Immunity in neuromodulation: probing neural and immune pathways in brain disorders

Abstract

Immunity finely regulates brain function. It is directly involved in the pathological processes of neurodegenerative diseases such as Parkinson’s and Alzheimer’s disease, post-stroke conditions, multiple sclerosis, traumatic brain injury, and psychiatric disorders (mood disorders, major depressive disorder (MDD), anxiety disorders, psychosis disorders and schizophrenia, and neurodevelopmental disorders (NDD)). Neuromodulation is currently a leading therapeutic strategy for the treatment of these disorders, but little is yet known about its immune impact on neuronal function and its precise beneficial or harmful consequences. We review relevant clinical and preclinical studies and identify several specific immune modifications. These data not only provide insights into how neuromodulation acts to optimize immune-brain interactions, but also pave the way for a better understanding of these interactions in pathological processes.

Highlights

• Few studies have yet been conducted on the immune effects of neuromodulation strategies.

• Brain disorders should be considered as systemic diseases involving central-peripheral immune interaction.

• Immune parameters vary depending on age, sex, environment and disease progression, requiring a tailored approach for each pathology.

• Preferential immune pathways must be investigated, as hub genes and shared immune pathways influence the effects of neuromodulation across disorders.

• A critically important concept is emerging to control the effects of neuromodulation on the brain: the study of the dynamic response of the extracellular matrix (ECM).

• Future studies of DBS-induced immunomodulation in neurodevelopmental disorders such as autism spectrum disorder and dystonia will provide a better understanding of the impact of neuromodulation on neuroimmune circuits.

Introduction

As the nervous system and immune factors use and produce immune and neuroendocrine factors respectively, the neuroimmune system is important not only for brain health but also for the overall health of the body.Critical processes are influenced by the neuro-immune system not only at the neuronal level with neurotransmission and neuroplasticity, but also throughout the body, where the nervous system controls immune function and the immune system defends the whole body, particularly in the event of infection [1]. Recognition of this neuro-immune crosstalk has grown significantly with research into neurological conditions such as Parkinson’s disease (PD), Alzheimer’s disease (AD), multiple sclerosis (MS), post-stroke syndromes, traumatic brain injury (TBI), and psychiatric disorders, including MDD, anxiety disorders, psychosis disorders and schizophrenia, as well as neurodevelopmental disorders [2]. Neuromodulation is one of the most important current strategies for many of these disorders- including repetitive transcranial magnetic stimulation (rTMS), transcranial direct current stimulation (tDCS), electroconvulsive therapy (ECT), focused ultrasound (FUS), deep brain stimulation (DBS), and valgus nerve stimulation (VNS). To optimise neuromodulation interventions, the main current work is aimed at adjusting and improving signal processing to obtain better neurostimulation parameters. These techniques, which act on the cortex, modify not only the signal but also the components of the neurotransmission system, i.e. neuronal receptors and neuro-immune activity, and can also act on the extracellular support matrix which is fundamental to neuronal processes. Neuromodulation has the potential to modify the neuro-immune profile [3]. These effects may have long-term implications, including the promotion of therapeutic immune function—particularly in neurodegenerative diseases—and the enhancement of extracellular matrix dynamics [1, 4]. Peripheral and central immunity—mediated by macrophages, monocytes, microglia, and cytokine networks—contribute to the therapeutic effects of neuromodulation via neuroendocrine pathways [5]. Despite these advances, it is still essential to understand how this complex interaction can be exploited for therapeutic purposes. Few, if any, neuromodulation protocols take into account the patient’s baseline immune status or immune response, nor do they focus on activating specific immune pathways [6, 7].

Given that evidence is still developing at the research level and has not yet been translated into direct application with protocols that include neuroimmune function in neuromodulation treatments, this review aims to support this hypothesis by extracting from the literature the strong elements currently known about the modification of neurotransmission and immune activity in relation to neurostimulation. This review identifies and synthesizes all preclinical and clinical animal and human studies of neuromodulation techniques in the context of the above-mentioned diseases. It provides a detailed account of the immunomodulatory and consequent neural effects of neuromodulation currently known, highlighting their role in disease progression and therapeutic outcomes. It highlights the need to understand the precise impact of neuromodulation on the underlying neurobiological processes involved in signal processing, and the gaps in current knowledge in this area. It underscores the importance of patient-specific immune responses in determining therapeutic outcomes and proposes a framework for integrating immune biomarkers into future neuromodulation strategies.

It proposes a direction for emerging, as yet unexplored research, in neuromodulation: the dynamic and adaptive behavior of the extracellular matrix (ECM), which further deepens the complexity of studying the brain’s response to neuromodulation.

Methods

We searched for articles cited in this review using the NCBI Pubmed and ScienceDirect databases and proceeded methodically to collect articles of interest, following the strategy outlined in Fig. 1. Recent decades have seen a boom in neuromodulation studies thanks to the development and improvement of new technologies and therapeutic strategies, a better understanding of how the brain works and the diversification of therapeutic applications, particularly in neurological pathologies such as neurodegenerative disorders, pain processes, movement disorders or psychiatric disorders, among others. Considering that these new advances are based on the knowledge acquired and accumulated since the use of neuromodulation, we have given priority to the most recent studies in this field, enabling us to present the most recent data on advances in neuromodulation research and the studies currently being developed on immunity. The key mesh terms used were the association of the following diseases: “Parkinson”, “Alzheimer”, “multiple sclerosis”, “traumatic brain injury”, “post-stroke”, “mood disorders”, “depression”, “anxiety”, “psychosis”, and “schizophrenia” with the neuromodulation therapies: “rTMS” studied in Alzheimer disease and post-stroke (Fig. 2A and B), “tDCS” studied in multiple sclerosis (Fig. 3A) and in mood disorders (Fig. 3B), “ECT” studied in psychiatric disorders such as schizophrenia (Fig. 4A) and MDD (Fig. 4B), “focused ultrasound” studied in Alzheimer disease (Fig. 5), “DBS” studied in multiple sclerosis and Parkinson disease (Fig. 6A and B), and “VNS” studied in MDD (Fig. 7). Only pre-clinical and clinical trial studies were retained. Systematic reviews and meta-analyses targeting the immune response in each disease were selected to discuss the data using the previous mesh terms and the following: “immunity”, “neuromodulation”, “microglia”, “astrocytes”, “oligodendrocytes”, “cytokines”, “chemokines”, “NK (natural killer)”, and “lymphocytes”. Given the limited number of publications and data available on the specific and targeted topic of the immune response in neuromodulation, we selected the most recent studies that present the key and most significant results in the field. In addition, we specifically included studies that have been replicated and whose results are widely recognized and accepted within the scientific community. We selected them specifically because these data make it possible to identify precisely the next steps and the studies to be undertaken to advance knowledge in this field with discernment.

Fig. 1

Flowchart of the selection process for studies included

Fig. 2

A Summary of neuronal and immune changes with rTMS in preclinical models. B. Summary of neuronal and immune changes with rTMS in clinical models

Fig. 3

A Summary of neuronal and immune changes with tDCS in preclinical models. B Summary of neuronal and immune changes with tDCS in clinical models

Fig. 4

A Summary of neuronal and immune changes with ECT in preclinical models. B Summary of neuronal and immune changes with ECT in clinical models

Fig. 5

Summary of neuronal and immune changes with FUS in preclinical models

Fig. 6

A Summary of neuronal and immune changes with DBS in preclinical models. B Summary of neuronal and immune changes with DBS in clinical models

Fig. 7

Summary of neuronal and immune changes with VNS in preclinical models

Immunity and neurodegenerative diseases

Aging leads to systemic dysregulation of immune and inflammatory processes, particularly in T and B lymphocytes, which are key players in adaptive immune. This creates a chronic low-level inflammatory state known as"inflammaging”, which characterises many neurodegenerative diseases [7]. T-cell senescence is a central factor in this process, driven by the activity of telomerase reverse transcriptase (TERT), the regulator of telomere length. Targeting TERT using advanced gene neuromodulation techniques such as focused ultrasound (FUS), a non-invasive image-guided surgical technology that precisely directs ultrasound energy to specific regions of the brain, offers a promising route to counter T-cell senescence, reduce neuroinflammatory damage and slow the progression of neurodegeneration [8].

Peripheral immune cells, such as Th17 or CD8 T cells, can cross the blood–brain barrier (BBB) and directly influence the immune status of the brain. Certain pathways are involved in the regulation of CD8 T-cell trafficking, such as CXCL16-CXCR6 signalling, which is upregulated in the cerebrospinal fluid (CSF) of patients with cognitive aging, illustrating the systemic-regulatory crosstalk between the peripheral and central immune systems in neurodegeneration. Thus, CSF immunity may reflect central nervous system (CNS) dysregulation during cognitive aging [9]. Therefore, the current paradigm presents neurodegenerative diseases as systemic diseases with fine regulatory crosstalk between peripheral and central immune systems.

The pathological processes associated with neurodegenerative diseases result in activation of the immune system and microglia, leading to activation of the inflammasome and production of inflammatory cytokines. Neuromodulation strategies, such as DBS, that modify neurotransmitter expression can directly alter inflammasome activation and curb the disease state [10, 11]. Microglia is a central effector linking inflammation and neurodegeneration, and microglial activation is a direct result of their close, bidirectional interactions with astrocytes and neurons via ligands through cell–cell contacts and released mediators. The high frequency stimulation parameters of DBS negatively influence astrocyte metabolism, resulting in the secretion of matricellular proteins. Therefore, astrocytes are the long-term immune regulators of altered cell–cell interactions in perineuronal environments following DBS neuromodulation [12]. Specific signalling pathways are involved in the swith of microglia to a pro or anti-inflammatory state: we will see that these pathways are involved in the effects described after the application of neuromodulation techniques in the studies analyzed.

