Introduction

Refinements in clinical diagnosis and advances in cancer treatment have contributed to tremendous improvement in cancer survival rates and outcomes in cancer patients [1]. Nonetheless, longitudinal studies have documented the emergence of cognitive deficits in approximately 19–78% of cancer survivors who underwent chemotherapy as part of their treatment regimen [2,3,4,5]. The debilitating neurological sequelae in several cognitive domains following chemotherapy have currently become well-recognized and colloquially referred to as chemobrain which sometimes can be long-lasting and have irreversible symptoms [6,7,8]. With an estimation of 70 million cancer survivors around the world by the year 2020, understanding the pathological basis and findings of novel therapeutic approaches to treat chemobrain is urgent [9, 10].

Although doxorubicin (DOX) is a remarkable chemotherapeutic drug extensively used to treat various types of cancer, it has been well-documented that it causes chemobrain across patients with numerous types of cancer [11, 12]. Neuroinflammation and mitochondrial dysfunction have been proposed as crucial contributors to DOX-induced chemobrain [13,14,15,16,17,18]. The cross-link between these cellular candidates is an intricate pathophysiological threat, resulting in neuronal apoptosis, which is commonly acknowledged as the ultimate characteristic of DOX-induced chemobrain [13, 14]. Aberrant activation of alternative lytic pro-inflammatory programmed cell death (PCD) including necroptosis and pyroptosis has been recently observed in response to inflammatory processes, resulting in the impact of neuronal death and a vicious cycle of chronic neuroinflammation in certain specific brain regions [19]. These two types of PCD, distinct from apoptosis, have been shown to implicate the progression of neurodegenerative and neuroinflammatory disorders, contributing to the release of immunogenic cellular content and ultimately undesirable loss of neuronal cells and function [19, 20]. However, the contribution of PCD in response to chemotherapeutic agents is still inconclusive based on a very limited number of studies. Although various types of PCD including apoptosis, necroptosis, and pyroptosis, have been documented in experimental models of chemobrain [21,22,23], the molecular phenotypes of PCD have never been examined simultaneously in the same study model. Therefore, addressing the characteristics of the types of PCD underlying the pathophysiology of chemobrain possibly facilitates the development of better therapeutic interventions against chemobrain in cancer patients.

In addition to PCD, the potential Alzheimer’s lesions and accelerated age‑related tauopathy have been suggested as hallmarks of chemobrain pathology [21, 24, 25]. An elevation of Alzheimer’s disease (AD) biomarkers, including Tau protein in the peripheral circulation has been observed in patients who received cancer treatments [26]. The physiologic functions of Tau in the brain are to facilitate the formation and stabilization of microtubules by adhering to microtubules, thereby enabling the axonal transport of cellular vesicles and organelles [27]. Notably, the pathological post-translational modification of Tau, especially of hyperphosphorylation, is mainly responsible for its loss of normal physiological function and gain of neurotoxicity, contributing to cognitive deterioration in patients with AD [27]. In this aspect, glycogen synthase kinase-3 (GSK3) is traditionally considered a constitutively active kinase implicated in the hyperphosphorylation of Tau. There are two isoforms of GSK3, GSK3-α, and GSK3β [28]. GSK3β is the most ubiquitous, and it is found to be hyperactive in the brains of AD patients, providing empirical support that it contributes to AD pathogenesis through distinct mechanisms [28, 29]. Even though we have previously demonstrated that long-term administration of DOX led to the hyperphosphorylation of Tau at Thr181 [21], the molecular involvement of GSK3β and hyperphosphorylation of Tau following chemotherapy has never been investigated.

The coordination of different cognitive functions, especially in hippocampus-dependent learning and memory, requires appropriate cholinergic signaling via the activation of acetylcholine receptors (AChRs) [30, 31]. Cholinergic hypofunction has been identified as one of the key consequences of AD; therefore, correspondingly, the cholinergic hypothesis of AD proposes that therapy targeting cholinergic enhancement could be useful in ameliorating cognitive decline [30]. Importantly, cholinergic modulation using AChR agonists has been shown to effectively alleviate the impairment of cognitive and behavioral performance induced by lesions of cholinergic circuitry or antagonism of cholinergic receptors [32,33,34]. Additionally, there is accumulating evidence to indicate the anti-inflammatory roles of cholinergic stimulation in the peripheral and central nervous system (CNS) through both muscarinic receptors (mAChR) and nicotinic receptors (nAChR), especially in the case of α7 subtype (α7nAChR) [35,36,37]. However, several gaps of knowledge still exist since mechanistic insights regarding the effects of AChR agonists on inflammatory types of PCD and hyperphosphorylation of Tau following DOX administration have never been demonstrated. Therefore, the present study aimed to test the hypothesis that enhancing systemic cholinergic signaling with mAChR and α7nAChR agonists provides neuroprotection against DOX-induced chemobrain via the reduction of mitochondrial dysfunction, neuroinflammation, hyperphosphorylation of Tau, and pro-inflammatory PCD, thereby improving hippocampal-dependent learning and memory.

Materials and Methods

Animal Model

Adult male Wistar rats (n = 40, 350–400 g, 8-week-old) were sourced from the Nomura Siam International Co, Ltd., Thailand, and were maintained at the laboratory animal center of Chiangmai University in controlled conditions (21 ± 1 °C, 50 ± 10% humidity, 12-h light:dark cycle). All experimental procedures were conducted in accordance with a protocol authorized by the Institutional Animal Care and Use Committee (IACUC) of Chiang Mai University (permit no. 2563/RT-0012) and NIH recommendations (Guide for the Care and Use of Laboratory Animals).

