Baronti Dario, Tomov Nikola, Hupp Sabrina, Mitchell Timothy J, Iliev Asparouh I
Institute of Anatomy, University of Bern, Bern, Switzerland.
School of Immunity and Infection, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom.
Front Neurosci. 2023 Jan 10;16:912445. doi: 10.3389/fnins.2022.912445. eCollection 2022.
(pneumococcus) meningitis is a serious disease with substantial lethality and long-term disability in survivors. Loss of synaptic staining in the superficial layers of the neocortex in rodent models and in humans, and pneumolysin (a major pneumococcal toxin)-dependent dendritic spine collapse in brain slices have been described. It remains unclear how deep in the neocortex more discrete changes are present, how soon after disease onset these changes occur, and whether other properties of dendrites are also affected.
Using a mouse model of pneumococcal meningitis, we studied changes in the neocortex shortly (3-6 h) after the onset of clinical symptoms modified Golgi-Cox silver staining.
Dendritic changes were present in areas with otherwise unchanged cell numbers and no signs of necrosis or other apparent neuronal pathology. Mature dendritic spines were reduced in the pyramidal neurons running through layers 1-5. Additionally, spine morphology changes (swelling, spine neck distortion), were also observed in the deeper layers 4 and 5 of the neocortex. Immature spines (filopodia) remained unchanged between groups, as well as the dendritic arborization of the analyzed neurons. In a third of the animals with meningitis, massive mechanical distortion of the primary dendrites of most of the pyramidal neurons through layers 1-5 was observed. This distortion was reproduced in acute brain slices after exposure to pneumolysin-containing bacterial lysates ( D39 strain), but not to lysates of pneumolysin-deficient bacteria, which we explain by the tissue remodeling effect of the toxin. Experimental mechanical dendrite distortion in primary neural cultures demonstrated diminished FRAP diffusion of neuronally-expressed enhanced green fluorescent protein (eGFP), indicative of disturbed dendritic diffusion.
Our work extends earlier knowledge of synaptic loss in the superficial cortical layers during meningitis to deeper layers. These changes occurred surprisingly early in the course of the disease, substantially limiting the effective therapeutic window. Methodologically, we demonstrate that the dendritic spine collapse readout is a highly reliable and early marker of neural damage in pneumococcal meningitis models, allowing for reduction of the total number of animals used per a group due to much lower variation among animals.
肺炎球菌性脑膜炎是一种严重疾病，幸存者有很高的致死率和长期残疾风险。在啮齿动物模型和人类中，已发现新皮层表层的突触染色缺失，以及脑片中肺炎溶血素（一种主要的肺炎球菌毒素）依赖性的树突棘塌陷。目前尚不清楚新皮层中更细微的变化出现在多深的位置，疾病发作后多久会出现这些变化，以及树突的其他特性是否也会受到影响。
利用肺炎球菌性脑膜炎小鼠模型，我们在临床症状出现后不久（3 - 6小时），采用改良的高尔基-考克斯银染色法研究新皮层的变化。
在细胞数量未变且无坏死迹象或其他明显神经元病理特征的区域出现了树突变化。贯穿第1 - 5层的锥体神经元中成熟树突棘减少。此外，在新皮层较深的第4和第5层也观察到了树突棘形态变化（肿胀、树突棘颈部扭曲）。各组之间未成熟树突棘（丝状伪足）以及所分析神经元的树突分支保持不变。在三分之一的脑膜炎动物中，观察到贯穿第1 - 5层的大多数锥体神经元的初级树突出现大量机械性扭曲。在暴露于含肺炎溶血素的细菌裂解物（D39菌株）后的急性脑片中也出现了这种扭曲，但暴露于缺乏肺炎溶血素的细菌裂解物时未出现，我们将其解释为毒素的组织重塑作用。原代神经培养中的实验性机械性树突扭曲表明，神经元表达的增强型绿色荧光蛋白（eGFP）的荧光恢复率（FRAP）扩散减弱，这表明树突扩散受到干扰。
我们的工作将之前关于脑膜炎期间皮层表层突触丢失的认识扩展到了更深层。这些变化在疾病过程中出现得惊人地早，极大地限制了有效的治疗窗口。在方法上，我们证明树突棘塌陷读数是肺炎球菌性脑膜炎模型中神经损伤的高度可靠且早期的标志物，由于动物之间的变异性低得多，每组使用的动物总数得以减少。
