Adult spinal cord mixed cultures were first developed using rats with grant support from an NIH/NIDA R01 award (PI De Leo) and an NIH T32 training grant (PI Green). These methods were further adapted for mouse spinal cords with support from the following grants: NIH/NIDA 5K01DA023503 (PI Cao), NIH/NINDS 5R21NS066130 (PI Cao), and NIH/NIGMS P20GM103643 (PI Meng). The authors would also like to thank Dr. Alejandro M. S. Mayer, Department of Pharmacology, Chicago College of Osteopathic Medicine, for his technical help in obtaining microglia-rich mixed glial cells.

The following protocol was approved by the Institutional Animal Care and Use Committee (IACUC) at the University of New England. The following protocol is for preparing glial cultures from 4 adult mouse spinal cords.
1. Preparation of Solutions in a Culture Hood under Aseptic Conditions
<ol>
	<li>Prepare culture media: complete Dulbecco&#39;s modification of Eagle&#39;s media (cDMEM) containing DMEM (with 4.5 g/L glucose), 10% Fetal Bovine Serum (FBS), 2 mM L-glutamine, 100 IU/mL penicillin, 100 mg/mL streptomycin, 250 ng/mL Amphotericin B, and 50 &#181;M 2-mercaptoethanol (2-ME; see 1.1.1). Mix and filter sterilize all other components and then add FBS. Store the culture media at 4 oC.
	<ol>
		<li>Prepare 50 mM 2-ME/phosphate-buffered saline (PBS) stock solution: add 100 &#181;L of concentrated 2-ME (14.3 M) into 28.6 mL of 1x PBS, and filter sterilize (the solution can be stored at 4 oC for several months). Use 1 mL of 2-ME stock solution for every 1,000 mL of culture media.</li>
	</ol>
	</li>
	<li>Preparation of the density gradient media
	<ol>
		<li>Prepare a stock isotonic density gradient media solution (e.g., 100% Percoll) from 1 part 10x regular sterile PBS and 9 parts sterile stock density gradient media (e.g., Percoll).</li>
		<li>Dilute the 100% density gradient media (made in 1.2.1) with regular 1x sterile PBS to make a 20% density gradient media (e.g., 10 mL of 100% density gradient media with 40 mL of 1x PBS) and store it at 4 oC. Bring the 20% density gradient media to room temperature (RT) before preparing the cell culture.</li>
	</ol>
	</li>
	<li>Preparation of solutions from the papain dissociation system 
	NOTE: The papain dissociation system can be identified in the Materials Table. Other similar dissociation kits (including those assembled in-house) can also be used. All components must be sterile.
	<ol>
		<li>Add 32 mL of Earle&#39;s Balanced Salt Solution (EBSS) to the ovomucoid inhibitor mixture (powder).</li>
		<li>Add 5 mL of EBSS to one papain vial (powder). Place the papain vial in a 37 oC water bath for 10 min or until the papain is dissolved.</li>
		<li>Add 500 &#181;L of EBSS to one DNase vial (powder). Mix gently.</li>
		<li>Add 250 &#181;L of the DNase solution (above) to the papain vial.</li>
		<li>Calculate the total amount of the above papain/DNase mixture needed (approximately 800 &#181;L of papain/DNase mixture per mouse spinal cord) and transfer the needed mixture into a 50 mL tube for tissue digestion (step 3.2). Store the leftover in the original vial at 4 &#176;C for at least two weeks.</li>
		<li>Keep all components from the kits on ice until needed.</li>
	</ol>
	</li>
	<li>Prepare spinal cord collection tubes: one sterile 15 mL tube with 5 mL of Hank&#39;s balanced Salt Solution (HBSS; from the papain dissociation system,&#160;for collecting spinal cords) and one sterile 15 mL tube with 1x PBS (for filling up the 5 mL syringe, see step 2.4). In addition, prepare one sterile petri dish (35 mm or 60 mm) with 5 mL of HBSS and keep it in the culture hood.</li>
</ol>
2. Spinal Cord Collection
NOTE: All equipment must be sterile. Perform the collection in a designated area approved by the IACUC.
<ol>
	<li>Obtain the following tools for harvesting mouse spinal cords: straight sharp/blunt 18-cm surgical scissors, a 12-cm standard scalpel (#3 solid), carbon steel sterile scalpel blades #10, a small animal decapitator, 12-cm standard curved forceps, and a sterile 20G needle attached to a 5-mL&#160;syringe.</li>
	<li>Euthanize the animal with CO2 and disinfect the mouse head and the back region with 70% ethanol (EtOH).</li>
	<li>Decapitate animal with the decapitator. Make a middle longitudinal incision on the lower back to expose the muscle layer. Make a clean transection through the vertebrate column at the hip level (also cutting through the muscle layer that covers the vertebrate column) with the sterile straight surgical scissors (18 cm).</li>
	<li>Place the animal on a sheet of fresh disposable wipe. Carefully insert the 20 G needle (attached to a 5 mL syringe filled with sterile 1x PBS (step 1.4)) into the spinal column towards the rostral side. Hold the animal down tightly and push the syringe quickly. The whole spinal cord should come out from the cervical end. Collect the spinal cord into the 15 mL tube containing HBSS.</li>
	<li>Repeat step 2.4 for the rest of the mice. Between each animal, disinfect all used instruments with 70% EtOH. 
	NOTE: Typically, a single 12-well plate can be established for every 4 mouse spinal cords (see 4.3).</li>
</ol>
3. Preparation of the Single Cell Suspension