Other pathways, such as nicotinamide adenine dinucleotide (NAD) metabolism, involved in decline with aging, affect metabolic homeostasis, protein post-translational modification, DNA repair and immune responses. Modulation of NAD pathways could be a powerful therapeutic strategy for age-related neurodegenerative disorders, for example by modulating related pathways that stimulate NAD [13]. Similarly, cGAS–STING is an innate immune pathway that detects double-stranded DNA in the cytosol and, in response, triggers the expression of inflammatory genes that can lead to senescence or immune activation. This signalling pathway is mainly involved in age-related decline. cGAS-STING, predominantly expressed in microglia, is an essential molecular link in innate immunity that orchestrates the inflammatory framework in the progression of AD and constitutes an effective therapeutic target [14].

Emerging molecular biomarkers, such as miRNAs and exosomes detected in CSF or blood, are promising tools for the diagnosis, prognosis, and treatment of neurodegenerative diseases. Pre-clinical studies of miRNA-based drugs administrated in CSF or blood, such as exosomes and recombinant adeno-associated virus (rAAV)-based systems that express miRNA injected intracranially, are currently underway [15]. Moreover, certain miRNAs have been found to share similar functions in different neurodegenerative diseases, particularly in the neuronal and immune compartments. These predominant miRNAs regulate major pathways, mostly T-cell activation and differentiation, highlighting the presence of a strong immune response in neurodegenerative diseases that requires further analyses. These complementary explorations will help to identify potential common therapeutic pathways for different diseases [16].

Therefore, these insights demonstrate how neuroimmune mechanisms and cellular senescence contribute to neurodegeneration. We will analyze how neuromodulation techniques can be used to modulate these processes. By addressing the interplay between neuroinflammation, immune regulation, and neuromodulation, we present the currently known elements on which potential immune-informed therapies for neurodegenerative diseases could be developed.

Parkinson’s disease (Fig. 6A and B)

The regions of the cerebral cortex affected by PD are the substantia nigra, the basal ganglia (including the striatum and globus pallidus), the thalamus and the motor cortex. Immune changes occur during the emergence and progression of PD: astrocytes participate in the clearance of α-synuclein, inducing a proinflammatory state and a distinct pathological phenotype. Monocytes and T cells also play an essential role at different stages of disease progression, with cytotoxic CD8 T cells infiltrating the brain in the early stages, followed by CD4 helper T cells. These disease states activate innate and adaptive immune responses, leading to the death of dopaminergic neurons [17]. The dopaminergic pathway itself serves as a central modulator of innate and adaptive immune activation and regulation.

One of the main mechanisms underlying PD is mitochondrial dysfunction, which leads to excessive fragmentation of mitochondria and triggers an innate immune response in neurons through the release of danger-associated molecular patterns (DAMPs). Mitochondrial-derived vesicles (MDVs), embedded in extracellular vesicles, further modify cellular signalling pathways. At the same time, α-synuclein aggregation activates innate immunity. This communication of MDV and α-synuclein between neurons and glial cells drives neuroinflammation [18].

Preclinical studies (Table 1)

Seven preclinical studies have been conducted on rTMS, aVNS, ECT, and DBS, and all demonstrated a significant reduction in neuroinflammation or a return to a normal state.

Immune pathways Concerning glial activation, a significant downregulation of astrocytic and microglial responses was reported after intermittent rTMS theta-burst stimulation in 6-OHDA-hemilesioned rats [19] and the level of CX3 CL1 (fractalkine, a chemokine secreted at membrane level), synthesized by neurons and targeting microglia expressing CX3 CR1, was reduced, as were those of CX3 CR1 and Iba-1 (ionized calcium-binding adaptor molecule 1) after one week of ipsilateral DBS stimulation of the subthalamic nucleus in a rat model of PD [20]. Brains of naive rats stimulated in the STN with DBS also showed less highly activated microglia [21]. High-frequency stimulation (HFS) was applied to cultured astrocytes, along with cytokine induction and NF-κB activation. HFS-DBS treatment regulated the inflammatory response by attenuating astrocyte activation [22].

Concerning cytokine expression, aVNS, ECT, DBS, STN-DBS, and HSF-DBS inhibit the increase in pro-inflammatory cytokine: an overall decrease in proinflammatory cytokine levels after ECT [23], a decrease in IL-1β and TNF-α levels in the ventral midbrain of 6-OHDA-treated rats after aVNS [24], a decrease in IL-1β and IL-6 levels and their mRNA released by microglia involved in PD pathogenesis via NF-κB signaling but not TNF-α after DBS [20], and a decrease in MCP-1 levels via TNF-α and NF-κB activation after HFS-DBS [25]. IL-1β levels were decreased in substantia nigra microglia and the increase in IL-1 receptor (IL-1R), the receptor for IL-1β in dopaminergic neurons, was reversed. Levels of anti-inflammatory cytokines (IL-4 and IL-13) were reduced to normal levels by DBS and an increase in the p-ERK/ERK ratio (phosphorylated extracellular signal-regulated kinase pathway) and levels of cleaved-caspase3 were abolished [20]. STN-DBS significantly reduces neuroinflammation modulated by immune chemokine pathways [21]. DBS may act by suppressing glial cell activation and neuroinflammation, with striatal cytokine expression, reducing inflammatory reactivity and suppressing morphological changes in astrocytes [25].

Concerning lymphocyte activation, Tregs were upregulated and the number of Th17 cells decreased after treatment with aVNS. The neuroprotective effect of aVNS may be due to modulation of the innate immune response and suppression of progressive inflammation, resulting in protection of dopaminergic neurons [24].

A study of the electrode-human brain tissue interface by visualization of the tissue contact point using scanning electron microscopy (SEM) or transmission electrode microscopy (TEM) has shown that the polyurethane component of the electrode surface can influence the clinical response to DBS in PD and other diseases treated with this modality. A multinucleated giant cell foreign body response was observed, with a number of giant cells arising from the fusion of parenchymal microglia, resident perivascular macrophage precursors and circulating blood monocytes or macrophages, as well as mononuclear macrophages containing lysosomes with filopodia. Both cell types contained phagocytosed material [26].

Neuronal pathways rTMS, aVNS, ECT, and STN-DBS improve dopaminergic functioning. rTMS increases striatal dopamine levels, with full restoration of plasticity, improved motor performance and early gene activation in striatal spinous neurons [19]. aVNS administered 7 days after treatment with 6-OHDA in adult male Wistar rats (administered unilaterally to the medial forebrain bundle, every two days for 8 days–500 ms train of 15 biphasic pulses every 30 s (0.8 mA, 30 Hz) delivered for a total time of 30 min) significantly increased the number of tyrosine hydroxylase-positive neurons in the 6-OHDA-lesioned substantia nigra, the number of which was reduced on the damaged side, with significant protection of dopaminergic neurons in 6-OHDA rats [24]. ECT and mesenchymal stem cells (MSCs) have been shown to have a synergistic effect in the treatment of PD, with ECT enhancing the differentiation of MSCs into dopaminergic neurons in a mouse model of PD, as well as upregulating MSC dopamine levels [23]. DBS stimulation of the ipsilateral subthalamic nucleus in a rat model of PD (unilateral injection of 6-hydroxydopamine into the left striatum) for one week increased the survival of dopaminergic neurons in the substantia nigra [20].

Other protective neuronal events occur and modulate neuronal excitability and synaptic communication. aVNS increases activation of the α7 nicotinic acetylcholine receptor (nAChR) with protective effects on neurons [24]. STN-DBS increases the number of neural precursor cells expressing cell cycle, plasticity, and precursor cell markers (Ki67, MCM2) in the brains of naive rats compared with microinjured and sham animals [21]. DBS in a rat model of 6-OHDA disease reduces extracellular glutamate concentrations in the striatum, with increased levels of the excitatory amino acid transporter (EAAT)−2, promoting glutamate clearance and maintaining synaptic balance [25].

Clinical studies (Table 2)

Immune and vascular pathways DBS has an immunomodulatory and neuroprotective effect in PD, as evidenced by significantly higher serum concentrations of pro-hepcidin (a hormone involved in oxidative stress (involved in the systemic metabolism of iron) and a member of the family of protein mediators of the acute inflammatory response and induced in response to IL-6) in PD patients than in those treated pharmacologically, with a greater therapeutic effect [27]. A significant increase in the thickness, length and density of microvessel endothelial cells and adherens junction- and tight junction-associated proteins, as well as a decrease in the density of activated microglia, were observed post-mortem in the subthalamic nucleus (STN) of PD patients treated with DBS. Vascular endothelial growth factor (VEGF) expression was also significantly increased. The observed DBS-induced microvascular changes may have been caused by the increase in VEGF and the suppression of inflammatory pathways [28].

The recent study of McFleder in humans and animals highlights DBS induces a shift from pro-inflammatory CD4 + T helper 17 cells, which contribute to neurodegeneration, to anti-inflammatory CD4 + regulatory T cells. RNA sequencing and immunohistochemistry on the brains of the A53 T alpha-synuclein rat model of Parkinson’s disease showed that DBS also reduces neuroinflammation. These findings support the idea that DBS can help modify the disease by halting inflammatory processes [29].

