Inhaled nitric oxide reduces injury and microglia activation in porcine hippocampus after deep hypothermic circulatory arrest.

OBJECTIVE: Dysregulation of local nitric oxide (NO) synthetases occurs during ischemia and reperfusion associated with cardiopulmonary bypass, deep hypothermic circulatory arrest (DHCA), and reperfusion. Rapid fluctuations in local NO occurring in neonates and infants probably contribute to inflammation-induced microglial activation and neuronal degeneration after these procedures, eventually impairing neurodevelopment. We evaluated the anti-inflammatory efficacy of inhaled NO (iNO) in a piglet model emulating conditions during pediatric open-heart surgery with DHCA. METHODS: Infant Yorkshire piglets underwent DHCA (18°C) for 30 minutes, followed by reperfusion and rewarming either with or without iNO (20 ppm) in the ventilator at the onset of reperfusion for 3 hours (n = 5 per group, DHCA-iNO and DHCA). Through craniotomy, brains were extracted after perfusion fixation for histology. RESULTS: Plasma NO metabolites were elevated 2.5 times baseline data before DHCA by iNO. Fluoro-Jade C staining identified significantly lower number of degenerating neurons in the hippocampus of the DHCA-iNO group (P = .02) compared with the DHCA group. Morphologic analyses of ionized calcium-binding adapter molecule-1 stained microglia, evaluating cell body and dendritic process geometry with Imaris imaging software, revealed subjectively less microglial activation in the hippocampus of pigs receiving iNO. CONCLUSIONS: Using DHCA for 30 minutes, consistent with clinical exposure, we noted that iNO reduces neuronal degeneration in the hippocampus. In addition, iNO reduces microglial activation in the hippocampus after DHCA. The data suggest that iNO reduces neuronal degeneration by ameliorating inflammation and may be a practical mode of neuroprotection for infants undergoing DHCA.

Tbr2 expression in Cajal-Retzius cells and intermediate neuronal progenitors is required for morphogenesis of the dentate gyrus.

The dentate gyrus (DG) is a unique cortical region whose protracted development spans the embryonic and early postnatal periods. DG development involves large-scale reorganization of progenitor cell populations, ultimately leading to the establishment of the subgranular zone neurogenic niche. In the developing DG, the T-box transcription factor Tbr2 is expressed in both Cajal-Retzius cells derived from the cortical hem that guide migration of progenitors and neurons to the DG, and intermediate neuronal progenitors born in the dentate neuroepithelium that give rise to granule neurons. Here we show that in mice Tbr2 is required for proper migration of Cajal-Retzius cells to the DG; and, in the absence of Tbr2, formation of the hippocampal fissure is abnormal, leading to aberrant development of the transhilar radial glial scaffold and impaired migration of progenitors and neuroblasts to the developing DG. Furthermore, loss of Tbr2 results in decreased expression of Cxcr4 in migrating cells, leading to a premature burst of granule neurogenesis during early embryonic development accompanied by increased cell death in mutant animals. Formation of the transient subpial neurogenic zone was abnormal in Tbr2 conditional knock-outs, and the stem cell population in the DG was depleted before proper establishment of the subgranular zone. These studies indicate that Tbr2 is explicitly required for morphogenesis of the DG and participates in multiple aspects of the intricate developmental process of this structure.

Patterned assembly and neurogenesis in the chick dorsal root ganglion.

The birth of small-diameter TrkA+ neurons that mediate pain and thermoreception begins approximately 24 hours after the cessation of neural crest cell migration from progenitors residing in the nascent dorsal root ganglion. Although multiple geographically distinct progenitor pools have been proposed, this study is the first to comprehensively characterize the derivation of small-diameter neurons. In the developing chick embryo we identify novel patterns in neural crest cell migration and colonization that sculpt the incipient ganglion into a postmitotic neuronal core encapsulated by a layer of proliferative progenitor cells. Furthermore, we show that this outer progenitor layer is composed of three spatially, temporally, and molecularly distinct progenitor zones, two of which give rise to distinct populations of TrkA+ neurons.

Stimulation of neural regeneration in the mouse retina.

Müller glia can serve as a source of new neurons after retinal damage in both fish and birds. Investigations of regeneration in the mammalian retina in vitro have provided some evidence that Müller glia can proliferate after retinal damage and generate new rods; however, the evidence that this occurs in vivo is not conclusive. We have investigated whether Müller glia have the potential to generate neurons in the mouse retina in vivo by eliminating ganglion and amacrine cells with intraocular NMDA injections and stimulating Müller glial to re-enter the mitotic cycle by treatment with specific growth factors. The proliferating Müller glia dedifferentiate and a subset of these cells differentiated into amacrine cells, as defined by the expression of amacrine cell-specific markers Calretinin, NeuN, Prox1, and GAD67-GFP. These results show for the first time that the mammalian retina has the potential to regenerate inner retinal neurons in vivo.

PACAP promotes sensory neuron differentiation: blockade by neurotrophic factors.

Developing neurons encounter a panoply of extracellular signals as they differentiate. A major goal is to identify these extrinsic cues and define the mechanisms by which neurons simultaneously integrate stimulation by multiple factors yet initiate one specific biological response. Factors that are known to exert potent activities in the developing nervous system include the NGF family of neurotrophic factors, ciliary neurotrophic factor (CNTF), and pituitary adenylate cyclase-activating peptide (PACAP). Here we demonstrate that PACAP promotes the differentiation of nascent dorsal root ganglion (DRG) neurons in that it increases both the number of neural-marker-positive cells and axonogenesis without affecting the proliferation of neural progenitor cells. This response is mediated through the PAC1 receptor and requires MAP kinase activation. Moreover, we find that, in the absence of exogenously added PACAP, blockade of the PAC1 receptor inhibits neuronal differentiation. These data coupled with our finding that both PACAP and the PAC1 receptor are expressed during the peak period of neuronal differentiation in the DRG suggest that PACAP functions in vivo to promote the differentiation of nascent sensory neurons. Interestingly, we also demonstrate that the neurotrophic factors NT-3 and CNTF completely block the PACAP-induced neuronal differentiation. This points to the intricate integration of cellular signals by nascent neurons and, to our knowledge, is the first evidence for neurotrophic factor abrogation of a pathway regulated by G-protein-coupled receptors (GPCRs).