Inflammation modifies breathing phenotype in mice with epilepsy

Inflammation modifies breathing phenotype in mice with epilepsy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Inflammation modifies breathing phenotype in mice with epilepsy Amol Mohan Bhandare, Adwoa Boaten, Dylan Dunkwu, Jade Hill, Biborka Balazs, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7707048/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 06 Jan, 2026 Read the published version in Journal of Neuroinflammation → Version 1 posted 10 You are reading this latest preprint version Abstract Impaired CO₂ responsiveness in epilepsy can result in hypoventilation and hypercapnia and these respiratory disturbances are key contributors to Sudden Unexpected Death in Epilepsy (SUDEP). While mild to moderate inflammation is known to modulate respiratory function, its specific role in regulating respiratory responses in the context of epilepsy remains unclear. We studied the effects of lipopolysaccharide (LPS)-induced glial priming and microglial inhibition via minocycline during the acute and chronic phases of epilepsy on hypercapnic ventilatory responses (HCVR) in the intrahippocampal kainic acid model of temporal lobe epilepsy in male C57BL/6 mice. LPS treatment during acute seizures and minocycline during spontaneous seizures in the chronic phase of epilepsy restored the impaired HCVR in mice. Notably, LPS treatment during acute seizures also reduced the frequency of spontaneous seizures. In contrast, minocycline given during acute seizures and LPS administered during chronic epilepsy further exacerbated HCVR impairment. Immunohistochemical analysis of chemosensitive retrotrapezoid nucleus (RTN) revealed varied effects of different treatments in epileptic mice on microglia density, morphology and their expression of triggering receptor expressed on myeloid cells 2 (TREM2), P2Y12 receptor, and astrocytic adenosine 2A receptor (A2AR). Overall, inflammation, along with associated changes in microglial and astrocytic receptor expression, plays a central role in the reduction of HCVR in epilepsy and may represent a key mechanistic target in SUDEP. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Epileptic seizures, characterized by neuronal hyperexcitability, disrupt brain networks and function, and are associated with significant comorbidities in people with epilepsy such as cognitive impairment and sudden unexpected death in epilepsy (SUDEP) 1 – 3 . While the molecular mechanisms underlying SUDEP remain unclear, emerging evidence implicates postictal breathing irregularities - ether brain-mediated 4 , 5 or due to airway obstruction 6 - and abnormal slow heartbeats occurring immediately after seizures significantly alter breathing or heart function 7 leading to SUDEP. The proposed mechanisms include brain-driven alterations in cardiorespiratory control 4 , 6 , 8 – 15 , genetic factors such as Kv1.1 potassium channel deficiency 7 , 16 , and the involvement of neurotransmitters like serotonin 17 , 18 and adenosine 19 , 20 , as well as glial cell activity 21 . Targeting of serotonin and adenosine pathways has shown promise in preventing seizure-induced respiratory arrest in animal models 19 , 22 , and inflammation-driven microglial activation can modulate these pathways 23 , 24 . Microglia play a dual role in epilepsy 25 , 26 , where acute activation is protective 9 , 27 , while chronic activation contributes to pathology 28 , 29 . Despite these insights, the roles of inflammation, glial activation, and serotonin-adenosine signalling in the mechanisms underlying impaired breathing in epilepsy remain unclear, and SUDEP incidence continues to be high. Studies have demonstrated impaired CO₂-induced respiratory responses in both epileptic rats 30 and humans following generalized convulsive seizures 31 . Our own research has shown that mice with chronic epilepsy exhibit reduced hypercapnic ventilatory responses (HCVR), correlating with altered activity of chemosensitive neurons of the retrotrapezoid nucleus (RTN) 5 , 32 . Inflammation, while essential for defence against injury and infection, can become detrimental in chronic neurological conditions as shown in Alzheimer’s 33 and Parkinson’s disease 34 . Neuroinflammation modifies breathing responses in animal models 23 , 35 , and in the context of epilepsy, these responses could contribute the mechanism of SUDEP. We studied the role of inflammation and microglial activation in the development of breathing phenotype in epilepsy using a kainic acid (KA)-induced mouse model of temporal lobe epilepsy. Systemic lipopolysaccharide (LPS) and oral minocycline - a non-specific microglial activation inhibitor - were administered at two distinct time points. LPS treatment during acute seizures (early-LPS) and minocycline treatment during chronic epilepsy (late-mino) was beneficial in preventing development of breathing phenotype in mice with epilepsy. In contrast, LPS treatment during chronic epilepsy (late-LPS) and minocycline during acute seizures (early-mino) exacerbated the phenotype. Given the central role of microglia and astrocytes in neuroinflammatory responses, we performed immunohistochemical analysis of chemosensitive neurons and glial cells in the RTN 32 . Our findings revealed that in epileptic group: there was elevated microglial density compared to epileptic mice treated with early LPS and both late- and early-mino groups; reduced triggering receptor expressed on myeloid cells 2 (TREM2) expression compared to control; and increased astrocytic adenosine A2A receptor (A2AR) expression compared to epileptic mice treated with early LPS. Notably, microglial P2Y12 expression did not change in any groups. Although C1q, a key mediator of synaptic pruning, is typically upregulated in inflammation 36 and epilepsy 37 , its expression remained unchanged or was reduced in minocycline- and LPS-treated epileptic mice. Our results, therefore, hint at a possible link of microglial density, TREM2, and astrocytic A2AR expression to the emergence of disordered breathing in epilepsy where acute inflammation appears protective, in contrast to its detrimental role in chronic epilepsy. Besides microglia priming with LPS during acute seizures also reduced spontaneous seizure frequency. Findings highlight the therapeutic potential of targeting inflammation and inhibiting microglial activation in chronic epilepsy, which mitigate respiratory dysfunction and potentially help to reduce the risk of SUDEP. Results Inflammation during acute seizures and inhibition of microglia activation during chronic epilepsy rescues development of breathing phenotype in mice with epilepsy. Changes in breathing responses to increased CO 2 (3 and 6%) were studied at week-3, -5 and -7 after induction of status epilepticus (SE) using intrahippocampal injection of KA in mice. We replicated the previous findings 5 , where epileptic mice showed reduced tidal volume (V T ) at room air and their responses to increased CO 2 were also reduced after induction of epilepsy ( Fig. 2B ). In epileptic mice, V T in room air was reduced at week-3 (0.87 µL/g), -5 (0.89 µL/g) and -7 (0.95 µL/g) post-SE compared to pre-SE (1.07 µL/g). Breathing responses were also reduced at week-3 (0.98 µL/g at 3% and 1.34 µL/g at 6%), -5 (1.08 µL/g at 3% and 1.50 µL/g at 6%) and -7 (1.11 µL/g at 3% and 1.62 µL/g at 6%) compared to pre-SE responses (1.29 µL/g at 3% and 1.74 µL/g at 6%). The slope to breathing response was maximally reduced at week-3 post-SE (58% and 25% reduction at 3 and 6% CO 2 , respectively, compared to pre-SE) ( Fig. 2I and Supplementary Fig. 1 ). Both the tidal volume in room air and the responses to increased CO 2 were recovered and were similar to pre-SE V T in the group of epileptic mice treated with early-LPS and minocycline during chronic seizures (late-mino). Breathing responses in early-LPS group were at week-3 (1.03 µL/g at room air, 1.23 µL/g at 3% and 1.71 µL/g at 6%), -5 (1.05 µL/g at room air, 1.20 µL/g at 3% and 1.62 µL/g at 6%) and -7 (1.04 µL/g at room air, 1.21 µL/g at 3% and 1.68 µL/g at 6%) compared to pre-SE responses (1.08 µL/g at room air, 1.37 µL/g at 3% and 1.82 µL/g at 6%) ( Fig. 2C ) and in late-mino group were at week-3 (1.01 µL/g at room air, 1.22 µL/g at 3% and 1.61 µL/g at 6%), -5 (1.01 µL/g at room air, 1.21 µL/g at 3% and 1.63 µL/g at 6%) and -7 (0.97 µL/g at room air, 1.19 µL/g at 3% and 1.64 µL/g at 6%) compared to pre-SE responses (1.08 µL/g at room air, 1.28 µL/g at 3% and 1.71 µL/g at 6%) ( Fig. 2D ). There was no major change in the slope of breathing responses to increased CO 2 at any time points in late-mino group, but early-LPS group showed 48% reduction in the slope of breathing response to 3% CO 2 at week-5 post-SE ( Fig. 2I and Supplementary Fig. 1 ). Contrarily, group of mice treated with early-minocycline and late-LPS exacerbated the breathing phenotype. In the mice treated with early-minocycline V T in room air was reduced at week-3 (0.94 µL/g), -5 (0.99 µL/g) and -7 (0.92 µL/g) post-SE compared to pre-SE (1.10 µL/g). Breathing responses were also reduced at week-3 (1.15 µL/g at 3% and 1.60 µL/g at 6%), -5 (1.13 µL/g at 3% and 1.53 µL/g at 6%) and -7 (1.12 µL/g at 3% and 1.58 µL/g at 6%) compared to pre-SE responses (1.41 µL/g at 3% and 1.89 µL/g at 6%) ( Fig. 2E ). The slope of breathing responses was maximally reduced at week-5 (55% at 3% CO 2 compared to pre-SE) ( Fig. 2I and Supplementary Fig. 1 ). Similarly in mice treated with late-LPS V T in room air was reduced at week-3 (0.80 µL/g), -5 (1.03 µL/g) and -7 (0.94 µL/g) post-SE compared to pre-SE (1.19 µL/g). Breathing responses were also reduced at week-3 (1.07 µL/g at 3% and 1.51 µL/g at 6%), -5 (1.36 µL/g at 3% and 1.68 µL/g at 6%) and -7 (1.35 µL/g at 3% and 1.66 µL/g at 6%) compared to pre-SE responses (1.51 µL/g at 3% and 2.08 µL/g at 6%) ( Fig. 2F ). The slope of breathing responses was equally reduced at week-5 and 7 (44% at 6% CO 2 compared to pre-SE) ( Fig. 2I and Supplementary Fig. 1 ). In control mice (intrahippocampal PBS) treated with early-LPS, we did notice reduction in V T in room air and reduced responses to 3% and 6% CO 2 at week-5. In control early-LPS treated mice V T in room air was at week-3 (0.99 µL/g), -5 (0.88 µL/g) and -7 (0.99 µL/g) post-SE compared to pre-SE (1.11 µL/g). Breathing responses were at week-3 (1.26 µL/g at 3% and 1.70 µL/g at 6%), -5 (1.14 µL/g at 3% and 1.43 µL/g at 6%) and -7 (1.35 µL/g at 3% and 1.82 µL/g at 6%) compared to pre-PBS responses (1.32 µL/g at 3% and 1.93 µL/g at 6%) ( Fig. 2G ). The slope of breathing responses was increased at 3% CO 2 at all time points (72% increase at week-7) but was maximally reduced at 6% CO 2 week-5 (52% at 6% CO 2 compared to pre-PBS) ( Fig. 2I and Supplementary Fig. 1 ). The reduction in breathing responses in control mice treated with early-LPS could be due to the impact of inflammation on the brainstem breathing circuitry and previous findings are in lined with our observations 23,35 . We did not notice any changes to breathing responses or room air V T in control mice treated with late-mino. In control late-mino treated mice V T in room air was at week-3 (1.00 µL/g), -5 (0.96 µL/g) and -7 (0.96 µL/g) post-SE compared to pre-SE (1.00 µL/g). Breathing responses were at week-3 (1.34 µL/g at 3% and 1.76 µL/g at 6%), -5 (1.33 µL/g at 3% and 1.73 µL/g at 6%) and -7 (1.25 µL/g at 3% and 1.73 µL/g at 6%) compared to pre-PBS responses (1.35 µL/g at 3% and 1.87 µL/g at 6%) ( Fig. 2H ). The slope of breathing responses was not much affected at any time point ( Fig. 2I and Supplementary Fig. 1 ). The breathing frequency (F R ) in mice did not change following the induction of epilepsy and responses during hypercapnic challenge remained unchanged before and after epilepsy ( Supplementary Fig. 2A ). This pattern was also observed in mice treated with early-LPS and late-mino ( Supplementary Fig. 2B-C ). In contrast, mice treated with early-mino and late-LPS showed a reduction in breathing frequency in response to hypercapnia; however, these changes were not statistically significant ( Supplementary Fig. 2D-E ). Control mice that received intrahippocampal PBS injections also showed no significant changes in breathing frequency in response to hypercapnia ( Supplementary Fig. 2F-G ). Minute ventilatory response (V E ) were similar to the tidal volume response in almost all group ( Fig. 2B-H and Supplementary Fig. 2H-N ). V E was reduced in epileptic mice at week 3 ( Supplementary Fig. 2H ), but this reduction was reversed in the late-mino and early-LPS treatment groups ( Supplementary Fig. 2I-J ), with a significant decrease observed at week 5 post-SE in early-LPS group. Expectedly, V E responses to both 3% and 6% CO₂ challenges were significantly reduced in mice treated with early-mino and late-LPS ( Supplementary Fig. 2K-L ). Among control mice, those treated with early-LPS exhibited a reduced V E response to 6% CO₂ at week 5 post-PBS injection, whereas V E responses remained unchanged in control mice treated with late-mino ( Supplementary Fig. 2M-N ). Early inflammation reduces spontaneous seizure frequency in epileptic mice. Automated detection and analysis of seizures frequency in different groups of mice after induction of SE showed reduced number of seizures in group of mice treated with early-LPS compared to epileptic mice ( Fig. 3A ). Although this reduction in number of seizures was not significant compared to epileptic mice (p= 0.2854; Fig. 3B ). None of the other treatment groups showed changes in seizure frequency compared with epileptic group. To validate that the induction of SE was equivalent in all groups of mice, we