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Background: Brain aging is an important risk factor in many human diseases, such as Alzheimer’s disease (AD). The production of excess reactive oxygen species (ROS) mediated by nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) and the maturation of inflammatory cytokines caused by activation of the NOD-like receptor protein 1 (NLRP1) inflammasome play central roles in promoting brain aging. However, it is still unclear when and how the neuroinflammation appears in the brain during aging process. Methods: In this study, we observed the alterations of learning and memory impairments, neuronal damage, NLRP1 inflammasome activation, ROS production and NOX2 expression in the young 6-month-old (6 M) mice, presenile 16 M mice, and older 20 M and 24 M mice. Results: The results indicated that, compared to 6 M mice, the locomotor activity, learning and memory abilities were slightly decreased in 16 M mice, and were significantly decreased in 20 M and 24 M mice, especially in the 24 M mice. The pathological results also showed that there were no significant neuronal damages in 6 M and 16 M mice, while there were obvious neuronal damages in 20 M and 24 M mice, especially in the 24 M group. Consistent with the behavioral and histological changes in the older mice, the activity of β-galactosidase (β-gal), the levels of ROS and IL-1β, and the expressions of NLRP1, ASC, caspase-1, NOX2, p47phox and p22phox were significantly increased in the cortex and hippocampus in the older 20 M and 24 M mice. Conclusion: Our study suggested that NLRP1 inflammasome activation may be closely involved in aging-related neuronal damage and may be an important target for preventing brain aging. Keywords: Brain aging, Learning and memory impairments, NLRP1 inflammasome, ROS, NADPH oxidase 2 understood. Growing evidence has indicated that neu- Introduction roinflammation plays an important role in the aging pro - Brain aging has been reported to be an important risk cess . Even the regulation of neuroinflammation by the factor in many human diseases, such as Alzheimer’s dis- peripheral immune system is involved in the develop- ease (AD) and Parkinson’s disease (PD) . At present, ment of aging and AD . According to a large amount the mechanism of brain aging is still not completely of epidemiological, clinical, and laboratory data, the rela- tionship between inflammation and aging-related dis - *Correspondence: firstname.lastname@example.org eases is inseparable . It has been reported that IFN-γ Dan Sun, Guofang Gao, Bihua Zhong contributed equally to this work and other proinflammatory cytokines interact with pro - Department of Pharmacology, Basic Medicine College, Anhui Medical University, No. 81 Meishan Road, Hefei 230032, Anhui, China cessing and production of Aβ peptide, suggesting that Full list of author information is available at the end of the article © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecom- mons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Sun et al. Behavioral and Brain Functions (2021) 17:11 Page 2 of 14 inflammation may be a "prodrome" to AD . However, inflammasome activation during aging process. To it is still unclear when and how the neuroinflammation confirm our hypothesis, we investigated the learning takes place during aging process. and memory function, the changes of NLRP1 inflam - Inflammasomes are multiprotein complexes in the masome activation, and the ROS production and cytoplasm that are responsible for the formation of pro- NOX2 expression in the cortex and hippocampus in inflammatory molecules. Inflammasomes have a central 6-, 16-, 20-, and 24-month-old (M) mice. The study role in the inflammatory response and can be activated has the potential to confirm when the neuroinflamma - by diverse stimuli, leading to the maturation of proin- tion appears in the brain and whether NOX2-NLRP1 flammatory cytokines . The nucleotide-binding oli - inflammasome is involved in aging-related neuronal gomerization domain NOD-like receptor protein 1 damage during aging. (NLRP1) is the first family of sensor proteins discovered to form inflammasomes. Recent studies demonstrated that the NLRP1 inflammasome is closely associated with Materials and methods neurological diseases such as AD . NLRP1 immunopo- Animals and treatment sitive neurons were increased 25–30-fold in brains of AD A total of 48 male 6-month-old ICR mice weighing patients compared to brains of normal elderly . Our (33–40 g) were obtained from the Center of Laboratory previous study showed that the NLRP1 inflammasome Animals of Anhui Medical University (Hefei, China) at was significantly increased in primary hippocampal neu - the same time. The animals were randomly divided into rons with prolongation of culture time . Down-regu- 4 groups (n = 12): 6 M, 16 M, 20 M, and 24 M. The con - lation of the NLRP1 inflammasome improves cognitive trol was 6 M group. All mice were housed (6 mice in deficits in different animal models . NLRP1 inflam - each cage) in a pathogen-free, temperature-controlled masome activation in hippocampal neurons significantly room with a 12-h light/12-h dark cycle and unrestricted exacerbates age-related cognitive impairment . How- access to food and water. After a week of acclimatiza- ever, it remains unclear when and how NLRP1 