Stanislava Pankratova

BACKGROUND: Metabolic and neurovascular disturbances contribute to brain injury after birth asphyxia. We previously showed that plasma feeding attenuated neuropathology after transient umbilical cord occlusion in piglets. This study explored the metabolic and molecular consequences of birth asphyxia and whether plasma feeding preserves metabolic homeostasis and brain barrier integrity. METHODS: Piglets (n=89) delivered by cesarean section on gestational day 113 underwent umbilical cord occlusion (asphyxia [ASP]) or remained nonasphyxiated (control [CON]). ASP and CON piglets were randomly assigned to receive formula reconstituted with either water (vehicle-treated group) or sow plasma (plasma-treated group). Animals were euthanized at 1 hour, 24 hours, or day 4 after insult. Cerebrospinal fluid metabolites were profiled by untargeted liquid chromatography–mass spectrometry metabolomics at 24 hours. Expression of metabolite transporters and structural components of the blood-brain barrier in the caudate nucleus and the blood–cerebrospinal fluid barrier at the choroid plexus was assessed by reverse transcription quantitative polymerase chain reaction and Western blotting. The CSF/plasma Na ⁺ ratio was assessed, and cell-specific AQP4 expression was confirmed with RNAscope. Data were analyzed using linear and linear mixed-effects models. RESULTS: Birth asphyxia dysregulated the CSF metabolic profile, with reductions in multiple metabolites and increases in a smaller subset, consistent with impaired amino acid and lipid metabolism. This was accompanied by reduced expression of substrate transporters, including xCT , LAT1 , ASCT2 , FATP1 , CAV1 , and MCTs . In the caudate nucleus, expression of blood-brain barrier structural proteins and ion transporters, including Na ⁺ /K ⁺ -ATPase, was reduced, whereas the choroid plexus blood-CSF barrier remained largely preserved. Plasma feeding partially restored transporter expression at both brain interfaces and increased AQP4 and ion transporter expression at the blood-brain barrier by day 4. Increased VE-cadherin (vascular endothelial cadherin) and ZO-1 (zonula occludens-1) expression and elevated CSF Na ⁺ concentrations suggested early modulation of the integrity of the 2 brain barriers. CONCLUSIONS: Plasma feeding preserves metabolic homeostasis and supports brain barrier integrity after birth asphyxia, highlighting plasma-derived factors as potential therapeutic targets to reduce neurodevelopmental impairment after perinatal asphyxia.

We present an integrated acoustofluidic plasmapheresis system designed for ultralow blood volume applications, such as neonatal care, enabling in-line sampling and plasma separation from whole blood. The system combines a two-stage acoustophoresis chip with microperistaltic pumps and PDMS-based flow pulsation dampeners to ensure continuous, stable operation. The input whole blood is acoustically separated into a cell-free plasma fraction and a returnable cell fraction, enabling closed-loop operation for neonates who have a circulating blood volume as low as 50 mL. The system achieves a plasma generation rate of 27.5 μL/min with ∼100% cell removal, outperforming previous microfluidic plasma separation approaches in terms of purity, throughput, and minimal sample volume. Plasma quality was validated by quantifying hemolysis and residual cellular content, while system robustness was demonstrated across hematocrit levels up to 50%, which is close to the average upper hematocrit value in neonates. Compared to previous microfluidic techniques, our system achieves the fastest generation of clinical quality undiluted plasma with the lowest required blood volume, making it highly suitable for point-of-care integration in neonatal intensive care units.

