Novel biomarkers of preterm brain injury from blood transcriptome in sheep model of intrauterine asphyxia.

BACKGROUND: Infants born preterm have a higher incidence of neurological deficits. A key step in finding effective treatments is to identify biomarkers that reliably predict outcome. METHODS: Following umbilical cord occlusion (UCO) in pregnant sheep, whole fetal blood RNA was sequenced pre- and post-UCO, brain injury outcome was determined by battery of neuropathology scoring and the transcriptome signature correlated to the degree of brain injury. Additionally, we developed a novel analytical procedure to deduce cell blood composition over time. RESULTS: Sixty-one genes were identified with significant altered expression after UCO. In pre-UCO blood, the level of three mRNAs (Trex2, Znf280b, novel miRNA) and in post-UCO, four mRNAs (Fam184a, Angptl2, novel lincRNA and an unknown protein-coding gene) were associated to brain injury (FDR < 0.01). Several of these mRNAs are related to inflammation and angiogenesis. Pathway analysis highlighted genes playing a role in perinatal death and growth failure. Results also indicate that several leukocyte populations undergo significant changes after UCO. CONCLUSION: We have used a whole transcriptomic approach to uncover novel biomarkers in fetal blood that correlate to neuropathology in the preterm sheep brain. The current data forms a basis for future studies to investigate mechanisms of these mRNAs in the injury progression. IMPACT: Trend analysis of genes following asphyxia reveal a group of genes associated with perinatal death and growth failure. Several pre-asphyxia transcripts were associated to brain injury severity suggesting genomic susceptibility to injury. Several post-asphyxia transcripts were correlated to brain injury severity, thus, serve as potential novel biomarkers of injury outcome. Successfully adaptation of cell profiling algorithms suggests significant changes in blood cell composition following asphyxia.

Treatment with RNase alleviates brain injury but not neuroinflammation in neonatal hypoxia/ischemia.

There is a need for new treatments to reduce brain injuries derived from neonatal hypoxia/ischemia. The only viable option used in the clinic today in infants born at term is therapeutic hypothermia, which has a limited efficacy. Treatments with exogenous RNase have shown great promise in a range of different adult animal models including stroke, ischemia/reperfusion injury, or experimental heart transplantation, often by conferring vascular protective and anti-inflammatory effects. However, any neuroprotective function of RNase treatment in the neonate remains unknown. Using a well-established model of neonatal hypoxic/ischemic brain injury, we evaluated the influence of RNase treatment on RNase activity, gray and white matter tissue loss, blood-brain barrier function, as well as levels and expression of inflammatory cytokines in the brain up to 6 h after the injury using multiplex immunoassay and RT-PCR. Intraperitoneal treatment with RNase increased RNase activity in both plasma and cerebropinal fluids. The RNase treatment resulted in a reduction of brain tissue loss but did not affect the blood-brain barrier function and had only a minor modulatory effect on the inflammatory response. It is concluded that RNase treatment may be promising as a neuroprotective regimen, whereas the mechanistic effects of this treatment appear to be different in the neonate compared to the adult and need further investigation.

A Neonatal Rodent Model of Retroorbital Vein Injection.

Intravenous (iv) injection is the most used route of drug administration in neonates in the clinical setting. Therefore, retroorbital vein injection is an important method for compound administration in research, where successful proof-of-concept studies can progress into much-needed neonatal clinical trials. Most intravenous studies in neonatal rodents use the superficial temporal/facial vein. However, retroorbital injection becomes unreliable in neonatal rodents older than 2 days after the skin darkens and the vein is no longer visible. In the present protocol, we describe the retroorbital injection of the venous sinus in both the neonatal mouse and rat at ages when the superficial temporal vein is no longer visible, but the eyes have not opened yet. Eye-opening facilitates retro-orbital injection by enabling the researcher to clearly see that they are not perforating the eye when inserting the needle. We demonstrate that this technique can be performed in a reliable and reproducible manner without adverse effects. Additionally, we show that it can be used in many studies, such as administering compounds to study neonatal brain injury.

