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Background:
Systematic Review

The Role of Microglial Activation in the Pathogenesis of Drug-Resistant Epilepsy: A Systematic Review of Clinical Studies

by
Abba Musa Abdullahi
1,*,
Shah Taha Sarmast
2 and
Usama Ishaq Abdulrazak
3
1
Department of Life Sciences, University of South Wales, Pontypridd CF48 1DL, UK
2
Chi Mercy Hospital Roseburg, Roseburg, OR 97470, USA
3
Children’s Emergency Department, Leicester Royal Infirmary, University Hospitals of Leicester NHS Trust, Leicester LE1 5WW, UK
*
Author to whom correspondence should be addressed.
BioChem 2025, 5(4), 43; https://doi.org/10.3390/biochem5040043 (registering DOI)
Submission received: 5 July 2025 / Revised: 26 October 2025 / Accepted: 6 November 2025 / Published: 1 December 2025
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Abstract

Background: Microglial cells are the resident immune cells in the central nervous system (CNS) and constitute the brain’s innate immune system. They are the smallest of the glial cells and are derived from phagocytic white blood cells, fetal monocytes, which migrate from the blood into the brain during development. On the other hand, epilepsy is a chronic condition defined as recurrent unprovoked seizures, with at least two seizures occurring over 24 h apart. Methods: To determine the role of microglial activation in the pathogenesis of drug-resistant epilepsy, we systematically searched published data for biomarkers of microglial activation from main databases including PubMed, PubMed Central, Scopus, Embase, Google Scholar, and Medline. Two research registries were also searched: the Cochrane Registry and clinicaltrial.gov. Data was collected after applying inclusion and exclusion criteria and studies were appraised critically. Both Medical Subject Headings (MeSH) and regular keyword search strategies were employed. Results: Our systematic review shows significant elevation of biomarkers of microglial activation in patients with drug-resistant epilepsy, suggesting its role in the disease’s pathogenesis. Conclusions: Microglia cells are therefore considered as a special type of mononuclear phagocytes found in the CNS that plays important roles in both the brain’s immunity and homeostatic functions. The role of microglial activation in the pathogenesis of drug-resistant epilepsy is an active area of study, with potential therapies for drug-resistant epilepsy that target microglia currently being investigated.

