Approaches to embryonic neurodevelopment: from neural cell to neural tube formation through mathematical models.

The development of the human central nervous system initiates in the early embryonic period until long after delivery. It has been shown that several neurological and neuropsychiatric diseases originate from prenatal incidents. Mathematical models offer a direct way to understand neurodevelopmental processes better. Mathematical modelling of neurodevelopment during the embryonic period is challenging in terms of how to 'Approach', how to initiate modelling and how to propose the appropriate equations that fit the underlying dynamics of neurodevelopment during the embryonic period while including the variety of elements that are built-in naturally during the process of neurodevelopment. It is imperative to answer where and how to start modelling; in other words, what is the appropriate 'Approach'? Therefore, one objective of this study was to tackle the mathematical issue broadly from different aspects and approaches. The approaches were divided into three embryonic categories: cell division, neural tube growth and neural plate growth. We concluded that the neural plate growth approach provides a suitable platform for simulation of brain formation/neurodevelopment compared to cell division and neural tube growth. We devised a novel equation and designed algorithms that include geometrical and topological algorithms that could fit most of the necessary elements of the neurodevelopmental process during the embryonic period. Hence, the proposed equations and defined mathematical structure would be a platform to generate an artificial neural network that autonomously grows and develops.

Corpora amylacea negatively correlate with hippocampal tau pathology in Alzheimer's disease.

Introduction: Severity and distribution of aggregated tau and neurofibrillary tangles (NFT) are strongly correlated with the clinical presentation of Alzheimer's disease (AD). Clearance of aggregated tau could decrease the rate of NFT formation and delay AD onset. Recent studies implicate corpora amylacea (CA) as a regulator of onset or accumulation of tau pathology. Normally, CA clear brain waste products by amassing cellular debris, which are then extruded into the cerebrospinal fluid to be phagocytosed. The proper functioning of CA may slow progression of AD-associated NFT pathology, and this relationship may be influenced by amount and distribution of phospho-tau (pTau) produced, age, sex, and genetic risk. Objective: The goal of this study was to determine if CA size and number are associated with hippocampal location and local pTau severity while accounting for variations in age, sex, and genetic risk. Methods: Postmortem brain hippocampal tissue sections from 40 AD and 38 unaffected donors were immunohistochemically stained with AT8 (pTau) and counter stained with periodic acid Schiff (PAS). Stained sections of the CA1 and CA3 regions of the hippocampus were analyzed. The percent area occupied (%AO) of CA, pTau, and NFT was calculated. Pairwise comparisons and regression modeling were used to analyze the influence of age, pTau %AO, and genetic risk on %AO by CA in each region, separately in donors with AD and unaffected donors. Results: CA %AO was significantly higher in the CA3 region compared to CA1 in both groups. A significant negative correlation of CA %AO with both pTau %AO and neurofibrillary tangle %AO in the CA3 region of AD brain donors was found. Regression analysis in the CA3 region revealed a significant negative association between CA with both pTau and age. Conclusion: We found an increase of CA in the CA3 region, compared to CA1 region, in AD and unaffected donors. This may suggest that the CA3 region is a hub for waste removal. Additionally, the negative correlation between %AO by CA and NFT in the CA3 region of the hippocampus in donors with AD suggests CA could play a role in AD pathologic progression by influencing tau clearance.

Association of region-specific hippocampal reduction of neurogranin with inflammasome proteins in post mortem brains of Alzheimer's disease.

INTRODUCTION: Neurogranin (Ng) is considered a biomarker for synaptic dysfunction in Alzheimer's disease (AD). In contrast, the inflammasome complex has been shown to exacerbate AD pathology. METHODS: We investigated the protein expression, morphological differences of Ng, and correlated Ng to hyperphosphorylated tau in the post mortem brains of 17 AD cases and 17 age- and sex-matched controls. In addition, we correlated the Ng expression with two different epitopes of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC). RESULTS: We show a reduction of Ng immunopositive neurons and morphological differences in AD compared to controls. Ng immunostaining was negatively correlated with neurofibrillary tangles, humanized anti-ASC (IC100) positive neurons and anti-ASC positive microglia, in AD. DISCUSSION: The finding of a negative correlation between Ng and ASC speck protein expression in post mortem brains of AD suggests that the activation of inflammasome/ASC speck pathway may play an important role in synaptic degeneration in AD. Highlights: We show the role that neurogranin plays on post-synaptic signaling in specific hippocampal regions.We demonstrate that there could be clinical implications of using neurogranin as a biomarker for dementia.We describe the loss of plasticity and neuronal scaffolding proteins in the present of AD pathology.We show the response of neuroinflammation when tau proteins phosphorylate in hippocampal neurons.We show that there is a potential therapeutic target for the inflammasome, and future studies may show that IC100, a humanized monoclonal antibody directed against ASC, may slow the progression of neurodegeneration.

