Influence of aromatase on neuromuscular plasticity resulting from testosterone plus locomotor training after spinal cord injury

脊髓损伤(SCI)引起的运动功能障碍是由神经损伤引起的 因其他阻碍运动神经元存活和肌肉恢复的因素而加剧，包括停用和低水平 睾酮(T)。体重支持的跑步机训练(BWSTT)的成功随着脊髓损伤的减少而减少 严重程度会恶化。我们开发了一种新的策略，将BWSTT与T-enanthate(TE)佐剂药物结合起来 部分通过保留脊髓损伤处的白质来促进依赖使用的神经可塑性的治疗 通过支持运动神经元的存活，刺激神经肌肉的恢复。然而， 我们使用的超生理剂量在我们的啮齿动物严重挫伤脊髓损伤模型中产生了前列腺肿大， 这限制了翻译。这项建议的目的是提高BWSTT+TE的翻译适用性 通过确定增强BWSTT介导的神经运动改善的最低TE剂量，努力 限制前列腺生长和其他雄激素副作用。其次，我们将确定雌二醇的影响 (E2)对BWSTT介导的运动恢复和神经肌肉可塑性的影响。后者在以下方面仍然很重要 翻译和机械论的观点，因为(1)T通过芳香酶在中央 (2)E_2治疗对大鼠脊髓损伤后有明显的神经保护作用。要提供 神经可塑性的综合证据，我们将评估我们的啮齿动物脊髓损伤模型的功能适应 对建议的治疗的反应和评估发生在脊髓病变和 病变远端，脊髓运动神经元和肌肉中。为支持这项建议，我们成立了一个 雄性啮齿动物严重中胸挫伤模型，表现为持续性后肢瘫痪和 与非脊髓损伤动物相比，进行性肌肉萎缩和循环T降低50%，类似于T缺乏 几乎存在于所有男性的脊髓损伤后。我们的数据表明，BWSTT+TE恢复了后肢地面行走 严重脊髓损伤后比BWSTT(单独)或TE(单独)更严重。此外，BWSTT+TE提高了回收率 肌纤维横截面积(FCSA)，肌力输出，并防止有害的慢(氧化) 到肌肉中快速的(糖酵解)纤维型转变。在目标1中，我们将进行剂量优化实验，以 确定最低有效TE剂量，我们将以原始数据为基础，通过综合评估 BWSTT+TE后的运动恢复和神经肌肉可塑性。主要结果是开放的-- 野战运动恢复、比目鱼肌功能、比目鱼肌FCSA和纤维类型分布。在实验1a中， SCI动物将继续不接受治疗(车辆)或将接受BWSTT与低、中、高剂量TE， 最低有效剂量提前。在实验1b中，脊髓损伤动物将接受Vehicle、BWSTT、TE或 BWSTT+TE。然后，目标2将确定雌二醇(E2)对BWSTT介导的运动恢复的影响 和神经肌肉的可塑性。在实验2a中，脊髓损伤大鼠接受Vehicle、BWSTT、BWSTT+AN(芳香酶 阻止T向E2转换的抑制剂)、BWSTT+TE或BWSTT+TE+An。实验2b将是一个剂量- 确定增强BWSTT介导的神经运动改善的最低E2剂量的优化研究。 脊髓损伤动物将接受低、中、高剂量E2的赋形剂或BWSTT。实验2c将是一个 脊髓损伤动物将接受赋形剂、BWSTT或E2(单独和联合使用)的比较疗效研究， 或BWSTT+TE。我们假设，神经肌肉可塑性的功能和解剖学测量将 在接受BWSTT+TE的脊髓损伤动物中，最明显的是E2部分介导了神经肌肉的可塑性 BWSTT+TE的结果，并且将出现更强大的神经运动改善 BWSTT+TE与BSWTT+E2。我们预计这些实验将提供必要的数据来 为我们的多模式治疗建立临床前原则证明。如果成功，这将为 将我们的临床前发现转化为未来的临床试验，旨在加速运动恢复和改善 脊髓损伤退伍军人的神经肌肉功能。

The motor dysfunction resulting from spinal cord injury (SCI) is precipitated by the neural insult and is exacerbated by other factors that hinder motoneuron survival and muscle recovery, including disuse and low testosterone (T). The success of bodyweight-supported treadmill training (BWSTT) diminishes as the SCI severity worsens. We have developed a novel strategy involving BWSTT with adjuvant T-enanthate (TE) drug treatment that promotes use-dependent neuroplasticity, in-part, by preserving white matter at the spinal lesion and by supporting motoneuron survival, which stimulates neuromuscular recovery. However, the supraphysiologic TE dose we used produced prostate enlargement in our rodent severe contusion SCI model, which limits translation. The purpose of this proposal is to improve the translational applicability of BWSTT+TE by identifying the lowest TE dose that enhances BWSTT-mediated neuromotor improvement, in an effort to limit prostate growth and other androgenic side-effects. Secondly, we will determine the influence of estradiol (E2) on BWSTT-mediated locomotor recovery and neuromuscular plasticity. The latter remains important from translational and mechanistic perspectives because (1) T is converted to E2, via aromatase, within the central nervous system and (2) E2 treatment produces