Background: Ischemic stroke is the second leading cause of death globally. The narrow time window for administering effective thrombolytic therapy motivates the search for alternative prevention strategies. Microglia and astrocyte activation-mediated inflammation play a pivotal role in ischemic stroke injury. Cottonseed oil (CSO) has been shown to exert anti-inflammatory effects against peripheral tissue injury, although CSO is mostly used as a solvent for lipid-soluble drugs. However, the role of CSO in neuroprotection against stroke has not been previously reported. Methods: We treated adult male rats with CSO (1.3 ml/kg, subcutaneous injection, once every other day for 3 weeks) and then constructed a middle cerebral artery occlusion (MCAO) model followed by 24 h of reperfusion. Then, we measured the neurological scores, infarction volume, neuronal injury, and brain edema; we also measured the levels of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α), degree of microglial and astrocytic activation, protein expression levels of Toll-like receptor 4 (TLR4), nuclear factor kappa B (NF-κB), C3d and S100A10, and the presence of A1 type astrocytes and A2 type astrocytes. Results: We found that CSO treatment significantly improved the neurological deficit, reduced infarction volume, and alleviated neuronal injuries, blood–brain barrier (BBB) disruption, and brain edema. Additionally, CSO treatment significantly reduced microglial and astrocytic activation, inhibited TLR4 and NF-κB protein expression, and reduced the release of IL-1β, IL-6, and TNF-α. Finally, CSO treatment significantly decreased the number of C3d/glial fibrillary acidic protein (GFAP)-positive cells and C3d protein expression, and increased the number of S100A10/GFAP-positive cells and S100A10 protein expression. Conclusion: Our results first found that CSO treatment alleviated ischemic stroke injury by reducing microglial and astrocytic activation and inflammation, which was related to the inhibition of TLR4/NF-κB pathway and the reduction of A1 phenotype neurotoxic astrocyte activation, suggesting that CSO could be a new strategy in the prevention of ischemic stroke.

The steroid hormone estrogen (17-β-estradiol, E2) provides neuroprotection against cerebral ischemic injury by activating estrogen receptors. The novel estrogen receptor G protein-coupled receptor 30 (GPR30) is highly expressed in the brain and provides acute neuroprotection against stroke. However, the underlying mechanisms remain unclear. In this study, ovariectomized female mice were subjected to middle cerebral artery occlusion (MCAO), and E2, G1, and ICI182780 were administered immediately upon reperfusion. The infarction volume, neurological scores, and neuronal injuries were examined. Primary microglial cells were subjected to oxygen-glucose deprivation (OGD), and the drugs were administered immediately upon reintroduction. The pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 in penumbra and microglia were assessed by ELISA. The cell viability and lactose dehydrogenase (LDH) release of neurons co-cultured with microglia were analyzed using cell counting kit-8 (CCK8) and LDH release assays. Microglial activation as well as GPR30, Iba1, and Toll-like receptor 4 (TLR4) protein expression and TLR4 mRNA expression were detected. Additionally, NF-κB activity was detected in lipopolysaccharide (LPS)-activated microglia after the activation of GPR30. GPR30 was highly expressed in microglia and significantly increased after ischemic injury. The activation of GPR30 significantly reduced the infarction volume, improved the neurological deficit, and alleviated neuronal injuries. Moreover, GPR30 activation significantly reduced the release of TNF-α, IL-1β, and IL-6 from ischemic penumbra and microglia subjected to OGD and alleviated neuronal injury as assessed using the CCK8 and LDH assays. Finally, the activation of GPR30 relieved microglial activation, reduced Iba1 and TLR4 protein expression and TLR4 mRNA levels, and inhibited NF-κB activity. Microglial GPR30 exerts acute neuroprotective effects by inhibiting TLR4-mediated microglial inflammation, which indicates that GPR30 may be a potential target for the treatment of ischemic stroke.

