Lipopolysaccharide (LPS) increases susceptibility to epilepsy via interleukin-1 type 1 receptor signaling - ScienceDirect
1. Introduction
Epilepsy is a disorder of the central nervous system (CNS) with predisposition to aberrant recurrence of neuronal activity in the brain, with approximately 50 million people afflicted worldwide (Pauletti et al., 2019). This disease is characterized by abnormal firing of neurons in the brain, with main clinical manifestations presented as convulsion, loss of consciousness, myoclonus, etc. (Bosco et al., 2020, Eyo et al., 2017). Around 30 % of epileptic patients fail to respond to treatment with anti-epileptic drugs (AEDs) for seizure (Hu et al., 2020, Kwan et al., 2010). At present, the panorama of the epileptic pathogenesis has not been fully revealed. Recently, increasing attention has been focused on the relationship between inflammation and epilepsy, which is deemed to be pivotal in the pathogenesis of epilepsy (Paudel et al., 2018, Terrone et al., 2019).
Emerging evidence has suggested the interplay between inflammation and epilepsy. For one thing, epilepsy is accompanied by inflammation response, characterized by activation of glial cells and release of inflammatory factors. Increased levels of pro-inflammatory cytokines such as IL-1β, IL-6, and rapid inflammation response were observed in brain regions of epileptic patients and animal models(Alapirtti et al., 2018, Choi et al., 2009, Plata-Salaman et al., 2000, Ravizza and Vezzani, 2006, Van Vliet et al., 2018). Conversely the cumulative inflammatory factors in the brain increase aberrant neuronal excitability, contributing to progression of epilepsy and aggravation of seizure. It has been reported that upregulation of pro-inflammatory cytokine levels can increase the susceptibility to epilepsy (Galic et al., 2008, Vezzani et al., 2008). Despite consensus regarding CNS inflammation in promoting epilepsy, little attention was paid to the effect of peripheral inflammation on epilepsy and the underlying mechanism, on which we focused in this study.
Peripheral inflammation may not only gain entry into the brain via the destruction of blood brain barrier (BBB), but also activate endothelial signaling to the brain via leukocytic rolling and adhesion to the vasculature and activate endothelial signaling to the brain, eventually leading to inflammation in the brain (Cluny et al., 2022, D'Mello et al., 2013, Vezzani and Viviani, 2015). The elevation of peripheral and central inflammatory mediators results in the collapse of BBB, which further facilitates leukocytic infiltration, thus leading to neuronal hyper-excitability and exacerbation of seizure (Perry and Teeling, 2013). Experimental studies have validated that autoimmune diseases or systemic inflammation can increase the risk of epilepsy to varying degrees (Ho et al., 2015, Rana and Musto, 2018).
LPS treatment can cause the generation of inflammation cytokines in the periphery and concomitant synthesis in the CNS (Verma et al., 2006). A few experimental studies have reported administration of LPS reduced seizure threshold, increasing susceptibility of seizure (Heida et al., 2005, Sayyah et al., 2003, Stéphane et al., 2009). Nevertheless, there is still a paucity of relevant reports with respect to how LPS increases susceptibility to epilepsy.
Pro-inflammatory cytokine IL-1β, a key cytokine released from activated microglia and astrocytes in the diseased brain tissue, tending to bind to its receptor IL-1R1, is reportedly activated in epileptic brain. A research from Karolina Kołosowska et al. described the elevated expression of IL-β and IL-1R1 in the rat hippocampus of pentylenetetrazole (PTZ)-induced kindling (Kolosowska et al., 2014), whereas IL-1R1 knock-out mice showed less potential to seizure (Dube et al., 2005), supporting the hypothesis that IL-1R1 plays a vital role in the process of epilepsy and pathophysiology of seizure. Albeit there is much debate as to the cell typology of IL-1R1 expression, it is indisputable that IL-1R1 expression is abundant on endothelial cells at the BBB. Moreover, there is evidence that brain IL-1R1 is also identified in endothelial and ventricular cells, astrocytes, and DG neurons, with endothelial IL-1R1 mediating sickness behavior (Liu et al., 2019). Therefore, IL-1R1 may act as a pivot in peripheral-central inflammatory signal transduction.