inflamma - tion, the 6 M group were sacrificed after behavioral some is involved in aging-associated neuronal damage tests for subsequent experiments. Animals in other during aging process. groups were bred separately to 16 months, 20 months Reactive oxygen species (ROS) accumulation report- and 24 months old and used for experiments. The entire edly plays a crucial role in the induction of inflammatory experiment lasted for 18 months. Three mice died at the cascades . Excessive release of ROS further promotes age of 23–24 months. All procedures were performed neuronal damage and subsequent inflammation result - based on the guidelines approved by the Animal Eth- ing in a feed-forward loop of neurodegeneration . ics and Care Committee of Anhui Medical University NADPH oxidase (NOX), a multi-protein enzyme, is cur- (LLSC20160183). The experimental procedure was rently the only enzyme family known to produce ROS as showed in Additional file 1: Fig. S1. its sole function. It has been reported that NOX-medi- ated ROS production involves in NLRP3 activation in metabolic and cognitive diseases, such as type 2 diabetes Open field test (OFT) mellitus, obesity, and AD . Excessive NOX-derived The OFT was performed to study changes of motor ROS also contributes to neuronal loss via oxidative stress and exploratory behavior when the mice reached their damage or disruption of redox signaling circuits . The specified age. The OFT equipment (Shanghai Biotech - NOX family comprises membrane components, includ- nology Co., Ltd.) comprised a computer-tracked cage ing p22phox and gp91phox homologues of NOX1–5, (60 × 60 × 50 cm) divided into nine squares (one cen- and several other cytosolic proteins, including p47phox, tral and eight peripheral) by two perpendicular trans- p40phox, and p67phox . NOX2 is widely expressed verse lines and vertical lines as previously described . throughout the brain in both microglia and neurons . Twelve mice in the 6 M, 16 M, and 20 M group and 9 The NOX2 has been studied mainly in microglia, where mice in the 24 M group were allowed 24 h to adjust the it plays a role in inflammation and may also contribute to environment. For the OFT, each mouse was placed in the neuronal death in pathological conditions , but it is cage for 2 min to adapt to the environment. Then, the still not completely understood whether NOX2-derived motor and exploration paths were recorded for 3 min by ROS production involves in NLRP1 inflammasome acti - ANY-maze Behavioral Tracking Software (Stoelting Co., vation during aging process. Wood Dale, IL, USA). The total moving distance (m), the In the present study, we hypothesized that NLRP1 mean speed (m/s), the number of lines crossing, and the inflammasome involves in aging-related neuronal dam - number of standing were calculated by the software to ages and NOX2 plays an important role in NLRP1 evaluate the motor and exploration behavior . Sun et al. Behavioral and Brain Functions (2021) 17:11 Page 3 of 14 Morris water maze (MWM) Determination of ROS production The next day after the OFT, the mice were subjected The ROS level was evaluated by using dihydroethidium to the MWM (in the same room with OFT) to detect (DHE) fluorescence staining as previously described learning and memory functions. The MWM test . Briefly, DHE (Beyotime Biotechnology, Shanghai, included four consecutive daily training trials and a China) was injected via the tail vein (100 μM, 0.1 ml/10 g spatial probe trial on the fifth day . The test pool body weight) for 30 min. Then the mice (n = 3) were (120 cm in diameter and 60 cm high) filled with water sacrificed by cervical dislocation, and the brains were (depth 30 cm, temperature 24 ± 2 °C) was divided into carefully removed and embedded in OCT (Sakura Fine- four quadrants. There was a hidden escape platform tek, Torrance, CA, USA). The brains were sectioned (9 cm in diameter) that was submerged 1 cm below the into 10 μm slices at − 20 °C with a freezing microtome surface of the water in the third quadrant. For training (Leica CM3050, Nussloch, Germany). The sections were trials, the mice with head facing towards the wall were mounted onto slides and washed three times with PBS. individually placed into the tank from the four quad- Then, the sections were incubated with Hoechst 33258 rants each day, each mouse was allowed to climbed (Sigma, 5 mg/L) for 5 min and washed three times with onto the platform within 60 s. If the mice failed to phosphate-buffered saline (PBS). The sections were find the hidden platform within 60 s, they were gently examined using a fluorescence microscope (Olympus guided to the platform by the experimenter. Animals IX71) and photographed for DHE (excitation: 480 nm, were allowed to remain there for 10 s to familiarize emission: 590 nm) and Hoechst 33258 (excitation: the location of the platform every time after climbing 360 nm, emission: 450 nm). The mean fluorescence den - the platform. Each mouse underwent four trials per sity of the DHE staining was quantified double-blindly day, and the delay between trial is at least 20 min. The from three random fields (400 ×) in cortex, hippocampal mean escape latency (MEL, s) of each day was recorded CA1 and CA3 areas by using Image-Pro Plus 6.0 auto- to evaluate learning ability. On the fifth day, the plat - matic analysis system to assess the