Insights in neuromodulation for PD

rTMS, aVNS, ECT and DBS are effective in attenuating neuroinflammation by modulating glial activation, cytokine expression, inflammasome activity, dopaminergic function, oxidative stress and microvascular function.

Research protocols to be developed could focus on targeting two factors in PD: α-synuclein aggregation and mitochondrial dysfunction. In this line, two therapeutic options should be considered: firstly, optimizing strategies using tDCS, rTMS, and VNS and developing strategies using transcranial alternating current stimulation (tACS); secondly, combining pharmacological therapies (such as molecules promoting mitochondrial biogenesis) or genetic interventions and neurostimulation techniques with a view to potentiating them.

Alzheimer disease’s (Figs. 2A, B, 5)

The brain regions affected by AD are the hippocampus, entorhinal cortex, temporal and frontal cortex, basal nucleus of Meynert, thalamus and limbic system. The involvement of peripheral immune cells plays a central role in the pathogenesis of AD. Peripheral T cells and NK cells can either disrupt the BBB and infiltrate the CNS, or indirectly modulate microglia through systemic inflammation. Immune-related genes regulate the infiltration of immune cells and their activity is altered in AD. In addition, regulatory transcription factors play a key role in the expression of these immune genes [30].

A large number of AD-related risk loci are linked to immune cell function, in particular the apolipoprotein E (APOE) variant through its interaction with the trigger receptor expressed on myeloid cells 2 (TREM2). TREM2, on microglial surfaces, performs essential immunomodulatory functions and is considered a central therapeutic target due to its neuroprotective role in the activation of efferocytosis in microglia. Modulating of TREM2 expression can maintain neuroinflammation associated with Aβ accumulation through microglial activation [31]. Pathogenic variants of TREM2 affect central macrophage functions such as endocytosis/phagocytosis, cholesterol metabolism, immune response, and efferocytosis. Other pathways under investigation include MS4 A4 A/MS4 A6 A, CD33, PU.1, PLCG2, and INPP5D/SHIP1 [32].

The innate immune system actively regulates brain homeostasis by eliminating pathogens through the release of proinflammatory factors. However, chronic inflammation contributes to neurodegeneration. Therefore, tight regulation of the elimination process could be an effective strategy to prevent damage caused by sustained neuroinflammation [33]. The innate immune system is also involved in tau-mediated pathologies, with innate immune genes, including tauopathy risk alleles, regulating tau kinases and tau aggregates. Notably, the expression of these innate immune pathways increases as the disease progresses [34].

Future progress should focus on clarifying the immunogenetic profile and genotype/phenotype relationship of innate and adaptive immune cells in the periphery and CNS in AD. This requires the identification of genetic variants and their impact on immune functions. The role of innate and adaptive immune cells evolves with the progression of AD. Innate immune cells, such as microglia can switch from a protective function to increased inflammation and neurodegeneration following chronic activation. Similarly, adaptive immune cells, such as T and B lymphocytes, have different implications depending on the stage of the disease. These dynamic changes are essential parameters to consider when developing targeted therapies aimed at modulating immune responses to slow disease progression.

Preclinical studies (Table 3)

A number of rTMS and FUS stimulation paradigms have been shown to reduce the amount of amyloid in the brain, under the effect of various immune responses, notably by downregulating microglial activation.

Immune pathways Concerning glial and leucocyte activation and cytokine release, a significant reduction in microglia (Iba⁺) and astrocyte (GFAP⁺/VIM⁺) activation was demonstrated using three different rTMS paradigms: (1) high frequency, wide-field rTMS in 4-to 5-month old 5xFAD mice (a dual transgenic Aβ precursor protein (an APP)/presenilin-1 (PS1) mouse model that develops severe amyloidopathy, with rapid gliosis of cerebral amyloid plaque and cognitive deficits [35]), (2) intermittent theta-burst stimulation (iTBS) treatment in the hippocampus and periventricular area of an AD mouse model injected intracerebroventricularly with streptozotocin (STZ) and showing mild reactive microgliosis in the dorsal hippocampus, medial habenula, the prefrontal cortex, and caudoputamen [36], and [3] a 20-Hz high-frequency rTMS protocol administered to double transgenic 5xFAD mice, resulting in the release of pro-inflammatory cytokines, such as TNF-α and IL-6. Indeed, an excess of pro-inflammatory cytokines can ligate cell membrane receptors and act directly on intracellular signaling pathways, such as the PI3 K/Akt/NF-κB pathway, with the nuclear transfer of NF-κB. Its subsequent binding to DNA leads to the production of pro-inflammatory cytokines. 20-Hz rTMS treatment regulates the PI3 K/Akt/NF-κB pathway in the hippocampus and cortex by modulating protein levels of Akt and phosphorylated-Akt (p-Akt), a downstream molecule of PI3 K signaling. This results in a decrease in TNF-α levels [37]. Furthermore, in a 10-Hz rTMS protocol applied to organotypic mouse brain tissue cultures of both sexes, microglial morphology and dynamics remained unchanged, while there was a significant release of plasticity-promoting cytokines from microglia. Indeed, the addition of TNF-α and IL6 to microglia-depleted cultures maintained rTMS-induced synaptic plasticity. In-vivo microglia depletion abolished rTMS-induced changes in neurotransmission in mPFC in anesthetized mice. Thus, cytokine release from microglia may be the primary target of rTMS effects on neuronal excitability and plasticity [38].

Fewer effects on microglia were observed with the FUS paradigms: aged APP/PS1 dE9 mice, a model of AD-like amyloidogenesis, were treated with FUS with microbubbles to enhance intravenous delivery of an anti-pGlu3 Aβ Fc-competent monoclonal antibody, mAb 07/2a, across the BBB (FUS-BBBD, Focused Ultrasound combined with Microbubble for Blood–Brain Barrier Disruption). Bilateral hippocampal FUS-BBBD significantly increased mAb 07/2a delivery and improved spatial learning and memory initiated by mAb injection alone. FUS-BBBD did not alter the presence of plaque-associated microglia, while it had an exclusive effect on plaque-associated Ly6G⁺ monocytes. Treatment with mAb 07/2a combined with FUS-BBBD increased monocyte infiltration and recruitment into plaques, leading to greater plaque clearance, synapse preservation, and improved cognitive function [39]. rTg4510 mice (a model for early stages of tau pathology) received unilateral focused ultrasound-induced BBB disruption treatment. This resulted in reduced phosphorylated tau levels in the hippocampi, particularly in CA1 pyramidal neurons, correlated with an immune response and microglial colocalization [40]. Twenty-four month-old APP/PS1 dE9 transgenic mice were treated with FUS-BBBD, unilaterally or bilaterally, and injection of 07/2a mAb. Significant immune responses were detected, with upregulation of IgG2a mAb levels in the cortex, immunoreactivity of resident Iba1⁺ and phagocytic CD68⁺ microglial cells, and infiltration of Ly6G⁺ immune cells [41]. Pulsed focused ultrasound (pFUS) combined with microbubbles (MB) is known to induce a sterile inflammatory response (SIR) in rats. The long-term effects of SIR on the brain were studied by MRI in female Sprague Dawley rats stimulated in the left cortex and four regions of the right hippocampus. Rats subjected to a single sonication showed little microglia activation compared to the controlateral hemisphere. Longer pFUS-MB treatment (6 weeks) resulted in cortical atrophy, and most rats showed persistent BBB disruption and astrogliosis on MRI. Rats subjected to multiple sonications showed more phagocytic stigmata in the parenchyma, more activated astrocytes and microglial regions, and more systemically infiltrating CD68⁺ macrophages on the treated side than on the controlateral side [42]. TgCRND8 mice expressing Swedish (KM670/671 NL) and Indiana (V717 F) APP mutations under the control of the hamster prion (PrP) promoter received either scyllo-inositol or an initial gFUS MRI treatment administering BAM-10 prior to scyllo-inositol treatment. Both treatments resulted in decreased amyloid concentrations and astrocyte activation in the hippocampus and cortex through microglia activation and upregulation of phagocytosis. The effects were similar for both treatments [43].

Other regulatory mechanisms may act indirectly through other cells influenced by rTMS in the pathological region, such as the enhanced glymphatic drainage system in the brain parenchyma and meningeal lymphatics. In the prefrontal cortex and hippocampus, c-FOS expression was also increased. Therefore, the treatment significantly reduced long-term memory loss for novel objects and locations [35].

Neuronal pathways Regulation of immune parameters consequently suppresses plaque accumulation. High-frequency wide-field rTMS reduces both intraneuronal Aβ deposition and plaque-like Aβ deposition in the mPFC, dental gyrus, and cornu ammonis 3 (CA3) of the dorsal hippocampus and S1 cortex [35]. Treatment with 20-Hz rTMS was found to significantly reduce Aβ accumulation in the cortex and dental gyrus region. It inhibited microglia activity, reduced Aβ plaques, and blocked neuroinflammation through the PI3 K/Akt/NF-κB signaling pathway, leading to increased levels of synapse-associated proteins and consequent neuronal synaptic plasticity [37]. Unilateral FUS-BBBD stimulation was found to lead to a bilateral effect, with increased suppression of phosphorylated tau levels [40] and treatment with mAb 07/2a combined with FUS-BBBD significantly decreased hippocampal plaque burden while increasing hippocampal synaptosome levels and CA3 synapses [39]. Intravascular leukocyte activity was analyzed in the FUS-BBBD-treated TgCRND8 mouse model of AD. Transendothelial migration and cell aggregate formation, used as markers of leukocyte responses to acute inflammation, were increased, with the participation of neutrophils. The peripheral immune response induced by FUS-BBBD may act through the participation of leukocytes to reduce β-amyloid pathology [44]. Repeated pFUS-MB treatment has been shown to result in an increase in the number of hyperphosphorylated Tau (pTau)-positive neurons containing neurofibrillary tangles (NFTs) in the treated cortex, but not in the hippocampus. The long-term consequences of pFUS + MB treatment need to be further investigated [42].

