Name: extraction and dissection of the domesticated pig brain
Uploaded: 2021-04-25T14:00:00
Description: Watch this Scientific Journal Video about Extraction and Dissection of the Domesticated Pig Brain at JoVE.com

The authors would like to acknowledge Jim Knoblauch and Martin-Booth Hodges of the College of Agricultural, Consumer and Environmental Sciences Information Technology and Communication Services for their expertise in shooting, recording, and editing audio and video.

Procedures involving animal subjects have been approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Illinois at Urbana-Champaign
NOTE: Prior to euthanasia, the pig was anesthetized via intramuscular injection with a combination of telazol:ketamine:xylazine (50.0 mg of tiletamine HCl plus 50.0 mg of zolazepam HCl reconstituted with 2.50 mL of ketamine HCl (100 g/L) and 2.50 mL of xylazine (100 g/L) and administered at 0.06 mg/kg BW). Once anesthetized, the pi...

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Suckling+Piglet+as+an+Agrimedical+Model+for+the+Study+of+Pediatric+Nutrition+and+Metabolism.">The Suckling Piglet as an Agrimedical Model for the Study of Pediatric Nutrition and Metabolism.</a> Annual Review of Animal Biosciences. 2 (1), 419-444 (2014).</li><li>Larsen, M. O., Rolin, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+the+G&#246;ttingen+minipig+as+a+model+of+diabetes,+with+special+focus+on+type+1+diabetes+research.">Use of the G&#246;ttingen minipig as a model of diabetes, with special focus on type 1 diabetes research.</a> ILAR Journal. 45 (3), 303-313 (2004).</li><li>Lehmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+minipig+in+general+toxicology.">The minipig in general toxicology.</a> Scandinavian Journal of Laboratory Animal Science. 25, 59-62 (1998).</li><li>Richer, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sacrococcygeal+and+transsacral+epidural+anesthesia+in+the+laboratory+pig.">Sacrococcygeal and transsacral epidural anesthesia in the laboratory pig.</a> Surgical Radiologic Anatomy. 20, 431-435 (1998).</li><li>Lind, N. 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Alternative for neurosurgical education.</a> Acta Cirurgica Brasileira. 29 (6), 405-409 (2014).</li><li>Bassi, T., Rohrs, E., Fernandez, K., Ornowska, M., Reynolds, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+brain+excision:+An+easier+method+to+harvest+the+pig's+brain.">Direct brain excision: An easier method to harvest the pig's brain.</a> Interdisciplinary Neurosurgery. 14, 37-38 (2018).</li><li>Bjarkam, C. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exposure+of+the+pig+CNS+for+histological+analysis:+A+manual+for+decapitation,+skull+opening,+and+brain+removal.">Exposure of the pig CNS for histological analysis: A manual for decapitation, skull opening, and brain removal.</a> Journal of Visualized Experiments. 122, e55511 (2017).</li><li>Pascalau, R., Szabo, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibre+dissection+and+sectional+study+of+the+major+porcine+cerebral+white+matter+tracts.">Fibre dissection and sectional study of the major porcine cerebral white matter tracts.</a> Anatomia, Histologia, Embryologia. 46, 378-390 (2017).</li><li>McFadden, W. 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The techniques described herein were designed for pigs approximately 4 weeks of age. It is critical to perform these steps immediately after the pig has been humanely euthanized to ensure the integrity of brain tissue structure is maintained, especially when considering subsequent biochemical assays. It is helpful to use an atlas or fiber16 dissection guides when first learning the techniques. It is recommended that the experimenter practice several brain extractions and dissections prior to obtai...

