AIMS:Adenosine 2A receptor (A2A R) is widely expressed in the brain and plays important roles in neuroinflammation, and the nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain-containing protein 3 (NLRP3) inflammasome is a crucial component of the innate immune system while the regulation of A2A R on it in the central nervous system (CNS) has not been clarified. METHODS:The effects of microglial A2A R on NLRP3 inflammasome assembly and activation were investigated in wild-type, A2A R- or NLRP3-knockout primary microglia with pharmacological treatment. Microglial A2A R or NLRP3 conditional knockout mice were used to interrogate the effects of this regulation on neuroinflammation posttraumatic brain injury (TBI). RESULTS:We found that A2A R directly interacted with NLRP3 and facilitated NLRP3 inflammasome assembly and activation in primary microglia while having no effects on mRNA levels of inflammasome components. Inhibition of the interaction via A2A R agonist or knockout attenuated inflammasome assembly and activation in vitro. In the TBI model, microglial A2A R and NLRP3 were co-expressed at high levels in microglia next to the peri-injured cortex, and abrogating of this interaction by microglial NLRP3 or A2A R conditional knockout attenuated the neurological deficits and neuropathology post-TBI via reducing the NLRP3 inflammasome activation. CONCLUSION:Our results demonstrated that inhibition of the interaction between A2A R and NLRP3 in microglia could mitigate the NLRP3 inflammasome assembly and activation and ameliorate the neuroinflammation post-TBI. It provides new insights into the effects of A2A R on neuroinflammation regulation post-TBI and offers a potential target for the treatment of NLRP3 inflammasome-related CNS diseases.

Traumatic brain injury (TBI) survivors suffer from long-term disability and neuropsychiatric sequelae due to irreparable brain tissue destruction. However, there are still few efficient therapies to promote neurorestoration in damaged brain tissue. This study aimed to investigate whether the pro-oncogenic gene ski can promote neurorestoration after TBI. We established a ski-overexpressing experimental TBI mouse model using adenovirus-mediated overexpression through immediate injection after injury. Hematoxylin-eosin staining, MRI-based 3D lesion volume reconstruction, neurobehavioral tests, and analyses of neuronal regeneration and astrogliosis were used to assess neurorestorative efficiency. The effects of ski overexpression on the proliferation of cultured immature neurons and astrocytes were evaluated using imaging flow cytometry. The Ski protein level increased in the perilesional region at 3 days post injury. ski overexpression further elevated Ski protein levels up to 14 days post injury. Lesion volume was attenuated by approximately 36-55% after ski overexpression, with better neurobehavioral recovery, more newborn immature and mature neurons, and less astrogliosis in the perilesional region. Imaging flow cytometry results showed that ski overexpression elevated the proliferation rate of immature neurons and reduced the proliferation rate of astrocytes. These results show that ski can be considered a novel neurorestoration-related gene that effectively promotes neurorestoration, facilitates neuronal regeneration, and reduces astrogliosis after TBI.

Pyroptosis plays a significant role in neuroinflammation after traumatic brain injury (TBI). However, the role of pyroptosis executor Gasdermin D (GSDMD) in neurological deficits and neuropathological alterations after TBI have not been elucidated. Our results demonstrated that GSDMD-KO exerted striking neuroprotective effects on motor dysfunction and neuropathological alterations (loss of synaptic proteins, microglia activation, astrogliosis, dendrite injury, and neuron death) at 3 days after TBI. GSDMD-KO inhibited the expression and release of pro-inflammatory cytokine releases (IL-1β and TNF-α) while promoting those of anti-inflammatory cytokines (IL-10 and TGF-β1). The temporal pattern of diverse inflammasome signals showed long-lasting elevations of NLRP3, caspase 1, and caspase 1 p20 after TBI, rather than NLRP1, NLRC4 or AIM2, similar to the change in GSDMD postinjury; and NLRP3-KO not only inhibited the expression and cleavage of GSDMD but also attenuated the loss of synaptic proteins and neurological deficits. Notably, RNA sequencing showed both GSDMD-KO and NLRP3-KO reversed the global expression of neuroinflammation- and neuropathology-related genes after TBI. Our findings proved that the inhibition of GSDMD exerts neuroprotective effects after TBI and is mainly driven by the NLRP3 inflammasome. GSDMD serves as a potent therapeutic target for the treatment of TBI.

