Frequencies and phenotypes of immune cells differ between neonates and adults in association with age-specific immune responses. Lymph nodes (LN) are critical tissue sites to quantify and define these differences. Advances in flow cytometry have enabled more multifaceted measurements of complex immune responses. Tissue processing can affect the immune cells under investigation that influence key findings. To understand the impact on immune cells in the LN after processing for single-cell suspension, we compared three dissociation protocols: enzymatic digestion, mechanical dissociation with DNase I treatment, and mechanical dissociation with density gradient separation. We analyzed cell yields, viability, phenotypic and maturation markers of immune cells from the lung-draining LN of neonatal and adult mice two days after intranasal respiratory syncytial virus (RSV) infection. While viability was consistent across age groups, the protocols influenced the yield of subsets defined by important phenotypic and activation markers. Moreover, enzymatic digestion did not show higher overall yields of conventional dendritic cells and macrophages from the LN. Together, our findings show that the three dissociation protocols have similar impacts on the number and viability of cells isolated from the neonatal and adult LN. However, enzymatic digestion impacts the mean fluorescence intensity of key lineage and activation markers that may influence experimental findings.

Perinatal and neonatal ischemic stroke is a significant cause of cognitive and behavioral impairments. Further research is needed to support models of neonatal ischemic stroke and advance our understanding of the mechanisms of infarction formation following such strokes. We used two different levels of photothrombotic stroke (PTS) models to assess stroke outcomes in neonatal mice. We measured brain damage, dynamic changes in glial cells, and neuronal expression at various time points within two weeks following ischemic injury. Our results from 2,3,5-Triphenyltetrazolium chloride (TTC) staining and immunofluorescence staining showed that in the severe group, a dense border of astrocytes and microglia was observed within 3 days post infarct. This ultimately resulted in the formation of a permanent cortical cavity, accompanied by neuronal loss in the surrounding tissues. In the mild group, a relatively sparse arrangement of glial borders was observed 7 days post infarct. This was accompanied by intact cortical tissue and the restoration of viability in the brain tissue beyond the glial boundary. Additionally, neonatal ischemic injury leads to the altered expression of key molecules such as Aldh1L1 and Olig2 in immature astrocytes. In conclusion, we demonstrated the dynamic changes in glial cells and neuronal expression following different degrees of ischemic injury in a mouse model of PTS. These findings provide new insights for studying the cellular and molecular mechanisms underlying neuroprotection and neural regeneration after neonatal ischemic injury.

Neonatal spinal cord injury (SCI) shows better functional outcomes than adult SCI. Although the regenerative capability in the neonatal spinal cord may have cues in the treatment of adult SCI, the mechanism underlying neonatal spinal cord regeneration after SCI is unclear. We previously reported age-dependent variation in the pathogenesis of inflammation after SCI. Therefore, we explored differences in the pathogenesis of inflammation after SCI between neonatal and adult mice and their effect on axon regeneration and functional outcome. We established two-day-old spinal cord crush mice as a model of neonatal SCI. Immunohistochemistry of the spinal cord revealed that the nuclear translocation of NF-κB, which promotes the expression of chemokines, was significantly lower in the astrocytes of neonates than in those of adults. Flow cytometry revealed that neonatal astrocytes secrete low levels of chemokines to recruit circulating neutrophils (e.g., Cxcl1 and Cxcl2) after SCI in comparison with adults. We also found that the expression of a chemokine receptor (CXCR2) and an adhesion molecule (β2 integrin) quantified by flow cytometry was lower in neonatal circulating neutrophils than in adult neutrophils. Strikingly, these neonate-specific cellular properties seemed to be associated with no neutrophil infiltration into the injured spinal cord, followed by significantly lower expression of inflammatory cytokines (Il-1β, Il-6 and TNF-α) after SCI in the spinal cords of neonates than in those of adults. At the same time, significantly fewer apoptotic neurons and greater axonal regeneration were observed in neonates in comparison with adults, which led to a marked recovery of locomotor function. This neonate-specific mechanism of inflammation regulation may have potential therapeutic applications in controlling inflammation after adult SCI.