The pig has been well documented and accepted as a translatable animal model for research in cardiovascular systems1, gastrointestinal systems2, nutrition3,4, diabetes5, toxicology6, and surgical techniques7. Use of the pig in neuroscience is beginning to increase, as PubMed searches for the keywords &#34;swine brain animal model&#34; result in four-times more results from 1996-2005 than the preceding 10 year period8, and even more results at present. A primary reason that the popularity of the pig model is expanding is due to its similarities in growth, structure, and function of the brain when compared with humans. In comparison to the human brain, the pig brain exhibits similar gyral patterning, vascularization and distribution of gray and white matter9. Moreover, the pig brain has been used in neuroimaging procedures, evoked potential recording, and in establishing neurosurgery techniques8,9. Unlike other animal models, however, the pig and human experience perinatal brain growth spurts, as opposed to pre- or post-natal growth spurts. At birth, the human and pig brain weigh approximately 27 and 25 percent of their adult brain weight, respectively, compared to the rat brain that weighs 12 percent of its adult brain weight and the rhesus monkey brain at 76 percent of adult weight10.
One reason the pig has been only slowly adopted as an animal model for neuroscience is because many researchers are unfamiliar with the animal in this context. Researchers may not be aware of its potential uses in the field or may not know the proper techniques required to use such a model. As use of the pig as a biomedical and preclinical model gains attention and use in neuroscience, it is necessary to establish standardized procedures of tissue removal to ensure accurate comparison of data across studies. Although dissection and surgical techniques involving the pig brain have been published elsewhere11,12,13, there is a need for simple and standardized protocols to collect pig brain tissue, especially for use in biochemical assays. As such, the aim of this video is to provide the knowledge necessary to allow researchers to perform a standardized brain extraction and dissection. This video illustrates one proper technique to remove the pig brain while keeping the cortex and brainstem intact, and subsequently review methods to dissect several key brain regions.

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extraction and dissection of the domesticated pig brain

Use of the pig as a preclinical and translatable animal model has been well-documented and accepted by research fields investigating cardiovascular systems, gastrointestinal systems, and nutrition, and the pig is increasingly being used as a large animal model in neuroscience. Furthermore, the pig is an accepted model to study neurodevelopment as it displays brain growth and development patterns similar to what occurs in humans. As a less common animal model in neuroscience, surgical and dissection procedures on pigs may not be as familiar or well-practiced among researchers. Therefore, a standardized visual protocol detailing consistent extraction and dissection methods may prove valuable for researchers working with the pig. The following video showcases a technique to remove the pig brain while keeping the cortex and brainstem intact and reviews methods to dissect several commonly investigated brain regions including the brainstem, cerebellum, midbrain, hippocampus, striatum, thalamus, and medial prefrontal cortex. The purpose of this video is to provide researchers with the tools and knowledge necessary to consistently perform a brain extraction and dissection on the four-week-old pig.

This section describes examples of results obtained after correct extraction and dissection of a 4-week-old pig brain. Figure 1 outlines the shape of each brain region for use as a guide during dissection. Part of the brainstem may remain in the skull after removal of the cerebellum (Figure 1B). This can be removed while isolating the desired region of the cerebellum. Table 1 displays the average weight (mean &#177; standard error of the mean) f...

Watch this Scientific Journal Video about Extraction and Dissection of the Domesticated Pig Brain at JoVE.com

Extraction and Dissection of the Domesticated Pig Brain

This protocol details the technique for removal of the pig brain in its entirety and dissection of several brain regions commonly studied in neuroscience.

Use of the pig as a preclinical and translatable animal model has been well-documented and accepted by research fields investigating cardiovascular ...

