Flow cytometry analysis of inflammatory cells isolated from the sciatic nerve and DRG after chronic constriction injury in mice

Liu, Liping; Yin, Yan; Li, Fēi; Malhotra, Charvi; Cheng, Jianguo

doi:10.1016/j.jneumeth.2017.04.012

Cited by 13 publications

(10 citation statements)

References 28 publications

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“…A modification of the sciatic nerve chronic constriction injury (CCI) model developed by Bennett and Xie (Bennett and Xie, 1988) was used for application in the mouse (Costa et al, 2008; Martucci et al, 2008; Liu et al, 2017) and briefly described here. Under isoflurane anesthesia (induction at 3.0 followed by 2.0–2.5 vol.% in oxygen, 2.0L/min), the lower back and dorsal left thigh were shaved and then cleaned with diluted Bacti-Stat AE (EcoLab Health Care Division, Mississauga, Ontario, Canada), followed by water, and lastly swabbed with 70% EtOH that was allowed to air dry before proceeding.…”

Section: Methodsmentioning

confidence: 99%

Effects of spinal non-viral interleukin-10 gene therapy formulated with d -mannose in neuropathic interleukin-10 deficient mice: Behavioral characterization, mRNA and protein analysis in pain relevant tissues

Vanderwall

Noor

Sun

et al. 2018

Brain, Behavior, and Immunity

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Studies show that spinal (intrathecal; i.t.) interleukin-10 (IL-10) gene therapy reverses neuropathic pain in animal models, and co-administration with the mannose receptor (MR; CD206) ligand d-mannose (DM) greatly improves therapeutic efficacy. However, the actions of endogenous IL-10 may be required for enduring pain control observed following i.t. IL-10 gene therapy, potentially narrowing the application of this non-viral transgene delivery approach. Here, we show that i.t. application of naked plasmid DNA expressing the IL-10 transgene co-injected with DM (DM/pDNA-IL-10) for the treatment of peripheral neuropathic pain in IL-10 deficient (IL-10 KO) mice results in a profound and prolonged bilateral pain suppression. Neuropathic pain is induced by unilateral sciatic chronic constriction injury (CCI), and while enduring relief of light touch sensitivity (mechanical allodynia) in both wild type (WT) and IL-10 KO mice was observed following DM/pDNA-IL-10 co-therapy, transient reversal from allodynia was observed following i.t. DM alone. In stably pain-relieved IL-10 KO mice given DM/pDNA-IL-10, mRNA for the IL-10 transgene is detected in the cauda equina and ipsilateral dorsal root ganglia (DRG), but not the lumbar spinal cord. Further, DM/pDNA-IL-10 application increases anti-inflammatory TGF-β1 and decreases pro-inflammatory TNF mRNA in the ipsilateral DRG compared to allodynic controls. Additionally, DM/pDNA-IL-10 treated mice exhibit decreased spinal pro-inflammatory mRNA expression for TNF, CCL2 (MCP-1), and for the microglial-specific marker TMEM119. Similarly, DM/pDNA-IL-10 treatment decreases immunoreactivity for the astrocyte activation marker GFAP in lumbar spinal cord dorsal horn. Despite transient reversal and early return to allodynia in DM-treated mice, lumbar spinal cord revealed elevated TNF, CCL2 and TMEM119 mRNA levels. Both MR (CD206) and IL-10 receptor mRNAs are increased in the DRG following CCI manipulation independent of injection treatment, suggesting that pathological conditions stimulate upregulation and availability of relevant receptors in critical anatomical regions required for the therapeutic actions of the DM/pDNA-IL-10 co-therapy. Taken together, the current report demonstrates that non-viral DM/pDNA-IL-10 gene therapy does not require endogenous IL-10 for enduring relief of peripheral neuropathic pain and does not require direct contact with the spinal cord dorsal horn for robust and enduring relief of neuropathic pain. Spinal non-viral DM/pDNA-IL-10 co-therapy may offer a framework for the development of non-viral gene therapeutic approaches for other diseases of the central nervous system.

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Section: Methodsmentioning

confidence: 99%

Effects of spinal non-viral interleukin-10 gene therapy formulated with d -mannose in neuropathic interleukin-10 deficient mice: Behavioral characterization, mRNA and protein analysis in pain relevant tissues

Vanderwall

Noor

Sun

et al. 2018

Brain, Behavior, and Immunity

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“…Total leukocytes obtained from the 30–70% Percoll interface were collected and counted on a hemocytometer using trypan blue dye exclusion method. To isolate mononuclear cells from DRG, we employed a non-enzymatic dissociation protocol described previously [ 41 ]. Briefly, six ganglia (L3-L5) were collected in a solution containing 1× HBSS/25 mM HEPES/10% FBS/10 μg/ml DNase ( n = 4–6 animals/group/experiment).…”

