movements of water molecules, to the visualization of peripheral nerves after injury. The rat sciatic nerve was subjected to contusive injury, and the data obtained from diffusion tensor imaging (DTI) were used to determine the tracks of nerve fibers (DTT). The DTT images obtained using the fractional anisotropy (FA) threshold value of 0.4 clearly revealed the recovery process of the contused nerves. Immediately after the injury, fiber tracking from the designated proximal site could not be continued beyond the lesion epicenter, but the intensity improved thereafter, returning to its pre-injury level by 3 weeks later. We compared the FA value, a parameter computed from the DTT data, with the results of histological and functional examinations of the injured nerves, during recovery. The FA values of the peripheral nerves were more strongly correlated with axon-related (axon density and diameter) than with myelin-related (myelin density and thickness) parameters, supporting the theories that axonal membranes play a major role in anisotropic water diffusion and that myelination can modulate the degree of anisotropy. Moreover, restoration of the FA value at the lesion epicenter was strongly correlated with parameters of motor and sensory functional recovery. These correlations of the FA values with both the histological and functional changes demonstrate the potential usefulness of DTT for evaluating clinical events associated with Wallerian degeneration and the regeneration of peripheral nerves.

Pre-evaluated safe human iPSC-derived neural stem cells promote functional recovery after spinal cord injury in common marmoset without tumorigenicity.

Murine and human iPSC-NS/PCs (induced pluripotent stem cell-derived neural stem/progenitor cells) promote functional recovery following transplantation into the injured spinal cord in rodents. However, for clinical applicability, it is critical to obtain proof of the concept regarding the efficacy of grafted human iPSC-NS/PCs (hiPSC-NS/PCs) for the repair of spinal cord injury (SCI) in a non-human primate model. This study used a pre-evaluated “safe” hiPSC-NS/PC clone and an adult common marmoset (Callithrix jacchus) model of contusive SCI. SCI was induced at the fifth cervical level (C5), followed by transplantation of hiPSC-NS/PCs at 9 days after injury. Behavioral analyses were performed from the time of the initial injury until 12 weeks after SCI. Grafted hiPSC-NS/PCs survived and differentiated into all three neural lineages. Furthermore, transplantation of hiPSC-NS/PCs enhanced axonal sparing/regrowth and angiogenesis, and prevented the demyelination after SCI compared with that in vehicle control animals. Notably, no tumor formation occurred for at least 12 weeks after transplantation. Quantitative RT-PCR showed that mRNA expression levels of human neurotrophic factors were significantly higher in cultured hiPSC-NS/PCs than in human dermal fibroblasts (hDFs). Finally, behavioral tests showed that hiPSC-NS/PCs promoted functional recovery after SCI in the common marmoset. Taken together, these results indicate that pre-evaluated safe hiPSC-NS/PCs are a potential source of cells for the treatment of SCI in the clinic.

The common marmoset as a novel animal model system for biomedical and neuroscience research applications

The common marmoset (Callithrix jacchus), a small New World primate, has been attracting much attention in the research field of biomedical science and neuroscience, based on its (i) cross-reactivity with human cytokines or hormones, (ii) comparative ease in handling due to its small size, (iii) high reproductive efficiency, (iv) establishment of basic research tools, and (v) advantages of its unique behavioral and cognitive characters. Various neurological disease models have been developed in the common marmoset, including Parkinsons disease, Huntingtons disease, Alzheimers disease, stroke, multiple sclerosis and spinal cord injury. We recently developed transgenic common marmoset with germline transmission, which is expected to provide a new animal model for the study of human diseases. In this review, we summarize the recent progress of biomedical research and neuroscience using common marmoset as an excellent model system.