An early report indicated that LPS-induced stimulation elevated IL-1R1 mRNAs expression in the brain (Gabellec et al., 1996). Blockage of central IL-1β signaling attenuates sickness behaviors in response to peripheral injection of the bacterial endotoxin LPS (Laye et al., 2000). Increasing pre-clinical, clinical and experimental findings suggest that peripheral inflammation can affect epilepsy. We thus hypothesized that LPS treatment activates IL-1R1 signal, thereby stimulating central inflammatory response, leading to the elevation of seizure susceptibility.
By intraperitoneal injection of LPS to mediate peripheral inflammation, we identified that LPS can significantly elevate the seizure susceptibility, and increase hippocampal inflammation and hippocampal neurogenesis. Furthermore, IL-1R1 was upexpressed under LPS treatment in the hippocampus, while inhibition of central IL-1R1 reduced seizure susceptibility, which justified our hypothesis.
2. Results
2.1. LPS injection increases susceptibility and severity to epilepsy
Adult mice were intraperitoneally injected with LPS at doses of 0.5 mg/kg, 1 mg/kg, and 2 mg/kg, respectively. KA-induced epileptic mouse model was adopted to record the seizure onset time (SOT) of the initial seizure and seizure threshold (ST) of KA-induced epilepsy. Statistical analysis revealed that LPS significantly decreased the ST and SOT at the doses of 1 mg/kg and 2 mg/kg (P < 0.05, P < 0.01 respectively; Fig. 1 A, B) versus the control group, with better effect at 2 mg/kg, showing LPS injection increased susceptibility to epileptic seizure. Afterwards, Racine score of epileptic seizure and the number of mice with Ⅳ-Ⅴ seizure were recorded with the percentage calculated at LPS-injected dose of 2 mg/kg. The results presented that LPS significantly increased the Racine score and the percentage of mice with stage Ⅳ-Ⅴ seizure (P < 0.01, P < 0.05, P < 0.01 respectively; Fig. 1C, D), indicating LPS treatment aggravated the severity of epilepsy.
Fig. 1. Effect of LPS treatment on epileptic seizure susceptibility and severity of epilepsy. KA-induced epileptic seizure threshold (ST) in mouse model injected with different concentrations of LPS (0.5 mg/kg, 1 mg/kg, and 2 mg/kg) (A); The latency to the initial behavioral seizure, defined as the seizure onset time (SOT) with different concentrations of LPS (0.5 mg/kg, 1 mg/kg, and 2 mg/kg) (B); Racine score in epilepsy mouse model injected with concentrations of LPS as 2 mg/kg (C); The percentage of mice with stage Ⅳ-Ⅴ seizure in epileptic mouse model injected with concentrations of LPS as 2 mg/kg (D). Data are expressed as mean ± SEM; n = 15/group, **P < 0.01, *P < 0.05.
2.2. LPS injection increases hippocampal inflammation in epileptic mice
Herein, for assessment of the hippocampal neuroinflammation in LPS mice, we detected microglial cells and astrocytes in the hippocampus by GFAP and IBA-1 immunofluorescent staining after LPS procedures in KA-induced epilepsy model. Our immunofluorescence data confirmed the elevation of the count of IBA-1+ and GFAP+ cells in CA1 and CA3 regions of the hippocampus in LPS group versus the KA + Saline group, and they presented evident activation state (P < 0.05; Fig. 2). To probe into the proinflammatory effect of LPS in the epileptic hippocampus, after epilepsy induction the inflammatory cytokines were detected for neuroinflammation analysis. ELISA results revealed that the levels of inflammatory cytokines IL-1β and IL-6 were progressively elevated after LPS injection compared to the KA + Saline group (P < 0.05; Fig. 2). Briefly, these data suggested that LPS increased hippocampal neuroinflammation following KA-induced epilepsy.
Fig. 2. Effect of LPS on hippocampal inflammation after KA-induced epilepsy. Representative images of the immunostained IBA-1 and GFAP in the hippocampal CA1 and CA3 regions in the control, KA + Saline and KA + LPS mice (A). Graph shows number of IBA-1+ and GFAP + cells normalized to Control (B).The concentrations of hippocampal inflammatory cytokines (IL-1β, IL-6) were measured by ELISA (C). Data are expressed as mean ± SEM; n = 5/group, **P < 0.01, *P < 0.05.