Chemotherapy and Treatment Paradigms

After 1 week of acclimatization, all rats were divided into a control group (n = 8) and a DOX-treated group (n = 32) to receive six doses of either 0.9% NSS or 3 mg/kg of DOX (Fresenius Kabi Oncology Ltd., India) via intraperitoneal injection (i.p.). The sample size was calculated by using the G*Power program (version 3.1.9.4) according to our preliminary study. The initial three doses were administered every 4 days, followed by the final three doses administered once per week. The dosage and administration protocol of DOX in this study are determined based on our preliminary data, which indicates that this specific dose can induce cognitive impairments in rats. Furthermore, this dosage is comparable to those employed in our prior investigations [21, 38]. Rats in the DOX-treated group were subdivided into four groups (n = 8/groups) to be intraperitoneally treated with either 0.9% normal saline as the vehicle, PNU-282987 (α7nAChR agonist; 3 mg/kg/day, Alomone, Israel) [39], bethanechol (mAChR agonist; 12 mg/kg/day, Sigma-Aldrich, USA) [40], or a combined administration of PNU-282987 and bethanechol for 30 consecutive days, starting with the first dose of DOX injection. The dosages of PNU-282987 and bethanechol used were based on previous studies [39, 40]. Twenty-four hours following the administration of the last dose of all treatments, animals were cognitively assessed.

Behavioral Tests

After the completion of the treatment paradigm, all rats were subjected to a series of tests to assess cognitive function consisting of a novel object location task (NOLT) and a novel object recognition task (NORT) as illustrated in Fig. 1A. To confirm the results and impose a greater cognitive load on long-term learning and memory, the test scenario was modified so that the NOLT and NORT were conducted with a 24-h interval between each test [41, 42]. Briefly, rats were allowed to explore an arena (a 70-cm-diameter and 50-cm-height made of an opaque plastic tank) for 10 min during the habituation phase. The following day, rats were given 10 min to explore the testing arena containing two identical objects during the familiarization phase prior to being transferred back to their home cages for 24 h. In the NOLT phase, each rat was positioned in the center of an arena containing the same two objects, one at the same location as in the familiarization phase and the other at the opposite end. The rats were given 10 min to explore the objects. The rats were returned to the arena 24 h after the NOLT, where one of the familiar objects at the novel location was replaced with a novel object. The percentage of preference indices for NOLT and NORT was calculated by dividing the amount of time the rat spent exploring the object at the novel location in NOLT or the novel object in NORT by the total amount of time spent exploring both objects during a 10-min testing period.

Fig. 1
figure 1

The effects of an α7nAChR agonist and mAChR agonist on cognitive function in DOX-induced chemobrain. A The behavioral test procedure. B The percentage preference index of two objects during the familiarization phase in the modified novel object location and recognition tests. C The percentage preference index of the object in a novel location during the novel object location test. D The percentage preference index of a novel object during the novel object recognition test; n = 6–8/group; *p < 0.05 vs. control; †p < 0.05 vs. DOX + vehicle (one-way ANOVA followed by an LSD post hoc test). NOLT, novel object location test; NORT, novel object recognition test

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Western Blot Analysis

The hippocampal tissues were homogenized with an ice-cold non-ionizing lysis buffer containing 100 mM NaCl (RCI Labscan Limited., Thailand), 25 mM EDTA (Ajax Finechem™, Australia), 10 mM Tris (HiMedia Laboratories Pvt. Ltd., India), 1% v/v Triton X-100 (Sigma-Aldrich, USA), 1% v/v NP-40 (Thermo Fisher Scientific, USA), and 1 × protease inhibitor (Merck KGaA, Germany). Equal amounts of protein at 30 μg were separated by 10% hand-casting SDS–polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. Then, membranes were blocked for 1 h with 5% bovine serum albumin (BSA) or 5% non-fat dry milk Tris-buffered saline with 0.1% Tween buffer at room temperature for 1 h. They were then probed overnight at 4 °C with primary antibodies as shown in supplementary Table 1. All immunoblots were then probed with secondary antibodies conjugated with horseradish peroxidase as shown in supplementary Table 1 at room temperature for 1 h. Protein band immunoreactivities were detected using the ChemiDoc™ Touch Gel Imaging System (Bio-Rad Laboratories, USA).

Morphological Analysis of Microglia and Astrocytes

Prior to cryoprotection by immersion in 30% sucrose, the brains were postfixed in 4% paraformaldehyde (PFA) in PBS overnight. The brain tissues were sectioned with a cryostat (Leica CM1950, Leica, Germany) into 20-μm coronal sections. After 1 h in permeabilizing solution (3% H2O2 and 1% Triton X-100 in distilled water), sections were blocked in 5% BSA in PBS for 1 h. To label microglia and astrocytes, primary antibodies against goat anti-Iba-1 (1:1000 dilution; ab5076, Abcam, USA) and rabbit anti-GFAP (1:1000 dilution; ab16997, Abcam, USA) were applied to the brain slices, respectively. The sections were then rinsed three times and probed with Alexa Flour 488-labeled donkey anti-goat IgG (1:1000; ab150129, Abcam, USA) and Alexa Flour 647-labeled goat anti-rabbit IgG (1:500; ab150079, Abcam, USA) for 1 h, followed by nuclear staining with DAPI (TOCRIS, UK) for 15 min. The hippocampal CA1 region was imaged using 60 × objectives with a confocal microscope (Olympus FLUOVIEW FV3000, Japan). Morphological analyses of microglia and astrocytes were carried out utilizing Imaris software (Oxford Instruments) [43,44,45,46].

The Response of Brain Mitochondria to Oxidative Stress

After decapitation, the brains were rapidly removed and homogenized with 0.05% bacterial proteinase (Sigma-Aldrich, USA) in ice-cold MSE solution consisting of 25 mM mannitol (Elago Enterprises Pty Ltd., Australia), 75 mM sucrose (RCI Labscan Limited., Thailand), 1 mM EGTA (Sigma-Aldrich, USA), 5 mM HEPES (Sigma-Aldrich, USA), and 1 mg/ml BSA (HiMedia Laboratories Pvt. Ltd., India) at 600 rpm. Differential centrifugation was performed to isolate the mitochondrial fraction [18, 47]. The isolated mitochondrial fraction was stained with dichlorohydrofluorescein diacetate (DCFH-DA) dye (Sigma-Aldrich, USA) for 20 min at room temperature to measure the level of mitochondrial reactive oxygen species (ROS). To determine the change in mitochondrial membrane potential (ΔΨm), the isolated mitochondrial suspension was incubated with JC-1 dye (Sigma-Aldrich, USA) for 15 min at 37 °C. A reduction in the proportion of red to green fluorescence intensity indicates depolarization of ΔΨm. 2 mmol/L H2O2 was co-incubated with either DCFH-DA or JC-1 dye to ascertain the potential of mitochondria to neutralize ROS. The percentage change in emission intensity between mitochondria stimulated with and without H2O2 was evaluated. By measuring the absorbance of mitochondrial fraction at a wavelength of 540 nm, the swelling of mitochondria was determined [48]. A decrease in the absorbance signifies mitochondrial swelling. All mitochondrial studies were performed in triplicate.

