Mika Takarada-Iemata

Hemorrhagic transformation (HT) after ischemic stroke reflects spatially heterogeneous neurovascular pathology, but the regional distribution of individual hemorrhagic lesions and their relationship to ischemic injury severity remain incompletely defined. Conventional histological and imaging approaches do not readily combine non-destructive preservation of whole-brain three-dimensional anatomical continuity with object-based quantification of individual hemorrhagic lesions alongside infarction and edema in the same experimental brain. Here, we applied ex vivo phosphotungstic acid-enhanced micro-computed tomography (micro-CT) to 121 mouse brains collected 1, 3, 7, and 14 days after transient middle cerebral artery occlusion. Whole-brain three-dimensional analyses were used to quantify infarction, hemispheric volume changes, HT, and individual microhemorrhagic lesions. Micro-CT findings were validated against immunohistological markers and a genetic edema model, and associations with sensorimotor outcomes were assessed. CT-defined hypodense lesions corresponded histologically to regions of neuronal loss surrounded by glial scar, and micro-CT detected aggravated edema in Ndrg2 knockout mice. Hyperdense regions corresponded to Ter119-positive erythrocyte clusters, supporting their hemorrhagic nature, and HT incidence increased with longer ischemia duration. Mice with HT exhibited greater edema and poorer performance on rotarod and grid-walk tests. Object-based three-dimensional analysis of individual microhemorrhagic lesions characterized a region-specific pattern, with declining cortical and persistent striatal microhemorrhagic burden during the first week after ischemia-reperfusion. At day 7, microhemorrhagic burden was greater in the caudate putamen than in the cortex, and caudate putamen microhemorrhage number correlated positively with both infarct volume and neurological deficit score, whereas no significant correlations were observed in the cortex. These findings demonstrate spatial heterogeneity of post-ischemic microhemorrhagic burden, and highlight contrast-enhanced micro-CT as a quantitative platform for integrated, spatially resolved 3D assessment of infarction, edema, and hemorrhagic lesions after cerebral ischemia.

BACKGROUND: Subarachnoid hemorrhage (SAH) induces inflammatory responses and subsequent immune cell activation, which may contribute in cerebral vasospasm, microcirculatory impairment and poor neurological outcomes. Although cerebral vasospasm has traditionally been considered a major cause of delayed cerebral ischemia after SAH, therapies targeting angiographic vasospasm have not consistently improved functional outcomes. Early inflammatory responses may contribute to microcirculatory impairment, cerebral vasospasm, and subsequent neurological injury. Herein, we investigated whether interleukin-10 (IL-10), an anti-inflammatory cytokine, improves these outcomes in an experimental SAH model. METHODS: Mice received intramuscular injections of either an adeno-associated virus encoding IL-10 (AAV/IL-10) vector or an AAV expressing green fluorescent protein (AAV/GFP) vector (control). India ink angiography was performed to assess the diameter of the sphenoidal segment of the middle cerebral artery (MCA), the total length of the visible cortical arteries, and cortical staining intensity, as indices of cerebral vasospasm, microcirculatory impairment, and cerebral perfusion, respectively. Perivascular inflammatory cell infiltration and cytokine levels were assessed using immunohistochemistry and ELISA. We also evaluated the therapeutic efficacy of the AAV/IL-10 vector when administered immediately after SAH induction. RESULTS: IL-10 overexpression significantly improved neurological outcomes after SAH and was associated with attenuated cerebral vasospasm and microcirculatory impairment, as well as preservation of cerebral perfusion. It also significantly reduced neutrophil and macrophage infiltration around the internal carotid artery and attenuated SAH-induced elevations in IL-6 and matrix metalloproteinase-3 levels. Mice treated with the AAV/IL-10 vector immediately after SAH induction showed significant improvements in neurological scores and cerebral perfusion. CONCLUSIONS: AAV-mediated IL-10 overexpression improves neurological outcomes after SAH, likely by attenuating inflammatory responses, cerebral vasospasm, and microcirculatory impairment. These findings suggest that IL-10-based anti-inflammatory therapy is a promising therapeutic strategy for SAH.

