Status epilepticus, a seizure lasting longer than 30 minutes or a series of seizures without recovery between them, is one of the most dangerous emergencies in neurology. When it is not brought under control, it can permanently damage the brain, disrupt the blood–brain barrier, and inflame the hippocampus, the structure that anchors learning and memory. Even with aggressive rescue treatment, 30 to 40 percent of patients go on to develop cognitive impairment as the condition evolves into a chronic epileptic state.
In this protocol, rats receive lithium chloride followed by pilocarpine 18 hours later, with atropine given beforehand to blunt peripheral cholinergic side effects. Repeated low doses of pilocarpine were administered until animals reached stage 4 or 5 seizures on the Racine scale, and diazepam terminated the seizures 60 minutes after onset.
Western blotting of hippocampal tissue collected one, three, and seven days after seizure induction revealed a coordinated shift in both pathways. Gasdermin D, the executioner protein of canonical pyroptosis, rose significantly on day one and climbed further on days three and seven, with P values of 0.030, 0.010, and 0.000 respectively compared with controls. Glutathione peroxidase 4, or GPX4, the central enzyme that consumes glutathione to neutralize toxic lipid peroxides and thereby acts as a primary brake on ferroptosis, moved in the opposite direction, progressively declining across the same three time points with P values of 0.015, 0.001, and 0.000.
To test whether Gasdermin D sits upstream of both processes, the team delivered an adeno-associated virus carrying GSDMD-targeting RNA interference directly into the hippocampus through stereotaxic injection, using coordinates from a rat brain atlas. Control animals received a scrambled AAV construct. One week after viral delivery, the rats were subjected to the lithium-pilocarpine protocol, producing four experimental groups: sham, status epilepticus alone, status epilepticus plus control RNAi, and status epilepticus plus GSDMD RNAi, each containing 30 animals. Western blotting confirmed the intervention worked, showing a substantial reduction of GSDMD protein in the knockdown group relative to the control-RNAi group, with a P value of 0.002.
The neuroprotective effect was visible at the cellular level. Nissl staining, which labels the rough endoplasmic reticulum of viable neurons, showed that status epilepticus caused a dramatic loss of Nissl-positive cells compared with sham-operated rats, with a P value of 0.000. In the GSDMD knockdown group, the number of surviving neurons rose markedly, also reaching P = 0.000 in comparison, while the control-RNAi group showed no rescue at all, with a P value of 0.969 indicating no difference from untreated status epilepticus animals.
The knockdown also quieted the inflammatory output of pyroptosis. Quantitative reverse transcription polymerase chain reaction revealed that messenger RNA levels of the cytokines interleukin-1β, interleukin-6, and tumor necrosis factor-α surged in the hippocampus after status epilepticus, with P values of 0.009, 0.003, and 0.007 versus sham. After GSDMD silencing, all three cytokines fell significantly relative to both the untreated seizure group and the control-RNAi group. Against the control-RNAi comparison, the reported P values were 0.021 for IL-1β, 0.010 for IL-6, and 0.015 for TNF-α. This matters mechanistically because in the classical pyroptosis pathway, inflammasome-activated caspase-1 cleaves GSDMD into a pore-forming fragment and simultaneously matures the precursors of IL-1β and IL-18, which then escape through the membrane pores to fuel inflammation and tissue injury.
Strikingly, the same intervention also dampened ferroptosis. Prussian blue staining exposed heavy iron deposition in the hippocampus of seizure animals compared with shams, and GSDMD knockdown significantly reduced that deposition relative to both the seizure group and the control-RNAi group. Enzyme-linked immunosorbent assay showed that malondialdehyde, a toxic aldehyde generated during lipid peroxidation, was substantially elevated after status epilepticus and dropped sharply after knockdown, while reduced glutathione, the antioxidant cofactor consumed by GPX4, was depleted after seizures and rebounded after knockdown. GPX4 protein itself, which had fallen after seizures, rose again in the knockdown group relative to both comparison groups.
The functional consequences showed up in behavior. In the spontaneous-activity test, which counts how many grid lines a rat crosses in five minutes, seizure animals moved far less than shams, and GSDMD knockdown significantly increased crossings relative to both the untreated and control-RNAi seizure groups, with P values of 0.000 in each comparison. The novel object-recognition test, a hippocampus-dependent memory task in which rats explore a familiar object and a new one, told a similar story. The discrimination index, calculated from the difference in exploration time between the new and old objects, collapsed in seizure animals but rose significantly after GSDMD knockdown, again with P values of 0.000 against both comparison groups. Control-RNAi animals were indistinguishable from untreated seizure rats in both tasks.
Why would a pyroptosis protein influence ferroptosis at all? The authors propose an indirect route mediated by inflammation. Inflammatory cytokines released during pyroptosis are not merely waste products; prior work shows they can actively induce ferroptosis. For example, studies cited in the paper report that activation of the NLRP3/JAK2/STAT3 pathway increases lipid peroxidation and disrupts antioxidant balance in asthma, that the NLRP3 inhibitor MCC950 reverses both neuronal pyroptosis and ferroptosis after cerebral ischemia–reperfusion, and that interleukin-1β can suppress the cystine transporter xCT in endothelial cells, cutting glutathione synthesis and thereby promoting ferroptosis. The authors also point to converging evidence from other disease models, including diabetic cardiomyopathy, where NLRP3 knockdown simultaneously suppressed pyroptosis and ferroptosis, and from psoriasis, where GSDMD knockdown inhibited the NLR pathway and improved lesion severity.
The team is careful about the limits of the work. The precise mechanism linking the two cell death pathways remains unresolved, and the authors acknowledge that inflammatory and mTOR pathways may contribute indirectly but are unconfirmed in this study. Sample sizes were small, with three rats per group for most molecular experiments and four per group for behavioral testing, and the authors call for larger studies to strengthen reliability and generalizability. The lithium-pilocarpine model, though extensively validated, does not fully reproduce the complexity of human status epilepticus, so any therapeutic strategy targeting GSDMD would need validation in more clinically relevant models before translation.
Subject of Research: Gasdermin D knockdown and pyroptosis-ferroptosis crosstalk in a rat model of status epilepticus
Article Title: Knockdown of Gasdermin D protects hippocampal neurons by regulating both pyroptosis and ferroptosis in a rat model of status epilepticus
Article References: Feng, F., Wang, H., Tian, Y., Luo, R., & Cai, Q. (2026). Knockdown of Gasdermin D protects hippocampal neurons by regulating both pyroptosis and ferroptosis in a rat model of status epilepticus. Acta Epileptologica, 8(1), Article 20. https://doi.org/10.1186/s42494-026-00255-5
Image Credits: AI Generated
DOI: 10.1186/s42494-026-00255-5
Keywords: status epilepticus, Gasdermin D, pyroptosis, ferroptosis, hippocampus, neuroinflammation, GPX4, lithium-pilocarpine model, cognitive function, AAV RNA interference, neuronal survival, epilepsy
News Source: Cassandra Pierce. (October 30, 2026). Silencing Gasdermin D Shields Hippocampal Neurons in Rat Status Epilepticus. Scienmag.
