Brain microglia are related to peripheral macrophages but undergo a highly specific process of regional maturation and differentiation inside the brain. Here, we examined this deactivation and morphological differentiation in cerebral cortex and periventricular subcortical white matter, the main "fountain of microglia" site, during postnatal mouse development, 0-28 days after birth (P0-P28). Only macrophages in subcortical white matter but not cortical microglia exhibited strong expression of typical activation markers alpha5, alpha6, alphaM, alphaX, and beta2 integrin subunits and B7.2 at any postnatal time point studied. White matter phagocyte activation was maximal at P0, decreased linearly over P3 and P7 and disappeared at P10. P7 white matter phagocytes also expressed high levels of IGF1 and MCSF, but not TNFalpha mRNA; this expression disappeared at P14. This process of deactivation followed the presence of ingested phagocytic material but correlated only moderately with ramification, and not with the extent of TUNEL+ death in neighboring cells, their ingestion or microglial proliferation. Intravenous fluosphere labeling revealed postnatal recruitment and transformation of circulating leukocytes into meningeal and perivascular macrophages as well as into ramified cortical microglia, but bypassing the white matter areas. In conclusion, this study describes strong and selective activation of postnatally resident phagocytes in the P0-P7 subcortical white matter, roughly equivalent to mid 3rd trimester human fetal development. This presence of highly active and IGF1- and MCSF-expressing phagocytes in the neighborhood of vulnerable white matter could play an important role in the genesis of or protection against axonal damage in the fetus and premature neonate.

Non-integrating lentiviral vectors show considerable promise for gene therapy applications as they persist as long-term episomes in non-dividing cells and diminish risks of insertional mutagenesis. In this study, non-integrating lentiviral vectors were evaluated for their use in the adult and fetal central nervous system of rodents. Vectors differentially pseudotyped with vesicular stomatitis virus, rabies and baculoviral envelope proteins allowed targeting of varied cell populations. Efficient gene delivery to discrete areas of the brain and spinal cord was observed following stereotactic administration. Furthermore, after direct in utero administration (E14), sustained and strong expression was observed 4 months into adulthood. Quantification of transduction and viral copy number was comparable when using nonintegrating lentivirus and conventional integrating vector. These data support the use of non-integrating lentiviral vectors as an effective alternative to their integrating counterparts in gene therapy applications, and highlight their potential for treatment of inherited and acquired neurological disorders.

In the current study, we explored the role of TNF cluster cytokines on the lipopolysaccharide (LPS)-mediated, synergistic increase in brain injury after hypoxic ischemic insult in postnatal day 7 mice. Pretreatment with moderate doses of LPS (0.3 mg/g) resulted in particularly pronounced synergistic injury within 12 h. Systemic application of LPS alone resulted in a strong upregulation of inflammation-associated cytokines TNFa, LTb, interleukin (IL) 1b, IL6, chemokines, such as CXCL1, and adhesion molecules E-Selectin, P-Selectin and intercellular adhesion molecule-1 (ICAM1), as well as a trend toward increased LTa levels in day 7 mouse forebrain. In addition, it was also associated with strong activation of brain blood vessel endothelia and local microglial cells. Here, deletion of the entire TNF gene cluster, removing TNFa, LTb and LTa completely abolished endotoxin-mediated increase in the volume of cerebral infarct. Interestingly, the same deletion also prevented endothelial and microglial activation following application of LPS alone, suggesting the involvement of these cell types in bringing about the LPS-mediated sensitization to neonatal brain injury. KEYWORDS: encephalopathy; hypoxia; inflammation; ischemia; neonate; TNF Although bacteria and viruses can directly infect and injure developing brain, infections occurring outside the brain frequently will also have a damaging effect. Congenital infections appear to contribute up to 5% of cerebral palsy cases 1,2 and may sensitize the brain to perinatal hypoxic ischemic (HI) insult. [3][4][5] This synergistic effect was also reproduced in mammalian and avian animal models, combining HI insult and the lipopolysaccharide break-down product of bacteria in HI animal models.6-10 However, the molecular mediators of this endotoxin effect in vivo are currently still unknown.Both in vitro and in vivo studies show that endotoxin will upregulate numerous cytokines and chemokines, 11 upregulate signaling enzymes, such as inducible nitrogen oxide synthase (iNOS), and cyclo-oxygenase-2 (COX2) and enhance the expression of adhesion molecules on parenchymal microglia and the brain vascular endothelium. 12-15Follow on molecular studies reveal that these effects are transmitted through the classical endotoxin receptors, primarily the toll-like receptor 4 (TLR4), on blood vessel endothelia and microglia, 16,17 and involve MyD88 and NF-kappa-B components of the innate immunity cascade. 17In particular, endotoxin-induced pro-inflammatory cytokines, including TNFa and interleukin 1b (IL1b) are known to have a number of deleterious effects, including a direct toxic effect on neurones and vulnerable oligodendrocyte precursors, 18,19 astrogliosis with release of nitric oxide, and mitochondrial dysfunction, 20 as well as microglial activation with release of nitric oxide, superoxide and a panel of other inflammation-associated molecules.

Generation of new axonal sprouts plays an important role in neural repair. In the current study, we examined the appearance, composition and effects of gene deletions on intrabrainstem sprouts following peripheral facial nerve axotomy. Axotomy was followed by the appearance of galanin+ and calcitonin gene-related peptide (CGRP)+ sprouts peaking at day 14, matching both large, neuropeptide+ subpopulations of axotomized facial motoneurons, but with CGRP+ sprouts considerably rarer. Strong immunoreactivity for vesicular acetylcholine transporter (VAChT) and retrogradely transported MiniRuby following its application on freshly cut proximal facial nerve stump confirmed their axotomized motoneuron origin; the sprouts expressed CD44 and alpha7beta1 integrin adhesion molecules and grew apparently unhindered along neighboring central white matter tracts. Quantification of the galanin+ sprouts revealed a stronger response following cut compared with crush (day 7–14) as well as enhanced sprouting after recut (day 8 + 6 vs. 14; 14 + 8 vs. 22), arguing against delayed appearance of sprouting being the result of the initial phase of reinnervation. Sprouting was strongly diminished in brain Jun-deficient mice but enhanced in alpha7 null animals that showed apparently compensatory up-regulation in beta1, suggesting important regulatory roles for transcription factors and the sprout-associated adhesion molecules. Analysis of inflammatory stimuli revealed a 50% reduction 12–48 hours following systemic endotoxin associated with neural inflammation and a tendency toward more sprouts in TNFR1/2 null mutants (P = 10%) with a reduced inflammatory response, indicating detrimental effects of excessive inflammation. Moreover, the study points to the usefulness of the facial axotomy model in exploring physiological and molecular stimuli regulating central sprouting. J. Comp. Neurol. 518:699–721, 2010. © 2009 Wiley-Liss, Inc.