The p75 neurotrophin receptor attenuates secondary thalamic damage after cortical infarction by promoting angiogenesis.

BACKGROUND: Angiogenesis is crucial in neuroprotection of secondary thalamic injury after cortical infarction. The p75 neurotrophin receptor (p75NTR) plays a key role in activating angiogenesis. However, the effects of p75NTR on angiogenesis in the thalamus after cortical infarction are largely unknown. Herein we investigate whether p75NTR facilitates angiogenesis to attenuate secondary thalamic damage via activating hypoxia-inducible factor 1α (HIF-1α)/vascular endothelial growth factor (VEGF) pathway mediated by Von Hippel-Lindau (VHL) after distal middle cerebral artery occlusion (dMCAO). METHODS: The male rat model of dMCAO was established. The effects of p75NTR on the angiogenesis was evaluated using RNA-sequencing, immunohistochemistry, western blot, quantitative real-time polymerase chain reaction, magnetic resonance imaging, behavior tests, viral and pharmacological interventions. RESULTS: We found that the p75NTR and vessel density were decreased in ipsilateral thalamus after dMCAO. The p75NTR-VHL interaction was reduced, which promoted the ubiquitination degradation of HIF-1α and reduced VEGF expression after dMCAO. Notably, p75NTR overexpression restrained the ubiquitination degradation of HIF-1α by inhibiting VHL-HIF-1α interaction, further promoted angiogenesis, increased cerebral blood flow of ipsilateral thalamus and improved neurological function after dMCAO. CONCLUSION: For the first time, we highlighted that the enhancement of p75NTR-VHL interaction promoted angiogenesis in attenuating secondary thalamic damage after dMCAO.

Transplantation of hESCs-Derived Neural Progenitor Cells Alleviates Secondary Damage of Thalamus After Focal Cerebral Infarction in Rats.

Human embryonic stem cells-derived neural progenitor cells (hESCs-NPCs) transplantation holds great potential to treat stroke. We previously reported that delayed secondary degeneration occurs in the ventroposterior nucleus (VPN) of ipsilateral thalamus after distal branch of middle cerebral artery occlusion (dMCAO) in adult male Sprague-Dawley (SD) rats. In this study, we investigate whether hESCs-NPCs would benefit the neural recovery of the secondary damage in the VPN after focal cerebral infarction. Permanent dMCAO was performed with electrocoagulation. Rats were randomized into Sham, dMCAO groups with or without hESCs-NPCs treatment. HESCs-NPCs were engrafted into the peri-infarct regions of rats at 48 h after dMCAO. The transplanted hESCs-NPCs survive and partially differentiate into mature neurons after dMCAO. Notably, hESCs-NPCs transplantation attenuated secondary damage of ipsilateral VPN and improved neurological functions of rats after dMCAO. Moreover, hESCs-NPCs transplantation significantly enhanced the expression of BDNF and TrkB and their interaction in ipsilateral VPN after dMCAO, which was reversed by the knockdown of TrkB. Transplantated hESCs-NPCs reconstituted thalamocortical connection and promoted the formation of synapses in ipsilateral VPN post-dMCAO. These results suggest that hESCs-NPCs transplantation attenuates secondary damage of ipsilateral thalamus after cortical infarction, possibly through activating BDNF/TrkB pathway, enhancing thalamocortical projection, and promoting synaptic formation. It provides a promising therapeutic strategy for secondary degeneration in the ipsilateral thalamus post-dMCAO.

Stabilization of nuclear ß-catenin by inhibiting KDM2A mediates cerebral ischemic tolerance.

