IL-1 modulation preserves biomolecular, structural and functional integrity of the somatosensory cortex after fetal inflammation

IL-1 modulation preserves biomolecular, structural and functional integrity of the somatosensory cortex after fetal inflammation | 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 Article IL-1 modulation preserves biomolecular, structural and functional integrity of the somatosensory cortex after fetal inflammation Robert Galinsky, Sharmony Kelly, Steven Cho, Valerie Zahra, Mira Menyen, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7882525/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Perinatal inflammation, often caused by infection, is strongly linked with lifelong disability. Human and experimental studies identify interleukin-1 (IL-1), a pro-inflammatory cytokine, as a key mediator. We tested the hypothesis that systemic administration of IL-1 receptor antagonist (IL-1Ra) could attenuate cortical inflammation and improve neuronal development in late gestation fetal sheep exposed to lipopolysaccharide (LPS)-induced inflammation. Fetal sheep, instrumented for continuous EEG, were randomised to: (1) saline infusion, (2) repeated intravenous LPS + vehicle infusions or (3) the same LPS regimen plus intravenous IL-1Ra infusions one hour after each LPS dose. Four-days later, brains were examined using RNAseq, Golgi staining and immunohistochemistry. On EEG, LPS-exposure reduced beta power compared to control, particularly in REM sleep. In the somatosensory cortex, LPS-exposure decreased expression of genes involved in dendritogenesis and synaptogenesis, and increased genes involved in immune activation via LPS and IL-1 signalling. LPS-exposed fetuses had increased microglial activation and reduced neuronal arborisation. IL-1Ra treatment improved EEG band power, normalised expression of genes involved in synaptogenesis, dendritogenesis and immune activation, reduced microglial activation, and restored neuronal arborisation. In summary, IL-1Ra reduced LPS-induced inflammation and improved biomolecular, structural and functional markers of neurodevelopment. Thus, IL-1Ra may improve neurodevelopmental outcomes following perinatal infection/inflammation. Biological sciences/Neuroscience/Regeneration and repair in the nervous system Health sciences/Neurology/Neurological disorders/Brain injuries/Neonatal brain damage Neuroinflammation neurophysiology interleukin 1 interleukin 1 receptor antagonist neuronal dendrites RNAseq Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Exposure to inflammation, often caused by infection, during the perinatal period is strongly associated with a greater risk of perinatal mortality, brain injury and neurodevelopmental impairments; including learning difficulties, autism spectrum disorder, attention deficit and hyperactivity, schizophrenia and cerebral palsy 1 , 2 , 3 . The risk of cerebral palsy (CP) is several-fold greater after perinatal infection/inflammation (odds ratio 2.5–9.3) 4, 5, 6 . The cumulative lifetime cost of CP in the US was estimated to be over USD11.5 billion in 2003 2 , which equates to approximately USD20 billion in 2025 after adjusting for changes in the CPI, not including the wider burden on affected individuals, their families and society 7 . Structurally, MRI studies have shown that exposure to antenatal infection is independently associated with reductions in functional connectivity 8 and impaired cortical development, including reduced sulcal depth and cortical volume (particularly in the parietal lobe/somatosensory cortex), without evidence of overt grey matter injury 9 , 10 . Reduced neonatal cortical volume has been shown to persist into adolescence and is associated with impaired cognition 11 . Preclinical studies indicate that these associations between exposure to antenatal infection / inflammation and impaired cortical development are likely causal. For example, in fetal sheep, lipopolysaccharide (LPS)-induced inflammation was associated with increased gliosis and reduced neuronal complexity within the somatosensory cortex but did not affect overall numbers of neurons 12 . In neonatal rats, LPS-induced inflammation from postnatal days 1–3 led to reductions in neuronal complexity and cortical volume without affecting numbers of neurons in the motor cortex 13 . Moreover, maternal injections of group B Streptococcus in pregnant rats between gestational days 19 and 22 led to thinning of the cerebral cortex and impaired motor function in offspring at postnatal day 40 14 . Although multiple pathways are involved in the pathogenesis of neurological injury after exposure to perinatal infection and/or inflammation, the pro-inflammatory cytokine interleukin-1β (IL-1β) is consistently upregulated in blood and brain samples from human and animal studies of perinatal encephalopathy 15 , 16 , 17 . Further, elevated cord blood IL-1β concentration has been linked to impaired brain metabolism and developmental delay in early childhood 17 , 18 , and increased circulating IL-1β concentration in fetal sheep, neonatal mice and piglets is associated with both white and grey matter injury 19 , 20 , 21 , 22 , 23 . Collectively, these data support the hypothesis that controlling IL-1β bioactivity during perinatal inflammation could help attenuate neural injury and improve neuronal function. We tested the hypothesis that IL-1 inhibition starting one hour after LPS exposure with an FDA approved IL-1 receptor antagonist (IL-1Ra), anakinra, mitigates molecular, electrophysiological, and histological markers of inflammation and neuronal injury in the somatosensory cortex of late gestation fetal sheep. Results Baseline period and fetal growth Before LPS exposure, EEG spectra did not differ between groups and were within the normal range for our laboratory. There were no differences in subject brain and body weights or sex between the groups (Control: body weight = 4.6 ± 0.6 Kg, brain weight = 51.3 ± 3.7 g, 34% female; LPS + vehicle: body weight = 4.6 ± 0.5 Kg, brain weight = 49.8 ± 3.4 g, 25% female; and LPS + IL-1Ra: body weight = 4.7 ± 0.9 Kg, brain weight = 49.2 ± 5.2 g, 44% female). Spectral band analysis Delta, theta, and alpha band power were not different did not differ between the groups (Supplementary Figure 3A - C). In the LPS + vehicle group, beta band power was increased from 4 – 20 hours after the first LPS infusion ( P < 0.05 vs controls, Figure 1). After the second LPS infusion, beta band power was higher in the LPS + vehicle group between 28 and 48 hours (i.e 4 – 24 hours after the second LPS infusion; P < 0.05 vs controls, Figure 1). After the third LPS infusion, beta band power was higher in the LPS + vehicle group between 60 and 64 hours, and 80 and 92 hours (i.e. 12 – 16 hours, and 32 – 44 hours after the third LPS infusion; P < 0.05 vs controls, Figure 1). Beta band power was increased in the LPS + IL-1Ra group from 4 – 24 hours after the first LPS infusion ( P < 0.05 vs LPS + vehicle, Figure 1). After the second LPS infusion, beta band power was higher in the LPS + IL-1Ra group between 28 and 48 hours (i.e 4 – 24 hours after the second LPS infusion; P < 0.05 vs LPS + vehicle, Figure 1). After the third LPS infusion, beta band power was higher in the LPS + IL-1Ra group from 52 – 64 hours, and from 80 – 92 hours (i.e. 8 – 20 and 32 – 44 hours after the third LPS infusion; P < 0.05 vs LPS + vehicle, Figure 1). Sleep state cycling Sleep state cycling was observed in all subjects throughout the experimental period (Figures 2A - C). During the final 24 hours of the experimental period, EEG power was increased in the LPS + vehicle group compared to the control and LPS + IL-1Ra groups during REM sleep between 1200 hours and 0300 hours ( P < 0.05; Figure 2D). There was no difference in EEG power during nREM sleep (Figure 2E). Spectral edge frequency did not differ between groups during REM and nREM sleep (Figures 2F and G). There were no differences in delta band power between the groups during REM and nREM sleep (Figures 2H and I). In the LPS + vehicle group, the proportion of beta band power was decreased during REM sleep compared to control between 0900 hours and 0600 hours ( P < 0.05, Figure 2J). In the LPS + IL-1Ra group, the proportion of beta band power was increased compared to LPS + vehicle between 1200 and 1500 and 0300-0600 hours ( P < 0.05, Figure 2J). There were no differences in beta band power between the groups during nREM (Figure 2K). The proportions of theta and alpha band power did not differ between groups during REM and nREM sleep states throughout the last 24 hours of the experimental period (Supplementary Figure 4 A-D). RNA expression (RNA sequencing) To gain insight into the biomolecular changes associated with inflammation-induced changes in cortical neuronal growth we performed RNA sequencing on frozen tissue sections collected from the lateral cortex adjacent to the regions analysed for neuronal morphology and histopathology. We identified 281 genes that were differentially expressed in the LPS + vehicle ( n = 8) compared to the control group ( n = 7, 2 samples did not contain sufficient RNA), with 86 genes (31%) being of unknown function (Figure 3A, Supplementary Table 1). 326 genes were differentially expressed between LPS + IL-1Ra ( n = 9) vs LPS + vehicle ( n = 8). Pathway over-representation analysis demonstrated that altered gene expression between LPS + vehicle vs control was predominantly associated with the toll-like receptor 4 (TLR4) ligand binding and IL-1 family signalling pathways, as well as inflammatory pathways linked to the G protein coupled receptor (GPCR) cascade (Figures 3C and D, Supplementary Figure 5A). Genes mapped to GPCR ligand binding pathways were predominantly decreased in the LPS + vehicle group when compared to controls, with the most differentially expressed genes being largely hormones such as corticotropin-releasing hormone ( CRH ), parathyroid hormone ( PTH ) and thyrotropin-releasing hormone ( TRH ) or GPCRs for neuropeptides such as neuropeptide FF receptor 2 ( NPFFR2 , reduced expression in LPS + vehicle vs. control) and tachykinin receptor 3 ( TACR3 , increased expression in LPS + vehicle vs. control) (Figure 3A, Supplementary Table 1). Taken together these observations suggests LPS-activation of a pro-inflammatory response that is accompanied by changes to the expression of neuropeptides linked to GABAergic interneuron development and regulation of neuronal branching ( CRH ), synapse formation ( TRH ), neural excitation ( PTH and TRH ) 24, 25, 26 , and neurodegeneration ( NPFFR2 and TACR3 ) 27, 28 . When comparing LPS + vehicle to the LPS + IL-1Ra group, we did not observe significant difference to genes related to the TLR4 cascade that were upregulated by LPS (Figures 3B and C, Supplementary Table 2). Similarly, most GPCR ligand binding genes modulated by LPS also remained unchanged versus LPS + IL-1Ra, except for endocrine genes where we observe a significantly upregulated TRH and CRH (LPS + IL-1Ra vs. LPS + vehicle, Figure 3D). Pathway over-representation analysis showed the dominant gene changes between LPS + vehicle and LPS + IL-1Ra groups were primarily associated with modulated expression of extracellular matrix organisation genes that regulate synaptogenesis and spine development ( FBLN2) 29 , dendrite growth ( AGRN) 30 , IL-1 signalling and microglial activation ( LOX and ADAMTS4) 31, 32 (Figure 3E) or overlapping pathways such as collagen formation, including genes such as COL13A1 , COL19A1 and COL21A1 (Supplementary Figure 5B). We identified 56 genes that were differentially expressed between the LPS + vehicle group ( n = 8) compared to the control ( n = 7) and LPS + IL-1Ra groups ( n = 9) (Figures 4A and B and Supplementary Table 3) to represent genes that may be related to IL-1Ra's mechanistic actions or genes that are indicative of IL-1Ra action. After excluding genes of unknown function and genes with limited specificity in brain tissue, 42 genes remained in the analysis. These 42 genes were divided into two key pathways: neuronal structure and function (20) and immune pathways and function (22) and are described in Table 1. In summary, in the LPS + IL-1Ra group we observed normalised expression of genes relating primarily to dendritogenesis (e.g. NRG4, COL13A1, KIF16B ), synaptogenesis (e.g. CIC2 , CTXN3, FBLN2, VGLUT1, TRH ), neurorepair (e.g. DEPTOR, ESR2, STAR, IGF1, IGF-R1 ), neurodegeneration (e.g. NEMP1, PPP1R42, RABEP2 ) and immune activation (e.g. GVIN1, H1-6, IL-27, TRIM47, TRIM55 ) ( P <0.05 vs. LPS + vehicle and NS vs. control, Table 1). Of the 56 genes that were differentially expressed between the LPS + vehicle group compared to the control and LPS + IL-1Ra groups, 18 genes were not classified. Using Ensemble genome browse and Basic Local Alignment Search Tool (BLAST), we identified 10 of these 18 genes that had a known classification in ovine tissue: (ENSOARG00020034656: MFSD14A, ENSOARG00020039350: PNPLA3, ENSOARG00020033980: ubiquitin D, ENSOARG00020037154: IL27, ENSOARG00020006863: serpin B6, ENSOARG00020005819: APOC2, ENSOARG00020034724: C13H20orf96, ENSOARG00020038681: GIMAP8, ENSOARG00020037893: WWP1, and ENSOARG00020009042: GVIN1). Thus, 8 genes were not included for further analysis due to no classification/unknown function of these genes in ovine brain (ENSOARG00020040904, ENSOARG00020031121, ENSOARG00020032393, ENSOARG00020037506, ENSOARG00020038936, ENSOARG00020037822, ENSOARG00020036053, ENSOARG00020037194). Six genes were not subject to further analysis due to limited specificity relating to their role in brain tissue (C13H20orf96, CHTF18, GYG2, OSBPL3). Neuronal dendrite morphology To determine the effects of inflammation on neuronal morphology, we assessed Golgi-stained basal dendrites collected four days after the start of LPS exposure. In cortical neurons of the lateral parietal lobe, the number of dendritic terminals on basal dendrites was significantly reduced in the LPS + vehicle group compared to controls ( P < 0.05, Figures 5A, E, F). The number of dendritic terminals in the LPS + IL-1Ra group was significantly increased compared to LPS + vehicle ( P < 0.05, Figure 5A and G). In the LPS + vehicle group, the summated length of basal dendrites was significantly reduced compared to controls ( P < 0.05 vs controls, Figure 5B). In the LPS + IL-1Ra group, summated dendritic length did not differ from controls but was not significantly increased compared to LPS + vehicle ( P = 0.07 vs LPS + vehicle, Figure 5B). Sholl analysis of pyramidal neuron complexity showed reduced dendritic arborisation in the LPS + vehicle group compared to control at 40 – 230 μm away from the soma ( P < 0.05; Figure 5C). In the LPS + IL-1Ra group, dendritic arborisation was increased compared to LPS + vehicle at 5 – 75 μm away from the soma ( P < 0.05; Figure 5C). In the LPS + IL-1Ra group, dendritic arborisation was increased compared to control at 5 – 45 μm away from the soma ( P < 0.05, Figure 5C). In the LPS + IL-1Ra group, dendritic arborisation was reduced compared to control at 65 – 220 μm away from the soma. Neuronal dendritic spine number and morphology The total number of dendritic spines was reduced in the LPS + vehicle and LPS + IL-1Ra groups compared to control ( P < 0.05, Supplementary Figure 6). Numbers of long thin spines were significantly reduced in the LPS + vehicle group compared to control ( P < 0.05, Figure 5G). There were no significant differences in numbers of stubby, mushroom, or filipodia spines between the groups (Supplementary Figure 6). Histopathology IL-1β immunoreactivity was increased in the somatosensory cortex of the LPS + vehicle group compared to controls ( P < 0.05, Figures 6A and F). In the LPS + IL-1Ra group, IL-1β immunoreactivity was reduced compared to the LPS + vehicle group ( P < 0.05, Figures 6A and