12/15-lipoxygenase inhibition attenuates neuroinflammation by suppressing inflammasomes.

Introduction: Lipoxygenases (LOXs) have essential roles in stroke, atherosclerosis, diabetes, and hypertension. 12/15-LOX inhibition was shown to reduce infarct size and brain edema in the acute phase of experimental stroke. However, the significance of 12/15-LOX on neuroinflammation, which has an essential role in the pathophysiology of stroke, has not been clarified yet. Methods: In this study, ischemia/recanalization (I/R) was performed by occluding the proximal middle cerebral artery (pMCAo) in mice. Either the 12/15-LOX inhibitor (ML351, 50 mg/kg) or its solvent (DMSO) was injected i.p. at recanalization after 1 h of occlusion. Mice were sacrificed at 6, 24, and 72-h after ischemia induction. Infarct volumes were calculated on Nissl-stained sections. Neurological deficit scoring was used for functional analysis. Lipid peroxidation was determined by the MDA assay, and the inflammatory cytokines IL-6, TNF-alpha, IL-1beta, IL-10, and TGF-beta were quantified by ELISA. The inflammasome proteins NLRP1 and NLRP3, 12/15-LOX, and caspase-1 were detected with immunofluorescence staining. Results: Infarct volumes, neurological deficit scores, and lipid peroxidation were significantly attenuated in ML351-treated groups at 6, 24, and 72-h. ELISA results revealed that the pro-inflammatory cytokines IL-1beta, IL-6, and TNF-alpha were significantly decreased at 6-h and/or 24-h of I/R, while the anti-inflammatory cytokines IL-10 and TNF-alpha were increased at 24-h or 72-h of ML351 treatment. NLRP1 and NLRP3 immunosignaling were enhanced at three time points after I/R, which were significantly diminished by the ML351 application. Interestingly, NLRP3 immunoreactivity was more pronounced than NLRP1. Hence, we proceeded to study the co-localization of NLRP3 immunoreactivity with 12/15-LOX and caspase-1, which indicated that NLRP3 was co-localized with 12/15-LOX and caspase-1 signaling. Additionally, NLRP3 was found in neurons at all time points but in non-neuronal cells 72 h after I/R. Discussion: These results suggest that 12/15-LOX inhibition suppresses ischemia-induced inflammation in the acute and subacute phases of stroke via suppressing inflammasome activation. Understanding the mechanisms underlying lipid peroxidation and its associated pathways, like inflammasome activation, may have broader implications for the treatment of stroke and other neurological diseases characterized by neuroinflammation.

Extracellular-Vesicle-Based Cancer Panels Diagnose Glioblastomas with High Sensitivity and Specificity.

Glioblastoma is one of the most devastating neoplasms of the central nervous system. This study focused on the development of serum extracellular vesicle (EV)-based glioblastoma tumor marker panels that can be used in a clinic to diagnose glioblastomas and to monitor tumor burden, progression, and regression in response to treatment. RNA sequencing studies were performed using RNA isolated from serum EVs from both patients (n = 85) and control donors (n = 31). RNA sequencing results for preoperative glioblastoma EVs compared to control EVs revealed 569 differentially expressed genes (DEGs, 2XFC, FDR < 0.05). By using these DEGs, we developed serum-EV-based biomarker panels for the following glioblastomas: wild-type IDH1 (96% sensitivity/80% specificity), MGMT promoter methylation (91% sensitivity/73% specificity), p53 gene mutation (100% sensitivity/89% specificity), and TERT promoter mutation (89% sensitivity/100% specificity). This is the first study showing that serum-EV-based biomarker panels can be used to diagnose glioblastomas with a high sensitivity and specificity.

Confocal reflectance microscopy for metal and lipid nanoparticle visualization in the brain.

Aim: A requirement for nanoparticle (NP) research is visualization of particles within cells and tissues. Limitations of electron microscopy and low yields of NP fluorescent tagging warrant the identification of alternative imaging techniques. Method: Confocal reflectance microscopy (CRM) in combination with fluorescence imaging was assessed for visualizing rhodamine B-conjugated silver and fluorescein isothiocyanate-conjugated lipid core-stearylamine NP uptake in vitro and in vivo. Results: CRM successfully identified cellular uptake and blood-brain barrier penetration of NPs owing to their distinguishing refractive indices. NP-dependent reflectance signals in vitro were dose and incubation time dependent. Finally, CRM facilitated the distinction between nonspecific fluorescence signals and NPs. Conclusion: These findings demonstrate the value of CRM for NP visualization in tissues, which can be performed with a standard confocal microscope.

