Detecting Migration and Infiltration of Neutrophils in Mice

Lu, Qing‐Yi; Yuan, Kai; Li, Xiaohong; Jiang, Haixu; Huo, Guiyang; Jia, Wenrui; Huang, Guangrui; Xu, Anlong

doi:10.3791/60543

“…Renal malondialdehyde (MDA) level was assessed by the evaluation of lipid peroxidation. 10 Kidney tissue samples were prepared by homogenizing in 150 mM potassium chloride buffer on ice. MDA levels were assessed by spectrophotometric analysis.…”

Section: Methodsmentioning

confidence: 99%

Quantitative Increase in Short-Chain Fatty Acids, Especially Butyrate Protects Kidney from Ischemia/Reperfusion Injury

Sun

¹

,

Zhou

²

,

Chen

³

et al. 2022

Journal of Investigative Medicine

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Short-chain fatty acids (SCFAs), the end products of fermentation carried out by the intestinal microbiota, were demonstrated to produce anti-oxidant and anti-inflammatory effects. Butyrate, part of the SCFAs, also shows the same effect. Renal ischemia/reperfusion (I/R) injury commonly occurs in renal transplantation and is often accompanied by oxidative stresses and inflammatory responses. In this study, we explore butyrate effect on renal I/R injury and SCFAs changes in renal transplant. Male Sprague-Dawley rats were pretreated with butyrate as research, and underwent the surgery of renal ischemia for 45 min followed by reperfusion. 90 rats were randomly divided into 3 groups (n=30 each group): (1) sham-operated group; (2) butyrate-treated group; (3) control group. The samples of blood and renal were collected immediately for further studies. Thirty-two patients were enrolled to investigate the levels of SCFAs after the renal transplantation. Rats model showed that butyrate treatments significantly enhanced the function and structure of kidney, as evidenced by the lower serum creatinine levels and less pathological damages of renal tissue. With the recovery of renal function after renal transplantation, SCFAs increased, which were negatively correlated with creatinine. Butyrate expressed like SCFAs. In this study, we demonstrated that butyrate increased with the recovery of renal function after renal transplantation. Most importantly, butyrate treatments alleviated the renal damages caused by I/R via the upregulation of intracellular oxidant stress and inflammations.

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“…Eight-week-old mice were used after one week of adaptive maintenance. The AA model was induced by FCA injection into ankle joints as described previously [ 22 ] on Day 0. 20 microlitres of FCA was injected into the ankle cavity, and 80 μ L of FCA was injected around the joint.…”

Section: Methodsmentioning

confidence: 99%

“…After decalcification, paraffin sections of the mouse joints were prepared as described previously [ 22 ]. HE and Safranin O-fast green staining were conducted using standard procedures.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Tetrandrine Ameliorates Rheumatoid Arthritis in Mice by Alleviating Neutrophil Activities