TDP-43 and Alzheimer's Disease Pathology in the Brain of a Harbor Porpoise Exposed to the Cyanobacterial Toxin BMAA.

Cetaceans are well-regarded as sentinels for toxin exposure. Emerging studies suggest that cetaceans can also develop neuropathological changes associated with neurodegenerative disease. The occurrence of neuropathology makes cetaceans an ideal species for examining the impact of marine toxins on the brain across the lifespan. Here, we describe TAR DNA-binding protein 43 (TDP-43) proteinopathy and Alzheimer's disease (AD) neuropathological changes in a beached harbor porpoise (Phocoena phocoena) that was exposed to a toxin produced by cyanobacteria called ß-N-methylamino-L-alanine (BMAA). We found pathogenic TDP-43 cytoplasmic inclusions in neurons throughout the cerebral cortex, midbrain and brainstem. P62/sequestosome-1, responsible for the autophagy of misfolded proteins, was observed in the amygdala, hippocampus and frontal cortex. Genes implicated in AD and TDP-43 neuropathology such as APP and TARDBP were expressed in the brain. AD neuropathological changes such as amyloid-ß plaques, neurofibrillary tangles, granulovacuolar degeneration and Hirano bodies were present in the hippocampus. These findings further support the development of progressive neurodegenerative disease in cetaceans and a potential causative link to cyanobacterial toxins. Climate change, nutrient pollution and industrial waste are increasing the frequency of harmful cyanobacterial blooms. Cyanotoxins like BMAA that are associated with neurodegenerative disease pose an increasing public health risk.

Identification of inflammasome signaling proteins in neurons and microglia in early and intermediate stages of Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disease that destroys memory and cognitive function. Inflammasome activation has been suggested to play a critical role in the neuroinflammatory response in AD progression, but the cell-type expression of inflammasome proteins in the brain has not been fully characterized. In this study, we used samples from the hippocampus formation, the subiculum, and the entorhinal cortex brain from 17 donors with low-level AD pathology and 17 intermediate AD donors to assess the expression of inflammasome proteins. We performed analysis of hippocampal thickness, ß-amyloid plaques, and hyperphosphorylated tau to ascertain the cellular pathological changes that occur between low and intermediate AD pathology. Next, we determined changes in the cells that express the inflammasome sensor proteins NOD-like receptor proteins (NLRP) 1 and 3, and caspase-1. In addition, we stained section with IC100, a humanized monoclonal antibody directed against the inflammasome adaptor protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and a commercially available anti-ASC antibody. Our results indicate that hippocampal cortical thickness did not significantly change between low and intermediate AD pathology, but there was an increase in pTau and ß-amyloid clusters in intermediate AD cases. NLRP3 was identified mainly in microglial populations, whereas NLRP1 was seen in neuronal cytoplasmic regions. There was a significant increase of ASC in neurons labeled by IC100, whereas microglia in the hippocampus and subiculum were labeled with the commercial anti-ASC antibody. Caspase-1 was present in the parenchyma in the CA regions where amyloid and pTau were identified. Together, our results indicate increased inflammasome protein expression in the early pathological stages of AD, that IC100 identifies neurons in early stages of AD and that ASC expression correlates with Aß and pTau in postmortem AD brains.

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.

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.

White matter injury but not germinal matrix hemorrhage induces elevated osteopontin expression in human preterm brains.