Maternal n-3 enriched diet reprograms neurovascular transcriptome and blunts inflammation in neonate.

Infection during perinatal period can adversely affect brain development, predispose infants to ischemic stroke and have lifelong consequences. We previously demonstrated that diet enriched in n-3 polyunsaturated fatty acids (PUFA) transforms brain lipid composition and protects from neonatal stroke. Vasculature is a critical interface between blood and brain providing a barrier to systemic infection. Here we examined whether maternal PUFA-enriched diets exert reprograming of endothelial cell signalling in 9-day old mice after endotoxin (LPS)-induced infection. Transcriptome analysis was performed on brain microvessels from pups born to dams maintained on 3 diets: standard, n-3 or n-6 enriched. N-3 diet enabled higher immune reactivity in brain vasculature, while preventing imbalance of cell cycle regulation and extracellular matrix cascades that accompanied inflammatory response in standard diet. LPS response in blood and brain was blunted in n-3 offspring. Cerebral angioarchitecture analysis revealed modified vessel complexity after LPS. Thus, n-3-enriched maternal diet partially prevents imbalance in homeostatic processes and alters inflammation rather than affects brain vascularization during early life. Importantly, maternal diet may presage offspring neurovascular outcomes later in life.

Long-term impact of maternal obesity on the gliovascular unit and ephrin signaling in the hippocampus of adult offspring.

BACKGROUND: Children born to obese mothers are at increased risk of developing mood disorders and cognitive impairment. Experimental studies have reported structural changes in the brain such as the gliovascular unit as well as activation of neuroinflammatory cells as a part of neuroinflammation processing in aged offspring of obese mothers. However, the molecular mechanisms linking maternal obesity to poor neurodevelopmental outcomes are not well established. The ephrin system plays a major role in a variety of cellular processes including cell-cell interaction, synaptic plasticity, and long-term potentiation. Therefore, in this study we determined the impact of maternal obesity in pregnancy on cortical, hippocampal development, vasculature and ephrin-A3/EphA4-signaling, in the adult offspring in mice. METHODS: Maternal obesity was induced in mice by a high fat/high sugar Western type of diet (HF/HS). We collected brain tissue (prefrontal cortex and hippocampus) from 6-month-old offspring of obese and lean (control) dams. Hippocampal volume, cortical thickness, myelination of white matter, density of astrocytes and microglia in relation to their activity were analyzed using 3-D stereological quantification. mRNA expression of ephrin-A3, EphA4 and synaptic markers were measured by qPCR in the brain tissue. Moreover, expression of gap junction protein connexin-43, lipocalin-2, and vascular CD31/Aquaporin 4 were determined in the hippocampus by immunohistochemistry. RESULTS: Volume of hippocampus and cortical thickness were significantly smaller, and myelination impaired, while mRNA levels of hippocampal EphA4 and post-synaptic density (PSD) 95 were significantly lower in the hippocampus in the offspring of obese dams as compared to offspring of controls. Further analysis of the hippocampal gliovascular unit indicated higher coverage of capillaries by astrocytic end-feet, expression of connexin-43 and lipocalin-2 in endothelial cells in the offspring of obese dams. In addition, offspring of obese dams demonstrated activation of microglia together with higher density of cells, while astrocyte cell density was lower. CONCLUSION: Maternal obesity affects brain size, impairs myelination, disrupts the hippocampal gliovascular unit and decreases the mRNA expression of EphA4 and PSD-95 in the hippocampus of adult offspring. These results indicate that the vasculature-glia cross-talk may be an important mediator of altered synaptic plasticity, which could be a link between maternal obesity and neurodevelopmental/neuropsychiatric disorders in the offspring.

Immune-Neurovascular Interactions in Experimental Perinatal and Childhood Arterial Ischemic Stroke.