F). Numbers of Iba-1+ microglia were increased in the somatosensory cortex of the LPS + vehicle group compared to controls ( P < 0.05, Figure 6B, G). In the LPS + IL-1Ra group, numbers of Iba-1+ microglia were reduced compared to the LPS + vehicle group ( P < 0.05, Figures 6B and G). Numbers of reactive (phosphorylated (p)STAT3+/Iba-1+) microglia were increased in the somatosensory cortex in the LPS + vehicle group compared to control ( P < 0.05; Figures 6C, H). In the LPS + IL-1Ra group, numbers of (p)STAT3+/Iba-1+ microglia were reduced in the LPS + IL-1Ra group compared to LPS + vehicle ( P < 0.05; Figures 6C and H). Numbers of GFAP+ astrocytes and NeuN+ neurons did not differ between the groups in the somatosensory cortex (Figures 6D and I and 6E, J, respectively). Discussion This study demonstrates that competitive IL-1 receptor inhibition, using the FDA approved IL-1Ra anakinra (identical to endogenous human IL-1Ra except for one amino acid), improved neuronal complexity (arborisation) of pyramidal neurons and normalised RNA expression of genes that regulate neuronal development and neuroinflammation during LPS-induced inflammation in late gestation fetal sheep. Functionally, the improvements in histological and biomolecular outcomes in IL-1Ra treated subjects were associated with improved development of EEG spectral band power and normalisation of fetal sleep state architecture. To our knowledge, this is the first in vivo study to integrate bulk RNA sequencing, histopathology and electrophysiology to demonstrate the molecular, cellular and functional effects of IL-1Ra on cortical pyramidal neurons in a large animal translational model of perinatal inflammation. Clinically, perinatal infection/inflammation is associated with an increased risk of impaired neurodevelopment 4 , 33 , 34 , 35 , 36 . Moreover, Gram negative infections including E. coli continue to be among the most common pathogens linked to perinatal infection/inflammation and increased risk of perinatal brain injury 37 , 38 . In this study, we reproduced key features of Gram-negative infection-induced inflammation using repeated and escalating doses of LPS infusions to promote a progressive fetal inflammatory response, including increased circulating concentrations of IL-1β (previously published in 19 ). Elevated systemic and cerebrospinal fluid concentrations of IL-1β within the first few days after birth were associated with impaired brain metabolism and developmental delay in early childhood 17 . Similarly, in preterm infants elevated circulating IL-1β within the first 2 weeks after birth were associated with impaired neurodevelopment at 2 years of age 18 . Polymorphisms in the IL1B gene that promote increased production of the IL-1β protein are associated with an increased risk of intraventricular haemorrhage and periventricular leukomalacia 39 . Moreover, polymorphisms in the gene encoding IL-1Ra, known as IL1RN , that reduce production of IL-1Ra and increase proinflammatory signalling are associated with an increased risk of stillbirth 40 . In post-mortem brain tissue from human neonates (gestational age range: 29–37 weeks), accumulation of IL-1β was localised to areas of parenchyma where markers of gliosis and injury were highest 15 . Furthermore, in areas of tissue injury, accumulation of IL-1Ra was reduced compared to IL-1β, thus reducing the IL-1Ra:IL-1β ratio. This imbalance of pro- and anti-inflammatory cytokine concentrations was more pronounced in infants with worse histological outcomes. Mechanistic studies in small and large animals have shown a strong link between elevated systemic and cerebral IL-1β with neuroinflammation and perinatal brain injury 41 . Consistent with these observations, in the present study we show that progressive LPS exposure increased RNA expression of genes related to E. Coli -induced inflammatory signalling, including the TLR4 and IL-1 associated pathway genes: IRAK4, CD14, IRAK2, MAPKAPK2 LY86 and PNPLA3 . Collectively, these data confirm that inflammation, induced with repeated increasing doses of E. coli LPS in fetal sheep, promotes neuroinflammation and disturbances in neuronal development and function through activation of the TLR4 and IL-1 signalling pathways within the somatosensory cortex. Intravenous treatment with anakinra reduced IL-1β immunoreactivity in the somatosensory cortex compared to LPS + vehicle. Consistent with this, numbers of total and activated (phosphoSTAT3+) microglia were reduced in the somatosensory cortex of LPS + IL-1Ra treated subjects compared to LPS + vehicle. Further, there was increased RNA expression of genes responsible for IL-1β signalling and immune activation in the somatosensory cortex of the LPS + vehicle group compared to controls. Moreover, in the LPS + IL-1Ra group we observed restored RNA expression (P < 0.05 vs. LPS + vehicle but not control) of multiple genes linked to microglial activation and neuroinflammation, indicating IL-1Ra treatment normalised RNA expression of these genes in the somatosensory cortex. These data are consistent with evidence that excessive systemic and / or locally produced IL-1β contributes directly to brain immune activation and neural injury, at least in part, through microglial activation which can further increase secretion of IL-1β and other pro-inflammatory cytokines into the brain parenchyma to promote tissue damage 41 . Together, these data suggest that mitigation of microglial activation with IL-1Ra mediated the reduction in IL-1β accumulation in the somatosensory cortex of LPS + IL-1Ra treated subjects compared to LPS + vehicle. Moreover, in vitro studies have shown that microglial processes interact with synapses to eliminate dendritic spines suggesting a direct effect of microglial activation on dendritic spine density 42 , 43 . However, IL-1Ra treatment did not significantly ameliorate the LPS-induced reduction in numbers of dendritic spines (LPS + vehicle vs. LPS + IL-1Ra, P = 0.08). Preclinical and human cohort studies have shown that elevated circulating levels of IL-1β are associated with impaired cerebral oxidative metabolism and suppression of EEG amplitude and frequency in fetuses and neonates 12 , 17 , 19 . Consistent with these studies, we now show that LPS-exposed fetuses had disturbed distribution of EEG spectra throughout the experimental period compared to controls. This included an increase in the proportion of EEG activity in the delta (slow wave) band and a reduction in the proportion of EEG activity in beta (fast wave) band. This was also associated with higher total EEG power and a reduction in the proportion of EEG activity in the beta band (reduced high frequency activity) during phases of rapid eye movement (REM) sleep in LPS-exposed fetuses during the penultimate 24 hours of the experimental period. REM sleep is defined by cycling between low power and high frequency activity with bursts of high frequency beta waves 44 . It is postulated to be critical for maintenance and development of neuronal circuits through strengthening of neuronal dendrites and dendritic spines to facilitate synapse formation 45 . This notion is supported by the finding of the present study, that LPS exposed fetuses had reduced neuronal dendritic arborisation and fewer numbers of dendritic spines. In humans and sheep, the marked cortical expansion that occurs during the last trimester of gestation reflects a prolific increase in neuronal dendritic growth and spine development during this stage of development. Our observations suggest that, at this stage of late gestation, neuronal development within the somatosensory cortex is vulnerable to inflammation-induced impairments in dendritic arborisation, and that these structural abnormalities are manifest by functional disturbances that can be detected electrographically. In line with the reduced neuronal arborisation in the LPS + vehicle group compared to controls, we observed a reduced RNA expression of vesicular glutamate transporter 1 (VGLUT1, gene name: SLC17A7 ) (Fig. 4 B, Table 1 ) which is known to be expressed in presynaptic terminals and dendrites of excitatory pyramidal neurons in the somatosensory cortex 46 . VGLUT1 is integral for transporting glutamate into synaptic vesicles and has a key role in glutamate mediated neurotransmission and synaptic function 47 . Collectively these data suggest that inflammation-induced disturbances to pre or post synaptic connections between glutamatergic neurons or impaired glutamatergic signalling could contribute to the impairments observed in neuronal arborisation and EEG activity in the somatosensory cortex of LPS-exposed fetuses. Moreover, in the LPS + vehicle group compared to controls, we observed reduced RNA expression of neuregulin-4 ( NRG4 ), steroidogenic acute regulatory protein ( STAR ) and thyrotropin releasing hormone ( TRH ), parathyroid hormone ( PTH ). The genes are known to play critical roles in the development and maintenance of neuronal signalling through supporting dendritic arborisation, spine development and synaptogenesis of cortical neurons 25 , 48 , 49 . In the LPS + IL-1Ra treated fetuses, the distribution of EEG spectral band power was comparable to controls throughout the experimental period. Moreover, the improved EEG activity in the LPS + IL-1Ra treated subjects was associated with an improvement in numbers of dendritic terminals and intermediate improvements in dendritic length and dendritic arborisation compared to the LPS + vehicle group. Indeed, in vitro studies show that IL-1β inhibits growth and branching of neurons and this can be reversed with IL-1β blockade 50 . RNA sequencing showed IL-1Ra treatment restored RNA expression of genes controlling dendritogenesis and synaptogenesis, including COL13A1, CTXN3, NRG4, VGLUT1 and TRH 47 , 48 , 49 , 51 , 52 , 53 . Moreover, gene pathway analysis showed genes relating to extracellular matrix organisation and collagen formation were the most differentially expressed in the LPS + IL-1Ra group compared to LPS + vehicle. Specifically, in the LPS + IL-1Ra group compared to LPS + vehicle there was increased RNA expression of AGRN, FNLN2, COL19A1 and COL21A1 which have all been linked to development, maintenance and maturation of neuronal dendrites and synapses 29 , 54 , 55 , 56 , 57 , 58 , and reduced expression of ADAMTS4 and LOX which has been linked to neurodegeneration through promotion of perineuronal net degradation and suppression of dendrite development, respectively 59 , 60 , 61 . Collectively, these data suggest that IL-1Ra promotes upregulation and maintenance of neuroprotective and regenerative qualities, independent of its direct effects on IL-1 and immune signalling. Under basal conditions IL-1ɑ and β abundance is low in the brain (Rothwell 2003). Indeed, a low concentration of IL-1, particularly IL-1β, is important for regulating neurodevelopment 62 ; however, it is rapidly induced in pathological inflammation (Rothwell 2003). In the present study we used the commercially available IL-1Ra, anakinra, to inhibit IL-1 mediated systemic inflammation and brain pathology in late gestation fetal sheep. Anakinra is a non-glycosylated form of the human IL-1Ra that exerts its physiological effects by competitively binding to the IL-1 receptor and attenuating the effects of IL-1 to prevent downstream inflammatory signalling 41 . Anakinra has been in clinical use for over 20 years (FDA approved in 2001) in a range of auto immune conditions, as well as sepsis and other hyper-inflammatory syndromes to reduce inflammation related morbidity. It has an established safety profile in adults and adolescents, and its safety, feasibility and pharmacokinetics is currently being investigated in a phase 1b/2a clinical trial in preterm infants 63 . Anakinra has a molecular weight of 17 kDa and has been shown to penetrate the blood brain barrier in humans and preclinical animal studies 64 . We and others have shown that repeated intravenous dosing with IL-1Ra does not completely abolish IL-1 signalling, with IL-1β concentrations in plasma and brain tissue being similar to controls 19 , 23 . Moreover, in neonatal rodents, repeated IL-1Ra administration had no deleterious effects on brain anatomy or behavioural outcomes 65 , 66 . In contrast, only complete loss of IL-1 signalling in knockout and transgenic animal has been associated with impaired neurodevelopment 65 , 67 . Collectively, these data suggest that allowing for restoration of IL-1 signalling via intermittent IL-1Ra treatment does not adversely affect makers of brain structure and function in small and large animal models. Although we did not serially sample CSF throughout the experimental period to confirm IL-1Ra concentrations in the sheep brain, previous studies in adults demonstrated that intravenous IL-1Ra crosses the BBB and achieves therapeutic concentrations in the brain within approximately 45-minutes 64 . This study, together with others, demonstrates that IL-1 is implicated in the pathophysiology of infection/inflammation related injury in the developing perinatal brain, and that restoring IL-1 homeostasis with IL-1Ra is a promising therapeutic option for infants with acute infection around the time of birth. However, the optimal timing of treatment remains uncertain. Critically emerging evidence suggests that ongoing neuroinflammation contributes to the sub-acute and chronic phases of neural injury, which may develop several days to weeks after the insult 68 , 69 . This raises the possibility that early or delayed use of IL-1Ra alone or in combination with complementing interventions targeting cell damage or repair (e.g. anti-excitotoxicity, trophic factors, stem cells, essential amino acids) 70 , 71 , 72 , could be an effective strategy to mitigate perinatal brain injury and warrants interrogation in future translational studies. There are no clinically proven treatments to prevent brain injury related to perinatal infection or inflammation. Current immunomodulatory therapies used in routine clinical care (e.g. antibiotics and corticosteroids) are limited by their broad mechanisms of action and their potential to cause deleterious effects in both animals and humans. For example, corticosteroids are associated with an increased risk of cerebral palsy, intraventricular haemorrhage and hyperactivity in childhood 73 , 74 ; the underlying mechanisms remain unknown. Prophylactic antibiotics have been linked to an increased risk of neonatal death and disability 75 , the reason for this is unclear but animal studies speculate it could be linked to bacterial lysis promoting the release of bacterial fragments that augment inflammation-induced illness and tissue injury 72 , 76 , 77 , 78 . Thus, a more targeted anti-inflammatory approach to modulate the effects of unbridled inflammation in the perinatal brain could mitigate these harmful off target effects. Here, we show that IL-1Ra administered to late gestation fetal sheep exposed to progressive LPS-induced inflammation restored expression of genes related to dendritogenesis, synaptogenesis and immune activation in the somatosensory cortex. Moreover, In IL-1Ra treated subjects we observed reduced markers of cortical inflammation and improved cortical neuronal complexity and electrophysiological recovery. Collectively, these findings suggest that IL-1Ra could be a promising anti-inflammatory intervention to prevent perinatal brain injury in infants exposed to infection/inflammation during the perinatal period. Further translational studies are needed to determine the optimal dosing regimen, assess the efficacy of delayed administration and whether use of IL-1Ra with interventions targeting other mechanisms of injury can augment neuroprotection in the perinatal brain. Materials and methods All procedures were approved by the Hudson Institute of Medical Research