Osteopontin (OPN) is a matricellular protein that mediates various physiological functions and is implicated in neuroinflammation, myelination, and perinatal brain injury. However, its expression in association with brain injury in preterm infants is unexplored. Here we examined the expression of OPN in postmortem brains of preterm infants and explored how this expression is affected in brain injury. We analyzed brain sections from cases with white matter injury (WMI) and cases with germinal matrix hemorrhage (GMH) and compared them to control cases having no brain injury. WMI cases displayed moderate to severe tissue injury in the periventricular and deep white matter that was accompanied by an increase of microglia with amoeboid morphology. Apart from visible hemorrhage in the germinal matrix, GMH cases displayed diffuse white matter injury in the periventricular and deep white matter. In non-injured preterm brains, OPN was expressed at low levels in microglia, astrocytes, and oligodendrocytes. OPN expression was significantly increased in regions with white matter injury in both WMI cases and GMH cases. The main cellular source of OPN in white matter injury areas was amoeboid microglia, although a significant increase was also observed in astrocytes in WMI cases. OPN was not expressed in the germinal matrix of any case, regardless of whether there was hemorrhage. In conclusion, preterm brain injury induces elevated OPN expression in microglia and astrocytes, and this increase is found in sites closely related to injury in the white matter regions but not with the hemorrhage site in the germinal matrix. Thus, it appears that OPN takes part in the inflammatory process in white matter injury in preterm infants, and these findings facilitate our understanding of OPN's role under both physiological and pathological conditions in the human brain that may lead to greater elucidation of disease mechanisms and potentially better treatment strategies.

Association of neurogranin gene expression with Alzheimer's disease pathology in the perirhinal cortex.

INTRODUCTION: Synaptic damage is a key pathology of Alzheimer's disease (AD). The mechanism underlying synaptic vulnerability in AD remains elusive. METHODS: Using a large-scale transcriptomic dataset, we analyzed the neurogranin-centered integrative gene network and assessed the correlation of neurogranin (NRGN) gene expression with AD pathology in post mortem brains. We studied the association of NRGN expression with Clinical Dementia Rating (CDR) and neuropathological diagnosis of AD. RESULTS: We find that the genes positively correlated with NRGN expression in AD are involved in synaptic transmission and cation channel pathways. NRGN expression is correlated with amyloid and tau pathology in the perirhinal cortex of post mortem brains. NRGN expression is associated with the diagnosis of AD and correlated with CDR. DISCUSSION: Transcriptional regulation of the gene encoding for synaptic protein is involved in selective synaptic damage in AD. Identifying the genes associated with synaptic damage pathways in AD may provide targets for intervention.

Assessment of radial glia in the frontal lobe of fetuses with Down syndrome.

Down syndrome (DS) occurs with triplication of human chromosome 21 and is associated with deviations in cortical development evidenced by simplified gyral appearance and reduced cortical surface area. Radial glia are neuronal and glial progenitors that also create a scaffolding structure essential for migrating neurons to reach cortical targets and therefore play a critical role in cortical development. The aim of this study was to characterise radial glial expression pattern and morphology in the frontal lobe of the developing human fetal brain with DS and age-matched controls. Secondly, we investigated whether microstructural information from in vivo magnetic resonance imaging (MRI) could reflect histological findings from human brain tissue samples. Immunohistochemistry was performed on paraffin-embedded human post-mortem brain tissue from nine fetuses and neonates with DS (15-39 gestational weeks (GW)) and nine euploid age-matched brains (18-39 GW). Radial glia markers CRYAB, HOPX, SOX2, GFAP and Vimentin were assessed in the Ventricular Zone, Subventricular Zone and Intermediate Zone. In vivo diffusion MRI was used to assess microstructure in these regions in one DS (21 GW) and one control (22 GW) fetal brain. We found a significant reduction in radial glial progenitor SOX2 and subtle deviations in radial glia expression (GFAP and Vimentin) prior to 24 GW in DS. In vivo, fetal MRI demonstrates underlying radial projections consistent with immunohistopathology. Radial glial alterations may contribute to the subsequent simplified gyral patterns and decreased cortical volumes observed in the DS brain. Recent advances in fetal MRI acquisition and analysis could provide non-invasive imaging-based biomarkers of early developmental deviations.

Interneuron Development Is Disrupted in Preterm Brains With Diffuse White Matter Injury: Observations in Mouse and Human.