Emerging clinical and preclinical data have demonstrated that the pathophysiology of arterial ischemic stroke in the adult, neonates, and children share similar mechanisms that regulate brain damage but also have distinct molecular signatures and involved cellular pathways due to the maturational stage of the central nervous system and the immune system at the time of the insult. In this review, we discuss similarities and differences identified thus far in rodent models of 2 different diseases-neonatal (perinatal) and childhood arterial ischemic stroke. In particular, we review acquired knowledge of the role of resident and peripheral immune populations in modulating outcomes in models of perinatal and childhood arterial ischemic stroke and the most recent and relevant findings in relation to the immune-neurovascular crosstalk, and how the influence of inflammatory mediators is dependent on specific brain maturation stages. Finally, we discuss the current state of treatments geared toward age-appropriate therapies that signal via the immune-neurovascular interaction and consider sex differences to achieve successful translation.

Therapeutic Effect of Nicotinamide Mononucleotide for Hypoxic-Ischemic Brain Injury in Neonatal Mice.

SUMMARY STATEMENT: Neonatal hypoxia-ischemia reduces nicotinamide adenine dinucleotide (NAD+) and SIRT6 levels in the injured hippocampus.Hippocampal high mobility group box-1 (HMGB1) release is significantly increased after neonatal hypoxia-ischemia.Nicotinamide mononucleotide (NMN) treatment normalizes hippocampal NAD+ and SIRT6 levels, with significant decrease in caspase-3 activity and HMGB1 release.NMN improves early developmental behavior, as well as motor and memory function.

Two different isoforms of osteopontin modulate myelination and axonal integrity.

Abnormal myelination underlies the pathology of white matter diseases such as preterm white matter injury and multiple sclerosis. Osteopontin (OPN) has been suggested to play a role in myelination. Murine OPN mRNA is translated into a secreted isoform (sOPN) or an intracellular isoform (iOPN). Whether there is an isoform-specific involvement of OPN in myelination is unknown. Here we generated mouse models that either lacked both OPN isoforms in all cells (OPN-KO) or lacked sOPN systemically but expressed iOPN specifically in oligodendrocytes (OLs-iOPN-KI). Transcriptome analysis of isolated oligodendrocytes from the neonatal brain showed that genes and pathways related to increase of myelination and altered cell cycle control were enriched in the absence of the two OPN isoforms in OPN-KO mice compared to control mice. Accordingly, adult OPN-KO mice showed an increased axonal myelination, as revealed by transmission electron microscopy imaging, and increased expression of myelin-related proteins. In contrast, neonatal oligodendrocytes from OLs-iOPN-KI mice compared to control mice showed differential regulation of genes and pathways related to the increase of cell adhesion, motility, and vasculature development, and the decrease of axonal/neuronal development. OLs-iOPN-KI mice showed abnormal myelin formation in the early phase of myelination in young mice and signs of axonal degeneration in adulthood. These results suggest an OPN isoform-specific involvement, and a possible interplay between the isoforms, in myelination, and axonal integrity. Thus, the two isoforms of OPN need to be separately considered in therapeutic strategies targeting OPN in white matter injury and diseases.

Proteomics identifies lipocalin-2 in neonatal inflammation associated with cerebrovascular alteration in mice and preterm infants.

Staphylococcus (S.) epidermidis is the most common nosocomial coagulase-negative staphylococci infection in preterm infants. Clinical signs of infection are often unspecific and novel markers to complement diagnosis are needed. We investigated proteomic alterations in mouse brain after S. epidermidis infection and in preterm infant blood. We identified lipocalin-2 (LCN2) as a crucial protein associated with cerebrovascular changes and astrocyte reactivity in mice. We further proved that LCN2 protein expression was associated with endothelial cells but not astrocyte reactivity. By combining network analysis and differential expression approaches, we identified LCN2 linked to blood C-reactive protein levels in preterm infants born <28 weeks of gestation. Blood LCN2 levels were associated with similar alterations of cytokines and chemokines in both infected mice and human preterm infants with increased levels of C-reactive protein. This experimental and clinical study suggests that LCN2 may be a marker of preterm infection/inflammation associated with cerebrovascular changes and neuroinflammation.

Transcriptome network analysis links perinatal Staphylococcus epidermidis infection to microglia reprogramming in the immature hippocampus.

