Cytomegalovirus initiates infection selectively from high-level ß1 integrin-expressing cells in the brain.

Cytomegalovirus (CMV) is a prevalent pathogen in intrauterine infections that causes congenital anomalies such as CMV encephalitis, which is characterized by the focal areas of reactive gliosis, reactive mononuclear cells, microglial nodules, and ventriculoencephalitis. To elucidate the mechanisms of CMV susceptibility in the developing brain, cell tropism and the infectious dynamics of CMV infection were investigated. We evaluated intraventricular and intravascular infections from the perspective of the distribution of CMV and its receptor (ß1 integrin) in the earliest phase of infection. Murine CMV (MCMV) immediate early 1-positive cells were colocalized mainly with meninges and choroid plexus (after intraventricular infection) or with endothelial cells and pericytes (after intravascular infection). Using green fluorescent protein-expressing recombinant MCMV particles and fluorescent microbeads (100 to 300 nm), we revealed that CMV particle size is the primary factor determining the initial CMV distribution. ß1 Integrin inhibition using a shRNA and functional blocking antibody significantly reduced MCMV infection. IHC analysis, flow cytometric, and brain slice analyses strongly support that high-level ß1 integrin-expressing cells (eg, endothelial cells, pericytes, meninges, choroid plexus, and neural stem progenitor cells) are the first targets of MCMV. Therefore, our data demonstrate that the initial distributions of MCMV particles and ß1 integrin determine the distinct pattern of infection in the brain in the acute phase.

Intracerebroventricular and Intravascular Injection of Viral Particles and Fluorescent Microbeads into the Neonatal Brain.

In the study on the pathogenesis of viral encephalitis, the infection method is critical. The first of the two main infectious routes to the brain is the hematogenous route, which involves infection of the endothelial cells and pericytes of the brain. The second is the intracerebroventricular (ICV) route. Once within the central nervous system (CNS), viruses may spread to the subarachnoid space, meninges, and choroid plexus via the cerebrospinal fluid. In experimental models, the earliest stages of CNS viral distribution are not well characterized, and it is unclear whether only certain cells are initially infected. Here, we have analyzed the distribution of cytomegalovirus (CMV) particles during the acute phase of infection, termed primary viremia, following ICV or intravascular (IV) injection into the neonatal mouse brain. In the ICV injection model, 5 µl of murine CMV (MCMV) or fluorescent microbeads were injected into the lateral ventricle at the midpoint between the ear and eye using a 10-µl syringe with a 27 G needle. In the IV injection model, a 1-ml syringe with a 35 G needle was used. A transilluminator was used to visualize the superficial temporal (facial) vein of the neonatal mouse. We infused 50 µl of MCMV or fluorescent microbeads into the superficial temporal vein. Brains were harvested at different time points post-injection. MCMV genomes were detected using the in situ hybridization method. Fluorescent microbeads or green fluorescent protein expressing recombinant MCMV particles were observed by fluorescent microscopy. These techniques can be applied to many other pathogens to investigate the pathogenesis of encephalitis.

The Possible Link between GABAergic Dysfunction and Cognitive Decline in a Patient with Idiopathic Hypoparathyroidism.

Idiopathic hypoparathyroidism (IHP) is accompanied by cognitive impairment. We report the case of a 70-year-old IHP patient with cognitive disturbance. Brain computed tomography showed bilateral calcification in basal ganglia, thalamus, and cerebellum. Neuropsychological assessment revealed low scores for intelligence, memory, and perseverative errors. Brain positron emission tomography showed a significant reduction in [(18)F]-Fludeoxyglucose (FDG) uptake in bilateral frontal, left temporal and parietal cortices, along with a marked reduction in [(11)C]-flumazenil binding in left frontal, temporal, parietal, and bilateral cerebellum. These findings suggest cognitive impairment in IHP may be ascribed to GABAergic dysfunction, thus leading to, or coexisting with, cerebral hypometabolism.

Aberrant fetal macrophage/microglial reactions to cytomegalovirus infection.

OBJECTIVE: Congenital cytomegalovirus (CMV) infection is the leading viral cause of neurodevelopmental disorders in humans, with the most severe and permanent sequelae being those affecting the cerebrum. As the fetal immune reactions to congenital CMV infection in the brain and their effects on cerebral development remain elusive, our aim was to investigate primitive innate immunity to CMV infection and its effects on cerebral corticogenesis in a mouse model for congenital CMV infection using a precise intraplacental inoculation method. METHODS: At 13.5 embryonic days (E13.5), pregnant C57BL/6 mice were intraplacentally infected with murine CMV (MCMV). Placentas and fetal organs were collected at 1, 3, and 5 days postinfection and analyzed. RESULTS: MCMV antigens were found frequently in perivascular macrophages, and subsequently in neural stem/progenitor cells (NSPCs). With increased expression of inducible nitric oxide synthase and proinflammatory cytokines, activated macrophages infiltrated into the infectious foci. In addition to the infected area, the numbers of both meningeal macrophages and parenchymal microglia increased even in the uninfected areas of MCMV-infected brain due to recruitment of their precursors from other sites. A bromodeoxyuridine (BrdU) incorporation experiment demonstrated that MCMV infection globally disrupted the self-renewal of NSPCs. Furthermore, BrdU-labeled neurons, particularly Brn2(+) neurons of upper layers II/III in the cortical plate, decreased in number significantly in the MCMV-infected E18.5 cerebrum. INTERPRETATION: Brain macrophages are crucial for innate immunity during MCMV infection in the fetal brain, while their aberrant recruitment and activation may adversely impact on the stemness of NSPCs, resulting in neurodevelopmental disorders.