Hypoxic postconditioning (HPC) with 8% oxygen increases nuclear accumulation of ß-catenin through activating the classical Wnt pathway, thereby alleviating transient global cerebral ischemia (tGCI)-induced neuronal damage in the hippocampal CA1 subregion of adult rats. However, little is understood about the regulatory mechanism of nuclear ß-catenin in HPC-mediated cerebral ischemic tolerance. Although lysine(K)-specific demethylase 2A (KDM2A) has been known as a crucial regulator of nuclear ß-catenin destabilization, whether it plays an important role through modulating nuclear ß-catenin in cerebral ischemic tolerance induced by HPC remains unknown. In this study, we explored the molecular mechanism of stabilizing nuclear ß-catenin by inhibiting KDM2A-mediated demethylation in the HPC-offered neuroprotection against tGCI. In addition, we confirmed that nuclear methylated-ß-catenin in CA1 decreased and nuclear ß-catenin turnover increased after tGCI, which were reversed by HPC. The administration with methyltransferase inhibitor AdOx abrogated HPC-induced methylation and stabilization of nuclear ß-catenin in CA1, as well as the neuroprotection against tGCI. Notably, HPC downregulated the expression of KDM2A in CA1 and reduced the interaction between KDM2A and ß-catenin in the nucleus after tGCI. The knockdown of KDM2A with small-interfering RNA could upregulate nuclear methylated-ß-catenin and stabilize ß-catenin, thereby increasing survivin in CA1 and improving the cognitive function of rats after tGCI. Opposite results were observed by the administration of KDM2A-carried adenovirus vector. Furthermore, we demonstrated that KDM2A mediates the demethylation of nuclear ß-catenin through jumonji C (JmjC) domain of KDM2A in HEK-293T and SH-SY5Y cells. Our data support that the inhibition of KDM2A-mediated demethylation of nuclear ß-catenin contributes to HPC-induced neuroprotection against tGCI.

Hypoxic postconditioning restores mitophagy against transient global cerebral ischemia via Parkin-induced posttranslational modification of TBK1.

Hypoxic postconditioning (HPC) has been reported to enhance Parkin-catalyzed mitochondrial ubiquitination to restore mitophagy in hippocampal CA1 against transient global cerebral ischemia (tGCI). However, the molecular mechanism leading ubiquitinated mitochondria to final clearance during HPC-mediated mitophagy after tGCI is unclear. This study aims to investigate whether HPC restores mitophagy after tGCI through Parkin-induced K63-linked poly-ubiquitination (K63-Ub) to activate tumor necrosis factor associated factor family member associated nuclear factor κB activator -binding kinase 1 (TBK1) in CA1 of male rats. We found that HPC maintained TBK1 expression, promoted p62 and TBK1 phosphorylation in mitochondria, and enhanced their recruitments to mitochondria in CA1 after tGCI. However, these effects were partially abolished by TBK1 inhibitor BX795. K63-Ub of mitochondrial TBK1 was disturbed at 26 h of reperfusion after tGCI, which was reversed by HPC. The maintenance of K63-Ub of mitochondrial TBK1 induced by HPC was counteracted under Parkin knockdown with AAV-mediated Prkn small-interfering RNA, accompanied by the suppression on TBK1 activation and the reduction of mitochondrial p62 phosphorylation. This innovative study indicated that HPC maintained K63-Ub of TBK1 in a Parkin-dependent manner to promote TBK1 phosphorylation, and then phosphorylated TBK1 activated p62 to restore mitophagy, thereby alleviating neuronal damage in CA1 after tGCI.

Neuroprotective Effects of Rhodiola Sacra on Transient Global Cerebral Ischemia Through Activating AMPK/Nrf2 Pathway in Rats.

Aims: Rhodiola sacra is a widely used pharmaceutical component with multiple functions, including anti-oxidation and anti-inflammation. However, the exact mechanisms involved in neuroprotection against transient global cerebral ischemia (tGCI) remain to be elucidated. Herein, we aim at closing the gap in understanding on whether rhodiola sacra reduces neuronal death in hippocampal CA1 and at demonstrating how rhodiola sacra offers neuroprotection after tGCI. Results: The results show that rhodiola sacra (2.4 g/kg/d by feeding) pretreatment or/and postreatment significantly alleviated neuronal injury, inhibited glial activation, and improved cognitive function in male rats subjected to tGCI. The neuroprotection of prophylaxis with rhodiola sacra is equivalent to that of therapeutics. The binding mode of adenosine monophosphate-activated protein kinase (AMPK) α2-subunit with rhodiola sacra was predicted by molecular docking. Further, rhodiola sacra upregulates phosphorylated AMPK and promotes nuclear translocation of nuclear factor erythroid 2 related factor 2 (Nrf2). In addition, rhodiola sacra increases heme oxygenase-1 (HO-1) expression and activity and reduces malondialdehyde (MDA) content in CA1 after tGCI. However, the neuroprotection of rhodiola sacra is abolished by Nrf2 knockdown with small interfering RNA (siRNA) after tGCI. Similarly, the inhibition of AMPK with Compound C or siRNA against AMPK α2 aggravates neuronal death after tGCI through decreasing nuclear Nrf2 and the expression and activity of HO-1, and by increasing the release of MDA. Innovation and Conclusion: For the first time, this study demonstrates that as a prophylactic or therapeutic agent rhodiola sacra prevents oxidant stress, protects neurons, and improves cognitive function through activating the AMPK/Nrf2 pathway in tGCI rats. Antioxid. Redox Signal. 36, 567-591.