Insights in neuromodulation for AD

Peripheral immune cells, such as T lymphocytes and NK cells, actively contribute to pathogenesis and neuroinflammatory processes by disrupting the BBB.

Research protocols could focus on:

• modulating peripheral immune responses through VNS, tDCS, tACS, rTMS and modulating the sympathetic nervous system.

• targeting APOE and TREM2 through rTMS, tDCS, VNS or through combined protocols such as rTMS with drugs that modify APOE expression or agents that stimulate microglia via TREM2, or through genetic therapies using gene expression modulators using RNA interference or CRISP-Cas9.

• targeting tau protein aggregation and β-amyloid accumulation with rTMS and tDCS or combining neuromodulation strategies with drugs such as GSK-3β kinase inhibitors, agents that modulate tau phosphorylation processes, or small molecules such as LMTX.

• targeting the PI3 K/Akt/NF-κB pathway with rTMS and FUS. Combining FUS-BBBD with monoclonal antibodies can improve cognitive function and mitigate pathological processes. However, long-term adverse effects, such as cortical atrophy and astrogliosis, remain challenging to address.

Diffuse demyelination disorders: multiple sclerosis (Figs. 3 A, 6 A and B) and traumatic brain injury (TBI)

MS and TBI lesions are directly related to diffuse demyelination, with myelin damage and diffuse axonal damage, respectively, being associated with secondary demyelination. They also share some degree, albeit limited, of remyelination.

Multiple sclerosis

Multiple sclerosis primarly affects the regions surrounding the cerebral ventricles, particularly the corpus callosum and periventricular areas, the white matter with corticospinal tracts and sensory pathways, the brainstem, optic nerves, the cerebral cortex with grey matter, particularly in the motor cortex and spinal cord. MS pathological processes have been shown to be related to lymphocyte activity, including abnormal T lymphocytes function crossing the BBB and B lymphocyte activity leading to the secretion of autoantibodies against neuronal proteins [45]. These pathogenic processes are quite similar to those of AD, with a common pathogenic mechanism involving regulatory T cells that control neuroinflammation throughout disease progression. Modulation of leucocyte function to prevent peripheral leukocyte activation and transmigration to the brain could be an effective therapeutic target [46]. Moreover, NK cells contribute significantly to the pathogenesis of MS, exhibiting significant heterogeneity that blurs the boundary between adaptive and innate immunity. Further studies are essential to elucidate NK cells functions and their modulation, along with their pathophysiological impact [47]. Although disruption of immune tolerance against neuronal antigens is a major factor explaining the pathological process, demyelination occurs, with altered expression of neuronal adaptor receptors (AdRs) and subsequent impairment of their interaction with adaptor ligands (AdLs) in the CNS. Therefore, therapeutic strategies should focus on modulating the AdR/AdLs axis [48]. Different immunological and transcriptional profiles have been reported depending on the form and progression of MS. Therefore, new perspectives in personalized and precision therapy are needed, including neuromodulation strategies [49].

Preclinical studies (Table 4)

Immune pathways Experimental autoimmune encephalomyelitis (EAE) mice preemptively stimulated with tDCS with cathodal or anodal polarity for five days (intensity of 325 μA for 10 min with constant current stimulator) three days after immunization showed significant upregulation of microglia/macrophage cell density in anodal and Sham mice, while the microglia/macrophage ratio decreased in cathodal mice [50]. Similar microglia/macrophage activation in the sonicated right hemisphere was found in FUS-BBBD. The activation of inflammation in the sonicated brain may impact the structure of the entire tissue matrix, with differences due to changes in biomechanical parameters [51].

Neuronal pathways In the aforementioned study by Marenna et al., axonal loss was lower and the number of complete paranodic domains higher in the cathodal groups than in the anodal and Sham groups. Indeed, cathodal tDCS is known to prevent myelin damage (assessed by delayed visual evoked potentials (VEPs), a prognostic biomarker of neuroaxonal damage in MS), with fewer inflammatory cells. tDCS may have an anti- inflammatory therapeutic effect in autoimmune demyelinating disorders [50].

tDCS and FUS-BBBD are two promising therapies for the modulation of immune and neuronal functions, with notable consequences on the biomechanical properties of brain tissue and the extracellular matrix (ECM). FUS-BBBD generates local mechanic stress altering the composition of the ECM. This leads to changes in immune cell migration, cytokine release, and cell–cell interactions in the brain, with regenerative outcomes or neuroinflammatory responses, depending on the treatment parameters. Cathodal tDCS could also stabilize ECM components by reducing myelin damage and inflammatory cell infiltration, with neuroprotective effects. In addition to immune activation and neuronal integrity, the biomechanical properties of the ECM must be integrated into the therapeutic effect, highlighting the complexity of neuromodulation therapies.

Insights in neuromodualtion for MS

T and B lymphocytes cross the BBB, producing autoantibodies against neuronal proteins. NK cells also play a central role in pathogenesis.

Therapeutic approaches could focus on disrupting immune tolerance, responsible for demyelination and altered receptor-ligand (AdR/AdL) interactions, thereby improving rTMS, VNS, tACS and tDCS protocols. Preclinical studies demonstrate that cathodal tDCS decreases microglial and macrophage activation, reduces inflammatory cell infiltration, preserves myelin and axonal integrity, and minimizes neuroaxonal injury. FUS-BBBD also targets ECM functionality, modulating immune cell migration and cytokine release, highlighting the complexity of neuromodulation strategies.

Traumatic brain injury (TBI)

Increase in chronic central and peripheral inflammation after TBI is a breeding ground for cognitive and psychiatric disorders through microglia activation and cytokines contribution [52]. Specific immune [53,54,55,56,57] and genetic pathways [58,59,60] can be modulated for the treatment of TBI, with profound neurometabolic changes [61]. An efficient glymphatic-meningeal lymphatic system is also essential for the proper functioning of the immune system [62,63,64]. The initial inflammatory phenotype of patients after TBI is crucial for personalized treatment aimed at regulating inflammation [65].

Preclinical studies (Table 7)

Immune pathways Concerning glial activation, eight weeks of 25-Hz rTMS treatment in lamina II and III and the dorsal horn of the spinal cord resulted in a significant reduction in the number of microglia and astrocytes (Iba1 and GFAP) in the dorsal and ventral horns, respectively, at the L4-L5 level in a rat spinal cord injury-induced pain model [66]. Treatment with 1.5 MHz high-intensity focused ultrasound (HIFU) pulse trains on mouse skulls resulted in an increase in GFAP density in the parietal and temporal cortex, corpus callosum, and hippocampus, as well as an increase in the density of the ionized calcium-binding adaptor molecule, a microglial marker, with astrocyte reactivity. Thus, IFU could be used to study the biological effects of some mild non-impact traumatic brain injuries [67].

Concerning the sterile inflammatory response (SIR), an acute damage-associated molecular response (DAMP), including increased levels of heat-shock protein 70, IL-1, IL-18, and TNFα, indicating SIR in the parenchyma, was induced by pFUS + MB, similar to ischemia or mild traumatic brain injury. Levels of pro-inflammatory, anti-inflammatory, and trophic factors increased, as well as those of neurotrophic and neurogenesis factors. In addition, SIR may occur through induction of the NFκB pathway. An innate immune response, with infiltration of CD68⁺ macrophages, increased albumin levels in the cortex, which disappeared withi 24 h, combined with TUNEL⁺ neurons, activated astrocytes, microglia, and increased cell adhesion molecules in the vasculature was described. pFUS + MB may result in SIR similar to that seen in ischemia or mild TBI [68].

Although studies reported conflicting results regarding the ability of rTMS to modulate cognition, a systematic review of 820 patients with post-traumatic brain injury cognitive disorder (PTBICD) revealed that rTMS combined with cognitive training (CT) significantly improved cognitive function, mental state, and performance in daily activities. Improvements on cognitive scales were correlated with downregulation of P300 latency and amplitude [69].

A novel therapeutic approach, transcranial photobiomodulation (PBM), which uses specific wavelengths of red to near-infrared light to modulate brain functions, affects the electrical properties and polymerization dynamics of neuronal microstructures, such as microtubules and tubulins. Each parameter, including wavelength, power density, dose, light source positioning, and pulse frequency, can alter the results [70].

Insights in neuromodulation for TBI

Cytokine dysregulation and microglial activation promote chronic inflammation via the glymphatic and meningeal lymphatic systems, contributing to cognitive and psychiatric disorders. Preclinical studies demonstrate that rTMS reduces glial activation, while HIFU enhances astrocyte reactivity and increases microglial density. Furthermore, the pFUS + MB combination induces a sterile inflammatory response mediated by NFκB signaling, similar to responses observed in ischemia or mild TBI. Clinical studies show that rTMS combined with CT significantly improves cognitive and daily functioning in post-TBI patients, as evidenced by improvements in P300 latency and amplitude. PBM is a promising therapy in the treatment of TBI.

