Enriched Environment Inhibits Neurotoxic Reactive Astrocytes via JAK2-STAT3 to Promote Glutamatergic Synaptogenesis and Cognitive Improvement in Chronic Cerebral Hypoperfusion Rats.

Chronic cerebral hypoperfusion (CCH) is a primary contributor to cognitive decline in the elderly. Enriched environment (EE) is proved to improve cognitive function. However, mechanisms involved remain unclear. The purpose of the study was exploring the mechanisms of EE in alleviating cognitive deficit in rats with CCH. To create a rat model of CCH, 2-vessel occlusion (2-VO) surgery was performed. All rats lived in standard or enriched environments for 4 weeks. Cognitive function was assessed using the novel object recognition test and Morris water maze test. The protein levels of glutamatergic synapses, neurotoxic reactive astrocytes, reactive microglia, and JAK2-STAT3 signaling pathway were measured using Western blot. The mRNA levels of synaptic regulatory factors, C1q, TNF-α, and IL-1α were identified using quantitative PCR. Immunofluorescence was used to detect glutamatergic synapses, neurotoxic reactive astrocytes, and reactive microglia, as well as the expression of p-STAT3 in astrocytes in the hippocampus. The results demonstrated that the EE mitigated cognitive impairment in rats with CCH and enhanced glutamatergic synaptogenesis. EE also inhibited the activation of neurotoxic reactive astrocytes. Moreover, EE downregulated microglial activation, levels of C1q, TNF-α and IL-1α and phosphorylation of JAK2 and STAT3. Our results suggest that inhibition of neurotoxic reactive astrocytes may be one of the mechanisms by which EE promotes glutamatergic synaptogenesis and improves cognitive function in rats with CCH. The downregulation of reactive microglia and JAK2-STAT3 signaling pathway may be involved in this process.

Enriched environment attenuates ferroptosis after cerebral ischemia/reperfusion injury by regulating iron metabolism.

Preventing neuronal death after ischemic stroke (IS) is crucial for neuroprotective treatment, yet current management options are limited. Enriched environment (EE) is an effective intervention strategy that promotes the recovery of neurological function after cerebral ischemia/reperfusion (I/R) injury. Ferroptosis has been identified as one of the mechanisms of neuronal death during IS, and inhibiting ferroptosis can reduce cerebral I/R injury. Our previous research has demonstrated that EE reduced ferroptosis by inhibiting lipid peroxidation, but the underlying mechanism still needs to be investigated. This study aims to explore the potential molecular mechanisms by which EE modulates iron metabolism to reduce ferroptosis. The experimental animals were randomly divided into four groups based on the housing environment and the procedure the animals received: the sham-operated + standard environment (SSE) group, the sham-operated + enriched environment (SEE) group, the ischemia/reperfusion + standard environment (ISE) group, and the ischemia/reperfusion + enriched environment (IEE) group. The results showed that EE reduced IL-6 expression during cerebral I/R injury, hence reducing JAK2-STAT3 pathway activation and hepcidin expression. Reduced hepcidin expression led to decreased DMT1 expression and increased FPN1 expression in neurons, resulting in lower neuronal iron levels and alleviated ferroptosis. In addition, EE also reduced the expression of TfR1 in neurons. Our research suggested that EE played a neuroprotective role by modulating iron metabolism and reducing neuronal ferroptosis after cerebral I/R injury, which might be achieved by inhibiting inflammatory response and down-regulating hepcidin expression.

Enriched Environment Attenuates Ferroptosis after Cerebral Ischemia/Reperfusion Injury via the HIF-1α-ACSL4 Pathway.

Enriched environment (EE) has been proven to be an effective intervention strategy which can improve neurofunctional recovery following cerebral ischemia/reperfusion (I/R) injury. However, it still needs further investigation for the underlying mechanisms. Recently, it has been shown that ferroptosis played an essential role in the pathophysiological development of ischemic stroke (IS). This study is aimed at investigating whether EE plays a neuroprotective role by attenuating ferroptosis after cerebral I/R injury. We used middle cerebral artery occlusion/reperfusion (MCAO/R) to build a model of cerebral I/R injury. To evaluate the effect of EE on neurological recovery, we used the modified neurological severity score (mNSS) and the Morris water maze (MWM). We used the western blot to detect the protein levels of glutathione peroxidase 4 (GPX4), hypoxia-inducible factor-1α (HIF-1α), and acyl-CoA synthetase long-chain family member 4 (ACSL4). We used the quantitative real-time PCR (qRT-PCR) to measure the mRNA levels of ACSL4 and inflammatory cytokines including tumor necrosis factor alpha (TNFα), interleukin-6 (IL-6), and interleukin 1 beta (IL-1ß). The occurrence of ferroptosis was detected by TdT-mediated dUTP nick-end labeling (TUNEL) assay, diaminobenzidine- (DAB-) enhanced Perls' staining, iron level assays, and malondialdehyde (MDA) level assays. The results verified that EE enhanced functional recovery and attenuated ferroptosis and neuroinflammation after cerebral I/R injury. EE increased the expression of HIF-1α while inhibited the expression of ACSL4. Our research indicated that EE improved functional recovery after cerebral I/R injury through attenuating ferroptosis, and this might be related to its regulation of the neuroinflammation and HIF-1α-ACSL4 pathway.