Concomitant Isolation of Primary Astrocytes and Microglia for Protozoa Parasite Infection

Pacheco, Aline de Oliveira Lima; Amaral, Marcelo Pires; Farias, Ingrid Sancho de; Bottino, Luiza Zainotti Miguel Fahur; Bortoluci, Karina Ramalho

doi:10.3791/60680

Cited by 5 publications

(4 citation statements)

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“…The extraction of microglia refers to the method of Pacheco ( Pacheco et al, 2020 ), and the specific operations are as follows: Take three 1–2 days old neonatal SD rats, quickly cut the skull under sterile conditions, take out the cerebral hemisphere tissue into a petri dish containing HBSS (GIBCO, Invitrogen Corp, Carlsbad, CA, USA) + 10% fetal bovine serum (FBS, GIBCO, Invitrogen Corp, Carlsbad, CA, USA) solution. Then carefully peel off the meninges under the microscope.…”

Section: Methodsmentioning

confidence: 99%

Xenon ameliorates chronic post-surgical pain by regulating mitophagy in microglia and rats mediated by PINK1/Parkin pathway

Lv,

Huang,

Zhang

et al. 2024

PeerJ

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Background Chronic post-surgical pain (CPSP) is one of the important causes of poor postoperative outcomes, the activation of microglia in the spinal cord is closely related to the generation, transmission and maintenance of CPSP. Xenon (Xe), an anesthetic gas, has been reported to be able to significantly reduce intraoperative analgesia and postoperative pain sensation at low doses. However, the mechanism of the regulatory effect of xenon on activated microglia after CPSP remains unclear. Methods In this study, CPSP model rats were treated with 50% Xe inhalation for 1 h following skin/muscle incision and retraction (SMIR), once a day for 5 consecutive days, and then the painbehavioraltests (pain behavior indexes paw withdrawal mechanical threshold, PWMT and thermal withdrawal latency, TWL), microglial activation, oxidative stress-related indexes (malondialdehyde, MDA; superoxide dismutase, SOD; hydrogen peroxide, H2O2; and catalase, CAT), mitophagy and PINK1/Parkin pathway were examined. Results The present results showed that a single dose of Xe treatment in SMIR rat model could significantly improve PWMT and TWL in the short-term at a single treatment and long-term at multiple treatments. Xe treatment inhibited microglia activation and oxidative stress in the spinal dorsal horn of SMIR rats, as indicated by the decrease of Iba1 and MDA/H2O2 levels and the increase of SOD/CAT levels. Compared with the control group, Xe further increased the CPSP promoted Mito-Tracker (a mitochondrial marker) and LC3 (an autophagy marker) co-localization positive spots and PINK1/Parkin/ATG5/BECN1 (autophagy-related proteins) protein expression levels, and inhibited the Mito-SOX (a mitochondrial reactive oxygen species marker) positive signal, indicating that Xe promoted microglia mitophagy and inhibited oxidative stress in CPSP. Mechanistically, we verified that Xe promoted PINK1/Parkin signaling pathway activation. Conclusion Xe plays a role in ameliorating chronic post-surgical pain by regulating the PINK1/Parkin pathway mediated microglial mitophagy and provide new ideas and targets for the prevention and treatment of CPSP.

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Section: Methodsmentioning

confidence: 99%

Xenon ameliorates chronic post-surgical pain by regulating mitophagy in microglia and rats mediated by PINK1/Parkin pathway

Lv,

Huang,

Zhang

et al. 2024

PeerJ

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“…For the in vitro experiments parasite load was checked as previously described ( 32 ). Briefly, after each time point supernatant was collected and reserved.…”

Section: Methodsmentioning

confidence: 99%

NAIP/NLRC4 inflammasome participates in macrophage responses to Trypanosoma cruzi by a mechanism that relies on cathepsin-dependent caspase-1 cleavage

Amaral,

Cardoso,

de Farias

et al. 2023

Front. Immunol.