Subarachnoid hemorrhage (SAH) due to the rupture of an intracranial aneurysm is a highly fatal type of stroke. Cerebral vasospasm (CVS) is a major post-SAH complication leading to delayed ischemic neurological deficits, thereby worsening patient outcomes. Previously, we found that lower plasma levels of the soluble receptor for advanced glycation end products (RAGE) predict symptomatic CVS in patients with SAH. However, the molecular mechanisms underlying CVS remain unclear. Here, using an SAH mouse model with endovascular perforation, we found that neurological deficits, CVS in the circle of Willis, and impaired cortical microarterial perfusion were markedly ameliorated in RAGE-deficient mice. Neutrophils accumulated in the cerebral perivascular space of WT mice after SAH but were markedly reduced in RAGE-deficient mice. Myeloid lineage-targeted deletion of RAGE, including neutrophils, improved CVS after SAH. Inhibition of the high mobility group box 1 (HMGB1)/RAGE axis or neutrophil elastase ameliorated CVS. In a transwell assay, the HMGB1/RAGE axis drives neutrophil migration and NETosis. These findings indicate that neutrophil RAGE signaling contributes to cerebrovascular dysfunction after SAH and suggest that RAGE-mediated neutrophil inflammation may be a therapeutic target in the hyperacute phase to mitigate early brain injury.

Endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) contribute to the pathogenesis of traumatic brain injury (TBI), yet the cell type-specific roles of UPR pathways remain poorly understood. We previously identified endothelial cells (ECs) as a primary site of IRE1 pathway activation following brain injury. In this study, we investigated the role of endothelial IRE1 signaling in TBI using EC-specific IRE1 conditional knockout mice subjected to cortical ablation. Loss of IRE1 in ECs exacerbated blood-brain barrier (BBB) disruption, enhanced immune cell infiltration, amplified neuroinflammation, and expanded neuronal damage, ultimately leading to worsened neurological outcomes. RNA-sequencing revealed enrichment of interferon-related programs and identified Cxcl10 as an endothelial chemokine linked to the exacerbated leukocyte recruitment in endothelial IRE1 deficiency. Treatment with the chemical chaperone tauroursodeoxycholic acid (TUDCA) suppressed Cxcl10 expression both in vitro and in vivo, and significantly improved motor function following TBI. These findings reveal a critical role for endothelial IRE1 signaling in maintaining BBB integrity and restraining inflammation during the acute phase of TBI. Modulation of ER stress in brain ECs may represent a promising and accessible therapeutic strategy for reducing secondary injury after TBI.

Social behavior is essential for health, survival, and reproduction of animals; however, the role of astrocytes in social behavior remains largely unknown. The transmembrane protein CD38, which acts both as a receptor and ADP-ribosyl cyclase to produce cyclic ADP-ribose (cADPR) regulates social behaviors by promoting oxytocin release from hypothalamic neurons. CD38 is also abundantly expressed in astrocytes in the postnatal brain and is important for astroglial development. Here, we demonstrate that the astroglial-expressed CD38 plays an important role in social behavior during development. Selective deletion of CD38 in postnatal astrocytes, but not in adult astrocytes, impairs social memory without any other behavioral abnormalities. Morphological analysis shows that depletion of astroglial CD38 in the postnatal brain interferes with synapse formation in the medial prefrontal cortex (mPFC) and hippocampus. Moreover, astroglial CD38 expression promotes synaptogenesis of excitatory neurons by increasing the level of extracellular SPARCL1 (also known as Hevin), a synaptogenic protein. The release of SPARCL1 from astrocytes is regulated by CD38/cADPR/calcium signaling. These data demonstrate a novel developmental role of astrocytes in neural circuit formation and regulation of social behavior in adults.

Background: Although early brain injury (EBI) is recognized as a critical step following subarachnoid hemorrhage (SAH), its pathophysiology and underlying mechanisms remain poorly understood. Herein, we investigated the role of cerebral circulation in the acute phase using patient data and a mouse SAH model and evaluated its regulation via the sympathetic nervous system. Methods: The cerebral circulation time and neurological outcomes in the human body were retrospectively examined in 34 SAH cases with ruptured anterior circulation aneurysms and 85 cases with unruptured anterior circulation cerebral aneurysms at Kanazawa University Hospital from January 2016 to December 2021. In a mouse study, a SAH model was created via endovascular perforation, and India-ink angiography was performed over time. Additionally, bilateral superior cervical ganglionectomy was performed immediately before surgery, and neurological scores and brain water content were evaluated after SAH. Results: Cerebral circulation time was prolonged in the acute phase of SAH compared with that in the unruptured cerebral aneurysm group, especially in those with electrocardiographic changes. Furthermore, it was more prolonged in the poor prognosis group (modified Rankin Scale scores 3–6) than in the good prognosis group (modified Rankin Scale scores 0–2) at discharge. In mice, cerebral perfusion was significantly reduced at 1 and 3 hours after SAH and recovered at 6 hours. superior cervical ganglionectomy improved cerebral perfusion without altering the diameter of the middle cerebral artery at 1 hour and improved neurological outcomes at 48 hours after SAH. Consistently, brain edema, quantified by brain water content, was improved by superior cervical ganglionectomy 24 hours after SAH. Conclusions: Sympathetic hyperactivity may play a critical role in the development of EBI by impairing cerebral microcirculation and edema in the acute phase following SAH.