Post-stroke (Fig. 2A, B)

Post-stroke lesions are caused by cerebral ischaemia leading to neuronal degeneration with death and ischemic neuronal loss. Reduced and localized demyelination may occur secondarily. Stoke affects the neurovascular unit by causing damage to blood vessels and altering the interactions between glial cells, neurons, extracellular matrix components and vascular cells. Neuroinflammation follows an acute stroke, with cell death releasing DAMPs (molecular motifs associated with danger signals) and alarmins, which activate pattern recognition receptors (PPRs). Activation of PPRs leads to the infiltration of inflammatory cells but also promotes tissue remodeling for recovery. However, this pro-inflammatory activation is time-dependent and affects several pathological signaling pathways, including oxidative stress, the transforming growth factor-β activated kinase 1 (TAK1) a member of the MAPK kinase kinase family, NFkB signaling, matrix metalloproteinases (MMPs), high mobility group box 1 (HMGB1), mitogen-activated protein kinases (MAKs), and post-translational modifications that support ischemic brain injury [71]. Microglia also undergo phenotypic changes in their polarization after stroke by assuming a dual role: perform phagocytosis to help restore neuroplasticity and release cytokines with critical immunomodulatory functions [71].

Post-stroke immune dysfunction requires close regulation due to changes in the central and peripheral immune systems. Several pathways participate in the regulation of the immune-inflammatory process such as the metabolism of indoleamine 2,3-dioxygenase (IDO) by its reactivity to cytokines, especially TNF-α. IDO is strongly associated with post-stroke MDD [72]. Non-coding RNA, as epigenetic factors, regulates the dysfunction of neuroimmune inflammatory cascades. The modulation of their expression may have a relevant impact on post-stroke phenomena [73].

Understanding the stages of pro-inflammatory signal production is crucial to developing therapeutic strategies and determining how neuromodulation strategies could alter this immune signaling [74]. Critical sex-based differences play an important role in the inflammatory response to stroke and therapeutic strategies should be finely tuned to these differences [75].

Preclinical studies (Table 5)

Immune pathways Concerning glial activation and immune regulatory pathways, a significant increase in let-7b-5p levels in microglia was observed on the one hand and a suppression of ischemia/reperfusion induced by microglia M1 on the other after a 10-Hz rTMS protocol applied to transient middle cerebral artery occlusion (MCAO) rats and oxygen and glucose deprivation/reoxygenation (OGD/R) injured BV2 cells. Let-7b-5p directly modulated HMGA2 and the NF-kB signaling pathway, which were inhibited by rTMS [76]. Another rTMS protocol in MCAO rats with long-term treatment had no effect on the proliferation rate but reduced the inflammatory polarization of Iba-1⁺ CD86⁺ microglia and promoted the anti-inflammatory polarization of Iba-1⁺ CD206⁺ microglia. It improved neuronal differentiation following microglia polarization and neurogenesis. Treatment also resulted in decreased levels of cleaved caspase3 and p-IκBα, p-NF-κB, and p-STAT6, which participate in neuroinflammation caused by cerebral ischemia, with activation of NF-κB and STAT6 [77]. Microglia were significantly activated in the tMCAO group after high-frequency rTMS (HF-rTMS) at 10- and 20-Hz, with high levels of double positive cells for Iba-1 and CD68 phagocytosis markers, hypertrophic morphology and thickened and retracted processes. Treatment at 20-Hz had a positive effect on neuroinflammation, with the suppression of microglial activation, with fewer CD68⁺ Iba-1⁺ cells with smaller cell bodies and thinner processes. Transformation of a M1 to M2 phenotype via the JAK2-STAT3 pathway occurred, with a reduction in the number of CD68⁻ and CD16⁺ CD32⁺ microglia and an increase in the number of CD206⁺ microglia. It therefore improved neurological function, with significant beneficial effects on post-stroke cognitive impairment (PSCI) [78]. Microglial switches M1 to M2 phenotype and astrocytic switches A1 to A2 occurred with suppression of micro/astrogliosis after 5-min daily continuous theta-burst rTMS (3 pulses of 50 Hz repeated every 200 ms, intensity at 200 G) stimulation applied to the hemisphere infarct after the generation of a photothrombotic lesion of stroke in rats. Synaptic loss and neuronal degeneration in the peri-infarct cortical region as well as infarction volume were significantly reduced after treatment. Treatment inhibits the production of 3-NT (a marker for peroxynitrite production) and increases the production of MnSOD, resulting in decreased oxidative neuronal damage and preservation of cellular redox homeostasis. It promoted the preservation of mitochondrial membrane integrity and inhibited the mitochondrial caspase-9/3 pathway in the peri-infarct cortex. Treatment was highly effective, especially when applied within 3 h of ischemic stroke [79]. Cathodal polarity was particularly effective for functional recovery, neurogenesis, and reduction of microglia activation, with a limited therapeutic window after tDCS in the subacute and chronic phases in murine models after induced focal cerebral ischemia [80]. A sex-dependent reaction to treatment of rTMS was shown, with a difference in microglia activation. Female mice showed a lower density of GFAP⁺ astrocytes and IBA1⁺ microglia near the lesion following low and high frequency ipsilateral stimulation, whereas the density of these cells increased in male mice [81].

The study of the effects of VNS on ischemic stroke models in rats by occlusion of the middle cerebral artery reveals that VNS induces a change from the pro-inflammatory phenotype to the regulatory phenotype in ischemic microglia. VNS inhibits the expression of TLR 4/NF-kappa B signaling by activating the α7 nicotinic acetylcholine receptor. This leads to a reduction of pro-inflammatory markers, such as inducible nitric oxide synthase and TNFα, while increasing regulatory markers like arginase 1 and TGFβ [82].

Concerning cytokine expression, TNF-α concentration was downregulated and IL-10 concentration increased in microglia culture medium (MCM) in rTMS-treated MCAO rats, with reduced ischemic volume and neurological improvements [76]. tMCAO rats had high levels of pro-inflammatory cytokines (TNF-α, IL-1β, and iNOS) and low levels of anti-inflammatory cytokines (Arg-1, IL-10, TGF-β), which were reversed by treatment at 20-Hz and white matter lesions in the peri-infarct region, callosum, and thalamus decreased [78].

Clinical studies (Table 6)

Immune pathways In one study, rTMS treatment was administered to post-stroke MDD patients and a control group. After eight weeks of treatment, serum levels of IL-1b, IL-6 and TNF-α decreased, with a more pronounced effect in patients with post-stroke (p < 0.05) [83]. rTMS not only significantly reduce the inflammatory response in patients with post-stroke MDD, which is essential to improve clinical outcomes, but also restore and strengthen immune function of PSD patients, promote overall recovery and resilience to recurrent or secondary complications. This is essential to improve clinical outcomes.

Neuronal pathways In the same study, a significant increase in dopamine, norepinephrine, and serotonin levels was detected in serum, particularly in patients with post-stroke MDD. Serum glycine and glutamate levels also increased after eight weeks of treatment. At the same time, PHQ-9 and MBI scores improved in both groups, with a greater effect in post-stroke patients. The rTMS treatment of patients with post-stroke MDD is promising, as it effectively promotes the synthesis and release of monoamine neurotransmitters, with regulation of inhibitory/excitatory balance, decreased neuroinflammation, improved immune function and clinical effects of treatment [83].

The effectiveness and precision of the cortical effects of non-invasive brain stimulation (NIBS) techniques after a stroke need further improvement, especially with respect to motor outcomes.

NIBS by modulating the facilitatory or inhibitory mechanisms, are thought to influence the spontaneous neuronal activity and cerebral plasticity [84]. Patients with a subacute stroke receiving an anodic tDCS have short- and long-term effects on postural stability and only short-term effects on trunk stability. Long-term benefits are seen in physical performance and anticipatory postural adjustments. However, there are no short- or long-term effects on quality of life [85]. Although NIBS techniques such as tDCS and rTMS can be used as complementary treatments to improve cognitive and communicational rehabilitation in patients with chronic stroke, their impact on behavioral outcomes is not yet sufficiently demonstrated [84]. Another study demonstrates the superiority of rTMS over tDCS and sham treatments to improve walking cadence, speed and functional balance, with tDCS having only a short-term effect on motor rehabilitation [86].

In contrast, deep brain neuromodulation and transcranial single-pulse magnetic stimulation (TMS) provide more precise tools to target deep brain structures while providing functional and structural assessments of cortical condition. Techniques such as transcranial time-interference stimulation (tTIS) and low-intensity focused ultrasound (LIFUS) are more recent strategies with significant potential to modulate deeper brain structures, showing strong promise for rehabilitation. In addition, motor evoked potentials (MEPs) obtained by single-pulse TMS could provide valuable prognostic information, allowing the customization of NIBS treatments to meet the specific needs of each patient [87]. Similarly, the synergistic use of bilateral rTMS and VR-BCI (a closed-loop neurofeedback with brain-computer interfaces (BCIs) and proprioceptive feedback delivered by embodied virtual reality (VR)) has demonstrated significant effectiveness in improving patient recovery. In addition, as MEPs obtained with single-pulse TMS, the electroencephalographic signal IAF (individual alpha frequency) can be used as a reliable biomarker for recovery assessment due to its strong correlation with rehabilitation outcomes [88].

