Fluoxetine attenuates pain-like and depression-related behaviors via reducing neuroinflammation and synaptic deficits in bone cancer mice

doi:10.21203/rs.3.rs-2188826/v1

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Fluoxetine attenuates pain-like and depression-related behaviors via reducing neuroinflammation and synaptic deficits in bone cancer mice

https://doi.org/10.21203/rs.3.rs-2188826/v1

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Background

Bone cancer patients are often accompanied with pain and depression, which seriously affects their quality of life and survival time. Fluoxetine, a selective serotonin reuptake inhibitor, has been reported to be effective not only in reducing depression-like behaviors but also in alleviating cancer pain. However, the specific mechanisms involved remain obscure.

Methods

Bone cancer mice were treated with fluoxetine for 7 consecutive days after the formation of pain and depression symptoms. Neuroinflammation and synaptic changes at the basolateral amygdala (BLA) after treatment were examined with western blotting, immunofluorescence and Golgi-Cox staining.

Results

Compared with the tumor group, fluoxetine significantly improved the mechanical allodynia and sugar water preference ratio, and reduced the immobility time of forced swimming. In addition, we found fluoxetine had an inhibitory effect on reactive glial cells and neurotoxic glial cells of bone cancer pain (BCP) mice. Meanwhile, fluoxetine could improve synaptic function in the bone cancer mice basolateral amygdala regions.

Conclusions

Fluoxetine can effectively alleviate pain-like and depression-related behaviors in BCP model. The concerned mechanisms may be related to reducing neurotoxic glial cells activation and promoting synapse formation at BLA.

bone cancer pain

depression

fluoxetine

neuroinflammation

glial cells

Bone cancer pain (BCP) is a common destructive pain in clinic and about 50% of patients with advanced bone cancer experience moderate to severe pain who also suffer from negative emotions such as depression and anxiety [1, 2]. A clinical study indicates that antidepressants can be effective in reducing cancer pain and emotional burden [3]. Meanwhile, there is good relief of pain symptom in chemotherapy pain mice treated with antidepressants [4, 5]. Both clinical and basic studies suggest that in addition to antidepressant effects, antidepressants also exhibit satisfactory analgesic effect although the mechanism remain unclear.

Fluoxetine, a selective serotonin reuptake inhibitor, is widely used in the treatment of depression, panic or anxiety. Not only those, it has also displayed powerful effects of anti-inflammatory and neuroprotection in many nervous system diseases [6]. Although some guidelines for the treatment of chronic pain include fluoxetine, how it reduces pain is not well understood. Recent studies indicate that fluoxetine can inhibit the activation of reactive glial cells and the appearance of pro-inflammatory factors in vitro and vivo experiments [7, 8]. In addition, fluoxetine also displays a critical role in synaptic remodeling of hippocampus in postpartum model of depression [9]. Although current researches suggest that neuroinflammation and remodeling of neuronal synapses or dendritic spine structures simultaneously engaged in the regulation of pain or depression, whether the antidepressant fluoxetine is involved in the regulation of BCP through above mechanisms remain unclear [10, 11]. Meanwhile, studies have shown that basolateral amygdala (BLA) is the transit station for pain and negative emotional processing [12, 13], and its high inhibited state in circuits is of great significance under normal physiological conditions [14]. Therefore, we established BCP model in this study to determine the effect of the antidepressant fluoxetine on pain and the correlation between neuroinflammation and neuroprotection at the BLA.

Experimental Animals

60 male mice of C3H (4–6 weeks old) were used for research. Mice were housed in rooms with constant temperature and suitable humidity, lighting time was 12 h per day. Animal experiments were carried out according to the principles of animal ethics and obtained agreement of the ethics committee of Nanjing Drum Tower Hospital (Project number: 2020AE01025).

Establishment of Mouse Model

Mice were performed the arthrotomy of right knee joint and drilled through the femoral bone marrow cavity with the needle after anesthesia. 20µl MEM (ATCC, USA) containing 2×10⁵ NCTC cells (ATCC, USA) or 20µl MEM was injected into the femoral bone marrow cavity. Block the needle hole with bone wax and suture the incision.

Pharmacological Treatment

According to the previous study[15], fluoxetine was administered via an intraperitoneal injection for 7 consecutive days after the formation of pain and depressive-like behaviors on day 14 after BCP molding.

Paw Withdrawal Mechanical Threshold (PWMT)

The plantar of mouse was stimulated with different intensities of Von Frey filaments after the mouse was left of 30 min rest. Mouse rapid retracting paw was considered to a positive response of stimulate. In five repetitions of each stimulus, three or more times the lowest Von Frey filament stimulation intensity was considered the PWMT of the mouse.

