Non-human Primate Brain Tissue Research Articles

Integrative analysis of human and non-human primate brain tissue for alcohol consumption

Integrative analysis of human and non-human primate brain tissue for alcohol consumption

Preparation of Non-human Primate Brain Tissue for Pre-embedding Immunohistochemistry and Electron Microscopy.

Despite all the technological advances at the light microscopylevel, electron microscopy remains the only tool in neuroscience to examine and characterize ultrastructural and morphological details of neurons, such as synaptic contacts. Good preservation of brain tissue for electron microscopy can be obtained by rigorous cryo-fixation methods, but these techniques are rather costly and limit the use of immunolabeling, which is crucial to understand the connectivity of identified neuronal systems. Freeze-substitution methods have been developed to allow the combination of cryo-fixation with immunolabeling. However, the reproducibility of these methodological approaches usually relies on costly freezing devices. Moreover, achieving reliable results with this technique is very time-consuming and skill-challenging. Hence, the traditional chemically fixed brain, particularly with acrolein fixative, remains a time-efficient and low-cost method to combine electron microscopy with immunohistochemistry. Here, we provide a reliable experimental protocol using chemical acrolein fixation that leads to the preservation of primate brain tissue and is compatible with pre-embedding immunohistochemistry and transmission electron microscopic examination.

Preparation of Non-human Primate Brain Tissue for Pre-embedding Immunohistochemistry and Electron Microscopy

Preparation of Non-human Primate Brain Tissue for Pre-embedding Immunohistochemistry and Electron Microscopy

Constitutive and inducible expression of the neurokinin-1 receptor for the inflammatory neuropeptide substance P in human microglia and astrocytes

Abstract We have previously demonstrated that murine microglia and astrocytes express the neurokinin-1 high affinity receptor (NK-1R) for the neuropeptide substance P. Importantly, we have also shown that substance P/NK-1R interactions augment the inflammatory responses of isolated murine glia to bacterial challenge and exacerbate in vivo neuroinflammatory damage within the central nervous system in mouse models of meningitis. Here, we show the constitutive expression of NK-1R in acutely isolated non-human primate (NHP) brain tissue and ex vivo brain slices. Interestingly, NK-1R protein levels were increased in brain tissue as rapidly as 4 hours following ex vivo infection with Borrelia burgdorferi, the causative agent of Lyme neuroborreliosis. Furthermore, we observed higher NK-1R protein levels in NHP brain tissue at 2 and 4 weeks following in vivo infection, increases that correlated with decreases in the expression of glial fibrillary acidic protein. These in vivo changes were prevented by treatment with a specific NK-1R antagonist suggesting a key role for substance P/NK-1R interactions in these effects. We then investigated the expression of NK-1R in human glial cells. While human microglia showed robust NK-1R expression at rest, astrocytes constitutively expressed only low levels. However, challenge with bacteria or their products elicited rapid increases in whole cell and cell surface NK-1R protein expression by primary human astrocytes and cell lines. Together, these data indicate that human microglia and astrocytes show constitutive and/or inducible expression of the substance P receptor and may therefore be susceptible to this pro-inflammatory neuropeptide.

Discovery and Preclinical Validation of [(11)C]AZ13153556, a Novel Probe for the Histamine Type 3 Receptor.

The histamine type 3 receptor (H3) is a G protein-coupled receptor implicated in several disorders of the central nervous system. Herein, we describe the radiolabeling and preclinical evaluation of a candidate radioligand for the H3 receptor, 4-(1S,2S)-2-(4-cyclobutylpiperazine-1-carbonyl)cyclopropyl]-N-methyl-benzamide (5), and its comparison with one of the frontrunner radioligands for H3 imaging, namely, GSK189254 (1). Compounds 1 and 5 were radiolabeled with tritium and carbon-11 for in vitro and in vivo imaging experiments. The in vitro binding of [(3)H]1 and [(3)H]5 was examined by (i) saturation binding to rat and nonhuman primate brain tissue homogenate and (ii) in vitro autoradiography on tissue sections from rat, guinea pig, and human brain. The in vivo binding of [(11)C]1 and [(11)C]5 was examined by PET imaging in mice and nonhuman primates. Bmax values obtained from Scatchard analysis of [(3)H]1 and [(3)H]5 binding were in good agreement. Autoradiography with [(3)H]5 on rat, guinea pig, and human brain slices showed specific binding in regions known to be enhanced in H3 receptors, a high degree of colocalization with [(3)H]1, and virtually negligible nonspecific binding in tissue. PET measurements in mice and nonhuman primates demonstrated that [(11)C]5 binds specifically and reversibly to H3 receptors in vivo with low nonspecific binding in brain tissue. Whereas [(11)C]1 showed similar binding characteristics in vivo, the binding kinetics appeared faster for [(11)C]5 than for [(11)C]1. [(11)C]5 has suitable properties for quantification of H3 receptors in nonhuman primate brain and has the potential to offer improved binding kinetics in man compared to [(11)C]1.