1. Introduction

Epilepsy is a long-term neurological disorder that impacts millions globally and is associated with high morbidity and mortality. It is defined by the International League Against Epilepsy (ILAE) as a chronic disorder of the brain characterized by an enduring disposition towards recurrent unprovoked seizures. The condition is diagnosed when a person has had at least two unprovoked seizures more than 24 h apart, or one unprovoked seizure along with abnormal findings on an EEG or brain imaging scan that indicate a high probability of a second seizure [1]. Globally about 50 million people have epilepsy, with virtually 80% of them living in developing countries where access to medical services is grossly inadequate. An estimated 2.4 million people are diagnosed with epilepsy each year [2]. A major and serious issue in epilepsy care is the emergence of drug-resistant epilepsy (DRE), which the ILAE defines as ‘the failure of adequate trials of two tolerated and appropriately chosen and used AED schedules (whether as monotherapies or in combination) to achieve sustained seizure freedom’ [3]. Approximately 30% of people with epilepsy develop drug resistance, which can result in early death, brain injury, or a decline in quality of life [4]. Numerous studies have indicated that neuroinflammation—particularly neuroinflammation driven by the activation of microglia—plays a key role in both the onset and persistence of DRE [5].
Microglial cells serve as the central nervous system’s (CNS) resident immune cells. They are the smallest type of glial cells and originate from fetal monocytes—phagocytic white blood cells that migrate into the brain during early development. As such, they are regarded as a specialized form of mononuclear phagocyte unique to the CNS [6]. Microglial cells exist in two states: a resting branched (ramified) form and an active, amoeboid form. In normal conditions, they adopt a ramified shape with long extensions used to monitor their environment, support neural development, and maintain CNS homeostasis. When activated by factors like seizures, trauma, infection, or the build-up of harmful substances, they enlarge and shift into an amoeboid, phagocytic form [7]. In this state, they release a cascade of inflammatory mediators and clear pathogens, waste, and damaged cells through phagocytosis. These mediators include pro-inflammatory cytokines (like IL-1β, TNF-α, and IL-6), chemokines (like CCL2 and CXCL10), and markers of oxidative stress like reactive oxygen species (ROS). These molecules can disrupt neuronal function by increasing neuronal excitability, altering synaptic plasticity, inducing cell death and gliosis, and weakening blood–brain barrier (BBB) integrity [8]. Inflammatory cytokines influence the function of neurotransmitter receptors and ion channels. For example, IL-1β increases calcium entry through NMDA receptors, contributing to excitotoxic damage. Both IL-1β and TNF-α enhance excitatory glutamate signaling while reducing inhibitory GABAergic activity, leading to increased neuronal excitability [9,10]. These cytokines also compromise the blood–brain barrier (BBB), making it more permeable and allowing immune cells and inflammatory molecules from the bloodstream to enter the CNS, thereby perpetuating and amplifying local inflammation [11]. Moreover, microglial cells are involved in aberrant synaptic pruning, disrupting normal neural connections and promoting seizure recurrence. Persistent inflammation and excitotoxicity can ultimately cause neuronal damage and death, further strengthening the network of hyperexcitable neurons associated with epilepsy [10].
Microglial activation contributes significantly to the development of drug-resistant epilepsy (DRE) by promoting a pro-inflammatory environment that increases neuronal excitability and undermines the effectiveness of antiepileptic drugs (AEDs). In DRE, microglia remain persistently activated, leading to perpetual cycles of neuroinflammation, creating a vicious cycle of repeated seizures and brain damage [12]. Many researchers have identified several ways in which chronic inflammation may reduce AED efficacy. Firstly, by increased efflux of transporters, cytokines such as IL-1β stimulate the production of P-glycoprotein (P-gp) at the blood–brain barrier and in astrocytes, which actively pump AEDs out of brain tissue, limiting their therapeutic action [13,14]. Secondly, by receptor and ion channel alteration, inflammatory mediators interfere with GABA and glutamate receptor function, which are primary targets for many AEDs, thereby reducing their drug-binding abilities and effectiveness [15]. Thirdly, through sustained epileptogenic networks, chronic microglia-induced inflammation preserves hyperexcitable neural circuits that resist drug treatment [7]. Therefore, neuroinflammation is a key factor in the development of drug resistance in epilepsy. Identifying peripheral biomarkers that reflect the severity of neuroinflammation may help determine which patients could benefit from anti-inflammatory therapies.
There is currently no systematic review, to the best of our knowledge, which explores the role of microglial activation in the development of DRE. However, we found two systematic reviews in PROSPERO that were related to but not similar to our review. The first one was a study by Keezer et al. (available at https://www.crd.york.ac.uk/PROSPERO/view/CRD42016051814, accessed on 20 August 2025) entitled ‘The burden and predictors in drug-resistant epilepsy: a systematic review and meta-analysis’. This study is different from ours as it examined the incidence and prevalence of drug-resistant epilepsy in people with epilepsy and also assessed the predictors and correlates of drug resistance in people with epilepsy. The second one was a study by Yow et al. (available at https://www.crd.york.ac.uk/PROSPERO/view/CRD42024560628, accessed on 20 August 2025) entitled ‘Exploring the metabolic biomarkers in pharmacoresistant epilepsy: A systematic review’. This study mainly examined the metabolic biomarkers of pharmacoresistance, while our study examines neuroinflammatory biomarkers that are specific to microglia. The aim of our study is to systematically investigate how microglial activation contributes to the pathogenesis of DRE, with the hope of paving ways for new therapeutic strategies for patients with refractory epilepsy.

2. Materials and Methods

2.1. Study Design and Protocol

We conducted a systematic review of the published literature on the role of microglial activation in the pathogenesis of drug resistance in epilepsy involving a synthesis of both graphical and narrative information. The protocol employed for this study was based on the ‘Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA)” guidelines [16]. The research team prepared the review protocol, which was then reviewed by two experts in the field who were not part of the research team. The review protocol was prepared and registered on PROSPERO (the International Prospective Register Of Systematic Reviews) “Ref-2025: CRD420251136116” and is available online at https://www.crd.york.ac.uk/PROSPERO/view/CRD420251136116 (accessed on 20 August 2025).

2.2. Sources of Data Collection and Search Strategy

We systematically searched major electronic databases for published data including PubMed, PubMed Central, Scopus, Embase, Google Scholar, and Medline. Two research registries comprising the Cochrane Central Register (Cochrane Library, all issues; www.cochranelibrary.com accessed 15 August 2024) and clinicaltrial.gov (www.clinicaltrials.gov, accessed on 20 August 2024) were searched for relevant clinical trials. A search of the reference lists of the included articles for related articles was conducted and effort was made to search for unpublished studies by contacting experts. Both MeSH and regular keyword search strategies were used for the identification of the relevant articles. The MeSH strategy was mainly used for searching PubMed, PubMed Central, and Medline while both MeSH and regular keywords were employed for searching the other databases.

