The Jun-dependent axon regeneration gene program: Jun promotes regeneration over plasticity.

The regeneration-associated gene (RAG) expression program is activated in injured peripheral neurons after axotomy and enables long-distance axon re-growth. Over 1000 genes are regulated, and many transcription factors are upregulated or activated as part of this response. However, a detailed picture of how RAG expression is regulated is lacking. In particular, the transcriptional targets and specific functions of the various transcription factors are unclear. Jun was the first-regeneration-associated transcription factor identified and the first shown to be functionally important. Here we fully define the role of Jun in the RAG expression program in regenerating facial motor neurons. At 1, 4 and 14 days after axotomy, Jun upregulates 11, 23 and 44% of the RAG program, respectively. Jun functions relevant to regeneration include cytoskeleton production, metabolic functions and cell activation, and the downregulation of neurotransmission machinery. In silico analysis of promoter regions of Jun targets identifies stronger over-representation of AP1-like sites than CRE-like sites, although CRE sites were also over-represented in regions flanking AP1 sites. Strikingly, in motor neurons lacking Jun, an alternative SRF-dependent gene expression program is initiated after axotomy. The promoters of these newly expressed genes exhibit over-representation of CRE sites in regions near to SRF target sites. This alternative gene expression program includes plasticity-associated transcription factors and leads to an aberrant early increase in synapse density on motor neurons. Jun thus has the important function in the early phase after axotomy of pushing the injured neuron away from a plasticity response and towards a regenerative phenotype.

Extracellular signal-regulated kinase 2 has duality in function between neuronal and astrocyte expression following neonatal hypoxic-ischaemic cerebral injury.

KEY POINTS: This study identifies phosphorylated extracellular signal-regulated kinase (ERK) to be immediately diminished followed by a rapid if transient increase for up to 4 h following hypoxic-ischaemic insult (HI) in the neonatal mouse. Phosphorylated ERK up-regulation was prevented with systemic injection of the mitogen-activated protein kinase kinase (MEK) inhibitor SL327. Treatment with SL327 both pre- and post-HI gave a strong reduction in the number of dying cells and microgliosis. By utilising transgenic mouse mutations, we observe that neuronal ERK2 significantly contributes to tissue damage, while ERK1 and astrocytic ERK2 are neuroprotective. Compared to global inactivation, selective cell-specific interference with ERK activity could result in stronger neuroprotection. ABSTRACT: Hypoxia-ischaemia (HI) is a major cause of neonatal brain injury resulting in cerebral palsy, epilepsy, cognitive impairment and other neurological disabilities. The role of extracellular signal-regulated kinase (ERK) isoforms and their mitogen-activated protein kinase kinase (MEK)-dependent phosphorylation in HI has previously been explored but remains unresolved at cellular level. This is pertinent given the growing awareness of the role of non-neuronal cells in neuroprotection. Using a modified Rice-Vannucci model of HI in the neonatal mouse we observed time- and cell-dependent ERK phosphorylation (pERK), with strongly up-regulated pERK immunoreactivity first in periventricular white matter axons within 15-45 min of HI, followed by forebrain astrocytes and neurons (1-4 h post-HI), and return to baseline by 16 h. We explored the effects of pharmacological ERK blockade through the MEK inhibitor SL327 on neonatal HI-brain damage following HI alone (30 or 60 min) or lipopolysaccharide (LPS)-sensitised HI insult (30 min). Global inhibition of ERK phosphorylation with systemically applied SL327 abolished forebrain pERK immunoreactivity, and significantly reduced cell death and associated microglial activation at 48 h post-HI. We then explored the effects of cell-specific ERK2 deletion alone or in combination with global ERK1 knockout under the same conditions of HI insult. Neuronal ERK2 deletion strongly decreased infarct size, neuronal cell death and microglial activation in grey matter following both HI alone or LPS-sensitised HI. ERK1 deletion attenuated the protective effect of neuronal ERK2 deletion. Removal of astroglial ERK2 produced a reverse response, with a 3- to 4-fold increase in microglial activation and cell death. Our data suggest a cell-specific and time-dependent role of ERK in neonatal HI, with a predominant, neurotoxic effect of neuronal ERK2, which is counteracted by neuroprotection by ERK1 and astrocytic ERK2. Overall, global pharmacological inhibition of ERK phosphorylation is strongly neuroprotective.