Lu

¹

,

Jiang

²

,

Zhu

³

et al. 2022

Evidence-Based Complementary and Alternative Medicine

Self Cite

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Rheumatoid arthritis (RA) is a common autoimmune disease worldwide. Neutrophils play critical roles in the onset and development of RA and are the promising target for RA treatment. Tetrandrine is a bis-benzyl isoquinoline alkaloid derived from the traditional Chinese herbal Stephania tetrandra S. Moore. Tetrandrine is effective in alleviating RA by inhibiting macrophage inflammatory response, fibroblast overproliferation, and pannus formation. However, whether tetrandrine regulates the activities of neutrophils in RA is largely unknown. In this study, we adopted adjuvant-induced arthritis (AA) murine model to explore the effect of tetrandrine on RA and neutrophils. Twenty-eight mice were divided into four groups. The control group was injected with PBS in the limbs and treated with PBS by intraperitoneal injection (i.p.) from Day 10 to Day 37. The arthritis murine model was induced by injecting FCA into the ankle joints of hind limbs. The AA group, the AA + TET group, and the AA + DEX group mice were treated with PBS, tetrandrine (6 mg/kg), or dexamethasone (1 mg/kg) i.p. daily, respectively. Arthritic scores were evaluated, and the joint diameter was measured every three days. A cytometric bead assay was performed to measure the concentrations of IFN-γ, TNF-α, and IL-6 in the serum. H&E staining and Safranin O-fast staining were adopted to monitor the tissue changes in the joint. Immunohistochemistry assays were applied to detect the MPO, NE, CitH3, and PAD4 expression levels. To assess the effect of tetrandrine on neutrophil activities in vitro, CCK8 tests were applied to determine cell viability. The qPCR and ELISA were performed to determine IL-1β and IL-6 expression levels. Immunofluorescence assays were performed to measure the formation of NETs. The results indicated that tetrandrine significantly alleviated the symptoms of RA in terms of the ankle diameter (from 4.629 ± 2.729 to 3.957 ± 0.257; P < 0.01 ) and ankle score (from 4.000 ± 0.000 to 3.286 ± 0.756; P < 0.05 ). Tetrandrine treatment significantly increased the cartilage areas and decreased serum IL-6 significantly (from 5.954 ± 2.127 to 2.882 ± 2.013; P < 0.01 ). The immunohistochemistry assays also showed decreased expression levels of NE, MPO, PAD4, and CitH3 induced by tetrandrine in comparison with the AA group ( P < 0.01 ). The qPCR assays and ELISAs showed that tetrandrine had an anti-inflammatory effect in vitro by significantly inhibiting IL-6 ( P < 0.01 ). The immunofluorescence assays showed that NET formation induced by PMA could be reduced by tetrandrine ( P < 0.01 ). In conclusion, tetrandrine has good efficacy in treating RA by regulating neutrophil-involved inflammation and NET formation.

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“…In addition, a number of other agents that evoke an inflammatory or immune response can be injected into the air pouch in lieu of carrageenan. These include, but are not limited to, bovine serum albumin (Sin, Sedgwick, & Willoughby, 1984; Yoshino et al., 1984), mycobacteria (Castro, Esaguy, Macedo, Aguas, & Silva, 1991) or streptococcal bacteria (Chen et al., 2018), lipopolysaccharide (Baliou et al., 2021; Lu et al., 2020; Nakamura et al., 2001; Stubelius, Andersson, Islander, & Carlsten, 2017), zymosan (Kapellos et al., 2019; Yoon et al., 2005), sodium urate crystals (Iverson et al., 2018), and lipids (Kadl, Galkina, & Leitinger, 2009; Zhao et al., 2011). For this protocol, however, we focus on a single methodology for rats, with modifications for mice, that uses carrageenan as the inflammatory stimulus.…”

Section: Introductionmentioning

confidence: 99%

Models of Inflammation: Carrageenan Air Pouch

Fehrenbacher

¹

,

McCarson

²

2021

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The subcutaneous air pouch is an in vivo model that can be used to study the components of acute and chronic inflammation, the resolution of the inflammatory response, the oxidative stress response, and potential therapeutic targets for treating inflammation. Injection of irritants into an air pouch in rats or mice induces an inflammatory response that can be quantified by the volume of exudate produced, the infiltration of cells, and the release of inflammatory mediators. The model presented in this article has been extensively used to identify potential anti‐inflammatory drugs. © 2021 Wiley Periodicals LLC. Basic Protocol: Air pouch model in the rat Alternate Protocol: Air pouch model in the mouse

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Detecting Migration and Infiltration of Neutrophils in Mice

Cited by 7 publications

References 0 publications

Quantitative Increase in Short-Chain Fatty Acids, Especially Butyrate Protects Kidney from Ischemia/Reperfusion Injury

Quantitative Increase in Short-Chain Fatty Acids, Especially Butyrate Protects Kidney from Ischemia/Reperfusion Injury

Tetrandrine Ameliorates Rheumatoid Arthritis in Mice by Alleviating Neutrophil Activities

Models of Inflammation: Carrageenan Air Pouch

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