ROS production. form was removed from the pool, and each mouse per- formed a spatial probe test for 60 s. The latency of first Determination of senescence‑associated β‑galactosidase entry to the platform (LFP, s), the swimming time in the (β‑gal) activity quadrant of the platform (STP, s), and the number of OCT-embedded brains (n = 3) were cut into 10 µm crossing the platform (NCP) were recorded to indicate sections. The sections were stained with a β-gal kit the memory results. At the end of each test, remove (Beyotime Biotechnology, Shanghai, China). All of the the mouse from the pool, wipe dry and return it to the operations were performed strictly in accordance with home cage. During the test, keep the position of the the instructions. β-gal staining will generate blue prod- spatial reference objects such as lights and objects in ucts under the catalysis of senescence-associated β-gal the laboratory unchanged, and eliminate the influence with X-gal as the substrate. Thus, the density of blue cells of interference factors on the experimental results. can be used to examine the β-gal activity. The results were observed under a light microscope (Olympus IX71). The density was analyzed double-blindly from three ran - Histological examination dom fields (400 ×) in cortex, hippocampal CA1 and CA3 After behavior tests, the mice (n = 4) were killed by cervi- areas by using Image-Pro Plus 6.0 automatic analysis cal dislocation, the brains were removed and fixed in 4% system. paraformaldehyde. The brain tissues were embedded in paraffin and sliced into 5 μm sections using a microtome Immunohistochemistry (Leica, Nussloch, Germany). The sections were stained For immunohistochemical staining, paraffin-embed - with hematoxylin and eosin (H&E) or Nissl staining ded brain sections (n = 4) were deparaffinized and then (Beyotime Institute of Biotechnology, China) to examine were incubated with 3% H O for 15 min at room tem- 2 2 the neuronal morphology in the cortex and hippocam- perature to remove the endogenous peroxidase activity. pal CA1 and CA3 regions using a microscope (Olym- Subsequently, the sections were heated in a microwave pus IX71; Olympus, Tokyo, Japan). Nissl bodies appear oven with a sodium citrate antigen retrieval solution for blue-purple, and Nissl staining is often used to show the 15 min and were blocked with non-immune goat serum Nissl bodies in neurons. The density of the Nissl bodies for 30 min at 37 °C. Thereafter, the sections were washed was analyzed double-blindly from three random fields with PBS and then incubated with a mouse anti-MAP2 (400 ×) in cortex, hippocampal CA1 and CA3 areas by antibody (1:200; ab11268; Abcam, Cambridge, UK) over- using the Image-Pro Plus 6.0 automatic analysis system night at 4 °C. The next day, the sections were washed with to assess the amount of Nissl bodies . Sun et al. Behavioral and Brain Functions (2021) 17:11 Page 4 of 14 PBS three times and then incubated with a general sec- statistical software. The data were analyzed with one- ondary antibody (ZSGB-BIO; Beijing, China) at room way global analysis of variance (ANOVA). If it shows sig- temperature for 1 h. The sections were performed with nificant effect, then Bonferroni’s post hoc test is used to a DAB kit (ZSGB-BIO; Beijing, China). Finally, the tis- compare differences between groups. Statistical signifi - sues were counterstained with hematoxylin and were cance is defined as P < 0.05. viewed under a microscope (Olympus IX72). The density was analyzed double-blindly from three random fields Results (400 ×) in cortex, hippocampal CA1 and CA3 areas by Eec ff ts of aging on motor and exploratory behavior in mice using Image-Pro Plus 6.0 automatic analysis system to In the study, the OFT was used to observe the effects indicate the changes of MAP2 expression during the of aging on motor activity and exploratory behavior in aging process. mice. The results showed that the mean moving distance (m) (Fig. 1A; F(3,41) = 7.698, P < 0.01), the mean moving speed (m/s) (Fig. 1B; F(3,41) = 7.685, P < 0.01), the num- Immunoblot analysis ber of line crossing (Fig. 1C; F(3,41) = 7.035, P < 0.01), Immunoblotting was performed according to previous and standing up (Fig. 1D, F(3,41) = 15.72, P < 0.01) had description . The hippocampal tissues (n = 3) were significant effects, and they were significantly decreased homogenized in radioimmunoprecipitation assay (RIPA) in 20 M and 24 M mice compared to 6 M mice. In addi- buffer (Beyotime Biotechnology, Shanghai, China) with tion, the moving distance and the mean moving speed protease and phosphatase inhibitors to extract total pro- were significantly decreased, while the number of lines tein. The BCA Protein Assay Kit (Beyotime Biotechnol - crossing and number of standing were not significantly ogy, Shanghai, China) was used to determine protein decreased in 16 M compared to 6 M mice. These data concentration. An equal amount (30 μg) of protein from demonstrated an aging phenomenon: 16 M mice showed each sample was separated by using sodium dodecyl a mild decrease in motor ability, while the effects were sulfate–polyacrylamide gel (10%) electrophoresis (SDS- much more pronounced in 20 M and 24 M mice. PAGE). The separated protein was transferred to a polyvi - nylidene fluoride (PVDF) membrane (Millipore, Bedford, Eec ff ts of aging on learning