Disease Of Human Central Nervous System Research Articles

Limited expression of Nrf2 in neurons across the central nervous system

Nrf2, encoded by the gene Nfe2l2, is a broadly expressed transcription factor that regulates gene expression in response to reactive oxygen species (ROS) and oxidative stress. It is commonly referred to as a ubiquitous pathway, but this generalization overlooks work indicating that Nrf2 is essentially unexpressed in some neuronal populations. To explore whether this pattern extends throughout the central nervous system (CNS), we quantified Nfe2l2 expression and chromatin accessibility at the Nfe2l2 locus across multiple single cell datasets. In both the mouse and human CNS, Nfe2l2 was repressed in almost all mature neurons, but highly expressed in non-neuronal support cells, and this pattern was robust across multiple human CNS diseases. A subset of key Nrf2 target genes, like Slc7a11, also remained low in neurons. Thus, these data suggest that while most cells express Nfe2l2, with activity determined by ROS levels, neurons actively avoid Nrf2 activity by keeping Nfe2l2 expression low.

Platinum/carbon dots nanocomposites from palm bunch hydrothermal synthesis as highly efficient peroxidase mimics for ultra-low H2O2 sensing platform through dual mode of colorimetric and fluorescent detection

Detection of hydrogen peroxide and glucose in nanomolar level is crucial for point-of-care medical diagnosis. It has been reported that human's central nervous system diseases such as Alzheimer's disease, Parkinson's disease, and even amyotrophic lateral sclerosis, are presumably caused H2O2 or reactive radical species (ROS). Sensing of H2O2 released from human biofluids, tissues, organ from metabolism disorder at ultra-low concentration assists for early identification of severe diabetis mellitus related to glucose, and heart attack, as well as stroke related to cholesterol. In this work, carbon dots (CDs) having an average diameter at 6.99 nm with highly photoluminescence performance were successfully synthesized from palm empty fruit bunch (EFB) using green and environmentally friendly process via hydrothermal condition. CDs acted well on peroxidase-like activity for H2O2 detection at room temperature, however their sensitivity on ultra-low H2O2 concentration needed to be improved. To enhance their reactivity on H2O2 nanozyme activity at room temperature, synthesis of hybrid metal nanoparticles (AgNPs and PtNPs) on CDs surface was established. The findings exhibited that CDs/PtNPs was the most suitable nanozyme achieving highly efficient peroxidase mimic for dual mode of colorimetric and fluorescent H2O2 sensing platform at very low limit of detection of 0.01 mM (10 nM) H2O2.

Bioengineering the human spinal cord.

Three dimensional, self-assembled organoids that recapitulate key developmental and organizational events during embryogenesis have proven transformative for the study of human central nervous system (CNS) development, evolution, and disease pathology. Brain organoids have predominated the field, but human pluripotent stem cell (hPSC)-derived models of the spinal cord are on the rise. This has required piecing together the complex interactions between rostrocaudal patterning, which specifies axial diversity, and dorsoventral patterning, which establishes locomotor and somatosensory phenotypes. Here, we review how recent insights into neurodevelopmental biology have driven advancements in spinal organoid research, generating experimental models that have the potential to deepen our understanding of neural circuit development, central pattern generation (CPG), and neurodegenerative disease along the body axis. In addition, we discuss the application of bioengineering strategies to drive spinal tissue morphogenesis in vitro, current limitations, and future perspectives on these emerging model systems.

Toward the next generation of vascularized human neural organoids.

Thanks to progress in the development of three-dimensional (3D) culture technologies, human central nervous system (CNS) development and diseases have been gradually deciphered by using organoids derived from human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs). Selforganized neural organoids (NOs) have been used to mimic morphogenesis and functions of specific organs in vitro. Many NOs have been reproduced in vitro, such as those mimicking the human brain, retina, and spinal cord. However, NOs fail to capitulate to the maturation and complexity of in vivo neural tissues. The persistent issues with current NO cultivation protocols are inadequate oxygen supply and nutrient diffusion due to the absence of vascular networks. In vivo, the developing CNS is interpenetrated by vasculature that not only supplies oxygen and nutrients but also provides a structural template for neuronal growth. To address these deficiencies, recent studies have begun to couple NO culture with bioengineering techniques and methodologies, including genetic engineering, coculture, multidifferentiation, microfluidics and 3D bioprinting, and transplantation, which might promote NO maturation and create more functional NOs. These cutting-edge methods could generate an ever more reliable NO model in vitro for deciphering the codes of human CNS development, disease progression, and translational application. In this review, we will summarize recent technological advances in culture strategies to generate vascularized NOs (vNOs), with a special focus on cerebral- and retinal-organoid models.