Inhibition of Signal Transducer and Activator of Transcription 3 (STAT3) reduces neonatal hypoxic-ischaemic brain damage.

Hypoxic-ischaemic encephalopathy is a leading cause of child death, with high mortality and morbidity, including cerebral palsy, epilepsy and cognitive disabilities. Hypoxia-ischaemia (HI) strongly up-regulates Signal Transducer and Activator of Transcription 3 (STAT3) in the immature brain. Our aim was to establish whether STAT3 up-regulation is associated with neonatal HI-brain damage and evaluate the phosphorylated STAT3-contribution from different cell types in eliciting damage. We subjected postnatal day seven mice to unilateral carotid artery ligation followed by 60 min hypoxia. Neuronal STAT3-deletion reduced cell death, tissue loss, microglial and astroglial activation in all brain regions. Astroglia-specific STAT3-deletion also reduced cell death, tissue loss and microglial activation, although not as strongly as the deletion in neurons. Systemic pre-insult STAT3-blockade at tyrosine 705 (Y705) with JAK2-inhibitor WP1066 reduced microglial and astroglial activation to a more moderate degree, but in a pattern similar to the one produced by the cell-specific deletions. Our results suggest that STAT3 is a crucial factor in neonatal HI-brain damage and its removal in neurons or astrocytes, and, to some extent, inhibition of its phosphorylation via JAK2-blockade reduces inflammation and tissue loss. Overall, the protective effects of STAT3 inactivation make it a possible target for a therapeutic strategy in neonatal HI. Current data show that neuronal and astroglial STAT3 molecules are involved in the pathways underlying cell death, tissue loss and gliosis following neonatal hypoxia-ischaemia, but differ with respect to the target of their effect. Y705-phosphorylation contributes to hypoxic-ischaemic histopathology. Protective effects of STAT3 inactivation make it a possible target for a therapeutic strategy in neonatal hypoxia-ischaemia.

Peptidylarginine deiminases: novel drug targets for prevention of neuronal damage following hypoxic ischemic insult (HI) in neonates.

Neonatal hypoxic ischaemic (HI) injury frequently causes neural impairment in surviving infants. Our knowledge of the underlying molecular mechanisms is still limited. Protein deimination is a post-translational modification caused by Ca(+2) -regulated peptidylarginine deiminases (PADs), a group of five isozymes that display tissue-specific expression and different preference for target proteins. Protein deimination results in altered protein conformation and function of target proteins, and is associated with neurodegenerative diseases, gene regulation and autoimmunity. In this study, we used the neonatal HI and HI/infection [lipopolysaccharide (LPS) stimulation] murine models to investigate changes in protein deimination. Brains showed increases in deiminated proteins, cell death, activated microglia and neuronal loss in affected brain areas at 48 h after hypoxic ischaemic insult. Upon treatment with the pan-PAD inhibitor Cl-amidine, a significant reduction was seen in microglial activation, cell death and infarct size compared with control saline or LPS-treated animals. Deimination of histone 3, a target protein of the PAD4 isozyme, was increased in hippocampus and cortex specifically upon LPS stimulation and markedly reduced following Cl-amidine treatment. Here, we demonstrate a novel role for PAD enzymes in neural impairment in neonatal HI Encephalopathy, highlighting their role as promising new candidates for drug-directed intervention in neurotrauma. Hypoxic Ischaemic Insult (HI) results in activation of peptidylarginine deiminases (PADs) because of calcium dysregulation. Target proteins undergo irreversible changes of protein bound arginine to citrulline, resulting in protein misfolding. Infection in synergy with HI causes up-regulation of TNFα, nuclear translocation of PAD4 and change in gene regulation as a result of histone deimination. Pharmacological PAD inhibition significantly reduced HI brain damage.