Staphylococcus epidermidis (S. epidermidis) is the most common nosocomial pathogen in preterm infants and associated with increased risk of cognitive delay, however, underlying mechanisms are unknown. We employed morphological, transcriptomic and physiological methods to extensively characterize microglia in the immature hippocampus following S. epidermidis infection. 3D morphological analysis revealed activation of microglia after S. epidermidis. Differential expression combined with network analysis identified NOD-receptor signaling and trans-endothelial leukocyte trafficking as major mechanisms in microglia. In support, active caspase-1 was increased in the hippocampus and using the LysM-eGFP knock-in transgenic mouse, we demonstrate infiltration of leukocytes to the brain together with disruption of the blood-brain barrier. Our findings identify activation of microglia inflammasome as a major mechanism underlying neuroinflammation following infection. The results demonstrate that neonatal S. epidermidis infection share analogies with S. aureus and neurological diseases, suggesting a previously unrecognized important role in neurodevelopmental disorders in preterm born children.

The Alpha 7 Nicotinic Acetylcholine Receptor Does Not Affect Neonatal Brain Injury.

Inflammation plays a central role in the development of neonatal brain injury. The alpha 7 nicotinic acetylcholine receptor (α7nAChR) can modulate inflammation and has shown promising results as a treatment target in rodent models of adult brain injury. However, little is known about the role of the α7nAChR in neonatal brain injury. Hypoxic-ischemic (HI) brain injury was induced in male and female C57BL/6 mice, α7nAChR knock-out (KO) mice and their littermate controls on postnatal day (PND) 9-10. C57BL/6 pups received i.p. injections of α7nAChR agonist PHA 568487 (8 mg/kg) or saline once daily, with the first dose given directly after HI. Caspase-3 activity and cytokine mRNA expression in the brain was analyzed 24 h after HI. Motor function was assessed 24 and 48 h after HI, and immunohistochemistry was used to assess tissue loss at 24 h and 7 days after HI and microglial activation 7 days after HI. Activation of α7nAChR with the agonist PHA 568487 significantly decreased CCL2/MCP-1, CCL5/RANTES and IL-6 gene expression in the injured brain hemisphere 24 h after HI compared with saline controls in male, but not female, pups. However, α7nAChR activation did not alter caspase-3 activity and TNFα, IL-1ß and CD68 mRNA expression. Furthermore, agonist treatment did not affect motor function (24 or 48 h), neuronal tissue loss (24 h or 7 days) or microglia activation (7 days) after HI in either sex. Knock-out of α7nAChR did not influence neuronal tissue loss 7 days after HI. In conclusion, targeting the α7nAChR in neonatal brain injury shows some effect on dampening acute inflammatory responses in male pups. However, this does not lead to an effect on overall injury outcome.

Geometrical modelling of neuronal clustering and development.

The dynamic geometry of neuronal development is an essential concept in theoretical neuroscience. We aimed to design a mathematical model which outlines stepwise in an innovative form and designed to model neuronal development geometrically and modelling spatially the neuronal-electrical field interaction. We demonstrated flexibility in forming the cell and its nucleus to show neuronal growth from inside to outside that uses a fractal cylinder to generate neurons (pyramidal/sphere) in form of mathematically called 'surface of revolution'. Furthermore, we verified the effect of the adjacent neurons on a free branch from one-side, by modelling a 'normal vector surface' that represented a group of neurons. Our model also indicated how the geometrical shapes and clustering of the neurons can be transformed mathematically in the form of vector field that is equivalent to the neuronal electromagnetic activity/electric flux. We further simulated neuronal-electrical field interaction that was implemented spatially using Van der Pol oscillator and taking Laplacian vector field as it reflects biophysical mechanism of neuronal activity and geometrical change. In brief, our study would be considered a proper platform and inspiring modelling for next more complicated geometrical and electrical constructions.

Sex-Dependent Gliovascular Interface Abnormality in the Hippocampus following Postnatal Immune Activation in Mice.