Note: Perform steps 3 and 4 in a culture hood to keep everything sterile.
<ol>
	<li>Transfer all spinal cords into the HBSS-containing petri dish (step 1.4). Cut each of the spinal cords into many fine, small pieces with sterile scissors and forceps. Transfer the spinal cord tissue to the 50-mL&#160;conical tube containing the prepared papain/DNase enzyme mixture (step 1.3.5) using sterile forceps or a 10 mL pipette (avoid adding HBSS to the enzyme mixture, as this will further dilute the prepared enzyme solution and may result in decreased performance of the enzyme).</li>
	<li>Vortex the tube gently to mix. Incubate the tube at 37 oC for 1 h in an incubator/shaker with orbital shaking at 150 rpm.</li>
	<li>Vortex the tube again and vigorously triturate the enzyme solution with the tissue using a 5 mL pipette to promote further dissociation.</li>
	<li>Transfer the cell suspension into a 15-mL&#160;tube and centrifuge at 300 x g for 5 min at RT.</li>
	<li>During centrifugation, mix 2.7 mL EBSS with 300 &#181;L of reconstituted albumin-ovomucoid inhibitor solution (1.3.1) in a sterile tube. Add 150 &#181;L of the DNase solution (step 1.3.3).</li>
	<li>Following centrifugation, remove the supernatant and resuspend the cell pellet with the solution prepared above (step 3.5). Vortex well to break the cell pellet.</li>
	<li>Add 3 mL of reconstituted albumin-ovomucoid inhibitor solution (step 1.4.1) to the cell suspension. Centrifuge cells at 70 x g for 6 min at RT. Remove the supernatant (which contains membrane fragments).</li>
</ol>
4. Further Removal of Myelin from the Single Cell Suspension
<ol>
	<li>Add 8 mL of 20% density gradient media (prepared in step 1.2.2) into the tube containing the cell pellet, vortex gently to disrupt the pellet, and centrifuge the cells at 800 x g for 30 min at RT without braking. Carefully remove the top layer of debris (mostly myelin) and the supernatant, but keep the pellet.</li>
	<li>To remove remnants of the density gradient, wash the cells by resuspending the cell pellet with 8 mL of a diluted cDMEM (1 part cDMEM and 2 parts HBSS). Centrifuge the cells at 400 x g for 10 min at 4 oC. Remove the supernatant and wash the cells again with the diluted cDMEM (above) in the same manner. Keep the cells on ice until seeding them.</li>
	<li>Remove the supernatant and resuspend the cell pellet in culture media (cDMEM supplied with 2-ME (prepared in step 1.1)). For one 12-well plate, use 3 mL x 4 (number of mice used) + 2 mL = 14 mL media. This will ensure that there is sufficient cell suspension for the entire plate (12 wells) and will provide for extra wells that can be used to determine the average cell number per well and the microglial content of the culture. If other types of culture vessels will be used, calculate the total volume of needed culture media proportionally.</li>
	<li>Add 1 mL of the cell suspension into each well of a 12-well plate.</li>
	<li>Incubate the cells at 35.9 oC with 5% CO2.</li>
	<li>Change the media (remove old media via aspiration) on D 1 and then every 3 - 4 d thereafter (typically change the media on D: 1, 4, 8, and 11, and then use the cells on day 12). 
	NOTE: On day 1, the culture may contain significant amounts of debris due to the residue myelin from the spinal cord tissue; thus, changing the media on day 1 is recommended (please see the Discussion for more information). Cultures are ready for treatment between D 12 - 14. Cells usually are 80% confluent at D&#160;12 and can be near 100% confluent by D 14. Typically, on D 12, there are about 100,000 cells per well in a 12-well plate.</li>
</ol>
<img alt="Figure 4" src="/files/ftp_upload/54801/54801fig4.jpg" /> 
Figure 2: Representative images of mixed glial cells at different times after the establishment of the mixed glia culture. Primary spinal cord mixed glia were prepared from adult C57Bl/6 mice. Representative images show the progress of the glial culture after plating. At day 1, some cells are attached to the culture plate, but they are still mostly round. There are also many floating cells and significant debris. At day 4, the majority of the cells are attached to the culture plate. Cells appear ramified with visible processes. Cells are sporadically distributed and cultures are about 20-30% confluent at this time. At day 8, cultures are between 50-60% confluent. Some areas of the cultures have large patches of cells. Other areas of the cultures have sparse growth of cells. Some cells within the patches take on a &#8220;square-like&#8221; appearance. At day 12, cultures are at or above 80% confluent (about 90% confluent in the image shown here). Cell are in the log phase of growth at this time and between days 12-14 is the optimal time for using the glia cells for experiments. <a href="http://cloudfront.jove.com/files/ftp_upload/54801/54801fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="7">
	<li>Examine the microglial content using cells from the extra wells (step 4.3) via standard fluorescence-activated cell sorting (FACS) staining protocol11 using the combination of anti-mouse-CD45 and anti-mouse CD11b monoclonal antibodies. CD45+CD11b+ cells are identified as microglia.</li>
</ol>

