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- Springer Journals
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- Copyright © The Author(s) 2023
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- 10.1186/s12993-023-00205-y
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Abstract
Background The pristane-induced lupus (PIL) model is a useful tool for studying environmental-related systemic lupus erythematosus (SLE). However, neuropsychiatric manifestations in this model have not been investigated in detail. Because neuropsychiatric lupus (NPSLE) is an important complication of SLE, we investigated the neuropsychi- atric symptoms in the PIL mouse model to evaluate its suitability for NPSLE studies. Results PIL mice showed olfactory dysfunction accompanied by an anxiety- and depression-like phenotype at month 2 or 4 after pristane injection. The levels of cytokines (IL-1β, IFN-α, IFN-β, IL-10, IFN-γ, IL-6, TNF-α and IL-17A) and chemokines (CCL2 and CXCL10) in the brain and blood–brain barrier (BBB) permeability increased significantly from week 2 or month 1, and persisted throughout the observed course of the disease. Notably, IgG deposition in the choroid plexus and lateral ventricle wall were observed at month 1 and both astrocytes and microglia were activated. Persistent activation of astrocytes was detected throughout the observed course of the disease, while microglial acti- vation diminished dramatically at month 4. Lipofuscin deposition, a sign of neuronal damage, was detected in cortical and hippocampal neurons from month 4 to 8. Conclusion PIL mice exhibit a series of characteristic behavioral deficits and pathological changes in the brain, and therefore might be suitable for investigating disease pathogenesis and for evaluating potential therapeutic targets for environmental-related NPSLE. Keywords Neuropsychiatric lupus, Mouse model, Behavioral deficit, Cytokine, IgG, Glia cells, Lipofuscin Nervous system involvement in SLE affecting cognition, Background mood and the level of consciousness is termed neuropsy- Systemic lupus erythematosus (SLE) is an intractable, chiatric lupus (NPSLE), and is associated with a worse multisystemic and relapsing disease characterized pri- prognosis and more cumulative organ damage [2, 3]. As marily by the loss of tolerance to self-antigens, immune for the pathogenesis of NPSLE, it is generally believed complex formation and diverse end-organ damages [1]. that several pathogenic factors, such as autoantibodies, immune cells and inflammatory mediators, may disrupt *Correspondence: the blood–brain barrier (BBB) and promote an inflamma - Ling Qin qinlingling@yahoo.com tory process causing glial activation, neurodegeneration Pingting Yang and behavioral deficits [4–6]. However, the mechanisms yangpingtingting@163.com 1 underlying NPSLE remain largely unknown. Department of Nephrology, Shengjing Hospital of China Medical University, Shenyang, China Human research on NPSLE is limited by the diffi - Department of Physiology, China Medical University, Shenyang, China culty of brain biopsy. Although histological analysis can Department of Rheumatology and Immunology, First Affiliated Hospital, be conducted on autopsy, rapid decomposition of brain China Medical University, Shenyang, China tissue severely impacts the findings. Therefore, animal © The Author(s) 2023. 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:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Yun et al. Behavioral and Brain Functions (2023) 19:3 Page 2 of 19 models of NPSLE are indispensable tools for exploring for research on the cellular and molecular mechanisms the pathogenic mechanisms [7]. MRL/lpr and NZB/W underlying environmental-related NPSLE. F1 mice are commonly used as NPSLE animal models. These mice spontaneously develop neuropsychiatric Results symptoms such as cognitive dysfunction [8, 9], anxiety Lupus‑like manifestations in PIL mice and/or depression-like behavior [8, 10, 11] and decreased Consistent with previous studies [18, 20, 21], PIL mice locomotor activity [12]. However, these models rely on exhibited several representative lupus-like manifes- inbred mouse strains, exhibit delayed and inconsistent tations, including lipogranuloma, elevated serum onset of SLE-related symptoms and progress slowly [13]. cytokines, chemokines and autoantibodies, immune The heterogeneous and mild neuropsychiatric symptoms complex-mediated nephritis with renal failure and limit the application of these models for NPSLE research. proteinuria, arthritis, as well as splenomegaly (Fig. 2). Additionally, the impact of environmental factors in the Lipogranuloma adherent to the abdominal surface of pathogenesis of NPSLE can not be fully reflected by these the diaphragm appeared at month 1 after pristane injec- genetically predisposed models. tion, and progressed over time (Fig. 2A). Meanwhile, Exposing wild-type mice to chemical agents is an alter- the serum levels of the cytokines interleukin-1β (IL-1β), native method for establishing SLE models, and can interferon-α (IFN-α), IFN-β, IFN-γ, IL-10, IL-6, IL-17A mimic the impact of environmental factors on the induc- and tumor necrosis factor-α (TNF-α) were elevated in tion of lupus-specific manifestations. Such models allow PIL mice compared with their age-matched controls, investigation of non-genetic factors initiating a break- from week 2, or month 1 or 2 (Fig. 2B). However, no sig- down of immune tolerance and can be used to evaluate nificant difference between PIL and control mice was the efficacy of SLE therapeutic drugs. Pristane is known observed in the levels of tumor necrosis factor-like weak as hydrocarbon oil (2,6,10,14-tetramethylpentadecane) inducer of apoptosis (TWEAK), B cell activating fac- that can induce a wide range of autoantibodies specific to tor (BAFF) or a proliferation-inducing ligand (APRIL) or associated with SLE in mice. In nature, this oil can be (Fig. 2B). Compared with control mice, the serum lev- found in small amounts in vegetables [14], in the liver of els of chemokines C–C motif chemokine 2 (CCL2) and some sharks [15], and as a byproduct of petroleum distil- C-X-C motif chemokine 10 (CXCL10) were significantly lation [14]. Administration of pristane into the abdominal elevated in PIL mice at week 2 or month 2, whereas no cavity of mice can cause a series of lupus-like manifesta- significant difference was found in serum CCL7 level tions, including ascitic fluid enriched with monoclonal (Fig. 2B). Similar to most cytokines, the levels of serum antibodies, lipogranulomas, rheumatoid-like erosive autoantibodies (anti-chromatin, anti-double stranded arthritis and diffuse proliferative glomerulonephritis [16]. DNA (anti-dsDNA), anti-nuclear ribonucleoprotein Though most of the major clinical and laboratory mani - (anti-nRNP) and anti-Sm) and serum total immuno- festations of SLE well described by the American College globin G (IgG) were significantly increased in PIL mice of Rheumatology are observed in this pristane-induced from month 1, 2 or 4 (Fig. 2B). Granular deposits of IgG lupus (PIL) model, there are few