Animal Ethics committee and were conducted in accordance with the ARRIVE guidelines 79 and the National Health and Medical Research Council Code of Practice for the Care and Use of Animals for Scientific Purposes (Eighth Edition). Twenty-six pregnant Border-Leicester ewes bearing singleton or twin fetuses of both sexes underwent aseptic surgery on 125 ± 1 days of gestation. At this age, cortical development in sheep is broadly comparable to the human brain at term 80 , 81 , 82 . Food but not water was withdrawn 18 hours before surgery. Anaesthesia was induced by intravenous (i.v.) injection of sodium thiopentone (20 mL) and maintained using 2–3% isoflurane and a fraction of inspired oxygen of 60% (Bomac Animal Health, New South Wales, Australia). Maternal prophylactic antibiotics (engemycin, 500 mg i.v.; Schering-Plough, Upper Hutt, New Zealand, and ampicillin, 1 g i.v.; Austrapen, Lennon Healthcare, St. Leonards, New South Wales, Australia) were given immediately before surgery. Maternal heart rate, respiratory rate and isoflurane levels, were continuously monitored by trained anaesthetic staff throughout surgery. Fetal instrumentation A midline maternal laparotomy was performed, the fetus was exposed, and polyvinyl catheters were inserted into the right brachiocephalic artery and axillary vein. In the case of a twin pregnancy, one twin was instrumented. Two pairs of electroencephalogram (EEG) electrodes (AS633-7SSF; Cooner Wire, Chatsworth, CA, USA) were placed through burr holes onto the dura over the parasagittal parietal cortex (10 and 20 mm anterior to bregma, and 10 mm lateral). Electrodes were secured using surgical bone wax and cyanoacrylate glue. The fetus was returned to the uterus in its original orientation, and all fetal leads were exteriorised through the maternal flank. A catheter was inserted into the maternal jugular vein for administration of post-operative antibiotics and humane culling at the end of the experimental period. At the completion of surgery, ewes received fentanyl for three days via a transdermal patch placed on the left hind leg (75 µg/hour; Janssen Cilag, North Ryde, New South Wales, Australia). Ewes were randomly housed together in separate metabolic crates in a temperature-controlled room (20 ± 2°C and relative humidity of 50 ± 10%) with a 12-hour light-dark cycle and ad libitum access to food and water. Four to five days of postoperative recovery was allowed before experiments commenced. Ewes and fetuses received daily i.v. infusions of ampicillin (800 mg, maternal i.v. and 200 mg, fetal i.v.) and engemycin (500 mg, maternal i.v.) for three consecutive days after surgery. Fetal catheters were maintained patent with a continuous infusion of heparinised saline (25 IU/mL) at a rate of 0.2 mL/hour. Experimental recordings The fetal EEG was continuously recorded from 24 hours prior to the first saline or LPS infusion (129 days of gestation) until the end of the experiment (133 days of gestation). The analogue fetal EEG signal was bandpass filtered with a cut-off frequency set at 1 and 22 Hz and digitised at a sampling frequency of 400 Hz. EEG power was derived from the analogue signal, whilst spectral edge was calculated as the frequency below which 90% of the intensity was present. Relative (%) spectral power in the delta (Δ, 0–3.9 Hz), theta (θ, 4–7.9 Hz), alpha (ɑ, 8–12.9 Hz), and beta (β, 13–22 Hz) frequency bands was quantified by calculating the power spectra over 4 hour epochs, using fast Fourier transform, of the EEG on sequential epochs using a 10-s Hanning window to minimise spectral leakage, as previously described 12 , 83 , 84 . Experimental protocol Experiments started at 129 days of gestation. Fetuses were randomly allocated, using an online random number generator, to three groups: 1. Control + vehicle (4 ml saline), n = 9 2. LPS (Escherichia coli, O55:B5, MilliporeSigma, MO, USA) + vehicle, n = 8 3. LPS + IL-1Ra (anakinra, 10 mg/kg i.v. dissolved in 4 ml saline), n = 9 The dose was guided by previous pharmacokinetic and neuroprotection trials in fetal sheep, non-human primates, and human cohorts which administered anakinra i.v. 19, 64, 85 . Fetuses received 300 ng, 600 ng and 1200 ng infusions of LPS diluted in 2 mL of saline i.v. (infusion rate: 1 mL/ minute) at 0 hours, 24 hours and 48 hours, respectively. This model is relevant to acute perinatal gram-negative infection and reproduces the associated pattern of acute systemic inflammation that is associated with adverse neurodevelopment 4 , 19 , 86 , 87 , 88 , 89 . Controls received an equivalent volume of saline at the same infusion rate. Infusions of IL-1Ra (Anakinra, Sobi, Stockholm, Sweden) began 1 hour after LPS administration on each consecutive day (i.e. 1, 25 and 49 hours, respectively), at a rate of 0.75 mL/hour over 4 hours. Four days after the start of infusions, sheep were euthanized by intravenous injection of pentobarbitone sodium (Lethabarb, Virbac, New South Wales, Australia). The study protocol is illustrated in Supplementary Fig. 1. The rate of fetal loss before the end of the experimental recording period was 4% and did not differ between the groups. In cases of fetal loss, the individual was excluded from the study. Sleep State Cycling Sleep state cycling was examined over the final 24 hours of the experimental period, beginning at 9 am (72 hours after the first LPS dose). Raw traces of EEG power, spectral edge frequency and spectral power in delta, theta, alpha and beta bands were examined by an assessor who was masked to the treatment group by independent coding of files. Sleep stage cycling was defined as a repetitive alternating pattern of low-voltage-high-frequency (rapid eye movement [REM]) activity, and high-voltage-low-frequency (non-REM [nREM]) activity, with, on average, each phase lasting approximately 20 minutes, as previously described 12 , 68 . A minimum duration of 3 minutes excluding periods of transitional sleep, was required to be classed as a sleep state 90 . EEG parameters were extracted for each sleep state and presented as the mean from eight 1-hour epochs across the 24-hours. Brain collection and processing The right hemisphere was immersion fixed with 10% phosphate-buffered formalin for four days before processing and embedding in paraffin. The right hemisphere was cut with a blocking blade into 8 mm thick coronal blocks, using a brain mould. Blocks from the forebrain, ~ 23 mm anterior to stereotaxic zero, with a clearly visible cortex were sectioned using a microtome (Leica Microsystems, Victoria, Australia) into 8-µm thick coronal sections. Region matched brain sections from the left hemisphere containing a clearly visible lateral parietal lobe/somatosensory cortex were immersion-fixed using a commercially available FD Rapid Golgi Stain Kit according to manufacturer instructions (FD Neurotechnologies Inc., MD, USA). Tissue sections were cut using a Leica VT1200S vibratome at 100 µm and mounted onto coverslips, processed for Golgi visualisation and cover slipped. RNA isolation, sequencing and analysis Cortical grey matter from the parietal lobe of the left hemisphere containing a clearly visible lateral parietal lobe containing the somatosensory cortex, adjacent to the section collected for Golgi visualisation, was homogenised and total mRNA was isolated using a RNeasy Midi Kit (QIAGEN, Venlo, Netherlands). RNA samples were submitted to the Hudson Genomics Facility (Hudson Institute of Medical Research; Clayton, Australia) for integrity and concentration assessment via capillary electrophoresis (Agilent Technologies) and fluorometric quantification (Qubit, Invitrogen). An RNA integrity number (RIN) ≥ 7.5 was used to confirm the integrity of all samples. RNA sequencing was performed using a custom in-house multiplex method, as previously described 91 , 92 . Samples were given a unique i7 index, in addition to a unique molecular identifier (UMI)), during individual pA priming and first strand synthesis which adds a template switch sequence to the 5’ end. Samples were then pooled into sets and amplified using P7 and an oligo which binds the template switch sequence. Final library construction was completed by tagmentation and addition of P5 by PCR. Sequencing was performed on an Illumina NSQ2k run with 111nt SR (cDNA). A 20nt i7 read contained the 10nt index and 10nt UMI. Samples were parsed using unique i7 indexes. Data analysis and bioinformatics were performed at the Monash Bioinformatics Platform (Clayton, Australia), whereby the nf-core/rnaseq Nextflow pipeline verion 3.10.1 was used to process the RNA sequencing reads 93 . ENSEMBL version 112 Sheep (Ovis Aries) was used as the reference genome. Briefly, reads were trimmed using Trim Galore and then aligned to the genome using STAR 94 , 95 . STAR produces alignments to transcripts that were then deduplicated using UMI-tools 96 and quantified using Salmon 97 . Genes with fewer than 10 UMIs in at least one sample were filtered. Raw gene read counts were imported into RStudio (v2023.06.1 + 524) with R v4.4.0 and converted into a DGEList object using edgeR 98 and normalised for library sizes using the trimmed-mean of M-values (TMM) method. Only genes expressed at > 1 counts per million (CPM) in at least 7 samples were retained. Differential gene expression analysis was performed using the limma-voom pipeline 99 . Voom transformation of count data to log2-counts per million (CPM) were analysed via linear models in limma 100 and contrasts between control versus LPS + vehicle and LPS + vehicle versus LPS + IL-1Ra were performed with empirical Bayes moderation. Genes were and considered to be differentially expressed if log2 fold change >|0.58| and P < 0.05. Pathway Over-Representation Analysis (ORA) of differentially expressed genes was then performed using the ClusterProfiler 101 and reactomePA packages 102 . Pathways were considered over-represented if P < 0.05 and gene count mapped was ≥ 3. Heatmap visualisations were performed using ComplexHeatmap 103 . Immunohistochemistry Slides were dewaxed in xylene, rehydrated in ethanol, and washed in phosphate-buffered saline (PBS). Antigen retrieval was performed by microwaving the slides in citrate buffer (pH 6) for 15 minutes. Endogenous peroxide quenching was performed by incubating slides in 0.1% H 2 O 2 in methanol. Blockade of non-specific antigens was performed using 3% normal goat serum. Sections were labelled with 1:250 rabbit anti-IL-1β (cat#: NB600-633, Novus, CO, USA), 1:200 rabbit anti-ionised calcium binding adaptor molecule 1 (Iba-1, Abcam, cat#: ab153696), 1:200 rabbit anti-neuronal nuclei (NeuN, Abcam, cat#: ab177487) and 1:200 rabbit anti-glial fibrillary acidic protein (GFAP; Abcam, cat#: ab68428) overnight at 4°C. Sections were incubated in biotin conjugated IgG (1:200, goat anti-rabbit (Dako, Victoria, Australia), for three hours at room temperature before being incubated in avidin-biotin complex (Sigma-Aldrich) for 45 minutes at room temperature. Sections were reacted with 3,3′-diamino- benzidine tetrahydrochloride (Sigma-Aldrich). The reaction was stopped in PBS before slides were dehydrated in xylene and increasing concentrations of ethanol, mounted in dibutyl phthalate polystyrene xylene and cover slipped. Astrocytes (GFAP + cells), microglia (Iba-1 + cells), and neurons (NeuN + cells) were visualised using light microscopy (Olympus, Tokyo, Japan) at 40× magnification and CellSens imaging software (Version 2.3, Olympus). The IL-1β immunoreactivity was scored using published protocols by us and others 12 , 19 , 21 . Scoring was based on the intensity of staining (1 = light, 2 = moderate, 3 = moderate-to-intense and 4 = intense). Numbers of positive cells or immunoreactivity were quantified within the lateral parietal lobe between cortical layers 3 and 5 from two sections per subject using ImageJ software (v2.00, LOCI, University of Wisconsin). An assessor who was blinded to the treatment group by slide coding performed all imaging and cell counts. Immunofluorescence Slides were baked at 60°C for one hour then dewaxed in xylene, rehydrated in increasing concentrations of ethanol and washed in PBS. Antigen retrieval was performed in citrate buffer (pH 6) using a microwave for 15 minutes. Non-specific antigen blocking was performed using 10% normal goat serum. Sections were labelled with 1:200 mouse anti-ionised calcium binding adaptor molecule 1 (Iba-1, Abcam, cat#: ab 283319) overnight at 4°C followed by incubation with an Alexa Fluor-488-conjugated 2 ∘ antibody (1:200, Jackson Research, cat#: 115-545-003) for 2 hours at room temperature. Sections were labelled with 1:200 rabbit anti-Signal transducer and activator of transcription 3 (Cell Signalling, Cat#: 9145) overnight at 4°C. Sections were incubated with an Alexa Fluor-594-conjugated secondary antibody (1:200, Jackson Research, cat#: 111-585-003) for two hours at room temperature. Sections were washed in PBS then incubated with the nuclear stain HOECHST (1:1000 diluted in PBS; Invitrogen, USA) for five minutes, then washed in PBS. Cover-slipping was performed using DAKO anti-fade fluorescent mounting medium (Agilent Technologies, Australia). Negative controls that did not contain the target antibody were included to confirm the absence of non-specific staining (Supplementary Fig. 2). Sections with fluorescently double labelled microglia (Iba-1+/STAT3+) were scanned using a VS120 Olympus virtual slide microscope and visualised at 20x magnification using QuPath imaging software (Version 0.4.3). Double labelled cells were quantified within the somatosensory cortex of the lateral parietal lobe between cortical layers 3 and 5 from two sections per subject. An assessor who was blinded to the treatment group by slide coding performed all imaging and cell counts. Assessment of Neuronal Morphology Coded Golgi-stained tissue sections (10 serial sections per subject) that were region matched to sections used for immunohistochemical and RNAseq analyses were used to assess basal dendrites from pyramidal neurons between layers 3 and 5 of the parietal lobe, as described in our previously published protocol 12 . The Golgi staining produced incomplete filling of the apical dendrites relative to the basal dendrites. Thus, apical dendrites were not analysed. Basal dendrites from pyramidal neurons were visualised on an Olympus BX61 stereology microscope equipped with an DP73 camera (×0.5 lens) at 60× magnification using CellSens imaging software (version 2.3; Olympus). Basal dendrites from a total of 20 pyramidal neurons, selected from 10 serial sections of the lateral parietal lobe from each subject, met the pre-defined inclusion criteria for imaging. Pyramidal neurons were selected based on established morphological criteria 12 , 104 , as follows: 1. A triangular shaped soma and apical dendrites perpendicular to the pial surface 2. Complete Golgi impregnation of the cell that permitted visualisation of the entire dendritic arbour and spines 3. Neuronal soma and processes not obscured by other neurons, glia or blood vessels 4. Neurons exhibiting a complete basilar dendritic tree without truncated or cut processes. Images were cropped, separated into individual channels using ImageJ and imported into Imaris (version 9.2.1, Bitplane, Oxford Instruments Company, Abington, UK). The Imaris filament tracer tool was used to measure numbers of dendritic spines and dendritic complexity, including summated dendritic length, numbers of dendritic terminals and Sholl analysis (numbers of dendrite intersections per Sholl ring). Sholl intersections were set at 5 µm concentric rings centred on the soma. The MATLAB spine classification extension was used to classify dendritic spine morphology (filopodia, long thin, stubby, mushroom) (MATLAB, R2019a, Mathworks Inc., CA, USA). Data analysis and statistics The Shapiro-Wilk test was used to test data for normality. EEG data were analysed using a two-way ANOVA, with the baseline, vehicle/LPS/IL-1Ra infusion and recovery periods analysed as separate time periods. For EEG data and Sholl analysis, when statistical significance was found between groups or between group and time, post hoc comparisons were made using the Benjamini-Hochberg correction. Differentially expressed genes, identified using the limma-voom method 99 , 105 , were defined as having a >|0.58| log2 fold-change and an unadjusted P value < 0.05. Neuronal dendrite morphology and histological data were analysed by one-way ANOVA. The Benjamini-Hochberg correction was used for post hoc comparisons. For non-parametric data, between group comparisons were performed using Kruskal-Wallis tests. Post hoc power analysis for summated dendritic length showed 85% power to detect a minimum difference of 977 µm, with an alpha of 0.05. Moreover, post hoc power analysis of EEG spectral bands showed 90% power to detect a 30% reduction in beta spectral band power, with an alpha of 0.05. Statistical significance was accepted when P < 0.05. Data are presented as means ± standard error of the mean (SE). Declarations Author contributions Sharmony B. Kelly and Robert Galinsky conceptualised and designed the study. Sharmony B. Kelly, Steven X Cho, Valerie Zahra, Mira Menyen, Rodney W Hunt, Claudia A Nold-Petry, Alistair J Gunn, Graeme R Polglase, Stuart B Hooper, Marcel F Nold, and Robert Galinsky undertook experiments and formal analysis of the data. Sharmony B. Kelly, Steven X Cho, and Valerie Zahra designed and undertook all RNA sequencing analyses. Sharmony B. Kelly undertook the Golgi analysis and Immunohistochemistry. Sharmony B Kelly and Robert Galinsky undertook all cell quantification, analysis and preparation of figures. Robert Galinsky provided overall oversight of the research. All authors critically reviewed the manuscript and approved the final manuscript as submitted and agree to be accountable for all aspects of the work. Funding support This study was supported by the National Health & Medical Research Council of Australia (1105526 to GRP, 1090890 and 1164954 to RG, 11173584 and 2033196 to CANP), Cerebral Palsy Alliance Grant (ERG02123 to RG), the Harold and Cora Brenan Benevolent Trust and the Victorian Government’s Operational Infrastructure Support Program. 