Sucrose Preference Test (SPT)

Mice were allowed to have two bottles of 1% sugar water in the cage for one day. One bottle of 1% sugar water and one bottle of pure water were obtained in the cage for the following day. After 24 h of fasting and drinking, the amount of sugar water and pure water intake per mouse within 12 h was recorded.

Forced Swimming Test (FST)

Transparent water cylinders were used for mice forced swimming test. Mice were adapted to swimming for 1 day in advance and forced to swim 6 min in the test. When the mouse did not move in the water and just keep its head above the water was considered immobility behavior.

Immunofluorescence

Whole brain tissues were excised from mice after perfusion with normal saline and formaldehyde transcardially, then fixed in formaldehyde for 24 h and gradient dehydrationed with 15% and 30% sucrose. Frozen sections of 20 µm in thickness were subjected to 3‰ Triton for 15 min after PBS rinsing and blocked with 10% goat serum. Then, sections were incubated with the following primary antibodies at 4°C overnight: GFAP (1:1000, Cell signaling) or IBA1 (1:500, Wako) or CD68 (1:1000, Abcam) or C3 (1:500, Novus). Sections were incubated with the corresponding secondary antibody for 1 hour and washed with PBS again. Finally, DAPI stain was added for blocking slices.

Western Blotting

Amygdala proteins of fresh brain tissue were extracted with lysate. Proteins were separated with 10-12.5% SDS-PAGE gels and transferred to PDVF membrane. The protein brands were incubated overnight with the following antibodies after blocked with 5% skim milk: rabbit anti-IL-6 (1:1000, Abcam, USA) or rabbit anti-IL-1β (1:1000, Abcam, USA) or mouse anti-SYN1 (1:1000, Millipore, USA) or mouse anti-PSD95 (1:1000, Abcam, USA) or mouse anti-SYN (1:1000, Abcam, USA) or rabbit anti-ACTIN (1:10000, Proteintech, China). TBST washed bands 3 times for 10 min each time. Then, brands were incubated with the corresponding secondary antibody for 1 hour and TBST washed 3×10 min again. Finally, ECL solution was used for proteins visualization.

Golgi-Cox Staining

According to the Rapid GolgiStain Kit (FD Neurotech, China) instructions, fresh mice brains were fixed and stained. Brain slices containing amygdala were used for image acquisition and analysis.

Statistical Analysis

SPSS 24.0 (IBM Corporation, USA) was used for statistical analysis. Data were analyzed by normality test and expressed as means ± SE. Two-way ANOVA and Bonferroni post-test were performed to analyze the research data. GraphPad Prism 8.0 was applied to draw graphs and p < 0.05 considered statistically significant.

Fluoxetine Treatment Schedule and Behavior Analysis

Our previous findings showed that mice exhibited pain-like and depression-like behaviors on day 14 after BCP molding [16]. To explore the effects of antidepressants on pain-like and depressive-like behaviors in mice with BCP, mice were treated for 7 consecutive days intraperitoneally with fluoxetine after the formation of cancer pain on day 14 (Fig. 1A). Compared with sham group, there was significant pain in tumor-bearing group on day 21 postoperatively. Although tumor-bearing mice could not obtain complete pain relief, fluoxetine administration increased the PWMT in tumor-bearing group (Fig. 1B). At the same time, the proportion of sucrose water intake in mice with tumor was increased and the immobility time for forced swimming was decreased, following fluoxetine treatment (Fig. 1C and D). These results showed that the antidepressant fluoxetine relieved both pain and depression in mice with bone cancer.

Fluoxetine Alleviated BCP-induced Reactive Gliosis in BLA

IBA1 and GFAP immunofluorescence staining were used to detect glial cells morphology and number changes at BLA. We found that microglia presented amoeboid-like morphology in BLA of tumor-bearing mice while astrocyte appeared an increase in branches (Fig. 2A). Glial cells in tumor group were significantly more than sham group, and the number of microglia is far more than astrocytes (Fig. 2B and C). Fluoxetine intervention had greatly reduction of reactive glial cells in tumor mice, but there was no significant difference between the sham group and the sham group after fluoxetine treatment.

Fluoxetine Alleviated the Effects of Neuroinflammation in BLA

Studies have shown that fluoxetine can alleviate neuroinflammation and glial cells polarization[6, 17]. Our immunofluorescence results showed a significant increase in M1 microglia (CD68⁺) in the BLA, which consistent with the tendency of reactive glial cells in tumor-bearing mice (Fig. 3A). Similarly, the expression of C3 (A1 astrocyte marker) showed increasing trends in BLA of tumor group (Fig. 3B). In addition, we found that the expression of inflammatory factors IL-6 and IL-1β were significantly increased in tumor group (Fig. 3C). Fluoxetine treatment reversed the polarization of microglia and astrocyte (Fig. 3A and B). Likewise, the expression of IL-6 and IL-1β were decreased due to the interventions of fluoxetine (Fig. 3D).