Insights in neuromodulation for post-stroke conditions

DAMPs induce neuroinflammation by infiltration of immune cells and activation of signaling cascades, including NF-κB, MMPs, and oxidative stress pathways. The dynamics of non-coding RNA and sex-based differences further emphasize the need for personalized therapeutic approaches. Preclinical studies show that rTMS and VNS facilitate microglial phenotypic switching and astrocytic polarization, effectively reducing inflammation and oxidative stress while promoting neurogenesis. For example, HF-rTMS protocols at 20-Hz are effective in reducing infarction volume and restoring mitochondrial integrity. VNS activates the α7 nicotinic acetylcholine receptor, inhibits TLR4/NF-kappa B signaling and reduces pro-inflammatory markers while increasing regulatory ones. In addition, the cathodic tDCS provides neuroprotection during the sub-acute phase. Clinical studies show that rTMS reduces pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α in patients after stroke, improving neurotransmitter synthesis (dopamine and norepinephrine) and promoting functional recovery. Advanced NIBS techniques, including tTIS, LIFUS, and closed-loop systems (VR-BCI), provide superior accuracy and improved rehabilitation results.

Immunity and psychiatric and neurodevelopmental disorders

Immune factors directly influence connectivity between anatomical and functional brain circuits. They are increasingly used as diagnostic and prognostic biomarkers of therapeutic response in various psychiatric pathologies, such as bipolar disorder, major depressive disorder (MDD) and anxiety disorders, and schizophrenia [89].

Major depressive disorder (MDD)/anxiety disorders

The brain regions affected by MDD are the prefrontal cortex, amygdala, hippocampus, anterior cingulate cortex and nucleus accumbens. The brain regions affected by anxiety disorders are the amygdala, dorsolateral prefrontal cortex, anterior cingulate cortex, hippocampus, nucleus accumbens and reward system, thalamus, brainstem and autonomic nervous system, the paraventricular region of the hypothalamus (PVN).

The depression phenotype is associated with changes in myeloid and lymphoid populations due to innate and adaptive immune changes. An increase in white blood cells, granulocytes, neutrophils, monocytes, CD4⁺ helper T cells, NK cells, B cells, and activated T cells has been reported [90]. In the CSF, white blood cell counts were also high [91]. Some types of dysregulation can also be important targets for modulation therapies: the PD-1 (programmed death-1) pathway inhibits immune mediators [92], the expression of Foxp3 transcription factor associated with anxiety disorders and MDD following the activation of inflammasome [93] and the infiltration of the enteric and peripheral immune system by the inflammasome NLRP3 [94], and the kynurenine pathway, with modulation of the neuroprotective and neurodegenerative balance. This pathway explains the link between oxidative stress and depression-related inflammation through the upregulation of proinflammatory cytokines, IDO production and an increase in the KYN/TRP ratio [95]. Immune-related genes are involved in the development of MDD, particularly the interferon signaling pathways such as interferon-related genes, which have been found to be differentially methylated [96]. Five genes have been reported to be involved in treatment-resistant MDD and could be targeted by neuromodulation strategies [97]. Polymorphic immune variants regulating the cytokines gene are specifically associated with the onset of certain psychiatric disorders [98]. Significant immune-related molecular activation has been reported in MDD and understanding the signature of inflammatory mRNA could facilitate the development of neuromodulation strategies [99]. Non-coding RNAs (ncRNAs) strongly contribute to the link between neuroinflammation and MDD by regulating the evolution of MDD through neuritis. Genetic regulation is therefore an important therapeutic area [100].

Schizophrenia

The brain regions affected by schizophrenia and psychotic disorders are the prefrontal cortex, basal ganglia, limbic system, thalamus, anterior cingulate cortex, temporal cortex, parietal cortex and cerebellum. Some immunophenotypes were significantly associated with schizophrenia, such as the presence of IgD⁺ B cells, CD4⁺CD8⁺ T cells, naive CD4⁺T cells, HLA DR expressed on CD14⁻ CD16⁻ cells, CD33^dim HLA DR⁺ CD11b⁻ cells and CD4 Tregs. Exploring the links between immune cells and the genetic risk of schizophrenia could provide new therapeutic targets [101]. Microglia also undergo morphological and functional changes in schizophrenia as the disease progresses [102].

Preclinical studies (Table 8)

Immune pathways Concerning glial activation, the level of microglia (Iba1⁺ cells) was reduced following a four-week 15-Hz-rTMS protocol in a mouse model of chronic unpredictable mild stress (CUMS)-induced depression (in the CA1 and dentate gyrus (DG) areas of the hippocampus and in the prefrontal cortex (PFC)), but astrocyte levels (GFAP⁺ cells) were upregulated in the hippocampal CA1, DG, and PFC [103]. Similar results were obtained in two ECT studies: in the brain of Gunn rats with schizophrenia-like behavior and microglial activation (higher levels of CD11b and GFAP than in Wistar rats), with lowered levels of CD11b and GFAP in the hippocampus [104] after ECS (an animal alternative to ECT) and in EAE with repression of microglial neurotoxicity by downregulation of inducible NOS, Cxcl9 and Il1b expression, nitric oxide and reactive oxygen species (ROS) production, and oxidative stress in the CNS. Treated EAE mice showed fewer microglia expressing T-cell stimulatory and chemoattractant factors. ECS may reduce chronic neuroinflammation in the injured CNS by a decrease in microglial toxicity, with fewer IBA1⁺ activated microglia/macrophages and NG2⁺ oligodendrocyte-progenitor cells [105]. Treatment appeared to have no impact on microglia, in particular, the level of Iba1, and astroglial activity (GFAP and C3 d) was downregulated in the CA1, CA3, and DG in the hippocampus. As serum GFAP levels in depressed-MS patients were significantly elevated in patients with moderate to major depressive symptoms, the inhibition of neurotoxic reactive astrocytes by rTMS could be a potential therapeutic strategy for MS patients with MDD. Indeed, in an EAE mouse model, anxiety- and depressive-related behaviors were alleviated by rTMS [106].

Concerning immune regulatory pathways, levels of pro-inflammatory cytokines were decreased, including IL-6, IL-1β, and TNF-α in the hippocampus and IL-6 and IL-1β in the PFC, and the TLR4/NF-kB/NLRP3 signaling pathway was restored, following the previously cited 15 Hz-rTMS protocol [103]. In the ECS study on EAE mice mentioned above, ECS reduced neuroinflammation, spinal immune cell infiltration, demyelination and axonal loss and directly affected the CNS, with inhibition of T cell-induced neuroinflammation, without affecting the adaptive immune response. In patients, the neutrophil–lymphocyte ratio (NLR) was found to be higher in psychotic than non-psychotic MDD, and the NLR, platelet-lymphocyte ratio (PLR), and systemic immune-inflammation index (SII) were higher in patients with late-onset than early-onset MDD after ECT. There was no correlation between ECT responders/non-responders and remitters/non-remitters and blood ratios. A significant negative correlation was observed between blood ratios (PLR and SII) and depression scores in all groups, with more severe depressive symptoms being negatively associated with systemic inflammation. In addition, higher initial blood cell ratios were associated with longer orientation recovery in some patients, implying that poorer cognitive effect post-ECT was proportional to increased peripheral inflammation [107]. In an antidepressant-resistant rat model, phospho-glycogen synthase kinase 3 (GSK3β) and phospho-mTOR were increased in infralimbic and ventral hippocampal subregions, with no effect on dorsal subregions after chronic nucleus accumbens (NAc) DBS. This demonstrates that DBS has an impact on immune function through changes in the expression of mTOR, which is a major regulator of immune function (differentiation, activation and function of T lymphocytes, B lymphocytes, and antigen-presenting cells) and metabolism [108].

LPS injection was used as model of inflammation in wild-type rats and mice and in transgenic mice with inhibition of IL-6 transsignaling (sgp130 Fc mice). Vagal nerve stimulation and peripheral administration of the nicotinic a7 receptor agonist PHA543613 were used to activate the anti-inflammatory reflex (a neural circuit for regulation of the peripheral immune response). After treatment with vagal nerve stimulation (0.5 l A, 1 s stimulation at 10 Hz, every 20 s, with 500 ls bipolar stimuli during periods for an acute anti-inflammatory reflex), animals were injected with LPS and stimulated once again for 45 min. Superinfusion of IL-6 into layer V pyramidal cell slices of rat medial prefrontal cortex (mPFC) resulted in a decrease in inhibitory post-synaptic current (IPCS) amplitude in control mice, but not in sgp130 Fc mice. Thus, neuronal transsignaling is responsible for the IL-6-induced decrease in IPCS amplitude. In a similar experiment, it was shown that Janus kinase/signal transducer activator of transcription (JAK/STAT) and glycogen synthase kinase type 3b (GSK3b) signaling are both involved in preventing the inhibitory effect of IL-6 on IPCS. IPCS in mPFC may be sensitive to IL-6-dependent JAK/STAT and GSK3b signaling. As a result, LPS-induced hyperexcitability is impaired by activation of the anti-inflammatory reflex. An IL-6-dependent mechanism with the GABAergic system is involved in the decrease of sI/E in the prefrontal cortex, a mechanism that plays a critical role in hyperexcitable neuropsychiatric conditions, such as epilepsy, autism spectrum disorder, MDD, anxiety disorders, and psychosis. In addition, an increase in IPCS saturation current was observed in GFAP sgp130 Fc mice (with or without LPS). Sgp130 Fc could be an effective therapy to improve stress-induced pathological conditions caused by IL-6 [109].