2.3. Search Content

The following terms were used for the identification of relevant articles: “neuroinflammation” AND “drug resistant epilepsy” AND “human” AND “clinical trials”, “neuroinflammation” AND “intractable epilepsy” AND “human” AND “clinical trials”, “neuroinflammation” AND “pharmacoresistance” AND “human” AND “clinical trials”, “cytokines” AND “drug-resistant epilepsy” AND “human” AND “clinical trials”, “chemokines” AND “drug-resistant epilepsy” AND “human” AND “clinical trials”, “oxidative stress markers” AND “drug-resistant epilepsy” AND “human” AND “clinical trials”, “reactive oxygen specie” AND “drug-resistant epilepsy” AND “human” AND “clinical trials”, “high mobility group box 1 protein” AND “drug-resistant epilepsy” AND “human” AND “clinical trials”, “toll like receptor 4” AND “drug-resistant epilepsy” AND “human” AND “clinical trials”.

2.4. Inclusion and Exclusion Criteria

The following inclusion criteria were used: (1) Any clinical study on the role of microglial activation in the pathogenesis of drug-resistant epilepsy. (2) Studies performed on microglia-induced neuroinflammation. (3) Studies performed exclusively on humans. (4) Studies performed exclusively on DRE or pharmacoresistance. (5) Any studies measuring the biomarkers of microglial activation in patients with DRE through imaging, immunohistochemistry, serum, or CSF analysis. (6) Studies published in English or translated into English. Any study that did not meet the above inclusion criteria was excluded.

2.5. Population

We included patients with drug-resistant epilepsy as defined by ILAE regardless of their age, sex, or country of origin.

2.6. Intervention/Exposure

The exposure here was microglial activation. This includes studies measuring the biomarkers of microglial activation through imaging, immunohistochemistry, serum, or CSF analysis of cytokines, chemokines, or oxidative stress markers.

2.7. Comparison

Patients with drug-resistant epilepsy who had neuroinflammatory biomarkers of microglial activation were compared with patients with drug-responsive epilepsy who did not have evidence of neuroinflammation.

2.8. Outcome

We evaluate evidence of microglial involvement in the pathogenesis of drug resistance among the study population. This includes assessing the correlation between markers of neuroinflammation related to microglial activation and perpetuation of seizure frequency or severity.

2.9. Data Extraction

We extracted our data from the included studies using a standard data extraction form and the information extracted included the following factors: authors’ details, publication year, sample size, demography of the study population, microglia-specific molecules, and neuroinflammatory markers analyzed in the study. These markers mainly included pro-inflammatory cytokines, chemokines, oxidative stress markers, HMGB1 protein, and TLR-4.

2.10. Level of Evidence

The level of the evidence was assessed using the Scottish Intercollegiate Guidelines Network (SIGN) grading system.

2.11. Quality Assessment

The CASP Checklist (2018) was used to assess the methodological quality and validity of the included studies. The checklist contains questions that assess the validity and effectiveness of the study results. A paper with a score of >7 was considered as a high quality paper; 5–7, a moderate quality paper; and <5 was considered as a low quality paper. Out of the 15 included studies, 13 studies had a score of >7, and the remaining 2 studies had a score between 5 and 7.

2.12. Risk of Bias Assessment

The Newcastle–Ottawa Scale (NOS) was used for the risk of bias assessment. The risk of bias was determined based on type of study. For case–control studies, risk of bias was assessed based on the following domains: independent selection of cases and control from the same population, homogeneity between cases and control, well-defined data collection method as either prospective or retrospective, and use of objective measures of exposure, identification and control of confounders. For cohort studies, risk of bias was determined using the following domains: assessment of comparability between the exposed and unexposed group, sufficient follow-up period, rate of loss to follow-up and/or drop-out in both exposed and unexposed groups, and use of appropriate instrument for measuring the exposure. For cross-sectional studies, the risk of bias was evaluated using the following domains: appropriate sampling method and use of valid measures in assessing exposure and outcome. Each domain was graded as high, moderate, or low, with information from the study paper justifying our judgment.

3. Results

3.1. Literature Search

A comprehensive search of multiple electronic databases yielded 418 articles: 21 from PubMed, 74 from PubMed Central, 102 from Medline, 50 from Scopus, 115 from Google Scholar, 51 from Embase, 3 from the Cochrane Database Registry, and 2 from clinicaltrials.gov. After removing 5 duplicates, 413 articles remained. Of these, 25 were selected for full-text review based on abstract screening, while the remaining 388 were excluded as irrelevant. Following a detailed review, only 15 articles met the inclusion and exclusion criteria and were included in the systematic review; the other 10 were excluded. All 15 included studies underwent critical appraisal and were assessed as high quality. A flowchart summarizing the study selection process is presented in Figure 1.