Development of a novel purification protocol to isolate and identify brain microglia.

Microglia, the tissue-resident macrophage of the central nervous system (CNS), play a paramount role in brain health and disease status. Here, we describe a novel method for enriching and isolating primary microglia from mouse brain tissue. This isolation method yields a high number of cells from either young or adult mice, and importantly, maintains the health and function of the cells for subsequent cell culture. We also describe flow cytometry methods using novel cell surface markers, including CX3CR1 and Siglec-H, to specifically label microglia while avoiding other bone marrow and/or non-CNS derived macrophages and monocytes, which has been historically difficult to achieve. As microglia are crucial in multiple aspects of biology, such as in normal brain development/function, immune response, neurodegeneration, and cancer, this isolation technique could greatly benefit a wide range of studies in human CNS biology, health, and disease mechanisms. Being able to isolate a largely pure population of microglia could also allow for a more comprehensive understanding of their functional dynamics and role in disease mechanisms, advancement of potential biomarkers, and development of novel therapeutic targets to improve prognosis and quality of life in multiple diseases.

Inhibition of Transient Receptor Potential Vanilloid 4 (TRPV4) Mitigates Seizures.

Astrocytes are critical regulators of the immune/inflammatory response in several human central nervous system (CNS) diseases. Emerging evidence suggests that dysfunctional astrocytes are crucial players in seizures. The objective of this study was to investigate the role of transient receptor potential vanilloid 4 (TRPV4) in 4-aminopyridine (4-AP)-induced seizures and the underlying mechanism. We also provide evidence for the role of Yes-associated protein (YAP) in seizures. 4-AP was administered to mice or primary cultured astrocytes. YAP-specific small interfering RNA (siRNA) was administered to primary cultured astrocytes. Mouse brain tissue and surgical specimens from epileptic patient brains were examined, and the results showed that TRPV4 was upregulated, while astrocytes were activated and polarized to the A1 phenotype. The levels of glial fibrillary acidic protein (GFAP), cytokine production, YAP, signal transducer activator of transcription 3 (STAT3), intracellular Ca2+([Ca2+]i) and the third component of complement (C3) were increased in 4-AP-induced mice and astrocytes. Perturbations in the immune microenvironment in the brain were balanced by TRPV4 inhibition or the manipulation of [Ca2+]iin astrocytes. Knocking down YAP with siRNA significantly inhibited 4-AP-induced pathological changes in astrocytes. Our study demonstrated that astrocytic TRPV4 activation promoted neuroinflammation through the TRPV4/Ca2+/YAP/STAT3 signaling pathway in mice with seizures. Astrocyte TRPV4 inhibition attenuated neuroinflammation, reduced neuronal injury, and improved neurobehavioral function. Targeting astrocytic TRPV4 activation may provide a promising therapeutic approach for managing epilepsy.

Differentiation of Human Induced Pluripotent Stem Cells into Cortical Neurons to Advance Precision Medicine.

A major obstacle in studying human central nervous system (CNS) diseases is inaccessibility to the affected tissue and cells. Even in limited cases where tissue is available through surgical interventions, differentiated neurons cannot be maintained for extended time frames, which is prohibitive for experimental repetition and scalability. Advances in methodologies for reprogramming human somatic cells into induced pluripotent stem cells (iPSC) and directed differentiation of human neurons in culture now allow access to physiological and disease relevant cell types. In particular, patient iPSC-derived neurons represent unique ex vivo neuronal networks that allow investigating disease genetic and molecular pathways in physiologically accurate cellular microenvironments, importantly recapitulating molecular and cellular phenotypic aspects of disease. Generation of functional neural cells from iPSCs relies on manipulation of culture formats in the presence of specific factors that promote the conversion of pluripotent stem cells into neurons. To this end, several experimental protocols have been developed. Direct differentiation of stem cells into post-mitotic neurons is usually associated with low throughput, low yield, and high technical variability. Instead, methods relying on expansion of the intermediate neural progenitor cells (NPCs) show incredible potential for posterior generation of suitable neuronal cultures for cellular and biochemical assays, as well as drug screening. NPCs are expandable, self-renewable multipotent cells that can differentiate into astrocytes, oligodendrocytes, and electrically active neurons. Here, we describe a protocol for generating iPSC-derived NPCs via formation of neural aggregates and selection of NPC precursor neural rosettes, followed by a simple and reproducible method for generating a mixed population of cortical-like neurons through growth factor withdrawal. Implementation of this protocol has the potential to advance the goals of precision medicineresearch for both neurological and psychiatric disorders.