The neuro-gliovascular unit is a crucial structure for providing a balanced well-functioning environment for neurons and their synapses. Activation of the immune system during the developmental period is believed to affect the gliovascular unit, which may trigger neurodevelopmental and neurological/neuropsychiatric diseases. In this study, we hypothesized that vulnerability of the male brain to a neonatal insult was conditioned by sex-dependent differences in the impairment of the hippocampal gliovascular unit. Male and female C57BL/6J pups received lipopolysaccharide (LPS) (1 mg/kg) or saline on postnatal day (P) 5. Brains were collected at P12 and morphological quantifications of hippocampal fibrillary glial acid protein (GFAP+) astrocytes and ionized calcium-binding adaptor molecule 1 protein (Iba1+) microglia were performed by using 3-D image analysis together with measuring the length of CD31+ and aquaporin-4 (AQP4+) vessels. We found a significant increase in the length of CD31+ capillaries in the male LPS group compared to the saline group; however, coverage of capillaries by astrocytic end-feet (AQP4+) was significantly reduced. In contrast, there was a significant increase in AQP4+ capillary length in female pups 1 week after LPS injection. GFAP+ astrocytes via morphological changes in the hippocampus showed significant enhancement in the activity 1 week following LPS injection in male mice. We propose that neonatal inflammation could induce susceptibility to neurodevelopmental disorders through modification of hippocampal gliovascular interface in a sex-dependent manner.

Reelin cells and sex-dependent synaptopathology in autism following postnatal immune activation.

BACKGROUND AND PURPOSE: Autism spectrum disorders (ASD) are heterogeneous neurodevelopmental disorders with considerably increased risk in male infants born preterm and with neonatal infection. Here, we investigated the role of postnatal immune activation on hippocampal synaptopathology by targeting Reelin+ cells in mice with ASD-like behaviours. EXPERIMENTAL APPROACH: C57/Bl6 mouse pups of both sexes received lipopolysaccharide (LPS, 1 mg·kg-1 ) on postnatal day (P) 5. At P45, animal behaviour was examined by marble burying and sociability test, followed by ex vivo brain MRI diffusion kurtosis imaging (DKI). Hippocampal synaptogenesis, number and morphology of Reelin+ cells, and mRNA expression of trans-synaptic genes, including neurexin-3, neuroligin-1, and cell-adhesion molecule nectin-1, were analysed at P12 and P45. KEY RESULTS: Social withdrawal and increased stereotypic activities in males were related to increased mean diffusivity on MRI-DKI and overgrowth in hippocampus together with retention of long-thin immature synapses on apical dendrites, decreased volume and number of Reelin+ cells as well as reduced expression of trans-synaptic and cell-adhesion molecules. CONCLUSION AND IMPLICATIONS: The study provides new insights into sex-dependent mechanisms that may underlie ASD-like behaviour in males following postnatal immune activation. We identify GABAergic interneurons as core components of dysmaturation of excitatory synapses in the hippocampus following postnatal infection and provide cellular and molecular substrates for the MRI findings with translational value.

An Optimized and Detailed Step-by-Step Protocol for the Analysis of Neuronal Morphology in Golgi-Stained Fetal Sheep Brain.

Antenatal brain development during the final trimester of human pregnancy is a time when mature neurons become increasingly complex in morphology, through axonal and dendritic outgrowth, dendritic branching, and synaptogenesis, together with myelin production. Characterizing neuronal morphological development over time is of interest to developmental neuroscience and provides the framework to measure gray matter pathology in pregnancy compromise. Neuronal microstructure can be assessed with Golgi staining, which selectively stains a small percentage (1-3%) of neurons and their entire dendritic arbor. Advanced imaging processing and analysis tools can then be employed to quantitate neuronal cytoarchitecture. Traditional Golgi-staining protocols have been optimized, and commercial kits are readily available offering improved speed and sensitivity of Golgi staining to produce consistent results. Golgi-stained tissue is then visualized under light microscopy and image analysis may be completed with several software programs for morphological analysis of neurons, including freeware and commercial products. Each program requires optimization, whether semiautomated or automated, requiring different levels of investigator intervention and interpretation, which is a critical consideration for unbiased analysis. Detailed protocols for fetal ovine brain tissue are lacking, and therefore, we provide a step-by-step workflow of computer software analysis for morphometric quantification of Golgi-stained neurons. Here, we utilized the commonly applied FD Rapid GolgiStain kit (FD NeuroTechnologies) on ovine fetal brains collected at 127 days (0.85) of gestational age for the analysis of CA1 pyramidal neurons in the hippocampus. We describe the step-by-step protocol to retrieve neuronal morphometrics using Imaris imaging software to provide quantification of apical and basal dendrites for measures of dendrite length (µm), branch number, branch order, and Sholl analysis (intersections over radius). We also detail software add-ons for data retrieval of dendritic spines including the number of spines, spine density, and spine classification, which are critical indicators of synaptic function. The assessment of neuronal morphology in the developing brain using Rapid-Golgi and Imaris software is labor-intensive, particularly during the optimization period. The methodology described in this step-by-step description is novel, detailed, and aims to provide a reproducible, working protocol to quantify neuronal cytoarchitecture with simple descriptions that will save time for the next users of these commonly used techniques.

