Methylprednisolone Reduces Persistent Post-ischemic Inflammation in a Rat Hypoxia-Ischemia Model of Perinatal Stroke.

In perinatal stroke, the initial injury results in a chronic inflammatory response caused by the release of proinflammatory cytokines, gliosis and microglia activation. This chronic and ongoing inflammatory response exacerbates the brain injury, often resulting in encephalopathy and cerebral palsy (CP). Using a neonatal rat model of hypoxia-ischemia (HI) at postnatal day (P)7, we demonstrated that chronic inflammation is persistent and continues into the tertiary phase of perinatal stroke and can be attenuated by the administration of methylprednisolone sodium-succinate (MPSS, 30 mg/kg), a US Food and Drug Administration (FDA) approved anti-inflammatory agent. The inflammatory response was assessed by real-time quantitative PCR and ELISA for markers of inflammation (CCL3, CCL5, IL18 and TNFα). Structural changes were evaluated by histology (LFB/H&E), while cellular changes were assessed by Iba-1, ED1, GFAP, NeuN, Olig2 and CC1 immunostaining. Functional deficits were assessed with the Cylinder test and Ladder Rung Walking test. MPSS was injected 14 days after HI insult to attenuate chronic inflammation. In neonatal conditions such as CP, P21 is a clinically relevant time-point in rodents, corresponding developmentally to a 2-year-old human. Administration of MPSS resulted in reduced structural damage (corpus callosum, cortex, hippocampus, striatum), gliosis and reactive microglia and partial restoration of the oligodendrocyte population. Furthermore, significant behavioural recovery was observed. In conclusion, we demonstrated that administration of MPSS during the tertiary phase of perinatal stroke results in attenuation of the chronic inflammatory response, leading to pathophysiological and functional recovery. This work validates the high clinical impact of MPSS to treat neonatal conditions linked to chronic inflammation.

Severe-combined immunodeficient rats can be used to generate a model of perinatal hypoxic-ischemic brain injury to facilitate studies of engrafted human neural stem cells.

Cerebral palsy (CP) encompasses a group of non-progressive brain disorders that are often acquired through perinatal hypoxic-ischemic (HI) brain injury. Injury leads to a cascade of cell death events, resulting in lifetime motor and cognitive deficits. There are currently no treatments that can repair the resulting brain damage and improve functional outcomes. To date, preclinical research using neural precursor cell (NPC) transplantation as a therapy for HI brain injury has shown promise. To translate this treatment to the clinic, it is essential that human-derived NPCs also be tested in animal models, however, a major limitation is the high risk of xenograft rejection. A solution is to transplant the cells into immune-deficient rodents, but there are currently no models of HI brain injury established in such a cohort of animals. Here, we demonstrate that a model of HI brain injury can be generated in immune-deficient Prkdc knockout (KO) rats. Long-term deficits in sensorimotor function were similar between KO and wildtype (WT) rats. Interestingly, some aspects of the injury were more severe in KO rats. Additionally, human induced pluripotent stem cell derived (hiPSC)-NPCs had higher survival at 10 weeks post-transplant in KO rats when compared to their WT counterparts. This work establishes a reliable model of neonatal HI brain injury in Prkdc KO rats that will allow for future transplantation, survival, and long-term evaluation of the safety and efficacy of hiPSC-NPCs for neonatal brain damage. This model will enable critical preclinical translational research using human NPCs.

Exogenous Neural Precursor Cell Transplantation Results in Structural and Functional Recovery in a Hypoxic-Ischemic Hemiplegic Mouse Model.

Cerebral palsy (CP) is a common pediatric neurodevelopmental disorder, frequently resulting in motor and developmental deficits and often accompanied by cognitive impairments. A regular pathobiological hallmark of CP is oligodendrocyte maturation impairment resulting in white matter (WM) injury and reduced axonal myelination. Regeneration therapies based on cell replacement are currently limited, but neural precursor cells (NPCs), as cellular support for myelination, represent a promising regeneration strategy to treat CP, although the transplantation parameters (e.g., timing, dosage, mechanism) remain to be determined. We optimized a hemiplegic mouse model of neonatal hypoxia-ischemia that mirrors the pathobiological hallmarks of CP and transplanted NPCs into the corpus callosum (CC), a major white matter structure impacted in CP patients. The NPCs survived, engrafted, and differentiated morphologically in male and female mice. Histology and MRI showed repair of lesioned structures. Furthermore, electrophysiology revealed functional myelination of the CC (e.g., restoration of conduction velocity), while cylinder and CatWalk tests demonstrated motor recovery of the affected forelimb. Endogenous oligodendrocytes, recruited in the CC following transplantation of exogenous NPCs, are the principal actors in this recovery process. The lack of differentiation of the transplanted NPCs is consistent with enhanced recovery due to an indirect mechanism, such as a trophic and/or "bio-bridge" support mediated by endogenous oligodendrocytes. Our work establishes that transplantation of NPCs represents a viable therapeutic strategy for CP treatment, and that the enhanced recovery is mediated by endogenous oligodendrocytes. This will further our understanding and contribute to the improvement of cellular therapeutic strategies.