The Impact of IgA and the Microbiota on CNS Disease.

Although anatomically distant from the central nervous system (CNS), gut-derived signals can dynamically regulate both peripheral immune cells and CNS-resident glial cells to modulate disease. Recent discoveries of specific microbial taxa and microbial derived metabolites that modulate neuroinflammation and neurodegeneration have provided mechanistic insight into how the gut may modulate the CNS. Furthermore, the participation of the gut in regulation of peripheral and CNS immune activity introduces a potential therapeutic target. This review addresses emerging literature on how the microbiome can affect glia and circulating lymphocytes in preclinical models of human CNS disease. Critically, this review also discusses how the host may in turn influence the microbiome, and how this may impact CNS homeostasis and disease, potentially through the production of IgA.

Quantification Analysis of Progesterone Based on Terahertz Spectroscopy

Progesterone is a natural progestational hormone secreted by the corpus luteum and placenta that protects the growth of the fetus in the early stage. It is also applied to the study on the treatment of human central nervous system diseases, which proved to have high clinical medicinal value. The present detection methods of progesterone concentration, such as high-performance liquid chromatography and enzyme-linked immunosorbent assay, have the disadvantages of high consumption, cumbersome procedures, and expensive equipment. Here, the terahertz spectroscopy is applied to detect the medicinal concentration of progesterone. First, the characteristic peaks of progesterone are 1.24, 1.64, and 2.12 THz, determined by the density functional theory calculation and experimental collection of terahertz spectra. Then, the peak height and peak area of the above characteristic peaks are used to establish a linear correlation model with the concentration of progesterone injection, and the coefficient of determination (R <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sup> ), in this model, is greater than 0.95. Meanwhile, we adopt Raman spectroscopy technology and got the R <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sup> of 0.71, which is not so good as terahertz spectroscopy. Furthermore, multivariate curve resolution-alternating least square is used to analyze the composition of progesterone in progesterone capsules. The result shows that the error rate between the predicted concentration of progesterone and the real one is less than 5%. Therefore, terahertz spectroscopy provides a new way for the detection of progesterone drug content.

MiR-124: A Promising Therapeutic Target for Central Nervous System Injuries and Diseases.

Central nervous system injuries and diseases, such as ischemic stroke, spinal cord injury, neurodegenerative diseases, glioblastoma, multiple sclerosis, and the resulting neuroinflammation often lead to death or long-term disability. MicroRNAs are small, non-coding, single-stranded RNAs that regulate posttranscriptional gene expression in both physiological and pathological cellular processes, including central nervous system injuries and disorders. Studies on miR-124, one of the most abundant microRNAs in the central nervous system, have shown that its dysregulation is related to the occurrence and development of pathology within the central nervous system. Herein, we review the molecular regulatory functions, underlying mechanisms, and effective delivery methods of miR-124 in the central nervous system, where it is involved in pathological conditions. The review also provides novel insights into the therapeutic target potential of miR-124 in the treatment of human central nervous system injuries or diseases.

Establishment of 3D Neuro-Organoids Derived from Pig Embryonic Stem-Like Cells.

Although the human brain would be an ideal model for studying human neuropathology, it is difficult to perform in vitro culture of human brain cells from genetically engineered healthy or diseased brain tissue. Therefore, a suitable model for studying the molecular mechanisms responsible for neurological diseases that can appropriately mimic the human brain is needed. Somatic cell nuclear transfer (SCNT) was performed using an established porcine Yucatan EGFP cell line and whole seeding was performed using SCNT blastocysts. Two Yucatan EGFP porcine embryonic stem-like cell (pESLC) lines were established. These pESLC lines were then used to establish an in vitro neuro-organoids. Aggregates were cultured in vitro until 61 or 102 days after neural induction, neural patterning, and neural expansion. The neuro-organoids were sampled at each step and the expression of the dopaminergic neuronal marker (TH) and mature neuronal marker (MAP2) was confirmed by reverse transcription-PCR. Expression of the neural stem cell marker (PAX6), neural precursor markers (S100 and SOX2), and early neural markers (MAP2 and Nestin) were confirmed by immunofluorescence staining. In conclusion, we successfully established neuro-organoids derived from pESLCs in vitro. This protocol can be used as a tool to develop in vitro models for drug development, patient-specific chemotherapy, and human central nervous system disease studies.