Fluoxetine Enhanced the Spine Density and Synaptic Protein Expression

It has been documented that pain and depression are often accompanied by synaptic remodeling. To determine synaptic changes after fluoxetine treatment, we detected synapses of BLA by Golgi staining and Western blotting experiments. The Golgi staining results showed that the number of dendritic spines was significantly reduced and appeared more immature dendritic spines in mice with tumor. The above phenomena improved after fluoxetine treatment (Fig. 4A and B). Likewise, we found that the synaptic proteins PSD95, SYN1 and SYN were significantly reduced in the tumor group. Fluoxetine administration significantly increased SYN1 and SYN protein levels, and PSD95 was also increased but not statistically significant (Fig. 4C and D).

Clinically, pain and depression are two symptoms with high rates and serious harm in cancer patients, and the comorbidity rate was up to 40% [18, 19]. Pain is a major hazard factor for depression [20], and depression can also exacerbate chronic pain and hinder its effective treatment. However, the mutual mechanisms between the two are unclear. Our previous study showed that BCP mice began to develop pain-related depressive behaviors on the 14th day after bone cancer molding. To explore the effects of depression on pain behavior, we use antidepressants to block depressive symptoms in mice to study the effect of antidepressants on pain.

In clinical practice, antidepressants have been widely used for the treatment of pain [21]. In this study, we demonstrated that the antidepressant fluoxetine was effective in relieving depressive mood in mice with BCP. In parallel, a mitigation effect was observed for pain. Clinical studies have shown that oral fluoxetine is effective in relieving the severity of cancer-associated depression and pain, suggesting that fluoxetine is a promising agent in the treatment of cancer pain and depression [22]. After 7 days of fluoxetine treatment, tumor-bearing mice not only alleviated depressive behavior, but also obtained pain relief, which was consistent with the results of neuropathic pain study[23]. This suggests that antidepressants have great potential in pain relief.

The common mechanism of fluoxetine antidepressant is through anti-inflammatory and promotes neurogenesis[24, 25, 26, 27]. M1-like microglia and A1-like astrocytes with neurotoxic phenotypes are often found in different brain regions of pain and depression model and serve as important sources of inflammatory factors [28, 29]. In this study, we found that reactive glial cells in BLA of BCP model were predominantly reactive microglia, presented with the polarization of M1 type, and the expression of inflammatory factors IL-1β and IL-6 were significantly increased. Unlike peripheral nerve injury-induced pain and depression, astrocytes and A1 astrocytes were indeed few in BLA of bone cancer model [7]. Here, we consider that was due to differences in pain models and mouse strains. Fluoxetine treatment could improve the neuroinflammation milieu of BLA in BCP mice by inhibiting the activation of M1-like microglia and A1-like astrocyte. These results suggested that the mechanism of fluoxetine in pain relief may be associated with inhibition of neurotoxic glial cells at the BLA.

Considering the role of fluoxetine in synaptic plasticity, we examined synapse loss in BCP. Studies have shown that changes of dendritic spines size, shape, and number often influence synaptic plasticity and neural circuitry, and play great significance for the development of pain and depression [30, 31]. We found that the number of dendritic spines and synaptic protein (PSD95, SYN1 and SYN) levels in mice with BCP were significantly reduced in BLA, which was related to their pain and depression levels. The treatment of antidepressant fluoxetine could significantly reduce neuroinflammation, and had a good restorative effect on synaptic proteins and the number of dendritic spines.

Certainly, there are some limitations in our study. We only explored the effects of fluoxetine on neuroinflammation, glial cells and synapses at BLA in current research, and the specific mechanism of how fluoxetine can affect them has not yet been explored. In addition, the upstream and downstream neural circuits of basolateral amygdala also deserve further exploration, for they play an indispensable role in in pain and depression[32, 33, 34]. Blocking depression can relieve pain, so does pain treatment have an effect on depression? The causal relationships between them are very interesting studies.

Taken together, fluoxetine can exert anti-inflammatory and synaptic remodeling effects by regulating glial cells reactivity and synaptic proteins. Our findings showed that fluoxetine can alleviate pain behavior while against depression in BCP. Antidepressant treatment alone also worked in pain relief with the comorbidity of pain and depression. Its mechanism might be associated with inhibiting reactive astrocytes and microglia at the BLA, reducing neuroinflammation, and promoting the development of synapses. Our findings can partially explain the underlying mechanisms of fluoxetine in pain and depression comorbidities, and we hope our researches can introduce a new horizon in the field of research on negative emotions such as pain and depression.