Dendritic Spine Staining

The fresh brain tissues were glued onto a metal block holder and coronally sectioned to a thickness of 400 μm by using a vibrating blade microtome (Vibratome Series 1000 Sectioning system, UK). Brain sections were then fixed in 4% PFA for 1 h and stained for 7 days with DiI dye (Invitrogen, USA). The secondary or tertiary dendritic segments of three pyramidal neurons were captured from the CA1 region per section using a confocal microscope (Olympus FLUOVIEW FV3000, Japan). The density and volume of dendritic spines were evaluated as previously described [49].

Quantification and Statistical Analysis

Data administration and representation were carried out using GraphPad Prism software (version 7, GraphPad Software, Inc., USA). All error bars shown indicate the standard error of the mean (SEM). A one-way ANOVA followed by an LSD post hoc test was performed to compare multiple groups. A p-value < 0.05 was considered a measure of statistical significance.

Results

AChR Agonists Attenuated Cognitive Dysfunction in Rats with DOX-Induced Chemobrain

To determine the therapeutic effects of parasympathetic activation on DOX-induced cognitive impairment, the NOLT and NORT were performed as shown in Fig. 1A. In this study, parasympathomimetic drugs including PNU-282987 and bethanechol were administered to activate the α7nAChR and mAChR, respectively. There was no significant difference in the amount of time spent interacting with two objects among all groups in the familiarization phase, indicating no preference between the two objects (Fig. 1B). During the NOLT, DOX-treated rats exhibited cognitive impairment as evidenced by a substantial reduction in the preference index during the NOLT, compared to that of the controls (Fig. 1C). Interestingly, PNU-282987, bethanechol, and the combined treatment equally improved the preference index in the NOLT (Fig. 1C). Consistent with the NOLT, DOX administration shortened the time interaction with the new object in the NORT (Fig. 1D). Noticeably, PNU-282987, bethanechol, or the two as a combined treatment substantially prolonged the time rats spent interacting with the novel object. These findings showed that PNU-282987, bethanechol, and the combined drugs equally prevented cognitive deficits in rats treated with DOX.

The α7nAChR Agonist and mAChR Agonist Maintained the Density and Volume of Dendritic Spines, Enhanced the Levels of Tight Junction Proteins, and Mitigated Hyperphosphorylation of Tau in Rats with DOX-Induced Chemobrain

The dendritic spine density/volume and the expression level of PSD-95 were then examined to determine dendritic spine density and volume as represented in Fig. 2A–E. Fluorescence analysis demonstrated that the number and volume of dendritic spines were remarkably reduced in DOX-treated rats compared with the controls (Fig. 2A–C). Consistent with these findings, the expression level of PSD-95 was also markedly reduced in the DOX-treated group, suggesting synaptic dysplasticity following DOX treatment (Fig. 2D, E). In particular, long-term treatment with PNU-282987, bethanechol, and the combined drugs effectively preserved dendritic spine density, spine volume, and PSD-95 expression in DOX-treated rats (Fig. 2A–E). Furthermore, the expression levels of tight junction proteins including claudin-5 and occludin in the hippocampus were substantially decreased in DOX-treated rats relative to the control group (Fig. 2D, F, G), implying weakening BBB integrity. Critically, the activation of α7nAChR and mAChR led to a significant upregulation of hippocampal tight junction proteins to the levels observed in the control group (Fig. 2D, F). The combined therapy of both AChR agonists contributed a similar outcome as either as a monotherapy (Fig. 2D, F, G). In addition, DOX administration resulted in hyperphosphorylation of Tau at Thr181, a well-established pathological feature of AD (Fig. 2D, I). Given that GSK3β is a key kinase enzyme responsible for the phosphorylation of Tau, which was inactivated by phosphorylation at Ser9, we subsequently investigated the phosphorylation level of GSK3β at Ser9 in the hippocampal tissues. The results demonstrated that there were no detectable alterations in the phosphorylation of GSK3β at Ser9 between the control and DOX groups (Fig. 2D, H). However, PNU-282987, bethanechol, or the two as a combined treatment ameliorated the hyperphosphorylation of Tau and augmented the phosphorylated GSK3β at Ser9 in DOX-treated rats, suggesting that activation of the AChR axis mediates the suppressed phosphorylation of Tau via the inactivation of GSK3β (Fig. 2D, H, I).

Fig. 2
figure 2

The effects of an α7nAChR agonist and mAChR agonist on dendritic spines, tight-junction protein expressions, and tau phosphorylation in rats with DOX-induced chemobrain. A Representative images of dendritic spine density using DiI staining. B The number of dendritic spines per 10-μm apical tertiary dendrite. C Dendritic spine volume. D Representative western blot bands. E PSD-95 protein expression. F Claudin-5 protein expression. G Occludin protein expression. H The expression ratio of p-GSK3βSer9/total GSK3β. I The expression ratio of p-Tau.Thr181/total Tau; n = 12 slices from six rats/group for DiI staining and n = 4–6/group for western blotting; *p < 0.05 vs. control; †p < 0.05 vs. DOX + vehicle (one-way ANOVA followed by an LSD post hoc test). GSK3β, glycogen synthase kinase-3β; PSD-95, postsynaptic density protein 95