reports on NPSLE and complement 3 (C3) were observed in the glomeruli of manifestations in this model [17–19]. For this, we com- PIL mice (Fig. 2C). The mean fluorescent intensity (MFI) bined behavioral, histomorphological and biochemical of IgG and C3 increased significantly at month 2, and fur - approaches to comprehensively evaluate brain dysfunc- ther increased with time (Fig. 2D). As important indexes tions and pathological changes in the PIL model (Fig. 1). of renal injury, the levels of serum creatinine (Scr) and Our findings highlight the significance of the PIL model blood urea nitrogen (BUN) were detected to assess renal Fig. 1 Experimental schedule. Mice were administered pristane (0.5 ml) or PBS via intraperitoneal injection. After a battery of behavioral tests, mice were sacrificed at month 1, 2, 4 or 8. Blood and tissue samples were collected for further histomorphological and molecular biological assays Y un et al. Behavioral and Brain Functions (2023) 19:3 Page 3 of 19 function. Compared with control mice, the levels of Scr difference became significant at month 4 (Fig. 3B). Con- and BUN were markedly elevated n PIL mice at month 4 sistent with the results in the open field test, PIL mice and further increased as the disease progressed (Fig. 2E). exhibited anxiety-like behavior in the elevated zero maze Throughout the observation period, the average 24 h test, as evidenced by significantly decreased total tracked urine protein level was higher in PIL mice compared with distance and time spent in the open arms from month 4, control mice from month 4, and the difference was sta - and further decreased with time (Fig. 3C). Furthermore, tistically significant (Fig. 2E). At month 4, symmetrical immobility time in the forced swim test was significantly swelling appeared in the hind paws of PIL mice (Fig. 2F). increased in PIL mice from month 4 to 8 (Fig. 3D). There Compared with control mice, the arthritis severity score were no differences between PIL mice and their age- showed a significant elevation in PIL mice from month matched controls in the novel object recognition test, 4 to 8 (Fig. 2F). In PIL mice, histologic sections of joint social novelty preference test, rotarod test or prepulse tissue showed the typical sign of severe arthritis with inhibition (PPI) test at any testing time point (Fig. 4). increased inflammatory cell infiltration in the synovial Therefore, these findings suggest that deficits in olfactory sub-lining (Fig. 2F). Similar to the arthritis severity score, function precede the anxiety- and depression-like pheno- the synovial inflammatory score in PIL mice was sig - type in PIL mice. nificantly increased at month 4, and gradually increased further as the disease progressed (Fig. 2F). Splenomegaly Overproduction of cytokines and chemokines in the brain was observed in PIL mice, and the increase in the spleen and BBB leakage in PIL mice index became significant at month 8 (Fig. 2G). Taken ELISA assays revealed that the levels of the cytokines together, these results suggest that PIL mice exhibit typi- IL-1β, IFN-α, IFN-β, IL-10, IFN-γ, IL-6, TNF-α and cal lupus-like manifestations during the observed course IL-17A and the chemokines CCL2 and CXCL10 were of the disease. significantly elevated in the brain tissues of PIL mice compared with their age-matched controls at week Olfactory dysfunction and an anxiety‑ and depression‑like 2 or month 1, and these high levels were maintained phenotype in PIL mice throughout the observed course of the disease (Fig. 5A, We used a battery of behavioral tests to assess the neu- B). We found no significant difference in the levels of the ropsychiatric phenotype of PIL mice. As shown in cytokines TWEAK, BAFF and APRIL or the chemokine Fig. 3A, PIL mice spent less time sniffing male or female CCL7 between PIL and control mice (Fig. 5A, B). No fecal dilute solution than their age-matched controls obvious pathological changes, such as parenchymal from month 2. Although not statistically significant lesions, ischemic lesions by vasculitis or thromboem- at any testing time point, an increase in the time spent bolism or brain atrophy, were observed in the brain of sniffing vinegar and alcohol was also observed in PIL PIL mice (data not shown). However, BBB permeabil- mice (Fig. 3A). These results indicate that PIL mice show ity, determined by evaluating the extravasation of Evans reduced interest toward attractants, especially biological blue, showed some abnormalities. Compared with age- odorants, as well as decreased sensitivity to repellants. In matched controls, there was a marked increase in Evans the open field test, both the total tracked distance and the blue content in the brain of PIL mice at month 1, and was time spent in the center decreased over the course of the maintained at a relatively stable level over the observed disease in PIL mice, compared with control mice, and the course of the disease (Fig. 5C). (See figure on next page.) Fig. 2 Examination for lupus-like symptoms in PIL mice. A Representative images showing lipogranulomas (arrow) adherent to the abdominal surface of the diaphragm in PIL mice. B Serum levels of cytokines (IL-1β, IFN-α, IFN-β, IFN-γ, IL-10, IL-6, IL-17A, TNF-α, TWEAK, BAFF and APRIL), chemokines (CCL2, CXCL10 and CCL7), autoantibodies (anti-chromatin, anti-dsDNA, anti-nRNP and anti-Sm) and total IgG, as detected by ELISA. IL-1β (group effect: F = 144.5, p < 0.0001; time effect: F = 5.593, p = 0.0005; interaction effect: F = 6.255, p < 0.0001); IFN-α 1,22 3.991,87.81 5,110 (p < 0.0001, 0.0472, 0.0754); IFN-β (p < 0.0001, < 0.0001, < 0.0001); IFN-γ (p < 0.0001, < 0.0001, < 0.0001); IL-10 (p < 0.0001, < 0.0001, < 0.0001); IL-6 (p < 0.0001, < 0.0001, < 0.0001); IL-17A (p < 0.0001, < 0.0001, < 0.0001); TNF-α (p < 0.0001, 0.3646, 0.3835); TWEAK (p = 0.0657, 0.7505, 0.9879); BAFF (p = 0.0538, 0.1978, 0.6215); APRIL (p = 0.2328, 0.8839, 0.8463); CCL2 (p < 0.0001, 0.0004, < 0.0001); CXCL10 (p < 0.0001, < 0.0001, 0.0037); CCL7 (p = 0.0985, 0.1661, 0.7891); anti-chromatin (p < 0.0001, 0.0004, < 0.0001); anti-dsDNA (p < 0.0001, < 0.0001, < 0.0001); anti-nRNP (p < 0.0001, < 0.0001, < 0.0001); anti-Sm (p < 0.0001, 0.0091, 0.0012) and total IgG (p < 0.0001, < 0.0001, < 0.0001). C Left panel showing representative images of glomeruli stained with H&E. Right panel showing representative images of frozen kidney sections stained for IgG and C3. D Quantitative analysis of the MFI of IgG (p < 0.0001, < 0.0001, < 0.0001) and C3 (p < 0.0001, < 0.0001, < 0.0001) in glomeruli. E Quantitative analysis of Scr (p < 0.0001, < 0.0001, < 0.0001), BUN (p < 0.0001, 0.0430, 0.0024) and 24 h proteinuria (p < 0.0001, 0.0001, < 0.0001). F Upper panel showing representative images of the hind paw, and quantitative analysis of arthritis severity score (p < 0.0001, 0.0004, < 0.0001). Lower panel showing representative images of joint tissues with H&E staining, and quantitative analysis of synovial inflammatory score (p < 0.0001, < 0.0001, < 0.0001). G Representative images of spleen, and quantitative analysis of spleen index (p = 0.0007, 0.0013, 