compared the Racine score during SE induction, which showed no difference between the groups except class-4 seizures, which were significantly lower in SE + early-mino ( p = 0.013) and SE + late-LPS ( p = 0.017) groups ( Fig. 3C ). Reduced microglial TREM2 expression, but not P2Y12, in the RTN of epileptic mice with breathing phenotype, which wasn’t consistent in early-mino and late-LPS groups To evaluate molecular changes in microglia associated with the chronic epilepsy breathing phenotype, we assessed the expression of key microglial receptors, TREM2 and P2Y12R, in the RTN. The immunohistochemistry findings showed significant reduction in colocalisation of TREM2 with the microglia marker, ionised calcium-binding adaptor molecule 1 (Iba1) in SE mice compared to healthy controls (p=0.0294) ( Fig. 4A-B ). However, these findings were not consistent in epileptic mice treated with early-mino and late-LPS where TREM2 expression was neither different from control nor SE group but showed exacerbation of breathing phenotype ( Fig. 4A and 2E-F) . Compared to SE group, TREM2-Iba1 colocalisation appears higher but not significantly different in epileptic mice treated with early-LPS (mean 0.713 median ρ =0.717) and late-mino (mean 0.705 median ρ =0.717) - the groups that experience recovery of the breathing phenotype compared to SE group (mean 0.531 median ρ =0.579) ( Fig. 4A-B ). Pearson’s correlation coefficient showed P2Y12R-Iba1 immunostaining within the RTN was not significantly different in any of the groups ( Fig. 4C ), which was evident through the representative micrographs ( Fig. 4D ). The Pearson’s coefficient of different groups is - Control (mean 0.284 median ρ =0.250), SE (mean 0.246 median ρ = 0.249), SE + early-LPS (mean 0.389 median ρ =0.356), SE + late-mino (mean 0.379 median ρ = 0.338), SE + early-mino (mean 0.332 median ρ = 0.315), SE + late-LPS (mean 0.326 median ρ = 0.280), ( Fig. 4C-D ). Reduced microglia density, but not C1q expression, in the RTN of early-LPS and late-mino treated mice that show breathing phenotype recovery compared to epileptic mice. We further evaluated microglia number and its morphology with Sholl analysis in the RTN of different groups of mice. Microglia density is lower in the brainstem compared to forebrain regions 38,39 . Our analysis showed significant reduction in microglia number in SE mice treated with early-LPS (p = 0.0365; mean 3.21 median ρ = 3.0) and late-mino (p <0.0001; mean 2.10 median ρ = 2.0) compared to SE group (mean 5.6 median ρ = 5.0) suggesting reduction in microglia density was linked to rescuing of breathing phenotype in mice with epilepsy ( Fig. 5A, C ). Although this wasn’t consistent with SE + early-mino group which showed significant reduction in microglia density (p <0.0001; mean 2.0 median ρ = 2.0) without the rescue of the breathing phenotype ( Fig. 5A, C ). Microglia Sholl analysis revealed reduced microglia branch length, but not-significantly in SE mice treated with mino (both early and late) and SE + early-LPS group ( Fig. 5B ). This trend was similar to microglia density comparison and showed reduction in both branch length and number of microglia in both minocycline treated group and group treated with early-LPS. We next investigated the expression of C1q complement factor, a protein that activates the classical complement pathway, promoting target opsonisation (“eat-me” signals) and microglia phagocytosis 40 . However, C1q immunolabelling in our study revealed decreased levels of C1q in the RTN of SE mice treated with late-mino (p = 0.0003), early-Mino (p=0.0072), late-LPS (p=0.003) but not early-LPS compared to control and SE groups ( Fig 5D-E ). We quantified expression levels of C1q in the RTN by measuring the mean fluorescence intensity (MFI) of C1q immunostaining. Our findings are contrary to previous studies where human refractory epileptic tissue demonstrated elevated C1q levels and its increased localisation to dendrites 37 . Additionally, in an LPS-induced mouse model of neuroinflammation, the C1q complement cascade was largely responsible for microglia-dependent synaptic loss in the hippocampus and cognitive impairment 36 . Astrocytic adenosine 2A receptor expression upregulated in epileptic mice compared to mice treated with early-LPS showing breathing phenotype recovery. We used immunohistochemical fluorescence staining to label the A2AR and reactive astrocyte marker, GFAP, in mouse brainstem tissue and analysed changes in A2AR expression in different groups of mice. To quantify and compare the level of A2AR-GFAP colocalisation, the Pearson’s linear correlation coefficient (ρ) was measured. Findings showed extensive colocalisation between GFAP and A2AR in the RTN of SE mice (mean 0.438 median ρ = 0.381), whereas this was significantly reduced in SE + early-LPS group of mice (p = 0.0263, mean 0.171 median ρ = 0.131) where we see the recovery of the breathing phenotype ( Fig. 6A-B ). However, statistical analysis revealed no significant difference in A2AR-GFAP colocalisation between SE and SE + late-mino group of mice (mean 0.195 median ρ =0.201) which also shows the breathing phenotype recovery. SE + early-mino (mean 0.323 median ρ =0.309) and SE + late-LPS (mean 0.309 median ρ =0.306) group also showed equivalent A2AR and GFAP colocalization to SE group. Results indicate a reduction of A2AR-GFAP colocalisation in epileptic mice with acute phase LPS-induced microglia priming, where we notice recovery of breathing phenotype and reduction in seizure frequency compared to SE mice (p=0.0263). As summarized in the table-1, our findings suggest a potential link between microglial density, TREM2 expression, and astrocytic A2AR expression with the onset of disordered breathing in epilepsy. Interestingly, acute inflammation appears to play a protective role, in contrast to its detrimental effects in chronic epilepsy. Notably, microglial priming with LPS during acute seizures led to a reduction in spontaneous seizure frequency. These results underscore the therapeutic potential of modulating inflammation and inhibiting microglial activation in chronic epilepsy, which may help alleviate respiratory dysfunction and ultimately reduce the risk of SUDEP. Discussion Inflammation is key aspect of animal biology and plays a significant role in modulating neuroglial circuitry within the brain. Its impact on neurological disorders is highly context-dependent, varying with the timing and nature of the condition 34 , 41 , 42 . While inflammation may be protective during early stages, it may also exacerbate disease progression. Recognized risk factors for SUDEP include recurrent, drug-resistant, and nocturnal seizures, prior brain injury, central nervous system infections, living or sleeping alone, and prolonged ictal or postictal central apnoea 4 , 14 , 43 – 45 . However, the precise mechanisms by which these factors contribute to cardiorespiratory arrest and SUDEP remain poorly understood. Brain-mediated central respiratory arrest is a leading cause of SUDEP 43 – 45 . Previous studies, including our own 5 , have demonstrated that both humans 31 and animal models with epilepsy 30 exhibit a reduced hypercapnic ventilatory response to elevated CO₂ levels, which may play a critical role in SUDEP pathophysiology 43 , 46 . We showed that mice with chronic spontaneous seizures have neural corelate in the RTN neurons of reduced respiratory chemosensitivity 5 . Mild to moderate inflammation, induced by agents such as LPS, can prime glial cells and modulate respiratory responses through distinct immune pathways 23 , 35 , 47 . However, the role of inflammation and glial activation in regulating respiratory function in epilepsy remains unclear. Our findings indicate that glial priming and activation via LPS during the acute phase of epilepsy is protective, preventing respiratory dysfunction and preserving chemosensitivity and hypercapnic ventilatory responses. In contrast, during chronic epilepsy, inflammation exerts detrimental effects, and inhibition of microglial activation during spontaneous seizures restores respiratory and chemosensitive responses in mice. Our findings have significant implications in understanding the mechanism of sudden death in people with epilepsy. This is especially more significant as we noticed exacerbated breathing phenotype in the cohort of epileptic mice treated with LPS during chronic seizures. Notably, mice from this group showed the most significant reduction in hypercapnic breathing responses (Fig. 2 F). The bidirectional role of inflammation in epilepsy, beneficial during acute seizure/epilepsy and detrimental during chronic spontaneous seizures, in modifying breathing responses in mice suggest longevity of the condition and time of inflammation could have different consequences and outcomes for people with epilepsy. Microglia, astrocytes, and other immune cells respond to inflammatory signals and mediate their downstream effects. Microglia and astrocytes are the key players in this inflammatory process 24 , 36 , 41 , 48 , and our findings demonstrate that inhibiting microglial activation during the chronic epilepsy with the non-specific inhibitor minocycline can prevent the emergence of respiratory dysfunction in mice (Fig. 2 ). Recent studies have shown that microglial fractalkine signalling promotes extracellular adenosine formation and regulates phrenic long-term facilitation following acute intermittent hypoxia 47 , suggesting that changes in microglial receptor expression may influence molecular pathways and respiratory function. We observed a reduction in microglial TREM2 - but not P2Y12 - receptor expression, which may contribute to impaired respiratory chemosensitivity in RTN neurons. TREM2 is an important immune surface receptor that allows microglia to recognise and respond appropriately to damage, infection, and inflammation. Its signalling is often involved in microglial phagocytosis of apoptotic cells 49 , and loss of TREM2 has been observed in human epileptic tissue 37 and impair microglial phagocytosis ability in epileptic mice 50 . Our findings imply that reduced microglial TREM2 in epilepsy alter microglia activity and contributes the development breathing phenotype. However, further investigation is required as neither the early minocycline nor the late LPS treatment groups despite exhibiting exacerbated breathing phenotypes showed reduced TREM2 expression. Future studies employing chemogenetic or pharmacological ablation of TREM2 during chronic seizures, alongside respiratory chemosensitivity assessments and histological analyses at the time of breathing impairment, will be essential to resolve this discrepancy. On the other hand, P2Y12R is a purinoreceptor, crucial in facilitating microglial process extension and surveillance of the CNS microenvironment 51 . In epilepsy, the P2Y12R is involved in limiting seizure activity and regulating neurogenesis 52 . However, our data show microglial P2Y12 expression does not correlate with breathing responses in epileptic mice. While P2Y12 has been implicated in seizure-induced neurogenesis 52 and microglial activity following status epilepticus and the outcome of seizures 53 , its role in long-term respiratory modulation appears limited. A key limitation of our immunohistochemical analysis is the temporal mismatch: histological assessments were conducted at week 8 post-SE, whereas the most severe respiratory phenotypes were observed between weeks 3 and 5. Future studies involving time-specific tissue collection will be essential to ascertain the molecular mechanisms underlying breathing impairment in epilepsy. Despite limitations, microglial morphology and density remain valuable indicators of functional state and are key to understanding their role in disease processes 54 . Activated microglia undergo morphological changes that reflect their molecular profile and can contribute to epileptogenesis and disease progression 48 . Our findings suggest a potential protective role for reduced microglial density and process length in mitigating respiratory dysfunction in epilepsy. However, this pattern did not hold in epileptic mice treated with early minocycline, which also showed reduced microglial number and process length but no improvement in respiratory phenotype. This discrepancy may reflect a secondary effect of minocycline on microglial morphology, independent of functional or molecular changes. Interestingly, microglial expression of complement factor C1q deviated from expected patterns reported in the literature 36 , 37 . We observed significantly reduced C1q expression in groups of mice exhibiting the most severe respiratory phenotypes, as well as in those treated with late minocycline, which showed recovery of respiratory phenotype. This unexpected finding suggests that C1q expression may not directly correlate with respiratory outcomes in epilepsy, or that its role is temporally dependent. These inconsistencies highlight the importance of evaluating molecular changes at time points that align with the onset of respiratory dysfunction. Further time-resolved studies are needed to clarify the contribution of C1q and other microglial markers to breathing impairment in epilepsy. Given the established role of adenosine and A2AR in phrenic nerve facilitation 23 , 47 and their implication in SUDEP mechanisms 19 – 21 , we examined A2AR expression across all experimental groups and its association with respiratory phenotypes in epilepsy. Although A2AR expression wasn’t significantly different between epileptic mice and those treated with late minocycline, our data hints that increased A2AR expression in astrocytes - rather than