Data Availability

Data will be made available on reasonable request.

Acknowledgements

We appreciate all members of Anesthesiology Laboratory of Nanjing Drum Tower Hospital for their helpful technical support and constructive discussions.

Funding

This work was supported by the China National Natural Science Foundation (82171225, 82071229, 81971044, 81870871).

Ethics declarations

Ethics Approval

All animal protocols were reviewed and approved by the ethics committee of Nanjing Drum Tower Hospital

Consent to Participate

All authors approved to participate.

Consent for Publication

All authors approved publication.

Contributions

Li Jiang, Xu-li Yang and Yue Qian: Investigation, Writing-Original draft preparation. Si-min Huang, Yu-lin Huang and Yan-ting Mao: Methodology, Data curation. Rui Xu, Hui-jie Zhu and Xiao-ping Gu: Visualization, Software. Yu-e Sun, Kun Ni and Zheng-liang Ma: Writing-Reviewing and Editing.

Delaney A, Fleetwood-Walker SM, Colvin LA et al (2008) Translational medicine: cancer pain mechanisms and management. Br J Anaesth 101:87–94
Bates N, Bello JK, Osazuwa-Peters N et al (2022) Depression and Long-Term Prescription Opioid Use and Opioid Use Disorder: Implications for Pain Management in Cancer. Curr Treat Options Oncol 23:348–358
Curry ZA, Dang MC, Sima AP et al (2021) Combination therapy with methadone and duloxetine for cancer-related pain: a retrospective study. Ann Palliat Med 10:2505–2511
Micov AM, Tomic MA, Todorovic MB et al (2020) Vortioxetine reduces pain hypersensitivity and associated depression-like behavior in mice with oxaliplatin-induced neuropathy. Prog Neuropsychopharmacol Biol Psychiatry 103:109975
Salat K, Furgala-Wojas A, Salat R (2021) The Microglial Activation Inhibitor Minocycline, Used Alone and in Combination with Duloxetine, Attenuates Pain Caused by Oxaliplatin in Mice. Molecules 26
Alboni S, Poggini S, Garofalo S et al (2016) Fluoxetine treatment affects the inflammatory response and microglial function according to the quality of the living environment. Brain Behav Immun 58:261–271
Liu L, Dai L, Xu D et al (2022) Astrocyte secretes IL-6 to modulate PSD-95 palmitoylation in basolateral amygdala and depression-like behaviors induced by peripheral nerve injury. Brain Behav Immun 104:139–154
Park SH, Lee YS, Yang HJ et al (2021) Fluoxetine Potentiates Phagocytosis and Autophagy in Microglia. Front Pharmacol 12:770610
Pedraza LK, Sierra RO, Giachero M et al (2019) Chronic fluoxetine prevents fear memory generalization and enhances subsequent extinction by remodeling hippocampal dendritic spines and slowing down systems consolidation. Transl Psychiatry 9:53
Bao H, Li H, Jia Y et al (2021) Ganoderic acid A exerted antidepressant-like action through FXR modulated NLRP3 inflammasome and synaptic activity. Biochem Pharmacol 188:114561
Gandhi PJ, Gawande DY, Shelkar GP et al (2021) Dysfunction of Glutamate Delta-1 Receptor-Cerebellin 1 Trans-Synaptic Signaling in the Central Amygdala in Chronic Pain. Cells 10
Zhang MM, Geng AQ, Chen K et al (2022) Glutamatergic synapses from the insular cortex to the basolateral amygdala encode observational pain. Neuron 110:1993–2008 e1996
Zhuang X, Zhan B, Jia Y et al (2022) IL-33 in the basolateral amygdala integrates neuroinflammation into anxiogenic circuits via modulating BDNF expression. Brain Behav Immun 102:98–109
Yu H, Chen L, Lei H et al (2022) Infralimbic medial prefrontal cortex signalling to calbindin 1 positive neurons in posterior basolateral amygdala suppresses anxiety- and depression-like behaviours. Nat Commun 13:5462
Oh JY, Kim YK, Kim SN et al (2018) Acupuncture modulates stress response by the mTOR signaling pathway in a rat post-traumatic stress disorder model. Sci Rep 8:11864
Jiang L, Hao J, Yang XL et al (2022) Basolateral Amygdala Reactive Microglia May Contribute to Synaptic Impairment and Depressive-Like Behavior in Mice with Bone Cancer Pain. Neurochem Res 47:3454–3463
Fang Y, Ding X, Zhang Y et al (2022) Fluoxetine inhibited the activation of A1 reactive astrocyte in a mouse model of major depressive disorder through astrocytic 5-HT2BR/beta-arrestin2 pathway. J Neuroinflammation 19:23