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1","display":"","copyAsset":false,"role":"figure","size":89463,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTime course changes in % beta activity. \u003c/strong\u003e% beta activity in the control (black, \u003cem\u003en\u003c/em\u003e = 9), LPS + vehicle (orange, \u003cem\u003en\u003c/em\u003e = 8), and LPS + IL-1Ra (blue, \u003cem\u003en\u003c/em\u003e = 9) groups. Data are 4 - hourly means ± standard error (SE). *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05 LPS + vehicle vs. control. #\u003cem\u003eP\u0026lt; 0.05 \u003c/em\u003eLPS + vehicle vs LPS + IL-1Ra.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7882525/v1/8cccf09ebd1d39a48099fa59.png"},{"id":95372566,"identity":"cdd1379d-ce5c-4e85-a15b-c03250e5482a","added_by":"auto","created_at":"2025-11-07 10:00:29","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":282421,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTime Course changes in fetal sleep state cycling (SSC).\u003c/strong\u003eRepresentative epochs of EEG power (µV) and % beta activity (Hz) in control (\u003cstrong\u003eA\u003c/strong\u003e), LPS + vehicle (\u003cstrong\u003eB\u003c/strong\u003e), and LPS + IL-1Ra (\u003cstrong\u003eC\u003c/strong\u003e) groups during the baseline period (\u003cstrong\u003e1\u003c/strong\u003e), and the final 24 hours of the experiment (\u003cstrong\u003e2\u003c/strong\u003e). Data shows changes in rapid eye movement (REM) and non-REM (nREM) cycling over eight epochs during the last 24 hours of the experimental recordings. Changes in EEG power during REM (\u003cstrong\u003eD\u003c/strong\u003e), and nREM (\u003cstrong\u003eE\u003c/strong\u003e), changes in spectral edge frequency during REM (\u003cstrong\u003eF\u003c/strong\u003e), and nREM (\u003cstrong\u003eG\u003c/strong\u003e), changes in % delta activity during REM (\u003cstrong\u003eH\u003c/strong\u003e, and nREM (\u003cstrong\u003eI\u003c/strong\u003e), and changes in % beta activity during REM (\u003cstrong\u003eJ\u003c/strong\u003e), and nREM (\u003cstrong\u003eK\u003c/strong\u003e) in the control (black, \u003cem\u003en\u003c/em\u003e = 9), LPS + vehicle (orange, \u003cem\u003en\u003c/em\u003e= 8), and LPS + IL-1Ra (blue, \u003cem\u003en\u003c/em\u003e = 9) groups. Data are hourly means ± standard error (SE). *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 LPS + vehicle vs. control. #\u003cem\u003eP\u0026lt; 0.05 \u003c/em\u003eLPS + vehicle vs LPS + IL-1Ra.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7882525/v1/54042d4f008050c837f8c33a.png"},{"id":95525069,"identity":"5925f7b5-019a-41ca-ab9f-dc846b08f8fd","added_by":"auto","created_at":"2025-11-10 10:04:10","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":218472,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferential gene expression analysis of the lateral cortex. \u003c/strong\u003eVolcano plot comparison of LPS + Vehicle vs control groups \u003cstrong\u003e(A)\u003c/strong\u003e, and LPS + IL-1Ra vs LPS + Vehicle groups \u003cstrong\u003e(B)\u003c/strong\u003e. Each circle point represents one gene. Data is the magnitude of change (log\u003csub\u003e2\u003c/sub\u003e fold change, x-axis) vs. p values (-log\u003csub\u003e10\u003c/sub\u003eP value, y-axis). A P value of 0.05 and a log\u003csub\u003e2\u003c/sub\u003efold change of |0.58| are indicated by grey dashed lines. Points which do not meet P value and/or log\u003csub\u003e2\u003c/sub\u003efold change thresholds to be considered differentially expressed are represented in grey. Points which are P value significant only are represented in yellow. Of the differentially expressed genes, points are depicted in pink or if the gene was mapped to the Reactome pathways; TLR4 Cascade in green, GPCR ligand binding pathway in dark blue, extracellular matrix organisation or collagen formation in brown or unknown genes represented in light blue. Gene expression of Toll-Like Receptor 4 (TLR4) cascade genes \u003cstrong\u003e(C)\u003c/strong\u003e, G-Protein couple receptor (GPCR) genes \u003cstrong\u003e(D)\u003c/strong\u003e, and extracellular matrix (ECM) genes \u003cstrong\u003e(E)\u003c/strong\u003e in control (black, \u003cem\u003en\u003c/em\u003e = 7, 2 samples did not contain sufficient RNA), LPS + vehicle (orange, \u003cem\u003en\u003c/em\u003e = 8), and LPS + IL-1Ra (blue, \u003cem\u003en\u003c/em\u003e = 9) groups. Data are means ± SEM, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7882525/v1/af42cf29765ff0cac163ed6c.png"},{"id":95372585,"identity":"ca1c5c13-7497-4c97-a772-24786004b306","added_by":"auto","created_at":"2025-11-07 10:00:30","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":665176,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChanges in RNA expression between the LPS and control/LPS + IL-1Ra groups. \u003c/strong\u003eVenn diagram showing the number of genes in the intersect which are differentially expressed in the LPS + vehicle group compared to the controls and LPS + IL-1Ra groups (\u003cstrong\u003eA\u003c/strong\u003e). Heatmap visualisation of the differentially expressed genes in the LPS + vehicle group compared to the controls and LPS + IL-1Ra group (\u003cstrong\u003eB\u003c/strong\u003e), where each row represents a gene, and each column represents a sample. The heatmap colour range is from red for positive Z-scores values to blue for Z-score negative values\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7882525/v1/f99bca8c2fcd4a62e48b27ef.png"},{"id":95526090,"identity":"09ef1001-843e-4b4d-8b81-a14d63c222b5","added_by":"auto","created_at":"2025-11-10 10:06:14","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":97127,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNeuronal dendrite and dendritic spine morphology. \u003c/strong\u003eSummated dendritic length (μm) (\u003cstrong\u003eA\u003c/strong\u003e), number of dendritic terminals (\u003cstrong\u003eB)\u003c/strong\u003e, dendritic Sholl analysis showing the number of dendritic intersections indicated by the number of intersections at 5 μm intervals away from the soma (\u003cstrong\u003eC\u003c/strong\u003e), and total spine number (\u003cstrong\u003eD\u003c/strong\u003e) in the control (black, \u003cem\u003en\u003c/em\u003e = 7; two subjects had limited Golgi penetration), LPS + vehicle (orange, \u003cem\u003en\u003c/em\u003e = 7; 1 subject had limited Golgi penetration), and LPS + IL-1Ra (blue, \u003cem\u003en\u003c/em\u003e = 9) groups. Data are means ± SE, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 vs control. Representative Golgi-stained traced images of basal dendrites from pyramidal neurons in the somatosensory cortex from control (\u003cstrong\u003eE\u003c/strong\u003e), LPS + vehicle (\u003cstrong\u003eF\u003c/strong\u003e), and LPS + IL-1Ra (\u003cstrong\u003eG\u003c/strong\u003e) subjects. Scale bar panel = 20 μm\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7882525/v1/caa27ce3a4e2c02c337fedc7.png"},{"id":95372571,"identity":"c0cc6332-4e2c-4762-a1e3-bb558848f9d8","added_by":"auto","created_at":"2025-11-07 10:00:29","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1189001,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistopathology data of the parietal / somatosensory cortex. \u003c/strong\u003eInterleukin 1 (IL)-1β immunoreactivity scores (\u003cstrong\u003eA\u003c/strong\u003e), numbers of total ionised calcium binding adaptor molecule (Iba-1) + microglia (\u003cstrong\u003eB\u003c/strong\u003e), Number of double labelled Iba-1+ signal transducer and activator of transcription 3 (STAT3)+ microglia (\u003cstrong\u003eC\u003c/strong\u003e), number of glial fibrillary acidic protein (GFAP) + astrocytes (\u003cstrong\u003eD\u003c/strong\u003e), and numbers of NeuN + neurons (\u003cstrong\u003eE\u003c/strong\u003e), in the lateral parietal lobe in control (black, \u003cem\u003en\u003c/em\u003e = 9), LPS + vehicle (orange, \u003cem\u003en\u003c/em\u003e = 8), and LPS + IL-1Ra (blue, \u003cem\u003en\u003c/em\u003e = 9) groups. Data are means ± SE, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 vs control.\u003cstrong\u003e \u003c/strong\u003eRepresentative photomicrographs showing immunohistochemical staining of IL-1β (\u003cstrong\u003eF\u003c/strong\u003e), Iba-1 (\u003cstrong\u003eG\u003c/strong\u003e), Iba-1+STAT3+ microglia (\u003cstrong\u003eH\u003c/strong\u003e), GFAP (\u003cstrong\u003eI\u003c/strong\u003e), and NeuN (\u003cstrong\u003eJ\u003c/strong\u003e) in the lateral parietal lobe of the control (\u003cstrong\u003e1\u003c/strong\u003e), LPS + vehicle (\u003cstrong\u003e2\u003c/strong\u003e), and LPS + IL-1Ra (\u003cstrong\u003e3\u003c/strong\u003e) groups. Scale bar = 100 μm\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7882525/v1/495ba207e78c32ebf135ae99.png"},{"id":95531083,"identity":"4ae0f6d6-da13-4855-a82d-f4981dd7d0a7","added_by":"auto","created_at":"2025-11-10 10:22:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3774082,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7882525/v1/945cf9c9-1cea-485a-b003-a24ebe34ddef.pdf"},{"id":95372586,"identity":"05d37262-7d72-40b3-ab2b-5c5ea7b741cc","added_by":"auto","created_at":"2025-11-07 10:00:30","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":179629,"visible":true,"origin":"","legend":"Supplementary Figure 1","description":"","filename":"SupFig1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7882525/v1/22c700a67cb2c33fed27e91c.pdf"},{"id":95372567,"identity":"157fa607-7cd8-48df-a614-83620263919b","added_by":"auto","created_at":"2025-11-07 10:00:29","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":14934,"visible":true,"origin":"","legend":"Supplementary table 3","description":"","filename":"SupplementaryTable3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7882525/v1/c74d60d19a137ec9078d2758.xlsx"},{"id":95525862,"identity":"8b9b79a9-9e39-498e-9362-647f97a0afbb","added_by":"auto","created_at":"2025-11-10 10:05:44","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":251208,"visible":true,"origin":"","legend":"Supplementary Figure 3","description":"","filename":"SupFig3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7882525/v1/2eb2a76dea942945ba6f5f2c.pdf"},{"id":95525718,"identity":"8062263c-8de4-4800-bb63-7a016722b90d","added_by":"auto","created_at":"2025-11-10 10:05:36","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":314961,"visible":true,"origin":"","legend":"Supplementary Figure 5","description":"","filename":"SupFig5.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7882525/v1/aceccd4ac3e30942808d35b0.pdf"},{"id":95525442,"identity":"e544d204-4944-46af-b1d6-f298c8f19947","added_by":"auto","created_at":"2025-11-10 10:05:01","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":120021,"visible":true,"origin":"","legend":"Supplementary Figure 4","description":"","filename":"SupFig4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7882525/v1/cf79b95ea5960ce383892bc5.pdf"},{"id":95372584,"identity":"c4a8704e-89c8-4d51-853e-d31846241fb8","added_by":"auto","created_at":"2025-11-07 10:00:30","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":195924,"visible":true,"origin":"","legend":"Supplementary Figure 2","description":"","filename":"SupFig2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7882525/v1/58b678c053ca7474cb7811b2.pdf"},{"id":95372575,"identity":"a70a7779-36d9-49c7-9218-1abbbca5731b","added_by":"auto","created_at":"2025-11-07 10:00:29","extension":"xlsx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":41186,"visible":true,"origin":"","legend":"Supplementary table 2","description":"","filename":"SupplementaryTable2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7882525/v1/08ea412d0c9bf4f16b085c4d.xlsx"},{"id":95372578,"identity":"7d42bdec-4a39-477c-a0ed-d08f7f2c7ff0","added_by":"auto","created_at":"2025-11-07 10:00:29","extension":"pdf","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":204853,"visible":true,"origin":"","legend":"Supplementary Figure 6","description":"","filename":"SupFig6.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7882525/v1/c8c779f61ea214fbfb548902.pdf"},{"id":95526209,"identity":"755a8c89-2bcb-4fea-a02c-9e13de56af98","added_by":"auto","created_at":"2025-11-10 10:06:30","extension":"xlsx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":37049,"visible":true,"origin":"","legend":"Supplementary table 1","description":"","filename":"SupplementaryTable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7882525/v1/8e34fbea116a3d8ff52e743c.xlsx"},{"id":95372580,"identity":"42af8c1c-2722-42b0-892c-22d55e0a6662","added_by":"auto","created_at":"2025-11-07 10:00:29","extension":"docx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":101808,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7882525/v1/64a3957b5aa57dcd1935b749.