and memory abilities in mice MA, USA). The membranes were immersed in blocking The MWM was used to investigate changes in learn - buffer (5% fat-free milk in Tris-buffered saline with 20% ing and memory abilities during the aging process in Tween [TBST]) for 1 h, then incubated with primary mice. In the orientation navigation experiment, com- antibodies against NLRP1 (1:1000, Abcam, ab3683), pared with the first day (d1), the escape latency in apoptosis-associated speck-like protein containing a cas- 6 M, 16 M and 20 M group had a decreasing trend and pase recruitment domain (ASC; 1:500, Santa Cruz Tech- significantly decreased on d4 in 6 M group (Fig. 2A; nology, SC-514414), caspase-1 (1:1000, Abcam, ab1872), F(3,44) = 4.746, P < 0.01), on d3 in 16 M group (Fig. 2A; interleukin (IL)-1β (1:1000, Abcam, ab9722), NOX2 (1:1000, Abcam, ab31092), p22phox (1:500, Bioworld Technology, BS60290), p47phox (1:1000, Bioworld Tech- nology, BS4852), or β-actin (1:1000; ZSGB-BIO, TA-09) overnight at 4 °C. The next day, the membranes were incubated with horseradish peroxidase–conjugated sec- ondary antibody (ZSGB-BIO, ZF-2301, 1:10,000) for 1 h at room temperature (24 °C). After washing three times with TBST, the proteins were visualized by an enhanced chemiluminescent reagent (Amersham Biosciences, UK). Images of the blots were obtained using a Chemi Q4800 mini-imaging system (Shanghai Bioshine Technology, Shanghai, China). The density of the protein band was measured using Image J 1.44 software; the density was then normalized to β-actin band. The relative density compared to the control group was calculated to indicate Fig. 1 Eec ff ts of aging on motor activity and exploratory behavior in the expression of the target protein. 6 M, 16 M, 20 M and 24 M mice (open field test). A The total moving distance (m). B The mean moving speed (m/s). C The number of lines crossing. D The number of standing. Results are expressed as Statistical analysis mean ± SD. 6 M, 16 M, 20 M, n = 12; 24 M, n = 9. **P < 0.01 vs 6 M All data are presented as mean ± standard deviation control (SD). Statistical analyses were performed using SPSS 17.0 Sun et al. Behavioral and Brain Functions (2021) 17:11 Page 5 of 14 Fig. 2 Eec ff t of aging on learning and memory impairments in 6 M, 16 M, 20 M and 24 M mice (Morris water maze). A The mean escape latency (s) in the orientation navigation experiment. B Representative path of probe trial experiments on day 5. C The latency of first entry to the platform (s). D The swimming time in the quadrant of platform (s). E The number of crossing the platform. Results are expressed as mean ± SD. 6 M, 16 M, 20 M, # ## n = 12; 24 M, n = 9. *P < 0.05, **P < 0.01 vs 6 M group; P < 0.05, P < 0.01 vs d1 in the orientation navigation experiment learning and memory ability was mild decreased in F(3,44) = 4.556, P < 0.05), and on d4 in 20 M group 16 M mice, but was significantly impaired in 20 M and (Fig. 2A; F(3,44) = 3.885, P < 0.05). However, the escape 24 M mice, especially in the 24 M group. latency in 24 M group had no decreasing trend from d1 to d4. Additionally, the escape latency had signifi - Eec ff ts of aging on neuronal degeneration in the cortex cant effects on d4 (Fig. 2A; F(3,41) = 3.57, P < 0.05). and hippocampus in mice And compared with 6 M group, the escape latency H&E and Nissl staining were performed to examine neu- was significantly prolonged in 20 M and 24 M mice ropathological changes in the cortex and hippocampus on d4 (Fig. 2A; P < 0.05), and had no significant effects during aging. Based on H&E staining, there were a few on d1-d3. In the space exploration experiment on d5, neuronal abnormalities in the cortex and hippocam- compared with 6 M mice, the latency of first entry to pal CA1 and CA3 regions in 6 M mice. Compared with the platform (LFP, s) was significantly increased in 6 M mice, there were no obvious increase of pathologi- 16 M, 20 M, and 24 M mice (Fig. 2B, C; F(3,41) = 3.617, cal damages in 16 M mice. However, there were obvious P < 0.05 or P < 0.01). The swimming time in the quad - pathological damages in the cortex and hippocampal rant with platform (STP, s) (Fig. 2D; F(3,41) = 4.416, CA1 and CA3 regions in 20 M and 24 M mice, espe- P < 0.05 or P < 0.01) and the number of crossing the cially in the 24 M group. More neurons exhibited nuclear platform (NCP) (Fig. 2E; F(3,41) = 3.55, P < 0.05) pyknosis and hyperchromatic nuclei in cortex and CA3 were significantly decreased in 20 M and 24 M com - regions in 24 M mice, and eosinophilic degeneration in pared with 6 M mice. These results suggested that the CA3 region in 24 M mice (Additional file 2: Fig. S2). Sun et al. Behavioral and Brain Functions (2021) 17:11 Page 6 of 14 Eec ff ts of aging on NLRP1, ASC, caspase‑1, and IL‑1β Nissl staining is a well-known method that specifi - expression in the hippocampus in mice cally stains Nissl bodies and is often used to identify In order to confirm whether NLRP1 inflammasome neuronal damage . Nissl staining showed abundant activation is involved in aging-related neuronal dam- Nissl bodies in the cortex and hippocampal CA1 and age, we further investigated the expressions of NLRP1, CA3 regions in 6 M and 16 M mice. When compared ASC, caspase-1, and IL-1β in the hippocampus. The with 6 M mice, the number of Nissl bodies was sig- results showed that the expression