0.0001). The data are expressed as the mean ± SEM (n = 12 in each group). Two-way ANOVA followed by Tukey’s post hoc test or Scheirer–Ray–Hare test: *p < 0.05, **p < 0.01 Yun et al. Behavioral and Brain Functions (2023) 19:3 Page 4 of 19 Fig. 2 (See legend on previous page.) Y un et al. Behavioral and Brain Functions (2023) 19:3 Page 5 of 19 Fig. 3 Behavioral assessments of olfactory function and anxiety- and depression-like phenotype. A Olfactory sensitivity test. Time spent sniffing male feces (p < 0.0001, 0.0037, < 0.0001), female feces (p < 0.0001, 0.0378, 0.0003), vinegar (p = 0.0035, 0.2964, 0.8263) or alcohol (p = 0.0055, 0.0248, 0.4079). B Open field test. Left panel showing representative images of the travelled path. Right panel showing quantitative analysis of the total distance travelled (p < 0.0001, 0.0001, 0.0925) and time spent in the center (p < 0.0001, 0.1251, 0.0007). C Elevated zero maze test. Left panel showing representative images of the travelled path. Right panel showing quantitative analysis of the total distance travelled (p < 0.0001, < 0.0001, 0.0056) and time spent in the open arms (%) (p < 0.0001, < 0.0001, 0.0201). D Forced swim test. Quantitative analysis of immobility time (p < 0.0001, 0.0009, 0.3390). The data are expressed as the mean ± SEM (n = 12 in each group). Two-way ANOVA followed by Tukey’s post hoc test or Scheirer–Ray–Hare test: **p < 0.01 Yun et al. Behavioral and Brain Functions (2023) 19:3 Page 6 of 19 Fig. 4 Behavioral assessments of novel object recognition, social novelty preference, rotarod and PPI tests. A Novel object recognition test. Quantitative analysis of the discrimination ratio (p = 0.1635, 0.0004, 0.6247). B Social novelty preference test. Quantitative analysis of the discrimination ratio (p = 0.6642, 0.0987, 0.6737). C Rotarod test. Quantitative analysis of the mean latency to fall (p = 0.0625, 0.0534, 0.5005). D Quantitative analysis of PPI% at 75 dB (p = 0.1491, 0.0777, 0.9946), 85 dB (p = 0.4376, 0.1693, 0.9995) or 95 dB (p = 0.4960, 0.1589, 0.8579). The data are expressed as the mean ± SEM (n = 12 in each group). Two-way ANOVA followed by Tukey’s post hoc test or Scheirer–Ray–Hare test IgG deposition in the brain of PIL mice for the microglial marker Iba-1. Compared with con- Immunofluorescence staining of brain sections revealed trol mice, the density of Iba-1-immunoreactive micro- IgG deposition in the choroid plexus and lateral ven- glia was increased significantly in PIL mice at month 1, tricular wall of PIL mice at month 1, and the deposits reached a peak at month 2, and then declined sharply gradually increased as the disease progressed (Fig. 6). (Fig. 7B, D). Notably, the microglia in PIL mice showed a Some IgGs co-localized with microglia (stained for ion- phenotypic transformation from the ramified to an acti - ized calcium binding adapter molecule 1 (Iba-1)) in PIL vated state characterized by short retracted processes mice (Fig. 6B, D). Because there was no difference in and a large irregular cell soma (Fig. 7B). Immunofluo - IgG deposition between the different testing time points rescence staining revealed that the density of glial fibril - in the age-matched controls, we show a representative lary acid protein (GFAP)-immunoreactive astrocytes was image for the control group (at month 1) in Fig. 6B, D for significantly increased in the hippocampus of PIL mice brevity. at month 1 and maintained throughout the observed disease course (Fig. 7C, E), accompanied by a change to Dynamic changes in microglia and astrocytes an activated morphology (Fig. 7C). These results suggest in the hippocampus of PIL mice that the behavioral deficits in PIL mice are accompanied Because the hippocampus plays a critical role in olfac- by dynamic changes in microglia and astrocytes. tory function [22] and mood regulation [23], our behav- ioral tests suggest an involvement of the hippocampus in Lipofuscin deposition in the brain of PIL mice PIL mice. We therefore conducted immunohistochemi- Lipofuscin, also called “age-pigment”, is frequently found cal staining on the brain slices to examine the effects as aggregates in neurons of subjects with neurodegenera- of pristane injection on glial cells in the hippocampus. tive disorders, caused by the accumulation of oxidized We compared the distribution of cells immunoreactive cross-linked proteins, lipids and sugars in the lysosomes Y un et al. Behavioral and Brain Functions (2023) 19:3 Page 7 of 19 Fig. 5 Levels of brain cytokines and chemokines, and BBB permeability. A Brain levels of cytokines (IL-1β, IFN-α, IFN-β, IL-10, IFN-γ, IL-6, TNF-α, IL-17A, TWEAK, BAFF and APRIL), as detected by ELISA. IL-1β (p < 0.0001, < 0.0001, < 0.0001); IFN-α (p < 0.0001, 0.0149, 0.0016); IFN-β (p < 0.0001, < 0.0001, < 0.0001); IL-10 (p < 0.0001, < 0.0001, < 0.0001); IFN-γ (p < 0.0001, 0.0002, 0.001); IL-6 (p < 0.0001, 0.0012, < 0.0001); TNF-α (p < 0.0001, < 0.0001, < 0.0001); IL-17A (p < 0.0001, < 0.0001, < 0.0001); T WEAK (p = 0.0562, 0.6937, 0.9245); BAFF (p = 0.0594, 0.6219, 0.5961) and APRIL (p = 0.9377, 0.0004, 0.4845). B Brain levels of chemokines (CCL2, CXCL10 and CCL7), as detected by ELISA. CCL2 (p < 0.0001, 0.0013, 0.0458); CXCL10 (p < 0.0001, 0.0016, 0.0295) and CCL7 (p = 0.0533, 0.6921, 0.9885). C Quantitative analysis of Evans blue dye extravasation (p < 0.0001, 0.6859, 0.8935). The data are expressed as the mean ± SEM (n = 12 in each group). Two-way ANOVA followed by Tukey’s post hoc test or Scheirer–Ray–Hare test: *p < 0.05, **p < 0.01 Yun et al. Behavioral and Brain Functions (2023) 19:3 Page 8 of 19 Fig. 6 Examination for IgG deposition in the choroid plexus and lateral ventricular wall. A, C Representative images of the choroid plexus and lateral ventricular wall stained with H&E. B, D Representative images of IgG deposition in the choroid plexus and lateral ventricular wall. White square showing the co-localization of an enlarged Iba-1-immunoreactive microglia (green) and IgG (red). DAPI staining for nuclei (blue). E, F Quantitative analysis of IgG deposition in the choroid plexus (p < 0.0001, < 0.0001, < 0.0001) and lateral ventricular wall (p < 0.0001, < 0.0001, < 0.0001) by MFI. The data are expressed as the mean ± SEM (n = 12 in each group). Two-way ANOVA followed by Tukey’s post hoc test or Scheirer–Ray–Hare test: **p < 0.01 Y un et al. Behavioral and Brain Functions (2023) 19:3 Page 9 of 19 Fig. 7 Density and morphology of microglia and astrocytes in the hippocampus. A Representative images of the hippocampus stained with H&E. B, C Representative images showing Iba-1-immunoreactive microglia and GFAP-immunoreactive astrocytes in the hippocampus. DAPI staining of nuclei (blue). White square showing an enlarged cell to compare morphology. D, E Quantitative analysis of the density of Iba-1-immunoreactive cells (p < 0.0001, < 0.0001, < 0.0001) and GFAP-immunoreactive cells (p < 0.0001, 0.5512, 0.3670). The data are expressed as the mean ± SEM (n = 12 in each group). Two-way ANOVA followed by Tukey’s post hoc test or Scheirer–Ray–Hare test: **p < 0.01 Yun et al. Behavioral and Brain Functions (2023) 19:3 Page 10 of 