3.2. Study Characteristics

A total of 15 studies met the inclusion criteria for this review. Of these, seven focused solely on pro-inflammatory cytokines [17,18,19,20,21,22,23], two on chemokines [24,25], two on oxidative stress markers [26,27], and two on both HMGB1 and TLR-4 [28,29]. One study examined both pro-inflammatory cytokines and chemokines [30], while another investigated cytokines alongside TLR-4 [31]. In the cytokine studies, the combined sample size was 331 participants, including 153 in the case group (78 males and 75 females) and 178 in the control group (90 males and 88 females), with participant ages ranging from 6 to 60 years. The chemokine studies included 87 participants: 42 in the case group (24 males and 18 females) and 45 in the control group (21 males and 24 females), aged 0–18 years. For oxidative stress markers, 129 participants were involved: 38 in the case group (26 males and 12 females) and 91 in the control group (59 males and 32 females), aged 20–61 years. In the HMGB1–TLR-4 group, 277 participants were included: 70 in the case group and 207 in the control group, with a total of 149 males and 128 females, aged 0–40 years. Across all studies, the case groups consisted of patients with drug-resistant epilepsy (DRE), while the control groups included individuals with drug-responsive epilepsy or healthy volunteers.

3.3. Risk of Bias Assessment

Overall, selection bias, recall bias, and confounding bias were assessed in the included case–control studies. Selection bias, attrition bias, and information bias were assessed in the included cohort studies. Selection bias, information bias, and confounding bias were assessed for the included cross-sectional studies. The included case–control studies clearly explained independent selection of cases and controls from a similar population, with both cases and control being similar in all aspects. Well-defined data collection methods were explained with objective measures of exposures. Additionally, important confounders were identified in most of the studies. Similarly, in the cohort studies, both exposed and unexposed groups were comparable across most studies and sufficient follow-up periods were used as well as appropriate use of instruments for measuring the exposure of interest. In the cross-sectional studies, a well-defined sampling method was used, along with the use of valid and reliable measures in assessing both the exposure and outcome. Therefore, we graded the included studies as having low risk of bias.

3.4. Pro-Inflammatory Cytokines

In this review, we identified nine studies that met our inclusion criteria focusing on pro-inflammatory cytokines: seven focused exclusively on cytokines, one examined both cytokines and chemokines, and one explored cytokines alongside TLR-4. Eight were case–control studies, and one was a cohort study. The cytokines identified across these studies included IL-1, IL-1β, IL-4, IL-5, IL-6, IL-7, IL-10, IL-17A, and TNF-α. IL-4, IL-5, and IL-7 were assessed only in plasma samples [21,30]. IL-1 and IL-17A were analyzed only in brain tissue [30]. The remaining cytokines, IL-1β [17,18,19,22,31], IL-6 [22,30], IL-10 [21,31], and TNF-α [19,20,21,22], were examined in both plasma and brain tissue samples. Across most studies, levels of all pro-inflammatory cytokines were significantly higher in DRE patients compared to controls, regardless of sample type. This consistent overexpression suggests these cytokines may play a role in the pathogenesis of drug-resistant epilepsy. However, two exceptions were observed: In one study [19], IL-1β levels were lower in DRE patients. This may be due to the younger age of the participants, where IL-1β might act as a protective factor rather than a pro-inflammatory one. In another study [23], TNF-α levels were reduced, potentially reflecting neurodegeneration from long-standing disease, as the patients had lived with DRE for over 20 years. Table 1 below summarizes the key findings for pro-inflammatory cytokines.

3.5. Chemokines

Our search identified three studies that met the inclusion criteria for investigating chemokines in drug-resistant epilepsy (DRE): one cross-sectional study, one case–control study, and one cohort study. Two studies focused solely on chemokines, while one examined both chemokines and cytokines. The chemokines reported were CCL2/MCP-1 and CX3CL1 [24]; CCL11, CCL2, and CCL4 [25]; and CCL5 [30]. All chemokines were analyzed using plasma samples, except for CCL5, which was measured in brain tissue. Most studies found elevated chemokine levels in patients with drug-resistant seizures compared to drug-responsive controls. However, in the study by Gakharia et al. [25], CCL2 and CCL4 levels did not differ significantly between the study groups. Overall, the consistent elevation of several chemokines across studies suggests a potential role in the pathogenesis of DRE. Table 2 below summarizes the key findings for chemokines.

