Viral entry and translation in brain endothelia provoke influenza-associated encephalopathy.

Influenza-associated encephalopathy (IAE) is extremely acute in onset, with high lethality and morbidity within a few days, while the direct pathogenesis by influenza virus in this acute phase in the brain is largely unknown. Here we show that influenza virus enters into the cerebral endothelium and thereby induces IAE. Three-weeks-old young mice were inoculated with influenza A virus (IAV). Physical and neurological scores were recorded and temporal-spatial analyses of histopathology and viral studies were performed up to 72 h post inoculation. Histopathological examinations were also performed using IAE human autopsy brains. Viral infection, proliferation and pathogenesis were analyzed in cell lines of endothelium and astrocyte. The effects of anti-influenza viral drugs were tested in the cell lines and animal models. Upon intravenous inoculation of IAV in mice, the mice developed encephalopathy with brain edema and pathological lesions represented by micro bleeding and injured astrocytic process (clasmatodendrosis) within 72 h. Histologically, massive deposits of viral nucleoprotein were observed as early as 24 h post infection in the brain endothelial cells of mouse models and the IAE patients. IAV inoculated endothelial cell lines showed deposition of viral proteins and provoked cell death, while IAV scarcely amplified. Inhibition of viral transcription and translation suppressed the endothelial cell death and the lethality of mouse models. These data suggest that the onset of encephalopathy should be induced by cerebral endothelial infection with IAV. Thus, IAV entry into the endothelium, and transcription and/or translation of viral RNA, but not viral proliferation, should be the key pathogenesis of IAE.

D-Asparagine is an Ideal Endogenous Molecule for Measuring the Glomerular Filtration Rate.

Introduction: An ideal endogenous molecule for measuring glomerular filtration rate (GFR) is still unknown. However, a rare enantiomer of serine, d-serine, is useful in GFR measurement. This study explored the potential of other d-amino acids for kidney function assessment. Methods: This was a cross-sectional observational study of 207 living kidney transplant donors and recipients, for whom GFR was measured using clearance of inulin (C-in). Associations between levels of d-amino acids and GFR were analyzed using multivariate factor analysis. Fractional excretion (FE), a ratio of the clearance of a substance to C-in as a standard molecule, was calculated to monitor the excretion ratio after glomerular filtration. Dissociation from an ideal FE of 100% was assessed as a bias. Proportional bias against C-in was calculated using Deming regression. Results: Multivariate analysis identified the blood level of d-asparagine to reflect GFR. Means of blood d-asparagine and clearance of d-asparagine (C-d-Asn) were 0.21 µM and 65.0 ml/min per 1.73 m2, respectively. Inulin-based FE (FEin) of d-asparagine was 98.67% (95% confidence interval [CI]: 96.43-100.90%) and less biased than those of known GFR markers, such as FEin of creatinine (147.93 [145.39-150.46]; P < 0.001) and d-serine (84.84 [83.22-86.46]; P < 0.001). A proportional bias of C-d-Asn to C-in was -7.8% (95% CI, -14.5 to -0.6%), which was minor compared to those of clearance of creatinine (-34.5% [-37.9 to -31.0%]) and d-serine (21.2% [13.9-28.9]). Conclusion: D-Asparagine acts similar to inulin in the kidney. Therefore, d-asparagine is an ideal endogenous molecule that can be used for GFR measurement.

Blood levels of d-amino acids reflect the clinical course of COVID-19.

d-Amino acids, rare enantiomers of amino acids, have been identified as biomarkers and therapeutic options for COVID-19. Methods for monitoring recovery are necessary for managing COVID-19. On the other hand, the presence of SARS-CoV2 virus in the blood is associated with worse outcomes. We investigated the potential of d-amino acids for assessing recovery from severe COVID-19. In patients with severe COVID-19 requiring artificial ventilation, the blood levels of d-amino acids, including d-alanine, d-proline, d-serine, and d-asparagine, which were lower than the normal range before treatment, quickly and transiently increased and surpassed the upper limit of the normal range. This increase preceded the recovery of respiratory function, as indicated by ventilation weaning. The increase in blood d-amino acid levels was associated with the disappearance of the virus in the blood, but not with inflammatory manifestations or blood cytokine levels. d-Amino acids are sensitive biomarkers that reflect the recovery of the clinical course and blood viral load. Dynamic changes in blood d-amino acid levels are key indicators of clinical course.

d-Alanine as a biomarker and a therapeutic option for severe influenza virus infection and COVID-19.