Cerebrospinal fluid metabolomics: detection of neuroinflammation in human central nervous system disease.

The high morbidity and mortality of neuroinflammatory diseases drives significant interest in understanding the underlying mechanisms involved in the innate and adaptive immune response of the central nervous system (CNS). Diagnostic biomarkers are important to define treatable neuroinflammation. Metabolomics is a rapidly evolving research area offering novel insights into metabolic pathways, and elucidation of reliable metabolites as biomarkers for diseases. This review focuses on the emerging literature regarding the detection of neuroinflammation using cerebrospinal fluid (CSF) metabolomics in human cohort studies. Studies of classic neuroinflammatory disorders such as encephalitis, CNS infection and multiple sclerosis confirm the utility of CSF metabolomics. Additionally, studies in neurodegeneration and neuropsychiatry support the emerging potential of CSF metabolomics to detect neuroinflammation in common CNS diseases such as Alzheimer's disease and depression. We demonstrate metabolites in the tryptophan–kynurenine pathway, nitric oxide pathway, neopterin and major lipid species show moderately consistent ability to differentiate patients with neuroinflammation from controls. Integration of CSF metabolomics into clinical practice is warranted to improve recognition and treatment of neuroinflammation.

Isolation, Culture and Functional Characterization of Glia and Endothelial Cells From Adult Pig Brain.

Primary cultures of glial and endothelial cells are important tools for basic and translational neuroscience research. Primary cell cultures are usually generated from rodent brain although considerable differences exist between human and rodent glia and endothelial cells. Because many translational research projects aim to identify mechanisms that eventually lead to diagnostic and therapeutic approaches to target human diseases, glia, and endothelial cultures are needed that better reflect the human central nervous system (CNS). Pig brain is easily accessible and, in many aspects, close to the human brain. We established an easy and cost-effective method to isolate and culture different primary glial and endothelial cells from adult pig brain. Oligodendrocyte, microglia, astrocyte, and endothelial primary cell cultures were generated from the same brain tissue and grown for up to 8 weeks. Primary cells showed lineage-specific morphology and expressed specific markers with a purity ranging from 60 to 95%. Cultured oligodendrocytes myelinated neurons and microglia secreted tumor necrosis factor alpha when induced with lipopolysaccharide. Endothelial cells showed typical tube formation when grown on Matrigel. Astrocytes enhanced survival of co-cultured neurons and were killed by Aquaporin-4 antibody positive sera from patients with Neuromyelitis optica. In summary, we established a new method for primary oligodendrocyte, microglia, endothelial and astrocyte cell cultures from pig brain that provide a tool for translational research on human CNS diseases.

Loss of homeoprotein Msx1 and Msx2 leading to athletic and kinematic impairment related to the increasing neural excitability of neurons in aberrant neocortex in mice

Although homeoproteins Msx1 and Msx2, the cell-specific transcription regulators, have been proven to play multiple roles in the embryogenesis of bone, muscle and tooth, the functions and mechanisms of Msx1 and Msx2 in the development of the central nervous system of mice after birth are not clear because of the death of Msx1 and Msx1/2 germline-deleted embryo at late gestation of mouse. In current research, Nestin-Cre mice was introduced to generate the central nervous system-specific knockout mice (Nestin-Cre;Msx1,Msx2fl/fl). We found that besides the falling of the body mass and the brain volume, the cortical tissue sections and staining showed the decreasing thickness of layer II-IV and declining number of vertebral cells in layer V resulting from Msx1/2 deletion. In addition, electrophysiological tests revealed the aberrant action potential parameters of deep pyramidal neurons in Nestin-Cre;Msx1,2 fl/fl mice, which may be related with the ethology impairment displayed in further experiments. We discovered Nestin-Cre;Msx1,2 fl/fl mice had severe impairment in their athletic ability and kinematic learning ability in rotate test, and exhibited hyperactivity in open-field test. Above all, our results revealed that deletion of homeoproteins Msx1 and Msx2 could lead to behavioral disorders and suggested that Msx1 and Msx2 played a crucial role in regulating the development and function of the neocortex. In addition, our current research provided a new mouse model for understanding the pathogenesis of human central nervous system disease.