3.6. Oxidative Stress Markers

To evaluate the role of oxidative stress in the development of drug-resistant epilepsy (DRE), we identified two studies that met our inclusion criteria: one retrospective case–control study [26] and one prospective case–control study [27]. Both studies examined three categories of oxidative stress markers: the first category is the reactive oxygen species (ROS) markers, including superoxide anion (O2−); the second category is the antioxidant enzyme activity markers, including superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), and glutathione reductase (GR); and the third category is the biomolecular damage markers, such as lipid peroxidation and DNA oxidation. In the first study, brain tissue samples were analyzed histologically. All three oxidative stress markers were significantly elevated in DRE patients compared to controls. Specifically, superoxide anions (O2−) were the only elevated ROS marker, catalase was the only antioxidant enzyme which significantly increased, and DNA oxidation was the only elevated marker of biomolecular damage [26]. However, this study also reported a significant decrease in GPx levels, possibly due to the analytical technique used. No significant differences were observed in SOD activity or lipid peroxidation. In the second study, oxidative stress markers were measured in blood samples. Here, two of the three marker categories showed significant elevation in DRE patients: lipid peroxidation (biomolecular damage) and SOD activity (antioxidant enzymes). Glutathione levels were similar between DRE patients and controls [27]. The consistent elevation of oxidative stress markers in both brain tissue and blood samples of DRE patients supports a strong link between oxidative stress and the pathogenesis of drug-resistant epilepsy. Table 3 below summarizes the key findings for oxidative stress markers.

3.7. High Mobility Group Box 1 (HMGB1) Protein

We identified two studies that met our inclusion criteria for the role of HMGB1 in DRE, one cohort study [28] and one case–control study [29]. In both studies, the HMGB1 was analyzed using serum samples from the participants. In both studies, the serum levels of HMGB1 were significantly higher in drug-resistant groups compared with drug-responsive groups [28,29]. These findings suggest a strong correlation between elevated HMGB1 expression and the development of drug resistance in epilepsy. Table 4 below summarizes the key findings for HMGB1 protein and TLR-4.

3.8. Toll-Like Receptor 4 (TLR-4)

We identified three studies that met our inclusion criteria for examining the role of TLR-4 in drug-resistant epilepsy (DRE): one cohort study [28] and two case–control studies [29,31]. Two studies [28,29] analyzed serum samples, while one study [31] examined brain tissue histologically. All three reported significantly higher levels and expression of TLR-4 in DRE patients compared to controls, suggesting a potential role of TLR-4 in the pathogenesis of drug resistance in epilepsy. Table 4 above summarizes the key findings for TLR-4. Figure 2 below illustrates key biomarkers of microglial activation that play roles in the pathogenesis of drug-resistant epilepsy.