The extent of intrauterine growth restriction determines the severity of cerebral injury and neurobehavioural deficits in rodents.

BACKGROUND: Cerebral Palsy (CP) is the most common physical pediatric neurodevelopmental disorder and spastic diplegic injury is its most frequent subtype. CP results in substantial neuromotor and cognitive impairments that have significant socioeconomic impact. Despite this, its underlying pathophysiological mechanisms and etiology remain incompletely understood. Furthermore, there is a need for clinically relevant injury models, which a) reflect the heterogeneity of the condition and b) can be used to evaluate new translational therapies. To address these key knowledge gaps, we characterized a chronic placental insufficiency (PI) model, using bilateral uterine artery ligation (BUAL) of dams. This injury model results in intrauterine growth restriction (IUGR) in pups, and animals recapitulate the human phenotype both in terms of neurobehavioural and anatomical deficits. METHODS: Effects of BUAL were studied using luxol fast blue (LFB)/hematoxylin & eosin (H&E) staining, immunohistochemistry, quantitative Magnetic Resonance Imaging (MRI), and Catwalk neurobehavioural tests. RESULTS: Neuroanatomical analysis revealed regional ventricular enlargement and corpus callosum thinning in IUGR animals, which was correlated with the extent of growth restriction. Olig2 staining revealed reductions in oligodendrocyte density in white and grey matter structures, including the corpus callosum, optic chiasm, and nucleus accumbens. The caudate nucleus, along with other brain structures such as the optic chiasm, internal capsule, septofimbrial and lateral septal nuclei, exhibited reduced size in animals with IUGR. The size of the pretectal nucleus was reduced only in moderately injured animals. MAG/NF200 staining demonstrated reduced myelination and axonal counts in the corpus callosum of IUGR animals. NeuN staining revealed changes in neuronal density in the hippocampus and in the thickness of hippocampal CA2 and CA3 regions. Diffusion weighted imaging (DWI) revealed regional white and grey matter changes at 3 weeks of age. Furthermore, neurobehavioural testing demonstrated neuromotor impairments in animals with IUGR in paw intensities, swing speed, relative print positions, and phase dispersions. CONCLUSIONS: We have characterized a rodent model of IUGR and have demonstrated that the neuroanatomical and neurobehavioural deficits mirror the severity of the IUGR injury. This model has the potential to be applied to examine the pathobiology of and potential therapeutic strategies for IUGR-related brain injury. Thus, this work has potential translational relevance for the study of CP.

Rodent Hypoxia-Ischemia Models for Cerebral Palsy Research: A Systematic Review.

Cerebral palsy (CP) is a complex multifactorial disorder, affecting approximately 2.5-3/1000 live term births, and up to 22/1000 prematurely born babies. CP results from injury to the developing brain incurred before, during, or after birth. The most common form of this condition, spastic CP, is primarily associated with injury to the cerebral cortex and subcortical white matter as well as the deep gray matter. The major etiological factors of spastic CP are hypoxia/ischemia (HI), occurring during the last third of pregnancy and around birth age. In addition, inflammation has been found to be an important factor contributing to brain injury, especially in term infants. Other factors, including genetics, are gaining importance. The classic Rice-Vannucci HI model (in which 7-day-old rat pups undergo unilateral ligation of the common carotid artery followed by exposure to 8% oxygen hypoxic air) is a model of neonatal stroke that has greatly contributed to CP research. In this model, brain damage resembles that observed in severe CP cases. This model, and its numerous adaptations, allows one to finely tune the injury parameters to mimic, and therefore study, many of the pathophysiological processes and conditions observed in human patients. Investigators can recreate the HI and inflammation, which cause brain damage and subsequent motor and cognitive deficits. This model further enables the examination of potential approaches to achieve neural repair and regeneration. In the present review, we compare and discuss the advantages, limitations, and the translational value for CP research of HI models of perinatal brain injury.

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.

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.

Peripheral facial nerve axotomy in mice causes sprouting of motor axons into perineuronal central white matter: time course and molecular characterization.

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.

Somato-dendritic distribution of 5-HT(1A) and 5-HT(1B) autoreceptors in the BDNF- and cAMP-differentiated RN46A serotoninergic raphe cell line.