Since the outbreak of coronavirus disease 2019 (COVID-19), biomarkers for evaluating severity, as well as supportive care to improve clinical course, remain insufficient. We explored the potential of d-amino acids, rare enantiomers of amino acids, as biomarkers for assessing disease severity and as protective nutrients against severe viral infections. In mice infected with influenza A virus (IAV) and in patients with severe COVID-19 requiring artificial ventilation or extracorporeal membrane oxygenation, blood levels of d-amino acids, including d-alanine, were reduced significantly compared with those of uninfected mice or healthy controls. In mice models of IAV infection or COVID-19, supplementation with d-alanine alleviated severity of clinical course, and mice with sustained blood levels of d-alanine showed favorable prognoses. In severe viral infections, blood levels of d-amino acids, including d-alanine, decrease, and supplementation with d-alanine improves prognosis. d-Alanine has great potentials as a biomarker and a therapeutic option for severe viral infections.

Clasmatodendrosis is associated with dendritic spines and does not represent autophagic astrocyte death in influenza-associated encephalopathy.

BACKGROUND: Influenza-associated encephalopathy (IAE) is one of the most serious CNS complications of an influenza virus infection, with unclear pathophysiology. Clasmatodendrosis is a complex of morphological changes in astrocytes characterized by fragmentation of the distal processes and swollen cell bodies. Although pathologists in Japan have long been aware of the presence of clasmatodendrosis in IAE brains, no details of the phenomenon have been published to date. We aimed to confirm the existence, and characterize the spatial distribution of clasmatodendrosis in postmortem IAE brains. METHODS: Autopsied brains from 7 patients with IAE and 8 non-IAE subjects were examined immunohistochemically. In addition, immunofluorescent staining and electron microscopy were performed. RESULTS: Clasmatodendrosis was present in all examined regions of the IAE brains, but none of the control brains. Fragmented processes of astrocytes in IAE brains were closely adjacent to synapses on the dendritic spines, with the fragmentation especially prominent in the cerebellar molecular layer. In addition, the clasmatodendrotic astrocytes were negative for autophagy markers. Furthermore, whereas aquaporin 4 was predominantly detected in the perivascular endfeet of astrocytes in the control brains, its primary localization site shifted to the fragmented perisynaptic processes in the IAE brains. CONCLUSION: Clasmatodendrosis was distributed diffusely in the IAE brains in close association with synapses, and was not caused by astrocyte autophagy. Clasmatodendrosis may be a suggestive pathological feature of IAE.

Vascular tight junction disruption and angiogenesis in spontaneously hypertensive rat with neuroinflammatory white matter injury.

Vascular cognitive impairment is a major cause of dementia caused by chronic hypoxia, producing progressive damage to white matter (WM) secondary to blood-brain barrier (BBB) opening and vascular dysfunction. Tight junction proteins (TJPs), which maintain BBB integrity, are lost in acute ischemia. Although angiogenesis is critical for neurovascular remodeling, less is known about its role in chronic hypoxia. To study the impact of TJP degradation and angiogenesis during pathological progression of WM damage, we used the spontaneously hypertensive/stroke prone rats with unilateral carotid artery occlusion and Japanese permissive diet to model WM damage. MRI and IgG immunostaining showed regions with BBB damage, which corresponded with decreased endothelial TJPs, claudin-5, occludin, and ZO-1. Affected WM had increased expression of angiogenic factors, Ki67, NG2, VEGF-A, and MMP-3 in vascular endothelial cells and pericytes. To facilitate the study of angiogenesis, we treated rats with minocycline to block BBB disruption, reduce WM lesion size, and extend survival. Minocycline-treated rats showed increased VEGF-A protein, TJP formation, and oligodendrocyte proliferation. We propose that chronic hypoxia disrupts TJPs, increasing vascular permeability, and initiating angiogenesis in WM. Minocycline facilitated WM repair by reducing BBB damage and enhancing expression of TJPs and angiogenesis, ultimately preserving oligodendrocytes.

