The Regulation Of Carotid Body In Hypoxic Injury Of The Chl1-null Mice

There are a series of compensatory reactions in vivo to maintain its oxygen concentration balance in hypoxia. Carotid body (CB) is a small chemoreceptor organ that senses partial pressure of oxygen in arterial blood. It releases neurotransmitters and initiates compensatory reflex responses via increased afferent nerve activity, resulting in relevant changes in ventilation. The CB is composed of clusters of neuron-like glomus, or type-I cells enveloped by glia-like sustentacular, or type-II cells. In response to hypoxia, glomus cells release one (or more) transmitter(s), which acting on the nerve terminals of chemosensory neurons, increases the afferent discharge. Among several molecules present in glomus cells, dopamine, acetylcholine and 5-adenosine-triphosphate have been proposed to be the excitatory transmitters in the CB.CHL1(close homologue of L1) is a member of the L1 family of neural adhension molecules, which is a subgroup of the Ig superfamily. The studies on CHL1 mainly focus on axonal growth, cell migration, fasciculation, synaptogenesis, synaptic remodeling, learning ability and memory. CHL1-null mice (CHL1 ) can grow and survival normally, but the synaptogenesis, learning and memory in adult have some defects. Our previous research showed that CHL1 plays a negative regulator in development of NPCs both in vivo and in vitro, and ERK1/2 is involoved in the function of CHL1-deficient NPCs. We also found that CHL1 gene deletion increases tolerance to acute hypoxia.How does the gene deletion enhance the anti-hypoxia ablity? Dose CB engage in this regulation? And what is the role of CHL1 play in this regulation? Here, we have investigated the functions of CHL1 in the regulation of hypoxic injury. 1. Anti-hypoxia activity of CHL1?/? miceCHL1~+/~+ and CHL1?/? male mice were exposed to normobaric hypoxia chamber (O2 concentration: 5%). Record the survival time of mice. Kaplan-Meier curves illustrate the difference in survival rates between CHL1~+/~+ and CHL1?/? mice. CHL1 deficiency show a statistically significant increase in hypoxic survival time. This result show that CHL1 deficiency increases tolerance to acute hypoxia in mice.2. Expression and location of CHL1 in CBCB is mainly composed by clusters of neuron-like glomus (TH) cells enveloped by glia-like sustentacular (GFAP or Nestin ) cells. Immunofluorescence staining of CHL1 in vivo and vitro, we found that:1. CHL1 were located on the glomus cells membrane and other cells membrane and cytoplasm. CHL1 express in two type cells in CB, it may play an important role in the function of CB.2. We then isolated primary cells from carotid bodies. We gain two type (TH~+ or GFAP~+) cells. CHL1 mainly located in the TH~+ and GFAP~+ cells'cytoplasm. But after 3 days culture, CHL1 expressed in TH~+ cells'cytoplasm and nucelus.3. Morphology of CB in CHL1?/? miceIn order to find the reason of CHL1 deficiency increases tolerance to acute hypoxia in mice, we study the impact of CHL1 deficiency on the morphology of carotid body.1. The total carotid body volume and the volume occupied by TH-positive cells were significantly increased in CHL1?/? mice compared with CHL1~+/~+ mice. The number of TH-positive cells were also significantly increased in CHL1?/? mice.2. CHL1 deficiency markedly enhanced expression of TH protein in the CB of CHL1?/? mice compared with CHL1~+/~+ mice. But the differences of GFAP and Nestin were not significant.4. CHL1 deficiency on the reaction of carotid body to acute hypoxiaWe performed the studies of carotid body sinus nerve activity and ventilatory responses to hypoxia.1. Using Buxco whole body plethysmography, we recorded the respiratory rate (RR) and the minute ventilation (VE) to hypoxia (O2 concentration: 5%) in CHL1?/? and control mice. The result showed that there are no significant differences of RR and VE between CHL1?/? and control mice. But the respiratory rate (RR) to hypoxia in CHL1?/? mice was significantly increased, and the minute ventilation(VE) to hypoxia in CHL1?/? mice was significantly enlarged.2. By a period hypoxic Krebs'solution superfusing, we recording of the afferent nerve responses to hypoxia in the isolated carotid body sinus nerve preparation taken from the CHL+/+ and CHL1?/? mice. The firing frequency of the afferent nerve responses is significantly higher in CHL1+/+ mice compared with CHL1?/? mice.5. Influence of acute hypoxia on of CHL1 expression in different tissues of mice To observe the effects of acute hypoxia on the CHL1 expression levels in different brain areas and main organs (heart,lung,kidney) of mice, and provide a basis for the role of CHL1 in hypoxia injury. We performed a study of CHL1 protein expression in different tissues by acute hypoxia (8% O2, 8h) treatment.1. In central nervous system, CHL1 protein expression was down-regulated in cerebral cortex, hypothalamus and brain stem by acute hypoxia and up-regulated in cerebellum.2. In heart and lung, CHL1 protein expression was down-regulated by acute hypoxia.CHL1 protein expressions were changed in different tissues after acute hypoxia, which suggested CHL1 might play an important role in hypoxia damage regulation.In summary, here we first investigate the regulation of CB in hypoxic injury of the CHL1?/? mice. Anti-hypoxia activity of CHL1?/? mice may regulated by the increase the number of TH+ cells. Because it enhanced the reaction of CB to acute hypoxia. And the Influence of acute hypoxia on of CHL1 expression in different tissues of mice provides new clues for the functions of CHL1 in the regulation of anti-hypoxia.