of NLRP1 was sig- nificantly reduced in cortex (Fig. 3A, B; F(2,9) = 243.9, nificantly increased in 20 M and 24 M mice, especially P < 0.01) and hippocampal CA1 (Fig. 3C; F(2,9) = 99.79, in the 24 M group, compared with 6 M (Fig. 6A, B; P < 0.05 or P < 0.01) and CA3 (Fig. 3D; F(2,9) = 71.02, F(3,8) = 8.872, P < 0.05). In addition, the expressions P < 0.05 or P < 0.01) regions in 20 M and 24 M mice, of ASC, caspase-1, and IL-1β gradually increased with especially the 24 M group. These results suggested aging; they were significantly increased in 20 M and that there were no significant neuronal damages before 24 M mice (ASC: Fig. 6A, C; F(3,8) = 5.362, P < 0.05; 16 M, but obvious neuronal damages were observed at caspase-1: Fig. 6D; F(3,8) = 7.365, P < 0.05; and IL-1β: the age of 20 M and 24 M in mice. Fig. 6E; F(3,8) = 6.496, P < 0.05) compared to 6 M mice. While in 16 M group, these parameters had no sig- nificant changes compared with 6 M mice. The data Eec ff ts of aging on senescence‑associated β‑gal expression suggested that NLRP1 inflammasome activation was in the cortex and hippocampus in mice closely involved in aging-related neuronal damage dur- The β-gal is an important biomarker for the senescence ing aging process. of neurons. β-gal activity is significantly increased in aging hippocampal neurons in vitro . Our results showed that β-gal activity was relatively low in the cor- Eec ff ts of aging on ROS production in the cortex tex and hippocampal CA1 and CA3 regions in 6 M and and hippocampus in mice 16 M mice. Compared with younger 6 M mice, β-gal Given that ROS plays crucial roles in neuroinflammation activity was significantly increased in the cortex (Fig. 4A, and neuronal damage, we also measured ROS production B; F(3,8) = 52.74, P < 0.05 or P < 0.01) and hippocampal in the cortex and hippocampus via DHE fluorescence CA1 (Fig. 4C; F(3,8) = 22.67, P < 0.05 or P < 0.01) and CA3 staining. The results showed that there was little ROS (Fig. 4D; F(3,8) = 25.16, P < 0.05 or P < 0.01) regions in production in the cortex and hippocampal CA1 and CA3 20 M and 24 M mice, especially in the 24 M group. The regions in 6 M mice. In 16 M mice, ROS production was results suggested that there was no significant neuronal slightly increased in the cortex, but it was significantly senescence in 16 M mice, but obvious neuronal senes- increased in hippocampal CA1 (Fig. 7B, E; F(3,8) = 31.36, cence was appeared at the age of 20 M and 24 M in mice. P < 0.05) and CA3 (Fig. 7C, F; F(3,8) = 16.56, P < 0.05) regions. In 20 M and 24 M mice, ROS production was significantly increased more than tenfold in the cor - Eec ff ts of aging on MAP2 expression in the cortex tex (Fig. 7A, C; F(3,8) = 22.17, P < 0.01) and hippocam- and hippocampus in mice pal CA1 (Fig. 7B, E; F(3,8) = 31.36, P < 0.01) and CA3 The MAP2 is an important biomarker located in neu - (Fig. 7C, F; F(3,8) = 16.56, P < 0.01) regions compared ronal dendrites. MAP2 expression in the hippocampus with 6 M mice. These results suggested that excessive and cortex is significantly decreased in old rats . ROS accumulation was closely involved in neuronal dam- Therefore, we further detected MAP2 expression in the age during aging. cortex and hippocampal CA1 and CA3 regions using immunohistochemistry. The results showed that the Eec ff ts of aging on NOX2, p22phox, and p47phox expression of MAP2 was abundant in the cortex and expression in the hippocampus in mice hippocampal CA1 and CA3 regions in 6 M and 16 M The NOX2 is a key enzyme in the process of ROS genera - mice (Fig. 5). Compared to 6 M mice, the expressions of tion in the brain. Hence, we further measured the effect MAP2 were significantly reduced in the cortex (Fig. 5A, of senescence on the expressions of NOX2, p22phox, and B; F(2,9) = 14.16, P < 0.05 or P < 0.01) and hippocampal p47phox proteins in the hippocampus via western blot- CA1 (Fig. 5C; F(2,9) = 10.67, P < 0.05 or P < 0.01) and CA3 ting. The results showed that the expressions of NOX2, (Fig. 5D; F(2,9) = 6.263, P < 0.05 or P < 0.01) regions in p22phox, and p47phox were relatively low in 6 M and in 20 M and 24 M mice, especially the 24 M group. These 16 M mice. Compared with 6 M mice, the expressions findings suggested that the expression of MAP2 in neu - of NOX2 (Fig. 8A, B; F(3,8) = 5.791, P < 0.05), p22phox rons might significantly decrease when the mice entered (Fig. 8C; F(3,8) = 10.06, P < 0.05), and p47phox (Fig. 8D; older age. Sun et al. Behavioral and Brain Functions (2021) 17:11 Page 7 of 14 Fig. 3 Eec ff ts of aging on changes of Nissl bodies in the cortex and hippocampus in mice (Nissl staining, 400 × , scale bar = 20 μm). A The results of Nissl staining in the cortex, hippocampus CA1 and CA3 in 6 M, 16 M, 20 M and 24 M mice. B The mean density of Nissl bodies in the cortex. C The mean density of Nissl bodies in hippocampus CA1. D The mean density of Nissl bodies in hippocampus CA3. Results are expressed as mean ± SD, n = 4. *P < 0.05, **P < 0.01 vs the 6 M control. AU presents an arbitrary unit Sun et al. Behavioral and Brain Functions (2021) 17:11 Page 8 of 14 Fig. 4 Eec ff ts of aging on β-gal activity in the cortex and hippocampus in mice. A The β-gal staining in the cortex, hippocampus CA1 and CA3 in the 6 M, 16 M, 20 M and 24 M mice (400 × , scale bar = 20 μm). B The mean density of β-gal in the