19 of post-mitotic cells. Compared with age-matched con- such as Parkinson’s disease [29], Alzheimer’s disease [30], trol mice, there was a significant increase in autofluores - or dementia with Lewy bodies [31], suggesting it may be cent lipofuscin deposits in the cortex and hippocampus helpful in the characterization of prodromal stages of of PIL mice at month 4, and these deposits further disease and the prediction of clinical outcomes of neuro- increased with time (Fig. 8). However, the density of neu- degenerative diseases. Additionally, affective deficits are ronal nuclei (NeuN)-immunoreactive neurons in the cor- frequent neuropsychiatric disturbances in NPSLE [32], tex and hippocampus did not significantly differ between and severely impact the patients’ quality of life. Simi- PIL and control mice (Additional file 1: Fig. S1). Further- larly, numerous genetically predisposed mouse models more, the terminal deoxynucleotidyl transferase dUTP of NPSLE also exhibit affective disorders. For example, nick end labeling (TUNEL) assay revealed no significant the MRL/lpr strain presents depression-like behavior and signs of apoptosis in either PIL or control mice (Addi- deficits in cognitive function without anxiety-like behav - tional file 2: Fig. S2). ior [8]. Furthermore, NZB/W F1 mice exhibit congeni- tal abnormalities, anxiety-like behavior and decreased Discussion locomotor activity [12], and B6.Nba2 mice show a strong To best represent human disease and investigate patho- anxiety phenotype and a mild depression phenotype genesis and novel treatment approaches, it is crucial [33]. Recently, several neuropsychiatric manifestations to take full advantage of the available mouse models of have been reported in PIL mice, including learning and spontaneous or inducible lupus-like disease. However, memory disturbance [17] and decreased spontaneous previous studies primarily focused on genetically sus- activities [18]. PIL mice in our current study also showed ceptible animals to investigate spontaneous NPSLE [24]. a decrease in total movement distance in the open field Neuropsychiatric symptoms in the inducible wild-type test and elevated zero maze test, indicating a reduc- models of lupus have been less studied. Here, we sys- tion in spontaneous activity caused by musculoskeletal tematically evaluated whether PIL mice can be used as changes. Nonetheless, the significant decrease in the an environmental-related inducible model of NPSLE. relative time spent in the center and open arms repre- Consistent with previous reports [18, 20, 21], PIL mice sents an anxiety-like phenotype in PIL mice from month presented several peripheral lupus-like manifestations 4 to 8 (Fig. 3B, C). Additionally, we observed a severe (Fig. 2). Notably, we identified some brain dysfunctions and consistent depression-like phenotype in PIL mice at and neuropathological changes, as follows: (a) olfactory month 4 to 8 (Fig. 3D). In line with previous findings [34, dysfunction and an anxiety- and depression-like pheno- 35], our results confirm an association between olfac - type; (b) cytokine and chemokine overproduction in the tory dysfunction and depression-like behavior. However, brain tissues; (c) BBB leakage; (d) IgG deposition in the no significant difference was found in the PPI, rotarod, choroid plexus and lateral ventricular wall, and co-local- novel object recognition or social novelty preference test ization of IgG with microglia; (e) changes in morphology (Fig. 4), indicating that sensory, motor coordination and and density of microglia and astrocytes in the hippocam- cognitive functions are not severely impaired in PIL mice. pus; and (f ) lipofuscin deposition in the cortical and hip- Our results are consistent with clinical data showing pocampal neurons. Our results highlight the potential that obvious olfactory dysfunction and mood disorders application of PIL mice for exploring the pathogenesis of mostly occur in NPSLE patients, while sensory, motor environmental-related NPSLE. and cognitive dysfunctions are infrequent or subtle [36]. In the present study, PIL mice lost their interest in Compared with genetic mouse models, such as MRL/ biological odorants and showed decreased sensitivity lpr mice with the MRL background, which itself may be to repellents (Fig. 3A). Spontaneous lupus-prone MRL/ involved in the severity of neurobehavioral abnormali- lpr mice display a constellation of behavioral deficits ties [24], our PIL mice, without a specific genetic back - dependent on olfaction [25, 26]. Human lupus patients ground, better mimic the clinical manifestations and are are also reported to have olfactory dysfunction [27] that therefore more suited for investigating the neuroimmune is associated with disease activity and anti-ribsomal P mechanisms of NPSLE. protein antibody levels [28]. One interesting point of our Several observations have suggested that cytokine dys- results is that the olfactory dysfunctions appeared earlier regulation may contribute to depression-like behavior than other behavioral deficits, suggesting that the olfac - in NPSLE. First, increased levels of cytokines, especially tory neural system is highly sensitive to immune attack. IL-6, IL-8, IL-1, TNF-α and IFN-α, are found in the cer- This highlights the possibility that olfactory function ebrospinal fluid of NPSLE patients [37–40]. Second, the tests might be an effective approach for early diagnosis of early dysregulation of cytokine production concords with NPSLE. Similarly, olfactory dysfunction has been shown the onset of depressive-like behavior in MRL/lpr mice to be an early marker for neurodegenerative diseases, [41] and other rodent strains [42, 43]. Third, anhedonia Y un et al. Behavioral and Brain Functions (2023) 19:3 Page 11 of 19 Fig. 8 Examination for lipofuscin deposition in the cortex and hippocampus. A and C Representative images of the cortex and hippocampus stained with H&E. B and D Representative images showing autofluorescent lipofuscin at 480 nm (green) and 550 nm (red) exciting light in the cortex and hippocampus. DAPI staining for nuclei (blue). White square showing the co-localization of an enlarged neuron (blue) with autofluorescence (green and red). E and F Quantification analysis of lipofuscin foci in the cortex (p < 0.0001, < 0.0001, < 0.0001) and hippocampus (p < 0.0001, < 0.0001, < 0.0001). The data are expressed as the mean ± SEM (n = 12 in each group). Two-way ANOVA followed by Tukey’s post hoc test or Scheirer–Ray–Hare test: **p < 0.01 Yun et al. Behavioral and Brain Functions (2023) 19:3 Page 12 of 19 and other behavioral indices of depressive-like behavior NPSLE. First, microglia can be activated by cytokines, in mice can be replicated by exogenous cytokines, such as such as TNF-α, and inhibited by downregulating mark- IL-6 and TNF-α [41, 44], and are prevented by knockout ers of microglial activation in MRL/lpr mice [48]. Second, of their receptors [44, 45]. In this study, we found sub- microglia may initially migrate to the BBB to protect its stantial increases in cytokine and chemokine expression integrity, and then transform