Harry Jung

We aimed to investigate the therapeutic effects of alpha-pinene (α-pinene), a major component of phytoncide, in in vitro and in vivo models of mild traumatic brain injury (mTBI), raising the possibility of treating mTBI, a condition currently lacking adequate therapies. An in vitro model was established using SH-SY5Y cells and a cell injury controller, and was treated with α-pinene (0.5 g/mL). An in vivo model was induced by a stereotaxic impactor in male C57BL/6J mice and treated with α-pinene intravenously (50 mg/kg and 100 mg/kg) for 3 days post-injury. Histopathological and immunohistochemical comparisons were conducted alongside cognitive function tests to evaluate -pinene treatment. In vitro analysis showed that alpha-pinene treatment significantly increased TUNEL-positive cells. Elevated NOX4 and p22phox mRNA expressions and a high Bax/Bcl-2 protein expression ratio were noted following alpha-pinene treatment. mTBI mice treated with alpha-pinene exhibited a notable decrease in brain water content with fewer FJB-positive neurons and lower protein expression of Bax and Bcl-2 compared to untreated mTBI mice. Immunofluorescence staining for NOX4 and GFAP-positive or Iba-1-positive cells demonstrated that the increased oxidative stress and astrogliosis or activated microglia triggered by mTBI were alleviated after alpha-pinene treatment. Cognitive function testing revealed a general improvement in mTBI mice treated with alpha-pinene, with statistical significance observed in the NOR test. Alpha-pinene appears to be beneficial for neuroprotection and enhancing cognitive function in the early phases of mTBI.

Background: We evaluated the potential neuroprotective effects of naloxone in moderate traumatic brain injury (TBI), focusing on its ability to alleviate neuroinflammation, reduce cognitive impairment, and to influence Janus tyrosine kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling markers. Methods: Male C57BL/6J mice were used to establish an in vivo model of moderate TBI using a stereotaxic impactor. Immediately post-injury, naloxone was administered intraperitoneally (1 mg/kg/day) for 7 days. A total of 72 mice were divided into four groups: Normal, normal with naloxone, TBI, and TBI with naloxone (18 mice in each group). Immunohistochemical analyses and cognitive functions were evaluated across the groups. Results: TBI mice treated with naloxone exhibited significantly reduced brain swelling and cortical tissue loss compared to untreated mice. Naloxone reduced Transforming growth factor beta 2 (TGF-β2) and increased interleukin 11 (IL-11) expression in the brain. Additionally, levels of JAK2, STAT3, and B-cell lymphoma 2 (Bcl-2) were significantly elevated following treatment, while expressions of Tumor protein p53 (p53), Caspase 3, Microtubule-associated proteins 1A/1B light chain 3B (LC3B), and Sequestosome 1 (p62) were reduced. Fluorescence intensities of ionized calcium binding adaptor molecule (Iba-1) and dichloro-dihydro-fluorescein diacetate (DCFH-DA) were enhanced, indicating decreased microglial activation and reactive oxygen species (ROS) production due to naloxone treatment. Cognitive function tests revealed improved performance in TBI mice treated with naloxone, demonstrated by decreased alteration rates in the Y-maze test and improved preference index scores in the novel object recognition (NOR) test. Conclusions: Naloxone shows potential for neuroprotection and enhanced cognitive performances, which may be associated with modulation of JAK2/STAT3 signaling in a mouse model of moderate TBI.

The gut–brain axis regulates brain functions and maintains central nervous system homeostasis and intestinal balance. Migraine patients often present with gastrointestinal (GI) comorbidities, with stronger associations observed in chronic migraine (CM) than in episodic migraine (EM). To investigate migraine-related GI alterations, nitroglycerin (NTG)-induced mouse models of EM (N = 15) and CM (N = 15) were established using single or repeated NTG injections (10 mg/kg). EM mice were euthanized 4 h after a single injection, whereas CM mice received NTG every other day for 9 days and were euthanized after the fifth injection. On the day of sacrifice, GI tissues were analyzed for morphological changes, cytokine expression, calcitonin gene-related peptide (CGRP) levels, and immune cell profiles. NTG-treated groups exhibited significant reductions in both food intake and body weight compared with controls. In addition, colon length was markedly shortened in the chronic migraine (CM) model (p < 0.01). Molecular analyses revealed distinct cytokine expression profiles between models: the episodic migraine (EM) model showed increased levels of proinflammatory cytokines (IL-1β, IL-6, and IL-8; p < 0.01), whereas the CM model displayed elevated anti-inflammatory cytokines (IL-4, IL-10, and TGF-β; p < 0.01), particularly in the colon. CGRP expression was also markedly upregulated throughout the gastrointestinal tract, with the highest expression observed in the colon of CM mice (p < 0.01). Flow cytometric immune profiling further demonstrated divergent immune cell patterns, with increased Th17 (p = 0.0085) and B cell (p = 0.0199) populations in EM, while CM was characterized by enrichment of T cells (p = 0.0221), regulatory T cells (Tregs) (p = 0.0114), and macrophages (p = 0.0062), indicating more pronounced immune alterations in the distal colon. These findings indicate that CM involves more severe gut–brain axis dysregulation than EM, supporting the potential of gut-targeted therapies as adjunct strategies in chronic migraine.