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Activation of AChRs Mitigated the Hippocampal Inflammation Induced by DOX in Rats

In contrast to the control group, DOX-treated rats exhibited neuroinflammation in the hippocampus as evidenced by increases in the phosphorylation of NF-κB at Ser536 and the expression of TNF-α (Fig. 3A–C). Consistently, DOX administration reduced the expression level of IL-10 and the phosphorylation of STAT3 at Tyr705, compared with the controls (Fig. 3A, C, D). Treatment with AChR agonists, either PNU-282987 or bethanechol, effectively counteracted these upregulated pro-inflammatory markers and increased the expression of IL-10 relative to those observed in rats treated with DOX alone (Fig. 3A–E). Notably, a substantial increase in the phosphorylation of STAT3 at Tyr705 was observed in rats treated with PNU-282987 and the combined treatment in comparison to the DOX-treated rats receiving the vehicle (Fig. 3A–E), suggesting that activation of α7nAChR, but not mAChR, effectively induced STAT3 anti-inflammatory signaling. Taken together, this suggested that activation of α7nAChR and mAChR attenuated neuroinflammation in the hippocampus of rats with DOX-induced chemobrain.

Fig. 3
figure 3

The effects of α7nAChR agonist and mAChR agonist on hippocampal inflammation in rats with DOX-induced chemobrain. A Representative western blot bands. B The expression ratio of p-NF-κBSer536/total NF-κB. C TNF-α protein expression. D IL-10 protein expression. E The expression ratio of p-STAT3.Tyr705/total STAT3. n = 4–6/group; *p < 0.05 vs. control; †p < 0.05 vs. DOX + vehicle (one-way ANOVA followed by an LSD post hoc test)

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The α7nAChR Agonist and mAChR Agonist Preserved the Morphologies of Microglia and Astrocytes in the Hippocampus of Rats with DOX-Induced Chemobrain

To investigate the modulatory effects of cholinergic activation on microglial reactivity, microglial morphology at the hippocampal CA1 region was further determined as shown in Fig. 4A. Fluorescence analysis revealed that DOX-treated rats exhibited microglial activation as indicated by increases in the number of Iba-1+ cells, Iba-1 immunofluorescence intensity, and soma volume of Iba-1+ cells (Fig. 4A–D). Microglial filament analysis was then performed, and the outcomes are illustrated in Fig. 4E. Administration of DOX reduced the length and branching of microglial processes (Fig. 4F–H), implying that DOX treatment caused a change in microglial morphology to an amoeboid-shaped neurotoxic phenotype. Remarkably, long-term treatments with α7nAChR, mAChR, and combined agonists in DOX-treated rats maintained all aspects of microglial morphology at levels comparable to those observed in the control group (Fig. 4A–H).

Fig. 4
figure 4

The effects of α7nAChR agonist and mAChR agonist on microglial morphology in rats with DOX-induced chemobrain. A Representative images of Iba-1 immunofluorescence under confocal microscopy at CA1 of the hippocampus. B The number of Iba-1-positive cells. C Mean intensity of Iba-1-positive cells. D Soma volume of Iba-1-positive cells. E Representative branch level of microglial processes. F Process length of Iba-1-positive cells. G Microglial process complexity as the number of Iba-1-positive cell process intersections using Sholl analysis. H The representative area under the curve of Sholl analysis. Each dot represents the average value of two slices from one animal, n = 6 rats per group; *p < 0.05 vs. control; †p < 0.05 vs. DOX + vehicle. FOV, field of vision

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Fig. 5 A shows the reactivation of astrocytes in rat CA1 after DOX administration. Astrocytic activation in response to DOX was demonstrated by a significant increase in mean fluorescence intensity, number, and cell volume of GFAP+ cells (Fig. 5B–D). In addition, the length and complexity of processes analyzed by Sholl analysis of GFAP+ cells dramatically decreased in comparison to the controls (Fig. 5E–H). Importantly, treatment with either PNU-282987 or bethanechol significantly reduced GFAP immunofluorescence intensity, the number of GFAP+ cells, and soma volume of GFAP+ cells in rats treated with DOX (Fig. 5A–D). The filament analysis also revealed that the administration of PNU-282987 or bethanechol significantly increased the length and branching of astrocytic processes in DOX-treated rats (Fig. 5E–H). However, no synergistic effect of these drugs was observed in the combined groups (Fig. 5A–H). Conclusively, activation of α7nAChR or mAChR using as the parasympathomimetic drugs was able to attenuate microglial and astrocytic activation in response to DOX administration.

Fig. 5
figure 5

The effects of an α7nAChR agonist and mAChR agonist on astrocytic morphology in rats with DOX-induced chemobrain. A Representative images of GFAP immunofluorescence under confocal microscopy at CA1 of the hippocampus. B The number of GFAP-positive cells. C Mean intensity of GFAP-positive cells. D Soma volume of GFAP-positive cells. E Representative branch level of astrocytic processes. F Process length of GFAP-positive cells. G Astrocytic process complexity as the number of GFAP-positive cell process intersections using Sholl analysis. H The representative area under the curve of Sholl analysis. Each dot represents the average value of two slices from one animal, n = 6 rats per group; *p < 0.05 vs. control; †p < 0.05 vs. DOX + vehicle. FOV, field of vision

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The Activation of α7nAChR and mAChR Improved Brain Mitochondrial Function and Suppressed Mitochondrial Fission in Rats with DOX-Induced Chemobrain

In this study, brain mitochondrial ROS-neutralizing ability was determined by co-incubation with H2O2. The results demonstrated that DOX administration impaired mitochondrial ROS-neutralizing ability as indicated by a significant increase in the percentage change in mitochondrial ROS level after H2O2 stimulation relative to that observed in the control group (Fig. 6A). In comparison to the control rats, DOX-treated rats also exhibited a significant increase in H2O2-stimulated membrane potential alterations, indicating mitochondrial membrane depolarization (Fig. 6B). Furthermore, DOX administration significantly decreased the absorbance of the isolated mitochondrial fraction in comparison to the control group, implying mitochondrial swelling in response to DOX treatment (Fig. 6B). PNU-282987, bethanechol, and the combined drugs had similar efficacy in preserving mitochondrial ROS-detoxifying capacity in DOX-treated rats (Fig. 6A–C).

