Microglial content-dependent inhibitory effects of calcitonin gene-related peptide (CGRP) on murine retroviral infection of glial cells

Malon, Jennifer; Grlickova-Duzevik, Eliza; Vaughn, James L.; Beaulac, Holly J.; Vunk, Tyler; Cao, Ling

doi:10.1016/j.jneuroim.2015.01.010

Cited by 3 publications

(8 citation statements)

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“…Microglial content can vary despite consistency in techniques and reagents. Recording this information prior to exper-imental usage of the cells can help with troubleshooting or uncover unexpected microglial content-dependent cellular effects, as we reported previously (Malon et al, 2015). Microglial content also can vary due to mouse strains, prior treatment of the animals, brands/types of cultural vessels, and even seasons.…”

Section: Cao Bean and Malonmentioning

confidence: 82%

“…This method can be used to study animal or human diseases/disorders that involve pathological changes, particularly the cellular responses of glial cells, in the spinal cord including various types of periphery neuropathies, ALS, MS, and spinal cord injury (Airas & Yong, 2022; Ban et al., 2019; Gaudet & Fonken, 2018; Liddelow et al., 2017; Orr & Gensel, 2018; Pitt & Ponath, 2019; Rodrigues Lima‐Junior et al., 2021), as well as environmental toxins or contaminant‐induced toxicological potential changes in the spinal cord (D'Mello et al., 2017; Ni et al., 2012). So far, we have used this in vitro preparation to study the effects of LPS, murine virus (LP‐BM5), CGRP, and HIV proteins (Tat and gp120) with or without dose responses (Cao et al., 2007; Malon et al., 2015; Malon & Cao, 2016; Malon et al., 2011) (and unpublished data) to study injury and HIV‐associated peripheral neuropathies. We suggest that any experiments performed using neonatal glia to study the mechanisms of adult spinal cord‐related disease/disorders should potentially be performed with adult spinal cord glial cell cultures either for confirmation or making more relevant new discoveries.…”

Section: Commentarymentioning

confidence: 99%

“…For example, the widely used microglial cell line BV2 was generated through a virus-mediated immortalization system and is known to actively produce murine viral products and thus may not be suitable for studying viral infections of microglia (Blasi et al, 1990;Bocchini et al, 1992) Here, we describe a protocol for preparing mixed glial cells from adult mouse spinal cord (Basic Protocol), which can be used in various in vitro evaluations directly or for further preparation of microglia-enriched and microglia-depleted cells. This method was first developed using adult rat spinal cord (Cao et al, 2007) and further refined using mouse spinal cord (Malon et al, 2015;Malon & Cao, 2016;Malon et al, 2011). In this protocol, spinal cord tissue is enzymatically dissociated, myelin debris are removed using a density gradient, and the resulting adult primary mixed glial cells are ready to be used in various assays between 12 and 14 days after the establishment of the culture.…”

Section: Introductionmentioning

confidence: 99%

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

2023

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Central nervous system glial cells are known to mediate many neurocognitive/neurodegenerative diseases, including Alzheimer's and Parkinson's diseases. Similar glial responses have been recognized as critical factors contributing to the development of diseases in the peripheral nervous system, including various types of peripheral neuropathies, such as peripheral nerve injury-induced neuropathic pain, diabetic neuropathy, and HIV-associated sensory neuropathy. Investigation of the central mechanisms of these peripherallymanifested diseases often requires the examination of spinal cord glial cells at cellular/molecular levels in vitro. When using rodent models to study these diseases, many investigators have chosen to use neonatal cerebral cortices to prepare glial cultures or immortalized cell lines in order to obtain sufficient numbers of cells for assessment. However, differences in responses between cell lines versus primary cultures, neonatal vs. adult cells, and brain vs. spinal cord cells may result in misleading data. Here, we describe a protocol for preparing mixed glial cells from adult mouse spinal cord that can be used for direct in vitro evaluations or further preparation of microglia-enriched and microgliadepleted cells. In this protocol, spinal cord tissue is enzymatically dissociated and adult mixed glial cells are ready to be used between 12 and 14 days after the establishment of the culture. This protocol may be further refined to prepare spinal cord glial cells from spinal cord tissues of adult rats and potentially other species. Mixed glial cultures can be prepared from animals of different strains or post-in vivo manipulations and therefore are suitable for studying a variety of diseases/disorders that involve spinal cord pathological changes, such as amyotrophic lateral sclerosis and multiple sclerosis, as well as toxin-induced changes.