The unfolded protein response (UPR) is a signal transduction network that responds to endoplasmic reticulum (ER) stress by coordinating protein homeostasis to maintain cell viability. The UPR can also trigger cell death when adaptive responses fail to improve protein homeostasis. Despite accumulating evidence suggesting that the UPR plays a role in neurodegenerative diseases and brain insults, our understanding of how ER stress is induced under neuropathological conditions is limited. Here, we investigated the cell‐ and time‐specific patterns of the ER stress response after brain injury using ER stress‐activated indicator (ERAI) mice, which enable monitoring of the UPR in vivo via increased fluorescence of a spliced XBP‐1 protein fused with the green fluorescent protein (GFP) variant Venus. Following cortical stab injury of ERAI mice, the GFP signal and number of GFP⁺ cells increased in the ipsilateral cortex throughout the observation period (6 h to 7 days post‐injury), confirming the induction of the UPR. GFP signals were observed in injured neurons early (from 6 h) after brain injury. However, non‐neuronal cells, mainly endothelial cells followed by astrocytes, accounted for the majority of GFP⁺ cells after brain injury. Similar results were obtained in a mouse model of focal cerebral ischemia. These findings suggest that activation of the UPR in both neuronal and non‐neuronal cells, especially endothelial cells and astrocytes, may play an important role in and could be a potential therapeutic target for acute brain injuries.

N-myc downstream-regulated gene 2 (NDRG2) is a member of the NDRG family, whose members have multiple functions in cell proliferation, differentiation, and stress responses. NDRG2 is widely distributed in the central nervous system and is uniquely expressed by astrocytes; however, its role in brain function remains elusive. The clinical relevance of NDRG2 and the molecular mechanisms in which it participates have been reported by studies using cultured cells and specimens of patients with neurological disorders. In recent years, genetic tools, including several lines of Ndrg2-knockout mice and virus-mediated gene transfer, have improved understanding of the roles of NDRG2 in vivo. This review aims to provide an update of recent growing in vivo evidence that NDRG2 is involved in brain function, focusing on research of Ndrg2-knockout mice with neurological disorders such as brain tumors, chronic neurodegenerative diseases, and acute brain insults including brain injury and cerebral stroke. These studies demonstrate that NDRG2 plays diverse roles in the regulation of astrocyte reactivity, blood–brain barrier integrity, and glutamate excitotoxicity. Further elucidation of the roles of NDRG2 and their molecular basis may provide novel therapeutic approaches for various neurological disorders.

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS). MS is characterized by extensive immune cell infiltration leading to inflammation, demyelination, and neurodegeneration. Recently, accumulating evidence has suggested that glial cells may contribute to the development of MS pathology. However, the molecular mechanism underlying the regulation of neuronal degeneration in MS remains largely unknown. N-myc downstream-regulated gene 2 (NDRG2) is a differentiation- and stress-associated molecule, and predominantly expressed in astrocytes in the CNS. In this study, we examined the relevance of NDRG2 in experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. The expression of NDRG2 was enhanced in the acute and chronic phase after induction of EAE. Genetic deletion of NDRG2 ameliorated the clinical course and demyelination after EAE induction. Although the loss of NDRG2 slightly affected the inflammatory response, it significantly reduced neurodegeneration both in the acute and chronic phase. Further analysis revealed that deletion of NDRG2 restored the EAE-related decreases in the expression of astrocytic glutamate transporters. Thus, our findings suggest that NDRG2, expressed in astrocytes, may play a key role in the pathology of MS by modulating neuronal vulnerability to glutamate toxicity.