Nanoparticles (NPs) are extremely small materials utilized in the healthcare sector mainly for the delivery of drugs into tissues that are not easily accessible with regular pharmaceuticals. One such tissue is the brain, which has a barrier between it and the bloodstream that prevents the passage of most drugs. For NP research, the successful entry of NPs into target tissues must be demonstrated, but this is complicated by the small size and weak labeling of NPs. In this article, the authors demonstrate a low-cost, complementary microscopy technique that is readily available in most biological research laboratories and that can be used to detect and analyze the entry of different NP types into brain tissue and their uptake by brain tumor cells. These data create new opportunities for research on NP-assisted drug delivery to the central nervous system.

Tailored Therapeutic Doses of Dexmedetomidine in Evolving Neuroinflammation after Traumatic Brain Injury.

BACKGROUND: Understanding the secondary damage mechanisms of traumatic brain injury (TBI) is essential for developing new therapeutic approaches. Neuroinflammation has a pivotal role in secondary brain injury after TBI. Activation of NLRP3 inflammasome complexes results in the secretion of proinflammatory mediators and, in addition, later in the response, microglial activation and migration of the peripheral immune cells into the injured brain are observed. Therefore, these components involved in the inflammatory process are becoming a new treatment target in TBI. Dexmedetomidine (Dex) is an effective drug, widely used over the past few years in neurocritical care units and during surgical operations for sedation and analgesia, and has anti-inflammatory effects, which are shown in in vivo studies. The aim of this original research is to discuss the anti-inflammatory effects of different Dex doses over time in TBI. METHODS: Brain injury was performed by using a weight-drop model. Half an hour after the trauma, intraperitoneal saline was injected into the control groups and 40 and 200 µg/kg of Dex were given to the drug groups. Neurological evaluations were performed with the modified Neurological Severity Score before being killed. Then, the mice were killed on the first or the third day after TBI and histopathologic (hematoxylin-eosin) and immunofluorescent (Iba1, NLRP3, interleukin-1ß, and CD3) findings of the brain tissues were examined. Nonparametric data were analyzed by using the Kruskal-Wallis test for multiple comparisons, and the Mann-Whitney U-test was done for comparing two groups. The results are presented as mean ± standard error of mean. RESULTS: The results showed that low doses of Dex suppress NLRP3 and interleukin-1ß in both terms. Additionally, high doses of Dex cause a remarkable decrease in the migration and motility of microglial cells and T cells in the late phase following TBI. Interestingly, the immune cells were influenced by only high-dose Dex in the late phase of TBI and it also improves neurologic outcome in the same period. CONCLUSIONS: In the mice head trauma model, different doses of Dex attenuate neuroinflammation by suppressing distinct components of the neuroinflammatory process in a different timecourse that contributes to neurologic recovery. These results suggest that Dex may be an appropriate choice for sedation and analgesia in patients with TBI.

Doxycycline alleviates acute traumatic brain injury by suppressing neuroinflammation and apoptosis in a mouse model.

Traumatic brain injury (TBI) is one of the significant causes of death among young people worldwide. Doxycycline (DOX), an antibiotic with anti-inflammatory effects, has not been used as a therapeutic agent to modify the inflammatory response after the traumatic brain injury. In this study, intraperitoneal administration of DOX reduced significantly the acute inflammatory markers like IL-6 and CD3, microglial migration to the damaged area marked with Iba-1, and neuronal apoptosis assessed with TUNEL assay at 72 h after the trauma. The low dose, 10 mg/kg of DOX had a dominant anti-inflammatory effect; while the high dose, 100 mg/kg of DOX, was more effective in decreasing neuronal apoptosis. In early hours after the head trauma, use of a low dose (10 mg/kg) of DOX for decreasing the acute form of inflammation followed by a high dose (100 mg/kg) for the anti-apoptotic effects particularly in severe head traumas, would be a promising approach to alleviate the brain injury.

Suppression of Aquaporin-4 By Antisense Oligonucleotides Reduces Brain Edema In Experimental Traumatic Brain Injury.