into a reactive phenotype in the brain of PIL mice as early as week 2 (Fig. 5A, B). that damages the BBB and triggers neuroinflammation in Because cytokine overexpression occurred earlier than NPSLE model mice [54]. Third, antagonism of microglial behavioral deficits, it may be an initiating factor in the activation significantly attenuates spatial memory defi - depressive-like behavior in PIL mice. It has been reported cits and depression-like behavior in MRL/lpr mice [55]. that neurons and glial cells in the brain are capable of In line with these observations, we found microglial acti- secreting cytokines, such as IFN-γ, IL-1, IL-6, TNF-α and vation during the initial stage of the disease in PIL mice, IL-10, in both their normal and pathological states [46]. indicated by an increase in microglial density and specific Cytokine production in the brain can be promoted by morphological changes (Fig. 7B, D). It has been suggested various cytokines and chemokines, cytotoxic agents and that activated microglia may play a neuroprotective role neuronal debris, which can trigger a cascade of inflam - during the early stage of neurodegenerative disorders matory processes. Overexpression of these cytokines has [56]. Microglia can respond to the stimulation of neuro- been suggested to be an important factor in the patho- transmitters, such as adenosine triphosphate, glutamate genesis of neurotoxic and neurodegenerative disorders and histamine, released by damaged neurons, and con- [47]. Pristane, known as a membrane-activating com- sequently increase phagocytic activity to clear unwanted pound, can induce apoptosis in peripheral tissues and debris, protein aggregates and soluble antigens, thereby produce sufficient autoantigen substrates for immune reducing their damage to neurons [57]. Notably, we also intolerance, which can lead to overproduction of found that microglial density declined sharply in the hip- cytokines and the development of lupus-like autoimmun- pocampus of PIL mice from month 4 to 8. The sustained ity. Cytokines, such as IL-1β and TNF-α, in the serum of immune attack during the protracted progression of SLE PIL mice are capable of stimulating microglia to produce may cause activation-mediated apoptosis of microglia inflammatory cytokines and further drive the inflamma - [58]. The loss of microglia may severely hamper their tory cascade [48]. The increased peripheral inflammatory ability to combat immune challenge and repair tissues, cytokines can disrupt the integrity of the BBB [49] and resulting in neural damage and behavioral deficits. Thus, enter into the brain [50, 51]. In this study, we detected loss of microglia over time following activation may play significant extravasation of Evans blue dye in the brain a critical role in the neuropathological changes in PIL tissues, suggesting BBB leakage in PIL mice (Fig. 5C). mice. A recent study showed that neuronal degenera- Moreover, cytokines produced in the brain can also enter tion or remodeling induced by the interaction between the serum across the damaged BBB. Thus, peripheral activated microglia and neurons may contribute to cog- and central cytokines appeared to be integrated in the nitive dysfunction [59]. A limitation of our study is the brain of PIL mice with a mutual promotion effect, which lack of detecting the markers of neuronal degeneration, is difficult to be separated. Furthermore, we found IgG such as neuronal complexity, length of dendrites and the deposition in the choroid plexus and lateral ventricu- number of spines in the brain. This shortcoming should lar wall (Fig. 6). A significant increase in IgG deposi - be addressed in future studies. Together with our previ- tion has been reported in the hippocampus of PIL mice ous results, our findings suggest that dynamic change [19]. IgG deposition in the brain may occur following the in microglia play an important role in the neuropathol- impairment of the blood-ventricular barrier and cho- ogy of NPSLE, and therefore, modulating microglia may roid plexus-vascular barrier caused by cytokines in PIL be a promising therapeutic strategy for NPSLE. In con- mice. IgG deposition can promote endothelial damage, trast, astrocytes were persistently activated throughout microglial activation and inflammatory mediator produc - the observed course of the disease in PIL mice (Fig. 7C, tion, resulting in a positive feedback loop that disrupts E). Astrocytes generally interact with both neural and immune homeostasis [52, 53]. Furthermore, we found a non-neural cells and perform dynamic activities crucial sign of phagocytosis of IgG by microglia (co-localization for neural circuit function, neurological function and of IgG with microglia), suggesting that microglia may behavior [60]. In neural disorders, astrocytes can be acti- play a neuroprotective role in PIL mice. vated and produce nerve growth factors and immune Microglia are innate immune cells in the brain, con- mediators, such as IL-1 and nitric oxide, in response necting the nervous system with the immune system. to brain inflammation or injury [61]. Histopathologi - Several studies have suggested that microglia are involved cal investigations of NPSLE patient’ brains confirm the in the development of neuropsychiatric symptoms in widespread presence of activated astrocytes along with Y un et al. Behavioral and Brain Functions (2023) 19:3 Page 13 of 19 microglia within the heterogeneous pathological tissue can induce the synthesis of oxidative stress products. changes [62]. Astrocytes are an important component of Accumulated oxidative stress products within neurons the BBB, and activated astrocytes are a typical hallmark may orchestrate inflammation, disrupt the metabolism of BBB dysfunction [63]. In addition to the physiological of lipids and metals, and thereby promote the genera- role of astrocytes in synaptic refinement, activated astro - tion of lipofuscin. Loss of neuroprotective effects from cytes have also been implicated in pathological synapse microglia may accelerate this pathological process dur- loss and dysfunction following injury or nervous system ing the progressive stage of SLE. Thus, lipofuscin deposi - degeneration in adults [64]. Overall, astrocyte activation tion in neurons may be an important factor involved in may contribute to the neural dysfunction in PIL mice; neuronal damage in PIL mice. Though we observed nei - however, further study is needed to clarify the underlying ther a significant decrease in neuronal density by NeuN mechanisms. staining nor a sign of neural apoptosis by TUNEL stain- Another important finding of our study is that lipofus - ing of cortical and hippocampal slices, it is premature to cin deposits were found in neurons in both the cortex and exclude the possibility of neural damage considering the hippocampus in PIL mice (Fig. 8). Lipofuscin, known as relatively low sensitivity of NeuN and TUNEL staining. age pigments, are autofluorescent lipopigments formed The detailed mechanisms of neural damage need to be by lipids, metals and misfolded proteins, which are espe- further explored in future