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"IL-1 modulation preserves biomolecular, structural and functional integrity of the somatosensory cortex after fetal inflammation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eExposure to inflammation, often caused by infection, during the perinatal period is strongly associated with a greater risk of perinatal mortality, brain injury and neurodevelopmental impairments; including learning difficulties, autism spectrum disorder, attention deficit and hyperactivity, schizophrenia and cerebral palsy \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. The risk of cerebral palsy (CP) is several-fold greater after perinatal infection/inflammation (odds ratio 2.5\u0026ndash;9.3) \u003csup\u003e4, 5, 6\u003c/sup\u003e. The cumulative lifetime cost of CP in the US was estimated to be over USD11.5\u0026nbsp;billion in 2003 \u003csup\u003e2\u003c/sup\u003e, which equates to approximately USD20\u0026nbsp;billion in 2025 after adjusting for changes in the CPI, not including the wider burden on affected individuals, their families and society \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eStructurally, MRI studies have shown that exposure to antenatal infection is independently associated with reductions in functional connectivity \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e and impaired cortical development, including reduced sulcal depth and cortical volume (particularly in the parietal lobe/somatosensory cortex), without evidence of overt grey matter injury \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Reduced neonatal cortical volume has been shown to persist into adolescence and is associated with impaired cognition \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Preclinical studies indicate that these associations between exposure to antenatal infection / inflammation and impaired cortical development are likely causal. For example, in fetal sheep, lipopolysaccharide (LPS)-induced inflammation was associated with increased gliosis and reduced neuronal complexity within the somatosensory cortex but did not affect overall numbers of neurons \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. In neonatal rats, LPS-induced inflammation from postnatal days 1\u0026ndash;3 led to reductions in neuronal complexity and cortical volume without affecting numbers of neurons in the motor cortex \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Moreover, maternal injections of group \u003cem\u003eB Streptococcus\u003c/em\u003e in pregnant rats between gestational days 19 and 22 led to thinning of the cerebral cortex and impaired motor function in offspring at postnatal day 40 \u003csup\u003e14\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAlthough multiple pathways are involved in the pathogenesis of neurological injury after exposure to perinatal infection and/or inflammation, the pro-inflammatory cytokine interleukin-1β (IL-1β) is consistently upregulated in blood and brain samples from human and animal studies of perinatal encephalopathy \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Further, elevated cord blood IL-1β concentration has been linked to impaired brain metabolism and developmental delay in early childhood \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, and increased circulating IL-1β concentration in fetal sheep, neonatal mice and piglets is associated with both white and grey matter injury \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Collectively, these data support the hypothesis that controlling IL-1β bioactivity during perinatal inflammation could help attenuate neural injury and improve neuronal function.\u003c/p\u003e\u003cp\u003eWe tested the hypothesis that IL-1 inhibition starting one hour after LPS exposure with an FDA approved IL-1 receptor antagonist (IL-1Ra), anakinra, mitigates molecular, electrophysiological, and histological markers of inflammation and neuronal injury in the somatosensory cortex of late gestation fetal sheep.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cem\u003eBaseline period and fetal growth\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eBefore LPS exposure, EEG spectra did not differ between groups and were within the normal range for our laboratory. There were no differences in subject brain and body weights or sex between the groups (Control: body weight = 4.6 \u0026plusmn; 0.6 Kg, brain weight = 51.3 \u0026plusmn; 3.7 g, 34% female; LPS + vehicle: body weight = 4.6 \u0026plusmn; 0.5 Kg, brain weight = 49.8 \u0026plusmn; 3.4 g, 25% female; and LPS + IL-1Ra: body weight = 4.7 \u0026plusmn; 0.9 Kg, brain weight = 49.2 \u0026plusmn; 5.2 g, 44% female).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSpectral band analysis\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eDelta, theta, and alpha band power were not different did not differ between the groups (Supplementary Figure 3A - C).\u003c/p\u003e\n\u003cp\u003eIn the LPS + vehicle group, beta band power was increased from 4 \u0026ndash; 20 hours after the first LPS infusion (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05 vs controls, Figure 1). After the second LPS infusion, beta band power was higher in the LPS + vehicle group between 28 and 48 hours (i.e 4 \u0026ndash; 24 hours after the second LPS infusion; \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 vs controls, Figure 1). After the third LPS infusion, beta band power was higher in the LPS + vehicle group between 60 and 64 hours, and 80 and 92 hours (i.e. 12 \u0026ndash; 16 hours, and 32 \u0026ndash; 44 hours after the third LPS infusion; \u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05 vs controls, Figure 1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBeta band power was increased in the LPS + IL-1Ra group from 4 \u0026ndash; 24 hours after the first LPS infusion (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05 vs LPS + vehicle, Figure 1). After the second LPS infusion, beta band power was higher in the LPS + IL-1Ra group between 28 and 48 hours (i.e 4 \u0026ndash; 24 hours after the second LPS infusion; \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 vs LPS + vehicle, Figure 1). After the third LPS infusion, beta band power was higher in the LPS + IL-1Ra group from 52 \u0026ndash; 64 hours, and from 80 \u0026ndash; 92 hours (i.e. 8 \u0026ndash; 20 and 32 \u0026ndash; 44 hours after the third LPS infusion; \u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05 vs LPS + vehicle, Figure 1).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSleep state cycling\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eSleep state cycling was observed in all subjects throughout the experimental period (Figures 2A - C). During the final 24 hours of the experimental period, EEG power was increased in the LPS + vehicle group compared to the control and LPS + IL-1Ra groups during REM sleep between 1200 hours and 0300 hours (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; Figure 2D). There was no difference in EEG power during nREM sleep (Figure 2E). Spectral edge frequency did not differ between groups during REM and nREM sleep (Figures 2F and G). There were no differences in delta band power between the groups during REM and nREM sleep (Figures 2H and I). In the LPS + vehicle group, the proportion of beta band power was decreased during REM sleep compared to control between 0900 hours and 0600 hours (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, Figure 2J). In the LPS + IL-1Ra group, the proportion of beta band power was increased compared to LPS + vehicle between 1200 and 1500 and 0300-0600 hours (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, Figure 2J). There were no differences in beta band power between the groups during nREM (Figure 2K). The proportions of theta and alpha band power did not differ between groups during REM and nREM sleep states throughout the last 24 hours of the experimental period (Supplementary Figure 4 A-D).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eRNA expression (RNA sequencing)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo gain insight into the biomolecular changes associated with inflammation-induced changes in cortical neuronal growth we performed RNA sequencing on frozen tissue sections collected from the lateral cortex adjacent to the regions analysed for neuronal morphology and histopathology.\u003c/p\u003e\n\u003cp\u003eWe identified 281 genes that were differentially expressed in the LPS + vehicle (\u003cem\u003en\u0026nbsp;\u003c/em\u003e= 8) compared to the control group (\u003cem\u003en =\u0026nbsp;\u003c/em\u003e7, 2 samples did not contain sufficient RNA), with 86 genes (31%) being of unknown function (Figure 3A, Supplementary Table 1). 326 genes were differentially expressed between LPS + IL-1Ra (\u003cem\u003en\u0026nbsp;\u003c/em\u003e= 9) vs LPS + vehicle (\u003cem\u003en\u0026nbsp;\u003c/em\u003e= 8).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePathway over-representation analysis demonstrated that altered gene expression between LPS + vehicle vs control was predominantly associated with the toll-like receptor 4 (TLR4) ligand binding and IL-1 family signalling pathways, as well as inflammatory pathways linked to the G protein coupled receptor (GPCR) cascade (Figures 3C and D, Supplementary Figure 5A).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGenes mapped to GPCR ligand binding pathways were predominantly decreased in the LPS + vehicle group when compared to controls, with the most differentially expressed genes being largely hormones such as corticotropin-releasing hormone (\u003cem\u003eCRH\u003c/em\u003e), parathyroid hormone (\u003cem\u003ePTH\u003c/em\u003e) and thyrotropin-releasing hormone (\u003cem\u003eTRH\u003c/em\u003e) or GPCRs for neuropeptides such as neuropeptide FF receptor 2 (\u003cem\u003eNPFFR2\u003c/em\u003e, reduced expression in LPS + vehicle vs. control) and tachykinin receptor 3 (\u003cem\u003eTACR3\u003c/em\u003e, increased expression in LPS + vehicle vs. control) (Figure 3A, Supplementary Table 1). Taken together these observations suggests LPS-activation of a pro-inflammatory response that is accompanied by changes to the expression of neuropeptides linked to GABAergic interneuron development and regulation of neuronal branching (\u003cem\u003eCRH\u003c/em\u003e), synapse formation (\u003cem\u003eTRH\u003c/em\u003e), neural excitation (\u003cem\u003ePTH\u003c/em\u003e and \u003cem\u003eTRH\u003c/em\u003e) \u003csup\u003e24, 25, 26\u003c/sup\u003e, and neurodegeneration (\u003cem\u003eNPFFR2\u003c/em\u003e and \u003cem\u003eTACR3\u003c/em\u003e) \u003csup\u003e27, 28\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eWhen comparing LPS + vehicle to the LPS + IL-1Ra group, we did not observe significant difference to genes related to the TLR4 cascade that were upregulated by LPS (Figures 3B and C, Supplementary Table 2). Similarly, most GPCR ligand binding genes modulated by LPS also remained unchanged versus LPS + IL-1Ra, except for endocrine genes where we observe a significantly upregulated \u003cem\u003eTRH\u003c/em\u003e and \u003cem\u003eCRH\u003c/em\u003e (LPS + IL-1Ra vs. LPS + vehicle, Figure 3D). Pathway over-representation analysis showed the dominant gene changes between LPS + vehicle and LPS + IL-1Ra groups were primarily associated with modulated expression of extracellular matrix organisation genes that regulate synaptogenesis and spine development (\u003cem\u003eFBLN2)\u003c/em\u003e\u003csup\u003e29\u003c/sup\u003e, dendrite growth (\u003cem\u003eAGRN)\u003c/em\u003e\u003csup\u003e30\u003c/sup\u003e, IL-1 signalling and microglial activation (\u003cem\u003eLOX\u003c/em\u003e and \u003cem\u003eADAMTS4)\u003c/em\u003e\u003csup\u003e31, 32\u003c/sup\u003e (Figure 3E)\u0026nbsp;or overlapping pathways such as collagen formation, including genes such as \u003cem\u003eCOL13A1\u003c/em\u003e, \u003cem\u003eCOL19A1\u003c/em\u003e and \u003cem\u003eCOL21A1\u0026nbsp;\u003c/em\u003e(Supplementary Figure 5B).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe identified 56 genes that were differentially expressed between the LPS + vehicle group (\u003cem\u003en\u003c/em\u003e = 8) compared to the control (\u003cem\u003en\u003c/em\u003e = 7) and LPS + IL-1Ra groups (\u003cem\u003en\u003c/em\u003e = 9) (Figures 4A and B and Supplementary Table 3) to represent genes that may be related to IL-1Ra\u0026apos;s mechanistic actions or genes that are indicative of IL-1Ra action.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAfter excluding genes of unknown function and genes with limited specificity in brain tissue, 42 genes remained in the analysis. These 42 genes were divided into two key pathways: neuronal structure and function (20) and immune pathways and function (22) and\u0026nbsp;are described in Table 1. In summary, in the LPS + IL-1Ra group we observed normalised expression of genes relating primarily to dendritogenesis (e.g. \u003cem\u003eNRG4, COL13A1, KIF16B\u003c/em\u003e), synaptogenesis (e.g. \u003cem\u003eCIC2\u003c/em\u003e, \u003cem\u003eCTXN3, FBLN2, VGLUT1, TRH\u003c/em\u003e), neurorepair (e.g. \u003cem\u003eDEPTOR, ESR2, STAR, IGF1, IGF-R1\u003c/em\u003e), neurodegeneration (e.g. \u003cem\u003eNEMP1, PPP1R42, RABEP2\u003c/em\u003e) and immune activation (e.g. \u003cem\u003eGVIN1, H1-6, IL-27, TRIM47, TRIM55\u003c/em\u003e) (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 vs. LPS + vehicle and NS vs. control, Table 1).\u003c/p\u003e\n\u003cp\u003eOf the\u0026nbsp;56 genes that were differentially expressed between the LPS + vehicle group compared to the control and LPS + IL-1Ra groups, 18\u0026nbsp;genes were not classified. Using Ensemble genome browse and Basic Local Alignment Search Tool (BLAST), we identified 10 of these 18 genes that had a known classification in ovine tissue: (ENSOARG00020034656: MFSD14A, ENSOARG00020039350: PNPLA3, ENSOARG00020033980: ubiquitin D, ENSOARG00020037154: IL27, ENSOARG00020006863: serpin B6, ENSOARG00020005819: APOC2, ENSOARG00020034724: C13H20orf96, ENSOARG00020038681: GIMAP8, ENSOARG00020037893: WWP1, and ENSOARG00020009042: GVIN1). Thus, 8 genes were not included for further analysis due to no classification/unknown function of these genes in ovine brain (ENSOARG00020040904, ENSOARG00020031121, ENSOARG00020032393, ENSOARG00020037506, ENSOARG00020038936, ENSOARG00020037822, ENSOARG00020036053, ENSOARG00020037194). Six genes were not subject to further analysis due to limited specificity relating to their role in brain tissue (C13H20orf96, CHTF18, GYG2, OSBPL3).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eNeuronal dendrite morphology\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo determine the effects of inflammation on neuronal morphology, we assessed Golgi-stained basal dendrites collected four days after the start of LPS exposure. In cortical neurons of the lateral parietal lobe, the number of dendritic terminals on basal dendrites was significantly reduced in the LPS + vehicle group compared to controls (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, Figures 5A, E, F). The number of dendritic terminals in the LPS + IL-1Ra group was significantly increased compared to LPS + vehicle (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, Figure 5A and G). In the LPS + vehicle group, the summated length of basal dendrites was significantly reduced compared to controls (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 vs controls, Figure 5B). In the LPS + IL-1Ra group, summated dendritic length did not differ from controls but was not significantly increased compared to LPS + vehicle (\u003cem\u003eP\u003c/em\u003e = 0.07 vs LPS + vehicle, Figure 5B). Sholl analysis of pyramidal neuron complexity showed reduced dendritic arborisation in the LPS + vehicle group compared to control at 40 \u0026ndash; 230 \u0026mu;m away from the soma (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; Figure 5C). In the LPS + IL-1Ra group, dendritic arborisation was increased compared to LPS + vehicle at 5 \u0026ndash; 75 \u0026mu;m away from the soma (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; Figure 5C). In the LPS + IL-1Ra group, dendritic arborisation was increased compared to control at 5 \u0026ndash; 45 \u0026mu;m away from the soma (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, Figure 5C). In the LPS + IL-1Ra group, dendritic arborisation was reduced compared to control at 65 \u0026ndash; 220 \u0026mu;m away from the soma.