Perioperative neurocognitive disorder (PND) is a concern following anesthesia, particularly in individuals at risk for Alzheimer’s disease (AD). This study compared the cognitive and pathological effects of propofol and remimazolam in a mouse model with AD following surgery. Five-month-old male ApoE4-KI mice underwent abdominal surgery under either propofol (170 mg/kg) or remimazolam (85 mg/kg) anesthesia. Cognitive function was assessed using the Morris water maze and Y-maze, and neuronal apoptosis and amyloid-beta (Aβ) deposition in the CA3 and dentate gyrus (DG) of the hippocampus were evaluated preoperatively and at 2, 4, and 7 days postoperatively. Both groups showed similar postoperative cognitive functions, with increased relative escape latency at day 2 and decreased relative spontaneous alternation at days 4 and 7. However, the neuropathological analysis revealed that propofol-induced significantly more neuronal death in the CA3 (days 4 and 7) and DG (days 2, 4, and 7), and greater Aβ accumulation in the CA3 (days 2 and 4) and DG (days 2 and 7) compared to remimazolam (p < 0.05). Propofol was associated with more pronounced neuropathologic changes in the hippocampus compared to remimazolam. These findings suggest remimazolam may be a safer anesthetic for patients at risk for neurodegenerative disorders, as it is associated with less severe hippocampal pathology, which is characteristic of AD.

One of the major obstacles to neural recovery following intracerebral hemorrhage (ICH) is the cavity-like lesion that occurs at the site of the hemorrhage, which impedes axonal regeneration. Here, we aim to address this challenge by investigating the migratory mechanisms of neural stem cells (NSCs) within the cavity in vitro using a hydrogel and endothelial cells. Mouse NSCs (mNSCs) isolated from the subventricular and subgranular zones using the 3D hydrogel culture were evaluated for their neurogenic, extracellular matrix (ECM), and adhesion-related mRNA expression compared to microglia (BV2) and secretory factors of human umbilical vein endothelial cells (HUVECs) in vitro and in vivo conditions. A hydrogel mesh combining mNSCs and HUVECs was developed for its therapeutic potential. mNSCs exhibit high stemness, neurogenesis, and ECM remodeling capabilities. mNSCs demonstrated close interaction with HUVECs and the surrounding vascular structures in in vitro and in vivo studies. Furthermore, mNSCs could degrade high concentrations of fibrin to facilitate migration and adhesion. mNSCs and HUVECs formed mesh networks through cell–cell contacts and maintained the structure through Matrigel support, potentially ensuring sufficient survival and regeneration capabilities. Our proposed hydrogel mesh platform with mNSCs and HUVECs demonstrated successful maintenance of cell survival and provision of structural support for the delivered cells by promoting ECM remodeling and neurogenesis, which may aid in axonal regeneration in the cavity lesions following ICH.

Background: Despite the important clinical issue of cognitive impairment after moderate traumatic brain injury (TBI), there is currently no suitable treatment. Here, we used in vitro and in vivo models to investigate the effect of Donepezil—an acetylcholinesterase (AChE) inhibitor—on cognitive impairment in the acute period following injury, while focusing on neuroinflammation and autophagy- and mitophagy-related markers. Methods: The purpose of the in vitro study was to investigate potential neuroprotective effects in TBI-induced cells after donepezil treatment, and the in vivo study, the purpose was to investigate therapeutic effects on cognitive impairment in the acute period after injury by analyzing neuroinflammation and autophagy- and mitophagy-related markers. The in vitro TBI model involved injuring SH-SY5Y cells using a cell-injury controller and then investigating the effect of donepezil at a concentration of 80 μM. The in vivo TBI model was made using a stereotaxic impactor for male C57BL/6J mice. Immuno-histochemical markers and cognitive functions were compared after 7 days of donepezil treatment (1 mg/kg/day). Mice were divided into four groups: sham operation with saline treatment, sham operation with donepezil treatment, TBI with saline treatment, and TBI with donepezil treatment (18 mice in each group). Donepezil treatment was administered within 4 h post-TBI. Results: In vitro, donepezil was found to lead to increased cell viability and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimi-dazolylcarbocyanine iodide (JC-1), along with decreased reactive oxygen species (ROS), lactate-dehydrogenase (LDH), 2′-7′-dichlorodihydrofluorescein diacetate (DCFH-DA)-positive cells, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells. The mRNA and protein expressions of neuroinflammation (Cyclooxygenase-2, COX-2; NOD-like receptor protein 3, NLRP3; Caspase-1; and Interleukin-1 beta, IL-1β), as well as autophagy- and mitophagy-related markers (death-associated protein kinase 1, DAPK1; PTEN-induced kinase 1, PINK1; BCL2/adenovirus E1B 19 kDa protein-interacting protein 3-like, BNIP3L; Beclin-1, BECN1; BCL2-associated X protein, BAX; microtubule-associated protein 1A/1B-light chain 3B (LC3B); Sequestosome-1; and p62) were all found to decrease after donepezil treatment. The in vivo study also showed that donepezil treatment resulted in decreased levels of cortical tissue losses and brain swelling in TBI compared to the TBI group without donepezil treatment. Donepezil treatment was also shown to decrease the mRNA and Western blotting expressions of all markers, and especially COX-2 and BNIP3L, which showed the most significant decreases. Moreover, TBI mice showed an decreased escape latency, increased alteration rate, and improved preference index, altogether pointing to better cognitive performance after donepezil treatment. Conclusions: Donepezil treatment may be beneficial in improving cognitive impairment in the early phase of moderate traumatic brain injury by ameliorating neuroinflammation, as well as autophagy and mitophagy.