Fig. 6
figure 6

The effects of an α7nAChR agonist and mAChR agonist on mitochondrial function and dynamics in rats with DOX-induced chemobrain. A The percentage changes in ROS production after H2O2 stimulation. B Brain mitochondrial membrane potential changes after H2O2 stimulation. C Mitochondrial swelling as indicated by absorbance at a wavelength of 540 nm. D Representative western blot bands. E The expression ratio of p-Drp-1Ser616/Drp-1. F MFN-1 protein expression. G MFN-2 protein expression. H OPA-1 protein expression; n = 5–8/group for mitochondrial function and n = 4–6/group for western blotting; *p < 0.05 vs. control; †p < 0.05 vs. Dox + vehicle (one-way ANOVA followed by an LSD post hoc test). ΔѰ, mitochondrial membrane potential; Dox, doxorubicin; Drp-1, dynamin-related protein-1; p-Drp-1Ser616, phosphorylated dynamin-related protein-1 at serine 616; MFN-1, mitofusin 1; MFN-2, mitofusin 2; OPA-1, optic atrophy 1; ROS, reactive oxygen species

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The effects of the drugs on mitochondrial dynamics were subsequently investigated. Western blotting analysis revealed that administration of DOX significantly upregulated the expression of both mitochondrial fission- and fusion-related proteins as indicated by significant upregulation of the fission marker (phosphorylated Drp-1 at Ser616 relative to total Drp-1) and fusion factors including MFN-1, MFN-2, and OPA-1 (Fig. 6D–H). Noticeably, treatment with PNU-282987, bethanechol, and the combined drugs equally attenuated excessive mitochondrial fission (Fig. 6D, E) in comparison to rats receiving DOX alone, while maintaining mitochondrial fusion as a compensatory mechanism (Fig. 6D, F–H). These findings demonstrated that PNU-282987, bethanechol, and the combined drugs improved brain mitochondrial ROS-detoxifying function and a balance of mitochondrial dynamics in rats with DOX-induced chemobrain.

The α7nAChR Agonist and mAChR Agonist Mitigated Hippocampal Apoptosis in Rats with DOX-Induced Chemobrain

Western blotting analysis revealed that DOX administration diminished the cell survival pathways as evidenced by significant decreases in the expression of phosphoinositide 3-kinase (PI3K) and phosphorylation of protein kinase B (AKT) and ERK at Ser473 and Thr202/Tyr204, respectively (Fig. 7A–D). These contributed to a substantial reduction of Bcl-2 expression and an increased ratio of cleaved caspase-3 to caspase-3, observed in the hippocampal tissues of DOX-treated rats (Fig. 7A, E, F). Strikingly, pharmacological intervention with PNU-282987, bethanechol, or the combined drugs enhanced the expression of PI3K and the phosphorylation of AKT and ERK equally, resulting in a decrease in the expression of apoptotic markers (Fig. 7A–F). Taken together, this suggested that AChR agonists provided neuroprotection against DOX-mediated neuronal apoptosis in part via PI3K/AKT and ERK signaling pathways.

Fig. 7
figure 7

The effects of an α7nAChR agonist and mAChR agonist on hippocampal apoptosis in rats with DOX-induced chemobrain. A Representative western blot bands. B PI3K protein expression. C The expression ratio of p-AKTSe473/total AKT. D The expression ratio of p-ERK1/2Thr202/Tyr204/total ERK1/2. G Bcl-2 protein expression. H The expression ratio of cleaved caspase-3/caspase-3; n = 4–6/group; *p < 0.05 vs. control; † p < 0.05 vs. DOX + vehicle (one-way ANOVA followed by an LSD post hoc test). AKT, protein kinase B; PI3K, phosphoinositide 3-kinases

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Long-Term Administration of DOX-Induced Hippocampal Pyroptosis and Necroptosis Which Were Diminished by Treatment with AChR Agonists

To support the hypothesis that DOX-induced chemobrain could lead to neuronal death via alternative forms of PCD, we further evaluated the pertinent proteins of each type of pro-inflammatory PCD including pyroptosis and necroptosis. Our results demonstrated that the expression of pyroptosis-related executioner protein, NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasomes, was markedly increased in DOX-treated rats (Fig. 8A, B). Consistently, higher levels of cleaved gasdermin D (GSDMD)/total GSDMD, and IL-1β were evident in the hippocampal tissues of DOX-treated rats, further confirming the induction of pyroptosis following DOX administration. In addition to pyroptosis, the ratios of p-RIPK1ser166/RIPK1, p-RIPK3ser232/RIPK3, and p-MLKLser358/MLKL were also markedly increased, indicating necroptosis in the hippocampus of DOX-treated rats (Fig. 8A, E–G). In summary, we showed unique molecular findings supporting that DOX-mediated forms of PCD in the hippocampus included apoptosis, necroptosis, and pyroptosis. Although treatment with AChR agonists did not alter the level of NLRP3 expression, they effectively reduced the expression IL-1β and the cleavage of GSDMD which are initiators of the pyroptosis cascade (Fig. 8A–D). Treatment with PNU-282987 and the combined drugs suppressed the activation of the RIPK1-RIPK3-MLKL axis to the levels observed in the healthy controls (Fig. 8A–G). Concomitant treatment with bethanechol decreased the expression of p-RIPK1ser166/RIPK1 and p-RIPK3ser232/RIPK3 but did not reverse an increased ratio of p-MLKLser358/MLKL in DOX-treated rats (Fig. 8A, E–G). These results suggested that intervention with α7nAChR or mAChR agonists effectively suppressed both pyroptosis and necroptosis in the hippocampus following DOX administration.