in microglia or neurons - within the RTN may contribute to impaired chemosensitivity in epilepsy. While the role of adenosine, and particularly A2AR, in respiratory regulation remains unclear 23 , 55 , emerging evidence suggests it may act through serotonergic pathways 56 . These findings point to a potentially astrocyte-specific mechanism by which A2AR signalling modulates breathing dysfunction in epilepsy, however this needs further investigation. Overall, our findings underscore the critical importance of inflammation - and its timing - in shaping respiratory function in epilepsy, a key factor in the pathophysiology of SUDEP. The data highlight a potential role for the adenosinergic system, particularly astrocytic A2AR signalling, as well as microglial density and TREM2 receptor expression, in modulating respiratory chemosensitivity. These insights provide promising therapeutic avenues, including the use of minocycline and other modulators of microglial activity, to prevent respiratory dysfunction and potentially reduce SUDEP risk. Materials and Methods Experiments were performed in accordance with the European Commission Directive 2010/63/EU (European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes) and the United Kingdom Home Office (Scientific Procedures) Act (1986) with project approval from the University of Warwick’s AWERB (PP1674884). Implantation of EEG electrodes and hippocampal cannula Adult male C57BL/6J mice (8–10 weeks old and 20–30 g) were anaesthetized with isoflurane (4%; Piramal Healthcare Ltd, Mumbai, India) in pure oxygen (4 L·min − 1 ). Adequate anaesthesia was maintained with 0.5-2% isoflurane in pure oxygen (1 L·min − 1 ) throughout the surgery. Mice received a presurgical subcutaneous injection of atropine (120 µg·kg − 1 ; Westward Pharmaceutical Co., Eatontown, NJ, USA) and meloxicam (2 mg·kg − 1 ; Norbrook Inc., Lenexa, KS, USA). Mice were placed in a prone position into a digital stereotaxic apparatus (Kopf Instruments, Tujunga, CA, USA) on a heating pad (TCAT 2-LV: Physitemp, Clifton, NJ, USA) and body temperature was maintained at a minimum of 33°C via a thermocouple. The head was levelled, at bregma and 2 mm caudal to bregma. Four holes were drilled for implantation of 2 EEG recording, a ground and a reference electrode. EEG electrodes were PFA-coated silver wire of diameter: 0.254 mm (Bilaney Consultants Ltd, UK) (coordinates: recording electrodes- ±1.5 mm lateral and 1.0 mm rostral from Bregma, ground and reference electrodes- ±2.5 mm lateral and − 1.5 mm caudal from Bregma) (Fig. 1 A). Silver wires were implanted and secured in position with SuperBond™ (Prestige Dental, Bradford, UK). EEG wires were passed through the pedestal (Bilaney Consultants Ltd, UK), and pedestal was implanted and secured in place over the head with SuperBond™. Another hole was drilled for implantation of unilateral hippocampal cannula (coordinates: 1.8 mm lateral and − 1.74 mm caudal from Bregma, and − 1.3 mm ventral from the surface of the dura). Stainless steel cannula, 26 gauge and 10 mm long, was implanted in the hippocampus and secured in place with SuperBond™. Postoperatively, mice received buprenorphine, and were allowed 1–2 weeks for recovery, with food and water ad libitum . Induction of status epilepsy (SE) and EEG recording in freely moving mice Instrumented mice were anaesthetized with isoflurane and placed into a stereotax as described above. Mice EEG electrodes were connected to wires. SE was induced in mice with total of 0.3 µg (in 100 nl volume) of unilateral kainic acid (KA) injection through hippocampal cannula with Hamilton Neuros syringe (Model 75 RN, Essex Scientific Laboratory Supplies Ltd., UK). Mice were taken off the anaesthesia and placed into open field chamber. EEG wires were connected to the amplifier (Digitimer, Welwyn Garden City, UK) via commutator (FL-2-C-MICRO-M; Dragonfly Research & Development, Inc.). The EEG recording was started in few minutes after KA injection. Mice were scored every minute for their behavioural seizures using Racine scale. 1- Sudden behavioral arrest and/or motionless staring; 2- Facial jerking with muzzle or stiffened and arched tail; 3- Neck jerks, head bobbing or partial body clonus; 4- Clonic seizure in a sitting position; 5- Convulsions including clonic and/or tonic–clonic seizures while lying on the belly and/or pure tonic seizures; 6- Convulsions including clonic and/or tonic–clonic seizures while lying on the side and/or wild jumping. The EEG frequency (0-100Hz) was increased during different classes of behavioural seizures (Fig. 1 B). At 60 minutes or recording 1–2 of class 6 scale seizure following injection of KA (whichever happened earlier), mice were anaesthetized with isoflurane and SE was terminated with intraperitoneal injection of ketamine (50 mg/kg) and diazepam (20 mg/kg). During SE, EEG activity was recorded, amplified and filtered using the NeuroLog system (Digitimer, Welwyn Garden City, UK) connected to a 1401 interface and acquired on a computer using Spike2 software (Cambridge Electronic Design, Cambridge, UK). The EEG activity raw data were DC removed. Video data during SE induction was recorded with Spike2 software and was synchronised with the EEG activity. Plethysmography Mice were placed into a custom-made 0.5 L plethysmography chamber, with an airflow rate of 1 L·min − 1 . The plethysmography chamber was heated to 31 0 C (thermoneutral for C57/BL6 mice). CO 2 concentrations were sampled via a Hitech Intruments (Luton, UK) GIR250 Dual Sensor Gas analyzer or ML206 gas analyzer (ADinstruments, Sydney, Australia) connected to the inflow immediately before entering the chamber. The analyser had a delay of ~ 15–20 sec to read-out the digital output of gas mixture. Pressure transducer signals and CO 2 measurements were amplified and filtered using the NeuroLog system (Digitimer, Welwyn Garden City, UK) connected to a 1401 interface and acquired on a computer using Spike2 software (Cambridge Electronic Design, Cambridge, UK). Video data was recorded with Spike2 software and was synchronised with the breathing trace. Airflow measurements were used to calculate: tidal volume (V T : signal converted to mL following calibration and 4–5 breathing sweeps were averaged to calculate area under curve (AUC) between two troughs), and respiratory frequency ( f R: breaths per minute). Minute ventilation (V E ) was calculated as V T x f R. Hypercapnia in freely behaving mice Mice were tested for hypercapnic challenge before and 3, 5 and 7-weeks after induction of SE (Fig. 2 A). Instrumented mice were allowed ~ 30 mins to acclimate to the plethysmograph. Using Spike2 , 3 min of baseline recordings were taken (gas mixture: 0% CO 2 21% O 2 79% N 2 ). The mice were then exposed to 3 min epochs of hypercapnic gas mixture at different concentrations of CO 2 (3, and 6% in 21% O 2 balanced N 2 ). Following exposure to the hypercapnic gas mixtures, CO 2 levels were reduced back to 0% and signals were recorded for a further 4 minutes recovery period (Fig. 1 C). Mice treatment Following the recording of pre-SE hypercapnic ventilatory responses and the induction of SE or control treatment, mice were assigned to different experimental groups as below. Mice weighing less than 20 g received an intraperitoneal injection of LPS at 0.5 mg/kg. For mice over 20 g, the LPS dose was capped at the amount equivalent to that for a 20 g mouse (0.5 mg/kg), regardless of their actual weight. This initial injection was followed by three additional daily injections at the same dose. The dosing protocol was established based on pilot studies aimed at identifying a dose that was both effective and well-tolerated, without causing significant weight loss. To inhibit microglial activation, minocycline - a non-specific microglial inhibitor - was administered orally via drinking water at a concentration of 0.5 mg/mL, provided ad libitum. The groups of mice studied were: SE: Epileptic mice ( n = 11) SE + early-LPS: Epileptic mice treated with LPS for four consecutive days following SE induction ( n = 12) SE + late-mino: Epileptic mice treated with minocycline from day 11 post-SE induction and continued until the end of the study ( n = 12) SE + early-mino: Epileptic mice treated with minocycline from day 1 to 10 post-SE induction ( n = 6) SE + late-LPS: Epileptic mice treated with four consecutive daily LPS injections from day 15 to 18 following SE induction ( n = 6) Control + early-LPS: Control mice treated with LPS for four consecutive days following intrahippocampal vehicle ( n = 5) Control + late-mino: Control mice treated with minocycline from day 11 following intrahippocampal vehicle and continued until the end of the study ( n = 5) Control (only for immunohistochemistry study): were healthy 8–12 weeks old C57BL/6 male and female mice. Behavioural seizure analysis To analyse the seizure frequency after induction of SE in different groups of mice, 6 mice in each group were continuously recorded from day 1–2 post-SE till week-7. Due to limitations in the number of cages that could be recorded simultaneously, it was not possible to video-monitor all mice for behavioural seizure analysis. We used MiniCAM, an open-source behavioural camera, for imaging animal movement and behaviour supported with MiniCAM LED ring to provide infrared illumination of the animal and clear behavioural image quality without interfering with animal behaviour (Open Ephys, Lisbon, Portugal). Video data was processed for behavioural seizure analysis using an open-source python-based package for semi-automated detection of convulsive seizures in mice 57 . This allowed us to process video data from all singly housed mice recorded over 50 days and identify and validate the convulsive tonic-clonic seizures. Seizure detection was blindly performed, and frequency was compared between different groups. Immunohistochemistry At the end of the study and around week-8 post intrahippocampal vehicle or KA treatment, mice were humanely killed by pentobarbital overdose (> 100 mg·kg − 1 ) and transcardially perfused with paraformaldehyde solution (4% PFA; Sigma-Aldrich, St Louis, MO, USA). The brains were removed and postfixed in PFA (4°C) overnight. Brainstems were serially sectioned at 50–70 µm. Free-floating sections were incubated for 1 hour in a blocking solution (PBS containing 0.1% Triton X-100 and 5% BSA). Primary antibodies (goat anti- ionized calcium binding adapter molecule 1 (Iba1) [Iba1; 1:100; PA5-18039; Invitrogen, Waltham, MA USA], or chicken anti- glial fibrillary acidic protein (GFAP) [GFAP; 1:1000; PA1-10004; Invitrogen, Waltham, MA USA], or rabbit anti-ChAT [ChAT; 1:2000; ab178850; Abcam PLC, Cambridge, UK], or mouse anti-P2Y12 [P2Y12; 1:5000; 476011; Synaptic systems GmbH, Goettingen, Germany], or mouse anti-TREM2 [TREM2; 1:200; AF1729; R&D Systems, Inc., Minneapolis, MN, Canada], or mouse anti-A2AR [A2AR; 1:1000; 05-717; Sigma-Aldrich, St. Louis, MO, USA], or rabbit anti-C1q [C1q; 1:500; ab182451; Abcam PLC, Cambridge, UK]) were added and tissue was incubated for 2 nights at 4 0 C. Slices were washed in PBS with 0.1% triton (4 × 15 mins) and then the secondary antibodies were added: either donkey anti-mouse Alexa Fluor 405 (1:250; Invitrogen, Waltham, MA USA), or donkey anti-chicken Alexa Fluor 680 (1:250; Jackson ImmunoResearch, Cambridge, UK), or donkey anti-goat Alexa Fluor 680 (1:250; Invitrogen, Waltham, MA USA), or donkey anti-rabbit Alexa Fluor 680 (1:250; Jackson ImmunoResearch, Cambridge, UK), or donkey anti-rabbit Alexa Fluor 594 (1:250; Invitrogen, Waltham, MA USA), or goat anti-rabbit Alexa Fluor 488 (1:250; Invitrogen, Waltham, MA USA), or donkey anti-goat Alexa Fluor 488 (1:250; Invitrogen, Waltham, MA USA) and tissue was then incubated overnight at 4 0 C. Tissue was washed in PBS with 0.1% triton (4 × 15 min). Slices were mounted, coverslipped and examined using a Zeiss 880 confocal microscope with ZEN acquisition software (Zeiss, Oberkochen, Germany). 