Nurses' knowledge and attitudes towards palliative care and death: a learning intervention.

BACKGROUND: In many countries, nurses are ill-prepared to provide care to patients with terminal illnesses. Limited education and training affect their ability to deliver proper palliative care. Only a few studies have explored appropriate and effective training methods of palliative care in China. Therefore, we aimed to provide evidence for a palliative care training system by appraising the effects of a mixed-method intervention on participants' knowledge of palliative care and attitudes towards dying patients and death. METHODS: An e-learning intervention approach was adopted for 97 nurses from oncology departments across five hospitals, using a mobile terminal combined with a virtual forum and face-to-face interactions. We conducted a pre- and post-training evaluation through the Palliative Care Quiz of Nursing (PCQN), Frommelt Attitude Toward Care of the Dying Scale Form B (FATCOD-B), and Death Attitude Profile-Revised (DAP-R). RESULTS: After a three-week intervention, there was a significant increase in the PCQN and FATCOD-B scores as compared to the baseline. For PCQN, the total score increased from 10.3 ± 1.9 to 11.1 ± 2.2 (p = .011) and the score for management of pain and other symptoms increased from 7.7 ± 1.7 to 8.4 ± 1.7 (p = .003). FATCOD-B scores increased noticeably from 100.6 ± 7.9 to 102.9 ± 8.9 (p = .019). The DAP-R scores showed no obvious difference between pre- and post-intervention results. CONCLUSIONS: The mixed-method intervention was effective in improving participants' knowledge and attitudes about palliative care. The implementation of training for nurses at appropriate intervals during both education and professional life is required, especially regarding the improvement in participants' attitudes towards death. Therefore, palliative care training in China should receive more attention.

Hypoxic postconditioning activates the Wnt/ß-catenin pathway and protects against transient global cerebral ischemia through Dkk1 Inhibition and GSK-3ß inactivation.

The Wingless/Int (Wnt)/ß-catenin pathway plays an essential role in cell survival. Although postconditioning with 8% oxygen can alleviate transient global cerebral ischemia (tGCI)-induced neuronal damage in hippocampal CA1 subregion in adult rats as demonstrated by our previous studies, little is understood about the role of Wnt/ß-catenin pathway in hypoxic postconditioning (HPC)-induced neuroprotection. This study tried to investigate the involvement of Wnt/ß-catenin pathway in HPC-induced neuroprotection against tGCI and explore the underlying molecular mechanism thereof. We observed that HPC elevated nuclear ß-catenin level as well as increased Wnt3a and decreased Dickkopf-1 (Dkk1) expression in CA1 after tGCI. Accordingly, HPC enhanced the expression of survivin and reduced the ratio of B-cell lymphoma/lewkmia-2 (Bcl-2)-associated X protein (Bax) to Bcl-2 following reperfusion. Moreover, our study has shown that these effects of HPC were abolished by lentivirus-mediated overexpression of Dkk1, and that the overexpression of Dkk1 completely reversed HPC-induced neuroprotection. Furthermore, HPC suppressed the activity of glycogen synthase kinase-3ß (GSK-3ß) in CA1 after tGCI, and the inhibition of GSK-3ß activity with SB216763 increased the nuclear accumulation of ß-catenin, up-regulated the expression of survivin, and reduced the ratio of Bax to Bcl-2, thus preventing the delayed neuronal death after tGCI. Finally, the administration of LY294002, an inhibitor of PI3K, increased GSK-3ß activity and blocked nuclear ß-catenin accumulation, thereby decreasing survivin expression and elevating the Bax-to-Bcl-2 ratio after HPC. These results suggest that activation of the Wnt/ß-catenin pathway through Dkk1 inhibition and PI3K/protein kinase B pathway-mediated GSK-3ß inactivation contributes to the neuroprotection of HPC against tGCI.-Zhan, L., Liu, D., Wen, H., Hu, J., Pang, T., Sun, W., Xu, E. Hypoxic postconditioning activates the Wnt/ß-catenin pathway and protects against transient global cerebral ischemia through Dkk1 inhibition and GSK-3ß inactivation.

Neuroglobin mediates neuroprotection of hypoxic postconditioning against transient global cerebral ischemia in rats through preserving the activity of Na+/K+ ATPases.

