Mechanisms of postnatal cutaneous afferent development during inflammation

描述（由申请人提供）：许多儿科疾病和对儿童进行的外科手术都会导致疼痛状态，但众所周知，目前针对疼痛的药物治疗对儿童有不良影响，如轻度恶心或呕吐，但也有更严重的副作用，如呼吸抑制甚至死亡。因此，为了开发更适合儿童疼痛的治疗方法，更好地了解特定的感觉传入人群如何可能导致儿童疼痛状态是至关重要的。虽然对成人外周损伤后皮肤伤害感受器的功能特性和可塑性了解很多，但对发育过程中感觉传入的功能特性和外周损伤后致敏的机制知之甚少。本研究的主要目标是在幼年小鼠和出生后炎症模型的整个发育过程中对皮肤感觉传入神经进行功能表征，并开始确定与感觉传入神经变化有关的一些机制。为了确定这一点，我们在新生/产后小鼠中开发了离体毛状皮肤、隐神经、背根神经节（DRG）、脊髓记录准备，使我们能够在皮肤损伤前后全面表型感觉纤维。在Specific Aim 1中，我们将首先结合体外记录和免疫细胞化学分析，分析这些传入事件在出生后发育不同时期的功能、解剖和神经化学表型。功能的改变将与各种受体/通道的mRNA和蛋白质表达的改变相关，这些受体/通道被认为与DRGs的感觉功能有关。接下来，在Specific Aim 2中，我们将进行与SA1中描述的类似的研究，除了我们将分析出生后发育过程中不同时间毛状皮肤注射卡拉胶后感觉传入的综合表型，并将这些数据与DRGs基因表达的变化联系起来，以确定观察到的皮肤传入功能变化的潜在机制。这些实验将使我们能够表征所有类型的皮肤感觉神经元在整个发育过程和周围炎症期间的变化，并有可能确定与发育中的感觉神经元如何对损伤作出反应相关的独特的年龄依赖性机制。这些研究将为未来的实验奠定基础，使用体内siRNA介导的单个周围神经基因敲低，并结合离体记录准备，我们将能够分析在正常（未受伤）发育和出生后皮肤炎症的不同时期特定受体/通道变化的作用。这些和未来的研究将使我们更好地了解感觉纤维的变化如何影响儿童疼痛，也可能导致建立更合适的儿童疼痛治疗方法。

DESCRIPTION (provided by applicant): Numerous pediatric disorders and surgical procedures performed on children lead to pain states yet it is well known that current pharmacological treatments for pain have adverse effects in children such as mild nausea or vomiting but also more severe side effects like respiratory depression and even death. Thus in order to develop more appropriate therapies for pain in children, a better understanding of how particular sensory afferent populations may contribute to pediatric pain states is of utmost importance. While much is known about the functional properties and plasticity of cutaneous nociceptors following peripheral injuries in adults, relatively little is known about the functiona properties of sensory afferents during development and the mechanisms of sensitization after peripheral injury. The main goal of this proposal is to functionally characterize cutaneous sensory afferents throughout development in naive mice and in models of postnatal inflammation, and begin to determine some of the mechanisms involved with the changes in sensory afferents. In order to determine this, we developed an ex vivo hairy skin, saphenous nerve, dorsal root ganglion (DRG), spinal cord recording preparation in neonatal/ postnatal mice that enables us to comprehensively phenotype sensory fibers before and after cutaneous injury. In Specific Aim 1, we will first analyze the functional, anatomical, and neurochemical phenotypes of these afferents at various times during postnatal development using combinations of ex vivo recording and immunocytochemical analyses. Changes in function will then be correlated with alterations in mRNA and protein expression of various receptors/ channels thought to be involved in sensory function in the DRGs. Next, in Specific Aim 2, we will perform similar studies as described in SA1 except we will analyze the comprehensive phenotypes of sensory afferents after hairy skin injection of carrageenan at different times during postnatal development and correlate these data with changes in gene expression in the DRGs to determine the potential mechanisms of the observed functional changes in cutaneous afferents. These experiments will enable us to characterize the changes in all types of cutaneous sensory neurons throughout development and during peripheral inflammation, and potentially identify unique age-dependent mechanisms associated with how developing sensory neurons respond to injury. These studies will establish the foundation of future experimentation using in vivo siRNA- mediated knockdown of genes in single peripheral nerves in conjunction with ex vivo recording preparations where we will be able to analyze the roles of changes in specific receptors/ channels during normal (uninjured) development and during different times of postnatal cutaneous inflammation. These and future studies will allow us to better understand how changes in sensory fibers impact pediatric pain and may also lead to the establishment of more suitable treatments for pain in children.