Osamu Hori

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.

Pathological conditions in cochlea, such as ototoxicity, acoustic trauma, and age-related cochlear degeneration, induce cell death in the organ of Corti and degeneration of the spiral ganglion neurons (SGNs). Although macrophages play an essential role after cochlear injury, its role in the SGNs is limitedly understood. We analyzed the status of macrophage activation and neuronal damage in the spiral ganglion after kanamycin-induced unilateral hearing loss in mice. The number of ionized calcium-binding adapter molecule 1 (Iba1)-positive macrophages increased 3 days after unilateral kanamycin injection. Macrophages showed larger cell bodies, suggesting activation status. Interestingly, the number of activating transcription factor 3 (ATF3)-positive-neurons, an indicator of early neuronal damage, also increased at the same timing. In the later stages, the number of macrophages decreased, and the cell bodies became smaller, although the number of neuronal deaths increased. To understand their role in neuronal damage, macrophages were depleted via intraperitoneal injection of clodronate liposome 24 h after kanamycin injection. Macrophage depletion decreased the number of ATF3-positive neurons at day 3 and neuronal death at day 28 in the spiral ganglion following kanamycin injection. Our results suggest that suppression of inflammation by clodronate at early timing can protect spiral ganglion damage following cochlear insult.

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.

Activating transcription factor 6 (ATF6) is an endoplasmic reticulum (ER) stress-regulated transcription factor that induces expression of major molecular chaperones in the ER. We recently reported that ATF6β, a subtype of ATF6, promoted survival of hippocampal neurons exposed to ER stress and excitotoxicity, at least in part by inducing expression of calreticulin, an ER molecular chaperone with high Ca²⁺-binding capacity. In the present study, we demonstrate that ATF6β deficiency in mice also decreases calreticulin expression and increases expression of glucose-regulated protein 78, another ER molecular chaperone, in emotional brain regions such as the prefrontal cortex (PFC), hypothalamus, hippocampus, and amygdala. Comprehensive behavioral analyses revealed that Atf6b^−/− mice exhibit anxiety-like behavior in the light/dark transition test and hyperactivity in the forced swim test. Consistent with these results, PFC and hypothalamic corticotropin-releasing hormone (CRH) expression was increased in Atf6b^−/− mice, as was circulating corticosterone. Moreover, CRH receptor 1 antagonism alleviated anxiety-like behavior in Atf6b^−/− mice. These findings suggest that ATF6β deficiency produces anxiety-like behavior and hyperactivity via a CRH receptor 1-dependent mechanism. ATF6β could play a role in psychiatric conditions in the emotional centers of the brain.

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.