Fig. 8
figure 8

The effects of an α7nAChR agonist and mAChR agonist on hippocampal pyroptosis and necroptosis in rats with DOX-induced chemobrain. A Representative western blot bands. B NLRP3 protein expression. C Cleaved GSDMD/total GSDMD. D IL-1β protein expression. E The expression ratio of p-RIPK1Ser166/total RIPK1. F The expression ratio of p-RIPK3Ser232/total RIPK3. G The expression ratio of p-MLKLSer358/total MLKL; n = 4–6/group; *p < 0.05 vs. control; †p < 0.05 vs. DOX + vehicle (one-way ANOVA followed by an LSD post hoc test). GSDMD, gasdermin D; MLKL, mixed lineage kinase domain-like protein; NLRP3, NACHT, LRR, and PYD domain-containing protein 3; p-MLKLSer358, phosphorylation of MLKL at serine 358; p-RIPK1Ser166, phosphorylation of RIPK1 at serine 166; p-RIPK3.Ser232, phosphorylation of RIPK3 at serine 232; RIPK1, receptor-interacting protein kinase 1; RIPK3, receptor-interacting protein kinase 3

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Discussion

The incidence of chemobrain has been attracting more attention in the medical field since it limits dosage administration or even leads to the inevitable termination of the treatments before completion of the regimen [50,51,52]. Comprehensive studies of the biological bases underlying chemobrain would contribute to the discovery of potential therapies to enhance the quality of life of cancer patients and survivors. This study mainly investigated the therapeutic efficacy of cholinergic activation using AChR agonists on the neuropathology of DOX-induced chemobrain. Our results underlined and extended the notion that DOX administration simultaneously induced multiple types of PCD including apoptosis, pyroptosis, and necroptosis in the hippocampus via upregulated inflammation, imbalance in mitochondrial homeostasis, loss of dendritic spine and BBB integrity, and hyperphosphorylation of Tau. Our study also provides novel insights into the neuroprotective impact of cholinergic activation as a promising therapeutic approach for treatment or even prevention of chemobrain. Treatment with AChR agonists in rats exposed to DOX effectively protected against the widespread aspects of neuropathology and mitigated several forms of PCD induced by DOX, contributing to an improvement of learning and memory.

Cholinergic receptors are broadly categorized into two main classes: the ionotropic nAChRs and the metabotropic mAChR. Both nAChRs and mAChR are expressed by neuronal and non-neuronal cells throughout the CNS and the peripheral nervous system [53, 54]. In the hippocampus, the most abundant nAChR subtype is α7nAChR and it has been shown that the M1, M2, and M4 mAChRs are significantly expressed pre- or post-synaptically [55]. Neurophysiological evidence has established that the cholinergic nervous system is one of the key components participating in the maintenance of intact brain functions. It is thus unsurprising that abnormal alterations in the function of AChRs, particularly in the hippocampus, have been tightly orchestrated in the pathophysiology of AD [30]. In this line, a significant portion of hippocampal-dependent cognitive function is also acknowledged as one of the crucial cognitive domains affected by chemotherapy among cancer patients [56,57,58]. Previous in vivo studies demonstrated that DOX enhanced the activity of acetylcholinesterase (AChE) and impeded the synthesis of acetylcholine (ACh) by lowering choline levels and suppressing choline acetyltransferase activity, thereby impairing cognitive function in rodents [59, 60]. Collectively, these studies accentuated the cholinergic disturbances as significant contributory components in the pathophysiologic cascade of DOX-induced chemobrain. Therefore, in this study, the impact of chemotherapeutic DOX and AChR agonists as interventions on cognitive performance was investigated using a modified cognitive testing procedure consisting of NOLT and NORT. The NOLT was followed by the NORT within a 24-h resting period in order to impose a cognitive load on both long-term spatial and working memories, which are regulated by hippocampal activity [61]. DOX-treated rats exhibited an inability to recognize a new location or object in NOLT and NORT, respectively, reflecting a deficit in hippocampal-dependent learning and memory. Interestingly, we demonstrated that concomitant treatment with either an α7nAChR or mAChR agonist diminished DOX-induced hippocampal-dependent cognitive impairment as represented by increased preference indexes in NOLT and NORT. Consistent with these findings, we previously demonstrated that intervention with AChE inhibitor donepezil showed prominent cognitive benefits in preservation of brain function in rats with DOX-induced chemobrain which was similar to the cognitive outcomes observed in DOX-treated rats receiving AChR agonists [21]. Hence, our results further suggest that the elevated level of ACh in response to donepezil treatment possibly exerted neuroprotection against DOX-mediated neurotoxicity via the activation of either α7nAChR or mAChR. Together, these data comprehensively spotlighted the neuroprotective roles of AChR activation in preservation of hippocampal-dependent cognitive function in a rat model of DOX-induced chemobrain.

Our investigation discovered reductions in dendritic spine density and volume in the hippocampal CA1 region as well as a decline in post-synaptic protein marker PSD-95 following DOX administration, implying weakening synaptic integrity. Unfortunately, the mechanism underlying the reduction of synaptic structure after chemotherapy is still largely unknown. Of significance, dendritic spines contain a substantial number of mitochondria, causing this neuronal compartment to be the most vulnerable to oxidative stress [62]. Therefore, DOX-mediated oxidative damage to these synaptosomal mitochondria possibly leads to a loss of synaptic integrity, which has been considered an early pathological manifestation of neurodegeneration [62]. In addition to oxidative stress, a previous study showed that inflammatory mediators secreted from activated microglia diminished the modulatory signals of brain-derived neurotrophic factor (BDNF) and elicited excitotoxicity, resulting in dendritic spine damage [63]. Remarkably, pharmacological intervention with all AChR agonists sustained all aforementioned synaptic parameters to equivalent levels observed in the control groups. We therefore speculated that cholinergic activation of either α7nAChR or mAChR preserved synaptic integrity in rats with DOX-induced chemobrain via amelioration of neuroinflammation and brain mitochondrial ROS-neutralizing function.