<ol>
	<li>Gaskin, D. J., Richard, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+economic+costs+of+pain+in+the+United+States.">The economic costs of pain in the United States.</a> J Pain. 13 (8), 715-724 (2012).</li><li>Ji, R. R., Berta, T., Nedergaard, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glia+and+pain:+Is+chronic+pain+a+gliopathy?.">Glia and pain: Is chronic pain a gliopathy?.</a> Pain. 154 (01), S10-S28 (2013).</li><li>Ni, M., Aschner, M., Maines, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+rat+primary+icroglia:+isolation,+culturing+and+selected+applications.">Neonatal rat primary icroglia: isolation, culturing and selected applications.</a> Curr Protoc Toxicol. , (2010).</li><li>Cao, L., Fei, L., Chang, T. T., DeLeo, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+interleukin-1beta+by+interleukin-4+in+lipopolysaccharide-treated+mixed+glial+cultures:+microglial-dependent+effects.">Induction of interleukin-1beta by interleukin-4 in lipopolysaccharide-treated mixed glial cultures: microglial-dependent effects.</a> J Neurochem. 102 (2), 408-419 (2007).</li><li>Malon, J. T., Maddula, S., Bell, H., Cao, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+calcitonin+gene-related+peptide+and+CCL2+production+in+CD40-mediated+behavioral+hypersensitivity+in+a+model+of+neuropathic+pain.">Involvement of calcitonin gene-related peptide and CCL2 production in CD40-mediated behavioral hypersensitivity in a model of neuropathic pain.</a> Neuron Glia Biol. 7 (2-4), 117-128 (2011).</li><li>Blasi, E., Barluzzi, R., Bocchini, V., Mazzolla, R., Bistoni, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalization+of+murine+microglial+cells+by+a+v-raf+/+v-myc+carrying+retrovirus.">Immortalization of murine microglial cells by a v-raf / v-myc carrying retrovirus.</a> J Neuroimmunol. 27 (2), 229-237 (1990).</li><li>Bocchini, V., 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=An+immortalized+cell+line+expresses+properties+of+activated+microglial+cells.">An immortalized cell line expresses properties of activated microglial cells.</a> J Neurosci Res. 31 (4), 616-621 (1992).</li><li>Alliot, F. O., Marty, M. C., Cambier, D., Pessac, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+spontaneously+immortalized+mouse+microglial+cell+line+expressing+CD4.">A spontaneously immortalized mouse microglial cell line expressing CD4.</a> Dev Brain Res. 95 (1), 140-143 (1996).</li><li>Alliot, F. O., Pessac, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytic+cell+clones+derived+from+established+cultures+of+8-day+postnatal+mouse+cerebella.">Astrocytic cell clones derived from established cultures of 8-day postnatal mouse cerebella.</a> Brain Res. 306 (1-2), 283-291 (1984).</li><li>Malon, J. T., 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=Microglial+content-dependent+inhibitory+effects+of+calcitonin+gene-related+peptide+(CGRP)+on+murine+retroviral+infection+of+glial+cells.">Microglial content-dependent inhibitory effects of calcitonin gene-related peptide (CGRP) on murine retroviral infection of glial cells.</a> J Neuroimmunol. 279, 64-70 (2015).</li><li>Cao, L., DeLeo, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CNS+Infiltrating+CD4(+)+T+lymphocytes+Contribute+to+Murine+Spinal+Nerve+Transection-Induced+Neuropathic+Pain.">CNS Infiltrating CD4(+) T lymphocytes Contribute to Murine Spinal Nerve Transection-Induced Neuropathic Pain.</a> Eur J Immunol. 38 (2), 448-458 (2008).</li><li>Mayer, A. M., 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=Effect+of+a+short-term+in+vitro+exposure+to+the+marine+toxin+domoic+acid+on+viability,+tumor+necrosis+factor-alpha,+matrix+metalloproteinase-9+and+superoxide+anion+release+by+rat+neonatal+microglia.">Effect of a short-term in vitro exposure to the marine toxin domoic acid on viability, tumor necrosis factor-alpha, matrix metalloproteinase-9 and superoxide anion release by rat neonatal microglia.</a> BMC Pharmacol. 1, 7 (2001).</li><li>Lawson, L. J., Perry, V. H., Dri, P., Gordon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+in+the+distribution+and+morphology+of+microglia+in+the+normal+adult+mouse+brain.">Heterogeneity in the distribution and morphology of microglia in the normal adult mouse brain.</a> Neuroscience. 39 (1), 151-170 (1990).</li><li>Rock, R. B., 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=Role+of+Microglia+in+Central+Nervous+System+Infections.">Role of Microglia in Central Nervous System Infections.</a> Clin Microbiol Rev. 17 (4), 942-964 (2004).</li><li>Yang, I., Han, S. J., Kaur, G., Crane, C., Parsa, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+Microglia+in+Central+Nervous+System+Immunity+and+Glioma+Immunology.">The Role of Microglia in Central Nervous System Immunity and Glioma Immunology.</a> J Clin Neurosci. 17 (1), 6-10 (2010).</li></ol>

The authors have nothing to disclose.