X-linked hypomyelination with spondylometaphyseal dysplasia (H-SMD) associated with mutations in AIFM1.

An X-linked condition characterized by the combination of hypomyelinating leukodystrophy and spondylometaphyseal dysplasia (H-SMD) has been observed in only four families, with linkage to Xq25-27, and recent genetic characterization in two families with a common AIFM1 mutation. In our study, 12 patients (6 families) with H-SMD were identified and underwent comprehensive assessment accompanied by whole-exome sequencing (WES). Pedigree analysis in all families was consistent with X-linked recessive inheritance. Presentation typically occurred between 12 and 36 months. In addition to the two disease-defining features of spondylometaphyseal dysplasia and hypomyelination on MRI, common clinical signs and symptoms included motor deterioration, spasticity, tremor, ataxia, dysarthria, cognitive defects, pulmonary hypertension, nystagmus, and vision loss due to retinopathy. The course of the disease was slowly progressive. All patients had maternally inherited or de novo mutations in or near exon 7 of AIFM1, within a region of 70 bp, including synonymous and intronic changes. AIFM1 mutations have previously been associated with neurologic presentations as varied as intellectual disability, hearing loss, neuropathy, and striatal necrosis, while AIFM1 mutations in this small region present with a distinct phenotype implicating bone. Analysis of cell lines derived from four patients identified significant reductions in AIFM1 mRNA and protein levels in osteoblasts. We hypothesize that AIFM1 functions in bone metabolism and myelination and is responsible for the unique phenotype in this condition.

Rodent Models of Vascular Cognitive Impairment.

Vascular cognitive impairment dementia (VCID), which is an increasingly important cause of dementia in the elderly, lacks effective treatments. Many different types of vascular disease are included under the diagnosis of VCID, including large vessel disease with multiple strokes and small vessel disease with lacunar infarcts and white matter disease. Animal models have been developed to study the multiple forms of VCID. Because of its progressive course, small vessel disease (SVD) is thought to be the optimal form of VCID for treatment. One theory is that the pathophysiology involves hypoxic hypoperfusion resulting in injury to the white matter and neuronal death. Bilateral occlusion of the common carotid arteries (BCAO) in a normotensive rat, which reduces cerebral blood flow, induces hypoxia with white matter damage; this model has been used to test drugs to block the injury. Another model is the spontaneously hypertensive/stroke prone rat (SHR/SP). Hypertension leads to small vessel disease resulting in progressive damage to the white matter, cortex, and hippocampus. Bilateral carotid artery stenosis (BCAS) with coils or ameroid constrictors produces a slower development of changes than BCAO, avoiding the acute ischemia. A few studies have been done with the two-clip, two-vessel occlusion renal model for induction of hypertension. There are benefits and drawbacks to each of these models with the model selected depending on the type of vascular damage that is to be studied. This review describes the most commonly used models, and the drugs that have been used to reduce the damage.

Oxidative DNA Damage Mediated by Intranuclear MMP Activity Is Associated with Neuronal Apoptosis in Ischemic Stroke.

Evidence of the pathological roles of matrix metalloproteinases (MMPs) in various neurological disorders has made them attractive therapeutic targets. MMPs disrupt the blood-brain barrier and cause neuronal death and neuroinflammation in acute cerebral ischemia and are critical for angiogenesis during recovery. However, some challenges have to be overcome before MMPs can be further validated as drug targets in stroke injury. Identifying in vivo substrates of MMPs should greatly improve our understanding of the mechanisms of ischemic injury and is critical for providing more precise drug targets. Recent works have uncovered nontraditional roles for MMPs in the cytosol and nucleus. These have shed light on intracellular targets and biological actions of MMPs, adding additional layers of complexity for therapeutic MMP inhibition. In this review, we discussed the recent advances made in understanding nuclear location of MMPs, their regulation of intranuclear sorting, and their intranuclear proteolytic activity and substrates. In particular, we highlighted the roles of intranuclear MMPs in oxidative DNA damage, neuronal apoptosis, and neuroinflammation at an early stage of stroke insult. These novel data point to new putative MMP-mediated intranuclear actions in stroke-induced pathological processes and may lead to novel approaches to treatment of stroke and other neurological diseases.

Transient increase of fractional anisotropy in reversible vasogenic edema.