studies. cially abundant in neural cells. Lipofuscin within the As shown in Fig. 9, the levels of cytokines and brain increase not only with age, but also with pathologi- chemokines simultaneously increased in the brain and cal processes, such as neuronal dysfunction and a rep- serum as early as week 2 after pristane injection. Ini- ertoire of cellular alterations, including oxidative stress, tial histological changes in the brain, such as BBB leak- and proteasomal, lysosomal and mitochondrial dysfunc- age, IgG deposition and glial cell activation, appeared at tions [65–69]. Recent evidence suggests that lipofuscin month 1 after pristane injection, accompanied by periph- may participate in the pathogenesis of various neurode- eral signs of lipogranuloma formation, autoantibody generative disorders [70]. Furthermore, senescent neural production and total IgG elevation in the serum. Tissue cells accumulate in the hippocampus of the MRL/lpr SLE damage commenced as olfactory dysfunction and IgG model mice with depressive behavior [71]. The increase and C3 deposition in the glomeruli at month 2, and grad- in lipofuscin deposition in PIL mice may be attributable ually progressed to anxiety- and depression-like behav- to the overexpression of cytokines in the brain, which ior, renal failure and arthritis at month 4. Splenomegaly Fig. 9 Peripheral manifestations, behavioral changes and brain pathogenic changes in PIL mice over the trial period Yun et al. Behavioral and Brain Functions (2023) 19:3 Page 14 of 19 appeared at month 8. Therefore, the pathological changes the animal experimental protocols and the treatment in PIL mice may initiate from pristane-induced innate procedures (No. KT2018060). immune responses, which cause the overproduction of cytokines and chemokines, and enhance the produc- Pristane injection tion and release of autoantibodies. Cytokines in serum As shown in Fig. 1, at the age of 8 weeks, mice were can disrupt the integrity of the BBB and facilitate the randomly divided into the following eight groups invasion of autoantibody IgGs into the brain, result- (n = 12 per group): 4 control groups (1 m, 2 m, 4 m and ing in glial cell activation and neural dysfunction. Simi- 8 m) and 4 PIL groups (1 m, 2 m, 4 m and 8 m), which lar pathological mechanisms involving cytokines and received a single intraperitoneal injection of 0.5 ml IgGs also work in the glomerulonephritis and arthritis phosphate buffer saline (PBS) or pristane (Sigma- in PIL mice. Further study, using the transcriptomic pro- Aldrich, St. Louis, MO, USA), respectively. The dose file comparisons between central and peripheral organs, of pristane used in this study was based on a previous such as brain, spleen, kidney and joint, may help eluci- research [73]. Mice were sacrificed at month 1, 2, 4 or date the overlapping and distinct immune mechanisms 8 after a battery of behavioral tests. Blood samples were in PIL mice and provide potential therapeutic targets for obtained from the eyeball. Tissue samples of spleen, environmental-related SLE. kidney, joint and brain were harvested for further examination. There was no early euthanasia of animals during the study. Conclusions We found that pristane can induce mice to exhibit olfac- tory dysfunction and an anxiety- and depression-like ELISA for brain cytokines and chemokines, and serum phenotype, along with increased expression of cytokines, cytokine, total IgG and autoantibody detection BBB leakage, activation of microglia and astrocytes and Mice were anesthetized and transcardially perfused aberrant deposition of IgG and lipofuscin in the brain. with 0.1 M PBS (pH 7.5, 4 °C). Brains were harvested Our results suggest that brain dysfunction in PIL mice and dissected into the left and right hemispheres. One may initiate from the dysregulation of cytokines, which hemisphere was snap-frozen in liquid nitrogen and subsequently triggers BBB impairment, IgG deposition, subsequently made into frozen sections for immuno- glial activation and neuronal damage. These findings fluorescence staining, while the other hemisphere was suggest that PIL mice are a promising model for NPSLE homogenized on ice in PBS and centrifuged at 12,000 rpm study, and highlight the important roles of glial cells in for 15 min at 4 °C to remove cell debris. Supernatants the pathogenesis of NPSLE. Glial cells, may therefore were collected and stored at − 80 °C until assay. Serum serve as a potential therapeutic target for NPSLE. Future was separated by centrifugation at 5000 rpm for 15 min studies should testify whether anti-inflammatory drugs at 4 °C. Cytokines and chemokines in brain tissues and such as retinoic acid [72] and coptisine [21], which have serum were detected at week 1 or 2, or month 1, 2, 4 or been shown to ameliorate renal and cardiovascular dys- 8 using IL-1β, IFN-α, IFN-β, IL-10, IFN-γ, IL-6, TNF-α, funciton in PIL mice, can reverse the neuroinflammation IL-17A, BAFF, CCL2 and CXCL10 ELISA kits (Boster and attenuate phenotypic changes in NPSLE. & Biological Technology, Wuhan, China), TWEAK and APRIL DuoSet kits (R&D Systems, Minneapolis, MN, Methods USA) and CCL7 ELISA kit (CUSABIO, Wuhan, China). Animals Autoantibodies (anti-chromatin IgG (Inova Diagnostics, Specific pathogen-free BALB/c mice were purchased San Diego, CA, USA), anti-dsDNA IgG and anti-Sm IgG from Vital River Laboratory (Beijing, China) at the age of (CUSABIO), and anti-nRNP IgG (Alpha Diagnostics, San 4 weeks. Female mice were used for the experiment at the Antonio, TX)) and total IgG (Boster & Biological Tech- age of 8 weeks. All animals were reared in standard ani- nology) in serum were determined using commercially mal cages under environmentally controlled laboratory available kits per manufacturer’s guidelines. Each sample conditions (12/12 h light/dark cycle, 22 ± 2 °C, 40–80% was tested at least three times, and the average value was humidity) with ad libitum access to food and water. All taken. efforts were made to minimize animal suffering. The ani - mals were maintained and treated in compliance with the policies and procedures detailed in the “Guide for Renal function and 24 h proteinuria assessment the Care and Use of Laboratory Animals” of the National The levels of Scr and BUN were detected using Scr and Institutes of Health. The Animal Care and Use Commit - BUN assay kits (Jiancheng, Nanjing, China). Experimen- tee of China Medical University reviewed and approved tal procedures were strictly followed according to the Y un et al. Behavioral and Brain Functions (2023) 19:3 Page 15 of 19 manufacturer’s protocols. Urine samples from mice in (45 cm × 24 cm × 20 cm), each mouse was introduced to a metabolic cage were collected over 24 h. Proteinuria a 5 cm × 5 cm piece of filter paper scented with 0.25 ml of was measured with a BCA kit (Beyotime biotechnology, an odorant for 2 min. Then, the scented filter paper was Shanghai, China). Each sample was tested at least three removed, and the mouse was allowed to rest for 1 min. times and the average value was taken. This procedure was repeated three times. Each mouse was presented