Tau hyperphosphorylation is a characteristic alteration present in a range of neurological conditions, such as traumatic brain injury (TBI) and neurodegenerative diseases. Treatments targeting high-mobility group box protein 1 (HMGB1) induce neuroprotective effects in these neuropathologic conditions. However, little is known about the interactions between hyperphosphorylated tau and HMGB1 in neuroinflammation. We established a model of TBI with controlled cortical impacts (CCIs) and a tau hyperphosphorylation model by injecting the virus encoding human P301S tau in mice, and immunofluorescence, western blotting analysis, and behavioral tests were performed to clarify the interaction between phosphorylated tau (p-tau) and HMGB1 levels. We demonstrated that p-tau and HMGB1 were elevated in the spatial memory-related brain regions in mice with TBI and tau-overexpression. Animals with tau-overexpression also had significantly increased nucleotide-binding oligomerization domain-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome activation, which manifested as increases in apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), activating caspase-1 and interleukin 1 beta (IL-1β) levels. In addition, NLRP3 –/– mice and the HMGB1 inhibitor, glycyrrhizin, were used to explore therapeutic strategies for diseases with p-tau overexpression. Compared with wild-type (WT) mice with tau-overexpression, downregulation of p-tau and HMGB1 was observed in NLRP3 –/– mice, indicating that HMGB1 alterations were NLRP3-dependent. Moreover, treatment with glycyrrhizin at a late stage markedly reduced p-tau levels and improved performance in the Y- and T-mazes and the ability of tau-overexpressing mice to build nests, which revealed improvements in spatial memory and advanced hippocampal function. The findings identified that p-tau has a triggering role in the modulation of neuroinflammation and spatial memory in an NLRP3-dependent manner, and suggest that treatment with HMGB1 inhibitors may be a better therapeutic strategy for tauopathies.

NLRP3 inflammasome plays a crucial role in the innate immune system. Our group previously reported that the microglial adenosine 2A receptor (A(2A)R) regulates canonical neuroinflammation, which is affected by the glutamate concentration. However, the regulatory effect of A(2A)R on NLRP3 inflammasome and the effects of glutamate concentration remain unknown. Therefore, we aimed to investigate the regulatory effect of microglial A(2A)R on NLRP3 inflammasome assembly and activation as well as the effects of glutamate concentration on the inflammasome assembly and activation. Experiments were conducted on magnetically sorted primary microglia from P14 mice. The results showed that pharmacological A(2A)R activation ameliorated NLRP3 activation under no or low glutamate concentrations, but this effect was reversed by high glutamate concentrations. Moreover, the mRNA levels of NLRP3 inflammasome-related genes were not affected by A(2A)R activation or the glutamate concentration. We further demonstrated that A(2A)R activation inhibited the interaction between NLRP3 and caspase 1 under no or low glutamate concentrations while promoting their interaction under high glutamate concentrations. The oligomerization of ASC also showed a similar trend. In conclusion, our findings proved that the high glutamate concentration could reverse the inhibition of A(2A)R on NLRP3 inflammasome activation by modulating its assembly, which provides new insights into the regulatory effect of A(2A)R on neuroinflammation under different pathological conditions.

CLARITY is a hydrogel embedding clearing method that has the advantages of transparency, different tissue compatibility and immunostaining compatibility. However, there are also some limitations to CLARITY as it requires a long time to achieve transparency, and the electrophoresis clearing is complex. Therefore, we aimed to simplify the electrophoresis system and shorten the processing time of CLARITY. In our study, we developed a non-circulation electrophoresis system to achieve easier manipulation of electrophoresis clearing. We modified the original CLARITY protocol in hydrogel embedding methods, clearing buffer and immunostaining. When comparing brains processed by our modified method or the original protocol, we found our modifications permit faster and more efficient clearing and labeling. Moreover, we developed a new clearing method named Passive pRe-Electrophroresis CLARITY (PRE-CLARITY) and a new immunostaining method named Centrifugation-Expansion staining (CEx staining). PRE-CLARITY achieved faster clearing and higher transparency, and CEx staining accomplished intact mouse brain labeling faster. With our modifications to CLARITY, we accomplished intact mouse brain clearing and immunostaining within one week, while this requires weeks to months with the original CLARITY. Our studies would allow high-content tracing and analysis of intact brain or other large-scale samples in a short time.

CLARITY is a novel tissue clearing technique that transforms intact biological tissues into a nanoporous hydrogel-tissue hybrid, preserving anatomical structures, proteins and nucleic acids. The hydrogel-based structure is transparent after the removal of lipids and permits several rounds of immunostaining and imaging. This technique provides an ideal way for researchers to examine the central nervous system (i.e., mouse brain and spinal cord) intact. CLARITY was selected as one of ten breakthroughs in 2013 by Science. However, the original CLARITY technique still has severe technical limitations which impede its application in wider fields. Therefore, many modified clearing methods based on CLARITY have emerged. As all CLARITY-based tissue clearing techniques involve similar procedures, the present review attempted to divide these methods into individual procedures in order to provide new ways to test and combine tissue clearing methods. Furthermore, the combination of clearing methods could help to determine the optimal method for clearing and imaging large samples.