40x and 63x oil objective lenses were used for image acquisition. From the RTN region, 5 images were acquired along the ventral surface per mouse. Images consisted of 15–17 layer Z-stacks. All images were analysed using ImageJ-Fiji (version 2.16.0/1.54p). Blinded analysis, whereby file names were coded during analysis, was used throughout to prevent unconscious bias. Colocalisation analysis of glial fibrillary acidic protein (GFAP)-A2AR, P2Y12R-Iba1, and TREM2-Iba1 immunoreactivity was performed on maximum intensity projections (MIP) images. Regions of interest (ROI) were used to isolate the RTN. Colocalisation analysis was performed using the BIOP JACoP plugin in Fiji. Pearson’s linear correlation coefficient (ρ) was measured for each image, and statistical significance was assessed using Costes’ randomisation test (2D, 100 permutations). An automated Yen intensity thresholding algorithm was applied to select positive pixels before the analysis. Pearson’s coefficient measures the strength and direction of the linear relationship between fluorescence intensities in two images. Its value ranges from 1 to -1, with 1 representing a complete positive correlation, -1, a complete negative correlation, and 0 representing no correlation. Sholl analysis was done on MIP images of Iba1-immunoreactive microglia. Iba1 + microglia were randomly selected from the RTN (5 microglia per mouse). The number of Sholl intersections were quantified at radial intervals of 0.5 µm, starting from the microglia soma. Manual cell counting of Iba1 + microglia in the RTN was done using the inclusion criteria: microglia with visible cell bodies in the image frame. Mean fluorescence intensity (MFI) of C1q was measured within RTN ROIs and normalised based on ROI area. Statistical analysis Statistical analysis was performed in GraphPad Prism software (version 10.1.0). Statistical significance for changes in breathing was determined using two-way repeated measure ANOVA followed by t tests with the Tukey’s correction and shown as a line graph. Comparisons were done between pre- and post-treatment (SE, control or treatment groups). The Racine seizure scores and seizure frequency between different groups (violin plot with superimposed data points) and histological data, including Sholl analysis, differences between multiple groups were compared with Kruskal-Wallis non-parametric test. Significant statistical tests were followed by Dunn’s post hoc comparisons test. P ≤ 0.05 was considered significant. **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01 and * p ≤ 0.05. Results are presented as mean ± SD or median and interquartile ranges throughout the text. Declarations Acknowledgements Funding: This work was supported by the Epilepsy Research Institute (ERI) UK Project Grant (P1903), Emerging Leader Fellowship award (F2203), an MRC Discovery Award (MC_PC_15070), and Wellcome-Warwick QBP funds. AB is an ERI Emerging Leader Fellow and ND was a Royal Society Wolfson Research Merit Award Holder. The authors acknowledge use of imaging facility and support provided by the Bio-Analytical Shared Resource Laboratories within the School of Life Sciences, University of Warwick. Author contributions AMB (Amol M. Bhandare) designed the study, performed all in vivo experiments, recordings, assisted immunohistochemistry data analysis and wrote the manuscript; ND conceived and designed the study, and contributed to writing of the manuscript. AB (Adwoa Boaten) performed immunohistochemistry, data analysis and contributed to writing of the manuscript. DD performed behavioural seizure detection analysis from continuous video monitoring. 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10:25:09","extension":"html","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":177281,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7707048/v1/bd146bb43d9dcf8cf7597bd3.html"},{"id":93032795,"identity":"86758c63-e839-4c2e-a3b1-c5409cf3322e","added_by":"auto","created_at":"2025-10-08 10:25:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":325707,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSE induction and hypercapnic breathing responses in mice.\u003c/strong\u003e (A) Representation of hippocampal cannula placement for seizure induction and EEG (recording, reference and ground) electrodes. (B) Representative EEG recordings during intrahippocampal KA-induced SE in mice illustrate distinct stages of Racine seizure progression, accompanied by corresponding EEG traces, power band analyses, and observed behaviour. (C) Whole-body plethysmography (WBP) recordings in mice demonstrate the hypercapnic ventilatory response to elevated CO₂ levels. Breathing patterns were assessed under baseline room air conditions, followed by exposure to 3% and 6% CO₂, and post-recovery, with corresponding changes in respiratory frequency (breaths per minute, bpm).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7707048/v1/24d35556ae7a581539e82810.png"},{"id":93032799,"identity":"53b34333-852b-489e-8275-d2f31357a99e","added_by":"auto","created_at":"2025-10-08 10:25:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":305611,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEarly inflammation and late inhibition of microglial activation during epilepsy rescues breathing phenotype in mice.\u003c/strong\u003e (A) Experimental design illustrating pre- and post-intrahippocampal KA or PBS injection breathing assessments in epileptic and control mice, incorporating LPS and minocycline treatment paradigms. (B-H) Hypercapnic ventilatory response (HCVR) at defined time points across experimental groups: (B) SE (\u003cem\u003en\u003c/em\u003e = 11), (C) SE + early LPS (\u003cem\u003en\u003c/em\u003e = 12), (D) SE + late mino (\u003cem\u003en\u003c/em\u003e = 12), (E) SE + early mino (\u003cem\u003en\u003c/em\u003e = 6), (F) SE + late LPS (\u003cem\u003en\u003c/em\u003e = 6), (G) Control + early LPS (\u003cem\u003en\u003c/em\u003e = 5), (H) Control + late mino (\u003cem\u003en\u003c/em\u003e = 5). (I) Percentage change in chemosensitivity at 3% and 6% CO\u003csub\u003e2\u003c/sub\u003e in different groups compared to their pre-SE response. P-values derived from two-way repeated measure (mixed effects) ANOVA with Tukey’s multiple comparisons are *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, and ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7707048/v1/c09ea14415e0546cd3933184.png"},{"id":93032797,"identity":"e7f62fab-c6aa-49f2-a349-f0ba1f9d3308","added_by":"auto","created_at":"2025-10-08 10:25:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":394560,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePriming microglia with LPS in latent period reduce spontaneous seizure frequency in mice.\u003c/strong\u003e (A) Seizure frequency up to 50 days after intrahippocampal KA injection in epileptic mice (SE) and mice treated with LPS and minocycline at different stages; SE + early LPS, SE + late mino, SE + early mino, SE + late LPS groups. (B) Comparison of seizure frequency in different groups of mice. (C-H) Racine seizure scores 1 to 6 during SE induction in different groups of mice. P-values for seizure frequency and Racine score were derived from Kruskal-Wallis multiple comparisons and Dunn’s correction for multiple comparison with value *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7707048/v1/9106542ed9cefbfffbb78570.png"},{"id":93033799,"identity":"5fd28c0c-9a1f-4069-8252-453d0adefd1b","added_by":"auto","created_at":"2025-10-08 10:41:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":502247,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eReduced microglia TREM2 expression, but not P2Y12, in the RTN of epileptic mice with breathing phenotype but wasn’t correlated with early-mino and late-LPS groups.\u003c/strong\u003e (A) Comparison of Pearson’s correlation coefficient (ρ) values of TREM2 and Iba1 co-immunostaining within the RTN in different groups of mice. Each data point represents the Pearson’s coefficient obtained from one image (5 images per mouse (n); SE: n=2; Control, SE + early-LPS, SE + late-mino, SE + early-mino, SE + late-LPS: n=3). (B) Representative 63x maximum intensity projection (MIP) micrographs showing Iba1 (green) and TREM2 (magenta) immunostaining in the RTN of control, SE, SE + early-LPS, SE + late-mino, SE + early-mino, SE + late-LPS mice, 7-10 weeks after SE induction. Dotted line marks the ventral surface of the brainstem. (C) Pearson’s correlation coefficient (ρ) values of P2Y12R and Iba1 co-immunostaining within the RTN. Each data point represents the Pearson’s coefficient obtained from one image (5 images per mouse (n); SE: n=2; Control, SE + early-LPS, SE + late-mino, SE + early-mino, SE + late-LPS: n=3). (D) Representative 63x MIP micrographs showing Iba1 (green) and P2Y12R (blue) immunostaining in the RTN of control, SE, SE + early-LPS, SE + late-mino, SE + early-mino, SE + late-LPS mice, 7-10 weeks after SE induction. Dotted line marks the ventral surface of the brainstem. Statistical significance was derived from Kruskal-Wallis test and Dunn’s correction for multiple comparison; *\u003cem\u003ep\u003c/em\u003e ≤ 0.05. Scale bar = 20μm.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7707048/v1/432d333032f9a804ecae4ced.png"},{"id":93032803,"identity":"32453e43-74f2-4ccf-b39e-d1f1f19f8b00","added_by":"auto","created_at":"2025-10-08 10:25:09","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":460618,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMicroglia density reduced, but not C1q expression, in the RTN of early-LPS and late-mino treated mice compared to epileptic mice.\u003c/strong\u003e (A) Microglia number per 18,211 μm\u003csup\u003e2\u003c/sup\u003e in different groups of mice with the median (thick black line) and interquartile ranges (thin coloured line). (B) Sholl analysis of Iba1+ microglia in the RTN, showing process intersections plotted against radial distance from the soma (Bi) and comparison between groups at 10, 20 and 30 μm branch length (Bii). (C) Representative 63x maximum intensity projection (MIP) micrographs showing microglia (Iba1; green) immunostaining in the RTN of control, SE, SE + early-LPS, SE + late-mino, SE + early-mino, SE + late-LPS mice, 7-10 weeks after SE induction. Dotted line marks the ventral surface of the brainstem. (D) MFI of C1q immunostaining within the RTN. Each data point represents the C1q MFI within the RTN region of interest (ROI) from one image. (5 images per mouse (n); SE + early-LPS: n=2; control, SE, SE + late-mino, SE + early-mino, SE + late-LPS: n=3). (E) Representative 63x MIP micrographs showing C1q complement factor (yellow) and DAPI (blue) immunostaining in the RTN of Control and SE mice, 7-10 weeks after SE induction. Dotted lines outline the RTN with ventral surface at the bottom and facial nucleus on the top. Statistical significance was derived from Kruskal-Wallis test and Dunn’s correction for multiple comparison; *\u003cem\u003ep\u003c/em\u003e ≤ 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001. Scale bar = 20μm.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7707048/v1/c51755b6464e3b18f94b5b67.png"},{"id":93032814,"identity":"254d3e20-e8c9-4dab-b038-5b65b68a1e7b","added_by":"auto","created_at":"2025-10-08 10:25:09","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":714542,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUpregulated A2AR expression in epileptic mice compared to epileptic mice treated with early-LPS and showed breathing phenotype recovery.\u003c/strong\u003e (A) Comparison of Pearson’s correlation coefficient (ρ) values of GFAP and A2AR co-immunostaining within the RTN. Each data point represents the Pearson’s coefficient obtained from one image (5 images per mouse (n); SE: n=2; control, SE + early-LPS, SE + late-mino, SE + early-mino, SE + late-LPS; n=3). Violin plot displays the distribution of ρ values with the median (thick black line) and interquartile ranges (thin coloured line). (B) Representative 63x MIP micrographs showing GFAP (red) and A2AR (green) immunostaining in the RTN of control, SE, SE + early-LPS, SE + late-mino, SE + early-mino, SE + late-LPS mice 7-10 weeks post SE induction. Statistical significance was derived from Kruskal-Wallis test and Dunn’s correction for multiple comparison; *\u003cem\u003ep\u003c/em\u003e ≤ 0.05. Scale bar = 20μm.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7707048/v1/93cb5dd9ab67fbc168c3e857.png"},{"id":99894673,"identity":"a3d74517-2d82-43ab-983c-f6f7b7c4d375","added_by":"auto","created_at":"2026-01-09 14:28:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3947148,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7707048/v1/bb1625ea-3072-4d19-a1ac-0f2be0c4d018.pdf"},{"id":93033081,"identity":"a2737ae4-e1b0-4831-86eb-08b10d8a3d4f","added_by":"auto","created_at":"2025-10-08 10:33:09","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":745670,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFiguresTables.docx","url":"https://assets-eu.researchsquare.com/files/rs-7707048/v1/595f6291f4a2e41b4df858f9.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Inflammation modifies breathing phenotype in mice with epilepsy","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEpileptic seizures, characterized by neuronal hyperexcitability, disrupt brain networks and function, and are associated with significant comorbidities in people with epilepsy such as cognitive impairment and sudden unexpected death in epilepsy (SUDEP)\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. While the molecular mechanisms underlying SUDEP remain unclear, emerging evidence implicates postictal breathing irregularities - ether brain-mediated\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e or due to airway obstruction\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e - and abnormal slow heartbeats occurring immediately after seizures significantly alter breathing or heart function\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e leading to SUDEP. The proposed mechanisms include brain-driven alterations in cardiorespiratory control\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan additionalcitationids=\"CR9 CR10 CR11 CR12 CR13 CR14\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, genetic factors such as Kv1.1 potassium channel deficiency\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, and the involvement of neurotransmitters like serotonin\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e and adenosine\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, as well as glial cell activity\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Targeting of serotonin and adenosine pathways has shown promise in preventing seizure-induced respiratory arrest in animal models\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, and inflammation-driven microglial activation can modulate these pathways\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Microglia play a dual role in epilepsy\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, where acute activation is protective\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, while chronic activation contributes to pathology\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Despite these insights, the roles of inflammation, glial activation, and serotonin-adenosine signalling in the mechanisms underlying impaired breathing in epilepsy remain unclear, and SUDEP incidence continues to be high.