It is critical to perform all steps-including the media changing schedule-in a consistent manner in order to obtain reproducible results between experiments. The following steps are particularly critical in obtaining excellent quality adult mixed glia.
The enzymatic digestion is an important aspect of this protocol. It is crucial that the tissue pieces are well-digested in order to obtain a single cell suspension. However, over-digestion will result in significantly fewer cells. Each lab needs to perform pilot tests to determine the exact digestion time based on the types of vials used for incubation, the average tissue amount used each time, and the type of shaker (rotating versus orbital shaker). Furthermore, regardless of the papain dissociation system chosen, it is essential to consistently use the same components from the same sources, as the occasional substitution of components could lead to significant variations in the yield of total number of cells.
In this protocol, the myelin is removed using a density gradient medium. It is critical to make and use density gradient solutions at RT since the densities of these solutions are temperature-sensitive. Density gradient solutions are usually stored at 4 oC. If necessary, the density gradient solutions can be kept at RT overnight before the day of setting up the culture. In addition, following the 30 min centrifugation of cells in the 20% density gradient, cells should be removed immediately from the centrifuge to prevent large pieces of debris from shifting (i.e., moving away from the top of the solution; see step 4.1).
It is important to remove debris on D 1 post initiation of the culture. Although cells will survive and proliferate if the culture media is changed every 3 to 4 d, changing media on D 1 will provide a more &#34;quiescent&#34; and &#34;cleaner&#34; culture later on. This allows cells to grow in a less disturbed environment, and cells can be kept up to 21 d post-initiation of the culture.
Microglial content for each experiment can be examined prior to using the mixed glia. Even with the most consistent technique, microglial content may still vary significantly between experiments. Some cellular effects are dependent on microglial content4, 10; thus, it is crucial to monitor the microglial content of each set of cultures established. Data obtained from this practice can help to interpret variations between experiments, as well as to provide novel (sometimes unexpected) knowledge regarding the contribution of astrocytes versus microglia under a particular experimental condition. In addition, seasonal variations on microglial content have been observed. This may be an additional factor to consider when planning large sets of experiments. Furthermore, while neurons usually will not survive under our culture condition, as NeuN (a&#160;neuronal- marker)-positive cells have not been observed in our glial cultures after immunohistochemical staining, the culture may contain limited numbers of oligodendrocytes (this has not been routinely quantified). One should decide whether this population needs to be examined depending on the specific experimental conditions.
As with many experimental methods, this method can be modified according to the needs of individual researchers. It is essential to perform pilot experiments to fully test the specific modifications prior to using them routinely. Furthermore, it should be noted that the mixed glia can be used to obtain microglia-depleted and microglia-enriched cultures to study the responses of astrocyte-dominant versus microglia-dominant cells after specific stimulations, as previously described for neonatal mixed glial cells3, 4. However, the yield of microglia-enriched cells will be limited unless large numbers of spinal cords are used to establish the culture at the beginning. This is a limitation of this method, particularly if mice are used. When rat spinal cords are used, one individual spinal cord (instead of 4 mouse spinal cords) can be used to set up one 12-well plate. Finally, the microglial content does not appear to be significantly different between mouse and rat adult spinal cord glial cultures4, 5. Both mouse and rat adult spinal cord glial cultures produce a robust response to LPS stimulation in terms of cytokine production; however, differential responses may be observed when alternative stimuli are used.
Altogether, this rodent adult glial culture system provides an alternative method to study glial cells in vitro. Results obtained from this culture system more closely reflect what would be observed under in vivo conditions compared to results obtained from neonatal cultures or cell lines. In the current method, glial cells are collected from adult rodents, as adult glia respond to stimuli very differently when compared to neonatal glia4 (Figure 1). The cells are not manipulated significantly through immortalization procedures, which would be used to generate and maintain cell lines. Although this method was initially developed to be used in neuropathic pain studies, it can be used to study other neurological diseases that involve pathological changes within the spinal cord, such as ALS and MS.