4. Discussion

This review demonstrates a notable upregulation of inflammatory mediators associated with microglial activation in patients diagnosed with drug-resistant epilepsy (DRE). Of the fifteen studies included, twelve were case–control studies, two were cohort studies, and one was cross-sectional. In terms of biological sample analysis, seven studies focused exclusively on serum, five on brain tissue, one investigated both serum and tissue, and two analyzed both serum and cerebrospinal fluid (CSF). To ensure consistency, a primary inclusion criterion was a specific focus on DRE, referred to in some studies as refractory or pharmacoresistant epilepsy. Despite this diagnostic uniformity, considerable heterogeneity was observed in study populations with respect to sample size, age, duration of DRE, and the methodologies used for sample analysis. These methodological and demographic differences may account for the low variability in reported levels of certain inflammatory mediators. For example, Panina et al. reported significantly reduced concentrations of TNF-α in patients with DRE compared to controls [23], a finding potentially attributable to neurodegenerative changes associated with a prolonged disease course (greater than 20 years in this cohort).
Epilepsy is a significant trigger of microglial activation, leading to morphological changes in microglia, which adopt an enlarged, amoeboid, and phagocytic phenotype. This transformation is accompanied by the release of various inflammatory mediators into the neuronal microenvironment, including pro-inflammatory cytokines, chemokines, oxidative stress markers, lipid mediators, and proteins. These factors collectively disrupt cerebrovascular function and enhance neuronal hyperexcitability by modulating neurotransmitter receptors and ion channels [32]. Numerous studies have reported positive correlations between serum concentrations of interleukins IL-4, IL-8, and IL-17 and both the frequency and severity of seizures, suggesting a central role for cytokines in the pathogenesis of drug-resistant epilepsy (DRE) [33,34]. IL-1β, primarily produced by activated microglia, is typically expressed at low levels in the healthy brain. However, in response to brain injury such as that induced by seizures, IL-1β levels rise significantly, promoting acute inflammation. When overexpressed, IL-1β contributes to increased neuronal excitability and persistent neuroinflammation, establishing a self-perpetuating cycle of seizures and neuronal damage [35]. Elevated peripheral IL-1β levels may therefore serve as a biomarker of seizure severity and resistance to treatment [36]. Similarly, IL-6, another pro-inflammatory cytokine, facilitates the secretion of chemokines and adhesion molecules involved in neuronal survival and differentiation. Overexpression of IL-6 in the brain has been linked to widespread neuroinflammation, blood–brain barrier disruption, aberrant hippocampal activity, recurrent spontaneous seizures, and progressive neurodegeneration. These effects may underlie seizure refractoriness and pharmacoresistance in DRE [37,38]. TNF-α, secreted by activated astrocytes and microglia, also contributes to the inflammatory cascade. It elevates microglial glutamate release, increases intracellular calcium levels, and induces GABA receptor endocytosis, thereby reducing inhibitory signaling. This dual effect on excitatory and inhibitory pathways may exacerbate neuronal hyperexcitability and interfere with antiepileptic drug (AED) efficacy by altering drug targets and reducing binding affinity [37]. Our systematic review identified several studies that associate elevated serum TNF-α with DRE, reinforcing its potential role as a biomarker of disease severity and pharmacoresistance.
In addition to pro-inflammatory cytokines, microglial cells produce chemokines—signaling molecules that mediate communication between immune cells in the brain and the peripheral immune system. These chemokines regulate neuronal excitability by controlling neurotransmitter release through voltage-dependent or G-protein-coupled channels. They also facilitate immune cell entry into the brain via specific G-protein-coupled receptors (GPCRs) [39,40]. Most chemokines and their receptors contribute to neuroinflammation by promoting neuroglial activation and interacting with peripheral monocytes [37]. CCL2, also known as monocyte chemoattractant protein-1 (MCP-1), is a potent monocyte attractant and a key mediator linking peripheral inflammation to neuronal hyperexcitability [41]. Multiple studies have shown increased levels of CCL2/MCP-1 in patients with pharmacoresistant epilepsy, along with significant upregulation of CCL2/MCP-1, CCL3, and CCL4 in hippocampal sclerosis samples compared to controls [24]. Fractalkine (CX3CL1), a transmembrane chemokine secreted by neurons and primarily expressed on microglia, has also been implicated in epilepsy pathogenesis and may serve as a biomarker for pharmacoresistance [42,43]. Immunohistochemical studies of brain tissue from patients undergoing surgery for refractory seizures have shown high expression of chemokine–receptor pairs—including CCL2, CCL3, CCL4, and CCL11—in hippocampal tissue [44]. Together, these findings, along with the results of our systematic review, suggest that chemokines play a critical role in drug-resistant epilepsy (DRE).
Oxidative stress (OS) is a biochemical condition caused by an imbalance between reactive oxygen species (ROS) and the body’s antioxidant defenses. This imbalance—often involving oxidants, nitrosative stress, and reduced antioxidant capacity—disrupts redox regulation, interferes with cellular signaling, and leads to molecular damage [27]. OS is implicated in both normal physiological processes and various diseases, including epilepsy, where it contributes to neuronal injury [45]. A major ROS involved in OS is the superoxide anion (O2−), which plays roles in both healthy and pathological processes [46]. It is rapidly converted by superoxide dismutase (SOD) into hydrogen peroxide (H2O2), which is then detoxified into water by catalase or glutathione peroxidase (GPx)—the latter requiring glutathione. Cells naturally produce baseline levels of O2− and H2O2 during regular metabolism [47,48]. In the brain, mitochondria are the main source of O2− under normal conditions, but microglial cells also significantly contribute [49,50,51]. In drug-resistant epilepsy (DRE), microglial cells become activated and adopt a pro-inflammatory phenotype, producing ROS, including superoxide anions, as part of their immune response. A large body of evidence links oxidative stress to both the onset of seizures and the development of resistance to antiepileptic drugs. This is supported by observed changes in antioxidant enzyme activity [26,27] and increased levels of oxidative damage biomarkers such as malondialdehyde (MDA), protein carbonyls, and 8-hydroxy-2-deoxyguanosine, as well as the activation of NADPH oxidase [26,27]. Therefore, based on the findings of our systematic review where oxidative stress markers were found to be elevated in patients with DRE, we posit that these markers played significant roles in the pathogenesis of DRE.