OBJECTIVE: To study the protective effect of melatonin (Mel) against oxygen-induced retinopathy (OIR) in neonatal mice and the role of the HMGB1/NF-κB/NLRP3 axis.
METHODS: Neonatal C57BL/6J mice, aged 7 days, were randomly divided into a control group, a model group (OIR group), and a Mel treatment group (OIR+Mel group), with 9 mice in each group. The hyperoxia induction method was used to establish a model of OIR. Hematoxylin and eosin staining and retinal flat-mount preparation were used to observe retinal structure and neovascularization. Immunofluorescent staining was used to measure the expression of proteins and inflammatory factors associated with the HMGB1/NF-κB/NLRP3 axis and lymphocyte antigen 6G. Colorimetry was used to measure the activity of myeloperoxidase.
RESULTS: The OIR group had destruction of retinal structure with a large perfusion-free area and neovascularization, while the OIR+Mel group had improvement in destruction of retinal structure with reductions in neovascularization and perfusion-free area. Compared with the control group, the OIR group had significant increases in the expression of proteins and inflammatory factors associated with the HMGB1/NF-κB/NLRP3 axis, the expression of lymphocyte antigen 6G, and the activity of myeloperoxidase (P<0.05). Compared with the OIR group, the OIR+Mel group had significant reductions in the above indices (P<0.05). Compared with the control group, the OIR group had significant reductions in the expression of melatonin receptors in the retina (P<0.05). Compared with the OIR group, the OIR+Mel group had significant increases in the expression of melatonin receptors (P<0.05).
CONCLUSIONS: Mel can alleviate OIR-induced retinal damage in neonatal mice by inhibiting the HMGB1/NF-κB/NLRP3 axis and may exert an effect through the melatonin receptor pathway.
目的: 观察褪黑素（melatonin，Mel）对新生小鼠氧诱导视网膜病变（oxygen-induced retinopathy，OIR）的保护作用，并探讨HMGB1/NF-κB/NLRP3轴在其中的作用。方法: 7日龄C57BL/6J新生小鼠随机分为对照组、模型组（OIR组）及Mel处理组（OIR+Mel组），各组n=9。采用高氧诱导法制备OIR模型。苏木精-伊红染色和视网膜铺片法检测视网膜结构和新生血管；免疫荧光染色法检测HMGB1/NF-κB/NLRP3轴相关蛋白和炎性因子及淋巴细胞抗原6G表达；比色法检测髓过氧化物酶活性。结果: OIR组视网膜结构被破坏，出现大片无灌注区和新生血管，OIR+Mel组可见破坏的视网膜结构改善，新生血管和无灌注区减少。与对照组相比，OIR组HMGB1/NF-κB/NLRP3轴相关蛋白和炎性因子表达升高（均P<0.05），淋巴细胞抗原6G表达和髓过氧化物酶活性升高（均P<0.05）；Mel处理后，上述各指标降低（均P<0.05）。与对照组相比，OIR组视网膜中褪黑素受体表达降低；Mel处理后，褪黑素受体表达较OIR组升高（均P<0.05）。结论: Mel可能通过抑制HMGB1/NF-κB/NLRP3轴减轻OIR新生小鼠视网膜损伤，且可能通过褪黑素受体途径发挥作用。.

The limited information about how descending inputs from the brain and sensory inputs from the periphery use spinal cord interneurons (INs) is a major barrier to understanding how these inputs may contribute to motor functions under normal and pathologic conditions. Commissural interneurons (CINs) are a heterogeneous population of spinal INs that has been implicated in crossed motor responses and bilateral motor coordination (ability to use the right and left side of the body in a coordinated manner) and, therefore, are likely involved in many types of movement (e.g., dynamic posture stabilization, jumping, kicking, walking). In this study, we incorporate mouse genetics, anatomy, electrophysiology, and single-cell calcium imaging to investigate how a subset of CINs, those with descending axons called dCINs, are recruited by descending reticulospinal and segmental sensory signals independently and in combination. We focus on two groups of dCINs set apart by their principal neurotransmitter (glutamate and GABA) and identified as VGluT2+ dCINs and GAD2+ dCINs. We show that VGluT2+ and GAD2+ dCINs are both extensively recruited by reticulospinal and sensory input alone but that VGluT2+ and GAD2+ dCINs integrate these inputs differently. Critically, we find that when recruitment depends on the combined action of reticulospinal and sensory inputs (subthreshold inputs), VGluT2+ dCINs, but not GAD2+ dCINs, are recruited. This difference in the integrative capacity of VGluT2+ and GAD2+ dCINs represents a circuit mechanism that the reticulospinal and segmental sensory systems may avail themselves of to regulate motor behaviors both normally and after injury.SIGNIFICANCE STATEMENT The way supraspinal and peripheral sensory inputs use spinal cord interneurons is fundamental to defining how motor functions are supported both in health and disease. This study, which focuses on dCINs, a heterogeneous population of spinal interneurons critical for crossed motor responses and bilateral motor coordination, shows that both glutamatergic (excitatory) and GABAergic (inhibitory) dCINs can be recruited by supraspinal (reticulospinal) or peripheral sensory inputs. Additionally, the study demonstrates that in conditions where the recruitment of dCINs depends on the combined action of reticulospinal and sensory inputs, only excitatory dCINs are recruited. The study uncovers a circuit mechanism that the reticulospinal and segmental sensory systems may avail themselves of to regulate motor behaviors both normally and after injury.