Features of pancreatitis associated with ulcerative colitis: a case series analysis

Trimethyltin (TMT), a neurotoxic organotin compound, is selectively localized within the limbic system. The mechanisms of TMT-induced hippocampal neurodegeneration include inflammatory responses, oxidative stress, and neuronal death. Increasing evidence shows that the inflammatory response, mediated by activated inflammasomes, is involved in apoptosis and cellular dysfunction during brain injury. This study aimed to assess the role of the nucleotide-binding oligomerization domain-like receptor pyrin-domain-containing protein 3 (NLRP3) inflammasome in TMT-induced central nervous system (CNS) injury. In addition, the mechanisms underlying TMT neurotoxicity are similar to those involved in the pathogenesis of multiple neurodegenerative diseases; hence, a study on TMT cytotoxicity may be informative for the understanding of human CNS diseases. Microglia were significantly activated in the rat hippocampal dentate gyrus after TMT treatment. The mRNA expression of pro-inflammatory cytokines, interleukin-1β and interleukin-18, was induced both in vitro and in vivo. TMT treatment activated the NLRP3 inflammasome in the microglial cell line BV2. NLRP3 RNA interference significantly protected these cells from TMT-induced neuroinflammation. Our results demonstrate that the NLRP3 inflammasome is a key mediator of neuroinflammation and plays an important role in TMT-induced neuroinflammation.

NLRP3 inflammasome activation is involved in trimethyltin-induced neuroinflammation

Trimethyltin (TMT), a neurotoxic organotin compound, is selectively localized within the limbic system. The mechanisms of TMT-induced hippocampal neurodegeneration include inflammatory responses, oxidative stress, and neuronal death. Increasing evidence shows that the inflammatory response, mediated by activated inflammasomes, is involved in apoptosis and cellular dysfunction during brain injury. This study aimed to assess the role of the nucleotide-binding oligomerization domain-like receptor pyrin-domain-containing protein 3 (NLRP3) inflammasome in TMT-induced central nervous system (CNS) injury. In addition, the mechanisms underlying TMT neurotoxicity are similar to those involved in the pathogenesis of multiple neurodegenerative diseases; hence, a study on TMT cytotoxicity may be informative for the understanding of human CNS diseases. Microglia were significantly activated in the rat hippocampal dentate gyrus after TMT treatment. The mRNA expression of pro-inflammatory cytokines, interleukin-1β and interleukin-18, was induced both in vitro and in vivo. TMT treatment activated the NLRP3 inflammasome in the microglial cell line BV2. NLRP3 RNA interference significantly protected these cells from TMT-induced neuroinflammation. Our results demonstrate that the NLRP3 inflammasome is a key mediator of neuroinflammation and plays an important role in TMT-induced neuroinflammation.

Lymphatic system in central nervous system

The considerable metabolic activity of the central nervous system (CNS) requires an efficient system of tissue drainage and detoxification. The CNS is however devoid of lymphatic vessels, a vasculature ensuring interstitial fluid drainage and immune survey in other organs. A unique system of drainage has recently been identified between the cerebrospinal fluid (CSF), brain interstitial fluids and meningeal lymphatic vessels. This system is coupling a cerebral "glymphatic" flow with a meningeal lymphatic vasculature. The "glymphatic" system includes perivascular spaces and astrocytes, and drains interstitial fluids, from and towards the CSF. Meningeal lymphatic vessels are functionally linked to the cerebral "glymphatic" efflux by clearing intracerebral macromolecules and antigens towards the peripheral lymphatic system. The "glymphatic"-"meningeal lymphatics" system is potentially offering new therapeutic targets to improve cerebral drainage and immune survey in human CNS diseases.

