Long-Term Culture of Organotypic Hippocampal Slice from Old 3xTg-AD Mouse: An ex vivo Model of Alzheimer's Disease

OBJECTIVE: Conventional methods for organotypic hippocampal tissue slice culture (OHSC) have shown several disadvantages or limitations regarding age of animals used, duration of culture and difficulty using neurodegenerative models. Therefore, we tried to establish OHSC from old 3xTg-Alzheimer’s disease (AD) mice for longer period (over 4 weeks) and to validate utility of this system as a valid platform for translational neuroscience of AD. METHODS: OHSC was performed with old 3xTg-AD mice (12–14 months), old wild type mice (12–14 months) and young 3xTg-AD mice (2–4 months) using serum-free medium for 4 weeks. Hippocampal structure was evaluated by 4’, 6-diamidino-2-phenylindole (DAPI) intensity and neuronal metabolism was measured by Alamarblue assay. Pathologic characteristics of AD were also investigated; β-amyloid levels by ELISA, amyloid plaque deposition by Thioflavin-S staining, and glial activation by immunohistochemistry. RESULTS: Following 4-week culture in serum-free media, hippocampal cells and layers were well preserved in cultured slices from old AD mice as was in those from young AD and old wild type mice. On the contrary, excessive regression of total visible cells was observed in conventional serum-containing medium regardless of genotype of mice. In parallel with this well preserved structure, major pathologic characteristics of AD were also well manifested in hippocampal slices from old AD mice. CONCLUSION: Our findings suggest that long-term OHSC from old 3xTg-AD mouse can serve as a promising ex vivo system for studies on pathophysiology of AD, especially with the minimum number of sacrifice of experimental animals.

Organotypic Spinal Cord Slice Culture to Study Neural Stem/Progenitor Cell Microenvironment in the Injured Spinal Cord

The molecular microenvironment of the injured spinal cord does not support survival and differentiation of either grafted or endogenous NSCs, restricting the effectiveness of the NSC-based cell replacement strategy. Studying the biology of NSCs in in vivo usually requires a considerable amount of time and cost, and the complexity of the in vivo system makes it difficult to identify individual environmental factors. The present study sought to establish the organotypic spinal cord slice culture that closely mimics the in vivo environment. The cultured spinal cord slices preserved the cytoarchitecture consisting of neurons in the gray matter and interspersed glial cells. The majority of focally applied exogenous NSCs survived up to 4 weeks. Pre-exposure of the cultured slices to a hypoxic chamber markedly reduced the survival of seeded NSCs on the slices. Differentiation into mature neurons was severely limited in this co-culture system. Endogenous neural progenitor cells were marked by BrdU incorporation, and applying an inflammatory cytokine IL-1beta significantly increased the extent of endogenous neural progenitors with the oligodendrocytic lineage. The present study shows that the organotypic spinal cord slice culture can be properly utilized to study molecular factors from the post-injury microenvironment affecting NSCs in the injured spinal cord.

Organotypic slice culture of the hypothalamic paraventricular nucleus of rat

Organotypic slice cultures have been developed as an alternative to acute brain slices because the neuronal viability and synaptic connectivity in these cultures can be preserved well for a prolonged period of time. This study evaluated a stationary organotypic slice culture developed for the hypothalamic paraventricular nucleus (PVN) of rat. The results showed that the slice cultures maintain the typical shape of the nucleus, the immunocytochemical signals for oxytocin, vasopressin, and corticotropin-releasing hormone, and the electrophysiological properties of PVN neurons for up to 3 weeks in vitro. The PVN neurons in the culture expressed the green fluorescent protein gene that had been delivered by the adenoviral vectors. The results indicate that the cultured slices preserve the properties of the PVN neurons, and can be used in longterm studies on these neurons in vitro.

Organotypic Slice Culture of Rat Hippocampus for Neuroscience Research / 대한소아신경학회지