Induction of Mitochondrial Fragmentation and Mitophagy after Neonatal Hypoxia-Ischemia.

Hypoxia-ischemia (HI) leads to immature brain injury mediated by mitochondrial stress. If damaged mitochondria cannot be repaired, mitochondrial permeabilization ensues, leading to cell death. Non-optimal turnover of mitochondria is critical as it affects short and long term structural and functional recovery and brain development. Therefore, disposal of deficient mitochondria via mitophagy and their replacement through biogenesis is needed. We utilized mt-Keima reporter mice to quantify mitochondrial morphology (fission, fusion) and mitophagy and their mechanisms in primary neurons after Oxygen Glucose Deprivation (OGD) and in brain sections after neonatal HI. Molecular mechanisms of PARK2-dependent and -independent pathways of mitophagy were investigated in vivo by PCR and Western blotting. Mitochondrial morphology and mitophagy were investigated using live cell microscopy. In primary neurons, we found a primary fission wave immediately after OGD with a significant increase in mitophagy followed by a secondary phase of fission at 24 h following recovery. Following HI, mitophagy was upregulated immediately after HI followed by a second wave at 7 days. Western blotting suggests that both PINK1/Parkin-dependent and -independent mechanisms, including NIX and FUNDC1, were upregulated immediately after HI, whereas a PINK1/Parkin mechanism predominated 7 days after HI. We hypothesize that excessive mitophagy in the early phase is a pathologic response which may contribute to secondary energy depletion, whereas secondary mitophagy may be involved in post-HI regeneration and repair.

Scavenger receptor CD36 governs recruitment of myeloid cells to the blood-CSF barrier after stroke in neonatal mice.

BACKGROUND: Ischemic stroke induces the activation and recruitment of peripheral leukocytes to the injured brain. These cells can infiltrate the brain through multiple routes, either by penetrating blood-brain barrier or via blood-CSF barriers at the meninges or the choroid plexus (CP). We previously showed that myeloid cell trafficking via the CP occurs early after neonatal arterial stroke and modulates injury. CD36 is a receptor that mediates function of endothelial cells and cells of the monocyte lineage under various neurodegenerative conditions and can influence brain injury after neonatal stroke. Here we asked whether CD36 impacts injury by altering leukocyte trafficking through the CP in neonatal mice subjected to transient middle cerebral artery occlusion (tMCAO). METHODS: In neonatal mice with intact or globally disrupted CD36 signalling (CD36 KO), we characterized the phenotypes of myeloid cells by flow cytometry and the underlying gene expression signatures in the CPs contralateral and ipsilateral to tMCAO by RNA sequencing analyses, focussing on early post-reperfusion time window. RESULTS: Flow cytometry in the isolated CPs revealed that CD36 mediates stepwise recruitment of myeloid cells to the CP ipsilateral to tMCAO early after reperfusion, with a predominant increase first in inflammatory monocyte subsets and neutrophils followed by patrolling monocytes. RNA sequencing analyses demonstrated marked changes in gene expression in the CP ipsilateral compared to the CP contralateral to tMCAO in wild type mice. Changes were further modified by lack of CD36, including distinction in several clusters of genes involved in inflammatory, metabolic and extracellular matrix signalling in the CP ipsilateral to tMCAO. CONCLUSION: Altogether, our data suggest cooperation between blood-CSF-brain interface via the CP through CD36-mediated signalling following neonatal stroke with a key role for inflammatory monocytes and neutrophils.