\u003c/p\u003e\u003cp\u003eStudies have demonstrated impaired CO₂-induced respiratory responses in both epileptic rats\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e and humans following generalized convulsive seizures\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Our own research has shown that mice with chronic epilepsy exhibit reduced hypercapnic ventilatory responses (HCVR), correlating with altered activity of chemosensitive neurons of the retrotrapezoid nucleus (RTN)\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Inflammation, while essential for defence against injury and infection, can become detrimental in chronic neurological conditions as shown in Alzheimer\u0026rsquo;s\u003csup\u003e33\u003c/sup\u003e and Parkinson\u0026rsquo;s disease\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Neuroinflammation modifies breathing responses in animal models\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, and in the context of epilepsy, these responses could contribute the mechanism of SUDEP.\u003c/p\u003e\u003cp\u003eWe studied the role of inflammation and microglial activation in the development of breathing phenotype in epilepsy using a kainic acid (KA)-induced mouse model of temporal lobe epilepsy. Systemic lipopolysaccharide (LPS) and oral minocycline - a non-specific microglial activation inhibitor - were administered at two distinct time points. LPS treatment during acute seizures (early-LPS) and minocycline treatment during chronic epilepsy (late-mino) was beneficial in preventing development of breathing phenotype in mice with epilepsy. In contrast, LPS treatment during chronic epilepsy (late-LPS) and minocycline during acute seizures (early-mino) exacerbated the phenotype. Given the central role of microglia and astrocytes in neuroinflammatory responses, we performed immunohistochemical analysis of chemosensitive neurons and glial cells in the RTN\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Our findings revealed that in epileptic group: there was elevated microglial density compared to epileptic mice treated with early LPS and both late- and early-mino groups; reduced triggering receptor expressed on myeloid cells 2 (TREM2) expression compared to control; and increased astrocytic adenosine A2A receptor (A2AR) expression compared to epileptic mice treated with early LPS. Notably, microglial P2Y12 expression did not change in any groups. Although C1q, a key mediator of synaptic pruning, is typically upregulated in inflammation\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e and epilepsy\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, its expression remained unchanged or was reduced in minocycline- and LPS-treated epileptic mice. Our results, therefore, hint at a possible link of microglial density, TREM2, and astrocytic A2AR expression to the emergence of disordered breathing in epilepsy where acute inflammation appears protective, in contrast to its detrimental role in chronic epilepsy. Besides microglia priming with LPS during acute seizures also reduced spontaneous seizure frequency. Findings highlight the therapeutic potential of targeting inflammation and inhibiting microglial activation in chronic epilepsy, which mitigate respiratory dysfunction and potentially help to reduce the risk of SUDEP.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eInflammation during acute seizures and inhibition of microglia activation during chronic epilepsy rescues development of breathing phenotype in mice with epilepsy.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChanges in breathing responses to increased CO\u003csub\u003e2\u003c/sub\u003e (3 and 6%) were studied at week-3, -5 and -7 after induction of status epilepticus (SE) using intrahippocampal injection of KA in mice. We replicated the previous findings\u003csup\u003e5\u003c/sup\u003e, where epileptic mice showed reduced tidal volume (V\u003csub\u003eT\u003c/sub\u003e) at room air and their responses to increased CO\u003csub\u003e2\u003c/sub\u003e were also reduced after induction of epilepsy (\u003cstrong\u003eFig. 2B\u003c/strong\u003e). In epileptic mice, V\u003csub\u003eT\u003c/sub\u003e in room air was reduced at week-3 (0.87 \u0026micro;L/g), -5 (0.89 \u0026micro;L/g) and -7 (0.95 \u0026micro;L/g) post-SE compared to pre-SE (1.07 \u0026micro;L/g). Breathing responses were also reduced at week-3 (0.98 \u0026micro;L/g at 3% and 1.34 \u0026micro;L/g at 6%), -5 (1.08 \u0026micro;L/g at 3% and 1.50 \u0026micro;L/g at 6%) and -7 (1.11 \u0026micro;L/g at 3% and 1.62 \u0026micro;L/g at 6%) compared to pre-SE responses (1.29 \u0026micro;L/g at 3% and 1.74 \u0026micro;L/g at 6%). The slope to breathing response was maximally reduced at week-3 post-SE (58% and 25% reduction at 3 and 6% CO\u003csub\u003e2\u003c/sub\u003e, respectively, compared to pre-SE) (\u003cstrong\u003eFig. 2I and Supplementary Fig. 1\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eBoth the tidal volume in room air and the responses to increased CO\u003csub\u003e2\u003c/sub\u003e were recovered and were similar to pre-SE V\u003csub\u003eT\u003c/sub\u003e in the group of epileptic mice treated with early-LPS and minocycline during chronic seizures (late-mino). Breathing responses in early-LPS group were at week-3 (1.03 \u0026micro;L/g at room air, 1.23 \u0026micro;L/g at 3% and 1.71 \u0026micro;L/g at 6%), -5 (1.05 \u0026micro;L/g at room air, 1.20 \u0026micro;L/g at 3% and 1.62 \u0026micro;L/g at 6%) and -7 (1.04 \u0026micro;L/g at room air, 1.21 \u0026micro;L/g at 3% and 1.68 \u0026micro;L/g at 6%) compared to pre-SE responses (1.08 \u0026micro;L/g at room air, 1.37 \u0026micro;L/g at 3% and 1.82 \u0026micro;L/g at 6%) (\u003cstrong\u003eFig. 2C\u003c/strong\u003e) and in late-mino group were at week-3 (1.01 \u0026micro;L/g at room air, 1.22 \u0026micro;L/g at 3% and 1.61 \u0026micro;L/g at 6%), -5 (1.01 \u0026micro;L/g at room air, 1.21 \u0026micro;L/g at 3% and 1.63 \u0026micro;L/g at 6%) and -7 (0.97 \u0026micro;L/g at room air, 1.19 \u0026micro;L/g at 3% and 1.64 \u0026micro;L/g at 6%) compared to pre-SE responses (1.08 \u0026micro;L/g at room air, 1.28 \u0026micro;L/g at 3% and 1.71 \u0026micro;L/g at 6%) (\u003cstrong\u003eFig. 2D\u003c/strong\u003e). There was no major change in the slope of breathing responses to increased CO\u003csub\u003e2\u003c/sub\u003e at any time points in late-mino group, but early-LPS group showed 48% reduction in the slope of breathing response to 3% CO\u003csub\u003e2\u003c/sub\u003e at week-5 post-SE (\u003cstrong\u003eFig. 2I and Supplementary Fig. 1\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eContrarily, group of mice treated with early-minocycline and late-LPS exacerbated the breathing phenotype. In the mice treated with early-minocycline V\u003csub\u003eT\u003c/sub\u003e in room air was reduced at week-3 (0.94 \u0026micro;L/g), -5 (0.99 \u0026micro;L/g) and -7 (0.92 \u0026micro;L/g) post-SE compared to pre-SE (1.10 \u0026micro;L/g). Breathing responses were also reduced at week-3 (1.15 \u0026micro;L/g at 3% and 1.60 \u0026micro;L/g at 6%), -5 (1.13 \u0026micro;L/g at 3% and 1.53 \u0026micro;L/g at 6%) and -7 (1.12 \u0026micro;L/g at 3% and 1.58 \u0026micro;L/g at 6%) compared to pre-SE responses (1.41 \u0026micro;L/g at 3% and 1.89 \u0026micro;L/g at 6%) (\u003cstrong\u003eFig. 2E\u003c/strong\u003e). The slope of breathing responses was maximally reduced at week-5 (55% at 3% CO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003ecompared to pre-SE) (\u003cstrong\u003eFig. 2I and Supplementary Fig. 1\u003c/strong\u003e). Similarly in mice treated with late-LPS V\u003csub\u003eT\u003c/sub\u003e in room air was reduced at week-3 (0.80 \u0026micro;L/g), -5 (1.03 \u0026micro;L/g) and -7 (0.94 \u0026micro;L/g) post-SE compared to pre-SE (1.19 \u0026micro;L/g). Breathing responses were also reduced at week-3 (1.07 \u0026micro;L/g at 3% and 1.51 \u0026micro;L/g at 6%), -5 (1.36 \u0026micro;L/g at 3% and 1.68 \u0026micro;L/g at 6%) and -7 (1.35 \u0026micro;L/g at 3% and 1.66 \u0026micro;L/g at 6%) compared to pre-SE responses (1.51 \u0026micro;L/g at 3% and 2.08 \u0026micro;L/g at 6%) (\u003cstrong\u003eFig. 2F\u003c/strong\u003e). The slope of breathing responses was equally reduced at week-5 and 7 (44% at 6% CO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003ecompared to pre-SE) (\u003cstrong\u003eFig. 2I and Supplementary Fig. 1\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eIn control mice (intrahippocampal PBS) treated with early-LPS, we did notice reduction in V\u003csub\u003eT\u003c/sub\u003e in room air and reduced responses to 3% and 6% CO\u003csub\u003e2\u003c/sub\u003e at week-5. In control early-LPS treated mice V\u003csub\u003eT\u003c/sub\u003e in room air was at week-3 (0.99 \u0026micro;L/g), -5 (0.88 \u0026micro;L/g) and -7 (0.99 \u0026micro;L/g) post-SE compared to pre-SE (1.11 \u0026micro;L/g). Breathing responses were at week-3 (1.26 \u0026micro;L/g at 3% and 1.70 \u0026micro;L/g at 6%), -5 (1.14 \u0026micro;L/g at 3% and 1.43 \u0026micro;L/g at 6%) and -7 (1.35 \u0026micro;L/g at 3% and 1.82 \u0026micro;L/g at 6%) compared to pre-PBS responses (1.32 \u0026micro;L/g at 3% and 1.93 \u0026micro;L/g at 6%) (\u003cstrong\u003eFig. 2G\u003c/strong\u003e). The slope of breathing responses was increased at 3% CO\u003csub\u003e2\u003c/sub\u003e at all time points (72% increase at week-7) but was maximally reduced at 6% CO\u003csub\u003e2\u003c/sub\u003e week-5 (52% at 6% CO\u003csub\u003e2\u003c/sub\u003e compared to pre-PBS) (\u003cstrong\u003eFig. 2I and Supplementary Fig. 1\u003c/strong\u003e). The reduction in breathing responses in control mice treated with early-LPS could be due to the impact of inflammation on the brainstem breathing circuitry and previous findings are in lined with our observations\u003csup\u003e23,35\u003c/sup\u003e. We did not notice any changes to breathing responses or room air V\u003csub\u003eT\u003c/sub\u003e in control mice treated with late-mino. In control late-mino treated mice V\u003csub\u003eT\u003c/sub\u003e in room air was at week-3 (1.00 \u0026micro;L/g), -5 (0.96 \u0026micro;L/g) and -7 (0.96 \u0026micro;L/g) post-SE compared to pre-SE (1.00 \u0026micro;L/g). Breathing responses were at week-3 (1.34 \u0026micro;L/g at 3% and 1.76 \u0026micro;L/g at 6%), -5 (1.33 \u0026micro;L/g at 3% and 1.73 \u0026micro;L/g at 6%) and -7 (1.25 \u0026micro;L/g at 3% and 1.73 \u0026micro;L/g at 6%) compared to pre-PBS responses (1.35 \u0026micro;L/g at 3% and 1.87 \u0026micro;L/g at 6%) (\u003cstrong\u003eFig. 2H\u003c/strong\u003e). The slope of breathing responses was not much affected at any time point (\u003cstrong\u003eFig. 2I and Supplementary Fig. 1\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eThe breathing frequency (F\u003cem\u003e\u003csub\u003eR\u003c/sub\u003e\u003c/em\u003e) in mice did not change following the induction of epilepsy and responses during hypercapnic challenge remained unchanged before and after epilepsy (\u003cstrong\u003eSupplementary Fig. 2A\u003c/strong\u003e). This pattern was also observed in mice treated with early-LPS and late-mino (\u003cstrong\u003eSupplementary Fig. 2B-C\u003c/strong\u003e). In contrast, mice treated with early-mino and late-LPS showed a reduction in breathing frequency in response to hypercapnia; however, these changes were not statistically significant (\u003cstrong\u003eSupplementary Fig. 2D-E\u003c/strong\u003e). Control mice that received intrahippocampal PBS injections also showed no significant changes in breathing frequency in response to hypercapnia (\u003cstrong\u003eSupplementary Fig. 2F-G\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMinute ventilatory response (V\u003csub\u003eE\u003c/sub\u003e) were similar to the tidal volume response in almost all group (\u003cstrong\u003eFig. 2B-H and Supplementary Fig. 2H-N\u003c/strong\u003e). V\u003csub\u003eE\u003c/sub\u003e was reduced in epileptic mice at week 3 (\u003cstrong\u003eSupplementary Fig. 2H\u003c/strong\u003e), but this reduction was reversed in the late-mino and early-LPS treatment groups (\u003cstrong\u003eSupplementary Fig. 2I-J\u003c/strong\u003e), with a significant decrease observed at week 5 post-SE in early-LPS group. Expectedly, V\u003csub\u003eE\u003c/sub\u003e responses to both 3% and 6% CO₂ challenges were significantly reduced in mice treated with early-mino and late-LPS (\u003cstrong\u003eSupplementary Fig. 2K-L\u003c/strong\u003e). Among control mice, those treated with early-LPS exhibited a reduced V\u003csub\u003eE\u003c/sub\u003e response to 6% CO₂ at week 5 post-PBS injection, whereas V\u003csub\u003eE\u003c/sub\u003e responses remained unchanged in control mice treated with late-mino (\u003cstrong\u003eSupplementary Fig. 2M-N\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEarly inflammation reduces spontaneous seizure frequency in epileptic mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAutomated detection and analysis of seizures frequency in different groups of mice after induction of SE showed reduced number of seizures in group of mice treated with early-LPS compared to epileptic mice (\u003cstrong\u003eFig. 3A\u003c/strong\u003e). Although this reduction in number of seizures was not significant compared to epileptic mice (p= 0.2854; \u003cstrong\u003eFig. 3B\u003c/strong\u003e). None of the other treatment groups showed changes in seizure frequency compared with epileptic group. To validate that the induction of SE was equivalent in all groups of mice, we compared the Racine score during SE induction, which showed no