Melatonin augments hypothermic neuroprotection in a perinatal asphyxia model.

Despite treatment with therapeutic hypothermia, almost 50% of infants with neonatal encephalopathy still have adverse outcomes. Additional treatments are required to maximize neuroprotection. Melatonin is a naturally occurring hormone involved in physiological processes that also has neuroprotective actions against hypoxic-ischaemic brain injury in animal models. The objective of this study was to assess neuroprotective effects of combining melatonin with therapeutic hypothermia after transient hypoxia-ischaemia in a piglet model of perinatal asphyxia using clinically relevant magnetic resonance spectroscopy biomarkers supported by immunohistochemistry. After a quantified global hypoxic-ischaemic insult, 17 newborn piglets were randomized to the following: (i) therapeutic hypothermia (33.5°C from 2 to 26 h after resuscitation, n = 8) and (ii) therapeutic hypothermia plus intravenous melatonin (5 mg/kg/h over 6 h started at 10 min after resuscitation and repeated at 24 h, n = 9). Cortical white matter and deep grey matter voxel proton and whole brain (31)P magnetic resonance spectroscopy were acquired before and during hypoxia-ischaemia, at 24 and 48 h after resuscitation. There was no difference in baseline variables, insult severity or any physiological or biochemical measure, including mean arterial blood pressure and inotrope use during the 48 h after hypoxia-ischaemia. Plasma levels of melatonin were 10 000 times higher in the hypothermia plus melatonin than hypothermia alone group. Melatonin-augmented hypothermia significantly reduced the hypoxic-ischaemic-induced increase in the area under the curve for proton magnetic resonance spectroscopy lactate/N-acetyl aspartate and lactate/total creatine ratios in the deep grey matter. Melatonin-augmented hypothermia increased levels of whole brain (31)P magnetic resonance spectroscopy nucleotide triphosphate/exchangeable phosphate pool. Correlating with improved cerebral energy metabolism, TUNEL-positive nuclei were reduced in the hypothermia plus melatonin group compared with hypothermia alone in the thalamus, internal capsule, putamen and caudate, and there was reduced cleaved caspase 3 in the thalamus. Although total numbers of microglia were not decreased in grey or white matter, expression of the prototypical cytotoxic microglial activation marker CD86 was decreased in the cortex at 48 h after hypoxia-ischaemia. The safety and improved neuroprotection with a combination of melatonin with cooling support phase II clinical trials in infants with moderate and severe neonatal encephalopathy.

Investigation of cerebral autoregulation in the newborn piglet during anaesthesia and surgery.

The relationship between cerebral autoregulation (CA) and the neurotoxic effects of anaesthesia with and without surgery is investigated. Newborn piglets were randomly assigned to receive either 6 h of anaesthesia (isoflurane) or the same with an additional hour of minor surgery. The effect of the spontaneous changes in mean arterial blood pressure (MABP) on the cerebral haemodynamics (oxy- and deoxy-haemoglobin, HbO2 and Hb) was measured using transverse broadband near-infrared spectroscopy (NIRS). A marker for impaired CA, concordance between MABP and intravascular oxygenation (HbD = HbO2 - Hb) in the ultra-low frequency domain (0.0018-0.0083 Hz), was assessed using coherence analysis. Presence of CA impairment was not significant but found to increase with surgical exacerbation. The impairment did not correlate with histological outcome (presence of cell death, apoptosis and microglial activation in the brain).

Methyl-isobutyl amiloride reduces brain Lac/NAA, cell death and microglial activation in a perinatal asphyxia model.