Brain vasogenic edema, involving disruption of the blood-brain barrier, is a common pathological condition in several neurological diseases, with a heterogeneous prognosis. It is sometimes reversible, as in posterior reversible encephalopathy syndrome, but often irreversible and our current clinical tools are insufficient to reveal its reversibility. Here, we show that increased fractional anisotropy in magnetic resonance imaging is associated with the reversibility of vasogenic edema. Spontaneously, hypertensive rats-stroke prone demonstrated posterior reversible encephalopathy syndrome-like acute encephalopathy in response to high-dose cyclosporine A treatment; the deteriorating neurological symptoms and worsening scores in behavioral tests, which were seen in acute phase, dissappered after recovery by cessation of cyclosporine A. In the acute phase of encephalopathy, the fractional anisotropy and apparent diffusion coefficient increased in areas with IgG leakage. This increase of fractional anisotropy occurred in the absence of demyelination: fluid leakage into the myelinated space increased the axial, but not the radial, diffusivity, resulting in the increased fractional anisotropy. This increased fractional anisotropy returned to pre-encephalopathy values in the recovery phase. Our results highlight the importance of the fractional anisotropy increase as a marker for the reversibility of brain edema, which can delineate the brain areas for which recovery is possible.

A case of cerebral hypomyelination with spondylo-epi-metaphyseal dysplasia.

We reported on a male patient with rare leukoencephalopathy and skeletal abnormalities. The condition was first noticed as a developmental delay, nystagmus and ataxia at 1 year of age. At 4 years of age, he was diagnosed as hypomyelination with skeletal abnormalities from clinical features, brain magnetic resonance imaging (MRI) and skeletal X-rays. His brain MRI revealed diffuse hypomyelination. These findings suggested the classical type of Pelizaeus-Merzbacher disease (PMD) caused by proteolipid protein (PLP)-1 gene or Pelizaeus-Merzbacher-like disease (PMLD). However, we found neither mutation nor duplication of PLP-1. The patient had severe growth retardation and general skeletal dysplasia compatible with spondylo-epi-metaphyseal dysplasia; however the mutation of discoidin domain receptor (DDR) 2 gene was absent. The co-morbidity of hypomyelination with skeletal abnormalities is rare. We performed array CGH and no causal copy number variation was recognized. Alternatively, this condition may have been caused by a mutation of the gene encoding a molecule that functions in both cerebral myelination and skeletal development.

Evaluation of oral iron treatment in pediatric restless legs syndrome (RLS).

OBJECTIVE: We conducted a retrospective chart review of children with restless legs syndrome (RLS) to evaluate the efficacy of oral iron treatment, which was administered open-label during the course of clinical care. In addition, we provided detailed clinical information about RLS in this pediatric cohort. PATIENTS AND METHODS: The study included 30 consecutive Japanese children with RLS who visited the Pediatric Sleep Clinic at Osaka University Hospital, and consisted of 17 boys and 13 girls, aged 2-14 years (mean ± SD, 6.5 ± 2.8). All-night polysomnography was performed in 18 patients and serum ferritin levels were measured in all the patients. After the diagnosis of RLS, iron was administered at doses between 1.6 and 7.8 mg/kg/day (3.2 ± 1.3). Serum ferritin was re-evaluated 3-6 months after iron treatment, or when RLS symptoms had disappeared. RESULTS: The patient age at onset of RLS symptoms ranged from six months to 13 years (4.3 ± 3.6). A positive family history was recognized in 19 children (63.3%). Serum ferritin levels before therapy were 9-62 ng/ml (26.6 ± 12.8) and oral iron supplementation was reported to be highly effective in 17 children, effective in 10, and ineffective in three. The serum ferritin level at follow-up was 23-182 ng/ml (83.5 ± 49.8). The onset of treatment effect was within approximately three months. CONCLUSIONS: Iron treatment could be effective in Japanese pediatric RLS.

Progressive leukoencephalopathy with intracranial calcification, congenital deafness, and developmental deterioration.