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eNeuronal dendritic spine number and morphology\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe total number of dendritic spines was reduced in the LPS + vehicle and LPS + IL-1Ra groups compared to control (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, Supplementary Figure 6). Numbers of long thin spines were significantly reduced in the LPS + vehicle group compared to control (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, Figure 5G). There were no significant differences in numbers of stubby, mushroom, or filipodia spines between the groups (Supplementary Figure 6).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eHistopathology\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIL-1\u0026beta; immunoreactivity was increased in the somatosensory cortex of the LPS + vehicle group compared to controls (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, Figures 6A and F). In the LPS + IL-1Ra group,\u0026nbsp;IL-1\u0026beta; immunoreactivity was reduced compared to the LPS + vehicle group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, Figures 6A and F). Numbers of Iba-1+ microglia were increased in the somatosensory cortex of the LPS + vehicle group compared to controls (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, Figure 6B, G). In the LPS + IL-1Ra group, numbers of Iba-1+ microglia were reduced compared to the LPS + vehicle group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, Figures 6B and G). Numbers of reactive (phosphorylated (p)STAT3+/Iba-1+) microglia were increased in the somatosensory cortex in the LPS + vehicle group compared to control (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; Figures 6C, H). In the LPS + IL-1Ra group, numbers of (p)STAT3+/Iba-1+ microglia were reduced in the LPS + IL-1Ra group compared to LPS + vehicle (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; Figures 6C and H). Numbers of GFAP+ astrocytes and NeuN+ neurons did not differ between the groups in the somatosensory cortex (Figures 6D and I and 6E, J, respectively).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study demonstrates that competitive IL-1 receptor inhibition, using the FDA approved IL-1Ra anakinra (identical to endogenous human IL-1Ra except for one amino acid), improved neuronal complexity (arborisation) of pyramidal neurons and normalised RNA expression of genes that regulate neuronal development and neuroinflammation during LPS-induced inflammation in late gestation fetal sheep. Functionally, the improvements in histological and biomolecular outcomes in IL-1Ra treated subjects were associated with improved development of EEG spectral band power and normalisation of fetal sleep state architecture. To our knowledge, this is the first \u003cem\u003ein vivo\u003c/em\u003e study to integrate bulk RNA sequencing, histopathology and electrophysiology to demonstrate the molecular, cellular and functional effects of IL-1Ra on cortical pyramidal neurons in a large animal translational model of perinatal inflammation.\u003c/p\u003e\u003cp\u003eClinically, perinatal infection/inflammation is associated with an increased risk of impaired neurodevelopment \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Moreover, Gram negative infections including \u003cem\u003eE. coli\u003c/em\u003e continue to be among the most common pathogens linked to perinatal infection/inflammation and increased risk of perinatal brain injury \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. In this study, we reproduced key features of Gram-negative infection-induced inflammation using repeated and escalating doses of LPS infusions to promote a progressive fetal inflammatory response, including increased circulating concentrations of IL-1β (previously published in \u003csup\u003e19\u003c/sup\u003e). Elevated systemic and cerebrospinal fluid concentrations of IL-1β within the first few days after birth were associated with impaired brain metabolism and developmental delay in early childhood \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Similarly, in preterm infants elevated circulating IL-1β within the first 2 weeks after birth were associated with impaired neurodevelopment at 2 years of age \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Polymorphisms in the \u003cem\u003eIL1B\u003c/em\u003e gene that promote increased production of the IL-1β protein are associated with an increased risk of intraventricular haemorrhage and periventricular leukomalacia\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Moreover, polymorphisms in the gene encoding IL-1Ra, known as \u003cem\u003eIL1RN\u003c/em\u003e, that reduce production of IL-1Ra and increase proinflammatory signalling are associated with an increased risk of stillbirth \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. In post-mortem brain tissue from human neonates (gestational age range: 29\u0026ndash;37 weeks), accumulation of IL-1β was localised to areas of parenchyma where markers of gliosis and injury were highest \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Furthermore, in areas of tissue injury, accumulation of IL-1Ra was reduced compared to IL-1β, thus reducing the IL-1Ra:IL-1β ratio. This imbalance of pro- and anti-inflammatory cytokine concentrations was more pronounced in infants with worse histological outcomes.\u003c/p\u003e\u003cp\u003eMechanistic studies in small and large animals have shown a strong link between elevated systemic and cerebral IL-1β with neuroinflammation and perinatal brain injury\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Consistent with these observations, in the present study we show that progressive LPS exposure increased RNA expression of genes related to \u003cem\u003eE. Coli\u003c/em\u003e-induced inflammatory signalling, including the TLR4 and IL-1 associated pathway genes: \u003cem\u003eIRAK4, CD14, IRAK2, MAPKAPK2 LY86\u003c/em\u003e and \u003cem\u003ePNPLA3\u003c/em\u003e. Collectively, these data confirm that inflammation, induced with repeated increasing doses of \u003cem\u003eE. coli\u003c/em\u003e LPS in fetal sheep, promotes neuroinflammation and disturbances in neuronal development and function through activation of the TLR4 and IL-1 signalling pathways within the somatosensory cortex.\u003c/p\u003e\u003cp\u003eIntravenous treatment with anakinra reduced IL-1β immunoreactivity in the somatosensory cortex compared to LPS\u0026thinsp;+\u0026thinsp;vehicle. Consistent with this, numbers of total and activated (phosphoSTAT3+) microglia were reduced in the somatosensory cortex of LPS\u0026thinsp;+\u0026thinsp;IL-1Ra treated subjects compared to LPS\u0026thinsp;+\u0026thinsp;vehicle. Further, there was increased RNA expression of genes responsible for IL-1β signalling and immune activation in the somatosensory cortex of the LPS\u0026thinsp;+\u0026thinsp;vehicle group compared to controls. Moreover, in the LPS\u0026thinsp;+\u0026thinsp;IL-1Ra group we observed restored RNA expression (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs. LPS\u0026thinsp;+\u0026thinsp;vehicle but not control) of multiple genes linked to microglial activation and neuroinflammation, indicating IL-1Ra treatment normalised RNA expression of these genes in the somatosensory cortex. These data are consistent with evidence that excessive systemic and / or locally produced IL-1β contributes directly to brain immune activation and neural injury, at least in part, through microglial activation which can further increase secretion of IL-1β and other pro-inflammatory cytokines into the brain parenchyma to promote tissue damage \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Together, these data suggest that mitigation of microglial activation with IL-1Ra mediated the reduction in IL-1β accumulation in the somatosensory cortex of LPS\u0026thinsp;+\u0026thinsp;IL-1Ra treated subjects compared to LPS\u0026thinsp;+\u0026thinsp;vehicle. Moreover, \u003cem\u003ein vitro\u003c/em\u003e studies have shown that microglial processes interact with synapses to eliminate dendritic spines suggesting a direct effect of microglial activation on dendritic spine density \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. However, IL-1Ra treatment did not significantly ameliorate the LPS-induced reduction in numbers of dendritic spines (LPS\u0026thinsp;+\u0026thinsp;vehicle vs. LPS\u0026thinsp;+\u0026thinsp;IL-1Ra, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.08).\u003c/p\u003e\u003cp\u003ePreclinical and human cohort studies have shown that elevated circulating levels of IL-1β are associated with impaired cerebral oxidative metabolism and suppression of EEG amplitude and frequency in fetuses and neonates \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Consistent with these studies, we now show that LPS-exposed fetuses had disturbed distribution of EEG spectra throughout the experimental period compared to controls. This included an increase in the proportion of EEG activity in the delta (slow wave) band and a reduction in the proportion of EEG activity in beta (fast wave) band. This was also associated with higher total EEG power and a reduction in the proportion of EEG activity in the beta band (reduced high frequency activity) during phases of rapid eye movement (REM) sleep in LPS-exposed fetuses during the penultimate 24 hours of the experimental period. REM sleep is defined by cycling between low power and high frequency activity with bursts of high frequency beta waves \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. It is postulated to be critical for maintenance and development of neuronal circuits through strengthening of neuronal dendrites and dendritic spines to facilitate synapse formation \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. This notion is supported by the finding of the present study, that LPS exposed fetuses had reduced neuronal dendritic arborisation and fewer numbers of dendritic spines.\u003c/p\u003e\u003cp\u003eIn humans and sheep, the marked cortical expansion that occurs during the last trimester of gestation reflects a prolific increase in neuronal dendritic growth and spine development during this stage of development. Our observations suggest that, at this stage of late gestation, neuronal development within the somatosensory cortex is vulnerable to inflammation-induced impairments in dendritic arborisation, and that these structural abnormalities are manifest by functional disturbances that can be detected electrographically. In line with the reduced neuronal arborisation in the LPS\u0026thinsp;+\u0026thinsp;vehicle group compared to controls, we observed a reduced RNA expression of vesicular glutamate transporter 1 (VGLUT1, gene name: \u003cem\u003eSLC17A7\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) which is known to be expressed in presynaptic terminals and dendrites of excitatory pyramidal neurons in the somatosensory cortex \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. VGLUT1 is integral for transporting glutamate into synaptic vesicles and has a key role in glutamate mediated neurotransmission and synaptic function \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Collectively these data suggest that inflammation-induced disturbances to pre or post synaptic connections between glutamatergic neurons or impaired glutamatergic signalling could contribute to the impairments observed in neuronal arborisation and EEG activity in the somatosensory cortex of LPS-exposed fetuses. Moreover, in the LPS\u0026thinsp;+\u0026thinsp;vehicle group compared to controls, we observed reduced RNA expression of neuregulin-4 (\u003cem\u003eNRG4\u003c/em\u003e), steroidogenic acute regulatory protein (\u003cem\u003eSTAR\u003c/em\u003e) and thyrotropin releasing hormone (\u003cem\u003eTRH\u003c/em\u003e), parathyroid hormone (\u003cem\u003ePTH\u003c/em\u003e). The genes are known to play critical roles in the development and maintenance of neuronal signalling through supporting dendritic arborisation, spine development and synaptogenesis of cortical neurons \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn the LPS\u0026thinsp;+\u0026thinsp;IL-1Ra treated fetuses, the distribution of EEG spectral band power was comparable to controls throughout the experimental period. Moreover, the improved EEG activity in the LPS\u0026thinsp;+\u0026thinsp;IL-1Ra treated subjects was associated with an improvement in numbers of dendritic terminals and intermediate improvements in dendritic length and dendritic arborisation compared to the LPS\u0026thinsp;+\u0026thinsp;vehicle group. Indeed, \u003cem\u003ein vitro\u003c/em\u003e studies show that IL-1β inhibits growth and branching of neurons and this can be reversed with IL-1β blockade \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. RNA sequencing showed IL-1Ra treatment restored RNA expression of genes controlling dendritogenesis and synaptogenesis, including \u003cem\u003eCOL13A1, CTXN3, NRG4, VGLUT1\u003c/em\u003e and \u003cem\u003eTRH\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Moreover, gene pathway analysis showed genes relating to extracellular matrix organisation and collagen formation were the most differentially expressed in the LPS\u0026thinsp;+\u0026thinsp;IL-1Ra group compared to LPS\u0026thinsp;+\u0026thinsp;vehicle. Specifically, in the LPS\u0026thinsp;+\u0026thinsp;IL-1Ra group compared to LPS\u0026thinsp;+\u0026thinsp;vehicle there was increased RNA expression of \u003cem\u003eAGRN, FNLN2, COL19A1\u003c/em\u003e and \u003cem\u003eCOL21A1\u003c/em\u003e which have all been linked to development, maintenance and maturation of neuronal dendrites and synapses \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e, and reduced expression of \u003cem\u003eADAMTS4\u003c/em\u003e and \u003cem\u003eLOX\u003c/em\u003e which has been linked to neurodegeneration through promotion of perineuronal net degradation and suppression of dendrite development, respectively \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. Collectively, these data suggest that IL-1Ra promotes upregulation and maintenance of neuroprotective and regenerative qualities, independent of its direct effects on IL-1 and immune signalling.