Cha John, Filatov Gregory, Smith Steven J, Gammaitoni Arnold R, Lothe Amélie, Reeder Thadd
University of California San Francisco, San Francisco, California, USA.
Zogenix, Inc. (now a part of UCB), Emeryville, California, USA.
Epilepsia Open. 2024 Feb;9(1):300-313. doi: 10.1002/epi4.12873. Epub 2023 Dec 22.
In patients with Dravet syndrome (DS), fenfluramine reduced convulsive seizure frequency and provided clinical benefit in nonseizure endpoints (e.g., executive function, survival). In zebrafish mutant scn1 DS models, chronic fenfluramine treatment preserved neuronal cytoarchitecture prior to seizure onset and prevented gliosis; here, we extend these findings to a mammalian model of DS (Scn1a mice) by evaluating the effects of fenfluramine on neuroinflammation (degenerated myelin, activated microglia) and survival.
Scn1a DS mice were treated subcutaneously once daily with fenfluramine (15 mg/kg) or vehicle from postnatal day (PND) 7 until 35-37. Sagittal brain sections were processed for immunohistochemistry using antibodies to degraded myelin basic protein (D-MBP) for degenerated myelin, or CD11b for activated (inflammatory) microglia; sections were scored semi-quantitatively. Apoptotic nuclei were quantified by TUNEL assay. Statistical significance was evaluated by 1-way ANOVA with post-hoc Dunnett's test (D-MBP, CD11b, and TUNEL) or Logrank Mantel-Cox (survival).
Quantitation of D-MBP immunostaining per 0.1 mm unit area of the parietal cortex and hippocampus CA3 yielded significantly higher spheroidal and punctate myelin debris counts in vehicle-treated DS mice than in wild-type mice. Fenfluramine treatment in DS mice significantly reduced these counts. Activated CD11b + microglia were more abundant in DS mouse corpus callosum and hippocampus than in wild-type controls. Fenfluramine treatment of DS mice resulted in significantly fewer activated CD11b + microglia than vehicle-treated DS mice in these brain regions. TUNEL staining in corpus callosum was increased in DS mice relative to wild-type controls. Fenfluramine treatment in DS mice lowered TUNEL staining relative to vehicle-treated DS mice. By PND 35-37, 55% of control DS mice had died, compared with 24% of DS mice receiving fenfluramine treatment (P = 0.0291).
This is the first report of anti-neuroinflammation and pro-survival after fenfluramine treatment in a mammalian DS model. These results corroborate prior data in humans and animal models and suggest important pharmacological activities for fenfluramine beyond seizure reduction.
Dravet syndrome is a severe epilepsy disorder that impairs learning and causes premature death. Clinical studies in patients with Dravet syndrome show that fenfluramine reduces convulsive seizures. Additional studies suggest that fenfluramine may have benefits beyond seizures, including promoting survival and improving control over emotions and behavior. Our study is the first to use a Dravet mouse model to investigate nonseizure outcomes of fenfluramine. Results showed that fenfluramine treatment of Dravet mice reduced neuroinflammation significantly more than saline treatment. Fenfluramine-treated Dravet mice also lived longer than saline-treated mice. These results support clinical observations that fenfluramine may have benefits beyond seizures.
在患有德雷维特综合征（DS）的患者中，芬氟拉明可降低惊厥发作频率，并在非癫痫发作终点（如执行功能、生存率）方面提供临床益处。在斑马鱼突变体scn1 DS模型中，长期使用芬氟拉明治疗可在癫痫发作开始前保留神经元细胞结构并预防胶质增生；在此，我们通过评估芬氟拉明对神经炎症（变性髓鞘、活化小胶质细胞）和生存率的影响，将这些发现扩展到DS的哺乳动物模型（Scn1a小鼠）。
从出生后第7天（PND）至35 - 37天，Scn1a DS小鼠每天皮下注射一次芬氟拉明（15 mg/kg）或赋形剂。矢状脑切片用于免疫组织化学，使用针对变性髓鞘的降解髓鞘碱性蛋白（D-MBP）抗体或针对活化（炎性）小胶质细胞的CD11b抗体；切片进行半定量评分。通过TUNEL法对凋亡细胞核进行定量。采用单因素方差分析及事后Dunnett检验（D-MBP、CD11b和TUNEL）或Logrank Mantel-Cox检验（生存率）评估统计学意义。
在顶叶皮质和海马CA3每0.1 mm单位面积上，对D-MBP免疫染色进行定量分析，发现用赋形剂处理的DS小鼠中球形和点状髓鞘碎片计数显著高于野生型小鼠。DS小鼠用芬氟拉明治疗后，这些计数显著降低。在DS小鼠的胼胝体和海马中，活化的CD11b +小胶质细胞比野生型对照更丰富。在这些脑区，用芬氟拉明治疗的DS小鼠中活化的CD11b +小胶质细胞比用赋形剂处理的DS小鼠显著减少。与野生型对照相比，DS小鼠胼胝体中的TUNEL染色增加。与用赋形剂处理的DS小鼠相比，DS小鼠用芬氟拉明治疗降低了TUNEL染色。到PND 35 - �7时，55%的对照DS小鼠死亡，而接受芬氟拉明治疗的DS小鼠为24%（P = 0.0291）。
这是关于芬氟拉明治疗哺乳动物DS模型后抗神经炎症和促生存作用的首次报道。这些结果证实了先前在人类和动物模型中的数据，并表明芬氟拉明除了减少癫痫发作外还具有重要药理学活性。
德雷维特综合征是一种严重的癫痫疾病，会损害学习能力并导致过早死亡。对德雷维特综合征患者的临床研究表明，芬氟拉明可减少惊厥发作。其他研究表明，芬氟拉明可能除了癫痫发作外还有益处，包括提高生存率以及改善对情绪和行为的控制。我们的研究首次使用德雷维特小鼠模型来研究芬氟拉明的非癫痫发作结局。结果表明，与盐水治疗相比，芬氟拉明治疗德雷维特小鼠可显著降低神经炎症。用芬氟拉明治疗的德雷维特小鼠也比用盐水治疗的小鼠寿命更长。这些结果支持了芬氟拉明可能除癫痫发作外还有益处的临床观察。