Hypoxic postconditioning (HPC) is an innovative neuroprotective strategy with cytoprotective effects on the hippocampal neurons against transient global cerebral ischemia (tGCI) in adult rats. However, its molecular mechanisms have not yet been adequately elucidated. Neuroglobin (Ngb) is an endogenous neuroprotectant with hypoxia-inducible property, and its role in experimental stroke has been increasingly attractive. Hence, the purpose of this study is to explore the involvement of Ngb in HPC-mediated neuroprotection and to further investigate its underlying molecular mechanism. We found that HPC increased Ngb expression in CA1 subregion after tGCI. Also, the inhibition of Ngb expression with Ngb antisense oligodeoxynucleotide (AS-ODNs) eliminated the neuroprotective effect mediated by HPC, whereas overexpression of Ngb ameliorated neuronal damage in CA1 after tGCI, indicating that HPC conferred neuroprotective effects via upregulation of Ngb. We further showed that HPC increased the membranous level of Na+/K+ ATPases ß1 subunit (Atp1b1) in CA1 after tGCI. Furthermore, we demonstrated that Ngb upregulation in CA1 after HPC maintained the membranous level of Atp1b1 through Ngb-Atp1b1 interaction and reduced the glutathionylation of membranous Atp1b1 via suppression of reactive oxygen species (ROS), ultimately preserving the activity of NKA. Taken together, these data indicate that Ngb is involved in the neuroprotection of HPC against tGCI via maintenance of NKA activity in the hippocampal CA1.

Autophagosome maturation mediated by Rab7 contributes to neuroprotection of hypoxic preconditioning against global cerebral ischemia in rats.

Autophagy disruption leads to neuronal damage in hypoxic-ischemic brain injury. Rab7, a member of the Rab GTPase superfamily, has a unique role in the regulation of autophagy. Hypoxic preconditioning (HPC) provides neuroprotection against transient global cerebral ischemia (tGCI). However, the underlying mechanisms remain poorly understood. Thus, the current study explored the potential molecular mechanism of the neuroprotective effect of HPC by investigating how Rab7 mediates autophagosome (AP) maturation after tGCI in adult rats. We found that HPC attenuated AP accumulation in the hippocampal CA1 region after tGCI via restoration of autophagic flux. We also confirmed that this HPC-induced neuroprotection was not caused by the increase in lysosomes or the improvement of lysosomal function after tGCI. Electron microscopic analysis then revealed an increase in autolysosomes in CA1 neurons of HPC rats. Moreover, the inhibition of autophagosome-lysosome fusion by chloroquine significantly aggravated neuronal death in CA1, indicating that AP maturation contributes to HPC-induced neuroprotection against neuronal injury after tGCI. Furthermore, the activation of Rab7 was found to be involved in the neuroprotective effect of AP maturation after HPC. At last, the knockdown of ultraviolet radiation resistance-associated gene (UVRAG) in vivo disrupted the interaction between Vps16 and Rab7, attenuated the activation of Rab7, interrupted autophagic flux, and ultimately abrogated the HPC-induced neuroprotection against tGCI. Our results indicated that AP maturation was enhanced by the activation of Rab7 via UVRAG-Vps16 interaction, which further demonstrated the potential neuroprotective role of Rab7 in HPC against tGCI-induced neuronal injury in adult rats.

Inhibition of Cathepsin B Alleviates Secondary Degeneration in Ipsilateral Thalamus After Focal Cerebral Infarction in Adult Rats.

Secondary degeneration in areas beyond ischemic foci can inhibit poststroke recovery. The cysteine protease Cathepsin B (CathB) regulates cell death and intracellular protein catabolism. To investigate the roles of CathB in the development of secondary degeneration in the ventroposterior nucleus (VPN) of the ipsilateral thalamus after focal cerebral infarction, infarct volumes, immunohistochemistry and immunofluorescence, and Western blotting analyses were conducted in a distal middle cerebral artery occlusion (dMCAO) stroke model in adult rats. We observed marked neuron loss and gliosis in the ipsilateral thalamus after dMCAO, and the expression of CathB and cleaved caspase-3 in the VPN was significantly upregulated; glial cells were the major source of CathB. Although it had no effect on infarct volume, delayed intracerebroventricular treatment with the membrane-permeable CathB inhibitor CA-074Me suppressed the expression of CathB and cleaved caspase-3 in ipsilateral VPN and accordingly alleviated the secondary degeneration. These data indicate that CathB mediates a novel mechanism of secondary degeneration in the VPN of the ipsilateral thalamus after focal cortical infarction and suggest that CathB might be a therapeutic target for the prevention of secondary degeneration in patients after stroke.

