Amyotrophic lateral sclerosis still depicts an incurable and devastating disease. Drug development efforts are mostly based on SOD1-G93A mice that present a very strong and early phenotype, allowing only a short time window for intervention. An alternative mouse model is available, that is based on the same founder line but has a reduced SOD1-G93A copy number, resulting in a weaker and delayed phenotype. To be able to use these SOD1-G93A/low mice for drug testing, we performed a characterization of ALS-typical pathologies. All analyses were performed compared to non-transgenic littermates of the same sex and age. In vivo analysis of SOD1-G93A/low mice was performed by weekly body weight measurements, analysis of the survival rate and measurement of the muscle strength of 24 to 30 weeks old female and male SOD1-G93A/low mice. Immunofluorescent labeling of SOD1, GFAP and Iba1 protein was performed in the cervical, thoracic, and lumbar ventral horn of the spinal cord of 24 to 30 weeks old male and female SOD1-G93A/low mice. The musculus gastrocnemius of male SOD1-G93A/low mice was labeled with fluorophore conjugated α-bungarotoxin and antibody against phosphorylated neurofilaments. Fluorescent labeling was detected and quantified by macro-based image analysis. Although SOD1 protein levels were highly increased in both sexes and all age groups, levels strongly peaked in 30 weeks old male SOD1-G93A/low mice. Astrocytosis and activated microglia in the spinal cord ventral horn and phosphorylated neurofilaments in the motor unit of the musculus gastrocnemius progressively increased while muscle strength progressively decreased in male SOD1-G93A/low mice. In female SOD1-G93A/low mice, only activated microglia increased progressively while muscle strength was constantly reduced starting at 26 weeks. These differences result in a shorter survival time of male SOD1-G93A/low mice of about 3 weeks compared to female animals. Our results suggest, that male SOD1-G93A/low mice present a stronger pathology and are therefore better suitable to evaluate the efficacy of new drugs against ALS as most pathological features are developing progressively paralleled by a survival time that allows a treatment start before symptom onset.

中文翻译：

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肌萎缩侧索硬化 SOD1-G93A 低拷贝数转基因小鼠模型神经病理学特征的评估

肌萎缩侧索硬化仍然是一种无法治愈的毁灭性疾病。药物开发工作主要基于 SOD1-G93A 小鼠，这些小鼠表现出非常强的早期表型，只允许很短的时间窗口进行干预。可以使用另一种小鼠模型，该模型基于相同的创始人系，但 SOD1-G93A 拷贝数减少，导致表型较弱和延迟。为了能够使用这些 SOD1-G93A/低小鼠进行药物测试，我们对 ALS 典型病理进行了表征。所有分析均与相同性别和年龄的非转基因同窝仔猪进行比较。通过每周测量体重、分析存活率和测量 24 至 30 周龄雌性和雄性 SOD1-G93A/低小鼠的肌肉力量，对 SOD1-G93A/低小鼠进行体内分析。在 24 至 30 周龄雄性和雌性 SOD1-G93A/低小鼠脊髓的颈、胸和腰腹角进行 SOD1、GFAP 和 Iba1 蛋白的免疫荧光标记。雄性 SOD1-G93A/low 小鼠的腓肠肌用荧光团标记的 α-银环蛇毒素和针对磷酸化神经丝的抗体进行标记。通过基于宏观的图像分析检测和量化荧光标记。尽管 SOD1 蛋白水平在两性和所有年龄组中均显着增加，但在 30 周龄雄性 SOD1-G93A/低小鼠中，其水平强烈达到峰值。在雄性 SOD1-G93A/low 小鼠中，脊髓腹角中的星形细胞增多症和活化的小胶质细胞以及腓肠肌运动单位中的磷酸化神经丝逐渐增加，而肌肉力量逐渐降低。在雌性 SOD1-G93A/low 小鼠中，只有激活的小胶质细胞逐渐增加，而肌肉力量从 26 周开始不断降低。这些差异导致雄性 SOD1-G93A/低小鼠的存活时间比雌性动物短约 3 周。我们的结果表明，雄性 SOD1-G93A/low 小鼠表现出更强的病理学，因此更适合评估新药对 ALS 的疗效，因为大多数病理特征正在逐渐发展，并且存活时间允许在症状出现之前开始治疗.