We report on a 12-year-old male with a unique cerebral white matter disease. His initial symptoms were congenital hearing loss and multiple intracranial calcifications on head CT. He developed severe intellectual disability and epilepsy. MRI showed signal abnormalities in the posterior limbs of the internal capsules, thalami, and cerebral white matter. The abnormalities were progressive over time. The neuropathology revealed diffuse and severe disruption of myelin and axons of the cerebral white matter and cerebrospinal tracts. We performed various metabolic examinations, detailed pathological investigations and genetic analyses, but could not identify the cause. To our knowledge his clinical course has not been described in the literature.

Variations in early gross motor milestones and in the age of walking in Japanese children.

BACKGROUND: Gross motor development is usually assessed in terms of age of achievement of motor milestones. Although there is generally an impression of faster development if the milestones are achieved at younger ages, no longitudinal studies have been done on the associations between the milestones, especially in Japan. As a part of the Japan Children's Study, the purpose of the present study was to determine whether the achievement of gross motor milestones in infancy is related with the age of walking. METHODS: This was a prospective cohort study of 290 healthy and term infants born in a district of Osaka City, Japan. Three milestones (rolling over, sitting, and crawling) were observed in the laboratory for infants aged at 4 and 9 months by a pediatrician and a developmental psychologist, and the age of walking was confirmed in questionnaires filled in by the parents at 18 and 27 months. RESULTS: Children who could roll over at 4 months, and sit and crawl at 9 months, walked earlier than children who could not roll over, sit and crawl, respectively. With regard to crawling, children who were creeping had a 1 month delay in walking, and those who could not move forward had a 2 month delay compared to typical crawlers. On multiple regression analysis these three milestones were positively associated with walking: rolling over (ß= 0.567), sitting (ß= 1.973) and crawling (ß= 1.473). CONCLUSION: The age and the patterns of sitting, crawling and rolling over were all related to the age of independent walking among Japanese infants. Consideration of milestone definition and variations is essential in medical check-up.

Clinical aspects and adrenal functions in eleven Japanese children with X-linked adrenoleukodystrophy.

X-linked adrenoleukodystrophy (X-ALD) is a genetic disease associated with demyelination of the central nervous system, adrenocortical insufficiency and accumulation of very long chain fatty acids. It is a clinically heterogeneous disorder ranging from a severe childhood cerebral form to an asymptomatic form. The incidence in Japan is estimated to be between 1:30,000 and 1:50,000 boys as determined by a nationwide retrospective survey between 1990 and 1999, which found no cases with Addison's form. We reviewed the medical records of eleven Japanese boys with X-ALD from 1990 to 2010 in our institute. Eight patients were detected by neuropsychological abnormalities, whereas a higher prevalence of unrecognized adrenocortical insufficiency (5/11: 45%) was observed than previously recognized. While no neurological abnormalities were demonstrated in two brothers, the elder brother had moderate Addison's disease at diagnosis and the presymptomatic younger brother progressed to Addison's disease six months after the diagnosis of X-ALD. Early detection of impaired adrenal function as well as early identification of neurologically presymptomatic patients by genetic analysis is essential for better prognosis. Addison's form might be overlooked in Japan; therefore, X-ALD should be suspected in patients with adrenocortical insufficiency.

Head growth evaluation in early childhood, from the Japan Children's Study.

BACKGROUND: Head circumference (HC) trajectories are held to reflect neurological development and the acquirement of intelligence. It is important to assess HC growth accurately because atypical HC growth is an indicator of various developmental disorders. HC growth is determined by both familial and physical factors but, hitherto, no one has considered both factors together. The aim of the present study was thus to investigate the relationship between HC, physical growth, and parental HC. METHODS: The study group in the Japan Children's Study consisted of 192 healthy full-term Japanese children. HC (maximum occiptofrontal circumference), height and bodyweight were measured at the ages of 4, 9 and 18 months. Multiple regression analysis were conducted predicting the HC from the body measurements and mid-parental HC (defined as the average of standardized paternal and maternal HC). RESULTS: Adjusted multiple R(2) were 0.336, 0.307 and 0.259, measured at the aforementioned three stages. Bodyweight and mid-parental HC predicted the HC on each occasion (P < 0.01). Bodyweight was more relevant than mid-parental HC. CONCLUSIONS: HC growth is influenced by physical growth and parental HC; therefore, it is important to consider both physical and familial factors. A formula is herein proposed to assess HC using bodyweight and mid-parental HC.