Preterm brain injury, occurring in approximately 30% of infants born <32 weeks gestational age, is associated with an increased risk of neurodevelopmental disorders, such as autism spectrum disorder (ASD) and attention deficit hyperactivity disorder (ADHD). The mechanism of gray matter injury in preterm born children is unclear and likely to be multifactorial; however, inflammation, a high predictor of poor outcome in preterm infants, has been associated with disrupted interneuron maturation in a number of animal models. Interneurons are important for regulating normal brain development, and disruption in interneuron development, and the downstream effects of this, has been implicated in the etiology of neurodevelopmental disorders. Here, we utilize postmortem tissue from human preterm cases with or without diffuse white matter injury (WMI; PMA range: 23+2 to 28+1 for non-WMI group, 26+6 to 30+0 for WMI group, p = 0.002) and a model of inflammation-induced preterm diffuse white matter injury (i.p. IL-1ß, b.d., 10 µg/kg/injection in male CD1 mice from P1-5). Data from human preterm infants show deficits in interneuron numbers in the cortex and delayed growth of neuronal arbors at this early stage of development. In the mouse, significant reduction in the number of parvalbumin-positive interneurons was observed from postnatal day (P) 10. This decrease in parvalbumin neuron number was largely rectified by P40, though there was a significantly smaller number of parvalbumin positive cells associated with perineuronal nets in the upper cortical layers. Together, these data suggest that inflammation in the preterm brain may be a contributor to injury of specific interneuron in the cortical gray matter. This may represent a potential target for postnatal therapy to reduce the incidence and/or severity of neurodevelopmental disorders in preterm infants.

Post-mortem Characterisation of a Case With an ACTG1 Variant, Agenesis of the Corpus Callosum and Neuronal Heterotopia.

Cytoplasmic Actin Gamma 1 (ACTG1) gene variant are autosomal dominant and can cause CNS anomalies (Baraitser Winter Malformation Syndrome; BWMS). ACTG1 anomalies in offspring include agenesis of the corpus callosum (ACC) and neuronal heterotopia which are ectopic nodules of nerve cells that failed to migrate appropriately. Subcortical and periventricular neuronal heterotopia have been described previously in association with ACC. In this case report, we investigated a neonatal brain with an ACTG1 gene variant and a phenotype of ACC, and neuronal heterotopia (ACC-H) which was diagnosed on antenatal MR imaging and was consistent with band heterotopia seen on post-mortem brain images. Histologically clusters of neurons were seen in both the subcortical and periventricular white matter (PVWM) brain region that coincided with impaired abnormalities in glial formation. Immunohistochemistry was performed on paraffin-embedded brain tissue blocks from this case with ACTG1 variant and an age-matched control. Using tissue sections from the frontal lobe, we examined the distribution of neuronal cells (HuC/HuD, calretinin, and parvalbumin), growth cone (drebrin), and synaptic proteins (synaptophysin and SNAP-25). Additionally, we investigated how the ACTG1 variant altered astroglia (nestin, GFAP, vimentin); oligodendroglia (OLIG2) and microglia (Iba-1) in the corpus callosum, cortex, caudal ganglionic eminence, and PVWM. As predicted in the ACTG1 variant case, we found a lack of midline radial glia and glutamatergic fibers. We also found disturbances in the cortical region, in glial cells and a lack of extracellular matrix components in the ACTG1 variant. The caudal ganglionic eminence and the PVWM regions in the ACTG1 variant lacked several cellular components that were identified in a control case. Within the neuronal heterotopia, we found evidence of glutamatergic and GABAergic neurons with apparent synaptic connections. The data presented from this case study with BWMS with variants in the ACTG1 gene provides insight as to the composition of neuronal heterotopia, and how disturbances of important migratory signals may dramatically affect ongoing brain development.

Bestrophin-3 Expression in a Subpopulation of Astrocytes in the Neonatal Brain After Hypoxic-Ischemic Injury.