cortex. C The mean density of β-gal in hippocampus CA1. D The mean density of β-gal in hippocampus CA3. Results are expressed as mean ± SD, n = 3. *P < 0.05, **P < 0.01 vs the 6 M control Sun et al. Behavioral and Brain Functions (2021) 17:11 Page 9 of 14 Fig. 5 Eec ff ts of aging on MAP2 expression in the cortex and hippocampus in mice (immunohistochemistry, 400 × , scale bar = 20 μm). A The expression of MAP2 in the cortex, hippocampus CA1 and CA3 in 6 M, 16 M, 20 M and 24 M mice. B The mean density of MAP2 in the cortex. C The mean density of MAP2 in hippocampus CA1. D The mean density of MAP2 in hippocampus CA3. Results are expressed as mean ± SD, n = 4. *P < 0.05, vs the 6 M control Sun et al. Behavioral and Brain Functions (2021) 17:11 Page 10 of 14 NLRP1 inflammasome in the cortex and hippocampus in young (6 M) and older (16 M, 20 M, and 24 M) mice. The current study demonstrated a significant increase in β-gal activity and neuronal damage in aged mice, particularly 20 M and 24 M mice. These changes were accompanied by a significant reduction in locomotor activity and learn - ing and memory functions. Consistent with the patholog- ical changes in the brain, the expressions of NOX2 and NLRP1 inflammasome were significantly increased in the hippocampus in the older mice, especially 20 M and 24 M mice. Our results suggested that the NLRP1 inflam - masome might play an important role in aging-related neuronal damage. Aging is associated with cognitive impairment, and brain regions (including the cortex and hippocampus) that are responsible for learning and memory are par- ticularly vulnerable to aging . The OFT can measure exploration, anxiety, and locomotor behavior of animals . The MWM is an important method to detect learn - ing and memory impairment, which is sensitive to hip- pocampal damage . Previous studies have revealed the changes of learning and memory function in young Fig. 6 Eec ff ts of aging on the expressions of NLRP1, ASC, caspase-1and IL-1β in the hippocampus in mice. A The bands of and old animals, but there is little research on how the NLRP1, ASC, caspase-1, IL-1β and β-actin examined by immunoblot learning and memory function changes with age. Gil- in 6 M, 16 M, 20 M and 24 M mice. B The relative expression of NLRP1 Mohapel et al.  reported that hippocampal neuro- over 6 M. C The relative expression of ASC over 6 M. D The relative genesis affects the type of search strategies with age, expression of caspase-1 over 6 M. E The relative expression of IL-1β while the ability to learn the task is not further influ - over 6 M. Results are expressed as mean ± SD, n = 3. *P < 0.05 vs the 6 M control enced by the age-induced decrease in neurogenesis after 1.5 months of age in mice. In the study, we found that there was no significant difference in the learning test in 16 M compared to 6 M mice. However, in 20 M and F(3,8) = 14.75, P < 0.05 or P < 0.01) were significantly 24 M mice, especially the 24 M group, severe declines in increased in 20 M and 24 M mice. The change of NOX2 motor and exploratory abilities and learning and memory was consistent with the ROS production in the brain dur- functions were observed. Gil-Mohapel et al. proposed ing aging. These results suggested that NOX2 was closely that pre-existing neurons may compensate for the reduc- involved in ROS generation and accumulation in brain tion in neurogenesis, but they only detected the learning during aging. ability in 1.5–12-month-old mice . We found that there were significant learning and memory impairments Discussion in 20 M and 24 M mice. These changes may be induced Aging is an natural, progressive, and deleterious process by neuronal damages in the cortex and hippocampus. that can lead to a variety of age-related diseases . Hence, to confirm whether the behavioral changes are Although the basic mechanism of aging is not completely consistent with the neuronal pathological changes in clear, it is easy to show the aging-related changes, such aging process, we further observed the pathological as the decline in the function of various organs and sys- changes in the cortex and hippocampal CA1 and CA3 tems. The brain is not an exception. Significant cognitive regions in 6 M, 16 M, 20 M, and 24 M mice. H&E and decline and neuronal damage are observed with brain Nissl staining results suggested that 16 M mice had no senescence . As the mechanism of aging has not been obvious neuronal damages, while in 20 M and 24 M mice, fully elucidated and there are still no effective anti-aging there were significant neuronal damages in the cortex measures, it is very important to study the mechanisms and hippocampus especially in the 24 M mice, the pyk- that underlie neuronal damage during aging. Further- notic cells were significantly increased. more, it remains unclear when the brain begins to show β-gal is an important biomarker for cell aging, dur- obvious age-related damages during the course of aging. ing aging. We found that the expression of β-gal had no In the current study, we observed the correlation between significant increase in 6 M and 16 M mice. However, aging-related neuronal damage and alterations in the Sun et al. Behavioral and Brain Functions (2021) 17:11 Page 11 of 14 Fig. 7 Eec ff ts of aging on ROS production in the cortex and hippocampus in mice (DHE staining, 400 × , scale bar = 