Protective effects of the angiotensin II AT2 receptor agonist compound 21 in ischemic stroke: a nose-to-brain delivery approach

Significant neuroprotective effects of angiotensin II type 2 (AT2) receptor (AT2 receptor) agonists in ischemic stroke have been previously demonstrated in multiple studies. However, the routes of agonist application used in these pre-clinical studies, direct intracerebroventricular (ICV) and systemic administration, are unsuitable for translation into humans; in the latter case because AT2 receptor agonists are blood-brain barrier (BBB) impermeable. To circumvent this problem, in the current study we utilized the nose-to-brain (N2B) route of administration to bypass the BBB and deliver the selective AT2 receptor agonist Compound 21 (C21) to naïve rats or rats that had undergone endothelin 1 (ET-1)-induced ischemic stroke. The results obtained from the present study indicated that C21 applied N2B entered the cerebral cortex and striatum within 30 min in amounts that are therapeutically relevant (8.4-9 nM), regardless of whether BBB was intact or disintegrated. C21 was first applied N2B at 1.5 h after stroke indeed provided neuroprotection, as evidenced by a highly significant, 57% reduction in cerebral infarct size and significant improvements in Bederson and Garcia neurological scores. N2B-administered C21 did not affect blood pressure or heart rate. Thus, these data provide proof-of-principle for the idea that N2B application of an AT2 receptor agonist can exert neuroprotective actions when administered following ischemic stroke. Since N2B delivery of other agents has been shown to be effective in certain human central nervous system diseases, the N2B application of AT2 receptor agonists may become a viable mode of delivering these neuroprotective agents for human ischemic stroke patients.

Generation and Maintenance of Neural Progenitor Cell Lines Derived from Human Embryonic Stem Cells by Small Molecules

Background: A new research perspective for human neurodevelopment and neurological disease modeling involves the use of human embryonic stem cells (hESC). The development of robust protocols that yield adequate neural cell populations with definite regional identities were the prerequisites for these comparative studies. Materials and Methods: We used small molecules cocktail to generate two neural progenitor cell (NPC) lines from hESCs as following: the first experimental group included Noggin, Dorsomorphin, CHIR99021 and A83-01(NDCA) and the second group composed of Noggin, Dorsomorphin, CHIR99021 and SIS3 (NDCS). To validate our findings, we expanded both cell lines for over 20 passages in vitro and checked for chromosomal stability, as well as expressions of neural and regional identity markers by immunofluorescence staining. Gene expression analysis was quantified by RT-PCR at different passages up to passage 20. Results: Both cell lines proliferated in an adherent culture system in the presence of FGF2. They retained progenitor characteristics of NESTIN, SOX1, and PAX6 protein expression, formed rosette-like structures, and had the high neurogenic capacity. Importantly, the NPC populations in their first 10 passages expressed rostral markers (OTX2 and TH), and the next 10 passages (10-20) changed their specification toward the hindbrain where they expressed HOXA3and HOXB2, which correlated with a normal central nervous system development pattern. Conclusion: These NPCs offer a new system to study human central nervous system development and disease modeling of specific neurodegenerative diseases. [GMJ.2017;6(2):143-56]

Novel region of interest interrogation technique for diffusion tensor imaging analysis in the canine brain.

Purpose We describe a novel technique for measuring diffusion tensor imaging metrics in the canine brain. We hypothesized that a standard method for region of interest placement could be developed that is highly reproducible, with less than 10% difference in measurements between raters. Methods Two sets of canine brains (three seven-week-old full-brains and two 17-week-old single hemispheres) were scanned ex-vivo on a 7T small-animal magnetic resonance imaging system. Strict region of interest placement criteria were developed and then used by two raters to independently measure diffusion tensor imaging metrics within four different white-matter regions within each specimen. Average values of fractional anisotropy, radial diffusivity, and the three eigenvalues (λ1, λ2, and λ3) within each region in each specimen overall and within each individual image slice were compared between raters by calculating the percentage difference between raters for each metric. Results The mean percentage difference between raters for all diffusion tensor imaging metrics when pooled by each region and specimen was 1.44% (range: 0.01-5.17%). The mean percentage difference between raters for all diffusion tensor imaging metrics when compared by individual image slice was 2.23% (range: 0.75-4.58%) per hemisphere. Conclusion Our results indicate that the technique described is highly reproducible, even when applied to canine specimens of differing age, morphology, and image resolution. We propose this technique for future studies of diffusion tensor imaging analysis in canine brains and for cross-sectional and longitudinal studies of canine brain models of human central nervous system disease.