The rapid differentiating effects of brain-derived neurotrophic factor (BDNF) or dibutyryl-cAMP (dBcAMP) were characterized on RN46A, a rat raphe-derived neuronal cell line. After BDNF treatment, RN46A cells were serotonin-immunopositive and bipolar, and expressed the microtubule-associated-protein 2 (Map2). After dBcAMP treatment, the cells often became multipolar, bearing very long processes strongly immunopositive for serotonin and Map2. Under both conditions, the expression and distribution of 5-HT(1A) and 5-HT(1B) autoreceptors remained identical. 5-HT(1A) and Map2 immunolabelings were superimposable, as expected of their somato-dendritic targeting. Surprisingly, the distribution of 5-HT(1B) immunoreactivity was similar, in contrast with its usual localization in axons and nerve terminals in the brain. In conclusion, both BDNF and cAMP-differentiated RN46A cells towards a neuronal serotoninergic-like phenotype without the typical differential targeting of the 5-HT(1) autoreceptors, an interesting model to study the molecular mechanisms ensuing the targeting of 5-HT(1) autoreceptors to somas and dendrites.

Rapid up-regulation of the neuronal serotoninergic phenotype by brain-derived neurotrophic factor and cyclic adenosine monophosphate: relations with raphe astrocytes.

Up-regulation of the neuronal serotoninergic phenotype in relation to astrocytic population was studied in primary cultures of rat embryonic rostral raphe. Short treatments (18 hr at day in vitro 4) with brain-derived neurotrophic factor (BDNF) or dibutyryl-cAMP (dBcAMP) increased the number of serotoninergic neurons by approximately 80% and approximately 40%, respectively, and markedly enhanced the branching (by 11-fold and 5-fold, respectively) and total length (by 4-fold and 2.5-fold, respectively) of their neurites. Concomitantly, under BDNF treatment, the astrocyte population was decreased by half and became mostly protoplasmic-like. In contrast, dBcAMP treatment also reduced the astrocytic cell density (by one-third) but induced a stellate morphology. Similar short treatment with the astrocyte-derived S100beta factor induced no modification of the serotonin (5-HT) neuronal phenotype nor of astrocytes morphology. Both BDNF- and cAMP-induced effects were abolished by simultaneous treatment with the specific tyrosine kinase inhibitor genistein, suggesting a role for the high-affinity BDNF receptor tyrosine kinase (TrkB). These data suggest that BDNF and cAMP, but not S100beta, rapidly induce both an up-regulation of the 5-HT neuronal phenotype and modifications of the neighboring astrocytes in a TrkB-dependent manner.

Adaption of the serotoninergic neuronal phenotype in the absence of 5-HT autoreceptors or the 5-HT transporter: involvement of BDNF and cAMP.

Serotonin 5-HT1A and 5-HT1B receptors and the 5-HT transporter are key regulators of the serotoninergic neuronal phenotype. We show here that genetic deletion of any of these elements differentially regulates 5-HT neuronal number in rostral raphe cultures from E14 mice. Serotonin neuronal number was increased by almost four-fold and 1.8-fold in cultures from 5-HT1AR-/- and 5-HT1BR-/- mice, respectively. In contrast, the lack of serotonin transporter expression was associated with a 50% decrease in 5-HT neuronal number. In raphe cultures from the rat, BDNF and cAMP have been shown to up-regulate the neuronal serotoninergic phenotype through TrkB-dependent mechanisms [Rumajogee et al. (2002) J. Neurochem., 83, 1525-1528]. Similar tyrosine kinase-dependent up-regulating effects, in the absence of serotoninergic key-elements are reported here, on both 5-HT neuronal number and neurites length. However, the extents of BDNF-triggered and cAMP-triggered effects on serotoninergic neuritic length were approximately 1.5-fold higher in 5-HT1AR-/- mutants. These findings show that the up-regulatory mechanisms triggered by BDNF on serotoninergic neuronal number and neurite extension are different and that the latter are partially linked to 5-HT, probably through 5-HT1A autoreceptors. Together, these data suggest that serotonin autoreceptors, mainly 5-HT1A but also 5-HT1B, may be responsible for a tonic auto-inhibitory effect of 5-HT itself on the serotoninergic neuronal phenotype during embryonic development, particularly marked in the absence of the 5-HT transporter.

Up-regulation of the neuronal serotoninergic phenotype in vitro: BDNF and cAMP share Trk B-dependent mechanisms.

The effects of brain-derived neurotrophic factor (BDNF) and cAMP on the neuronal serotoninergic phenotype were studied in primary cultures of E14 rat embryonic rostral raphe. Short treatments (for 18 h) with BDNF or dibutyryl-cAMP induced an almost two-fold increase in the number of serotoninergic neurones and a dramatic extension and ramification of their neurites. These changes were associated with marked increases in the levels of mRNAs encoding the serotonin transporter, the 5-HT1A and 5-HT1B receptors and the BDNF receptor tyrosine kinase B (TrkB). Concomitant blockade of tyrosine kinases by genistein suppressed all the up-regulating effects of BDNF and cAMP on 5-hydroxytryptamine (5-HT) neurones. These findings suggest that an auto-amplifying mechanism underlies the promoting effect of BDNF on the differentiation of serotoninergic neurones through TrkB activation, which is also triggered by cAMP.