Afferent vagal neurons convey gut–brain signals related to the mechanical and chemical sensing of nutrients, with the latter also mediated by gut hormones secreted from enteroendocrine cells. Cell bodies of these neurons are located in the nodose ganglia (NG), with the right NG playing a key role in metabolic regulation. Notably, glucagon-like peptide-1 receptor (GLP1R) neurons primarily innervate the muscle layer of the stomach, distant from glucagon-like peptide-1 (GLP-1)-secreting gut cells. However, the co-expression of gut hormone receptors in these NG neurons remains unclear. Using RNAscope combined with immunohistochemistry, we confirmed GLP1R expression in a large population of NG neurons, with Glp1r, cholecystokinin A receptor (Cckar), and Neuropeptide Y Y2 Receptor (Npy2r) being more highly expressed in the right NG, while neurotensin receptor 1 (Ntsr), G protein-coupled receptor (Gpr65), and 5-hydroxytryptamine receptor 3A (5ht3a) showed equal expressions in the left and right NG. Co-expression analysis demonstrated the following: (i) most Glp1r, Cckar, and Npy2r neurons co-expressed all three receptors; (ii) nearly all Ntsr1- and Gpr65-positive neurons co-expressed both receptors; and (iii) 5ht3a was expressed in subpopulations of all peptide-hormone-receptor-positive neurons. Retrograde labeling demonstrated that the anterior part of the stomach was preferentially innervated by the left NG, while the right NG innervated the posterior part. The entire gastrointestinal (GI) tract, including the distal colon, was strongly innervated by NG neurons. Most importantly, dual retrograde labeling with two distinct tracers identified a population of neurons co-expressing Glp1r, Cckar, and Npy2r that innervated both the stomach and the colon. Thus, neurons co-expressing GLP-1, cholecystokinin (CCK), and peptide YY (PYY) receptors, predominantly found in the right NG, sample chemical, nutrient-induced signals along the entire GI tract and likely integrate these with mechanical signals from the stomach.

Introduction: Preterm infants have increased risk of impaired neurodevelopment to which reduced systemic levels of insulin-like growth factor 1 (IGF-1) in the weeks after birth may play a role. Hence, we hypothesized that postnatal IGF-1 supplementation would improve brain development in preterm pigs, used as a model for preterm infants.Methods: Preterm pigs delivered by cesarean section received recombinant human IGF-1/IGF binding protein-3 complex (rhIGF-1/rhIGFBP-3, 2.25 mg/kg/day) or vehicle from birth to postnatal day 19. Motor function and cognition were assessed by monitoring of in-cage and open field activities, balance beam test, gait parameters, novel object recognition and operant conditioning tests. Collected brains were subject to magnetic resonance imaging (MRI), immunohistochemistry, gene expression analyses and protein synthesis measurements.Results: The IGF-1 treatment increased cerebellar protein synthesis rates (both in vivo and ex vivo). Performance in the balance beam test was improved by IGF-1 but not in other neurofunctional tests. The treatment decreased total and relative caudate nucleus weights, without any effects to total brain weight or grey/white matter volumes. Supplementation with IGF-1 reduced myelination in caudate nucleus, cerebellum, and white matter regions and decreased hilar synapse formation, without effects to oligodendrocyte maturation or neuron differentiation. Gene expression analyses indicated enhanced maturation of the GABAergic system in the caudate nucleus (decreased NKCC1:KCC2 ratio) with limited effects in cerebellum or hippocampus.Conclusion: Supplemental IGF-1 during the first three weeks after preterm birth may support motor function by enhancing GABAergic maturation in the caudate nucleus, despite reduced myelination. Supplemental IGF-1 may support postnatal brain development in preterm infants, but more studies are required to identify optimal treatment regimens for subgroups of very or extremely preterm infants.

Very preterm infants show low levels of insulin-like growth factor-1 (IGF-1), which is associated with postnatal growth restriction and poor neurologic outcomes. It remains unknown whether supplemental IGF-1 may stimulate neurodevelopment in preterm neonates. Using cesarean-delivered preterm pigs as a model of preterm infants, we investigated the effects of supplemental IGF-1 on motor function and on regional and cellular brain development. Pigs were treated with 2.25 mg/kg/d recombinant human IGF-1/IGF binding protein-3 complex from birth until day 5 or 9 before the collection of brain samples for quantitative immunohistochemistry (IHC), RNA sequencing, and quantitative PCR analyses. Brain protein synthesis was measured usingin vivolabeling with [2H5] phenylalanine. We showed that the IGF-1 receptor was widely distributed in the brain and largely coexisted with immature neurons. Region-specific quantification of IHC labeling showed that IGF-1 treatment promoted neuronal differentiation, increased subcortical myelination, and attenuated synaptogenesis in a region-dependent and time-dependent manner. The expression levels of genes involved in neuronal and oligodendrocyte maturation, and angiogenic and transport functions were altered, reflecting enhanced brain maturation in response to IGF-1 treatment. Cerebellar protein synthesis was increased by 19% at day 5 and 14% at day 9 after IGF-1 treatment. Treatment had no effect on Iba1⁺microglia or regional brain weights and did not affect motor development or the expression of genes related to IGF-1 signaling. In conclusion, the data show that supplemental IGF-1 promotes brain maturation in newborn preterm pigs. The results provide further support for IGF-1 supplementation therapy in the early postnatal period in preterm infants.

