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.

Staphylococcus epidermidis Bacteremia Induces Brain Injury in Neonatal Mice via Toll-like Receptor 2-Dependent and -Independent Pathways.

BACKGROUND: Staphylococcus epidermidis causes late-onset sepsis in preterm infants. Staphylococcus epidermidis activates host responses in part via Toll-like receptor 2 (TLR2). Epidemiologic studies link bacteremia and neonatal brain injury, but direct evidence is lacking. METHODS: Wild-type and TLR2-deficient (TLR2-/-) mice were injected intravenously with S. epidermidis at postnatal day 1 prior to measuring plasma and brain cytokine and chemokine levels, bacterial clearance, brain caspase-3 activation, white/gray matter volume, and innate transcriptome. RESULTS: Staphylococcus epidermidis bacteremia spontaneously resolved over 24 hours without detectable bacteria in the cerebrospinal fluid (CSF). TLR2-/- mice demonstrated delayed S. epidermidis clearance from blood, spleen, and liver. Staphylococcus epidermidis increased the white blood cell count in the CSF, increased interleukin 6, interleukin 12p40, CCL2, and CXCL1 concentrations in plasma; increased the CCL2 concentration in the brain; and caused rapid (within 6 hours) TLR2-dependent brain activation of caspase-3 and TLR2-independent white matter injury. CONCLUSIONS: Staphylococcus epidermidis bacteremia, in the absence of bacterial entry into the CSF, impairs neonatal brain development. Staphylococcus epidermidis bacteremia induced both TLR2-dependent and -independent brain injury, with the latter occurring in the absence of TLR2, a condition associated with an increased bacterial burden. Our study indicates that the consequences of transient bacteremia in early life may be more severe than commonly appreciated, and our findings may inform novel approaches to reduce bacteremia-associated brain injury.

The effect of osteopontin and osteopontin-derived peptides on preterm brain injury.

BACKGROUND: Osteopontin (OPN) is a highly phosphorylated sialoprotein and a soluble cytokine that is widely expressed in a variety of tissues, including the brain. OPN and OPN-derived peptides have been suggested to have potential neuroprotective effects against ischemic brain injury, but their role in preterm brain injury is unknown. METHODS: We used a hypoxia-ischemia (HI)-induced preterm brain injury model in postnatal day 5 mice. OPN and OPN-derived peptides were given intracerebroventricularly and intranasally before HI. Brain injury was evaluated at 7 days after the insults. RESULTS: There was a significant increase in endogenous OPN mRNA and OPN protein in the mouse brain after the induction of HI at postnatal day 5. Administration of full-length OPN protein and thrombin-cleaved OPN did not affect preterm brain injury. This was demonstrated with both intracerebroventricular and intranasal administration of OPN as well as in OPN-deficient mice. Interestingly, both N134-153 and C154-198 OPN-derived peptides increased the severity of brain injury in this HI-induced preterm brain injury model. CONCLUSIONS: The neuroprotective effects of OPN are age-dependent, and, in contrast to the more mature brain, OPN-derived peptides potentiate injury in postnatal day 5 mice. Intranasal administration is an efficient way of delivering drugs to the central nervous system (CNS) in neonatal mice and is likely to be an easy and noninvasive method of drug delivery to the CNS in preterm infants.

The immune response after hypoxia-ischemia in a mouse model of preterm brain injury.

BACKGROUND: Preterm brain injury consists primarily of periventricular leukomalacia accompanied by elements of gray-matter injury, and these injuries are associated with cerebral palsy and cognitive impairments. Inflammation is believed to be an important contributing factor to these injuries. The aim of this study was to examine the immune response in a postnatal day (PND) 5 mouse model of preterm brain injury induced by hypoxia-ischemia (HI) that is characterized by focal white and gray-matter injury. METHODS: C57Bl/6 mice at PND 5 were subjected to unilateral HI induced by left carotid artery ligation and subsequent exposure to 10% O2 for 50 minutes, 70 minutes, or 80 minutes. At seven days post-HI, the white/gray-matter injury was examined. The immune responses in the brain after HI were examined at different time points after HI using RT-PCR and immunohistochemical staining. RESULTS: HI for 70 minutes in PND 5 mice induced local white-matter injury with focal cortical injury and hippocampal atrophy, features that are similar to those seen in preterm brain injury in human infants. HI for 50 minutes resulted in a small percentage of animals being injured, and HI for 80 minutes produced extensive infarction in multiple brain areas. Various immune responses, including changes in transcription factors and cytokines that are associated with a T-helper (Th)1/Th17-type response, an increased number of CD4+ T-cells, and elevated levels of triggering receptor expressed on myeloid cells 2 (TREM-2) and its adaptor protein DNAX activation protein of 12 kDa (DAP12) were observed using the HI 70 minute preterm brain injury model. CONCLUSIONS: We have established a reproducible model of HI in PND 5 mice that produces consistent local white/gray-matter brain damage that is relevant to preterm brain injury in human infants. This model provides a useful tool for studying preterm brain injury. Both innate and adaptive immune responses are observed after HI, and these show a strong pro-inflammatory Th1/Th17-type bias. Such findings provide a critical foundation for future studies on the mechanism of preterm brain injury and suggest that blocking the Th1/Th17-type immune response might provide neuroprotection after preterm brain injury.