difference between the groups except class-4 seizures, which were significantly lower in SE + early-mino (\u003cem\u003ep\u003c/em\u003e = 0.013) and SE + late-LPS (\u003cem\u003ep\u003c/em\u003e = 0.017) groups (\u003cstrong\u003eFig. 3C\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReduced microglial TREM2 expression, but not P2Y12, in the RTN of epileptic mice with breathing phenotype, which wasn\u0026rsquo;t consistent in early-mino and late-LPS groups\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate molecular changes in microglia associated with the chronic epilepsy breathing phenotype, we assessed the expression of key microglial receptors, TREM2 and P2Y12R, in the RTN. The immunohistochemistry findings showed significant reduction in colocalisation of TREM2 with the microglia marker, ionised calcium-binding adaptor molecule 1 (Iba1) in SE mice compared to healthy controls (p=0.0294) (\u003cstrong\u003eFig. 4A-B\u003c/strong\u003e). However, these findings were not consistent in epileptic mice treated with early-mino and late-LPS where TREM2 expression was neither different from control nor SE group but showed exacerbation of breathing phenotype (\u003cstrong\u003eFig. 4A and 2E-F)\u003c/strong\u003e. Compared to SE group, TREM2-Iba1 colocalisation appears higher but not significantly different in epileptic mice treated with early-LPS (mean 0.713 median \u0026rho; =0.717) and late-mino (mean 0.705 median \u0026rho; =0.717) - the groups that experience recovery of the breathing phenotype compared to SE group (mean 0.531 median \u0026rho; =0.579) (\u003cstrong\u003eFig. 4A-B\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePearson\u0026rsquo;s correlation coefficient showed P2Y12R-Iba1 immunostaining within the RTN was not significantly different in any of the groups (\u003cstrong\u003eFig. 4C\u003c/strong\u003e), which was evident through the representative micrographs (\u003cstrong\u003eFig. 4D\u003c/strong\u003e). The Pearson\u0026rsquo;s coefficient of different groups is - Control (mean 0.284 median \u0026rho; =0.250), SE (mean 0.246 median \u0026rho; = 0.249), SE + early-LPS (mean 0.389 median \u0026rho; =0.356), SE + late-mino (mean 0.379 median \u0026rho; = 0.338), SE + early-mino (mean 0.332 median \u0026rho; = 0.315), SE + late-LPS (mean 0.326 median \u0026rho; = 0.280), (\u003cstrong\u003eFig. 4C-D\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReduced microglia density, but not C1q expression, in the RTN of early-LPS and late-mino treated mice that show breathing phenotype recovery compared to epileptic mice.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe further evaluated microglia number and its morphology with Sholl analysis in the RTN of different groups of mice. Microglia density is lower in the brainstem compared to forebrain regions\u003csup\u003e38,39\u003c/sup\u003e. Our analysis showed significant reduction in microglia number in SE mice treated with early-LPS (p = 0.0365; mean 3.21 median \u0026rho; = 3.0) and late-mino (p \u0026lt;0.0001; mean 2.10 median \u0026rho; = 2.0) compared to SE group (mean 5.6 median \u0026rho; = 5.0) suggesting reduction in microglia density was linked to rescuing of breathing phenotype in mice with epilepsy (\u003cstrong\u003eFig. 5A, C\u003c/strong\u003e). Although this wasn\u0026rsquo;t consistent with SE + early-mino group which showed significant reduction in microglia density (p \u0026lt;0.0001; mean 2.0 median \u0026rho; = 2.0) without the rescue of the breathing phenotype (\u003cstrong\u003eFig. 5A, C\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eMicroglia Sholl analysis revealed reduced microglia branch length, but not-significantly in SE mice treated with mino (both early and late) and SE + early-LPS group (\u003cstrong\u003eFig. 5B\u003c/strong\u003e). This trend was similar to microglia density comparison and showed reduction in both branch length and number of microglia in both minocycline treated group and group treated with early-LPS.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe next investigated the expression of C1q complement factor, a protein that activates the classical complement pathway, promoting target opsonisation (\u0026ldquo;eat-me\u0026rdquo; signals) and microglia phagocytosis\u003csup\u003e40\u003c/sup\u003e. However, C1q immunolabelling in our study revealed decreased levels of C1q in the RTN of SE mice treated with late-mino (p = 0.0003), early-Mino (p=0.0072), late-LPS (p=0.003) but not early-LPS compared to control and SE groups (\u003cstrong\u003eFig 5D-E\u003c/strong\u003e). We quantified expression levels of C1q in the RTN by measuring the mean fluorescence intensity (MFI) of C1q immunostaining. Our findings are contrary to previous studies where human refractory epileptic tissue demonstrated elevated C1q levels and its increased localisation to dendrites\u003csup\u003e37\u003c/sup\u003e. Additionally, in an LPS-induced mouse model of neuroinflammation, the C1q complement cascade was largely responsible for microglia-dependent synaptic loss in the hippocampus and cognitive impairment\u003csup\u003e36\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAstrocytic adenosine 2A receptor expression upregulated in epileptic mice compared to mice treated with early-LPS showing breathing phenotype recovery.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe used immunohistochemical fluorescence staining to label the A2AR and reactive astrocyte marker, GFAP, in mouse brainstem tissue and analysed changes in A2AR expression in different groups of mice. To quantify and compare the level of A2AR-GFAP colocalisation, the Pearson\u0026rsquo;s linear correlation coefficient (\u0026rho;) was measured. Findings showed extensive colocalisation between GFAP and A2AR in the RTN of SE mice (mean 0.438 median \u0026rho; = 0.381), whereas this was significantly reduced in SE + early-LPS group of mice (p = 0.0263, mean 0.171 median \u0026rho; = 0.131) where we see the recovery of the breathing phenotype (\u003cstrong\u003eFig. 6A-B\u003c/strong\u003e). However, statistical analysis revealed no significant difference in A2AR-GFAP colocalisation between SE and SE + late-mino group of mice (mean 0.195 median \u0026rho; =0.201) which also shows the breathing phenotype recovery. SE + early-mino (mean 0.323 median \u0026rho; =0.309) and SE + late-LPS (mean 0.309 median \u0026rho; =0.306) group also showed equivalent A2AR and GFAP colocalization to SE group. Results indicate a reduction of A2AR-GFAP colocalisation in epileptic mice with acute phase LPS-induced microglia priming, where we notice recovery of breathing phenotype and reduction in seizure frequency compared to SE mice (p=0.0263).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs summarized in the table-1, our findings suggest a potential link between microglial density, TREM2 expression, and astrocytic A2AR expression with the onset of disordered breathing in epilepsy. Interestingly, acute inflammation appears to play a protective role, in contrast to its detrimental effects in chronic epilepsy. Notably, microglial priming with LPS during acute seizures led to a reduction in spontaneous seizure frequency. These results underscore the therapeutic potential of modulating inflammation and inhibiting microglial activation in chronic epilepsy, which may help alleviate respiratory dysfunction and ultimately reduce the risk of SUDEP.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eInflammation is key aspect of animal biology and plays a significant role in modulating neuroglial circuitry within the brain. Its impact on neurological disorders is highly context-dependent, varying with the timing and nature of the condition\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. While inflammation may be protective during early stages, it may also exacerbate disease progression. Recognized risk factors for SUDEP include recurrent, drug-resistant, and nocturnal seizures, prior brain injury, central nervous system infections, living or sleeping alone, and prolonged ictal or postictal central apnoea\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. However, the precise mechanisms by which these factors contribute to cardiorespiratory arrest and SUDEP remain poorly understood.\u003c/p\u003e\u003cp\u003eBrain-mediated central respiratory arrest is a leading cause of SUDEP\u003csup\u003e\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Previous studies, including our own\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, have demonstrated that both humans\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e and animal models with epilepsy\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e exhibit a reduced hypercapnic ventilatory response to elevated CO₂ levels, which may play a critical role in SUDEP pathophysiology\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. We showed that mice with chronic spontaneous seizures have neural corelate in the RTN neurons of reduced respiratory chemosensitivity\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Mild to moderate inflammation, induced by agents such as LPS, can prime glial cells and modulate respiratory responses through distinct immune pathways\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. However, the role of inflammation and glial activation in regulating respiratory function in epilepsy remains unclear. Our findings indicate that glial priming and activation via LPS during the acute phase of epilepsy is protective, preventing respiratory dysfunction and preserving chemosensitivity and hypercapnic ventilatory responses. In contrast, during chronic epilepsy, inflammation exerts detrimental effects, and inhibition of microglial activation during spontaneous seizures restores respiratory and chemosensitive responses in mice.\u003c/p\u003e\u003cp\u003eOur findings have significant implications in understanding the mechanism of sudden death in people with epilepsy. This is especially more significant as we noticed exacerbated breathing phenotype in the cohort of epileptic mice treated with LPS during chronic seizures. Notably, mice from this group showed the most significant reduction in hypercapnic breathing responses (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). The bidirectional role of inflammation in epilepsy, beneficial during acute seizure/epilepsy and detrimental during chronic spontaneous seizures, in modifying breathing responses in mice suggest longevity of the condition and time of inflammation could have different consequences and outcomes for people with epilepsy.\u003c/p\u003e\u003cp\u003eMicroglia, astrocytes, and other immune cells respond to inflammatory signals and mediate their downstream effects. Microglia and astrocytes are the key players in this inflammatory process\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e, and our findings demonstrate that inhibiting microglial activation during the chronic epilepsy with the non-specific inhibitor minocycline can prevent the emergence of respiratory dysfunction in mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Recent studies have shown that microglial fractalkine signalling promotes extracellular adenosine formation and regulates phrenic long-term facilitation following acute intermittent hypoxia\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e, suggesting that changes in microglial receptor expression may influence molecular pathways and respiratory function. We observed a reduction in microglial TREM2 - but not P2Y12 - receptor expression, which may contribute to impaired respiratory chemosensitivity in RTN neurons.\u003c/p\u003e\u003cp\u003eTREM2 is an important immune surface receptor that allows microglia to recognise and respond appropriately to damage, infection, and inflammation. Its signalling is often involved in microglial phagocytosis of apoptotic cells\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e, and loss of TREM2 has been observed in human epileptic tissue\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e and impair microglial phagocytosis ability in epileptic mice\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Our findings imply that reduced microglial TREM2 in epilepsy alter microglia activity and contributes the development breathing phenotype. However, further investigation is required as neither the early minocycline nor the late LPS treatment groups despite exhibiting exacerbated breathing phenotypes showed reduced TREM2 expression. Future studies employing chemogenetic or pharmacological ablation of TREM2 during chronic seizures, alongside respiratory chemosensitivity assessments and histological analyses at the time of breathing impairment, will be essential to resolve this discrepancy.\u003c/p\u003e\u003cp\u003eOn the other hand, P2Y12R is a purinoreceptor, crucial in facilitating microglial process extension and surveillance of the CNS microenvironment\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. In epilepsy, the P2Y12R is involved in limiting seizure activity and regulating neurogenesis\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. However, our data show microglial P2Y12 expression does not correlate with breathing responses in epileptic mice. While P2Y12 has been implicated in seizure-induced neurogenesis\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e and microglial activity following status epilepticus and the outcome of seizures\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e, its role in long-term respiratory modulation appears limited. A key limitation of our immunohistochemical analysis is the temporal mismatch: histological assessments were conducted at week 8 post-SE, whereas the most severe respiratory phenotypes were observed between weeks 3 and 5. Future studies involving time-specific tissue collection will be essential to ascertain the molecular mechanisms underlying breathing impairment in epilepsy.