Na⁺/H⁺ exchanger (NHE) blockade attenuates the detrimental consequences of ischaemia and reperfusion in myocardium and brain in adult and neonatal animal studies. Our aim was to use magnetic resonance spectroscopy (MRS) biomarkers and immunohistochemistry to investigate the cerebral effects of the NHE inhibitor, methyl isobutyl amiloride (MIA) given after severe perinatal asphyxia in the piglet. Eighteen male piglets (aged < 24 h) underwent transient global cerebral hypoxia-ischaemia and were randomized to (i) saline placebo; or (ii) 3 mg/kg intravenous MIA administered 10 min post-insult and 8 hourly thereafter. Serial phosphorus-31 (³¹P) and proton (¹H) MRS data were acquired before, during and up to 48 h after hypoxia-ischaemia and metabolite-ratio time-series Area under the Curve (AUC) calculated. At 48 h, histological and immunohistochemical assessments quantified regional tissue injury. MIA decreased thalamic lactate/N-acetylaspartate and lactate/creatine AUCs (both p < 0.05) compared with placebo. Correlating with improved cerebral energy metabolism, transferase mediated biotinylated d-UTP nick end-labelling (TUNEL) positive cell density was reduced in the MIA group in cerebral cortex, thalamus and white matter (all p < 0.05) and caspase 3 immunoreactive cells were reduced in pyriform cortex and caudate nucleus (both p < 0.05). Microglial activation was reduced in pyriform and midtemporal cortex (both p < 0.05). Treatment with MIA starting 10 min after hypoxia-ischaemia was neuroprotective in this perinatal asphyxia model.

Neuronal c-Jun is required for successful axonal regeneration, but the effects of phosphorylation of its N-terminus are moderate.

Although neural c-Jun is essential for successful peripheral nerve regeneration, the cellular basis of this effect and the impact of c-Jun activation are incompletely understood. In the current study, we explored the effects of neuron-selective c-Jun deletion, substitution of serine 63 and 73 phosphoacceptor sites with non-phosphorylatable alanine, and deletion of Jun N-terminal kinases 1, 2 and 3 in mouse facial nerve regeneration. Removal of the floxed c-jun gene in facial motoneurons using cre recombinase under control of a neuron-specific synapsin promoter (junΔS) abolished basal and injury-induced neuronal c-Jun immunoreactivity, as well as most of the molecular responses following facial axotomy. Absence of neuronal Jun reduced the speed of axonal regeneration following crush, and prevented most cut axons from reconnecting to their target, significantly reducing functional recovery. Despite blocking cell death, this was associated with a large number of shrunken neurons. Finally, junΔS mutants also had diminished astrocyte and microglial activation and T-cell influx, suggesting that these non-neuronal responses depend on the release of Jun-dependent signals from neighboring injured motoneurons. The effects of substituting serine 63 and 73 phosphoacceptor sites (junAA), or of global deletion of individual kinases responsible for N-terminal c-Jun phosphorylation were mild. junAA mutants showed decrease in neuronal cell size, a moderate reduction in post-axotomy CD44 levels and slightly increased astrogliosis. Deletion of Jun N-terminal kinase (JNK)1 or JNK3 showed delayed functional recovery; deletion of JNK3 also interfered with T-cell influx, and reduced CD44 levels. Deletion of JNK2 had no effect. Thus, neuronal c-Jun is needed in regeneration, but JNK phosphorylation of the N-terminus mostly appears to not be required for its function.

Downstream effector molecules in successful peripheral nerve regeneration.

The robust axon regeneration that occurs following peripheral nerve injury is driven by transcriptional activation of the regeneration program and by the expression of a wide range of downstream effector molecules from neuropeptides and neurotrophic factors to adhesion molecules and cytoskeletal adaptor proteins. These regeneration-associated effector molecules regulate the actin-tubulin machinery of growth-cones, integrate intracellular signalling and stimulatory and inhibitory signals from the local environment and translate them into axon elongation. In addition to the neuronally derived molecules, an important transcriptional component is found in locally activated Schwann cells and macrophages, which release a number of cytokines, growth factors and neurotrophins that support neuronal survival and axonal regeneration and that might provide directional guidance cues towards appropriate peripheral targets. This review aims to provide a comprehensive up-to-date account of the transcriptional regulation and functional role of these effector molecules and of the information that they can give us with regard to the organisation of the regeneration program.