The following video is a demonstration of removal and dissection of the pig brain. It may be helpful to refer to an atlas, or fiber dissection guide before or during the procedure. A large butcher knife is best for decapitation as it will allow for one clean cut between the atlas and axis.Ensure the knife is sharp before starting with a new subject. A meat hook is used to remove the skull. A long slender tool such as a scalpel handle is used to remove the brain from the skull.A vice with screws or protrusions is necessary to secure the head while cutting the skull. Once the animal has been humanely euthanized according to established protocols proceed as follows. Cut just above the nape of the neck, between the shoulders and the top of the head.Secure the pig and move the years out of the way during decapitation. Use a scalpel to make incisions to separate the skin from the skull. Make an initial cut on the middle of the skull that continues to the posterior of the head.The second transverse cut should be on the posterior of the head. The third cut also transverse will start at the posterior of this snout, and inline with the eyes to ensure easy access to the skull. Use a bone saw to make to anterior to posterior cuts lateral to the midline.Start by lightly marking the area for the cut with the saw, and then begin sawing until you meet less resistance from the skull. Slightly bevel the saw toward the midline while cutting as this will ensure that you are cutting the thinnest part of the skull. If the cut is too close to the ear it will be difficult to remove the skull.But if the cut is too close to the midline the opening will be too small to remove the brain. Repeat this process on the perpendicular sides to create a rectangular window. Ensure that each corner of the window is completely cut, so that part of the skull can be removed to reveal the brain beneath.Next, use a meat hook or curved instrument to pry the rectangular skull section out of position. Start by placing the hook into one of the corners, and applying pressure to pop the skullcap off. In some cases, the meninges will remain on the brain.And if this occurs carefully use forceps and blunt scissors, or alternatively a scalpel to remove the meninges without cutting the brain. For reference the brain should look as follows before and after the meningeal layers have been removed. Cut through the muscle and fat on the posterior of the head to prepare for exposure of the cerebellum.To expose the cerebellum make two transverse cuts along the posterior of the skull. When making these cuts take care to not cut into the exposed brain. Once the cuts have been made place one hand on the snout, and one on the skull, and apply backwards pressure to pull the posterior portion of the skull away from the rest of the head.As highlighted here, the cerebellum is tucked behind the remaining meningeal layers. Use the end of a blunt instrument like a scalpel handle to carefully separate the brain from the skull, taking care to avoid damaging the brain in the process. Slightly invert the head to help coax the brain out of the skull, and use a gentle scooping motion to sever the cranial nerves and remove the brain.This process requires patience and practice to eliminate all remaining connections between the brain and cranial cavity. Inverting the pig's head can be helpful during this process. Continue using sweeping motions with the blunt instrument to free the brain.As highlighted here, the cerebellum and brainstem may be difficult to remove as they are tucked into the skull cavity. Take care when removing the cerebellum and medulla, or brainstem. The brain should fall out on its own when it is completely detached from the skull.The skull cavity should look as shown after removal of the brain. In preparation for brain region dissection ensure pre-labeled containers are organized, and in close proximity. Example preservation methods include snap freezing samples in liquid nitrogen, or submerging them in a preservative solution.Once extracted from the cranial cavity the whole brain should look as follows. To remove the medulla or brainstem, make a coronal cut caudal to the cerebellum. To remove the cerebellum, make a coronal cut posterior to the cortex.To separate the hemispheres make a midsagittal cut along the longitudinal fissure using a continuous motion to prevent damage. For the purposes of this video we will only show dissections from the right hemisphere. To remove the midbrain look for the superior, and inferior colliculi which are two bumps on the posterior portion of the brain.Remove the desired amount of brain tissue just ventral to these regions. To remove the hippocampus position the scalpel handle on the posterior of the corpus callosum, and gently roll the hippocampus out. The entire hippocampus horn will be bean shaped.The hippocampus should easily roll out when scooping your finger in a j-motion, starting from the posterior corpus callosum. To remove the striatum or caudate nucleus clear away the anterior portion of the corpus callosum. The caudate nucleus is the striated or striped region that's colored gray and white and located just below the corpus callosum.With the scalpel handle angled remove this region which predominantly includes the caudate nucleus. After removing this tissue you should be able to see a striated pattern where the brain region had been located. To remove the medial prefrontal cortex dissected tissue from the frontal gyrus to the corpus callosum taking the most medial portion of that section.The right cortex should remain after removal of the medial prefrontal cortex. To remove the thalamus, remove the bulb like structure at the center of the midsagittal surface. The thalamus has an obvious spherical shape, and it will look slightly darker than the surrounding tissue.Upon completion of the dissection tissue samples should be preserved properly based on specific experimental outcomes. Shown here are the dissected brain regions, including the relative size and shape expected when working with young pigs. Upon mastering these techniques, the entire brain extraction, and dissection procedure will take less than 15 minutes per subject, and produce consistent results.

extraction-and-dissection-of-the-domesticated-pig-brain

Research

JoVE Journal

Neuroscience

A Visual Description of the Dissection of the Cerebral Surface Vasculature and Associated Meninges and the Choroid Plexus from Rat Brain