Convection Enhanced Delivery: A Comparison of infusion characteristics in ex vivo and in vivo non-human primate brain tissue.

Convection enhanced delivery (CED) is emerging as a promising infusion toolto facilitate delivery of therapeutic agents into the brain via mechanically controlled pumps. Infusion protocols and catheter design have an important impact on delivery. CED is a valid alternative for systemic administration of agents in clinical trials for cell and gene therapies. Where gel and ex vivo models are not sufficient in modeling the disease, in vivo models allow researchers to better understand the underlying mechanisms of neuron degeneration, which is helpful in finding novel approaches to control the process or reverse the progression. Determining the risks, benefits, and efficacy of new gene therapies introduced via CED will pave a way to enter human clinical trial. The objective of this study is to compare volume distribution (Vd)/ volume infused (Vi) ratios and backflow measurements following CED infusions in ex vivo versus in vivo non-human primate brain tissue, based on infusion protocols developed in vitro. In ex vivo infusions, the first brain received 2 infusions using a balloon catheter at rates of 1 μL/min and 2 μL/min for 30 minutes. The second and third brains received infusions using a valve-tip (VT) catheter at 1 μL/min for 30 minutes. The fourth brain received a total of 45 μL infused at a rate of 1 μL/min for 15 minutes followed by 2 μL/min for 15 minutes. Imaging was performed (SPGR FA34) every 3 minutes. In the in vivo group, 4 subjects received a total of 8 infusions of 50 μL. Subjects 1 and 2 received infusions at 1.0 μL/min using a VT catheter in the left hemisphere and a smart-flow (SF) catheter in the right hemisphere. Subjects 3 and 4 each received 1 infusion in the left and right hemisphere at 1.0 μL/min. MRI calculations of Vd/Vi did not significantly differ from those obtained on post-mortem pathology. The mean measured Vd/Vi of in vivo (5.23 + /-1.67) compared to ex vivo (2.17 + /-1.39) demonstrated a significantly larger Vd/Vi for in vivo by 2.4 times (p = 0.0017). We detected higher ratios in the in vivo subjects than in ex vivo. This difference could be explained by the extra cellular space volume fraction. Studies evaluating backflow and morphology use in vivo tissue as a medium are recommended. Further investigation is warranted to evaluate the role blood pressure and heart rate may play in human CED clinical trials.

Immunocytochemical localization of Fos in perfused nonhuman primate brain tissue: fixation and antisera selection.

Immunocytochemical localization of immediate early gene proteins, such as Fos, provides a powerful tool with which to demonstrate activated neuronal populations in response to specific stimuli. In contrast to studies using rat brain tissue that consistently show good Fos detection with a variety of antisera, studies using brain tissue from other species yield variable Fos detection. This may be partly due to differences in Fos protein sequences among species or to perfusion and fixation methods. To determine the ability of various Fos antisera to detect neuronal activation in nonhuman primate tissue, we tested nine Fos antisera and compared these antibodies under conditions of intense or physiological stimulation. Monkey brain tissue was either perfused and postfixed with 4% paraformaldehyde or perfused with 4% paraformaldehyde and postfixed with 2.5% acrolein in 4% paraformaldehyde. In rat tissue, stained for comparison, several antisera resulted in good to excellent Fos detection. However, few antisera tested in monkey tissue resulted in excellent Fos staining. We demonstrate that detection of Fos in monkey brain tissue perfused with 4% paraformaldehyde can be improved by postfixation in a dilute acrolein solution. Our findings emphasize the importance of choosing appropriate antisera and perfusion-fixation procedures to optimize Fos detection in nonhuman primate tissue.