Activation of Akt/FoxO and inactivation of MEK/ERK pathways contribute to induction of neuroprotection against transient global cerebral ischemia by delayed hypoxic postconditioning in adult rats.

Ischemic postconditioning, a series of mechanical interruptions of blood flow immediately after reperfusion, has been described in brain studies. However, hypoxic postconditioning (HPC) has never been reported in transient global cerebral ischemia (tGCI) adult rat model. The purpose of this study is to explore the effects of neuroprotection by delayed HPC against tGCI in adult rats and investigate underlying mechanisms involving the Akt/Forkhead transcription factor, class O (FoxO) and mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathways. Postconditioning with 60-120 min hypoxia significantly reduced cell death in hippocampal CA1 subregion after 10 min of tGCI. Postconditioning was effective only when applied 1-2 days after tGCI. Nevertheless, the combination of hypoxic preconditioning and postconditioning provided no additive protection. Additionally, postconditioning increased phosphorylation of Akt and FoxOs after tGCI. Inhibiting phosphorylation of Akt and FoxOs with LY294002 suppressed the postconditioning-induced neuroprotection. In addition, postconditioning blocked the increase of MEK and ERK phosphorylation after tGCI. Inhibiting phosphorylation of MEK and ERK with U0126 attenuated neuronal damage after tGCI. These results suggest that delayed HPC exerts neuroprotection against tGCI-induced injury in adult rats. The activation of Akt/FoxO and inactivation of MEK/ERK pathways by postconditioning contributed to the induction of neuroprotection against tGCI.

Hypoxic preconditioning induces neuroprotection against transient global ischemia in adult rats via preserving the activity of Na(+)/K(+)-ATPase.

We demonstrated previously that 30min of hypoxic preconditioning (HPC) applied 1day before 10min of transient global cerebral ischemia (tGCI) reduced neuronal loss in the hippocampal CA1 subregion in adult rats. The aim of the present study was to investigate the role of Na(+)/K(+)-ATPase and protein kinase Mζ (PKMζ) in the protective effect of HPC against tGCI in adult rats. We found that the activity of Na(+)/K(+)-ATPase decreased in the hippocampal CA1 subregion after 10min of tGCI. This effect was not seen after 30min of HPC in adult rats. Corresponding to the changes in Na(+)/K(+)-ATPase activity, the surface expression of Na(+)/K(+)-ATPase α1 subunit increased after HPC. Furthermore, HPC dramatically reduced the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells in the hippocampal CA1 subregion after tGCI. However, neither PKMζ nor phosphorylation of PKMζ was changed after tGCI or HPC. The results of the present study are consistent with the hypothesis that both enhanced recovery of Na(+)/K(+)-ATPase activity due to preserved the protein levels of Na(+)/K(+)-ATPase α1 subunit and reduced DNA fragmentation after tGCI contribute to the protection afforded by HPC. However, PKMζ activation does not appear to play a role in this neuroprotection.

Activation of Akt/FoxO signaling pathway contributes to induction of neuroprotection against transient global cerebral ischemia by hypoxic pre-conditioning in adult rats.

It is well established that pre-conditioning protects neuronal injury against ischemia. However, the molecular mechanisms underlying ischemic tolerance are not completely understood. The purpose of the present study was to investigate the role of Akt/forkhead transcription factor, class O (FoxO) pathway in hypoxic pre-conditioning (HPC) using a newly developed HPC to transient global cerebral ischemia (tGCI) model in adult rats. HPC for 30-120 min significantly reduced cell death in the CA1 subregion after 10 min of tGCI. HPC was effective only when applied 1-4 days before ischemia. The maximum protection was observed with 30 min of hypoxia and 1 day interval between hypoxia and ischemia. The phosphorylated Akt and FoxOs measured by western blot and immunohistochemistry were significantly increased after hypoxia-ischemia except for a transient decrease in the HPC group. Lateral ventricular infusion of LY294002 before HPC blocked the increase in phosphorylated Akt and FoxOs and increased neuronal damage in HPC animals. These results suggest that pre-exposure to hypoxia induces protection against tGCI in adult rats. Activation of Akt results in the inactivation of FoxOs which may mediate ischemic tolerance after HPC.