Reyes CC, Anderson KO, Gonzalez CE et al (2019) Depression and survival outcomes after emergency department cancer pain visits. BMJ Support Palliat Care 9:e36
Kang Y, Meghani SH, Bruner DW et al (2020) Factors Associated with Depression in African American Patients Being Treated for Cancer Pain. Pain Manag Nurs 21:410–415
Hooten WM (2016) Chronic Pain and Mental Health Disorders: Shared Neural Mechanisms, Epidemiology, and Treatment. Mayo Clin Proc 91:955–970
Bonilla-Jaime H, Sanchez-Salcedo JA, Estevez-Cabrera MM et al (2022) Depression and Pain: Use of Antidepressants. Curr Neuropharmacol 20:384–402
Dai J, Liao N, Shi J et al (2017) Study of prevalence and influencing factors of depression in tumor patients and the therapeutic effects of fluoxetine. Eur Rev Med Pharmacol Sci 21:4966–4974
Mizoguchi H, Fukumoto K, Sakamoto G et al (2019) Maternal separation as a risk factor for aggravation of neuropathic pain in later life in mice. Behav Brain Res 359:942–949
Mendonca IP, Paiva IHR, Duarte-Silva EP et al (2022) Metformin and fluoxetine improve depressive-like behavior in a murine model of Parkinsons disease through the modulation of neuroinflammation, neurogenesis and neuroplasticity. Int Immunopharmacol 102:108415
Aguilar-Valles A, Haji N, De Gregorio D et al (2018) Translational control of depression-like behavior via phosphorylation of eukaryotic translation initiation factor 4E. Nat Commun 9:2459
Zahrai A, Vahid-Ansari F, Daigle M et al (2020) Fluoxetine-induced recovery of serotonin and norepinephrine projections in a mouse model of post-stroke depression. Transl Psychiatry 10:334
Garcia-Garcia ML, Tovilla-Zarate CA, Villar-Soto M et al (2022) Fluoxetine modulates the pro-inflammatory process of IL-6, IL-1beta and TNF-alpha levels in individuals with depression: a systematic review and meta-analysis. Psychiatry Res 307:114317
Liu Q, Li R, Yang W et al (2021) Role of neuroglia in neuropathic pain and depression. Pharmacol Res 174:105957
Liu LR, Liu JC, Bao JS et al (2020) Interaction of Microglia and Astrocytes in the Neurovascular Unit. Front Immunol 11:1024
Cao P, Chen C, Liu A et al (2021) Early-life inflammation promotes depressive symptoms in adolescence via microglial engulfment of dendritic spines. Neuron 109:2573–2589
Cui W, Li Y, Wang Z et al (2021) Spinal caspase-6 regulates AMPA receptor trafficking and dendritic spine plasticity through netrin-1 in postoperative pain after orthopedic surgery for tibial fracture in mice. Pain 162:124–134
Ma H, Li C, Wang J et al (2021) Amygdala-hippocampal innervation modulates stress-induced depressive-like behaviors through AMPA receptors. Proc Natl Acad Sci U S A 118:e2019409118
Jeong Y, Cho HY, Kim M et al (2021) Synaptic plasticity-dependent competition rule influences memory formation. Nat Commun 12:3915
Smith ML, Asada N, Malenka RC (2021) Anterior cingulate inputs to nucleus accumbens control the social transfer of pain and analgesia. Science 371:153–159

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Version 1

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You are reading this latest preprint version

Fluoxetine attenuates pain-like and depression-related behaviors via reducing neuroinflammation and synaptic deficits in bone cancer mice

Status:

Version 1

Abstract

Background

Methods

Results

Conclusions

Figures

Introduction

Materials And Methods

Experimental Animals

Establishment of Mouse Model

Pharmacological Treatment

Paw Withdrawal Mechanical Threshold (PWMT)

Sucrose Preference Test (SPT)

Forced Swimming Test (FST)

Immunofluorescence

Western Blotting

Golgi-Cox Staining

Statistical Analysis

Results

Fluoxetine Treatment Schedule and Behavior Analysis

Fluoxetine Alleviated BCP-induced Reactive Gliosis in BLA

Fluoxetine Alleviated the Effects of Neuroinflammation in BLA

Fluoxetine Enhanced the Spine Density and Synaptic Protein Expression

Conclusion

Declarations

References

Status:

Version 1