AIM: In this study, suppression of Aquaporin-4 (AQP4) synthesis via intracerebroventricular (i.c.v.) injection of antisense oligonucleotide after focal cortical contusion injury in mice was investigated. MATERIAL AND METHODS: 12-week female Swiss albino mice of 20-25 g were used to create a focal cortical contusion model by the weight drop method (35 grams blunt weight, 70 cm height) onto the parietal cortex after craniectomy. The sham group underwent craniectomy without trauma. In the control group, weight was dropped onto the parietal cortex immediately after Dulbecco\'s Modified Eagle Medium (DMEM) i.c.v. injection following craniectomy. In addition, 1 nM of aquaporin-4 (AQP4) antisense oligonucleotide (ASO) was injected via i.c.v route immediately after trauma (0 hours) and 4 hours after trauma. All animals underwent magnetic resonance (MR) imaging and were sacrificed at 24 h. Brain water content was determined using the wet/dry weight method. RESULTS: In the sham group, the average percentage of brain water content was 77.75% compared to the control group with 79.87%. The difference was statistically significant (*p=0.017). In the therapy group, the average was 78.81% and significantly reduced compared to the control group (*p=0.026) at 0 hours. In the 4-hour treatment group, the average of 79.11% was not statistically significant (p=0.39). MR imaging findings also showed a substantial reduction in brain edema in the 0-hour treatment group. However, the 4-hour treatment results, when compared with the control trauma group, did not show a significant difference. CONCLUSION: This study demonstrated that AQP4 antisense oligonucleotide therapy, when administered early after diffuse traumatic brain injury, leads to a significant reduction in brain edema.

Post-stroke inflammatory response is linked to volume loss in the contralateral hemisphere.

OBJECTIVES: There is a delicate homeostatic balance between the central nervous system and immune system. Stroke triggers an immunodepressive state to suppress a potential immune reaction directed against neuroglial tissue; however, this supposedly protective response inadvertently results in an infection-prone, and thereby a pro-inflammatory setting. In this study, we assessed the magnitude of cerebral volume loss in the unaffected contralateral hemisphere following stroke, and determined its relationship with inflammatory cascades. METHODS: The volume of the hemisphere contralateral to the ischemic insult was measured on admission and follow-up MRI's in 50 ischemic stroke patients. Information related to clinical features, infectious complications, and markers of inflammation (erythrocyte sedimentation rate, neutrophil/lymphocyte ratio, C-reactive protein) were prospectively collected, and their relationship with hemispheric volume change was evaluated using bivariate and multivariate statistics. RESULTS: The contralateral hemisphere volume decreased by a median (interquartile range) of 14 (4-32) mL after a follow-up duration of 101 (63-123) days (p < .001); the volume reduction was 0.8 (0.2-1.8) % per month with respect to baseline. Old age, atrial fibrillation, stroke severity, C-reactive protein level, neutrophil/lymphocyte ratio, and development of infections during hospitalization were significantly associated with volume loss (p < .05). Stroke severity (NIHSS score or infarct volume) and inflammation related parameters (neutrophil/lymphocyte ratio or systemic infections) remained independently and positively associated with volume loss in multivariate regression models. CONCLUSIONS: Cerebral tissue changes following stroke are not limited to the ischemic hemisphere. Apart from stroke severity, a pro-inflammatory state and post-stroke infections contribute to cerebral volume loss in the non-ischemic hemisphere.

12/15 Lipoxygenase as a Therapeutic Target in Brain Disorders.

Lipoxygenases are a family of lipid-oxidizing enzymes, which generate eicosanoids and related compounds from arachidonic acid and other polyunsaturated fatty acids. These metabolites play important roles in physiology and pathogenesis of host defense mechanisms, cardiovascular diseases, cancer, inflammatory, allergic and neurodegenerative diseases. The 12/15-lipoxygenase (LOX) is special in that it can directly oxidize lipid membranes containing polyunsaturated fatty acids, without the preceding action of a phospholipase, leading to the direct attack on membranous organelles, such as mitochondria. The cytotoxic activity of human 12/15-LOX is up-regulated in neurons and endothelial cells especially after a stroke and thought to contribute to both neuronal cell death and blood-brain barrier leakage. The discovery of inhibitors that selectively target recombinant 12/15-LOX in vitro, as well as possessing activity against the murine orthologous ex vivo, could potentially support a novel therapeutic strategy for the treatment of stroke and other brain disorders related to 12/15-LOX. Here we reviewed 12/15-LOX chemistry shortly, and the diseases in which 12/15-LOX has a role in their pathophysiology and recent advances of 12/15-LOX inhibitors as a treatment option for neurological diseases.