This video presentation was created to show a method of harvesting the two most important highly vascular structures, not residing within the brain proper, that support forebrain function. They are the cerebral surface (superficial) vasculature along with associated meninges (MAV) and the choroid plexus which are necessary for cerebral blood flow and cerebrospinal fluid (CSF) homeostasis. The tissue harvested is suitable for biochemical and physiological analysis, and the MAV has been shown to be sensitive to damage produced by amphetamine and hyperthermia 1,2. As well, the major and minor cerebral vasculatures harvested in MAV are of potentially high interest when investigating concussive types of head trauma. The MAV dissected in this presentation consists of the pial and some of the arachnoid membrane (less dura) of the meninges and the major and minor cerebral surface vasculature. The choroid plexus dissected is the structure that resides in the lateral ventricles as described by Oldfield and McKinley3,4,5,6. The methods used for harvesting these two tissues also facilitate the harvesting of regional cortical tissue devoid of meninges and larger cerebral surface vasculature, and is compatible with harvesting other brain tissues such as striatum, hypothalamus, hippocampus, etc. The dissection of the two tissues takes from 5 to 10 min total. The gene expression levels for the dissected MAV and choroid plexus, as shown and described in this presentation can be found at GSE23093 (MAV) and GSE29733 (choroid plexus) at the NCBI GEO repository. This data has been, and is being, used to help further understand the functioning of the MAV and choroid plexus and how neurotoxic events such as severe hyperthermia and AMPH adversely affect their function.

This video presentation shows a method of harvesting the two most important highly vascular structures that support forebrain function. They are the cerebral surface (superficial) vasculature along with associated meninges (MAV) and the choroid plexus which are necessary for cerebral blood flow and cerebrospinal fluid (CSF) homeostasis.

This video presentation was created to show a method of harvesting the two most important highly vascular structures, not residing within the brain ...

Consensus Brain-derived Protein, Extraction Protocol for the Study of Human and Murine Brain Proteome Using Both 2D-DIGE and Mini 2DE Immunoblotting

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or pathological conditions. Basically, this technique is based on the separation of proteins according to their isoelectric point in a first step, and secondly according to their molecular weights by SDS polyacrylamide gel electrophoresis (SDS-PAGE). In this report an optimized sample preparation protocol for little amount of human post-mortem and mouse brain tissue is described. This method enables to perform both two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mini 2DE immunoblotting. The combination of these approaches allows one to not only find new proteins and/or protein modifications in their expression thanks to its compatibility with mass spectrometry detection, but also a new insight into markers validation. Thus, mini-2DE coupled to western blotting permits to identify and validate post-translational modifications, proteins catabolism and provides a qualitative comparison among different conditions and/or treatments. Herein, we provide a method to study components of protein aggregates found in AD and Lewy body dementia such as the amyloid-beta peptide and the alpha-synuclein. Our method can thus be adapted for the analysis of the proteome and insoluble proteins extract from human brain tissue and mice models too. In parallel, it may provide useful information for the study of molecular and cellular pathways involved in neurodegenerative diseases as well as potential novel biomarkers and therapeutic targets.

A common protein extraction protocol using urea/thiourea/SDS buffer for human and mice brain tissue allows indentification of proteins by 2D-DIGE and their subsequent characterization by mini 2DE immunoblotting. This method enables one to obtain more reproducible and reliable results from human biopsies and experimental models.

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or ...

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the cochlea.

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated proteins and subsequent manipulation of neural activity by light. Channelrhodopsins (ChRs) are light-gated cation-channels and when fused to a fluorescent protein their expression allows for visualization and concurrent activation of specific cell types and their axonal projections in defined areas of the brain. Via stereotactic injection of viral vectors, ChR fusion proteins can be constitutively or conditionally expressed in specific cells of a defined brain region, and their axonal projections can subsequently be studied anatomically and functionally via ex vivo optogenetic activation in brain slices. This is of particular importance when aiming to understand synaptic properties of connections that could not be addressed with conventional electrical stimulation approaches, or in identifying novel afferent and efferent connectivity that was previously poorly understood. Here, a few examples illustrate how this technique can be applied to investigate these questions to elucidating fear-related circuits in the amygdala. The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories. Many lines of evidence suggest that the medial prefrontal cortex (mPFC) participates in different aspects of fear acquisition and extinction, but its precise connectivity with the amygdala is just starting to be understood. First, it is shown how ex vivo optogenetic activation can be used to study aspects of synaptic communication between mPFC afferents and target cells in the basolateral amygdala (BLA). Furthermore, it is illustrated how this ex vivo optogenetic approach can be applied to assess novel connectivity patterns using a group of GABAergic neurons in the amygdala, the paracapsular intercalated cell cluster (mpITC), as an example.