Chronic pain is a serious health problem that affects approximately 100 million adults in the United States, with an estimated annual cost of up to $635 billion1. Mounting evidence has indicated a significant contribution of spinal cord glial cells in the development of neuropathic pain, one of the most devastating kinds of chronic pain2. A proper in vitro culture system would help significantly in further investigating the roles of glial cells at the cellular and molecular levels.
Currently, the in vitro glial culture systems utilized by researchers include mainly glial cells derived from cortical tissues of neonatal mouse or rat brains and immortalized glial cell lines derived from neonatal mouse or rat primary glial cells. Neonatal cells contain significant numbers of undifferentiated cells that can be further differentiated into glial cells (mainly astrocytes and microglia), thus providing abundant cells for experimental usage3. However, our previous study has shown that neonatal brain glial cells responded significantly differently from adult spinal cord glial cells upon lipopolysaccharide (LPS) stimulation. For example, interleukin (IL)-4 displayed enhanced inhibitory effects on LPS-induced nitric oxide (NO) production in adult rat spinal cord glia compared to neonatal brain glia4. Furthermore, the profiles of chemokine production upon stimulation by a neuropeptide, calcitonin gene-related peptide (CGRP), are unarguably different between neonatal mouse brain glia (methods described in Ref. 5) and adult mouse spinal cord glia (Figure 1). Established cell lines are easy to use and maintain and can provide a large number of cells in a short time period. In general, immortalized cell lines are generated either using a virus-mediated immortalization system (such as the widely-used microglial cell line BV2)6, 7 or following the identification of spontaneous transformation (such as the astrocyte cell line C8-D1A and microglial cell line C8-B4)8, 9. Cell lines are excellent in studying the molecular characteristics of astrocytes and microglia individually; however, the results obtained from cell lines always require further validation in primary cells or in&#160;in vivo conditions. It should also be noted that there has not been a report of a glial cell line that is derived from a rodent spinal cord glia.
To help&#160;investigating&#160;the role of spinal cord glial cells in the development of neuropathic pain, a culture system that is derived from the adult mouse spinal cord has been developed by adapting a previously-reported method used to generate adult rat mixed glial cultures4, 5. The mouse spinal cord glial culture was further refined recently10 and is described in more detail in this article. Adult spinal cord mixed glial cultures can be established with a mouse from selected strains that fit the needs of the particular study, and cells can be maintained in culture for up to 21 d post initiation of the culture. This method can be used in neuropathic pain studies, as well as in investigations of various neurological diseases that involve pathological changes within the spinal cord, such as amyotrophic lateral sclerosis (ALS), human immunodeficiency virus (HIV)-associated sensory neuropathy, and multiple sclerosis (MS).

<table border="0" cellpadding="0" cellspacing="0" width="2053">
	<tbody>
		<tr height="24">
			<td height="24" style="height:24px;width:489px;">Dulbecco&#8217;s modification of Eagle&#8217;s media (DMEM) with 4.5g/L glucose</td>
			<td style="width:277px;">Lonza</td>
			<td style="width:119px;">12-709F</td>
			<td style="width:1168px;">The cDMEM media is a standard, widely used culture media. Individual researcher can decide where to purchase the DMEM and all other component used to make cDMEM media.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">L-Glutamine (100x)</td>
			<td style="width:277px;">Lonza</td>
			<td style="width:119px;">17-605E</td>
			<td>The L-glutamine is a standard, widely used component for various culture media. Individual researcher can decide where to purchase L-glutamine.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:489px;">Antibiotic-Antimycotic Solution (100x)</td>
			<td style="width:277px;">Corning-Mediatech</td>
			<td style="width:119px;">30-004-CI</td>
			<td>This is a combination of penicillin, streptomycin and Amphotericin formulated to contain 10,000 units/ml penicillin G, 10 mg/ml streptomycin sulfate and 25 &#181;g/ml amphotericin B. Individual researcher can decide where to purchase individual component.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">2-mercaptoethanol (2-ME)</td>
			<td style="width:277px;">Sigma-Aldrich</td>
			<td style="width:119px;">M3148</td>
			<td>A BioReagent, suitable for cell culture, molecular biology and electrophoresis.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Papain dissociation system</td>
			<td style="width:277px;">Worthington Biochemical Corporation</td>
			<td>LK003150</td>
			<td>Individual components of this kit can be purchased separately.&#160;</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:489px;">Percoll</td>
			<td>GE Health Care</td>
			<td style="width:119px;">17-0891-01</td>
			<td>Percoll is sold as sterile solution. Undiluted Percoll can be re-autoclaved if needed.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:489px;">Lab-line incubator/shaker&#160;</td>
			<td style="width:277px;">Barstead/Lab-line</td>
			<td style="width:119px;">MaxQ4000&#160;</td>
			<td>This is the incubator/shaker we have currently. Other types of shakers can be used instead.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:489px;">Lipopolysaccharides, Salmonella Minnesota Re595&#160;</td>
			<td style="width:277px;">Sigma-Aldrich</td>
			<td style="width:119px;">L-9764</td>
			<td>Other strains of LPS can also induce glial responses. The magnitude of the responses may vary.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:489px;">Mouse IL-6 DuoSet ELISA&#160;</td>
			<td style="width:277px;">R&#38;D Systems</td>
			<td style="width:119px;">DY406</td>
			<td>Each individual Researcher can select the ELISA kit he or she prefers.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:489px;">Mouse TNF-alpha DuoSet ELISA</td>
			<td style="width:277px;">R&#38;D Systems</td>
			<td style="width:119px;">DY410</td>
			<td>Each individual Researcher can select the ELISA kit he or she prefers.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:489px;">Mouse IP-10 (CXCL10) DuoSet ELISA</td>
			<td style="width:277px;">R&#38;D Systems</td>
			<td style="width:119px;">DY466</td>
			<td>Each individual Researcher can select the ELISA kit he or she prefers.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:489px;">Mouse MCP-1 (CCL2) ELISA Opteia set</td>
			<td style="width:277px;">BD Biosciences</td>
			<td style="width:119px;">555260</td>
			<td>Each individual Researcher can select the ELISA kit he or she prefers.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:489px;">Mouse chemokine Q-Plex</td>
			<td style="width:277px;">Quansys Biosciences</td>
			<td style="width:119px;">120251MS</td>
			<td>This product was available for in-house assay at the time. Instead, we sent out samples to the manufacturer for analysis.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:489px;">APC-anti-mouse-CD45 (clone 30-F11)</td>
			<td style="width:277px;">eBioscience</td>
			<td style="width:119px;">17-0451</td>
			<td>Antibody of the same clone but from other vendors can be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:489px;">FITC-anti-mouse-CD11b (clone M1/70)</td>
			<td style="width:277px;">eBioscience</td>
			<td style="width:119px;">11-0112</td>
			<td>Antibody of the same clone but from other vendors can be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:489px;">Accuri C6 flow cytometer</td>
			<td style="width:277px;">BD Biosciences</td>
			<td style="width:119px;">BD Accuri C6&#160;</td>
			<td>Each individual Researcher can use any flow cytometer he or she prefers.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:489px;">FlowJo software</td>
			<td style="width:277px;">Tree Star, Inc.</td>
			<td style="width:119px;">FlowJo7.6.5</td>
			<td>Each individual Researcher can use any analysis software he or she prefers.</td>
		</tr>
	</tbody>
</table>