Maternal n-3 Polyunsaturated Fatty Acid Enriched Diet Commands Fatty Acid Composition in Postnatal Brain and Protects from Neonatal Arterial Focal Stroke.

The fetus is strongly dependent on nutrients from the mother, including polyunsaturated fatty acids (PUFA). In adult animals, n-3 PUFA ameliorates stroke-mediated brain injury, but the modulatory effects of different PUFA content in maternal diet on focal arterial stroke in neonates are unknown. This study explored effects of maternal n-3 or n-6 enriched PUFA diets on neonatal stroke outcomes. Pregnant mice were assigned three isocaloric diets until offspring reached postnatal day (P) 10-13: standard, long-chain n-3 PUFA (n-3) or n-6 PUFA (n-6) enriched. Fatty acid profiles in plasma and brain of mothers and pups were determined by gas chromatography-mass spectrometry and cytokines/chemokines by multiplex protein analysis. Transient middle cerebral artery occlusion (tMCAO) was induced in P9-10 pups and cytokine and chemokine accumulation, caspase-3 and calpain-dependent spectrin cleavage and brain infarct volume were analyzed. The n-3 diet uniquely altered brain lipid profile in naïve pups. In contrast, cytokine and chemokine levels did not differ between n-3 and n-6 diet in naïve pups. tMCAO triggered accumulation of inflammatory cytokines and caspase-3-dependent and -independent cell death in ischemic-reperfused regions in pups regardless of diet, but magnitude of neuroinflammation and caspase-3 activation were attenuated in pups on n-3 diet, leading to protection against neonatal stroke. In conclusion, maternal/postnatal n-3 enriched diet markedly rearranges neonatal brain lipid composition and modulates the response to ischemia. While standard diet is sufficient to maintain low levels of inflammatory cytokines and chemokines under physiological conditions, n-3 PUFA enriched diet, but not standard diet, attenuates increases of inflammatory cytokines and chemokines in ischemic-reperfused regions and protects from neonatal stroke.

Microglia activation in postmortem brains with schizophrenia demonstrates distinct morphological changes between brain regions.

Schizophrenia (SCZ) is a psychiatric disorder that can include symptoms of disorganized speech and thoughts with uncertain underlying mechanisms possibly linked to over-activated microglia. In this study, we used brain samples from sixteen donors with SCZ and thirteen control donors to assess the differential activation of microglia by quantifying density and 3D reconstruction of microglia stained with ionized calcium-binding adaptor molecule-1 (Iba1). Our samples consisted of sections from the frontal, temporal, and cingulate cortical gray matter, subcortical white matter regions (SCWM), and included the anterior corpus callosum. In the first series of studies, we performed a density analysis followed by a spatial analysis to ascertain the microglial density, distribution, and soma size in SCZ brains. Second, we performed a series of morphological quantification techniques to investigate the arborization patterns of the microglia in SCZ. The results demonstrated an increase in microglia density in the cortical gray matter regions in SCZ cases, while in the SCWM, there was a significant increase in microglia density in the frontal and temporal, but not in the other brain regions of interest (ROIs). Spatial analysis using the "nearest neighbor" demonstrated that there was no effect in "clustering", but there were shorter distances between microglia seen in the SCZ cases. The morphological measures showed that there was a region-dependent increase in the microglia soma size in the SCZ cases while the Sholl analysis revealed a significant decrease in the microglia arborization in the SCZ cases across all the ROI's studied. An in-depth 3D reconstruction of microglia in Brodmann area 9 cortical region found that there was a significant association between age and reduced microglial arborization in the SCZ cases. This region-dependent age association can help determine whether longitudinal changes in microglial activation across age are brain region-dependent, which may point to potential therapeutic targets.