Xenon augmented hypothermia reduces early lactate/N-acetylaspartate and cell death in perinatal asphyxia.

OBJECTIVE: Additional treatments for therapeutic hypothermia are required to maximize neuroprotection for perinatal asphyxial encephalopathy. We assessed neuroprotective effects of combining inhaled xenon with therapeutic hypothermia after transient cerebral hypoxia-ischemia in a piglet model of perinatal asphyxia using magnetic resonance spectroscopy (MRS) biomarkers supported by immunohistochemistry. METHODS: Thirty-six newborn piglets were randomized (all groups n = 9), with intervention from 2 to 26 hours, to: (1) normothermia; (2) normothermia + 24 hours 50% inhaled xenon; (3) 24 hours hypothermia (33.5°C); or (4) 24 hours hypothermia (33.5°C) + 24 hours 50% inhaled xenon. Serial MRS was acquired before, during, and up to 48 hours after hypoxia-ischemia. RESULTS: Mean arterial blood pressure was lower in all treatment groups compared with normothermia (p < 0.01) (although >40mmHg); the combined therapy group required more fluid boluses (p < 0.05) and inotropes (p < 0.001). Compared with no intervention, both hypothermia and xenon-augmented hypothermia reduced the temporal regression slope magnitudes for phosphorus-MRS inorganic phosphate/exchangeable phosphate pool (EPP) and phosphocreatine/EPP (both p < 0.05); for lactate/N-acetylaspartate (NAA), only xenon-augmented hypothermia reduced the slope (p < 0.01). Xenon-augmented hypothermia also reduced transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)(+) nuclei and caspase 3 immunoreactive cells in parasagittal cortex and putamen and increased microglial ramification in midtemporal cortex compared with the no treatment group (p < 0.05). Compared with hypothermia, however, combination treatment did not reach statistical significance for any measure. Lactate/NAA showed a strong positive correlation with TUNEL; nucleotide triphosphate/EPP showed a strong negative correlation with microglial ramification (both p < 0.01). INTERPRETATION: Compared with no treatment, xenon-augmented hypothermia reduced cerebral MRS abnormalities and cell death markers in some brain regions. Compared with hypothermia, xenon-augmented hypothermia did not reach statistical significance for any measure. The safety and possible improved efficacy support phase II trials.

Experimental autoimmune encephalomyelitis repressed by microglial paralysis.

Although microglial activation occurs in inflammatory, degenerative and neoplastic central nervous system (CNS) disorders, its role in pathogenesis is unclear. We studied this question by generating CD11b-HSVTK transgenic mice, which express herpes simplex thymidine kinase in macrophages and microglia. Ganciclovir treatment of organotypic brain slice cultures derived from CD11b-HSVTK mice abolished microglial release of nitrite, proinflammatory cytokines and chemokines. Systemic ganciclovir administration to CD11b-HSVTK mice elicited hematopoietic toxicity, which was prevented by transfer of wild-type bone marrow. In bone marrow chimeras, ganciclovir blocked microglial activation in the facial nucleus upon axotomy and repressed the development of experimental autoimmune encephalomyelitis. We conclude that microglial paralysis inhibits the development and maintenance of inflammatory CNS lesions. The microglial compartment thus provides a potential therapeutic target in inflammatory CNS disorders. These results validate CD11b-HSVTK mice as a tool to study the impact of microglial activation on CNS diseases in vivo.

The ATM cofactor ATMIN protects against oxidative stress and accumulation of DNA damage in the aging brain.