Section: Methodsmentioning

confidence: 99%

Nitrosative damage during retrovirus infection-induced neuropathic pain

Chauhan

Sheng

et al. 2018

J Neuroinflammation

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BackgroundPeripheral neuropathy is currently the most common neurological complication in HIV-infected individuals, occurring in 35–50% of patients undergoing combination anti-retroviral therapy. Data have shown that distal symmetric polyneuropathy develops in mice by 6 weeks following infection with the LP-BM5 retrovirus mixture. Previous work from our laboratory has demonstrated that glial cells modulate antiviral T-cell effector responses through the programmed death (PD)-1: PD-L1 pathway, thereby limiting the deleterious consequences of unrestrained neuroinflammation.MethodsUsing the MouseMet electronic von Frey system, we assessed hind-paw mechanical hypersensitivity in LP-BM5-infected wild-type (WT) and PD-1 KO animals. Using multi-color flow cytometry, we quantitatively assessed cellular infiltration and microglial activation. Using real-time RT-PCR, we assessed viral load, expression of IFN-γ, iNOS, and MHC class II. Using western blotting, we measured protein nitrosylation within the lumbar spinal cord (LSC) and dorsal root ganglion (DRG). Histochemical staining was performed to analyze the presence of CD3, ionized calcium binding adaptor molecule (Iba)-1, MHCII, nitrotyrosine, isolectin B4 (IB4) binding, and neurofilament 200 (NF200). Statistical analyses were carried out using graphpad prism.ResultsHind-paw mechanical hypersensitivity observed in LP-BM5-infected animals was associated with significantly increased lymphocyte infiltration into the spinal cord and DRG. We also observed elevated expression of IFN-γ (in LSC and DRG) and MHC II (on resident microglia in LSC). We detected elevated levels of 3-nitrotyrosine within the LSC and DRG of LP-BM5-infected animals, an indicator of nitric oxide (NO)-induced protein damage. Moreover, we observed 3-nitrotyrosine in both small (IB4+) and large (NF200+) DRG sensory neurons. Additionally, infected PD-1 KO animals displayed significantly greater mechanical hypersensitivity than WT or uninfected mice at 4 weeks post-infection (p.i.). Accelerated onset of hind-paw hypersensitivity in PD-1 KO animals was associated with significantly increased infiltration of CD4+ and CD8+ T lymphocytes, macrophages, and microglial activation at early time points. Importantly, we also observed elevated levels of 3-nitrotyrosine and iNOS in infected PD-1 KO animals when compared with WT animals.ConclusionsResults reported here connect peripheral immune cell infiltration and reactive gliosis with nitrosative damage. These data may help elucidate how retroviral infection-induced neuroinflammatory networks contribute to nerve damage and neuropathic pain.

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“…Flow cytometry is a widely used approach for measuring macrophage infiltration or expansion in many studies [28,29,47]. Isolated live cells from SGs obtained by enzymatical digestion were incubated with a mix of fluorochromeconjugated antibodies against CD45 and CD11b and then analyzed by a LSRII cell analyzer.…”

Section: Flow Cytometry Analyses For Macrophage Infiltrationmentioning

confidence: 99%

Macrophage depletion in stellate ganglia alleviates cardiac sympathetic overactivation and ventricular arrhythmogenesis by attenuating neuroinflammation in heart failure

Zhang

et al. 2021

Basic Res Cardiol

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Cardiac sympathetic overactivation is involved in arrhythmogenesis in patients with chronic heart failure (CHF). Inflammatory infiltration in the stellate ganglion (SG) is a critical factor for cardiac sympathoexcitation in patients with ventricular arrhythmias. This study aims to investigate if macrophage depletion in SGs decreases cardiac sympathetic overactivation and ventricular arrhythmogenesis in CHF. Surgical ligation of the coronary artery was used for induction of CHF. Clodronate liposomes were microinjected into bilateral SGs of CHF rats for macrophage depletion. Using cytokine array, immunofluorescence staining, and Western blot analysis, we found that macrophage expansion and expression of TNFα and IL-1β in SGs were markedly increased in CHF rats. Flow cytometry data confirmed that the percentage of macrophages in SGs was higher in CHF rats than that in sham rats. Clodronate liposomes significantly reduced CHF-elevated proinflammatory cytokine levels and macrophage expansion in SGs. Clodronate liposomes also reduced CHF-increased N-type Ca2+ currents and excitability of cardiac sympathetic postganglionic neurons and inhibited CHF-enhanced cardiac sympathetic nerve activity. ECG data from 24-h, continuous telemetry recording in conscious rats demonstrated that clodronate liposomes not only restored CHF-induced heterogeneity of ventricular electrical activities, but also decreased the incidence and duration of ventricular tachycardia/fibrillation in CHF. Macrophage depletion with clodronate liposomes attenuated CHF-induced cardiac sympathetic overactivation and ventricular arrhythmias through reduction of macrophage expansion and neuroinflammation in SGs.

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Flow cytometry analysis of inflammatory cells isolated from the sciatic nerve and DRG after chronic constriction injury in mice

Abstract: We thus offer two simple and effective protocols that allow for application of flow cytometry to the investigation of cellular and molecular mechanisms of neuropathic pain.

Cited by 13 publications

References 28 publications

Effects of spinal non-viral interleukin-10 gene therapy formulated with d -mannose in neuropathic interleukin-10 deficient mice: Behavioral characterization, mRNA and protein analysis in pain relevant tissues

Effects of spinal non-viral interleukin-10 gene therapy formulated with d -mannose in neuropathic interleukin-10 deficient mice: Behavioral characterization, mRNA and protein analysis in pain relevant tissues

Nitrosative damage during retrovirus infection-induced neuropathic pain

Macrophage depletion in stellate ganglia alleviates cardiac sympathetic overactivation and ventricular arrhythmogenesis by attenuating neuroinflammation in heart failure

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