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that&#160;upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related circuits.

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

Exposure of the Pig CNS for Histological Analysis: A Manual for Decapitation, Skull Opening, and Brain Removal

Pigs have become increasingly popular in large-animal translational neuroscience research as an economically and ethically feasible substitute to non-human primates. The large brain size of the pig allows the use of conventional clinical brain imagers and the direct use and testing of neurosurgical procedures and equipment from the human clinic. Further macroscopic and histological analysis, however, requires postmortem exposure of the pig central nervous system (CNS) and subsequent brain removal. This is not an easy task, as the pig CNS is encapsulated by a thick, bony skull and spinal column. The goal of this paper and instructional video is to describe how to expose and remove the postmortem pig brain and the pituitary gland in an intact state, suitable for subsequent macroscopic and histological analysis.

The goal of this paper and instructional video is to describe how to expose and remove the postmortem pig brain and pituitary gland in an intact state, suitable for subsequent macroscopic and histological analysis.

Pigs have become increasingly popular in large-animal translational neuroscience research as an economically and ethically feasible substitute to ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is important to isolate these cells without changing their gene expression profile. The zebrafish model provides a large number of transgenic fish lines in which specific cell types are labelled; for example neurons in the NBT:DsRed line or macrophages/microglia in the mpeg1:eGFP line. Furthermore, antibodies have been developed to stain specific cells, such as microglia with the 4C4 antibody.
Here, we describe the isolation of neurons, macrophages and microglia from larval zebrafish brains. Central to this protocol is the avoidance of an enzymatic tissue digestion at 37 &#176;C, which could modify cellular profiles. Instead a mechanical system of tissue homogenization at 4 &#176;C is used. This protocol entails homogenization of brains into cell suspension, their immuno-staining and the isolation of neurons, macrophages and microglia by FACS. Afterwards, we extracted RNA from those cells and evaluated their quality/quantity. We managed to obtain RNA of high quality (RNA Integrity Number (RIN) &#62; 7) to perform qPCR on macrophages/microglia and neurons, and transcriptomic analysis on microglia. This approach enables a better characterization of these cells, as well as a clearer understanding about their role in development and pathologies.

We present a protocol to isolate neurons, macrophages and microglia from larval zebrafish brains under physiological and pathological conditions. Upon isolation, RNA is extracted from these cells to analyze their gene expression profile. This protocol allows for the collection of high-quality RNA for performing downstream analysis like qPCR and transcriptomics.

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is ...

Dissection and Isolation of Murine Glia from Multiple Central Nervous System Regions

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine neonates for the isolation of glial subpopulations. The purpose of the procedure is to dissociate microglia, oligodendrocyte progenitor cells (OPCs), and astrocytes from cortical, cerebellar, brainstem, and spinal cord tissue to facilitate further in vitro analysis. The CNS region isolation procedures allow for the determination of regional heterogeneity among glia in multiple cell culture systems. Rapid CNS region isolation is performed, followed by the mechanical removal of meninges to prevent meningeal cell contamination of glia. This protocol combines gentle tissue dissociation and plating on a specified matrix designed to preserve cell integrity and adherence. Isolating mixed glia from multiple CNS regions provides a comprehensive analysis of potentially heterogenous glia while maximizing the use of individual experimental animals. Additionally, following dissociation of regional tissue, mixed glia are further divided into multiple cell types including microglia, OPCs, and astrocytes for use in either single cell type, cell culture plate inserts, or co-culture systems. Overall, the demonstrated techniques provide a comprehensive protocol of broad applicability for careful dissection of four individual CNS regions from murine neonates and includes methods for the isolation of three individual glia cell types to examine regional heterogeneity in any number of in vitro cell culture systems or assays.

Here we present a protocol for in vitro isolation of multiple glial cell populations from a mouse CNS. This method allows for the segregation of regional microglia, oligodendrocyte precursor cells, and astrocytes to study the phenotypes of each in a variety of culture systems.

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine ...