50 μm). A–C The ROS production in the cortex, hippocampus CA1 and CA3 in 6 M, 16 M, 20 M and 24 M mice. D The mean density of ROS production in the cortex. E The mean density of ROS production in hippocampus CA1. F The mean density of ROS production in hippocampus CA3. Results are expressed as mean ± SD, n = 3. *P < 0.05, **P < 0.01 vs the 6 M control the β-gal expression was dramatically increased in 20 M decreased until the neurons show obvious aging-related and 24 M mice in the cortex and hippocampal CA1 damage. and CA3 regions, especially in 24 M mice. These results In recent years, increasing evidence has suggested that echo previous research  and suggest that the age neuroinflammation is an important event in brain aging of 16–20 months may be an important period during and senescence-associated neuronal damage . Neu- which aging-related neuronal damage occurs in mice. ronal inflammation also contributes to the pathogenesis MAP2 is a cytoskeletal and neuronal marker protein. of brain aging and neurodegenerative diseases . It has The expression of MAP2 is consistent with dendritic been reported that proinflammatory cytokines released growth, branching, and late dendritic remodeling . from microglia and astrocytes significantly induce neu - The expression of MAP2 is significantly reduced in the ronal damages and apoptosis in AD . Recent studies hippocampus and cortex in aged rats . However, to have shown that, in addition to microglia and astrocytes, date no study has systematically investigated how MAP2 neurons also contribute to the inflammatory response expression varies with age. In this study, we found that in the brain by releasing cytokines, such as IL-1β and the expression of MAP2 had no significant changes in IL-18 . Inflammasome is a multi-protein complex the presenile 16 M mice compared with the younger 6 M in the cytoplasm that is involved in maturation of pro- mice. While in aged 20 M and 24 M mice, MAP2 was sig- inflammatory molecules such as IL-1β, IL-6, and IL-18. nificantly decreased, and this phenomenon is consistent NLRP1 is the first reported member of the NLRP family with neuronal damage in aging mice. Therefore, we spec - to form an inflammasome, and is expressed ubiquitously ulated that the expression of MAP2 is not significantly in the brain, particularly in neurons [6, 37]. The NLRP1 Sun et al. Behavioral and Brain Functions (2021) 17:11 Page 12 of 14 Fig. 8 Eec ff ts of aging on the expressions of NOX2, p22phox and p47phox in the hippocampus in mice (immunoblot). A The bands of NOX2, p22phox, p47phox and β-actin in 6 M, 16 M, 20 M and 24 M mice. B The relative expression of NOX2 over 6 M. C The relative expression of p22phox over 6 M. D The relative expression of p47phox over 6 M. Results are expressed as mean ± SD, n = 3. *P < 0.05, **P < 0.01 vs the 6 M control inflammasome play an important role in many neuro - . Therefore, we speculated that excessive ROS pro - logical diseases, and studies in AD models indicate that duction might involve in NLRP1 inflammasome acti - the NLRP1 inflammasome is significantly upregulated in vation in neurons during aging process. In the present neurons . The NLRP1 inflammasome is composed of study, we found that only a small amount of ROS was NLRP-1, procaspase-1, and the adaptor protein ASC . produced in the cortex and hippocampus of 6 M mice. Caspase-1 activation is initiated by NLRP1, and ASC can The ROS production was slightly increased in 16 M enhance caspase-1 activity, which mediates the matura- mice, and the relative expressions of NOX2, p22phox, tion of IL-1β and IL-18 to trigger inflammation . We and p47phox were also slightly increased in CA1 and speculate that the NLRP1 inflammasome is involved CA3, but the difference has no significance. We thought in aging-related neuronal damage in aging process. The that there were other factors involved in ROS accumula- present study demonstrated that there was no significant tion such ROS scavenging system, which requires further difference in the expressions of NLRP1, ASC, caspase-1, study. But in 20 M and 24 M mice, the ROS production in and IL-1β in 6 M and 16 M mice. While in 20 M and the cortex and hippocampus was significantly increased 24 M mice, the expressions of NLRP1, ASC, caspase-1 over 10 times than that of in 6 M mice. These data sug - and IL-1β were significantly increased, especially in 24 M gest that excessive ROS accumulation is closely involved mice. These changes of NLRP1 inflammasome were con - in the brain aging process. Brain ROS production was sistent with the results of neuronal damage during the significantly increase from 16 to 20 M; hence, this period aging process in mice. might be important for preventing brain aging. It is well known that excessive ROS production is one Many enzymes are involved in intracellular ROS gen- of the main causes of NLRP1 inflammasome activation. eration. Among them, the NADPH oxidase (NOX) is cur- Increasing evidence has suggested that excessive ROS- rently the only enzyme family known to produce ROS as induced oxidative stress and neuroinflammation are its sole function. NOX is composed by several isoforms important events in brain aging and senescence-associ- such as NOX1-5. NOX2 is constitutively expressed in ated neuronal damage . ROS-induced oxidative stress many cells in the brain, especially