\u003c/p\u003e\u003cp\u003eUnder basal conditions IL-1ɑ and β abundance is low in the brain (Rothwell 2003). Indeed, a low concentration of IL-1, particularly IL-1β, is important for regulating neurodevelopment\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e; however, it is rapidly induced in pathological inflammation (Rothwell 2003). In the present study we used the commercially available IL-1Ra, anakinra, to inhibit IL-1 mediated systemic inflammation and brain pathology in late gestation fetal sheep. Anakinra is a non-glycosylated form of the human IL-1Ra that exerts its physiological effects by competitively binding to the IL-1 receptor and attenuating the effects of IL-1 to prevent downstream inflammatory signalling \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Anakinra has been in clinical use for over 20 years (FDA approved in 2001) in a range of auto immune conditions, as well as sepsis and other hyper-inflammatory syndromes to reduce inflammation related morbidity. It has an established safety profile in adults and adolescents, and its safety, feasibility and pharmacokinetics is currently being investigated in a phase 1b/2a clinical trial in preterm infants \u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. Anakinra has a molecular weight of 17 kDa and has been shown to penetrate the blood brain barrier in humans and preclinical animal studies \u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. We and others have shown that repeated intravenous dosing with IL-1Ra does not completely abolish IL-1 signalling, with IL-1β concentrations in plasma and brain tissue being similar to controls \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Moreover, in neonatal rodents, repeated IL-1Ra administration had no deleterious effects on brain anatomy or behavioural outcomes \u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. In contrast, only complete loss of IL-1 signalling in knockout and transgenic animal has been associated with impaired neurodevelopment \u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e. Collectively, these data suggest that allowing for restoration of IL-1 signalling via intermittent IL-1Ra treatment does not adversely affect makers of brain structure and function in small and large animal models. Although we did not serially sample CSF throughout the experimental period to confirm IL-1Ra concentrations in the sheep brain, previous studies in adults demonstrated that intravenous IL-1Ra crosses the BBB and achieves therapeutic concentrations in the brain within approximately 45-minutes \u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThis study, together with others, demonstrates that IL-1 is implicated in the pathophysiology of infection/inflammation related injury in the developing perinatal brain, and that restoring IL-1 homeostasis with IL-1Ra is a promising therapeutic option for infants with acute infection around the time of birth. However, the optimal timing of treatment remains uncertain. Critically emerging evidence suggests that ongoing neuroinflammation contributes to the sub-acute and chronic phases of neural injury, which may develop several days to weeks after the insult \u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e. This raises the possibility that early or delayed use of IL-1Ra alone or in combination with complementing interventions targeting cell damage or repair (e.g. anti-excitotoxicity, trophic factors, stem cells, essential amino acids) \u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e, could be an effective strategy to mitigate perinatal brain injury and warrants interrogation in future translational studies.\u003c/p\u003e\u003cp\u003eThere are no clinically proven treatments to prevent brain injury related to perinatal infection or inflammation. Current immunomodulatory therapies used in routine clinical care (e.g. antibiotics and corticosteroids) are limited by their broad mechanisms of action and their potential to cause deleterious effects in both animals and humans. For example, corticosteroids are associated with an increased risk of cerebral palsy, intraventricular haemorrhage and hyperactivity in childhood \u003csup\u003e\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e; the underlying mechanisms remain unknown. Prophylactic antibiotics have been linked to an increased risk of neonatal death and disability \u003csup\u003e\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e, the reason for this is unclear but animal studies speculate it could be linked to bacterial lysis promoting the release of bacterial fragments that augment inflammation-induced illness and tissue injury \u003csup\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e, \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e, \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e, \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e\u003c/sup\u003e. Thus, a more targeted anti-inflammatory approach to modulate the effects of unbridled inflammation in the perinatal brain could mitigate these harmful off target effects. Here, we show that IL-1Ra administered to late gestation fetal sheep exposed to progressive LPS-induced inflammation restored expression of genes related to dendritogenesis, synaptogenesis and immune activation in the somatosensory cortex. Moreover, In IL-1Ra treated subjects we observed reduced markers of cortical inflammation and improved cortical neuronal complexity and electrophysiological recovery. Collectively, these findings suggest that IL-1Ra could be a promising anti-inflammatory intervention to prevent perinatal brain injury in infants exposed to infection/inflammation during the perinatal period. Further translational studies are needed to determine the optimal dosing regimen, assess the efficacy of delayed administration and whether use of IL-1Ra with interventions targeting other mechanisms of injury can augment neuroprotection in the perinatal brain.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eAll procedures were approved by the Hudson Institute of Medical Research Animal Ethics committee and were conducted in accordance with the ARRIVE guidelines \u003csup\u003e\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/sup\u003e and the National Health and Medical Research Council Code of Practice for the Care and Use of Animals for Scientific Purposes (Eighth Edition).\u003c/p\u003e\u003cp\u003eTwenty-six pregnant Border-Leicester ewes bearing singleton or twin fetuses of both sexes underwent aseptic surgery on 125\u0026thinsp;\u0026plusmn;\u0026thinsp;1 days of gestation. At this age, cortical development in sheep is broadly comparable to the human brain at term \u003csup\u003e\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e, \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e, \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e\u003c/sup\u003e. Food but not water was withdrawn 18 hours before surgery. Anaesthesia was induced by intravenous (i.v.) injection of sodium thiopentone (20 mL) and maintained using 2\u0026ndash;3% isoflurane and a fraction of inspired oxygen of 60% (Bomac Animal Health, New South Wales, Australia). Maternal prophylactic antibiotics (engemycin, 500 mg i.v.; Schering-Plough, Upper Hutt, New Zealand, and ampicillin, 1 g i.v.; Austrapen, Lennon Healthcare, St. Leonards, New South Wales, Australia) were given immediately before surgery. Maternal heart rate, respiratory rate and isoflurane levels, were continuously monitored by trained anaesthetic staff throughout surgery.\u003c/p\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eFetal instrumentation\u003c/h2\u003e\u003cp\u003eA midline maternal laparotomy was performed, the fetus was exposed, and polyvinyl catheters were inserted into the right brachiocephalic artery and axillary vein. In the case of a twin pregnancy, one twin was instrumented. Two pairs of electroencephalogram (EEG) electrodes (AS633-7SSF; Cooner Wire, Chatsworth, CA, USA) were placed through burr holes onto the dura over the parasagittal parietal cortex (10 and 20 mm anterior to bregma, and 10 mm lateral). Electrodes were secured using surgical bone wax and cyanoacrylate glue. The fetus was returned to the uterus in its original orientation, and all fetal leads were exteriorised through the maternal flank. A catheter was inserted into the maternal jugular vein for administration of post-operative antibiotics and humane culling at the end of the experimental period. At the completion of surgery, ewes received fentanyl for three days via a transdermal patch placed on the left hind leg (75 \u0026micro;g/hour; Janssen Cilag, North Ryde, New South Wales, Australia).\u003c/p\u003e\u003cp\u003eEwes were randomly housed together in separate metabolic crates in a temperature-controlled room (20\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C and relative humidity of 50\u0026thinsp;\u0026plusmn;\u0026thinsp;10%) with a 12-hour light-dark cycle and \u003cem\u003ead libitum\u003c/em\u003e access to food and water. Four to five days of postoperative recovery was allowed before experiments commenced. Ewes and fetuses received daily i.v. infusions of ampicillin (800 mg, maternal i.v. and 200 mg, fetal i.v.) and engemycin (500 mg, maternal i.v.) for three consecutive days after surgery. Fetal catheters were maintained patent with a continuous infusion of heparinised saline (25 IU/mL) at a rate of 0.2 mL/hour.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eExperimental recordings\u003c/h2\u003e\u003cp\u003eThe fetal EEG was continuously recorded from 24 hours prior to the first saline or LPS infusion (129 days of gestation) until the end of the experiment (133 days of gestation). The analogue fetal EEG signal was bandpass filtered with a cut-off frequency set at 1 and 22 Hz and digitised at a sampling frequency of 400 Hz. EEG power was derived from the analogue signal, whilst spectral edge was calculated as the frequency below which 90% of the intensity was present. Relative (%) spectral power in the delta (Δ, 0\u0026ndash;3.9 Hz), theta (θ, 4\u0026ndash;7.9 Hz), alpha (ɑ, 8\u0026ndash;12.9 Hz), and beta (β, 13\u0026ndash;22 Hz) frequency bands was quantified by calculating the power spectra over 4 hour epochs, using fast Fourier transform, of the EEG on sequential epochs using a 10-s Hanning window to minimise spectral leakage, as previously described \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e, \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eExperimental protocol\u003c/h2\u003e\u003cp\u003eExperiments started at 129 days of gestation. Fetuses were randomly allocated, using an online random number generator, to three groups:\u003c/p\u003e\u003cp\u003e\u003col\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e1. Control\u0026thinsp;+\u0026thinsp;vehicle (4 ml saline), \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;9\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e2. LPS (Escherichia coli, O55:B5, MilliporeSigma, MO, USA)\u0026thinsp;+\u0026thinsp;vehicle, \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e3. LPS\u0026thinsp;+\u0026thinsp;IL-1Ra (anakinra, 10 mg/kg i.v. dissolved in 4 ml saline), \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;9\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003c/p\u003e\u003cp\u003eThe dose was guided by previous pharmacokinetic and neuroprotection trials in fetal sheep, non-human primates, and human cohorts which administered anakinra i.v. \u003csup\u003e19, 64, 85\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eFetuses received 300 ng, 600 ng and 1200 ng infusions of LPS diluted in 2 mL of saline i.v. (infusion rate: 1 mL/ minute) at 0 hours, 24 hours and 48 hours, respectively. This model is relevant to acute perinatal gram-negative infection and reproduces the associated pattern of acute systemic inflammation that is associated with adverse neurodevelopment \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e, \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e, \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e, \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eControls received an equivalent volume of saline at the same infusion rate. Infusions of IL-1Ra (Anakinra, Sobi, Stockholm, Sweden) began 1 hour after LPS administration on each consecutive day (i.e. 1, 25 and 49 hours, respectively), at a rate of 0.75 mL/hour over 4 hours. Four days after the start of infusions, sheep were euthanized by intravenous injection of pentobarbitone sodium (Lethabarb, Virbac, New South Wales, Australia). The study protocol is illustrated in Supplementary Fig.\u0026nbsp;1. The rate of fetal loss before the end of the experimental recording period was 4% and did not differ between the groups. In cases of fetal loss, the individual was excluded from the study.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eSleep State Cycling\u003c/h2\u003e\u003cp\u003eSleep state cycling was examined over the final 24 hours of the experimental period, beginning at 9 am (72 hours after the first LPS dose). Raw traces of EEG power, spectral edge frequency and spectral power in delta, theta, alpha and beta bands were examined by an assessor who was masked to the treatment group by independent coding of files. Sleep stage cycling was defined as a repetitive alternating pattern of low-voltage-high-frequency (rapid eye movement [REM]) activity, and high-voltage-low-frequency (non-REM [nREM]) activity, with, on average, each phase lasting approximately 20 minutes, as previously described \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e. A minimum duration of 3 minutes excluding periods of transitional sleep, was required to be classed as a sleep state \u003csup\u003e\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e\u003c/sup\u003e. EEG parameters were extracted for each sleep state and presented as the mean from eight 1-hour epochs across the 24-hours.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eBrain collection and processing\u003c/h2\u003e\u003cp\u003eThe right hemisphere was immersion fixed with 10% phosphate-buffered formalin for four days before processing and embedding in paraffin. The right hemisphere was cut with a blocking blade into 8 mm thick coronal blocks, using a brain mould. Blocks from the forebrain, ~\u0026thinsp;23 mm anterior to stereotaxic zero, with a clearly visible cortex were sectioned using a microtome (Leica Microsystems, Victoria, Australia) into 8-\u0026micro;m thick coronal sections.\u003c/p\u003e\u003cp\u003e Region matched brain sections from the left hemisphere containing a clearly visible lateral parietal lobe/somatosensory cortex were immersion-fixed using a commercially available FD Rapid Golgi Stain Kit according to manufacturer instructions (FD Neurotechnologies Inc., MD, USA). Tissue sections were cut using a Leica VT1200S vibratome at 100 \u0026micro;m and mounted onto coverslips, processed for Golgi visualisation and cover slipped.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eRNA isolation, sequencing and analysis\u003c/h2\u003e\u003cp\u003eCortical grey matter from the parietal lobe of the left hemisphere containing a clearly visible lateral parietal lobe containing the somatosensory cortex, adjacent to the section collected for Golgi visualisation, was homogenised and total mRNA was isolated using a RNeasy Midi Kit (QIAGEN, Venlo, Netherlands). RNA samples were submitted to the Hudson Genomics Facility (Hudson Institute of Medical Research; Clayton, Australia) for integrity and concentration assessment via capillary electrophoresis (Agilent Technologies) and fluorometric quantification (Qubit, Invitrogen). An RNA integrity number (RIN)\u0026thinsp;\u0026ge;\u0026thinsp;7.5 was used to confirm the integrity of all samples.\u003c/p\u003e\u003cp\u003eRNA sequencing was performed using a custom in-house multiplex method, as previously described \u003csup\u003e\u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e, \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e\u003c/sup\u003e. Samples were given a unique i7 index, in addition to a unique molecular identifier (UMI)), during individual pA priming and first strand synthesis which adds a template switch sequence to the 5\u0026rsquo; end. Samples were then pooled into sets and amplified using P7 and an oligo which binds the template switch sequence. Final library construction was completed by tagmentation and addition of P5 by PCR. Sequencing was performed on an Illumina NSQ2k run with 111nt SR (cDNA). A 20nt i7 read contained the 10nt index and 10nt UMI. Samples were parsed using unique i7 indexes.