2.3. LPS injection increases the hippocampal neuronal damage in epileptic mice
Neuroinflammation can lead to damage of hippocampal neurons. Accordingly, we performed Nissl staining in the hippocampus to detect neuronal distribution and quantity after epilepsy induction. Our results of Nissl staining revealed that the staining intensity was significantly decreased in CA1 and CA3 regions of the hippocampus in KA + Saline group versus the Contrl group (P < 0.05; Fig. 3). LPS treatment attenuated the Nissl staining intensity in CA1 and CA3 regions of the hippocampus as compared to the KA + Saline group (Fig. 3). Briefly, these findings suggested that the count of Nissl corpuscles was decreased in KA-induced epilepsy, and the cells became disarranged, indicating neuronal damage in epileptic hippocampus. In brief, LPS-induced inflammation increased hippocampal neuronal damage in epilepsy more critically.
Fig. 3. Nissl staining was performed to detect KA-induced neuronal damage and cell loss under LPS injection. Epilepsy can lead to neuronal damage and cell loss in hippocampus. Representative images of the Nissl staining in the hippocampal CA1 and CA3 regions in the Control, KA + Saline and KA + LPS mice (A). Graph shows number of neurons normalized to Control (B). Data are expressed as mean ± SEM; n = 5/group, *P < 0.05.
2.4. LPS injection increases hippocampal neurogenesis in epileptic mice
To validate the impact of LPS on hippocampal neurogenesis in epileptic mice, we applied double-label fluorescence immunocytochemistry of DCX and BrdU, Nestin-labeled neural stem cells in DG. The co-expression pattern of DCX and BrdU facilitated to identify new proliferative neurons in the hippocampus. The results confirmed the significant increase of DCX+/BrdU+ cell and Nestin + cell population of DG in KA-induced epilepsy. LPS treatment further significantly enhanced the count of DCX+/BrdU+ cells and Nestin+ cells versus the KA + Saline group (P < 0.05; Fig. 4). These results indicated LPS-induced inflammation increased hippocampal neurogenesis in KA-induced epilepsy model.
Fig. 4. Effect of LPS treatment on KA-induced hippocampal neurogenesis. Hippocampal neurogenesis increases markedly in a short time after epilepsy. Representative DCX+/BrdU+ and Nestin+ immunofluorescent staining of DG (A). Red fluorescence represents marker of new neurons, DCX; Green fluorescence represents marker of cell proliferation, BrdU; Red fluorescence represents marker of neural stem cells, Nestin. Graph shows number of DCX+/BrdU + cells and Nestin+ cells normalized to Control (B). Data are expressed as mean ± SEM; n = 6 in each group, Data are expressed as mean ± SEM; n = 5/group, *P < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
2.5. L-1R1 acts as a mediator in LPS-induced peripheral inflammation and central neuroinflammation
There is much discrepancy with respect to the role of IL-1R1 in the brain by conventional approaches. The dichotomy can be attributed to the disadvantages of IHC for determining low levels of IL-1R1 (Liu et al., 2015). Ning Quan et al. established a novel genetic mouse model to visualize IL-1R1-expressing cells. We were lucky to have the mouse model, and with RFP immunostaining we could label IL-1R1 expression in hippocampus. Here, the result showed elevated IOD of RFP in KA-induced epilepsy. LPS injection triggered even stronger fluorescence of RFP in epilepsy model versus the KA + Saline group (P < 0.05; Fig. 5). With central IL-1R1 suppressed, the seizure onset time of the first seizure and seizure threshold were significantly increased, and the Racine score and the percentages of mice with stage Ⅳ-Ⅴ seizure were notably decreased. Our results indicated LPS treatment increased hippocampal IL-1R1 expression under KA-induced epilepsy and epileptic seizure was suppressed by inhibition of central IL-1R1, showing that LPS promotes epilepsy, presumably via central IL-1R1 signaling. All these findings authenticate that IL-1R1 may act as a mediator in this process.Fig. 6.
Fig. 5. IL-1R1 expression under LPS treatment in KA-induced epilepsy model. We applied a mouse model by RFP immunostaining that can label IL-1R1 expression. Representative RFP immunofluorescent staining showed IL-1R1 mainly expressed in endothelial cells (A). Graph shows IOD of RFP fluorescence in Control (B). IL-1R1 protein expression of hippocampus was detected by western blotting. (B) The graph showed the protein expression levels as relative optical density (ROD) values. Data are expressed as mean ± SEM; n = 6 in each group. Data are expressed as mean ± SEM; n = 5/group, **P < 0.01.