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Section: Cao Bean and Malonmentioning

confidence: 82%

Section: Commentarymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

2023

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“…Adult male and female BALB/c mice were purchased from Charles River Laboratories (National Cancer Institute, Frederick, MD) and used in the experiment examining the effects of a CD137L neutralizing antibody. This strain of mice was selected because extensive studies regarding spinal cord inflammation and microglial responses following L5Tx, the chosen neuropathic pain model, have been performed with BALB/c mice ( Cao and DeLeo, 2008 ; Cao et al, 2009 , 2012 ; Malon et al, 2015 ; Cao and Malon, 2018 ). However, CD137L KO mice were only available on C57BL/6 (B6) background as CD137L KO mice on the B6 background (C57BL/6_CD137L KO mice) were originally generated by Amgen ( DeBenedette et al, 1999 ).…”

Section: Methodsmentioning

confidence: 99%

“…To determine CD137L’s window of action relative to the development of neuropathic pain-like behaviors, BALB/c mice were intrathecally injected daily with a CD137L neutralizing monoclonal antibody (clone: TKS-1, catalog #107108; BioLegend Inc.), starting on day 0 (immediately before surgery) through day 7 post-surgery (early regimen), or from day 6 through day 13 post-surgery (late regimen). Intrathecal injection was performed as previously described ( Yu et al, 1996 ; Malon et al, 2015 ). Two anti-CD137L doses, 0.1 or 0.5 µg, were tested in both regimens.…”

Section: Methodsmentioning

confidence: 99%

Contribution of CD137L to Sensory Hypersensitivity in a Murine Model of Neuropathic Pain

Wakley¹,

Leeming²,

Malon³

et al. 2018

eNeuro

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CD137L (4-1BBL) is a costimulatory molecule whose signaling can promote monocyte/macrophage functions; however, CD137L-mediated microglial response and its role in neuropathic pain remain unknown. We investigated CD137L following peripheral nerve injury-induced neuropathic pain using a spinal nerve L5 transection (L5Tx) murine model in both sexes. First, C57BL/6_CD137L knock-out (KO) mice displayed decreased mechanical and diminished heat hypersensitivity compared to wild-type (WT) controls, beginning on day 3 to up to day 35 post-L5Tx. Purified anti-mouse CD137L neutralizing monoclonal antibody (0.1 or 0.5 µg) was also used to identify CD137L’s window of action in BALB/c mice. Anti-CD137L antibody was intrathecally administered either from day 0 (before surgery) to day 7 (early treatment), or from day 6 to 13 post-L5Tx (late treatment), and nociceptive thresholds were assessed before surgery to up to day 35 post-surgery. Early treatment with anti-CD137L reduced L5Tx-induced mechanical but not heat hypersensitivity, while later treatment did not alter either sensitivity. Pro- versus anti-inflammatory responses within the lumbar spinal cord following L5Tx were further evaluated via quantitative real-time PCR (qRT-PCR) and immunohistochemistry (IHC) in time-course studies. Following L5Tx, female CD137L KO mice did not show increased iNOS mRNA and had reduced numbers of IL-1β+ cells compared to WT. At 21 d post-surgery, CD137L KO mice had higher total numbers of arginase (Arg)-1+ cells and Arg-1+ microglia. Altogether, results indicate that spinal cord CD137L contributes to the development of peripheral nerve injury-induced neuropathic pain, which may be in part mediated through CD137L’s modulation of the pro- and anti-inflammatory balance within the spinal cord.

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

Malon¹,

Cao²

2016

JoVE

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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's modified Eagle media (cDMEM) supplemented with 2-mercaptoethanol (2-ME) at 35.9 C. 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 (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.

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Microglial content-dependent inhibitory effects of calcitonin gene-related peptide (CGRP) on murine retroviral infection of glial cells

Cited by 3 publications

References 50 publications

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

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

Contribution of CD137L to Sensory Hypersensitivity in a Murine Model of Neuropathic Pain

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

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