\u003c/p\u003e\u003cp\u003eDespite limitations, microglial morphology and density remain valuable indicators of functional state and are key to understanding their role in disease processes\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. Activated microglia undergo morphological changes that reflect their molecular profile and can contribute to epileptogenesis and disease progression\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Our findings suggest a potential protective role for reduced microglial density and process length in mitigating respiratory dysfunction in epilepsy. However, this pattern did not hold in epileptic mice treated with early minocycline, which also showed reduced microglial number and process length but no improvement in respiratory phenotype. This discrepancy may reflect a secondary effect of minocycline on microglial morphology, independent of functional or molecular changes.\u003c/p\u003e\u003cp\u003eInterestingly, microglial expression of complement factor C1q deviated from expected patterns reported in the literature\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. We observed significantly reduced C1q expression in groups of mice exhibiting the most severe respiratory phenotypes, as well as in those treated with late minocycline, which showed recovery of respiratory phenotype. This unexpected finding suggests that C1q expression may not directly correlate with respiratory outcomes in epilepsy, or that its role is temporally dependent. These inconsistencies highlight the importance of evaluating molecular changes at time points that align with the onset of respiratory dysfunction. Further time-resolved studies are needed to clarify the contribution of C1q and other microglial markers to breathing impairment in epilepsy.\u003c/p\u003e\u003cp\u003eGiven the established role of adenosine and A2AR in phrenic nerve facilitation\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e and their implication in SUDEP mechanisms\u003csup\u003e\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, we examined A2AR expression across all experimental groups and its association with respiratory phenotypes in epilepsy. Although A2AR expression wasn\u0026rsquo;t significantly different between epileptic mice and those treated with late minocycline, our data hints that increased A2AR expression in astrocytes - rather than in microglia or neurons - within the RTN may contribute to impaired chemosensitivity in epilepsy. While the role of adenosine, and particularly A2AR, in respiratory regulation remains unclear\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e, emerging evidence suggests it may act through serotonergic pathways\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. These findings point to a potentially astrocyte-specific mechanism by which A2AR signalling modulates breathing dysfunction in epilepsy, however this needs further investigation.\u003c/p\u003e\u003cp\u003eOverall, our findings underscore the critical importance of inflammation - and its timing - in shaping respiratory function in epilepsy, a key factor in the pathophysiology of SUDEP. The data highlight a potential role for the adenosinergic system, particularly astrocytic A2AR signalling, as well as microglial density and TREM2 receptor expression, in modulating respiratory chemosensitivity. These insights provide promising therapeutic avenues, including the use of minocycline and other modulators of microglial activity, to prevent respiratory dysfunction and potentially reduce SUDEP risk.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e Experiments were performed in accordance with the European Commission Directive 2010/63/EU (European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes) and the United Kingdom Home Office (Scientific Procedures) Act (1986) with project approval from the University of Warwick\u0026rsquo;s AWERB (PP1674884).\u003c/p\u003e\n\u003ch3\u003eImplantation of EEG electrodes and hippocampal cannula\u003c/h3\u003e\n\u003cp\u003eAdult male C57BL/6J mice (8\u0026ndash;10 weeks old and 20\u0026ndash;30 g) were anaesthetized with isoflurane (4%; Piramal Healthcare Ltd, Mumbai, India) in pure oxygen (4 L\u0026middot;min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Adequate anaesthesia was maintained with 0.5-2% isoflurane in pure oxygen (1 L\u0026middot;min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) throughout the surgery. Mice received a presurgical subcutaneous injection of atropine (120 \u0026micro;g\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; Westward Pharmaceutical Co., Eatontown, NJ, USA) and meloxicam (2 mg\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; Norbrook Inc., Lenexa, KS, USA). Mice were placed in a prone position into a digital stereotaxic apparatus (Kopf Instruments, Tujunga, CA, USA) on a heating pad (TCAT 2-LV: Physitemp, Clifton, NJ, USA) and body temperature was maintained at a minimum of 33\u0026deg;C via a thermocouple. The head was levelled, at bregma and 2 mm caudal to bregma. Four holes were drilled for implantation of 2 EEG recording, a ground and a reference electrode. EEG electrodes were PFA-coated silver wire of diameter: 0.254 mm (Bilaney Consultants Ltd, UK) (coordinates: recording electrodes- \u0026plusmn;1.5 mm lateral and 1.0 mm rostral from Bregma, ground and reference electrodes- \u0026plusmn;2.5 mm lateral and \u0026minus;\u0026thinsp;1.5 mm caudal from Bregma) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Silver wires were implanted and secured in position with SuperBond\u0026trade; (Prestige Dental, Bradford, UK). EEG wires were passed through the pedestal (Bilaney Consultants Ltd, UK), and pedestal was implanted and secured in place over the head with SuperBond\u0026trade;. Another hole was drilled for implantation of unilateral hippocampal cannula (coordinates: 1.8 mm lateral and \u0026minus;\u0026thinsp;1.74 mm caudal from Bregma, and \u0026minus;\u0026thinsp;1.3 mm ventral from the surface of the dura). Stainless steel cannula, 26 gauge and 10 mm long, was implanted in the hippocampus and secured in place with SuperBond\u0026trade;. Postoperatively, mice received buprenorphine, and were allowed 1\u0026ndash;2 weeks for recovery, with food and water \u003cem\u003ead libitum\u003c/em\u003e.\u003c/p\u003e\n\u003ch3\u003eInduction of status epilepsy (SE) and EEG recording in freely moving mice\u003c/h3\u003e\n\u003cp\u003eInstrumented mice were anaesthetized with isoflurane and placed into a stereotax as described above. Mice EEG electrodes were connected to wires. SE was induced in mice with total of 0.3 \u0026micro;g (in 100 nl volume) of unilateral kainic acid (KA) injection through hippocampal cannula with Hamilton Neuros syringe (Model 75 RN, Essex Scientific Laboratory Supplies Ltd., UK). Mice were taken off the anaesthesia and placed into open field chamber. EEG wires were connected to the amplifier (Digitimer, Welwyn Garden City, UK) via commutator (FL-2-C-MICRO-M; Dragonfly Research \u0026amp; Development, Inc.). The EEG recording was started in few minutes after KA injection. Mice were scored every minute for their behavioural seizures using Racine scale. 1- Sudden behavioral arrest and/or motionless staring; 2- Facial jerking with muzzle or stiffened and arched tail; 3- Neck jerks, head bobbing or partial body clonus; 4- Clonic seizure in a sitting position; 5- Convulsions including clonic and/or tonic\u0026ndash;clonic seizures while lying on the belly and/or pure tonic seizures; 6- Convulsions including clonic and/or tonic\u0026ndash;clonic seizures while lying on the side and/or wild jumping. The EEG frequency (0-100Hz) was increased during different classes of behavioural seizures (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). At 60 minutes or recording 1\u0026ndash;2 of class 6 scale seizure following injection of KA (whichever happened earlier), mice were anaesthetized with isoflurane and SE was terminated with intraperitoneal injection of ketamine (50 mg/kg) and diazepam (20 mg/kg).\u003c/p\u003e\u003cp\u003eDuring SE, EEG activity was recorded, amplified and filtered using the NeuroLog system (Digitimer, Welwyn Garden City, UK) connected to a 1401 interface and acquired on a computer using \u003cem\u003eSpike2\u003c/em\u003e software (Cambridge Electronic Design, Cambridge, UK). The EEG activity raw data were DC removed. Video data during SE induction was recorded with \u003cem\u003eSpike2\u003c/em\u003e software and was synchronised with the EEG activity.\u003c/p\u003e\n\u003ch3\u003ePlethysmography\u003c/h3\u003e\n\u003cp\u003eMice were placed into a custom-made 0.5 L plethysmography chamber, with an airflow rate of 1 L\u0026middot;min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The plethysmography chamber was heated to 31\u003csup\u003e0\u003c/sup\u003eC (thermoneutral for C57/BL6 mice). CO\u003csub\u003e2\u003c/sub\u003e concentrations were sampled via a Hitech Intruments (Luton, UK) GIR250 Dual Sensor Gas analyzer or ML206 gas analyzer (ADinstruments, Sydney, Australia) connected to the inflow immediately before entering the chamber. The analyser had a delay of ~\u0026thinsp;15\u0026ndash;20 sec to read-out the digital output of gas mixture. Pressure transducer signals and CO\u003csub\u003e2\u003c/sub\u003e measurements were amplified and filtered using the NeuroLog system (Digitimer, Welwyn Garden City, UK) connected to a 1401 interface and acquired on a computer using \u003cem\u003eSpike2\u003c/em\u003e software (Cambridge Electronic Design, Cambridge, UK). Video data was recorded with \u003cem\u003eSpike2\u003c/em\u003e software and was synchronised with the breathing trace. Airflow measurements were used to calculate: tidal volume (V\u003csub\u003eT\u003c/sub\u003e: signal converted to mL following calibration and 4\u0026ndash;5 breathing sweeps were averaged to calculate area under curve (AUC) between two troughs), and respiratory frequency (\u003cem\u003ef\u003c/em\u003eR: breaths per minute). Minute ventilation (V\u003csub\u003eE\u003c/sub\u003e) was calculated as V\u003csub\u003eT\u003c/sub\u003e x \u003cem\u003ef\u003c/em\u003eR.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eHypercapnia in freely behaving mice\u003c/h2\u003e\u003cp\u003eMice were tested for hypercapnic challenge before and 3, 5 and 7-weeks after induction of SE (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Instrumented mice were allowed\u0026thinsp;~\u0026thinsp;30 mins to acclimate to the plethysmograph. Using \u003cem\u003eSpike2\u003c/em\u003e, 3 min of baseline recordings were taken (gas mixture: 0% CO\u003csub\u003e2\u003c/sub\u003e 21% O\u003csub\u003e2\u003c/sub\u003e 79% N\u003csub\u003e2\u003c/sub\u003e). The mice were then exposed to 3 min epochs of hypercapnic gas mixture at different concentrations of CO\u003csub\u003e2\u003c/sub\u003e (3, and 6% in 21% O\u003csub\u003e2\u003c/sub\u003e balanced N\u003csub\u003e2\u003c/sub\u003e). Following exposure to the hypercapnic gas mixtures, CO\u003csub\u003e2\u003c/sub\u003e levels were reduced back to 0% and signals were recorded for a further 4 minutes recovery period (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eMice treatment\u003c/h3\u003e\n\u003cp\u003eFollowing the recording of pre-SE hypercapnic ventilatory responses and the induction of SE or control treatment, mice were assigned to different experimental groups as below. Mice weighing less than 20 g received an intraperitoneal injection of LPS at 0.5 mg/kg. For mice over 20 g, the LPS dose was capped at the amount equivalent to that for a 20 g mouse (0.5 mg/kg), regardless of their actual weight. This initial injection was followed by three additional daily injections at the same dose. The dosing protocol was established based on pilot studies aimed at identifying a dose that was both effective and well-tolerated, without causing significant weight loss.\u003c/p\u003e\u003cp\u003eTo inhibit microglial activation, minocycline - a non-specific microglial inhibitor - was administered orally via drinking water at a concentration of 0.5 mg/mL, provided ad libitum. The groups of mice studied were:\u003c/p\u003e\u003cp\u003e\u003col\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eSE: Epileptic mice (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;11)\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eSE\u0026thinsp;+\u0026thinsp;early-LPS: Epileptic mice treated with LPS for four consecutive days following SE induction (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;12)\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eSE\u0026thinsp;+\u0026thinsp;late-mino: Epileptic mice treated with minocycline from day 11 post-SE induction and continued until the end of the study (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;12)\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eSE\u0026thinsp;+\u0026thinsp;early-mino: Epileptic mice treated with minocycline from day 1 to 10 post-SE induction (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eSE\u0026thinsp;+\u0026thinsp;late-LPS: Epileptic mice treated with four consecutive daily LPS injections from day 15 to 18 following SE induction (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eControl\u0026thinsp;+\u0026thinsp;early-LPS: Control mice treated with LPS for four consecutive days following intrahippocampal vehicle (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eControl\u0026thinsp;+\u0026thinsp;late-mino: Control mice treated with minocycline from day 11 following intrahippocampal vehicle and continued until the end of the study (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eControl (only for immunohistochemistry study): were healthy 8\u0026ndash;12 weeks old C57BL/6 male and female mice.