Clinical studies (Table 9)

Immune pathways Concerning cytokine expression and leucocyte activation, no significant differences in serum levels of inflammatory cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-12p40, and IP-10) was observed between the TBS group (theta-burst stimulation) and the sham group of patients with MDD stimulated with 600 bursts of continuous TBS (cTBS) over the right dorsolateral prefrontal cortex (DLPFC) followed by 600 bursts of iTBS over the left DLPFC (3 days per week, with 10 sessions over 22 days) [110]. However, serum CRP levels were increased in the sham group. Patients diagnosed with bipolar disorder type I and II in a depressive episode treated with tDCS (2 mA for 30 min per day for 10 consecutive days and at week 4 and week 6) applied to the left and right dorsolateral regions of the prefrontal cortex also did not show changes in plasma levels of BDNF, IL-2, IL-4, IL-8, IL-10, IL-18, IL-33, IL-12p70, IL-17a, or INF- γ but showed a significant decrease in plasma levels of IL-8 and IL-1β with an increase in TNF, GDNF, and IL17a levels at week 6 [111]. Except for TNF-α, levels of all plasma cytokines decreased (IL2, IL4, IL6, IL10, IL17a, INF- γ) in patients with acute depression with MDD without psychotic features stimulated by the tDCS protocol of the left bifrontal and right dorsolateral prefrontal cortex (F3 and F4) (0.8 A/m² per 30 min/day) [112]. Although IL6 and TNF-α levels were elevated in depressed patients at baseline and before ECT, no significant change in CRP, IL-6, IL-10, or TNF-α levels was found after treatment, with no correlation with neurocognitive scores [113]. In patients with a severe depressive episode resistant to treatment, IP10, IL5 and IL8 levels were significantly increased in CSF after ECT. A significant difference in CSF for IL6, INF- γ, IL2R, IL-1β, and Rantes was observed before and after ECT, depending on the number of ECT sessions. Similar differences were observed in serum for IL10, IL5, and IP-10. In addition, responders showed higher levels of MIP1α and IL2R and IP10 in CSF in treatment remission. The antidepressant effect of ECT correlated with serum MCP1 levels and CSF concentrations of the IL2R and MIP1 α levels. After ECT, only IL17 levels changed in CSF between responders and non-responders and Rantes, MIP1α, IL2R, and IP10 between remitters and non-remitters. Positive correlations between serum and CSF levels were shown for IL1R α, IFN α, IL2R, and IL2 before but not after ECT [114]. ECT significantly increased cytokine production (IL-6, TNF- α) by monocytes after LPS-stimulation in patients with treatment-resistant MDD or MDD with psychotic features. CD2/CD28 levels decreased, and only IFN-γ increased. The total number of leukocytes, monocytes, granulocytes, and CD16⁺/56⁺ cells (NK cells) increased significantly and the number of CD3⁺ cells decreased after treatment. The effect lasted no longer than 30 min after the electrical stimulus. The number of B cells did not change. The number and activity of NK cells increased transiently after ECT [115]. The proportion of CD56^highCD16^–/dim and cytotoxic CD56^dim CD16⁺ NK cells was different between patients xho underwent and those who did not undergo ECT, demonstrating a different cytotoxicity of NK cells. Indeed, NK cell cytotoxicity increased after a single session of ECT and played a role in ECT quality parameters and persistent cognitive changes (observed on depression scales). Ratios of immune cell subtypes (ICR) containing NK cells changed during ECT or between remitters and non-remitters. In addition, some ICR ratios have been associated with long-term cognitive changes [116]. The effects of VNS has been studied in patients with refractory MDD, and significant reductions in anxiety disorders and MDD severity were observed, as well as decreases in IL-7, CXCL8, CCL2, CCL13, CCL17, CCL22, Flt-1, and VEGFc levels, while bFGF levels increased. VNS may have long-term effects in reducing inflammation by improving the integrity of the blood–brain barrier and limiting the recruitment of inflammatory cell [117].

Concerning immune regulatory pathways, serum concentrations of the amino-acids phenylalanine and tyrosine, precursors of neurotransmitters, decreased significantly after rTMS stimulation of the frontal polar cortex in treatment-resistant MDD patients, with a decrease in the kynurenine/tryptophan ratio(Kyn/Trp), an indicator of immune activation or tolerance. Immune regulatory circuits and serum concentrations of neopterin and nitrite changed in proportion to BDNF concentrations after treatment, showing their direct action on BDNF levels, but leucocyte counts and levels of C-reactive protein and salivary amylase were not significantly altered [118]. Comparison of the impact of ketamine and ECT on kynurenine pathway metabolism (HPA axis hyperactivity and inflammation) in patients with treatment-resistant MDD versus healthy controls showed that salivary cortisol levels were lower in the ECT cohort and that plasma kynurenine concentrations and tryptophan levels were higher in the ketamine cohort, with a decrease in kynurenic acid levels and the kynurenic acid/kynurenine ratio in both groups [119]. A study of Guloksuz et al. showed an increase in kynurenic acid levels, the KYN/TRP ratio and the kynurenic acid/3-hydroxykynurenine ratio after ECT, which may correlate with its neuroprotective and antidepressant effects [120]. In another study, cortisol/ACTH levels were found to increase after stimulation, without the repetition of ECT having an impact on the levels of any of the parameters studied. ECT induced acute immunological and neuro-endocrine changes, but these were not sustained over time, with no additional effect of repetition [115]. NFκB and the inducible nitric oxide synthase were downregulated and plasma levels of nitrites (higher in females), PGE2, and 15 dPGJ2 significantly decreased in ECT-treated patients with MDD, bipolar disorder, schizophrenia, or schizoaffective disorder. Responders showed less lipid peroxidation, observed by measuring levels of thiobarbituric acid reactive substance. Thus, ECT appears to act directly on an important canonical pathway of regulation of inflammation and the innate immune system [121]. In addition, studies have shown a higher rate of remission in women than men after ECT.

Examination of correlations between immune parameters and clinical expression showed a direct association between IL-6 levels and changes in depression scores after tDCS [111], but no correlation was reported in another tDCS protocol between plasma cytokine concentrations and depression scores in acute depressive patients with MDD without psychotic features [112]. In addition, no correlation was found between a decrease in depressive symptoms and CSF immune markers, but such a correlation was observed in serum for IP10 after ECT [114]. Following rTMS, depressive symptoms and cognitive dysfunction were improved, but there was no significant difference in serum sTREM2 levels at the beginning and end of six-week treatment in patients with MDD [122]. In addition, no association between depressive symptoms and inflammatory cytokines levels was observed after cTBS-iTBS [110]. For patients with treatment-resistant depressive episodes in the context of MDD or bipolar disorder, the levels of CSF neurodegenerative markers (AEA (anandamide), Aβ1–40, T-tau protein and P-tau, Ng) and elements of the innate immune system (sCD14, IL6, neopterin, sCD163, phosphatidylcholines, and the total amount of sphingolipids) are positively correlated with the decrease in depressive symptoms after ECT. Baseline levels of sCD14, T-tau and P-tau could significantly predict the absolute reduction of depressive symptoms. Differences were observed between responders and non-responders for neurogranin, P-tau protein, T-tau protein and sCD163 [123]. Decreased innate cellular immune activity in the CSF may cause antidepressant effect of ECT therapy. This hypothesis was corroborated by a study comparing macrophage/microglia activation markers (IL-6, neopterin, sCD14, sCD163, MIF, and MCP1) in the CSF and blood of patients with treatment-resistant depressive episode. Compared to the pre-ECT analysis, only MIF levels decreased significantly in serum after treatment. A significant positive correlation with the number of ECT sessions was found for sCD14, neopterin and MIF CSF levels of before and after treatment. In addition, baseline levels of CSF sCD14 were shown to predict the reduction of depressive symptoms after ECT. For patients in remission, there was a difference in MIF CSF levels after ECT. A number of correlations between CSF and serum were found between the markers. Finally, there was a strong correlation between CSF sCD14 and sCD163 levels [124].

Neuronal changes Correlations between neuronal, clinical, and immune markers were observed in patients treated with ECT. IL6 levels decreased significantly after treatment and are correlated with an increase in total hippocampal volume. A slight decrease in TNF-α levels was correlated with a small increase in the volume of the left hippocampus [125]. No association was found in depressed patients between BDNF, IL-6, and TNF-α levels and the MADRS (depression scale) scores after ECT, but the effect of BDNF on the MADRS score was dependent on the level of TNF-α and the interaction between TNF-α and BDNF was significant. Higher levels of TNF-α made the relationship between BDNF and MADRS scores increasingly negative, while lower levels of TNF-α eliminated the relationship between BDNF and MADRS scores [126]. Correlations between IL8 levels and changes in the FAt (reflection of longitudinal and neurotrophic changes in white matter microstructure) were significant in the right upper longitudinal fasciculus and right cingulum II following ECT in responders, demonstrating their involvement in the therapeutic effect of ECT. A number of correlations have been observed in the left cingulum II and the left uncinate fasciculus [127]. ECT modulates the immune system and improves neurotrophin production. The impact of ECT on DNA methylation of gene regions coding for tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1), involved in the production of BDNF, has been studied in immune cell subtypes (B cells, monocytes, natural killer cells, and T cells) in treatment-resistant patients with MDD. No difference in DNA methylation by t-PA or PAI-1 was observed between ECT remitters and non-remitters, while major changes in DNA methylation of the t-PA regions of NK cells, T lymphocytes, B cells and monocytes were observed throughout the treatment. These changes were independent of clinical outcomes and identical throughout treatment [128]. Sadeck et al. proposed a theory linking ATP and purinergic signaling to P2X, P2Y, and P1 receptors. Neurons and glia secrete ATP during ECT. This pathway may have an impact on depressive symptoms by modulating excitatory transmitters and dopamine release. An increase in P2X7 receptor expression in glial cells would increase the secretion of cytokines, chemokines and neurotrophins. In addition, sequential ECT may have an impact on the expression of purinoceptors in mesolimbic and mesocortical regions involved in MDD and mood disorders [129].