Fig. 6. In the case of suppression of central IL-1R1, the changes of epileptic seizure susceptibility and severity of epilepsy are illustrated. To detect the effect of central IL-1R1 on epilepsy, anakinra was lateral cerebral ventricle injected to inhibit the central IL-1R1. We observed seizure threshold (ST) (A); the seizure onset time (SOT) (B); Racine score (C); The percentage of mice with stage Ⅳ-Ⅴ seizure in epilepsy mouse model (D). Data are expressed as mean ± SEM; n = 15/group, *P < 0.05.
3. Discussion
LPS is the cell wall component of Gram-negative bacteria, which can damage the BBB directly and indirectly (Peng et al., 2021). Vargas-Caraveo et al. discribed that LPS infiltrates in the brain in physiological conditions, possibly, via a lipoprotein transport mechanism, and binds to its receptors in blood–brain interfaces (Vargas-Caraveo et al., 2017), which showed LPS can penetrate the blood brain barrier. It was reported systemic injection of LPS in rats is a well-studied and described approach to precipitation of inflammatory reactions in the CNS (Vezzani and Granata, 2005), which is also feasible for investigation of links between immune system and epileptic activity (Kovács et al., 2006). In this study, we aimed to expound the implication of LPS in susceptibility to KA-induced seizure, specifically the effects on alteration of hippocampal inflammation, neuronal damage, hippocampal neurogenesis, further exploring the involvement of IL-1R1 in this process. Epilepsy is a dynamic process owing to aberrant firing of neurons. There are a series of apparent pathological changes in epileptic brain, including neuronal damage, presence of neuroinflammation and acute enhancement of neurogenesis of hippocampus. Neuroinflammation is not only a CNS response to epileptic brain damage, but also a stimulus to seizure, which have been confirmed in a wealth of literature. Evidence from humans and epileptic animal models has shown activation of neuroinflammatory response and enhancement of proinflammatory cytokines in serum and the brain tissues (Vezzani et al., 2011, Wang et al., 2019). Neuroinflammation can induce neuronal hyper-excitability and assist in lowering the threshold of seizure in susceptible brain areas (Mazarati et al., 2017). However, less is known about the contribution of peripheral inflammatory events to seizure modulation.
In the clinical scenario, the risk of epilepsy infection increases according to the severity of the patients with traumatic brain injury (TBI) (Pitkanen and Immonen, 2014), in which systemic inflammation may be involved in a key factor. In addition, a research with animal model suggested LPS-induced peripheral infection increases neuronal excitability in hippocampus and facilitates post-traumatic epileptogenesis (Wang et al., 2021), supporting the findings that infections can both increase seizure susceptibility and trigger the onset of epilepsy (Cusick et al., 2017, DePaula-Silva et al., 2018). Our findings suggested that LPS could reduce the latency for the initial seizure and the threshold for KA-induced epilepsy. Moreover, Racine score and percentage of mice with stage Ⅳ-Ⅴ seizure were higher in LPS-treated mice, i.e. inflammation induction via LPS injection in mice increased susceptibility to epileptic seizure and the severity of epilepsy. Our results were consistent with the report by Semple et al. that LPS can change neuronal excitability and increase seizure susceptibility (Semple et al., 2020).
Glial cells are prominent factors sensitive to brain disorder. Inflammatory changes in epileptic hippocampus include astrocytic proliferation, microglial activation, BBB injury and increased expression of proinflammatory cytokines (Morin-Brureau et al., 2018, Pitkanen and Sutula, 2002, Vargas-Sanchez et al., 2018, Vezzani et al., 2013). Enhanced levels of proinflammatory cytokines IL-1β and IL-6 were detected in activated astrocytes and microglia in brain regions immediately after epileptic onset (Abdallah, 2010, Arican et al., 2006). Incessant astrocytic activation can assist in glutamate excitotoxicity (Pekny et al., 2016), and microglials function as macrophages in the central nervous system, which reversely can aggravate brain inflammation, further exacerbating seizure. An interesting study from Rishabh Sharma et al. indicated the presence of a higher frequency of spontaneous seizure, and an increase in activation of IBA1+ microglia and GFAP+ astrocytes in LPS-treated mice, which was supportive of our findings that LPS treatment increased the release of the inflammatory cytokines of hippocampus as well as the number of microglia and astrocytes in KA-induced epilepsy mice, with glia cells appearing evidently activated (Rs et al., 2021). Thereby, LPS-induced peripheral inflammation can activate neuroinflammation, and aggravate epileptic seizure.