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003c/p\u003e\n\u003ch3\u003eBehavioural seizure analysis\u003c/h3\u003e\n\u003cp\u003eTo analyse the seizure frequency after induction of SE in different groups of mice, 6 mice in each group were continuously recorded from day 1\u0026ndash;2 post-SE till week-7. Due to limitations in the number of cages that could be recorded simultaneously, it was not possible to video-monitor all mice for behavioural seizure analysis. We used MiniCAM, an open-source behavioural camera, for imaging animal movement and behaviour supported with MiniCAM LED ring to provide infrared illumination of the animal and clear behavioural image quality without interfering with animal behaviour (Open Ephys, Lisbon, Portugal). Video data was processed for behavioural seizure analysis using an open-source python-based package for semi-automated detection of convulsive seizures in mice\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. This allowed us to process video data from all singly housed mice recorded over 50 days and identify and validate the convulsive tonic-clonic seizures. Seizure detection was blindly performed, and frequency was compared between different groups.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eImmunohistochemistry\u003c/h2\u003e\u003cp\u003eAt the end of the study and around week-8 post intrahippocampal vehicle or KA treatment, mice were humanely killed by pentobarbital overdose (\u0026gt;\u0026thinsp;100 mg\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and transcardially perfused with paraformaldehyde solution (4% PFA; Sigma-Aldrich, St Louis, MO, USA). The brains were removed and postfixed in PFA (4\u0026deg;C) overnight. Brainstems were serially sectioned at 50\u0026ndash;70 \u0026micro;m. Free-floating sections were incubated for 1 hour in a blocking solution (PBS containing 0.1% Triton X-100 and 5% BSA). Primary antibodies (goat anti- ionized calcium binding adapter molecule 1 (Iba1) [Iba1; 1:100; PA5-18039; Invitrogen, Waltham, MA USA], or chicken anti- glial fibrillary acidic protein (GFAP) [GFAP; 1:1000; PA1-10004; Invitrogen, Waltham, MA USA], or rabbit anti-ChAT [ChAT; 1:2000; ab178850; Abcam PLC, Cambridge, UK], or mouse anti-P2Y12 [P2Y12; 1:5000; 476011; Synaptic systems GmbH, Goettingen, Germany], or mouse anti-TREM2 [TREM2; 1:200; AF1729; R\u0026amp;D Systems, Inc., Minneapolis, MN, Canada], or mouse anti-A2AR [A2AR; 1:1000; 05-717; Sigma-Aldrich, St. Louis, MO, USA], or rabbit anti-C1q [C1q; 1:500; ab182451; Abcam PLC, Cambridge, UK]) were added and tissue was incubated for 2 nights at 4\u003csup\u003e0\u003c/sup\u003eC.\u003c/p\u003e\u003cp\u003eSlices were washed in PBS with 0.1% triton (4 \u0026times; 15 mins) and then the secondary antibodies were added: either donkey anti-mouse Alexa Fluor 405 (1:250; Invitrogen, Waltham, MA USA), or donkey anti-chicken Alexa Fluor 680 (1:250; Jackson ImmunoResearch, Cambridge, UK), or donkey anti-goat Alexa Fluor 680 (1:250; Invitrogen, Waltham, MA USA), or donkey anti-rabbit Alexa Fluor 680 (1:250; Jackson ImmunoResearch, Cambridge, UK), or donkey anti-rabbit Alexa Fluor 594 (1:250; Invitrogen, Waltham, MA USA), or goat anti-rabbit Alexa Fluor 488 (1:250; Invitrogen, Waltham, MA USA), or donkey anti-goat Alexa Fluor 488 (1:250; Invitrogen, Waltham, MA USA) and tissue was then incubated overnight at 4\u003csup\u003e0\u003c/sup\u003eC. Tissue was washed in PBS with 0.1% triton (4 \u0026times; 15 min). Slices were mounted, coverslipped and examined using a Zeiss 880 confocal microscope with ZEN acquisition software (Zeiss, Oberkochen, Germany). 40x and 63x oil objective lenses were used for image acquisition. From the RTN region, 5 images were acquired along the ventral surface per mouse. Images consisted of 15\u0026ndash;17 layer Z-stacks. All images were analysed using ImageJ-Fiji (version 2.16.0/1.54p). Blinded analysis, whereby file names were coded during analysis, was used throughout to prevent unconscious bias.\u003c/p\u003e\u003cp\u003eColocalisation analysis of glial fibrillary acidic protein (GFAP)-A2AR, P2Y12R-Iba1, and TREM2-Iba1 immunoreactivity was performed on maximum intensity projections (MIP) images. Regions of interest (ROI) were used to isolate the RTN. Colocalisation analysis was performed using the BIOP JACoP plugin in Fiji. Pearson\u0026rsquo;s linear correlation coefficient (ρ) was measured for each image, and statistical significance was assessed using Costes\u0026rsquo; randomisation test (2D, 100 permutations). An automated Yen intensity thresholding algorithm was applied to select positive pixels before the analysis. Pearson\u0026rsquo;s coefficient measures the strength and direction of the linear relationship between fluorescence intensities in two images. Its value ranges from 1 to -1, with 1 representing a complete positive correlation, -1, a complete negative correlation, and 0 representing no correlation.\u003c/p\u003e\u003cp\u003eSholl analysis was done on MIP images of Iba1-immunoreactive microglia. Iba1\u0026thinsp;+\u0026thinsp;microglia were randomly selected from the RTN (5 microglia per mouse). The number of Sholl intersections were quantified at radial intervals of 0.5 \u0026micro;m, starting from the microglia soma. Manual cell counting of Iba1\u0026thinsp;+\u0026thinsp;microglia in the RTN was done using the inclusion criteria: microglia with visible cell bodies in the image frame. Mean fluorescence intensity (MFI) of C1q was measured within RTN ROIs and normalised based on ROI area.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eStatistical analysis was performed in GraphPad Prism software (version 10.1.0). Statistical significance for changes in breathing was determined using two-way repeated measure ANOVA followed by t tests with the Tukey\u0026rsquo;s correction and shown as a line graph. Comparisons were done between pre- and post-treatment (SE, control or treatment groups). The Racine seizure scores and seizure frequency between different groups (violin plot with superimposed data points) and histological data, including Sholl analysis, differences between multiple groups were compared with Kruskal-Wallis non-parametric test. Significant statistical tests were followed by Dunn\u0026rsquo;s post hoc comparisons test. P\u0026thinsp;\u0026le;\u0026thinsp;0.05 was considered significant. ****\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.0001, ***\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.001, **\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.01 and *\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05. Results are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD or median and interquartile ranges throughout the text.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFunding: This work was supported by the Epilepsy Research Institute (ERI) UK Project Grant (P1903), Emerging Leader Fellowship award (F2203), an MRC Discovery Award (MC_PC_15070), and Wellcome-Warwick QBP funds. AB is an ERI Emerging Leader Fellow and ND was a Royal Society Wolfson Research Merit Award Holder. The authors acknowledge use of imaging facility and support provided by the Bio-Analytical Shared Resource Laboratories within the School of Life Sciences, University of Warwick.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAMB (Amol M. Bhandare) designed the study, performed all \u003cem\u003ein vivo\u003c/em\u003e experiments, recordings, assisted immunohistochemistry data analysis and wrote the manuscript; ND conceived and designed the study, and contributed to writing of the manuscript. AB (Adwoa Boaten) performed immunohistochemistry, data analysis and contributed to writing of the manuscript. DD performed behavioural seizure detection analysis from continuous video monitoring. BB performed breathing responses data analysis and JH performed immunohistochemistry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e The authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData and Materials availability:\u003c/strong\u003e Raw data available on request.\u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e\u003cp\u003eThis work was supported by the Epilepsy Research Institute (ERI) UK Project Grant (P1903), Emerging Leader Fellowship award (F2203), an MRC Discovery Award (MC_PC_15070), and Wellcome-Warwick QBP funds. AB is an ERI Emerging Leader Fellow and ND was a Royal Society Wolfson Research Merit Award Holder. The authors acknowledge use of imaging facility and support provided by the Bio-Analytical Shared Resource Laboratories within the School of Life Sciences, University of Warwick.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eNorton, A. 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A python-based package for long-lasting video acquisition and semi-automated detection of convulsive seizures in rodents. \u003cem\u003ebioRxiv\u003c/em\u003e, 2022.2004.2015.488472 (2022). https://doi.org/10.1101/2022.04.15.488472\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-neuroinflammation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jneu","sideBox":"Learn more about [Journal of Neuroinflammation](http://jneuroinflammation.biomedcentral.com)","snPcode":"12974","submissionUrl":"https://submission.nature.com/new-submission/12974/3","title":"Journal of Neuroinflammation","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7707048/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7707048/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eImpaired CO₂ responsiveness in epilepsy can result in hypoventilation and hypercapnia and these respiratory disturbances are key contributors to Sudden Unexpected Death in Epilepsy (SUDEP). While mild to moderate inflammation is known to modulate respiratory function, its specific role in regulating respiratory responses in the context of epilepsy remains unclear. We studied the effects of lipopolysaccharide (LPS)-induced glial priming and microglial inhibition via minocycline during the acute and chronic phases of epilepsy on hypercapnic ventilatory responses (HCVR) in the intrahippocampal kainic acid model of temporal lobe epilepsy in male C57BL/6 mice. LPS treatment during acute seizures and minocycline during spontaneous seizures in the chronic phase of epilepsy restored the impaired HCVR in mice. Notably, LPS treatment during acute seizures also reduced the frequency of spontaneous seizures. In contrast, minocycline given during acute seizures and LPS administered during chronic epilepsy further exacerbated HCVR impairment. Immunohistochemical analysis of chemosensitive retrotrapezoid nucleus (RTN) revealed varied effects of different treatments in epileptic mice on microglia density, morphology and their expression of triggering receptor expressed on myeloid cells 2 (TREM2), P2Y12 receptor, and astrocytic adenosine 2A receptor (A2AR). Overall, inflammation, along with associated changes in microglial and astrocytic receptor expression, plays a central role in the reduction of HCVR in epilepsy and may represent a key mechanistic target in SUDEP.\u003c/p\u003e","manuscriptTitle":"Inflammation modifies breathing phenotype in mice with epilepsy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-08 10:25:04","doi":"10.21203/rs.3.rs-7707048/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-24T05:40:32+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-20T12:39:42+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-16T18:59:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"102962934084993280640308941427605225687","date":"2025-10-01T05:36:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"305165598395880266972904081974044809861","date":"2025-09-29T13:53:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"224913257650240759791627536063180465570","date":"2025-09-26T12:20:40+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-26T05:31:43+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-26T05:14:23+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-26T01:26:29+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Neuroinflammation","date":"2025-09-24T21:29:06+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-neuroinflammation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jneu","sideBox":"Learn more about [Journal of Neuroinflammation](http://jneuroinflammation.biomedcentral.com)","snPcode":"12974","submissionUrl":"https://submission.nature.com/new-submission/12974/3","title":"Journal of Neuroinflammation","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"734dc321-04c4-4d12-b201-04c101c43a0a","owner":[],"postedDate":"October 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-09T14:27:43+00:00","versionOfRecord":{"articleIdentity":"rs-7707048","link":"https://doi.org/10.1186/s12974-025-03670-6","journal":{"identity":"journal-of-neuroinflammation","isVorOnly":false,"title":"Journal of Neuroinflammation"},"publishedOn":"2026-01-07 00:00:00","publishedOnDateReadable":"January 7th, 2026"},"versionCreatedAt":"2025-10-08 10:25:04","video":"","vorDoi":"10.1186/s12974-025-03670-6","vorDoiUrl":"https://doi.org/10.1186/s12974-025-03670-6","workflowStages":[]},"version":"v1","identity":"rs-7707048","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7707048","identity":"rs-7707048","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}