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Inflammasomes are large protein complexes that, once activated, initiate inflammatory responses by activating the caspase-1 protease. They play pivotal roles in host defense against pathogens. The well-established role of NAIP/NLRC4 inflammasome in bacterial infections involves NAIP proteins functioning as sensors for their ligands. However, recent reports have indicated the involvement of NLRC4 in non-bacterial infections and sterile inflammation, even though the role of NAIP proteins and the exact molecular mechanisms underlying inflammasome activation in these contexts remain to be elucidated. In this study, we investigated the activation of the NAIP/NLRC4 inflammasome in response to Trypanosoma cruzi, the protozoan parasite responsible for causing Chagas disease. This parasite has been previously demonstrated to activate NLRP3 inflammasomes. Here we found that NAIP and NLRC4 proteins are also required for IL-1β and Nitric Oxide (NO) release in response to T. cruzi infection, with their absence rendering macrophages permissive to parasite replication. Moreover, Nlrc4-/- and Nlrp3-/- macrophages presented similar impaired responses to T. cruzi, underscoring the non-redundant roles played by these inflammasomes during infection. Notably, it was the live trypomastigotes rather than soluble antigens or extracellular vesicles (EVs) secreted by them, that activated inflammasomes in a cathepsins-dependent manner. The inhibition of cathepsins effectively abrogated caspase-1 cleavage, IL-1β and NO release, mirroring the phenotype observed in Nlrc4-/-/Nlrp3-/- double knockout macrophages. Collectively, our findings shed light on the pivotal role of the NAIP/NLRC4 inflammasome in macrophage responses to T. cruzi infection, providing new insights into its broader functions that extend beyond bacterial infections.

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“…, HeLa, Vero, HFF, BeWo) and primary cell cultures ( Scheidegger et al., 2005 ; Müller and Hemphill, 2013 ), and among them, target cells or tissues ( e.g. , trophoblast and nervous cells, dendritic cells [DCs] or macrophages) should be highlighted ( Guimarães et al., 2008 ; Dellacasa-Lindberg et al., 2011 ; Mammari et al., 2014 ; Witola et al., 2014 ; Barbosa et al., 2015 ; da Silva et al., 2017 ; Pacheco et al., 2020 ). Most of publications on T. gondii implementing in vitro assays are focused on safety and efficacy assessment of potential antiparasitic drugs ( Basto et al., 2017 ; Murata et al., 2017 ; Radke et al., 2018 ) or on demonstrating the role of different host and parasite effectors in the T. gondii lytic cycle ( Camejo et al., 2014 ; Bai et al., 2018 ; Guo et al., 2019 ; Wang et al., 2020 ).…”

Section: Complementary Approaches On the Determination Of Virulence I...mentioning

confidence: 99%

Unifying Virulence Evaluation in Toxoplasma gondii: A Timely Task

Calero‐Bernal

Fernández‐Escobar

Katzer

et al. 2022

Front. Cell. Infect. Microbiol.

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Toxoplasma gondii, a major zoonotic pathogen, possess a significant genetic and phenotypic diversity that have been proposed to be responsible for the variation in clinical outcomes, mainly related to reproductive failure and ocular and neurological signs. Different T. gondii haplogroups showed strong phenotypic differences in laboratory mouse infections, which provide a suitable model for mimicking acute and chronic infections. In addition, it has been observed that degrees of virulence might be related to the physiological status of the host and its genetic background. Currently, mortality rate (lethality) in outbred laboratory mice is the most significant phenotypic marker, which has been well defined for the three archetypal clonal types (I, II and III) of T. gondii; nevertheless, such a trait seems to be insufficient to discriminate between different degrees of virulence of field isolates. Many other non-lethal parameters, observed both in in vivo and in vitro experimental models, have been suggested as highly informative, yielding promising discriminatory power. Although intra-genotype variations have been observed in phenotypic characteristics, there is no clear picture of the phenotypes circulating worldwide; therefore, a global overview of T. gondii strain mortality in mice is presented here. Molecular characterization has been normalized to some extent, but this is not the case for the phenotypic characterization and definition of virulence. The present paper proposes a baseline (minimum required information) for the phenotypic characterization of T. gondii virulence and intends to highlight the needs for consistent methods when a panel of T. gondii isolates is evaluated for virulence.

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Concomitant Isolation of Primary Astrocytes and Microglia for Protozoa Parasite Infection

Cited by 5 publications

References 0 publications

Xenon ameliorates chronic post-surgical pain by regulating mitophagy in microglia and rats mediated by PINK1/Parkin pathway

Xenon ameliorates chronic post-surgical pain by regulating mitophagy in microglia and rats mediated by PINK1/Parkin pathway

NAIP/NLRC4 inflammasome participates in macrophage responses to Trypanosoma cruzi by a mechanism that relies on cathepsin-dependent caspase-1 cleavage

Unifying Virulence Evaluation in Toxoplasma gondii: A Timely Task

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