with repellents (vinegar and alcohol) and Hematoxylin and eosin (H&E) staining attractants (male feces and female feces) diluted with Tissues from the hind limb, brain and kidney were har- PBS to the same concentration. Test chamber was cov- vested and post-fixed in a solution of 4% paraformal - ered with a clear piece of plexiglas to limit evaporation dehyde in PBS overnight at 4 °C. The joint tissues were and entry of external odorants. Active investigation was decalcified with ethylenediamine tetraacetic acid (EDTA, defined as directed sniffing within 0.5 cm of the odorant pH 8.0) for 2 weeks, and then embedded in paraffin after source and the sniffing time was recorded. Sniffing time dehydration, and sliced into 4 μm sections. The sections for each trial was summed to obtain a total value per were stained with H&E to observe synovial damage and animal. provide an anatomic reference for the glomerulus and brain areas. Open field test The open field chamber (40 cm × 28 cm × 40 cm) was Arthritis severity score and synovial inflammation score made up of black polyvinyl chloride panels with a For quantified scoring of arthritis severity, we used a pre - non-reflective base. The central zone was defined as a viously published scoring system [16], as follows: score 20 cm × 14 cm area. Each mouse was positioned individ- scale of 0–3, where 0 = normal, 1 = slight swelling or ery- ually in the center zone and allowed to freely explore the thema of the wrist/ankle joint or footpad, 2 = moderate arena for 30 min. Total distance travelled (km) and time swelling and erythema of the wrist/ankle joint or footpad, spent in the center (s) were digitally recorded and ana- and 3 = severe swelling and erythema of the paw. The lyzed using custom-built programs. scores for individual limbs were summed to obtain a total arthritis severity score of 12 per animal. Arthritis sever- Elevated zero maze test ity score was assessed twice by two independent observ- The elevated zero maze was a ring-shaped apparatus, ers. For synovial inflammation, we used a scoring system elevated 50 cm from the floor, and consisted of a circu - described previously [74], in which 5 high-power mag- lar platform (outer diameter 50 cm, width 10 cm) divided nification fields (HPF) were scored for the percentage of into four quadrants of equal length with two open arms infiltrative mononuclear inflammatory cells, as follows: and two closed arms (surrounded by a 20-cm wall from 0 = absent, 1 = mild (1–10%), 2 = moderate (11–50%), the surface of the maze). The test was conducted as pre - and 3 = severe (51–100%). The average score of the 5 viously described [75]. The test mouse was placed at the HPFs was used for analyses. open arm and was allowed to conduct a 10-min free exploration. Total distance travelled (m) and percentage Spleen index of time spent in the open arms were digitally recorded Spleen index was calculated as the ratio of spleen weight and analyzed by custom-built programs. to body weight (mg/g) as reported previously [18]. Behavioral assessments Novel object recognition test The mice were subjected to a series of behavioral tests in The novel object recognition test was used to evalu - the following order: olfactory sensitivity test, open field ate recognition memory and was conducted as pre- test, elevated zero maze test, novel object test, social viously described [76, 77]. During the acclimation novelty preference test, rotarod test, PPI test and forced phase, mice were allowed to habituate to the apparatus swim test. Mice were tested during the same lighting (55 cm × 40 cm × 30 cm) with no objects for 10 min, and and time-of-day conditions. All behavioral chambers then a test phase began 24 h later. On the trial day, two were cleaned with 70% ethanol as mice were changed. identical cylindrical objects were placed in the oppo- Researchers were blinded to the experimental groupings. site side of the apparatus, and mice were allowed to spend 10 min with the objects. One hour later, one of Olfactory sensitivity test the objects was replaced with a triangular object, and The paradigm used to assess olfactory sensitiv - time spent exploring the novel and familiar object were ity in this study was similar to that in an earlier report digitally recorded for 10 min. The time spent in close [26]. Following habituation to a new test chamber Yun et al. Behavioral and Brain Functions (2023) 19:3 Page 16 of 19 Forced swim test interaction with each object was converted into a dis- Each mouse was placed into a glass beaker containing crimination ratio, which was calculated as follows: time 3000 ml of water maintained at approximately 24 ± 1 °C. spent exploring the novel object/total time spent explor- Following habituation to swimming in this glass beaker ing both objects. for 2 min, a 4-min test session was digitally recorded. Mice placed in this situation had no way to escape, and Social novelty preference test began struggling and swimming, and eventually exhibited The social novelty preference test was performed as pre - behavioral despair, assessed as immobility [34]. Depres- viously described, with minor modification [78, 79]. Dur- sion-like behavior was defined assessed as the time spent ing the acclimation phase, a test mouse was allowed to immobile. habituate to the apparatus for 10 min. A stranger mouse was then placed in one of the wire cages. The test mouse was allowed to spend 10 min to explore the entire appa- Measurement of BBB permeability ratus. Subsequently, a novel stranger mouse was placed in To evaluate alterations in BBB permeability, Evans blue the other wire cage. The test mouse was allowed to freely dye was used as a marker of BBB leakage, as previously investigate the entire apparatus (the familiar mouse in described [82]. Briefly, mice were administered 2% Evans one corner and the novel stranger mouse in the opposite blue dye solution (4 ml/kg, Beyotime biotechnology) corner) for 10 min. The time spent in close interaction intravenously 30 min before sacrifice. Then, the brain with each mouse was digitally recorded and converted tissues were homogenized in 50% trichloroacetic acid at into a discrimination ratio, which was calculated as fol- a 1:3 v/v ratio. BBB permeability was assessed as Evans lows: time spent exploring the stranger mouse/total time blue extravasation, and was quantified in the supernatant spent exploring both mice. from each sample following addition of 90 μl of 95% etha- nol (absorbance, 620 nm). Rotarod test The rotarod test was used to evaluate motor coordination Immunofluorescence staining and was performed as previously described, with minor Kidney and brain tissues were dissected, fixed in 4% para - modification [18]. First, mice were placed on the station - formaldehyde for 24 h, followed by immersion in 30% ary bar to habituate to the apparatus for 2 min. Then, the sucrose (w/v) solution at 4 °C overnight. Tissues were rotarod began to accelerate from 4 to 40 rpm. Latency to then cut into 10-μm-thick coronal frozen sections, and fall off the rotating rod was recorded with a 5-min cut - blocked with 10% goat