potent neuroprotection in rats after SCI. To provide comprehensive evidence of neuroplasticity, we will evaluate functional adaptations in our rodent SCI model in response to the proposed treatments and assess anatomical changes that occur at the spinal cord lesion and distal to the lesion, and in spinal motoneurons and muscle. To support this proposal, we have established a male rodent severe mid-thoracic contusion SCI model that exhibits persistent hindlimb paralysis and progressive muscle decline and 50% lower circulating T than non-SCI animals, similar to the T deficiency present in nearly all men after SCI. Our data indicate that BWSTT+TE restored hindlimb overground walking after severe SCI more so than BWSTT (alone) or TE (alone). Moreover, BWSTT+TE improved recovery of muscle fiber cross-sectional area (fCSA), muscle force output, and prevented the deleterious slow (oxidative) to fast (glycolytic) fiber-type transition in muscle. In Aim 1, we will perform a dose-optimization experiment to identify the lowest effective TE dose and we will build upon our original data by comprehensively evaluating locomotor recovery and neuromuscular plasticity in response to BWSTT+TE. The primary outcomes are open- field locomotor recovery, soleus muscle function, soleus fCSA and fiber type distribution. In Experiment 1a, SCI animals will remain untreated (vehicle) or will receive BWSTT with low, moderate, or high-dose TE, with the lowest effective dose advancing. In Experiment 1b, SCI animals will receive vehicle, BWSTT, TE, or BWSTT+TE. Aim 2 will then determine the influence of estradiol (E2) on BWSTT-mediated locomotor recovery and neuromuscular plasticity. In Experiment 2a, SCI rats will receive vehicle, BWSTT, BWSTT+AN (aromatase inhibitor that blocks conversion of T to E2), BWSTT+TE, or BWSTT+TE+AN. Experiment 2b will be a dose- optimization study to identify the lowest E2 dose that enhances BWSTT-mediated neuromotor improvement. SCI animals will receive vehicle or BWSTT with low-, moderate-, or high-dose E2. Experiment 2c will be a comparative efficacy study in which SCI animals will receive vehicle, BWSTT or E2 (alone and in combination), or BWSTT+TE. We hypothesize that the functional and anatomical measures of neuromuscular plasticity will be most evident in SCI animals receiving BWSTT+TE, that E2 partially mediates neuromuscular plasticity resulting from BWSTT+TE, and that more robust neuromotor improvement will occur in response to BWSTT+TE versus BSWTT+E2. We anticipate that these experiments will provide the necessary data to establish preclinical proof-of-principle for our multimodal therapy. If successful, this will set the stage for translation of our preclinical findings to future clinical trials intended to hasten locomotor recovery and improve neuromuscular function in Veterans with SCI.