Multiple animal models of migraine have been used to develop new therapies. Understanding the transition from episodic (EM) to chronic migraine (CM) is crucial. We established models mimicking EM and CM pain and assessed neuropathological differences. EM and CM models were induced with single NTG or multiple injections over 9 days. Mechanical hypersensitivity was assessed. Immunofluorescence utilized c-Fos, NeuN, and Iba1. Proinflammatory and anti-inflammatory markers were analyzed. Neuropeptides (CGRP, VIP, PACAP, and substance P) were assessed. Mechanical thresholds were similar. Notable neuropathological distinctions were observed in Sp5C and ACC. ACC showed increased c-Fos and NeuN expression in CM (p < 0.001) and unchanged in EM. Sp5C had higher c-Fos and NeuN expression in EM (p < 0.001). Iba1 was upregulated in Sp5C of EM and ACC of CM (p < 0.001). Proinflammatory markers were strongly expressed in Sp5C of EM and ACC of CM. CGRP expression was elevated in both regions and was higher in CM. VIP exhibited higher levels in the Sp5C of EM and ACC of CM, whereas PACAP and substance P were expressed in the Sp5C in both models. Despite similar thresholds, distinctive neuropathological differences in Sp5C and ACC between EM and CM models suggest a role in the EM to CM transformation.

We evaluated the therapeutic effects of bone-marrow-derived mesenchymal stem cells (BMSCs) on behavioral and cognitive function in a mouse model of mild subarachnoid hemorrhage (SAH) and explored the underlying mechanisms in conjunction with the HMGB1–RAGE axis. The SAH models were generated in a total of 126 male C57BL/6J mice via endovascular perforation and evaluated 24 h and 72 h after the intravenous administration of BMSCs (3 × 10⁵ cells). The BMSCs were administered once, at 3 h, or twice, at 3 h and 48 h after the model induction. The therapeutic effects of the BMSCs were compared to those of the saline administration. Compared to saline-treated SAH-model mice, at 3 h, the mice with mild SAH treated with the BMSCs showed significant improvements in their neurological scores and cerebral edema. The administration of the BMSCs decreased the mRNA expression of HMGB1, RAGE, TLR4, and MyD88, as well as the protein expression of HMGB1 and phosphorylated NF-kB p65. Furthermore, the numbers of slips per walking time, impairments in short-term memory, and the recognition of novel objects were improved. There was some improvement in inflammatory-marker levels and cognitive function according to the BMSCs’ administration times, but no large differences were seen. The administration of BMSCs improved behavioral and cognitive dysfunction by ameliorating HMGB1–RAGE axis-mediated neuroinflammation after SAH.

Objective : The influence of moderate-to-severe traumatic brain injury (TBI) on acute pulmonary injury is well established, but the association between acute pulmonary injury and mild TBI has not been well studied. Here, we evaluated the histological changes and fluctuations in inflammatory markers in the lungs to determine whether an acute pulmonary inflammatory response occurred after mild TBI.Methods : Mouse models of mild TBI (n=24) were induced via open-head injuries using a stereotaxic impactor. The brain and lungs were examined 6, 24, and 72 hours after injury and compared to sham-operated controls (n=24). Fluoro-Jade B staining and Astra blue and hematoxylin staining were performed to assess cerebral neuronal degeneration and pulmonary histological architecture. Quantitative real-time polymerase chain reaction analysis was done to measure inflammatory cytokines.Results : Increased neuronal degeneration and the mRNA expression of interleukin (IL)-6, tumor necrosis factor (TNF)-α, IL-10, and transforming growth factor (TGF)-β were observed after mild TBI. The IL-6, TNF-α, and TGF-β levels in mice with mild TBI were significantly different compared to those of sham-operated mice 24 hours after injury, and this was more pronounced at 72 hours. Mild TBI induced acute pulmonary interstitial edema with cell infiltration and alveolar morphological changes. In particular, a significant infiltration of mast cells was observed. Among the inflammatory cytokines, TNF-α was significantly increased in the lungs at 6 hours, but there was no significant difference 24 and 72 hours after injury.Conclusion : Mild TBI induced acute pulmonary interstitial inflammation and alveolar structural changes, which are likely to worsen the patient’s prognosis.