preparation of primary mixed glial cultures from adult mouse spinal cord tissue

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information regarding glial activities at the cellular and molecular levels has been obtained through in vitro cell culture systems. The in vitro systems utilized, mainly include primary glia derived from neonatal brain cortical tissue and immortalized cell lines. However, these systems may not reflect the characteristics of spinal cord glial cells in vivo. In order to further investigate the roles of spinal cord glial cells in the development of peripheral nerve injury-induced neuropathic pain using a culture system that better reflects the in vivo condition, our laboratory has developed a method to establish primary spinal cord mixed glial cultures from adult mice. Briefly, spinal cords are collected from adult mice and processed through papain digestion followed by myelin removal with a density-gradient medium. Single cell suspensions are cultured in complete Dulbecco&#39;s modified Eagle media (cDMEM) supplemented with 2-mercaptoethanol (2-ME) at 35.9 oC. These culture conditions were optimized specifically for the growth of mixed glial cells. Under these conditions, cells are ready to be used for experimentation between 12 - 14 d&#160;(cells are usually in log phase during this time) after the establishment of the culture (D 0) and can be kept in culture conditions up to D 21. This culture system can be used to investigate the responses of spinal cord glial cells upon stimulation with various substances and agents. Besides neuropathic pain, this system can be used to study glial responses in other diseases that involve pathological changes of spinal cord glial cells.

This method can be used to prepare mixed glial cells from both mice and rats. The average total cell number per well in a 12-well plate on D 12 post-initiation of the culture should be relatively stable, with around 100,000 cells per well when cells are derived from mouse spinal cords. Glial cells obtained from this method can be used in experiments that are designed to examine adult spinal cord glial responses upon administration of substances and agents of interest. Figure 3&#160;provides an example of cytokine and chemokine responses from one typical experiment in which adult spinal cord microglia were obtained from adult BALB/c mice and treated with LPS (Salmonella Minnesota Re595) on D 13 post-initiation of the culture. Supernatants were collected 24 h post-LPS treatment for the determination of cytokine and chemokine levels via an enzyme-linked immunosorbent assay (ELISA) using commercially-available kits.
When this adult glial culture system was first established, cells were incubated in a standard culture environment at 37 oC. Under this condition, the average microglial content ranged from 5 - 10%. However, it was observed that despite the use of consistent culture techniques, cultures would in some instances have either very low microglial content (&#60;2%) or relatively high microglial content (15 -&#160;20%)10. It can be frustrating to obtain cultures that have very few microglia when the experimental results rely on the microglial responses within the mixed culture. Following the suggestion of Dr. Alejandro M. S. Mayer (Department of Pharmacology, Chicago College of Osteopathic Medicine)12, the method was modified by growing the mixed glial cells at 35.9 oC. This resulted in a more consistent and higher microglial yield (ranging between 10 - 40% and remaining mostly around 20%) within the majority of cultures. This improvement is illustrated in Figure 4. As reported previously, microglia are estimated to make up 5 - 21% of the CNS glial population in adult mice13-15. Although both culture conditions provide cultures that contain similar amounts of microglia as estimated in vivo, the modified culture condition (35.9 oC) is more appropriate for experiments in which it is critical to examine the responses from both astrocytes and microglia.
<img alt="Figure 1" src="/files/ftp_upload/54801/54801fig1.jpg" /> 
Figure 1: Calcitonin Gene-related Peptide (CGRP)-induced Chemokine Production by Mixed Glial Cells. Mixed glial cells prepared from neonatal BALB/c mouse brains (left) or adult BALB/c mouse spinal cords (right) were treated with various doses of CGRP. Levels of several chemokines in culture supernatants were determined via multiplex assay (performed by the manufacturer) at optimal times (mean &#177; SEM, n = 2 - 6). <a href="http://cloudflare.jove.com/files/ftp_upload/54801/54801fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54801/54801fig2.jpg" /> 
Figure 3: Cytokine and Chemokine Responses of Mouse Adult Spinal Cord Mixed Glia upon LPS Stimulation. Adult spinal cord mixed glial cells were prepared from BALB/c mice and stimulated with various doses of LPS. Levels of IL-6 (A), tumor necrosis factor (TNF)-alpha (B), interferon-gamma-inducible protein 10 (IP-10, also known as CXCL10) (C), and monocyte chemoattractant protein 1 (MCP-1, also known as CCL2) (D) in culture supernatants were determined via ELISAs at 24 h post-LPS treatment. <a href="http://cloudflare.jove.com/files/ftp_upload/54801/54801fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54801/54801fig3.jpg" /> 
Figure 4: Microglial Content in Adult Mixed Glial Cultures. Mixed glial cells were generated from adult C57B6/L mice and cultured at either 37 oC or 35.9 oC. Microglial content from each culture was analyzed via flow cytometry using APC-anti-mouse-CD45 (clone 30-F11) and FITC-anti-mouse-CD11b (clone M1/70). Representative plots from the flow cytometric analyses are shown. The total cell populations from the cultures were identified in plots A (37 oC) and C (35.9 oC), and the microglial populations (CD45+CD11b+ cells) were further isolated from the respective total cell populations in B (37 oC) and D (35.9 oC). Flow cytometric analysis was performed, and all data were analyzed as previously described5. <a href="http://cloudflare.jove.com/files/ftp_upload/54801/54801fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue at JoVE.com