Neuronal excitability is a critical feature of central nervous system development, playing a fundamental role in the functional maturation of brain regions, including the hippocampus, cerebellum, auditory and visual systems. The present study aimed to determine the mechanism by which hypoxia causes brain dysfunction through perturbation of neuronal excitability in a hypoxic neonatal mouse model. Functional brain development was assessed in humans using the Gesell Development Diagnosis Scale. In mice, gene transcription was evaluated via mRNA sequencing and quantitative PCR; furthermore, patch clamp recordings assessed potassium currents. Clinical observations revealed disrupted functional brain development in 6- and 18-month-old hypoxic neonates, and those born with normal hearing screening unexpectedly exhibited impaired central auditory function at 3 months. In model mice, CA1 pyramidal neurons exhibited reduced spontaneous activity, largely induced by excitatory synaptic input suppression, despite the elevated membrane excitability of hypoxic neurons compared to that of control neurons. In hypoxic neurons, Kcnd3 gene transcription was upregulated, confirming upregulated hippocampal Kv 4.3 expression. A-type potassium currents were enhanced, and Kv 4.3 participated in blocking excitatory presynaptic inputs. Elevated Kv 4.3 activity in pyramidal neurons under hypoxic conditions inhibited excitatory presynaptic inputs and further decreased neuronal excitability, disrupting functional brain development in hypoxic neonates.

Since the first evidence of cardiac regeneration was observed, almost 50 years ago, more studies have highlighted the endogenous regenerative abilities of several models following cardiac injury. In particular, analysis of cardiac regeneration in zebrafish and neonatal mice has uncovered numerous mechanisms involved in the regenerative process. It is now apparent that cardiac regeneration is not simply achieved by inducing cardiomyocytes to proliferate but requires a multifaceted response involving numerous different cell types, signaling pathways and mechanisms which must all work in harmony in order for regeneration to occur. In this review we will endeavor to highlight a variety of processes that have been identifed as being essential for cardiac regeneration.

The proliferative capacity of mammalian cardiomyocytes diminishes shortly after birth. In contrast, adult zebrafish and neonatal mice can regenerate cardiac tissues, highlighting new potential therapeutic avenues. Different factors have been found to promote cardiomyocyte proliferation in zebrafish and neonatal mice; these include maintenance of mononuclear and diploid cardiomyocytes and upregulation of the proto-oncogene c-Myc. The growth factor NRG-1 controls cell proliferation and interacts with the Hippo-Yap pathway to modulate regeneration. Key components of the extracellular matrix such as Agrin are also crucial for cardiac regeneration. Novel therapies explored in this review, include intramyocardial injection of Agrin or zebrafish-ECM and NRG-1 administration. These therapies may induce regeneration in patients and should be further explored.
The heart pumps blood across the body carrying nutrients and oxygen where they are needed. If the heart is damaged (e.g., after a heart attack), it may lose its ability to pump blood, and this can lead to heart failure, where the heart cannot meet the body\'s needs, leaving the affected person tired and breathless. This occurs because the human heart unfortunately has a limited ability to heal and regain function. Current therapies for heart injuries focus on minimizing the problems resulting from the injury but cannot recover damaged heart tissue. Scientists have found that in contrast to adult human hearts, the hearts of baby mice and zebrafish can repair themselves after injuries and recover normal function. This review highlights some important mechanisms that occur in the hearts of baby mice and zebrafish, which may help contribute to their regenerative abilities. These mechanisms involve small messenger chemicals that stimulate heart cells to replicate and reform normal heart tissues. Further research into these pathways may help develop new therapies for damaged human hearts and help them regain function.

Spontaneous electrical activity plays major roles in the development of cortical circuitry. This activity can occur highly localized regions or can propagate over the entire cortex. Both types of activity coexist during early development. To investigate how different forms of spontaneous activity might be temporally segregated, we used wide-field trans-cranial calcium imaging over an entire hemisphere in P1-P8 mouse pups. We found that spontaneous waves of activity that propagate to cover the majority of the cortex (large-scale waves; LSWs) are generated at the end of the first postnatal week, along with several other forms of more localized activity. We further found that LSWs are segregated into sleep cycles. In contrast, cortical activity during wake states is more spatially restricted and the few large-scale forms of activity that occur during wake can be distinguished from LSWs in sleep based on their initiation in the motor cortex and their correlation with body movements. This change in functional cortical circuitry to a state that is permissive for large-scale activity may temporally segregate different forms of activity during critical stages when activity-dependent circuit development occurs over many spatial scales. Our data also suggest that LSWs in early development may be a functional precursor to slow sleep waves in the adult, which play critical roles in memory consolidation and synaptic rescaling.

Enterovirus 71 can cause severe hand, foot, and mouth disease (HFMD) in children. However, little is known about the mechanism of inflammatory disorders caused by EV71 infection and why severe cases are mainly children aged under-three. In current study, using mRNA microarray assay, the differential expression of Placenta-specific 8 (PLAC8) was identified in mice brain. In addition, we found that PLAC8 expression was down-regulated with age in mice lung tissues and human peripheral blood. Then, we further proved that PLAC8 could promote inflammation progress and disturb Th1/Th2/Th17/Treg related cytokines release after EV71 infection using PLAC8 plasmid over-expressed neonatal mouse model. Our data suggest that PLAC8 might play a crucial role in Th cell differentiation and inflammatory damage caused by EV71 infection in infants. Thus, our findings would help understand the causes of severe inflammatory injury in infants during EV71 infection, and provide new insights into the prevention and control of severe HFMD.