There is a common consensus that neuroinflammation is the main cause of the pathogenesis of DOX-induced chemobrain [13]. A previous study demonstrated that microglial depletion with the colony-stimulating factor 1 receptor (CSF1R) inhibitor entirely abrogated neuroinflammation and cognitive decline in mice receiving DOX, further confirming the significant involvement of microglial activation as the central player in the perpetuation of DOX-induced chemobrain [17]. Similarly, we found an upregulation of the expression of pro-inflammatory cytokines including TNF-α and IL-1β as well as the phosphorylation of NF-κB in the hippocampus of rats receiving DOX. An anti-inflammatory cytokine IL-10 and the phosphorylation of STAT3 were also downregulated following DOX administration. Furthermore, the 3D morphological-based quantitative analysis demonstrated that microglia and astrocytes in the CA1 region were prone to polarize into pro-inflammatory, neurotoxic phenotypes as depicted by an amoeboid-like shaped morphology. Microglia also presented with decreased process length and complexity in response to DOX treatment, displaying transcriptomic alterations that indicate a neurodegenerative microglia phenotype largely approximating that of the stage 1 disease-associated microglia (DAM) [16]. Collectively, these data indicated the rigor of DOX-mediated neuroinflammation. Bidirectional communication between the cholinergic system and the immune system in the CNS has been identified as indicated by common receptors and ligands are expressed in cells of both systems [64, 65]. Using cholinergic activation as a therapeutic strategy in the current study, long-term treatment with PNU-282987, bethanechol, or the two drugs combined reversed pro-inflammatory profiles and altered microglial and astrocytic morphology in the CA1 region of DOX-treated rats, indicating the anti-inflammatory effect of AChR activation against DOX toxicity. Notably, PNU-282987, but not bethanechol, upregulated the phosphorylation of STAT3 at Tyr705, implying that α7nAChR activation conveyed anti-inflammatory signaling partly via the induction of the STAT3 pathway. Following this line of thought, α7nAChR has also previously been identified as a potent mediator of anti-inflammatory pathways via the activation of the Jak2/STAT3 pathway to facilitate the blockage of NF-κB translocation, further suppressing the production of pro-inflammatory cytokines [65, 66]. Treatment with selective α7nAChR agonists ameliorated neuroinflammation and cytokine production via attenuation of microglia and astrocyte reactivation [67,68,69,70]. In the present study, even though treatment with the mAChR agonist bethanechol did not alter the activation of STAT3, a decline in the pro-inflammatory cytokines was still observed, suggesting the possibility that mAChR activation regulates anti-inflammatory processes via other alternative pathways. A previous study demonstrated that selective optogenetic stimulation of cholinergic neurons caused the significant expression of M1 mAChR in the basal forebrain or M1 mAChR agonist therapy effectively reducing the level of serum TNFα during endotoxemia [37, 71]. Additionally, a non-selective mAChR agonist, oxotremorine, was shown to significantly hamper the expression of pro-inflammatory cytokines in the hippocampus of rats that underwent chronic restraint stress [72]. Therefore, it is possible that mAChR activation regulates anti-inflammatory effects in DOX-treated rats mainly via M1 mAChR signaling. However, further investigations using genetic modification or selective agonists/antagonists are needed to identify the specific receptors and mechanisms.

In addition to neuroinflammation, a substantial number of previous studies have reported brain mitochondrial abnormalities following systemic administration of DOX [15, 18, 21, 73, 74]. Prior works demonstrated that DOX administration in rodents impaired mitochondrial oxidative regulation and calcium homeostasis in the brain, ultimately leading to brain oxidative burst and impairment in hippocampal-dependent learning and memory [15, 21]. We recently demonstrated that Dox treatment caused a fluctuation in brain mitochondrial dynamics by promoting both fusion-related protein expression and phosphorylation of the fission protein Drp-1 [18]. Although the precise mechanism remains obscure, it has been suggested that excessive oxidative stress following DOX treatment is a key element driving the upregulated phosphorylation of the fission-related protein Drp-1 [18, 21]. Inhibition of mitochondrial fission has the capacity to rebalance these changes and improve cognitive function in rats treated with DOX [18]. Likewise, activation of either an α7nAChR or mAChR agonist could also be beneficial in protecting brain function against DOX-induced chemobrain through these mitochondrial protection processes. It is conceivable that activation of AChRs using their agonists provides anti-oxidant capacity, leading to a rebalance of mitochondrial dynamics in the hippocampus. This is corroborated by the fact that activation of α7nAChR induced the upregulation of the canonical Nrf2/HO-1 anti-oxidant pathway in both microglia and astrocytes [68, 70]. In addition, oxotremorine treatment, a non-selective mAChR agonist, was capable of mitigating oxidative damage and mitochondrial abnormalities in differentiated SH-SY5Y cells exposed to amyloid-β [75]. Insights into these subcellular mechanisms strongly illustrate the mitoprotective efficacy of cholinergic activation against DOX-induced chemobrain.

Post-translational modifications of Tau, particularly hyperphosphorylation of Tau at Thr181, have been broadly shown to regulate a spectrum of neurodegenerative diseases. Interestingly, treatment with α7nAChR or mAChR agonists effectively attenuated the hyperphosphorylation of Tau at Thr181 in the hippocampus following DOX treatment. The phosphorylated Thr181 residues can lead to microtubule instability as it is complicated by the binding kinetics of the Tau microtubule, resulting in the formation of neurofibrillary tangles (NFTs) [76]. In depth, we demonstrated that activation of α7nAChR or mAChR induced the activation of their downstream signaling kinases including PI3K, AKT, and ERK1/2. Although the ionotropic activity of α7nAChR is a pronounced property of neurons, the metabotropic ways are prevalent downstream of α7nAChR activation in non-neuronal cells. It has been reported that α7nAChR mediates neuroprotective effects through the involvement of various intracellular signaling pathways including PI3K/AKT [77]. Activation of M1 and M4 mAChRs is also associated with signal transduction via the engagement of PI3K and AKT [78]. Previous studies have demonstrated that AKT inhibited the activity of GSK3β, a kinase significantly responsible for Tau hyperphosphorylation in AD pathology [79, 80]. Consistently, activation of α7nAchR with PNU-282987 led to increased phosphorylation of AKT and GSK3β and a subsequent decline in phosphorylated Tau in hypothalamic neurons treated with a conditioned medium obtained from microglial cells previously stimulated with LPS [81]. The M1 mAChR agonist showed a protective effect on cognition by mitigating the hyperphosphorylated Tau and the number of neurons containing aggregated Tau [82]. From these results, we suggest that activation of α7nAChR or M1 and M4 mAChRs prevented hyperphosphorylation of Tau in the hippocampus of DOX-treated rats possibly via the activation of the PI3K/AKT/GSK3β axis.