Progressive accumulation of DNA damage is causally involved in cellular senescence and organismal aging. The DNA damage kinase ATM plays a central role in maintaining genomic stability. ATM mutations cause the genetic disorder ataxia telangiectasia, which is primarily characterized by progressive neurodegeneration and cancer susceptibility. Although the importance of ATM function to protect against oxidative DNA damage and during aging is well described, the mechanism of ATM activation by these stimuli is not known. Here we identify ATM interactor (ATMIN) as an essential component of the ATM signaling pathway in response to oxidative stress and aging. Embryos lacking ATMIN (atmin(Δ/Δ)) died in utero and showed increased numbers of cells positive for phosphorylated histone H2aX, indicative of increased DNA damage. atmin(Δ/Δ) mouse embryonic fibroblasts accumulated DNA damage and prematurely entered senescence when cultured at atmospheric oxygen levels (20%), but this defect was rescued by addition of an antioxidant and also by culturing cells at physiological oxygen levels (3%). In response to acute oxidative stress, atmin(Δ/Δ) mouse embryonic fibroblasts showed slightly lower levels of ATM phosphorylation and reduced ATM substrate phosphorylation. Conditional deletion of ATMIN in the murine nervous system (atmin(ΔN)) resulted in reduced numbers of dopaminergic neurons, as does ATM deficiency. ATM activity was observed in old, but not in young, control mice, but aging-induced ATM signaling was impaired by ATMIN deficiency. Consequently, old atmin(ΔN) mice showed accumulation of DNA damage in the cortex accompanied by gliosis, resulting in increased mortality of aging mutant mice. These results suggest that ATMIN mediates ATM activation by oxidative stress, and thereby ATMIN protects the aging brain by preventing accumulation of DNA damage.

TNF gene cluster deletion abolishes lipopolysaccharide-mediated sensitization of the neonatal brain to hypoxic ischemic insult.

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 µg/g) resulted in particularly pronounced synergistic injury within 12 h. Systemic application of LPS alone resulted in a strong upregulation of inflammation-associated cytokines TNFα, LTß, interleukin (IL) 1ß, 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 LTα 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 TNFα, LTß and LTα 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.

ERK activation causes epilepsy by stimulating NMDA receptor activity.

The ERK MAPK signalling pathway is a highly conserved kinase cascade linking transmembrane receptors to downstream effector mechanisms. To investigate the function of ERK in neurons, a constitutively active form of MEK1 (caMEK1) was conditionally expressed in the murine brain, which resulted in ERK activation and caused spontaneous epileptic seizures. ERK activation stimulated phosphorylation of eukaryotic translation initiation factor 4E (eIF4E) and augmented NMDA receptor 2B (NR2B) protein levels. Pharmacological inhibition of NR2B function impaired synaptic facilitation in area cornus ammonicus region 3 (CA3) in acute hippocampal slices derived from caMEK1-expressing mice and abrogated epilepsy in vivo. In addition, expression of caMEK1 caused phosphorylation of the transcription factor, cAMP response element-binding protein (CREB) and increased transcription of ephrinB2. EphrinB2 overexpression resulted in increased NR2B tyrosine phosphorylation, which was essential for caMEK1-induced epilepsy in vivo, since conditional inactivation of ephrinB2 greatly reduced seizure frequency in caMEK1 transgenic mice. Therefore, our study identifies a mechanism of epileptogenesis that links MAP kinase to Eph/Ephrin and NMDA receptor signalling.

Distribution of pH changes in mouse neonatal hypoxic-ischaemic insult.

We assessed the distribution in brain pH after neonatal hypoxic-ischaemic insult and its correlation with local injury. Postnatal day 7 mice were injected with neutral red and underwent left carotid occlusion and exposure to 8% oxygen. Images captured from the cut surface of snap-frozen brain were used to calculate the pH from the blue-green absorbance ratios. Carotid occlusion alone had no effect, but combined with hypoxia caused rapid, biphasic pH decline, with the first plateau at 15-30 min, and the second at 60-90 min. The ipsilateral dorsal cortex, hippocampus, striatum and thalamus were most affected. Contralateral pH initially showed only 30% of the ipsilateral decline, becoming more acidotic with increasing duration. Systemic blood analysis revealed, compared with hypoxia alone, that combined insult caused a 63% decrease in blood glucose (1.3 ± 0.2 mM), a 2-fold increase in circulating lactate (17.7 ± 2.9 mM), a reduction in CO(2) to 1.9 ± 0.1 kPa and a drop in pH (7.26 ± 0.06). Re-oxygenation resulted in the normalisation of systemic changes, as well as a global alkaline rebound in brain pH at 4-6 h. A topographic comparison of brain injury showed only a partial correlation with pH changes, with the severest injury occurring in the ipsilateral hippocampus and sparing acidic parts of the contralateral cortex.