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

The development of neuropathic pain involves pathological changes of spinal cord glial cells. A reliable glial culture system derived from adult spinal cord tissue and designed for studying these cells in vitro is lacking. Therefore, we show here how&#160;to establish primary mixed glial cultures from adult mouse spinal cord tissue.

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information ...

preparation-primary-mixed-glial-cultures-from-adult-mouse-spinal-cord

Nanyang Technological University

Research

JoVE Journal

Neuroscience

10.5K Views.  University of New England. The development of neuropathic pain involves pathological changes of spinal cord glial cells. A reliable glial culture system derived from adult spinal cord tissue and designed for studying these cells in vitro is lacking. Therefore, we show here how to establish primary mixed glial cultures from adult mouse spinal cord tissue.

Video: Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to network properties. We show how to prepare viable oblique slices of the spinal cord of young mice (P2 - P11). In this preparation, the motoneurons retain&#160;their axons coming out from the ventral roots of the&#160;spinal cord. Stimulation of these axons elicits back-propagating action potentials invading the motoneuron&#160;somas and exciting the motoneuron&#160;collaterals within the spinal cord. Recording of antidromic action potentials is an immediate, definitive and elegant way to characterize motoneuron identity, which surpasses&#160;other identification methods. Furthermore, stimulating the motoneuron collaterals is a simple and reliable way to excite the collateral targets of the motoneurons within the spinal cord, such as other motoneurons or Renshaw cells. In this protocol, we present antidromic recordings from the motoneuron somas as well as Renshaw cell excitation, resulting from ventral root stimulation.

We show how to prepare oblique slices of the spinal cord in young mice. This preparation allows for the stimulation of the ventral roots.

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to ...

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing organisms, combined with the embryonic optical clarity, served as initial compelling attributes of this model. Over the past two decades, the success of this model has been further propelled by its amenability to large-scale mutagenesis screens and by the ease of transgenesis. More recently, gene-editing approaches have extended the power of the model.
For neurodevelopmental studies, the zebrafish embryo and larva provide a model to which multiple methods can be applied. Here, we focus on methods that allow the study of an essential property of neurons, electrical excitability. Our preparation for the electrophysiological study of zebrafish spinal neurons involves the use of veterinarian suture glue to secure the preparation to a recording chamber. Alternative methods for recording from zebrafish embryos and larvae involve the attachment of the preparation to the chamber using a fine tungsten pin1,2,3,4,5. A tungsten pin is most often used to mount the preparation in a lateral orientation, although it has been used to mount larvae dorsal-side up4. The suture glue has been used to mount embryos and larvae in both orientations. Using the glue, a minimal dissection can be performed, allowing access to spinal neurons without the use of an enzymatic treatment, thereby avoiding any resultant damage. However, for larvae, it is necessary to apply a brief enzyme treatment to remove the muscle tissue surrounding the spinal cord. The methods described here have been used to study the intrinsic electrical properties of motor neurons, interneurons, and sensory neurons at several developmental stages6,7,8,9.

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

This manuscript describes methods for electrophysiological recordings from spinal neurons of zebrafish embryos and larvae. The preparation maintains neurons in situ and often involves minimum dissection. These methods allow for the electrophysiological study of a variety of spinal neurons, from the initial electrical excitability acquisition through the early larval stages.

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on postnatal day 1-3. A mouse litter, usually 4-10 pups born from one breeding pair, is gathered for one experiment, and spinal cords are collected individually from each mouse after euthanasia with isoflurane. The spinal column is dissected out and then the spinal cord is released from the column. The spinal cords are then minced to increase the surface area of delivery for an enzymatic protease that allows for the neurons and other cells to be released from the tissue. Trituration is then used to release the cells into solution. This solution is subsequently fractionated in a density gradient to separate the various cells in solution, allowing for neurons to be isolated. Approximately 1-2.5 x 106 neurons can be isolated from one litter group. The neurons are then seeded onto wells coated with adhesive factors that allow for proper growth and maturation. The neurons take approximately 7 days to reach maturity in the growth and culture medium and can be used thereafter for treatment and analysis.

