Abstract
Thergarajan P, Al-Hobaish G, Sutherland G, Tsantikos E, Jupp B, Haskali MB, Casillas-Espinosa PM, Hibbs ML, O’Brien TJ, Ali I, Jones NC. Brain Behav Immun. 2026 Jan;131:106183. doi: 10.1016/j.bbi.2025.106183. Epub 2025 Nov 13. PMID: 41241085. Background: Neuroinflammation is implicated in epilepsy pathogenesis, and microglia are key immune cells of the brain that participate in neuroinflammatory responses associated with epilepsy. This study investigated the role of early microglial activation following an epileptogenic brain injury on the incidence and severity of epilepsy and associated neurobehavioral impairments in a model of acquired epilepsy. Methods: Status epilepticus (SE) was induced in male C57BL/6 mice via electrical stimulation of the ventral hippocampus, while additional mice were enrolled as sham controls (n = 125 total). Following termination of SE, mice received injections of the colony-stimulating factor 1 (CSF1) receptor inhibitor PLX5622 (PLX; 50 mg/kg IP twice daily) to suppress microglial activation caused by SE, or vehicle, for seven days. At the end of treatment, the effect of microglial suppression on the neuroinflammatory response to SE was characterised using gene expression, immunohistochemistry, and flow cytometry. Additional mice were followed for four months and underwent a series of neurobehavioral tests and epilepsy assessment. Results: PLX treatment significantly reduced Iba1 + cell counts, reduced GFAP + immunoreactivity, and downregulated the expression of proinflammatory cytokines in the hippocampus compared to vehicle following SE, intimating that the neuroinflammatory response of SE was suppressed by PLX. Flow cytometry revealed that SE significantly reduced microglial expression of CX3CR1 and CD206, but increased expression of CD16/32, shifting microglia towards a pro-inflammatory state. However, PLX treatment did not influence the relative expression of these genes. In the chronic stage, SE mice treated with PLX exhibited improved spatial memory (Y-maze test: p = 0.0016) and reduced depressive-like behavior (tail suspension test: p = 0.04; sucrose preference: p = 0.14) compared to vehicle-treated SE mice. However, PLX treatment did not alter the incidence of epilepsy after SE (58% in vehicle-treated mice vs 50% PLX treated mice; p = 0.65) or seizure frequency in epileptic animals. Conclusion: Suppression of microgliosis with PLX eliminates the neuroinflammatory response after SE, and this is associated with prevention of long-term behavioral impairment. However, this intervention does not influence the development of epilepsy. These results demonstrate that acquired epilepsy and its behavioral comorbidities have different pathogenic mechanisms after SE, with early microglial driven neuroinflammation most relevant to the latter.
Commentary
Microglia are the resident immune cells of the central nervous system (CNS), with microglia being critical to normal brain development, synaptic plasticity, and immune surveillance. They participate in a variety of homeostatic functions in the healthy adult CNS and facilitate damage response and repair under pathological conditions. 1 For example, microglia sense changes in their surrounding environment to facilitate normal synaptic signaling, clear debris in response to neuronal and cellular damage, and prune neuronal synapses necessary to maintain normal homeostasis within the brain. In the context of an acute brain injury or epileptogenic insult (ie, status epilepticus [SE]), microglia will be the first to respond to the site of injury, transforming their morphology from a surveying phenotype defined by elongated processes into a reactive, amoeboid-like morphology characterized by an enlarged cell body and thickened processes. Reactive microglia release inflammatory cytokines and reactive oxygen species. Yet the role of microglia in response to a neurological insult and inflammatory cytokine release (such as interferon-γ) is dynamic: microglia transition between distinct phases of inflammatory response with distinct morphology, to a deactivation state, and finally morphing into a tissue repair phase. Inflammatory response prompts microglia to initiate tissue repair and suppress transcriptional induction of inflammatory signals, followed by activation of tissue repair signals until resolution. Notably, however, is that activated microglia continue to increase in numbers throughout the period after a neurological insult, but critically, these cells cannot be microscopically or immunohistochemically differentiated from peripherally invading macrophages. 2 Thus, microglia activation in discrete brain regions, such as the hippocampus, reflects a dynamic and transitional response to neuronal damage or damage-associated molecular signals until resolution of the insult. Yet the precise role that microglial activation plays in the early inflammatory cascade after an epileptogenic insult, such as traumatic brain injury or SE, as well as how microglia contribute to the onset and severity of the constellation of pathophysiological features of epilepsy, including the spontaneous recurrent seizures (SRS) and chronic behavioral deficits, is less clear.