In addition to neuroinflammatory response, cellular alterations characterized by neuronal damage and the excessive occurrence of neurogenesis are also supposed to be pathological modifications of the hippocampus in acute phase after epilepsy. Neuronal damage is considered to be typical in epileptic hippocampus, with neuroinflammatory storm leading to aberrant neural connectivity, enhancing neuron excitability, increasing neuronal death and altering the regeneration of neurons (Wolinski et al., 2022). Neuroinflammation is involved in the pathophysiology of epilepsy, not only leading to neuronal damage, but also affecting hippocampal neurogenesis. An increasing body of evidence suggests that neuroinflammation produced in epileptic brain largely affects hippocampal neurogenesis. Neurogenesis increases markedly in the weeks after epileptic insult (Danzer, 2019). The recent research from Xinjian Zhu et al. demonstrated that neurogenesis was suppressed in the case of inhibition of neuroinflammation in the pilocarpine-induced SE mouse model (Zhu et al., 2020). In this study, we observed neuronal damage and loss in CA1 and CA3 regions of epileptic hippocampus, with LPS treatment further exacerbating this outcome. In addition, multiplied BrdU+/DCX+ and Nestin+ cells were detected in KA-induced epilepsy, indicating increased neurogenesis in epileptic hippocampus. More significant increase of neurogenesis was displayed in LPS-treated mice. A plausible explication could be that LPS-induced peripheral inflammation leads to increased hippocampal neuroinflammation via a number of mediators, thereby aggravating neuronal injury and abnormal neurogenesis in acute stage.
IL-1β and its receptor IL-1R1 have been widely studied in brain disorders with respect to the involvement in neuroinflammation and pathological activity. Undoubtedly, the expression of IL-1R1 was unanimously reported to be elevated in the epileptic brain. Intriguingly, there is much controversy over the cell types of IL-1R1 expression. Recently, Ning Quan et al. established a genetic knockin reporter system in mice to track IL-1R1 expression, and identified brain IL-1R1 expressed in endothelial and ventricular cells, astrocytes, and DG neurons. Further, they demonstrated endothelial and ventricular IL-1R1 regulates microglial activation (Liu et al., 2019). It was well known that BBB is dependent on endothelial IL-1R1, which is a promoter of microglial activation and release of proinflammatory factors. Accordingly, IL-1R1 particularly BBB IL-1R1 may act as a mediating factor in the transition from peripheral inflammation to central neuroinflammation, with IL-1R1 signaling playing an essential role in this process. There is evidence that IL-1R1 on brain endothelial cells is involved in LPS-induced sickness responses (Matsuwaki et al., 2017). Further evidence is provided by Freria CM et al. that LPS pretreatment could up-regulate the expression of IL-1R1 in endothelial cells (Freria et al., 2020). Our results indicated LPS treatment increased hippocampal IL-1R1 expression under KA-induced epilepsy and epileptic seizure were suppressed by inhibition of central IL-1R1, suggesting that LPS promotes epilepsy presumably via IL-1R1 signaling.
In conclusion, our data in this study demonstrated that LPS-induced peripheral inflammation evoked upexpressed IL-1R1 and aggravated hippocampal neuroinflammation. Moreover, LPS treatment enhanced neuronal excitation, and increased neurogenesis of hippocampus, which could aggravate epileptic seizure activity in KA-induced mouse model of epilepsy. Our results are supportive of the potential of IL-1R1 as a mediating factor in peripheral-central inflammation, with further emphasis on the importance of IL-1R1 signaling in the regimen of LPS for epilepsy.
4. Materials and methods
4.1. Animals
Adult C57/BL6 male mice (aged 6–8 weeks, weighing 18–23 g), were provided by the Experimental Animal Center, Xuzhou Medical University and were housed in SPF level environment, with room temperature (r/t) maintained at 22 ± 2 °C. IL-1R1 globally restored mice (IL-1R1GR/GR) mice in which IL-1R1 expressing cells can be tracked by RFP fluorescence were obtained from Dr. Ning Quan at Florida Atlantic University. Mice were allowed for adaptation for 3 days with food and water ad libitum prior to experimentation.