Epileptic seizure is an important cause of neuronal injury and can occur as a result of excitotoxicity due to excessive and synchronized neuronal firing, oxidative stress and seizure-related chronic neuroinflammation, leading to neurotoxicity and cell loss. High Mobility Group Box 1 (HMGB1) is a nuclear non-histone protein and one of the damage-associated molecular patterns (DAMPs) that is released from injured tissues [52,53]. TLR-4 is an innate immune system receptor expressed on microglia that recognizes DAMPs like HMGB1. When HMGB1 is released from injured neuronal tissues into the extracellular space, it binds to TLR-4 receptor on microglia, forming the HMGB1-TLR-4 axis. This binding triggers microglial activation and consequently release of pro-inflammatory cytokines, leading to neuroinflammation [54,55]. The HMGB1-TLR4 signaling pathway plays a crucial role in promoting neuronal hyperexcitability by enhancing the phosphorylation of N-methyl-D-aspartate receptors (NMDARs) and increasing calcium channel permeability. This pathway also contributes to the upregulation of P-glycoprotein (Pgp), a blood–brain barrier (BBB) transporter found in drug-resistant epilepsy regions, which actively pumps anti-seizure medications out of the brain [56]. Moreover, activation of the HMGB1-TLR4 axis can compromise the integrity of the BBB, allowing immune cells, antibodies, inflammatory cytokines, and albumin from the peripheral blood to enter the central nervous system, further intensifying inflammation and contributing to refractoriness [57]. Elevated levels of HMGB1 and its receptor TLR4 have been observed in the hippocampus of both epileptic patients and animal models. Studies have revealed that both HMGB1 and TLR4 contribute to the occurrence and persistence of seizures [58,59,60]. Furthermore, few studies investigated the effects of HMGB1 and TLR4 on drug resistance in patients with epilepsy [61]. The results of our review demonstrated the role of these molecules in the pathogenesis of DRE.
Our study focused solely on the role of microglia without delving into the roles of other neuroglial cells like astrocytes in the pathogenesis of drug-resistant epilepsy because of their distinct primary functions in neuroinflammation based on its origin and specialization. While all glial cells contribute to the inflammatory response, microglial cells are the central nervous system’s (CNS) primary immune cells, whereas astrocytes and others primarily serve homeostatic and support roles that become altered during inflammation [62]. Microglial cells are the brain’s resident macrophages, originating from the embryonic yolk sac. They are the principal innate immune cells and the first responders to any pathological insult, infection, or injury in the CNS. Their primary function during neuroinflammation is to act as a direct line of defense, thereby initiating the neuroinflammatory response that initiates the process of DRE [63]. Unlike microglia, other neuroglial cells like astrocytes are not primarily immune cells. Their main role is to maintain CNS homeostasis. In neuroinflammation, their response is typically a secondary, but crucial, reaction to signals from microglia and other sources of damage. Therefore, they might not play a significant role in the initiation of DRE but instead contribute to sustenance of the process [64].
The role of microglial activation in the pathogenesis of drug-resistant epilepsy is the main topic of this review. The study examines the markers of microglial activation in patients with DRE. Our study is not without limitation. One of the major limitations is the supportive role played by other neuroglial cells, especially astrocytes as highlighted above, which also secrete some of these mediators and markers during epilepsy activity in the brain. Additionally, small sample size in some of the studies may increase the risk of both type I and type II errors. However, putting other studies with much larger sample sizes together may mitigate this risk significantly. Similarly, the younger age of some of the participants may influence the interpretation of our results, as certain chemokines may be elevated in children with DRE due to developmental immune differences, whereas the same markers may not be relevant in adults. Differences in sample collection (blood vs. tissue), processing, and analytical techniques (ELISA and immunohistochemistry) can lead to variable sensitivity, specificity, and quantification of biomarkers, which may affect the interpretation of our finding. These factors can yield conflicting findings even when examining the same targets. Therefore, this study acknowledges the need for more studies on the possibility of superimposed functions and additive roles of other neuroglial cells in the pathogenesis of DRE as well as the possibility of cell-to-cell interference in neuroinflammation. Similarly, to have robust and more reproducible findings, additional studies should be conducted using homogeneous studies in terms of sample size, patients’ demography, and methodological analysis. As some studies have shown promising results and good safety profiles for use of anti-pro-inflammatory biomarkers like anti-IL-1 and anti-IL-6, one of the main consequences of our study is the possible consideration of these microglial biomarkers and their mechanisms in causing refractoriness as therapeutic targets in DRE.