Thergarajan and colleagues set out to address this question and assess whether pharmacologically disrupting microglial activation immediately after an SE insult could change the neuroinflammatory and neuropathological damage immediately after SE, modify the onset of SRS, and influence the development of neurobehavioral deficits weeks later using a mouse model of electrically evoked self-sustaining SE insult. 3 Using the colony-stimulating factor 1 (CSF1) receptor inhibitor, PLX5622 (50 mg/kg, intraperitoneal [IP]), the group assessed whether suppression of microglial activation immediately after an SE insult would lead to any functional or neuropathological shifts in biomarkers of epilepsy in mice up to 15 weeks post-SE. 3 PLX5622 is a specific and selective small molecule inhibitor of CSF1, a receptor tyrosine kinase expressed on CNS microglia, that normally regulates their proliferation and survival, such that inhibiting CSF1 provides an opportunity to study the discrete microglial contributions to the postinjury pathological cascade, including epileptogenesis. PLX5622 can elicit concentration-related improvements in cognitive function and reduce microglial response in other neurological disease models, 4 but the specific impact in acquired epilepsy models is less well studied. 5 Thus, Thergarajan et al. tested how twice-daily IP PLX5622 administration (50 mg/kg) for the 7 days after SE insult in young adult (8 weeks old at study initiation) male mice influenced onset of neuropathology, microglial activation, and immune system inflammatory cytokine expression 7 days later, as well as behavioral comorbidity deficits at 9 weeks post-SE and SRS presentation from 11 to 15 weeks post-SE. This present study altogether provides intriguing insight into the role that microglia play in shaping the severity of neuropathological damage acutely after SE insult and chronic timing of neurobehavioral deficits weeks later. The authors found that PLX5622 administration led to generally reduced microglial and astroglial reactivity, regardless of SE status (main effect of PLX5622 treatment), but that this treatment generally suppressed SE-induced increases in proinflammatory gene expression. These acute effects were also expanded to investigate microglial phenotypes using flow cytometry, demonstrating significant shifts in proinflammatory gene expression profiles. There were surprising increases in the microglial activation marker, CD-11b, but the group failed to report CD-11b high/CD-45 low cell sorting profiles to differentiate CNS-resident microglia from infiltrating macrophages. 6 Thus, it is unclear whether this observed shift in microglial activation arose due to macrophage infiltration from the periphery in response to shifts in blood–brain barrier permeability with SE insult. 7 Nonetheless, the study provides interesting insight into the acute response by microglia to SE.
Yet acute neuropathological damage after SE was not the only physiological shift that the investigators assessed, considering that SE is a well-established method to evoke epileptogenesis and behavioral deficits long-term in laboratory rodents. Following SE, the surviving mice were allowed to recover for 9 weeks before undergoing a battery of behavioral tests to assess changes in anxiety-like behavior, spatial working memory, and anhedonia, showing that PLX5622-treated SE mice generally had attenuated behavioral deficits. However, a major gap in the experimental design is that the behavioral testing occurred prior to confirmation of SRS frequency and severity via video electroencephalogram (vEEG). In the absence of this confirmation, it is difficult to know whether the SRS burden in the two experimental groups was similar such that behavioral comorbidity deficits would be similar. Further, there was no investigation of the latency to SRS onset in the two post-SE groups using vEEG, which is a useful metric for disease modification studies in other post-SE rodent models. 8 Thergarajan and colleagues then monitored SRS frequency and duration from 11 to 15 weeks post-SE via vEEG, finding no difference in SRS frequency or duration between experimental groups, leading to the conclusion that microglial activation post-SE is necessary for behavioral comorbidities of epilepsy, but not SRS onset. Specifically, the group concluded that when neuroinflammation post-SE insult is suppressed, epilepsy development, as measured by vEEG-recorded SRS activity, is unaffected.
While there are unquestionably several gaps in the experimental design and interpretation of study findings, the implications of this work suggest that a more detailed investigation into the discrete pathophysiological features of epilepsy is necessary. Epilepsy is classically defined by increased neuroinflammation, and anti-inflammatory treatments are increasingly being investigated for symptomatic and disease-modifying benefit. Microglial response and activation play a key role in mediating the response to neurological insults, thus leaving them in a privileged position for pharmacological targeting to modify epilepsy trajectory. Yet, how microglia and associated neuroinflammation contribute to the trajectory of nonseizure symptoms of epilepsy is less well defined. Preclinical models of acquired epilepsy that are defined by inflammatory cytokine release or an autoimmune-mediated signaling cascade leading to microglial activation can indeed exhibit cognitive and neuropsychological deficits that more closely mimic the etiological origins of epilepsy around the world. 9 Future studies must therefore expand on the present observations by Thergarajan and colleagues to more thoroughly scrutinize how microglial activation across a diversity of epilepsy models shifts disease trajectory and response beyond the SRS alone. Only then can we come closer to developing and establishing impactful interventions to target the underlying disease biology and pathological symptoms that are often more detrimental to the patient's quality of life than the seizures themselves. 10
