METHODS: We induced pneumococcal meningitis in approximately 500 C57Bl6 adult mice using live Streptococcus pneumoniae (serotype 3, 1 × 105 colony forming units (cfu) in 10 µl) injected directly into the cisterna magna of anesthetized mice and treated these mice with ceftriaxone daily until recovered. We evaluated hearing thresholds over time, characterized the cochlear inflammatory response, and quantified the amount of new bone formation during meningitis recovery. We used microcomputed tomography (microCT) scans to quantify cochlear volume loss caused by neo-ossification. We also performed perilymph sampling in live mice to assess the integrity of the blood-perilymph barrier during various time intervals after meningitis. We then evaluated the effect of CX3CR1 or CCR2 deletion in meningitis symptoms, hearing loss, macrophage/monocyte recruitment, neo-ossification, and blood labyrinth barrier function.
RESULTS: Sixty percent of mice with pneumococcal meningitis developed hearing loss. Cochlear fibrosis could be detected within 4 days of infection, and neo-ossification by 14 days. Loss of spiral ganglion neurons was common, and inner ear anatomy was distorted by scarring caused by new soft tissue and bone deposited within the scalae. The blood-perilymph barrier was disrupted at 3 days post infection (DPI) and was restored by seven DPI. Both CCR2 and CX3CR1 monocytes and macrophages were present in the cochlea in large numbers after infection. Neither chemokine receptor was necessary for the induction of hearing loss, cochlear fibrosis, ossification, or disruption of the blood-perilymph barrier. CCR2 knockout (KO) mice suffered the most severe hearing loss. CX3CR1 KO mice demonstrated an intermediate phenotype with greater susceptibility to hearing loss compared to control mice. Elimination of CX3CR1 mononuclear phagocytes during the first 2 weeks after meningitis in CX3CR1-DTR transgenic mice did not protect mice from any of the systemic or hearing sequelae of pneumococcal meningitis.
CONCLUSIONS: Pneumococcal meningitis can have devastating effects on cochlear structure and function, although not all mice experienced hearing loss or cochlear damage. Meningitis can result in rapid progression of hearing loss with fibrosis starting at four DPI and ossification within 2 weeks of infection detectable by light microscopy. The inflammatory response to bacterial meningitis is robust and can affect all three scalae. Our results suggest that CCR2 may assist in controlling infection and maintaining cochlear patency, as CCR2 knockout mice experienced more severe disease, more rapid hearing loss, and more advanced cochlear ossification after pneumococcal meningitis. CX3CR1 also may play an important role in the maintenance of cochlear patency.
方法:我们使用活的肺炎链球菌(血清型3,10μl的1×105个菌落形成单位(cfu))直接注射到约500只C57Bl6成年小鼠中诱导了肺炎球菌性脑膜炎麻醉小鼠,并每天用头孢曲松治疗这些小鼠直至恢复。随着时间的推移,我们评估了听力阈值,以耳蜗炎症反应为特征,并量化脑膜炎恢复期间新骨形成的数量。我们使用显微计算机断层扫描(microCT)扫描来量化新骨化引起的耳蜗体积损失。我们还在活小鼠中进行了外淋巴采样,以评估脑膜炎后不同时间间隔内血液-外淋巴屏障的完整性。然后我们评估CX3CR1或CCR2缺失在脑膜炎症状中的作用,听力损失,巨噬细胞/单核细胞募集,新骨化,和血液迷宫屏障功能。
结果:60%的肺炎球菌性脑膜炎小鼠出现听力损失。在感染后4天内可检测到耳蜗纤维化,和新骨化14天。螺旋神经节神经元的丢失是常见的,内耳解剖结构由于新的软组织和骨沉积在鳞内而引起的疤痕而扭曲。在感染后3天(DPI),血-外淋巴屏障被破坏,并通过7个DPI恢复。CCR2和CX3CR1单核细胞和巨噬细胞在感染后大量存在于耳蜗中。两种趋化因子受体都不是诱发听力损失所必需的,耳蜗纤维化,骨化,或者血-外淋巴屏障的破坏。CCR2敲除(KO)小鼠遭受最严重的听力损失。与对照小鼠相比,CX3CR1KO小鼠表现出对听力损失具有更大易感性的中间表型。在CX3CR1-DTR转基因小鼠脑膜炎后的前2周内,CX3CR1单核吞噬细胞的消除不能保护小鼠免受肺炎球菌性脑膜炎的任何全身性或听力后遗症。
结论:肺炎球菌性脑膜炎可对耳蜗结构和功能产生破坏性影响,虽然不是所有的小鼠都经历了听力损失或耳蜗损伤。脑膜炎可导致听力损失的快速进展,从四个DPI开始出现纤维化,并在感染后2周内通过光学显微镜检测到骨化。对细菌性脑膜炎的炎症反应是强烈的,并且可以影响所有三种鳞片。我们的结果表明,CCR2可能有助于控制感染和维持耳蜗通畅,随着CCR2基因敲除小鼠经历更严重的疾病,更快的听力损失,肺炎球菌性脑膜炎后耳蜗骨化更严重。CX3CR1也可能在维持耳蜗通畅中起重要作用。