Human hepatocyte growth factor promotes functional recovery in primates after spinal cord injury

Many therapeutic interventions for spinal cord injury (SCI) using neurotrophic factors have focused on reducing the area damaged by secondary, post-injury degeneration, to promote functional recovery. Hepatocyte growth factor (HGF), which is a potent mitogen for mature hepatocytes and a mediator of the inflammatory responses to tissue injury, was recently highlighted as a potent neurotrophic factor in the central nervous system. We previously reported that introducing exogenous HGF into the injured rodent spinal cord using a herpes simplex virus-1 vector significantly reduces the area of damaged tissue and promotes functional recovery. However, that study did not examine the therapeutic effects of administering HGF after injury, which is the most critical issue for clinical application. To translate this strategy to human treatment, we induced a contusive cervical SCI in the common marmoset, a primate, and then administered recombinant human HGF (rhHGF) intrathecally. Motor function was assessed using an original open field scoring system focusing on manual function, including reach-and-grasp performance and hand placement in walking. The intrathecal rhHGF preserved the corticospinal fibers and myelinated areas, thereby promoting functional recovery. In vivo magnetic resonance imaging showed significant preservation of the intact spinal cord parenchyma. rhHGF-treatment did not give rise to an abnormal outgrowth of calcitonin gene related peptide positive fibers compared to the control group, indicating that this treatment did not induce or exacerbate allodynia. This is the first study to report the efficacy of rhHGF for treating SCI in non-human primates. In addition, this is the first presentation of a novel scale for assessing neurological motor performance in non-human primates after contusive cervical SCI.

Cellular composition and organization of the subventricular zone and rostral migratory stream in the adult and neonatal common marmoset brain.

The adult subventricular zone (SVZ) of the lateral ventricle contains neural stem cells. In rodents, these cells generate neuroblasts that migrate as chains toward the olfactory bulb along the rostral migratory stream (RMS). The neural‐stem‐cell niche at the ventricular wall is conserved in various animal species, including primates. However, it is unclear how the SVZ and RMS organization in nonhuman primates relates to that of rodents and humans. Here we studied the SVZ and RMS of the adult and neonatal common marmoset (Callithrix jacchus), a New World primate used widely in neuroscience, by electron microscopy, and immunohistochemical detection of cell‐type‐specific markers. The marmoset SVZ contained cells similar to type B, C, and A cells of the rodent SVZ in their marker expression and morphology. The adult marmoset SVZ had a three‐layer organization, as in the human brain, with ependymal, hypocellular, and astrocyte‐ribbon layers. However, the hypocellular layer was very thin or absent in the adult‐anterior and neonatal SVZ. Anti‐PSA‐NCAM staining of the anterior SVZ in whole‐mount ventricular wall preparations of adult marmosets revealed an extensive network of elongated cell aggregates similar to the neuroblast chains in rodents. Time‐lapse recordings of marmoset SVZ explants cultured in Matrigel showed the neuroblasts migrating in chains, like rodent type A cells. These results suggest that some features of neurogenesis and neuronal migration in the SVZ are common to marmosets, humans, and rodents. This basic description of the adult and neonatal marmoset SVZ will be useful for future studies on adult neurogenesis in primates. J. Comp. Neurol. 519:690–713, 2011.

Population-averaged standard template brain atlas for the common marmoset (Callithrix jacchus)

Advanced magnetic resonance (MR) neuroimaging analysis techniques based on voxel-wise statistics, such as voxel-based morphometry (VBM) and functional MRI, are widely applied to cognitive brain research in both human subjects and in non-human primates. Recent developments in imaging have enabled the evaluation of smaller animal models with sufficient spatial resolution. The common marmoset (Callithrix jacchus), a small New World primate species, has been widely used in neuroscience research, to which voxel-wise statistics could be extended with a species-specific brain template. Here, we report, for the first time, a tissue-segmented, population-averaged standard template of the common marmoset brain. This template was created by using anatomical T(1)-weighted images from 22 adult marmosets with a high-resolution isotropic voxel size of (0.2 mm)(3) at 7-Tesla and DARTEL algorithm in SPM8. Whole brain templates are available at International Neuroinformatics Japan Node website, http://brainatlas.brain.riken.jp/marmoset/.

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.