Aberrant activation of PCD in the neuronal cell population is a principal characteristic of pathologies involved in neurodegenerative diseases, culminating in detrimental loss of neuronal cells and cognitive deficit, respectively. As noted above, the deleterious effects of DOX on the CNS potentially orchestrate neuronal injury, ultimately leading to neuronal cell death. The importance of the present study is that DOX simultaneously induced several types of PCD including apoptosis, pyroptosis, and necroptosis. A series of extensive studies have comprehensively indicated that mitochondrial dysfunction is a key initiator of neuronal apoptosis following DOX administration [15, 74, 83]. We have also previously demonstrated that DOX administration provoked neuronal death in the hippocampus partly through necroptosis as affirmed by upregulated phosphorylation of RIPK1, RIPK3, and MLKL. It is therefore conceivable that the elevation of TNF-α level in response to DOX could induce the activation of the RIPK1–RIPK3–MLKL pathway via binding with tumor necrosis factor receptor 1 (TNFR1), eventually resulting in the assembly of necrosomes and subsequent necroptosis-mediated cell membrane rupture. Interestingly, concomitant intervention with either α7nAChR or mAChR agonists exerted influential anti-apoptotic, necroptotic, and pyroptotic properties in rats with DOX-induced chemobrain, indicating activation of AChRs protects against PCD following DOX administration in the brain by preventing mitochondrial dysfunction and neuroinflammation. Indeed, it has been previously described that the stimulation of α7nAChR initiates the survival PI3K/AKT/Bcl-2 pathway which may have contributed to the neuroprotection against DOX toxicity observed in the current study [84]. Similarly, it is known that mAChR inhibited apoptosis through the activation of PI3K and its downstream targets AKT and ERK1/2 [85]. Nonetheless, the direct anti-necroptotic effects of cholinergic activation in the brain are still poorly understood. We suggest that cholinergic anti-inflammatory effects could contribute to the suppression of TNF-α production, thereby preventing TNFR1 from initiating the necroptosis cascade in response to DOX administration. To our knowledge, this present study demonstrated for the first time that DOX monotherapy upregulated the expression of NLRP3, cleaved GSDMD, and IL-1β in the hippocampus, further indicating the existence of pyroptosis in response to DOX treatment. In correlation with the previous study, the effects of the combined therapy of cyclophosphamide and DOX have been shown to be associated with NLRP3-mediated oxidative stress and a debilitating neuroinflammation in the hippocampus [86]. However, further investigation is needed to determine whether damage-associated molecular patterns (DAMPs) or pathogen-associated molecular patterns (PAMPs) are responsible for the initiation of pyroptotic processes in DOX-induced chemobrain. Even though the molecular mechanism has not yet been identified, a previous study demonstrated that treatment with ACh or the α7nAChR agonist GTS-2 inhibited internalization of extracellular high mobility group box 1 protein (HMGB1), a potential initiator of pyroptosis, in cultured macrophages [87]. Additionally, the α7nAChR agonist also mimicked the benefits of vagus nerve stimulation by inhibiting neuronal pyroptosis after cerebral ischemia/reperfusion injury [88]. Therefore, we suggest that cholinergic activation possibly inhibited DOX-mediated pyroptosis via inhibition of DAMP or PAMPs internalization and the cholinergic anti-inflammatory pathway.

Surprisingly, the synergistic effects of the combined treatment were not observed in the DOX-induced chemobrain in the present study. It is possible that monotherapy with either α7nAChR agonist or mAChR agonist already reached the maximal benefits with regard to DOX-induced chemobrain. It is conceivable that the systemic activation of either α7nAChR or mAChR conveys the same common pathways to exert neuroprotection against DOX-induced chemobrain. Therefore, when the shared downstream pathways of these AChRs have already been thoroughly stimulated, the synergistic effects would not be discernible. Collectively, our study enhances our understanding of multiple types of PCD induced by chemotherapy, including apoptosis, necroptosis, and pyroptosis in the hippocampus, further gaining a comprehensive understanding of the molecular pathophysiology of chemobrain. These insights provide promising targets for the further development of effective interventions to treat chemobrain in the future. Furthermore, our research emphasizes the specific protective function mediated by each AChR receptor, thus providing in-depth neuroprotective mechanisms of AChR agonists that can be used as possible treatment options to alleviate neurological complications in cancer patients receiving chemotherapy, as well as for other neurodegenerative diseases. The proposed mechanisms in which α7nAChR and mAChR agonists exerted neuroprotective effects against DOX-induced chemobrain are illustrated in Fig. 9.

Fig. 9
figure 9

Schematic representation of the proposed mechanisms of AChR agonists against DOX-induced chemobrain. AChR agonists preserved long-term memory against DOX toxicity by reducing neuroinflammation and glial activation, restoring normal mitochondrial function and dynamics, and mitigating Tau hyperphosphorylation and various forms of programmed cell death including apoptosis, pyroptosis, and necroptosis. The effects of DOX administration in rats are denoted by red arrows, while the effects of AChR agonists in DOX-treated rats are represented by green arrows

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Conclusion

Our study demonstrated that cholinergic activation exerted neuroprotective effects in experimental rats with DOX-induced chemobrain through attenuation of neuroinflammation and glial activation, restoration of mitochondrial function and dynamics, and mitigation of several types of PCD. The current study substantiates the potential therapeutic advantages of parasympathetic therapy utilizing AChR agonists as a promising novel therapy to protect cancer patients against neurological sequelae.