neurons , and con- has been recognized as a contributing factor in aging and tributes to superoxide production in the cerebral circula- the progression of multiple neurodegenerative diseases tion under physiological conditions . It has been well such as AD . ROS production is increased in aging elucidated that NOX2 activation plays an important role mouse brain in association with significant cognitive in the pathogenesis of neurodegenerative diseases, such impairment, and SOD mimetic treatment reduces age- as AD and PD . However, the change of NOX2 in associated oxidative stress, significantly extended lifes - brain aging process remains unclear. The present results pan, and improves learning and memory performance showed that, consistent with the ROS production, the Sun et al. Behavioral and Brain Functions (2021) 17:11 Page 13 of 14 expressions of NOX2, p22phox, and p47phox only exhib- experiment (day7) of the MWM. Then, the mice were sacrificed (day8) and ited an increased trend in 16 M mice. By contrast, in the brain tissues were processed for other tests. 20 M and 24 M mice, the expressions of NOX2, p22phox, Additional file 2: Fig S2. Eec ff ts of aging on pathological changes in and p47phox were significantly increased compared to the cortex and hippocampus in mice (n = 4, H&E staining, 400 × , scale bar = 20 μm). Black arrows indicate nuclear pyknosis and hyperchromatic 6 M mice. These findings confirm that NOX2 plays an nuclei. Yellow arrows indicate eosinophilic degeneration. important role in ROS generation during brain aging, and redox imbalance is closely involved in aging-related Acknowledgements The authors thank Zhirui Fang in the Department of Pharmacology, Dake neuronal damage during aging process. Additionally, the Huang and Bao Li in the Synthetic Laboratory of Basic Medicine College for NOX2 expression and ROS production were consistent their technical assistance. with the results of aging-related neuronal damage and Authors’ contributions NLRP1 inflammasome activation during the brain aging DS, GG and BZ analysed data and was a major contributor in writing the process. manuscript. HZ collated the data, SD contributed to the immunoblot analysis Above all, brain aging is a complex process that and interpretation of the results. ZS and YZ was mainly responsible for the immunofluorescence detection. WL designed the study, critically revised the changes with time. During aging, the function of neu- manuscript for intellectually important content, supervised the study and robiological networks also showed obvious damage in wrote the manuscript. All authors read and approved the final submitted elderly mice, especially in 24 M mice. A large number of manuscript. studies have shown that there are significant changes in Funding molecules, cells, brain regions and learning and memory This study was supported by the National Natural Science Foundation of during aging. In the present study, we found that ROS China (81671384, 81970630). production and NOX2 enzyme systems, that promotes Availability of data and materials ROS production, were significantly increased; Inflam - The datasets used and analysed during the present study are available from matory cytokines and NLRP1 inflammasome were also the corresponding author on reasonable request. significantly increased in elderly mice, especially in 24 M mice. The excessive increase of these substances further Declarations damages the structure and function of neurons. The Ethics approval and consent to participate cortex and hippocampus are important neurobiological All experiments involving animals were approved by the Ethics Committee of networks involved in learning and memory. We found Laboratory Animals of Anhui Medical University. that the neurons showed obvious degeneration, and the Consent for publication Nissl bodies and MAP2 expression were significantly Not applicable. decreased in the cortex and hippocampus CA1 and CA3 Competing interests regions. These changes of neurobiological networks The authors declare that they have no competing interests. eventually lead to learning and memory impairments in elderly mice, especially in 24 M mice. Author details Department of Pharmacy, The First People’s Hospital of Xiaoshan District, 199 In conclusion, our study suggested that NOX2-NLRP1 Shixin South Road, Hangzhou 311200, Zhejiang, China. Department of Phar- inflammasome signaling is closely involved in aging- macology, Basic Medicine College, Anhui Medical University, No. 81 Meishan related neuronal damage during the aging process, and is Road, Hefei 230032, Anhui, China. Key Laboratory of Anti-Inflammatory and Immunopharmacology, Ministry of Education, Anhui Medical University, perhaps an important target for modulating brain aging. No. 81 Meishan Road, Hefei 230032, Anhui, China. However, there were many limitations of the methods in our study. We only observed the changes of 6 M, 16 M, Received: 5 August 2021 Accepted: 15 November 2021 20 M and 24 M mice in the process of aging, perhaps more time points are needed in the future study. 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Behavioral and Brain Functions – Springer Journals
Published: Dec 17, 2021
Keywords: Brain aging; Learning and memory impairments; NLRP1 inflammasome; ROS; NADPH oxidase 2
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