Bestrophin-3, a potential candidate for a calcium-activated chloride channel, recently was suggested to have cell-protective functions. We studied the expression and alternative splicing of bestrophin-3 in neonatal mouse brain and after hypoxic-ischemic (HI) injury and in human neonatal brain samples. HI brain injury was induced in 9-day old mice by unilateral permanent common carotid artery occlusion in combination with exposure to 10% oxygen for 50 min. Endoplasmic reticulum stress was induced by thapsigargin treatment in primary culture of mouse brain astrocytes. We also investigated expression of bestrophin-3 protein in a sample of human neonatal brain tissue. Bestrophin-3 protein expression was detected with immunohistochemical methods and western blot; mRNA expression and splicing were analyzed by RT-PCR. HI induced a brain tissue infarct and a pronounced increase in the endoplasmic reticulum-associated marker CHOP. Three days after HI a population of astrocytes co-expressed bestrophin-3 and nestin in a penumbra-like area of the injured hemisphere. However, total levels of Bestrophin-3 protein in mouse cortex were reduced after injury. Mouse astrocytes in primary culture also expressed bestrophin-3 protein, the amount of which was reduced by endoplasmic reticulum stress. Bestrophin-3 protein was detected in astrocytes in the hippocampal region of the human neonatal brain which had patchy white matter gliosis and neuronal loss in the Sommer's sector of the Ammon's horn (CA1). Analysis of bestrophin-3 mRNA in mouse brain with and without injury showed the presence of two truncated spliced variants, but no full-length mRNA. Total amount of bestrophin-3 mRNA increased after HI, but showed only minor injury-related change. However, the splice variants of bestrophin-3 mRNA were differentially regulated after HI depending on the presence of tissue injury. Our results show that bestrophin-3 is expressed in neonatal mouse brain after injury and in the human neonatal brain with pathology. In mouse brain bestrophin-3 protein is upregulated in a specific astrocyte population after injury and is co-expressed with nestin. Splice variants of bestrophin-3 mRNA respond differently to HI, which might indicate their different roles in tissue injury.

TWEAK Receptor Deficiency Has Opposite Effects on Female and Male Mice Subjected to Neonatal Hypoxia-Ischemia.

Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) is a multifunctional cytokine member of the TNF family. TWEAK binds to its only known receptor, Fn14, enabling it to activate downstream signaling processes in response to tissue injury. The aim of this study was to investigate the role of TWEAK signaling in neonatal hypoxia-ischemia (HI). We found that after neonatal HI, both TWEAK and Fn14 expression were increased to a greater extent in male compared with female mice. To assess the role of TWEAK signaling after HI, the size of the injury was measured in neonatal mice genetically deficient in Fn14 and compared with their wild-type and heterozygote littermates. A significant sex difference in the Fn14 knockout (KO) animals was observed. Fn14 gene KO was beneficial in females; conversely, reducing Fn14 expression exacerbated the brain injury in male mice. Our findings indicate that the TWEAK/Fn14 pathway is critical for development of hypoxic-ischemic brain injury in immature animals. However, as the responses are different in males and females, clinical implementation depends on development of sex-specific therapies.

Lymphocytes Contribute to the Pathophysiology of Neonatal Brain Injury.

BACKGROUND: Periventricular leukomalacia (PVL) is the most common form of preterm brain injury affecting the cerebral white matter. This type of injury involves a multiphase process and is induced by many factors, including hypoxia-ischemia (HI) and infection. Previous studies have suggested that lymphocytes play a significant role in the pathogenesis of brain injury, and the aim of this study was to determine the contribution of lymphocyte subsets to preterm brain injury. METHODS: Immunohistochemistry on brain sections from neonatal mice was performed to evaluate the extent of brain injury in wild-type and T cell and B cell-deficient neonatal mice (Rag1-/- mice) using a mouse model of HI-induced preterm brain injury. Flow cytometry was performed to determine the presence of different types of immune cells in mouse brains following HI. In addition, immunostaining for CD3 T cells and CD20 B cells was performed on postmortem preterm human infant brains with PVL. RESULTS: Mature lymphocyte-deficient Rag1-/- mice showed protection from white matter loss compared to wild type mice as indicated by myelin basic protein immunostaining of mouse brains. CD3+ T cells and CD20+ B cells were observed in the postmortem preterm infant brains with PVL. Flow cytometry analysis of mouse brains after HI-induced injury showed increased frequency of CD3+ T, αßT and B cells at 7 days after HI in the ipsilateral (injured) hemisphere compared to the contralateral (control, uninjured) hemisphere. CONCLUSION: Lymphocytes were found in the injured brain after injury in both mice and humans, and lack of mature lymphocytes protected neonatal mice from HI-induced brain white matter injury. This finding provides insight into the pathology of perinatal brain injury and suggests new avenues for the development of therapeutic strategies.