Magnetic Resonance-Guided Stereotaxy for Infusions to the Pig Brain

The overall goal of this procedure is to perform stereotaxy in the pig brain with real-time magnetic resonance (MR) visualization guidance to provide precise infusions. The subject was positioned prone in the MR bore for optimal access to the top of the skull with the torso raised, the neck flexed, and the head inclined downward. Two anchor pins anchored on the bilateral zygoma held the head steady using the head holder. A magnetic resonance imaging (MRI) flex-coil was placed rostrally across the head holder so that the skull was accessible for the intervention procedure. A planning grid placed on the scalp was used to determine the appropriate entry point of the cannula. The stereotactic frame was secured and aligned iteratively through software projection until the projected radial error was less than 0.5 mm. A hand drill was used to create a burr hole for insertion of the cannula. A gadolinium-enhanced co-infusion was used to visualize the infusion of a cell suspension. Repeated T1-weighted MRI scans were registered in real time during the agent delivery process to visualize the volume of gadolinium distribution. MRI-guided stereotaxy allows for precise and controlled infusion into the pig brain, with concurrent monitoring of cannula insertion accuracy and determination of the agent volume of distribution.

The protocol presented here demonstrates stereotaxy to the pig brain using convection-enhanced infusions, with real-time magnetic resonance imaging (MRI) visualization guidance and real-time infusion distribution visualization.

The overall goal of this procedure is to perform stereotaxy in the pig brain with real-time magnetic resonance (MR) visualization guidance to provide ...

Live Chick Embryo Brain Dissection and Vibratome Slicing

Use an egg candler to trace along the perimeter of the air pocket above the E14 or E15 embryo with a pencil and cover the outlined area with transparent tape. Using curved or fine scissors, gently cut around the traced area. Be careful not to cut into the embryo membrane or blood vessels.Then discard the top piece of the shell. After decapitating the chick embryo, place the head in a 10 centimeter dish with a cold, sterile CMF solution. Using sterile fine forceps, peel away the skin covering the brain to reveal the underlying dura mater.Remove the forming skull bones on the left and right sides of the brain. The forming skull bones do not yet cover the majority of the brain. Gently use fine pointed forceps to tear through the meninges, overlying the center of the brain and peel it to each side to uncover the brain.Using fine curved forceps, scoop the brain up from the bottom front and gently pull it out of its cavity. Dissect the brain into its three main parts forebrain, midbrain, or optic tecta and cerebellum. Remove the overlying pia mater from the tecta using fine forceps and place the dissected brain regions in a small sterile dish on ice.For embedding and slicing the brain, gently pick up either the optic tectum or forebrain region with curved forceps as a scoop and blot slightly on sterile gauze to remove extra liquid. Prepare a simple mold by forming aluminum foil around an appropriately sized object. Here, a small rectangular metal vibrating tissue slicer block is used as the object.Use a sterile transfer pipette and fill the mold with low melt agarose. Quickly place the brain region in agarose and let it solidify for approximately four to five minutes on ice. Cut the sections on a vibrating tissue slicer using a sapphire knife.Use the sterile spatula to scoop up and remove the slice from the tray. Gently slide the section off the spatula and onto a membrane insert using another sterile spatula. Place the six well plate of brain slices on membrane inserts in the cell culture incubator at 37 degrees Celsius and 5%carbon dioxide.On the day after platting, use a sterile Pasteur pipette to aspirate the media from below the insert and add one milliliter of fresh slice media to each well underneath the membrane insert. Next, to introduce the GBM cells onto the brain slices, remove one to several spheroids from their culture dish using a 20 microliter micro pipette set to five microliters. Gently expel the media with spheroids onto the desired brain slice and allow the spheroids to culture on the slice for two to five days.The viability results of x vivo optic tectum slices after one week in culture are shown in this figure. The confocal images of a brain slice stained for nuclei with bisbenzimide and immunostained for laminin clearly shows normal and intact blood vessels optically sectioned in various configurations. The maximum projection image of the confocal Z stack of the brain slice stained for SOX 2 transcription factor and total nuclei with bisbenzimide is shown here.These results suggest that chick embryo brain slices could be cultured on membrane inserts for approximately two weeks and remain viable with normal appearing blood vessels and transcription factor expression.