5. Conclusions

In this study, microglial activation was demonstrated to play an important role in the pathogenesis of DRE. We identified a number of neuroinflammation-related biomarkers that explain how microglial activation is connected to the epileptogenesis of DRE and how those markers could be important in the early detection of DRE. Such biomarkers could also be suggested as potential therapeutic targets for DRE. However, the currently available literature that we were able to examine on this topic does not show conclusive evidence on the critical role of microglial activation in the pathogenesis of DRE. However, it is clear that microglial activation has the ability to modulate neuronal excitability and contributes to perpetual cycles of neuroinflammation, which may explain refractoriness and pharmacoresistance observed in patients with DRE.

Author Contributions

Conceptualization, A.M.A. and S.T.S.; Formal analysis, A.M.A., S.T.S. and U.I.A.; Investigations, A.M.A. and S.T.S.; Methodology, A.M.A. and S.T.S.; Writing Original Draft, A.M.A., S.T.S. and U.I.A.; Review and Editing, A.M.A., S.T.S. and U.I.A. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Informed Consent Statement

Not applicable.

Data Availability Statement

All the articles used in the study were fully cited and referenced and can be traced easily. DOI were used if available.

Conflicts of Interest

The authors declare no conflicts of interest.

References

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Biochem 05 00043 g001
Figure 1. Flowchart of Study Selection Process.
Figure 1. Flowchart of Study Selection Process.
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Figure 2. The key biomarkers of microglial activation that play roles in the pathogenesis of drug-resistant epilepsy.
Figure 2. The key biomarkers of microglial activation that play roles in the pathogenesis of drug-resistant epilepsy.
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Table 1. The key findings for pro-inflammatory cytokines.
Table 1. The key findings for pro-inflammatory cytokines.
Study DesignSample Size (DRE/Control)Biomarkers StudiedAnalytical MethodMain Findings
Case–control (n = 8) and Cohort (n = 1)331 (153/178) combinedIL-1, IL-1β, IL-4, IL-5, IL-6, IL-7, IL-10, IL-17A, and TNF-αVaries (plasma and brain tissue analysis)Most studies showed significantly higher levels of pro-inflammatory cytokines in DRE patients. Exceptions include one study with lower IL-1β and another with lower TNF-α, possibly due to participant age and disease duration, respectively.
Table 2. The key findings for chemokines.
Table 2. The key findings for chemokines.
Study DesignSample Size (DRE/Control)Biomarkers StudiedAnalytical MethodMain Findings
Cross-sectional (n = 1), Case–control (n = 1), and Cohort (n = 1)87 (42/45) combinedCCL2/MCP-1, CX3CL1, CCL11, CCL4, and CCL5Varies (plasma and brain tissue analysis)Most studies found elevated chemokine levels in DRE patients compared to controls, except for one study where CCL2 and CCL4 levels were not significantly different.
Table 3. The key findings for oxidative stress markers.
Table 3. The key findings for oxidative stress markers.
Study DesignSample Size (DRE/Control)Biomarkers StudiedAnalytical MethodMain Findings
Retrospective Case–control (n = 1) and Prospective Case-control (n = 1)129 (38/91) combinedReactive oxygen species (O2−), antioxidant enzymes (SOD, catalase, GPx, GR), and biomolecular damage markers (lipid peroxidation, DNA oxidation)Varies (brain tissue and blood sample analysis)Both studies showed a significant elevation in some oxidative stress markers. One study found elevated O2−, catalase, and DNA oxidation in brain tissue, while the other found elevated lipid peroxidation and SOD activity in blood samples.
Table 4. The key findings for HMGB1 protein and TLR-4.
Table 4. The key findings for HMGB1 protein and TLR-4.
Study DesignSample Size (DRE/Control)Biomarkers StudiedAnalytical MethodMain Findings
HMGB1: Cohort (n = 1) & Case–control (n = 1)277 (70/207) combinedHigh Mobility Group Box 1 (HMGB1)Serum sample analysisBoth studies reported significantly higher serum HMGB1 levels in DRE patients, suggesting a strong correlation with drug resistance.
TLR-4: Cohort (n = 1) & Case–control (n = 2)277 (70/207) combinedToll-Like Receptor 4 (TLR-4)Varies (serum and brain tissue analysis)All three studies found significantly higher levels and expression of TLR-4 in DRE patients, suggesting a role in the pathogenesis of drug resistance.
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Abdullahi, A.M.; Sarmast, S.T.; Abdulrazak, U.I. The Role of Microglial Activation in the Pathogenesis of Drug-Resistant Epilepsy: A Systematic Review of Clinical Studies. BioChem 2025, 5, 43. https://doi.org/10.3390/biochem5040043

AMA Style

Abdullahi AM, Sarmast ST, Abdulrazak UI. The Role of Microglial Activation in the Pathogenesis of Drug-Resistant Epilepsy: A Systematic Review of Clinical Studies. BioChem. 2025; 5(4):43. https://doi.org/10.3390/biochem5040043

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Abdullahi, Abba Musa, Shah Taha Sarmast, and Usama Ishaq Abdulrazak. 2025. "The Role of Microglial Activation in the Pathogenesis of Drug-Resistant Epilepsy: A Systematic Review of Clinical Studies" BioChem 5, no. 4: 43. https://doi.org/10.3390/biochem5040043

APA Style

Abdullahi, A. M., Sarmast, S. T., & Abdulrazak, U. I. (2025). The Role of Microglial Activation in the Pathogenesis of Drug-Resistant Epilepsy: A Systematic Review of Clinical Studies. BioChem, 5(4), 43. https://doi.org/10.3390/biochem5040043

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