γδ T Cells Contribute to Injury in the Developing Brain.

Brain injury in premature infants, especially periventricular leukomalacia, is an important cause of neurologic disabilities. Inflammation contributes to perinatal brain injury development, but the essential mediators that lead to early-life brain injury remain largely unknown. Neonates have reduced capacity for mounting conventional αßT-cell responses. However, γδT cells are already functionally competent during early development and are important in early-life immunity. We investigated the potential contribution of γδT cells to preterm brain injury using postmortem brains from human preterm infants with periventricular leukomalacia and two animal models of preterm brain injury-the hypoxic-ischemic mouse model and a fetal sheep asphyxia model. Large numbers of γδT cells were observed in the brains of mice, sheep, and postmortem preterm infants after injury, and depletion of γδT cells provided protection in the mouse model. The common γδT-cell-associated cytokines interferon-γ and IL-17A were not detectable in the brain. Although there were increased mRNA levels of Il17f and Il22 in the mouse brains after injury, neither IL-17F nor IL-22 cytokines contributed to preterm brain injury. These findings highlight unique features of injury in the developing brain, where, unlike injury in the mature brain, γδT cells function as initiators of injury independently of common γδT-cell-associated cytokines. This finding will help to identify therapeutic targets for preventing or treating preterm infants with brain injury.

γδT cells but not αßT cells contribute to sepsis-induced white matter injury and motor abnormalities in mice.

BACKGROUND: Infection and sepsis are associated with brain white matter injury in preterm infants and the subsequent development of cerebral palsy. METHODS: In the present study, we used a neonatal mouse sepsis-induced white matter injury model to determine the contribution of different T cell subsets (αßT cells and γδT cells) to white matter injury and consequent behavioral changes. C57BL/6J wild-type (WT), T cell receptor (TCR) δ-deficient (Tcrd -/-, lacking γδT cells), and TCRα-deficient (Tcra -/-, lacking αßT cells) mice were administered with lipopolysaccharide (LPS) at postnatal day (PND) 2. Brain myelination was examined at PNDs 12, 26, and 60. Motor function and anxiety-like behavior were evaluated at PND 26 or 30 using DigiGait analysis and an elevated plus maze. RESULTS: White matter development was normal in Tcrd -/- and Tcrα -/- compared to WT mice. LPS exposure induced reductions in white matter tissue volume in WT and Tcrα -/- mice, but not in the Tcrd -/- mice, compared with the saline-treated groups. Neither LPS administration nor the T cell deficiency affected anxiety behavior in these mice as determined with the elevated plus maze. DigiGait analysis revealed motor function deficiency after LPS-induced sepsis in both WT and Tcrα -/- mice, but no such effect was observed in Tcrd -/- mice. CONCLUSIONS: Our results suggest that γδT cells but not αßT cells contribute to sepsis-induced white matter injury and subsequent motor function abnormalities in early life. Modulating the activity of γδT cells in the early stages of preterm white matter injury might represent a novel therapeutic strategy for the treatment of perinatal brain injury.

Effect of Trp53 gene deficiency on brain injury after neonatal hypoxia-ischemia.

Hypoxia-ischemia (HI) can result in permanent life-long injuries such as motor and cognitive deficits. In response to cellular stressors such as hypoxia, tumor suppressor protein p53 is activated, potently initiating apoptosis and promoting Bax-dependent mitochondrial outer membrane permeabilization. The aim of this study was to investigate the effect of Trp53 genetic inhibition on injury development in the immature brain following HI. HI (50 min or 60 min) was induced at postnatal day 9 (PND9) in Trp53 heterozygote (het) and wild type (WT) mice. Utilizing Cre-LoxP technology, CaMK2α-Cre mice were bred with Trp53-Lox mice, resulting in knockdown of Trp53 in CaMK2α neurons. HI was induced at PND12 (50 min) and PND28 (40 min). Extent of brain injury was assessed 7 days following HI. Following 50 min HI at PND9, Trp53 het mice showed protection in the posterior hippocampus and thalamus. No difference was seen between WT or Trp53 het mice following a severe, 60 min HI. Cre-Lox mice that were subjected to HI at PND12 showed no difference in injury, however we determined that neuronal specific CaMK2α-Cre recombinase activity was strongly expressed by PND28. Concomitantly, Trp53 was reduced at 6 weeks of age in KO-Lox Trp53 mice. Cre-Lox mice subjected to HI at PND28 showed no significant difference in brain injury. These data suggest that p53 has a limited contribution to the development of injury in the immature/juvenile brain following HI. Further studies are required to determine the effect of p53 on downstream targets.