Activation and deactivation of periventricular white matter phagocytes during postnatal mouse development.

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.

Three-dimensional noninvasive imaging of the vasculature in the mouse brain using a high resolution photoacoustic scanner.

The application of a novel photoacoustic imaging instrument based on a Fabry-Perot polymer film sensing interferometer to imaging the small animal brain is described. This approach provides a convenient backward mode sensing configuration that offers the prospect of overcoming the limitations of existing piezoelectric based detection schemes for small animal brain imaging. Noninvasive images of the vasculature in the mouse brain were obtained at different wavelengths between 590 and 889 nm, showing that the cerebral vascular anatomy can be visualized with high contrast and spatial resolution to depths up to 3.7 mm. It is considered that the instrument has a role to play in characterizing small animal models of human disease and injury processes such as stroke, epilepsy, and traumatic brain injury.

The AP-1 transcription factor c-Jun is required for efficient axonal regeneration.

Nerve injury triggers numerous changes in the injured neurons and surrounding nonneuronal cells that ultimately result in successful target reinnervation or cell death. c-Jun is a component of the heterodimeric AP-1 transcription factor, and c-Jun is highly expressed in response to neuronal trauma. Here we have investigated the role of c-jun during axonal regeneration using mice lacking c-jun in the central nervous system. After transection of the facial nerve, the absence of c-Jun caused severe defects in several aspects of the axonal response, including perineuronal sprouting, lymphocyte recruitment, and microglial activation. c-Jun-deficient motorneurons were atrophic, resistant to axotomy-induced cell death, and showed reduced target muscle reinnervation. Expression of CD44, galanin, and alpha7beta1 integrin, molecules known to be involved in regeneration, was greatly impaired, suggesting a mechanism for c-Jun-mediated axonal growth. Taken together, our results identify c-Jun as an important regulator of axonal regeneration in the injured central nervous system.

Endogenous transforming growth factor beta 1 suppresses inflammation and promotes survival in adult CNS.

Transforming growth factor beta1 (TGFbeta1) is a pleiotropic cytokine with potent neurotrophic and immunosuppressive properties that is upregulated after injury, but also expressed in the normal nervous system. In the current study, we examined the regulation of TGFbeta1 and the effects of TGFbeta1 deletion on cellular response in the uninjured adult brain and in the injured and regenerating facial motor nucleus. To avoid lethal autoimmune inflammation within 3 weeks after birth in TGFbeta1-deficient mice, this study was performed on a T- and B-cell-deficient RAG2-/- background. Compared with wild-type siblings, homozygous deletion of TGFbeta1 resulted in an extensive inflammatory response in otherwise uninjured brain parenchyma. Astrocytes increased in GFAP and CD44 immunoreactivity; microglia showed proliferative activity, expression of phagocytosis-associated markers [alphaXbeta2, B7.2, and MHC1 (major histocompatibility complex type 1)], and reduced branching. Ultrastructural analysis revealed focal blockade of axonal transport, perinodal damming of axonal organelles, focal demyelination, and myelin debris in granule-rich, phagocytic microglia. After facial axotomy, absence of TGFbeta1 led to a fourfold increase in neuronal cell death (52 vs 13%), decreased central axonal sprouting, and significant delay in functional recovery. It also interfered with the microglial response, resulting in a diminished expression of early activation markers [ICAM1 (intercellular adhesion molecule 1), alpha6beta1, and alphaMbeta2] and reduced proliferation. In line with axonal and glial findings in the otherwise uninjured CNS, absence of endogenous TGFbeta1 also caused an approximately 10% reduction in the number of normal motoneurons, pointing to an ongoing and potent trophic role of this anti-inflammatory cytokine in the normal as well as in the injured brain.