Astrocytes possess different morphological characteristics depending on the cerebral region in which they are found. However, none of the current astrocytic markers can label all subpopulations successfully. Thus, identifying the appropriate marker for a specific scientific investigation is critical. Here, we compared the distribution and protein expression of three astrocyte markers: NDRG2, GFAP, and S100β, in the cortex, hippocampus, and thalamus. NDRG2- and S100β-positive astrocytes were distributed more uniformly than GFAP-positive astrocytes throughout the whole cerebrum. NDRG2 and S100βimmunoreactivities were the strongest in the dorsal cortex and thalamus, while GFAP immunoreactivity was the strongest in the hippocampus. Moreover, protein expression levels of NDRG2, GFAP, and S100βin adult mice were the highest in the cortex, hippocampus, and thalamus, respectively. We also detected astrocyte morphology and found that, in the corpus callosum and cerebral peduncle, GFAP-positive astrocytes were found with more numerous and longer processes than NDRG2- and S100β-positive astrocytes. These results demonstrate that NDRG2 and S100βare more suitably used to visualize the overall distribution and changes in the number of astrocytes, as well as label astrocytes in the cortex and thalamus. GFAP, however, is more appropriately used to label astrocytes in the corpus callosum, cerebral peduncle, and the hippocampus. These results help to guide researchers in the choice of appropriate astrocyte marker and suggest differences in immunological qualities of astrocytes based on the tissue in which they are found.

CNS inflammation is known to be an important pathogenetic mechanism of perioperative neurocognitive disorder (PND), and iron overload was reported to participate in this process accompanied by oxidative stress. Ferroptosis is an iron-dependent form of cell death, and occurs in multiple neurodegenerative diseases with cognitive disorder. However, the effect of ferroptosis in inflammation-related PND is unknown. In this study, we found that the ferroptosis inhibitor liproxstatin-1 ameliorated memory deficits in the mouse model of lipopolysaccharide (LPS)-induced cognitive impairment. Moreover, liproxstatin-1 decreased the activation of microglia and the release of interleukin (IL)-6 and tumor necrosis factor-alpha (TNF)-α, attenuated oxidative stress and lipid peroxidation, and further weakened mitochondrial injury and neuronal damage after LPS exposure. Additionally, the protective effect of liproxstatin-1 was related to the alleviation of iron deposition and the regulation of the ferroptosis-related protein family TF, xCT, Fth, Gpx4, and FtMt. These findings enhance our understanding of inflammation-involved cognitive dysfunction and shed light on future preclinical studies.

17β-Estradiol (estradiol or E2) is a steroid hormone that has been broadly applied as a neuroprotective therapy for a variety of neurodegenerative and cerebrovascular disorders such as ischemic stroke, Alzheimer's disease, and Parkinson's disease. Several laboratory and clinical studies have reported that Estrogen Replacement Therapy (ERT) had no effect against these diseases in elderly postmenopausal women, and at worst, increased their risk of onset and mortality. This review focuses on the growing body of data from in vitro and animal models characterizing the potential underlying mechanisms and signaling pathways that govern successful neuroprotection by ERT, including the roles of E2 receptors in mediating neuroprotection, E2 genomic regulation of apoptosis- related pathways, membrane-bound receptor-mediated non-genomic signaling pathways, and the antioxidant mechanisms of E2. Also discussed is the current evidence for a critical period of effective treatment with estrogen following natural or surgical menopause and the outcomes of E2 administration within an advantageous time period. The known mechanisms governing the duration of the critical period include depletion of E2 receptors, the switch to a ketogenic metabolic profile by neuronal mitochondria, and a decrease in acetylcholine that accompanies E2 deficiency. Also the major clinical trials and observational studies concerning postmenopausal Hormone Therapy (HT) are summarized to compare their outcomes with respect to neurological disease and discuss their relevance to the critical period hypothesis. Finally, potential controversies and future directions for this field are discussed throughout the review.