PURPOSE: Organotypic slice cultures are suitable for morphological and electrophysiological studies, and a valuable method to evaluate physiological and pathological responses to external stimuli. This study was designed to establish a method and to assess its values of organotypic slice cultures of rats' hippocampus for neuroscience research. METHODS: 8-day-old neonatal Sprague-Dawley rat's brain was dissected quickly and the brain was removed. Both of the hippocampi were dissected out in Petri dishes and placed on a chopper or tissue cutter with Gey's balanced salt solution(GBSS). Slices of 450 micrometer were cut and separated by adding of a drop of GBBS. Extra GBBS was aspirated. Transfer tissue slices of Millicell membrane were inserted in six-well plates containing 1 mL of warmed Gahwiler's media. Six-well plates were placed in an incubator at 36 degrees C with 5% CO2, and the media were changed regularly every 2 to 3 days. Some tissues were placed in 4% paraformaldehyde for staining and others were placed in Phosphate buffered saline(PBS) for 30 min for Western blotting. Then, we stained the free-floating, cryocutting or paraffin embedded slices with H&E or the days of 6, 9, 14 and 24 in vitro(DIV), and evaluated any changes of tissues and neurons by an image analysis system RESULTS: Hippocampal cultures had well-defined cell body layers of dentate gyrus, CA1, 2, 3 and 4 as early as the day of 6 in vitro(DIV). After 14 DIV, the cultures became gradually thinner from 450 micrometer to 150 micrometer. After 21 DIV, there were migrations of cells away from the margins of the slices and degenerative changes of some neuronal cells occurred. But pyramidal cells always were organized in well-defined cellular layer even after several weeks in the culture. Therefore, the best results were obtained by culturing slices of 6 to 14 DIV. Slice cutures was maintained after 4 weeks in vitro, but the oppotunity of contamination and infection increased as the periods of cultures trolonged. In staining, after any tissue was cultured in 2 weeks in vitro, no differentiation of the morphology and distribution in dentate gyrus, CA1, 2 and 3 were seen. In organo-typic slice culture of rats' hippocampus, we witnessed growth of glial and neuronal cell, and found pyramidal and granular cells. NeuN proteins were identified by Western blotting, and the density of NeuN protein bands was at the maximum value on the day of 9 in vitro. CONCLUSION: Since the organotypic slice cultures of rats' hippocampus were similar to the hippocampus in vivo in terms of anatomical and cellular morphology, it will become a valuable method for neuroscience research.

Organotypic Slice Culture of Rat Hippocampus for Neuroscience Research / 대한소아신경학회지

PURPOSE: Organotypic slice cultures are suitable for morphological and electrophysiological studies, and a valuable method to evaluate physiological and pathological responses to external stimuli. This study was designed to establish a method and to assess its values of organotypic slice cultures of rats' hippocampus for neuroscience research. METHODS: 8-day-old neonatal Sprague-Dawley rat's brain was dissected quickly and the brain was removed. Both of the hippocampi were dissected out in Petri dishes and placed on a chopper or tissue cutter with Gey's balanced salt solution(GBSS). Slices of 450 micrometer were cut and separated by adding of a drop of GBBS. Extra GBBS was aspirated. Transfer tissue slices of Millicell membrane were inserted in six-well plates containing 1 mL of warmed Gahwiler's media. Six-well plates were placed in an incubator at 36 degrees C with 5% CO2, and the media were changed regularly every 2 to 3 days. Some tissues were placed in 4% paraformaldehyde for staining and others were placed in Phosphate buffered saline(PBS) for 30 min for Western blotting. Then, we stained the free-floating, cryocutting or paraffin embedded slices with H&E or the days of 6, 9, 14 and 24 in vitro(DIV), and evaluated any changes of tissues and neurons by an image analysis system RESULTS: Hippocampal cultures had well-defined cell body layers of dentate gyrus, CA1, 2, 3 and 4 as early as the day of 6 in vitro(DIV). After 14 DIV, the cultures became gradually thinner from 450 micrometer to 150 micrometer. After 21 DIV, there were migrations of cells away from the margins of the slices and degenerative changes of some neuronal cells occurred. But pyramidal cells always were organized in well-defined cellular layer even after several weeks in the culture. Therefore, the best results were obtained by culturing slices of 6 to 14 DIV. Slice cutures was maintained after 4 weeks in vitro, but the oppotunity of contamination and infection increased as the periods of cultures trolonged. In staining, after any tissue was cultured in 2 weeks in vitro, no differentiation of the morphology and distribution in dentate gyrus, CA1, 2 and 3 were seen. In organo-typic slice culture of rats' hippocampus, we witnessed growth of glial and neuronal cell, and found pyramidal and granular cells. NeuN proteins were identified by Western blotting, and the density of NeuN protein bands was at the maximum value on the day of 9 in vitro. CONCLUSION: Since the organotypic slice cultures of rats' hippocampus were similar to the hippocampus in vivo in terms of anatomical and cellular morphology, it will become a valuable method for neuroscience research.