A challenge in neurology is the lack of efficient brain-penetrable neuroprotectants targeting multiple disease mechanisms. Plasmonic gold nanostars are promising candidates to deliver standard-of-care drugs inside the brain but have not been trialed as carriers for neuroprotectants. Here, we conjugated custom-made peptide dendrimers (termed H3/H6), encompassing motifs of the neurotrophic S100A4-protein, onto star-shaped and spherical gold nanostructures (H3/H6-AuNS/AuNP) and evaluated their potential as neuroprotectants and interaction with neurons. The H3/H6 nanostructures crossed a model blood-brain barrier, bound to plasma membranes, and induced neuritogenesis with the AuNS, showing higher potency/efficacy than the AuNP. The H3-AuNS/NP protected neurons against oxidative stress, the H3-AuNS being more potent, and against Parkinson's or Alzheimer's disease (PD/AD)-related cytotoxicity. Unconjugated S100A4 motifs also decreased amyloid beta-induced neurodegeneration, introducing S100A4 as a player in AD. Using custom-made dendrimers coupled to star-shaped nanoparticles is a promising route to activate multiple neuroprotective pathways and increase drug potency to treat neurodegenerative disorders.

Alzheimer’s disease (AD) is a progressive and irreversible neurodegenerative disease characterized by the accumulation of aggregated amyloid beta (Aβ) and hyperphosphorylated tau along with a slow decline in cognitive functions. Unlike advanced AD, the initial steps of AD pathophysiology have been poorly investigated, partially due to limited availability of animal models focused on the early, plaque-free stages of the disease. The aim of this study was to evaluate the early behavioral, anatomical and molecular alterations in wild-type rats following intracerebroventricular injections of human Aβ oligomers (AβOs). Bioactive human AD and nondemented control brain tissue extracts were characterized using ELISA and proteomics approaches. Following a bilateral infusion, rats underwent behavioral testing, including the elevated plus maze, social recognition test, Morris water maze and Y-maze within 6 weeks postinjection. An analysis of brain structure was performed with manganese-enhanced MRI. Collected brain tissues were analyzed using stereology, immunohistochemistry, ELISA and qPCR. No sensorimotor deficits affecting motor performance on different maze tasks were observed, nor was spatial memory disturbed in AD rats. In contrast, a significant impairment of social memory became evident at 21 days postinjection. This deficit was associated with a significantly decreased volume of the lateral entorhinal cortex and a tendency toward a decrease in the total brain volume. Significant increase of cleaved caspase-3-positive cells, microglial activation and proinflammatory responses accompanied by altered expression of synaptic markers were observed in the hippocampus of AD rats with immunohistochemical and qPCR approaches at 6 weeks postinjection. Our data suggest that the social memory impairment observed in AβO-injected rats might be determined by neuroinflammatory responses and synaptopathy. An infusion of native oligomeric Aβ in the rat brain represents a feasible tool to model early plaque-free events associated with AD.

Infants with neonatal cholestasis are prone to neurodevelopmental deficits, however, the underlying pathogenesis is unclear. Lipid malabsorption and accumulation of potentially neurotoxic molecules in the blood such as bile acids are important yet relatively unexplored pathways. Here, we developed a translational piglet model to understand how the molecular bile acid and lipid composition of the brain is affected by this disease and relates to motor function. Piglets (8-days old) had bile duct ligation or sham surgery and were fed a formula diet for 3 weeks. Alongside sensory-motor deficits observed in bile duct-ligated animals, we found a shift toward a more hydrophilic and conjugated bile acid profile in the brain. Additionally, comprehensive lipidomics of the cerebellum revealed a decrease in total lipids including phosphatidylinositols and phosphatidylserines and increases in lysophospholipid species. This was paralleled by elevated cerebellar expression of genes related to inflammation and tissue damage albeit without significant impact on the brain transcriptome. This study offers new insights into the developing brain's molecular response to neonatal cholestasis indicating that bile acids and lipids may contribute in mediating motor deficits.