Insights in neuromodulation for MDD and anxiety disorders

Various immune populations, including CD4 + T cells, NK cells, B cells and monocytes, are significantly elevated, along with an increase in the number of white blood cells in the CSF. The activation of the inflammatory pathways is influenced by inhibition of the PD-1 pathway, activation of the NLRP3 inflammasome and alterations in the kynurenine pathway that impact on the balance between neuroprotective and neurodegenerative processes. Differentially methylated immune-related genes and polymorphic immune variants are also associated with MDD, while non-coding RNAs (ncRNAs) play a role in the regulation of depression-related neuroinflammation. Biomarkers such as cytokines, kynurenine levels and gene expression could help guide neuromodulation strategies, offering a promising route to target neurotoxic astrocytes and microglia. rTMS, ECT and VNS reduce inflammation by modulating immune pathways and cytokine levels (IL-6, IL-1β, TNF-α), correlated with improvements in MDD and anxiety disorders. rTMS and ECT influence the immune regulation pathways, with a decrease in microglial activation, changes in peripheral blood ratios and in immune markers such as CRP, IL-6 among others, associated with reduced symptoms in MDD and anxiety disorders. Concentrations of IL-7, CXCL8, and VEGFc decreased, confirming the role of inflammation in the pathophysiology of these conditions.

Insights in neuromodulation for schizophrenia

A strong association was identified with IgD + B cells, CD4 +/CD8 + T lymphocytes, naive CD4 + T lymphocytes and other subtypes of immune cells, as well as morphological and functional changes in microglia. Therapeutic strategies should focus on the interaction between immune cells and schizophrenia genes. Preclinical studies show that rTMS and ECS therapies modulate levels of microglia, astrocytes, and cytokine, thus reducing neuroinflammation. Vagal nerve stimulation and nicotinic receptor agonists activate anti-inflammatory pathways, particularly affecting IL-6 signaling. JAK/STAT and GSK3β are involved in the prevention of IL-6-induced decreases in inhibitory post-synaptic currents. In clinical studies, cytokine expression levels, such as IL-6 and TNF-α, vary with therapies such as tDCS, ECT and rTMS, but no consistent relationship was established between changes in depressive symptoms and plasma cytokine levels. However, positive correlations were observed between neurotrophin production (BDNF) and improvements in depression scales. In addition, high cytotoxicity of NK cells is associated with better cognitive and depressive outcomes after ECT. IL-6 and TNF-α downregulation are correlated to hippocampal volume growth after ECT. ECT also has immunomodulatory effects, as demonstrated by changes in sCD14, neopterin and kynurenine levels. A notable sex-specific effect has been identified, with higher remission rates observed in women after ECT. In addition, ECT has an impact on the methylation of DNA in immune cell subtypes, although this effect appears to be independent of clinical outcomes. Sequential ECT influences purinergic signaling, which may play a role in mood regulation.

Discussion

Communication between the brain and the immune system is essential for the therapeutic effect of neuromodulation. The brain is wired to detect immune signals and, in return, neurotransmitters exert an immunostimulatory or immunosuppressive effect on central and peripheral immune cells. This interaction involves multiple immune pathways, including the endocrine pathway, and the effects of a neurotransmitter may vary depending on receptor profile, signaling targets, and basal immune activation in the brain [130]. Additionally, the gut-brain axis in each of these pathologies may play a role in influencing neuromodulation strategies, and it represents an interesting complementary element that could further be considered [131].

Peripheral and central immune cells (T lymphocytes, NK cells, microglia) have critical functions in neuroinflammation associated with neuropathogenesis, and genetic factors may specifically influence immune responses. Neuromodulation therapies, such as rTMS, FUS, aVNS and DBS have a significant impact immune functions, reducing neuroinflammation by targeting pathways like PI3 K/Akt/NFkB and decreasing the pro-inflammatory cytokines TNF-α and IL-6. However, chronic inflammation contributing to neurodegeneration, such as tau aggregation in AD, highlights the importance of precisely balancing the protective and damaging roles of inflammation [3].Neuromodulation treatments also have the potential to improve synaptic function, neurogenesis, dopaminergic activity and even motor and cognitive rehabilitation, especially through therapies such as DBS and FUS-BBBD. But a blind field remains in terms of the risks and uncertainty surrounding their long-term effects.

Despite their potential, these therapies are limited. Accurately targeting inflammation remains a challenge, as do concerns about long-term adverse effects such as cortical atrophy and astrogliosis associated with FUS-BBBD. Techniques such as pFUS + MB can trigger sterile acute inflammatory responses, resembling ischemic or traumatic brain injuries with a disturbing iatrogenic potential. There is also mechanistic uncertainty: although some molecular pathways have been identified, the precise mechanisms of action of these therapies require further exploration. The great complexity of immune system regulation, influenced by specific genetic factors, highlights the challenge of standardizing therapies to adapt to each patient’s individual response.

This review highlights a little-explored yet fundamental field in neuromodulation to ensure that future studies will be based on a combined analysis of brain and immune responses with immune pathways targets (cytokine expression, glial activation, and lymphocyte response). These protocols should examine the genetic and phenotypic profiling of immune responses with the patient’s baseline immune status, data on peripheral and cerebral immunity and the sex/age to optimize therapeutic response according to disease stage and immune dynamics. They will involve precise and targeted stimulation patterns, with the adaptation of frequency, impedance, rhythmand current density to generate an optimal therapeutic beneficial neuroimmune response. The combination of neuromodulation with pharmacological approaches, such as monoclonal antibodies, offers a potential for synergistic effects in neuroprotection and immune regulation. Additionally, targeting shared immune pathways in neurodegenerative diseases—such as cGAS-STING and NAD metabolism, Jak/STAT and MAPK, HMGB1, kynurenine/IDO, SIRT/mTOR, TGFβ/SMAD and FoxP3, TLR 4/NF-kappa B and TNFα and α7 nicotinic acetylcholine receptor, Wnt/beta-catenin—can facilitate broad-spectrum neuromodulation strategies [132].

Early intervention in specific temporal and spatial windows could effectively halt or slow down pathological processes, supported by biomarkers that reflect the stage of the disease and its progression. These processes are therefore specific to each individual and pathology. Future neuromodulation studies should focus on preferred immune pathways to determine the most effective therapeutic approaches.

DBS holds particular promise for devastating NDDs, such as autism spectrum disorder or dystonia. Deciphering the interaction between innate and adaptive immune systems, neurotransmitters and neural circuits after neuromodulation in these debilitating conditions should be the next step to advance our understanding of neuro-immune circuits in age-related diseases [133, 134].

One of the most important and impactful questions that emerge from this work, which focuses on immune and neural pathways, is how neuromodulation techniques affect ECM. This is a major issue that could profoundly influence research on neuromodulation, offering substantial potential to improve the understanding and application of these techniques and ultimately achieve genuine therapeutic efficacy. Dynamic interactions between inflammation, tissue structure, neurocircuits and immune responses will be crucial in designing effective interventions for neuroinflammatory and neurodegenerative conditions, ultimately promoting neuronal repair and improving long-term outcomes. The study of the impact of neuromodulation techniques on the ECM currently presents a major technical and scientific challenge, requiring the development of new research strategies. This could be possible through the implementation of technologies based on artificial intelligence and combining high-precision data for optimal neurostimulation strategies.

Appendix

See. Tables 1, 2, 3, 4, 5, 6, 7, 8, 9.

Table 1 Summarizing the data of all preclinical studies in PD specifying the design, objectives, methodology, results, and conclusion

Table 2 Summarizing the data of all clinical studies in PD specifying the design, objectives, methodology, results, and conclusion

Table 3 Summarizing the data of all preclinical studies in AD specifying the design, objectives, methodology, results, and conclusion

Table 4 Summarizing the data of all preclinical studies in MS specifying the design, objectives, methodology, results, and conclusion

Table 5 Summarizing the data of all preclinical studies in post-stroke specifying the design, objectives, methodology, results, and conclusion

Table 6 Summarizing the data of all clinical studies in post-stroke specifying the design, objectives, methodology, results, and conclusion

Table 7 Summarizing the data of all preclinical studies in TBI specifying the design, objectives, methodology, results, and conclusion

Table 8 summarizing the data of all preclinical studies in anxiety disorders/MDD/schizophrenia specifying the design, objectives, methodology, results, and conclusion

Table 9 summarizing the data of all clinical studies in anxiety disorders/MDD/schizophrenia specifying the design, objectives, methodology, results, and conclusion