Role of microglia in a mouse model of paediatric traumatic brain injury.

The cognitive and behavioural deficits caused by traumatic brain injury (TBI) to the immature brain are more severe and persistent than TBI in the mature brain. Understanding this developmental sensitivity is critical as children under four years of age sustain TBI more frequently than any other age group. Microglia (MG), resident immune cells of the brain that mediate neuroinflammation, are activated following TBI in the immature brain. However, the type and temporal profile of this activation and the consequences of altering it are still largely unknown. In a mouse model of closed head weight drop paediatric brain trauma, we characterized i) the temporal course of total cortical neuroinflammation and the phenotype of ex vivo isolated CD11B-positive microglia/macrophage (MG/MΦ) using a battery of 32 markers, and ii) neuropathological outcome 1 and 5days post-injury. We also assessed the effects of targeting MG/MΦ activation directly, using minocycline a prototypical microglial activation antagonist, on these processes and outcome. TBI induced a moderate increase in both pro- and anti-inflammatory cytokines/chemokines in the ipsilateral hemisphere. Isolated cortical MG/MΦ expressed increased levels of markers of endogenous reparatory/regenerative and immunomodulatory phenotypes compared with shams. Blocking MG/MΦ activation with minocycline at the time of injury and 1 and 2days post-injury had only transient protective effects, reducing ventricular dilatation and cell death 1day post-injury but having no effect on injury severity at 5days. This study demonstrates that, unlike in adults, the role of MG/MΦ in injury mechanisms following TBI in the immature brain may not be negative. An improved understanding of MG/MΦ function in paediatric TBI could support translational efforts to design therapeutic interventions.

Does Caspase-6 Have a Role in Perinatal Brain Injury?

Apoptotic mechanisms are centre stage for the development of injury in the immature brain, and caspases have been shown to play a pivotal role during brain development and in response to injury. The inhibition of caspases using broad-spectrum agents such as Q-VD-OPh is neuroprotective in the immature brain. Caspase-6, an effector caspase, has been widely researched in neurodevelopmental disorders and found to be important following adult stroke, but its function in the neonatal brain has yet to be detailed. Furthermore, caspases may be important in microglial activation; microglia are required for optimal brain development and following injury, and their close involvement during neuronal cell death suggests that apoptotic cues such as caspase activation may be important in microglial activation. Therefore, in this study we aimed to investigate the possible apoptotic and non-apoptotic functions caspase-6 may have in the immature brain in response to hypoxia-ischaemia. We examined whether caspases are involved in microglial activation. We assessed cleaved caspase-6 expression following hypoxia-ischaemia and conducted primary microglial cultures to assess whether the broad-spectrum inhibitor Q-VD-OPh or caspase-6 gene deletion affected lipopolysaccharide (LPS)-mediated microglial activation and phenotype. We observed cleaved caspase-6 expression to be low but present in the cell body and cell processes in both a human case of white matter injury and 72 h following hypoxia-ischaemia in the rat. Gene deletion of caspase-6 did not affect the outcome of brain injury following mild (50 min) or severe (60 min) hypoxia-ischaemia. Interestingly, we did note that cleaved caspase-6 was co-localised with microglia that were not of apoptotic morphology. We observed that mRNA of a number of caspases was modulated by low-dose LPS stimulation of primary microglia. Q-VD-OPh treatment and caspase-6 gene deletion did not affect microglial activation but modified slightly the M2b phenotype response by changing the time course of SOCS3 expression after LPS administration. Our results suggest that the impact of active caspase-6 in the developing brain is subtle, and we believe there are predominantly other caspases (caspase-2, -3, -8, -9) that are essential for the cell death processes in the immature brain.