4.2. Animal model and administration procedure
Blinding and randomization principles were incorporated throughout the study and analyses. All experimental mice were randomly divided into three groups: Control group (injection of saline), KA + Saline group, KA + LPS group. Mice were injected with different doses of LPS (L2880; Sigma-Aldrich; 0.5, 1, 2 mg/kg, i.p.) dissolved in saline to induce inflammation before epilepsy modeling, with data recorded. 24 h later, the mice underwent a single i.p. injection of 30 mg/kg KA (K2389; Sigma-Aldrich.) dissolved in 0.9 % saline to induce generalized tonic-clonic seizure. Thereafter, mice seizure behavior was recorded until offset of convulsions. The criteria for the establishment of KA-induced epilepsy model were constituted as per Racine classification: Level 1: closed eyes, whisker movement, and facial twitching; Level 2: nodding, chewing with facial twitching; Level 3: paw raising and clonus in a forelimb; Level 4: standing with bilateral forelimb spasm; and Level 5: standing, twisting, and falling. The presence of the symptoms of grade 3 or above indicates successful establishment of the epileptic model. With respect to anakinra administration, anakinra (AMG-719, MedChemExpress) was injected at 1 h before KA treatment directly into the left lateral cerebral ventricle (position relative to bregma: posterior, –0.5 mm; lateral, –1.2 mm; depth, 2.5 mm) at a dose of 10 µg per mouse (Heida and Pittman, 2005, Nigel et al., 2005).
4.3. Tissue preparation
At 24 h post injection of KA, each mouse underwent deep anesthesia via an intraperitoneal injection of 30 mg/kg 3 % sodium pentobarbital, followed by transcardial perfusion with 4 % paraformaldehyde (MA0192, Meilunbio). The brain tissues were kept at 4 °C overnight, followed by refrigeration in 30 % sucrose. Some brains were systematically sliced into 15-μm thickness coronal sections on a freezing microtome (Leica CM1950). The slices containing the hippocampus were collected in PBS.
4.4. Nissl staining
With mice sacrificed four days after KA injection for Nissl staining to analyze neuronal death, the perfusion of each mouse heart was performed as described above. Following brain isolation and post-fixation with 4 % paraformaldehyde overnight at 4 °C, the tissues underwent rehydration and embedment in paraffin. The brain tissues were sectioned on a microtome at 5-μm and mounted on gelatinized slides prior to Nissl staining. The population of intact neurons in the CA1 and CA3 areas of hippocampus was calculated for quantitative analysis with ImageJ software (NIH, Bethesda, MD, USA).
4.5. Enzyme-linked immunosorbent assay (ELISA)
The inflammatory cytokines (IL-1β, IL-6) in the mouse hippocampus were analyzed with the use of ELISA kits (ExCell, China) as per the protocol. In brief, hippocampal homogenates were incubated with reaction buffers. The mixture was incubated for 2.5 h at r/t prior to detection on a microplate reader (BioTek, USA). ELISA of the samples was performed in duplicate, with each procedure repeated at least twice. The concentrations of the inflammatory cytokines were expressed as pg/mg of total protein content.
4.6. BrdU labeling
To explore the effects of LPS on neurogenesis in epilepsy, we injected 5-bromo-2′-deoxyuridine (BrdU) (B9285; Sigma-Aldrich; 100 mg/kg; i.p.) dissolved in 0.9 % NaCl into the mice after establishment of epilepsy model. Mice underwent transcardial perfusion 24 h after the injection, with matched mice subjected to the same procedure. Thereafter, transcardial perfusion was performed as described above, with the population of cells incorporating BrdU during the early period measured.