\u003c/p\u003e\u003cp\u003eData analysis and bioinformatics were performed at the Monash Bioinformatics Platform (Clayton, Australia), whereby the nf-core/rnaseq Nextflow pipeline verion 3.10.1 was used to process the RNA sequencing reads \u003csup\u003e\u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e\u003c/sup\u003e. ENSEMBL version 112 Sheep (Ovis Aries) was used as the reference genome. Briefly, reads were trimmed using Trim Galore and then aligned to the genome using STAR \u003csup\u003e\u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e94\u003c/span\u003e, \u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e95\u003c/span\u003e\u003c/sup\u003e. STAR produces alignments to transcripts that were then deduplicated using UMI-tools \u003csup\u003e\u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e96\u003c/span\u003e\u003c/sup\u003e and quantified using Salmon \u003csup\u003e\u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e97\u003c/span\u003e\u003c/sup\u003e. Genes with fewer than 10 UMIs in at least one sample were filtered. Raw gene read counts were imported into RStudio (v2023.06.1\u0026thinsp;+\u0026thinsp;524) with R v4.4.0 and converted into a DGEList object using edgeR \u003csup\u003e\u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e98\u003c/span\u003e\u003c/sup\u003e and normalised for library sizes using the trimmed-mean of M-values (TMM) method. Only genes expressed at \u0026gt;\u0026thinsp;1 counts per million (CPM) in at least 7 samples were retained. Differential gene expression analysis was performed using the limma-voom pipeline \u003csup\u003e\u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e99\u003c/span\u003e\u003c/sup\u003e. Voom transformation of count data to log2-counts per million (CPM) were analysed via linear models in limma \u003csup\u003e\u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e100\u003c/span\u003e\u003c/sup\u003e and contrasts between control versus LPS\u0026thinsp;+\u0026thinsp;vehicle and LPS\u0026thinsp;+\u0026thinsp;vehicle versus LPS\u0026thinsp;+\u0026thinsp;IL-1Ra were performed with empirical Bayes moderation. Genes were and considered to be differentially expressed if log2 fold change \u0026gt;|0.58| and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Pathway Over-Representation Analysis (ORA) of differentially expressed genes was then performed using the ClusterProfiler\u003csup\u003e\u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e101\u003c/span\u003e\u003c/sup\u003e and reactomePA packages \u003csup\u003e\u003cspan citationid=\"CR102\" class=\"CitationRef\"\u003e102\u003c/span\u003e\u003c/sup\u003e. Pathways were considered over-represented if \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and gene count mapped was \u0026ge;\u0026thinsp;3. Heatmap visualisations were performed using ComplexHeatmap \u003csup\u003e\u003cspan citationid=\"CR103\" class=\"CitationRef\"\u003e103\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eImmunohistochemistry\u003c/h2\u003e\u003cp\u003eSlides were dewaxed in xylene, rehydrated in ethanol, and washed in phosphate-buffered saline (PBS). Antigen retrieval was performed by microwaving the slides in citrate buffer (pH 6) for 15 minutes. Endogenous peroxide quenching was performed by incubating slides in 0.1% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in methanol. Blockade of non-specific antigens was performed using 3% normal goat serum. Sections were labelled with 1:250 rabbit anti-IL-1β (cat#: NB600-633, Novus, CO, USA), 1:200 rabbit anti-ionised calcium binding adaptor molecule 1 (Iba-1, Abcam, cat#: ab153696), 1:200 rabbit anti-neuronal nuclei (NeuN, Abcam, cat#: ab177487) and 1:200 rabbit anti-glial fibrillary acidic protein (GFAP; Abcam, cat#: ab68428) overnight at 4\u0026deg;C. Sections were incubated in biotin conjugated IgG (1:200, goat anti-rabbit (Dako, Victoria, Australia), for three hours at room temperature before being incubated in avidin-biotin complex (Sigma-Aldrich) for 45 minutes at room temperature. Sections were reacted with 3,3\u0026prime;-diamino- benzidine tetrahydrochloride (Sigma-Aldrich). The reaction was stopped in PBS before slides were dehydrated in xylene and increasing concentrations of ethanol, mounted in dibutyl phthalate polystyrene xylene and cover slipped.\u003c/p\u003e\u003cp\u003eAstrocytes (GFAP\u0026thinsp;+\u0026thinsp;cells), microglia (Iba-1\u0026thinsp;+\u0026thinsp;cells), and neurons (NeuN\u0026thinsp;+\u0026thinsp;cells) were visualised using light microscopy (Olympus, Tokyo, Japan) at 40\u0026times; magnification and CellSens imaging software (Version 2.3, Olympus). The IL-1β immunoreactivity was scored using published protocols by us and others\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Scoring was based on the intensity of staining (1\u0026thinsp;=\u0026thinsp;light, 2\u0026thinsp;=\u0026thinsp;moderate, 3\u0026thinsp;=\u0026thinsp;moderate-to-intense and 4\u0026thinsp;=\u0026thinsp;intense). Numbers of positive cells or immunoreactivity were quantified within the lateral parietal lobe between cortical layers 3 and 5 from two sections per subject using ImageJ software (v2.00, LOCI, University of Wisconsin). An assessor who was blinded to the treatment group by slide coding performed all imaging and cell counts.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eImmunofluorescence\u003c/h2\u003e\u003cp\u003eSlides were baked at 60\u0026deg;C for one hour then dewaxed in xylene, rehydrated in increasing concentrations of ethanol and washed in PBS. Antigen retrieval was performed in citrate buffer (pH 6) using a microwave for 15 minutes. Non-specific antigen blocking was performed using 10% normal goat serum. Sections were labelled with 1:200 mouse anti-ionised calcium binding adaptor molecule 1 (Iba-1, Abcam, cat#: ab 283319) overnight at 4\u0026deg;C followed by incubation with an Alexa Fluor-488-conjugated 2\u003csup\u003e∘\u003c/sup\u003e antibody (1:200, Jackson Research, cat#: 115-545-003) for 2 hours at room temperature. Sections were labelled with 1:200 rabbit anti-Signal transducer and activator of transcription 3 (Cell Signalling, Cat#: 9145) overnight at 4\u0026deg;C. Sections were incubated with an Alexa Fluor-594-conjugated secondary antibody (1:200, Jackson Research, cat#: 111-585-003) for two hours at room temperature. Sections were washed in PBS then incubated with the nuclear stain HOECHST (1:1000 diluted in PBS; Invitrogen, USA) for five minutes, then washed in PBS. Cover-slipping was performed using DAKO anti-fade fluorescent mounting medium (Agilent Technologies, Australia). Negative controls that did not contain the target antibody were included to confirm the absence of non-specific staining (Supplementary Fig.\u0026nbsp;2).\u003c/p\u003e\u003cp\u003eSections with fluorescently double labelled microglia (Iba-1+/STAT3+) were scanned using a VS120 Olympus virtual slide microscope and visualised at 20x magnification using QuPath imaging software (Version 0.4.3). Double labelled cells were quantified within the somatosensory cortex of the lateral parietal lobe between cortical layers 3 and 5 from two sections per subject. An assessor who was blinded to the treatment group by slide coding performed all imaging and cell counts.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eAssessment of Neuronal Morphology\u003c/h2\u003e\u003cp\u003eCoded Golgi-stained tissue sections (10 serial sections per subject) that were region matched to sections used for immunohistochemical and RNAseq analyses were used to assess basal dendrites from pyramidal neurons between layers 3 and 5 of the parietal lobe, as described in our previously published protocol\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. The Golgi staining produced incomplete filling of the apical dendrites relative to the basal dendrites. Thus, apical dendrites were not analysed. Basal dendrites from pyramidal neurons were visualised on an Olympus BX61 stereology microscope equipped with an DP73 camera (\u0026times;0.5 lens) at 60\u0026times; magnification using CellSens imaging software (version 2.3; Olympus). Basal dendrites from a total of 20 pyramidal neurons, selected from 10 serial sections of the lateral parietal lobe from each subject, met the pre-defined inclusion criteria for imaging.\u003c/p\u003e\u003cp\u003ePyramidal neurons were selected based on established morphological criteria \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR104\" class=\"CitationRef\"\u003e104\u003c/span\u003e\u003c/sup\u003e, as follows:\u003c/p\u003e\u003cp\u003e\u003col\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e1. A triangular shaped soma and apical dendrites perpendicular to the pial surface\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e2. Complete Golgi impregnation of the cell that permitted visualisation of the entire dendritic arbour and spines\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e3. Neuronal soma and processes not obscured by other neurons, glia or blood vessels\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e4. Neurons exhibiting a complete basilar dendritic tree without truncated or cut processes.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003c/p\u003e\u003cp\u003eImages were cropped, separated into individual channels using ImageJ and imported into Imaris (version 9.2.1, Bitplane, Oxford Instruments Company, Abington, UK). The Imaris filament tracer tool was used to measure numbers of dendritic spines and dendritic complexity, including summated dendritic length, numbers of dendritic terminals and Sholl analysis (numbers of dendrite intersections per Sholl ring). Sholl intersections were set at 5 \u0026micro;m concentric rings centred on the soma. The MATLAB spine classification extension was used to classify dendritic spine morphology (filopodia, long thin, stubby, mushroom) (MATLAB, R2019a, Mathworks Inc., CA, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eData analysis and statistics\u003c/h2\u003e\u003cp\u003eThe Shapiro-Wilk test was used to test data for normality. EEG data were analysed using a two-way ANOVA, with the baseline, vehicle/LPS/IL-1Ra infusion and recovery periods analysed as separate time periods. For EEG data and Sholl analysis, when statistical significance was found between groups or between group and time, post hoc comparisons were made using the Benjamini-Hochberg correction. Differentially expressed genes, identified using the limma-voom method \u003csup\u003e\u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e99\u003c/span\u003e, \u003cspan citationid=\"CR105\" class=\"CitationRef\"\u003e105\u003c/span\u003e\u003c/sup\u003e, were defined as having a \u0026gt;|0.58| log2 fold-change and an unadjusted \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Neuronal dendrite morphology and histological data were analysed by one-way ANOVA. The Benjamini-Hochberg correction was used for post hoc comparisons. For non-parametric data, between group comparisons were performed using Kruskal-Wallis tests. Post hoc power analysis for summated dendritic length showed 85% power to detect a minimum difference of 977 \u0026micro;m, with an alpha of 0.05. Moreover, post hoc power analysis of EEG spectral bands showed 90% power to detect a 30% reduction in beta spectral band power, with an alpha of 0.05. Statistical significance was accepted when \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Data are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SE).\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSharmony B. Kelly and Robert Galinsky conceptualised and designed the study. Sharmony B. Kelly,\u0026nbsp;Steven X Cho, Valerie Zahra, Mira Menyen, Rodney W Hunt, Claudia A Nold-Petry, Alistair J Gunn, Graeme R Polglase, Stuart B Hooper, Marcel F Nold, and Robert Galinsky\u0026nbsp;undertook experiments and formal analysis of the data. Sharmony B. Kelly, Steven X Cho, and Valerie Zahra designed and undertook all RNA sequencing analyses. Sharmony B. Kelly undertook the Golgi analysis and Immunohistochemistry. Sharmony B Kelly and Robert Galinsky undertook all cell quantification, analysis and preparation of figures. Robert Galinsky provided overall oversight of the research. All authors critically reviewed the manuscript and approved the final manuscript as submitted and agree to be accountable for all aspects of the work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding support\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the National Health \u0026amp; Medical Research Council of Australia (1105526 to GRP, 1090890 and 1164954 to RG, 11173584 and 2033196 to CANP), Cerebral Palsy Alliance Grant (ERG02123 to RG), the Harold and Cora Brenan Benevolent Trust and the Victorian Government\u0026rsquo;s Operational Infrastructure Support Program.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors gratefully acknowledge the technical assistance of the Monash Health Translation Precinct Histology Platform, the Monash Health Translation Precinct Micro Imaging Facility, the Hudson Institute of Medical Research Genomics Facility, and the Monash Genomics and Bioinformatics Platforms\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWu YW, Colford JM, Jr. Chorioamnionitis as a risk factor for cerebral palsy: A meta-analysis. \u003cem\u003eJAMA\u003c/em\u003e \u003cstrong\u003e284\u003c/strong\u003e, 1417-1424 (2000).\u003c/li\u003e\n\u003cli\u003eHoneycutt A, Dunlap L, Chen H, al Homsi G, Grosse S, Schendel DE. 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Differential lung gene expression identified Zscan2 and Bag6 as novel tissue repair players in an experimental COPD model. \u003cem\u003ePLoS One\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, e0309166 (2024).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e\n"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Neuroinflammation, neurophysiology, interleukin 1, interleukin 1 receptor antagonist, neuronal dendrites, RNAseq","lastPublishedDoi":"10.21203/rs.3.rs-7882525/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7882525/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePerinatal inflammation, often caused by infection, is strongly linked with lifelong disability. Human and experimental studies identify interleukin-1 (IL-1), a pro-inflammatory cytokine, as a key mediator. We tested the hypothesis that systemic administration of IL-1 receptor antagonist (IL-1Ra) could attenuate cortical inflammation and improve neuronal development in late gestation fetal sheep exposed to lipopolysaccharide (LPS)-induced inflammation. Fetal sheep, instrumented for continuous EEG, were randomised to: (1) saline infusion, (2) repeated intravenous LPS\u0026thinsp;+\u0026thinsp;vehicle infusions or (3) the same LPS regimen plus intravenous IL-1Ra infusions one hour after each LPS dose. Four-days later, brains were examined using RNAseq, Golgi staining and immunohistochemistry. On EEG, LPS-exposure reduced beta power compared to control, particularly in REM sleep. In the somatosensory cortex, LPS-exposure decreased expression of genes involved in dendritogenesis and synaptogenesis, and increased genes involved in immune activation via LPS and IL-1 signalling. LPS-exposed fetuses had increased microglial activation and reduced neuronal arborisation. IL-1Ra treatment improved EEG band power, normalised expression of genes involved in synaptogenesis, dendritogenesis and immune activation, reduced microglial activation, and restored neuronal arborisation. In summary, IL-1Ra reduced LPS-induced inflammation and improved biomolecular, structural and functional markers of neurodevelopment. Thus, IL-1Ra may improve neurodevelopmental outcomes following perinatal infection/inflammation.\u003c/p\u003e","manuscriptTitle":"IL-1 modulation preserves biomolecular, structural and functional integrity of the somatosensory cortex after fetal inflammation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-07 10:00:24","doi":"10.21203/rs.3.rs-7882525/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"985bdd1c-29ca-4a50-bac3-334c7c834e83","owner":[],"postedDate":"November 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":57453290,"name":"Biological sciences/Neuroscience/Regeneration and repair in the nervous system"},{"id":57453291,"name":"Health sciences/Neurology/Neurological disorders/Brain injuries/Neonatal brain damage"}],"tags":[],"updatedAt":"2025-11-07T10:00:24+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-07 10:00:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7882525","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7882525","identity":"rs-7882525","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}