serum for 30 min at room temper- off time for three trials per day over 3 consecutive days ature. Sections were incubated with primary antibodies, and the mean retention time on the rod per trial was including anti-C3 (1:100; Santa Cruz, CA, USA), anti- recorded. Iba-1 (1:200; Abcam, Cambridge, UK), anti-GFAP (1:500; Abcam) or anti-NeuN (1:500; Abcam) at 4 °C overnight. On the following day, sections were incubated with sec- PPI test ondary antibodies, including Alexa Fluor 488-conju- PPI test was measured using a startle chamber and was gated goat anti-mouse IgG (1:200; Proteintech, Wuhan, conducted as previously described, with minor modifica - China) or Alexa Fluor 488-conjugated goat anti-rabbit tion [80, 81]. The test mouse was given a 10-min acclima - IgG (1:200, Proteintech, Wuhan, CHN) for 2 h at room tion period in the startle chamber during which a 70 dB temperature in the dark. For locating cell nuclei, sec- background noise was presented, and then the test mouse tions were stained with DAPI (Beyotime biotechnology) was subjected to test trials consisting of four trial types; for an additional 8 min. TUNEL staining was performed that is, one type of startle stimulus only trial and three using a TUNEL Bright Green Apoptosis Detection kit types of PPI trials. White noise of 120 dB (40 ms) was (Vazyme, Nanjing, China) according to the manufactur- used as the startle stimulus for all trial types. The peak er’s instructions. Images were captured on a microscope startle amplitude was recorded with the onset of the star- (BX53, Olympus, JPN) at × 200 or × 400 magnification. tle sound. The prepulse stimulus was presented 100 ms The number of stained cells was automatically counted before the onset of the startle stimulus with an intensity in a defined area using Image J software. The MFI of C3 of 75, 85 or 95 dB (20 ms). Six blocks of the four trial deposition in the glomeruli was calculated using Image J types were presented in a pseudorandom order such that software. The data from three random sections for each each trial type was presented once within a block. The individual mouse were averaged to obtain a single value. intertrial interval had an average duration of 15 s. PPI For IgG staining, sections were incubated with Alexa responses were calculated as follows: PPI% = [1 − (pre- Fluor 594-conjugated goat anti-mouse IgG (1:200, Pro- pulse trials/startle only trials)] × 100. teintech) for 2 h at room temperature in the dark. The Y un et al. Behavioral and Brain Functions (2023) 19:3 Page 17 of 19 MFI of IgG deposition in the choroid plexus, lateral ven- and hippocampus stained with H&E. (B) and (D) Representative images tricular wall or glomeruli was calculated using Image J showing NeuN-immunoreactive cells in the cortex and hippocampus and quantitative analysis of neuronal density in the cortex (p = 0.5857, software. To examine autofluorescent lipofuscin, regions 0.1302, 0.7324) and hippocampus (p = 0.0650, 0.1922, 0.7277). The data of interest were captured at 480 nm and 550 nm exciting are expressed as the mean ± SEM (n = 12 in each group). Two-way ANOVA light. The mean gray value of autofluorescent lipofuscin followed by Tukey’s post hoc test or Scheirer–Ray–Hare test. was quantified with image J software. Additional file 2: Figure S2. TUNEL staining in the cortex and hippocam- pus. (A) and (C) Representative images of the cortex and hippocampus stained with H&E. (B) and (D) Representative images showing no TUNEL- immunoreactive cells in the cortex and hippocampus. DAPI staining for Statistical analysis nuclei (blue). GraphPad Prism V8 software (La Jolla, CA, USA) and SPSS 22.0 software (SPSS Inc., Chicago, USA) were used Author contributions for statistical analysis. Before applying parametric statis- PTY and LQ supervised the project. PTY, LQ and YY designed the project, tics, all data were checked for the assumptions of normal- wrote the manuscript and performed the statistical analysis and revised the ity using the D’Agostino-Pearson omnibus normality test. manuscript. YY, XJW, JYX and CYJ were involved in laboratory works and experimental design of the work. YY, JYC, XRW and JNW were involved in data All data were expressed as the mean ± SEM. Differences collection and lab assessments, and study designing. All authors read and in normally distributed data were detected by repeated approved the final manuscript. measures analysis of variance (ANOVA) (with time Funding as within factor and group [PBS, pristane] as between The study was funded by the Project for Construction of Key Platform, factor) followed by Tukey’s post hoc test for multiple Shenyang, China (19-109-4-15 to PTY ), “Xingliao Talent Plan” of Liaoning, China comparison. For non-parametric data, the Scheirer–Ray– (XLYC2002062 to PTY ), the Chinese National Key Technology R&D Program (2021YFC2501303 to PTY ), the Joint Funds of the National Natural Science Hare extension of the Kruskal–Wallis test [83] was used Foundation of China (U22A20309 to PTY ) the department of Science and as a non-parametric equivalent of the two-way ANOVA. Technology of Liaoning Province (2020JH2/10100014, 2021JH1/10400049 to p < 0.05 was considered a statistically significant differ - LQ), the “Xingliao Talent Plan” of Liaoning, China (XLYC2002094 to LQ) and the fellowship of China Postdoctoral Science Foundation (2021M703606 to XJW ). ence in all sampled groups. Availability of data and materials The data are available for any scientific use with kind permission. Abbreviations SLE Systemic lupus erythematosus NPSLE Neuropsychiatric lupus Declarations BBB Blood–brain barrier PIL P ristane induced lupus Ethics approval and consent to participate IL Interleukin The animals were maintained and treated in compliance with the policies and IFN Interferon procedures detailed in the “Guide for the Care and Use of Laboratory Animals” TNF T umor necrosis factors-α of the National Institutes of Health. The Animal Care and Use Committee of TWEAK Tumor necrosis factor-like weak inducer of apoptosis China Medical University reviewed and approved the animal experimental BAFF B cell activating factor protocols of the “Guide” and the treatment procedures (No. KT2018060). APRIL A proliferation inducing ligand CCL C–C motif chemokine Consent for publication CXCL C- X-C motif chemokine 10 Not applicable. Anti-dsDNA Anti-double stranded DNA Anti-nRNP Anti-nuclear ribonucleoprotein Competing interests IgG Immunoglobin G The authors declare no competing interests. C3 Complement 3 Scr Serum creatinine BUN Blood urea nitrogen Received: 9 July 2022 Accepted: 13 January 2023 MFI Mean fluorescent intensity PPI Prepulse inhibition Iba-1 Ionized calcium binding adapter molecule 1 GFAP Glial fibriliary acid protein Neun Neuronal nuclei References TUNEL Terminal deoxynucleotidyl transferase dUTP nick end labeling 1. Stock AD, Wen J, Doerner J, et al. 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Journal
Behavioral and Brain Functions – Springer Journals
Published: Feb 10, 2023
Keywords: Neuropsychiatric lupus; Mouse model; Behavioral deficit; Cytokine; IgG; Glia cells; Lipofuscin