4.7. Western blotting
Mice (n = 6/group) were sacrificed and hippocampal tissues were isolated for homogenization with lysis buffer containing RIPA (Beyotime, P0013B, China), and subsequently the supernatant after low-temperature centrifugation was absorbed. Protein concentration was determined by BCA protein assay kit (Beyotime, Shanghai, China) and was adjusted in consistency. Protein was separated and underwent sodium dodecyl sulfate polyacrylamide gel electrophoresis at 80 V constant voltage, followed by transfer onto nitrocellulose membranes (Millipore, Bedford, MA, USA), blocked with 5 % skim milk for 2 h. Subsequently, the NC membranes were incubated with antibodies against IL-1R1 (1: 400; R&D Systems, USA) overnight at 4 °C and β-actin (1: 2000; Abcam, USA), followed by goat anti-mouse 926-32210 IRDye 800 CW and anti-rabbit 926–68021 IRDye 680 RD secondary antibodies (LI-COR, Lincoln, NE, USA). Assessment was conducted on an Odyssey scanner (LI-COR, USA), and densitometric analysis of stripe was performed with ImageJ software ImageJ software (NIH, Bethesda, MD, USA).
4.8. Fluorescence staining
Slices containing the hippocampal tissues underwent fluorescence staining assays. Free-floating sections were rinsed in PBS (pH 7.2) prior to pre-incubation in a blocking solution (10 % of fetal sheep serum (FSS), 0.3 % of Triton X-100 in PBS at r/t for 40 min. Afterwards, these slices were incubated with rabbit anti-IBA-1 antibody (1:300, Abcam, UK), rabbit anti-GFAP antibody (1:500, Abcam, UK), rat anti-BrdU antibody (1:500, Abcam, UK), rabbit anti-Nestin antibody (1:600, Abcam, UK), rabbit anti-Doublecortin (1:500, Abcam, UK), rabbit anti-RFP antibody (1:500, Abcam, UK) overnight at 4 °C. After incubation, samples were rinsed thrice with PBS and probed with secondary antibody as follows: goat anti-rabbit IgG Alexa Fluor 594 conjugate (1:500, ab150080, Abcam, UK), goat anti-rat IgG Alexa Fluor 488 conjugate (1:500, ab150157, Abcam, UK) for 1 h at 37 °C. Slices were rinsed thrice with 0.01 mol/L PBS for 5 min and incubated with DAPI Staining Solution (C1005, Beyotime) at r/t for 10 min. These slices were rinsed with 0.01 mol/L PBS for 5 min, and sealed with glycerin for observation and photography under a fluorescence microscope. In addition, due to the specificity of BrdU immunofluorescence staining, DNA denaturation is required. Sections underwent denaturation in 2 N HCl for 30 min at 37 °C and neutralization in 0.1 M borate buffer for 10 min at 37℃. The primary antibodies were added on slices for incubation overnight at 4 °C, as previously described. Regions of interest were selected for photography, with the stained areas calculated with the ImageJ software (NIH, Bethesda, MD, USA).
4.9. Cell counting
The population of IBA-1+, GFAP+, BrdU+/DCX+ cells, Nestin+ cells and number of Nissl-positive cells in CA1 and CA 3 regions were manually counted at the area of 300 μm × 300 μm in a 20 × field with ImageJ software (NIH, Bethesda, MD, USA) by two independent technicians blinded to the sample grouping (Hu et al., 2020). Six pieces of hippocampal serial sections were selected and underwent immunofluorescence staining and cell counting in triplicate, with their average value calculated. All the data from statistical analysis were then normalized to those in the Control group (Wang et al., 2019).
4.10. Statistical analysis
All results are expressed as mean ± standard error of the mean (SEM). The data obtained were analyzed with one-way ANOVA analysis of variance followed by Tukey’s and Dunnet’s tests. The program used for the statistical analyses was GraphPad Prism software (GraphPad Software) that was tested normally.
5. Statement of Ethics
The experiments were approved by the Ethics Committee on Laboratory Animals of Xuzhou Medical University, China (L20210226446).
Funding sources
This work was supported by Natural Science Research Project for Universities of Jiangsu Province (21KJB180011).
CRediT authorship contribution statement
Ankang Hu: Conceptualization, Methodology, Methodology, Writing – original draft, Validation. Honghua Yuan: Data curation, Methodology, Writing – original draft, Investigation, Resources. Ying Qin: Data curation, Methodology, Writing – original draft, Investigation, Resources. Yuhua Zhu: Visualization, Investigation, Validation, Formal analysis. Lingzhi Zhang: Visualization, Investigation, Validation, Formal analysis. Quangang Chen: Validation, Formal analysis. Lianlian Wu: Supervision, Project administration.