This study presents a technique for the isolation of neurons from WT neonatal mice. It requires the careful dissection of the spinal cord from the neonatal mouse, followed by the separation of neurons from the spinal cord tissue through mechanical and enzymatic cleavage.

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

Laminectomy and Spinal Cord Window Implantation in the Mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated digital vaporizer is utilized to achieve a stable plane of anesthesia at a low-flow rate of isoflurane. A single vertebral spine is removed, and a commercially available cover-glass is overlaid on a thin agarose bed. A 3D-printed plastic backplate is then affixed to the adjacent vertebral spines using tissue adhesive and dental cement. A stabilization platform is used to reduce motion artifact from respiration and heartbeat. This rapid and clamp-free method is well-suited for acute multi-photon fluorescence microscopy. Representative data are included for an application of this technique to two-photon microscopy of the spinal cord vasculature in transgenic mice expressing eGFP:Claudin-5 &#8212; a tight junction protein.

This protocol describes implantation of a glass window onto the spinal cord of a mouse to facilitate visualization by intravital microscopy.

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...

Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a powerful tool for studying transcriptional profiles of cells, particularly in heterogeneous tissues such as the central nervous system. However, dissociation methods required for single cell sequencing can lead to experimental changes in the gene expression and cell death. Furthermore, these methods are generally restricted to fresh tissue, thus limiting studies on archival and bio-bank material. Single nucleus RNA sequencing (snRNA-Seq) is an appealing alternative for transcriptional studies, given that it accurately identifies cell types, permits the study of tissue that is frozen or difficult to dissociate, and reduces dissociation-induced transcription. Here, we present a high-throughput protocol for rapid isolation of nuclei for downstream snRNA-Seq. This method enables isolation of nuclei from fresh or frozen spinal cord samples and can be combined with two massively parallel droplet encapsulation platforms.

Here, we present a protocol to rapidly isolate high-quality nuclei from the fresh or frozen tissue for downstream massively parallel RNA sequencing. We include detergent-mechanical and hypotonic-mechanical tissue disruption and cell lysis options, both of which can be used for isolation of nuclei.

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...

Magnetic Isolation of Microglial Cells from Neonate Mouse for Primary Cell Cultures

Microglia, as brain resident macrophages, are fundamental to several functions, including response to environmental stress and brain homeostasis. Microglia can adopt a large spectrum of activation phenotypes. Moreover, microglia that endorse pro-inflammatory phenotype is associated with both neurodevelopmental and neurodegenerative disorders. In vitro studies are widely used in research to evaluate potential therapeutic strategies in specific cell types. In this context studying microglial activation and neuroinflammation in vitro using primary microglial cultures is more relevant than microglial cell lines or stem-cell-derived microglia. However, the use of some primary cultures might suffer from a lack of reproducibility. This protocol proposes a reproducible and relevant method of magnetically isolating microglia from neonate pups. Microglial activation using several stimuli after 4 h and 24 h by mRNA expression quantification and a Cy3-bead phagocytic assay is demonstrated here. The current work is expected to provide an easily reproducible technique for isolating physiologically relevant microglia from juvenile developmental stages.

Primary microglia cultures are commonly used to evaluate new anti-inflammatory molecules. The present protocol describes a reproducible and relevant method to magnetically isolate microglia from neonate pups.

Microglia, as brain resident macrophages, are fundamental to several functions, including response to environmental stress and brain homeostasis. ...

Electromagnetic Controlled Closed-Head Model of Mild Traumatic Brain Injury in Mice

Highly reproducible animal models of traumatic brain injury (TBI), with well-defined pathologies, are needed for testing therapeutic interventions and understanding the mechanisms of how a TBI alters brain function. The availability of multiple animal models of TBI is necessary to model the different aspects and severities of TBI seen in people. This manuscript describes the use of a midline closed head injury (CHI) to develop a mouse model of mild TBI. The model is considered mild because it does not produce structural brain lesions based on neuroimaging or gross neuronal loss. However, a single impact creates enough pathology that cognitive impairment is measurable at least 1 month after injury. A step-by-step protocol to induce a CHI in mice using a stereotaxically guided electromagnetic impactor is defined in the paper. The benefits of the mild midline CHI model include the reproducibility of the injury-induced changes with low mortality. The model has been temporally characterized up to 1 year after the injury for neuroimaging, neurochemical, neuropathological, and behavioral changes. The model is complementary to open skull models of controlled cortical impact using the same impactor device. Thus, labs can model both mild diffuse TBI and focal moderate-to-severe TBI with the same impactor.

The protocol describes mild traumatic brain injury in a mouse model. In particular, a step-by-step protocol to induce a mild midline closed head injury and the characterization of the animal model is fully explained.

Highly reproducible animal models of traumatic brain injury (TBI), with well-defined pathologies, are needed for testing therapeutic interventions and ...

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

Summary

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Chapters in this video

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

Laminectomy and Spinal Cord Window Implantation in the Mouse

Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Magnetic Isolation of Microglial Cells from Neonate Mouse for Primary Cell Cultures

Electromagnetic Controlled Closed-Head Model of Mild Traumatic Brain Injury in Mice