Invasive metastatic tumor-camouflaged ROS responsive nanosystem for targeting therapeutic brain injury after cardiac arrest.

Drug transmission through the blood-brain barrier (BBB) is considered an arduous challenge for brain injury treatment following the return of spontaneous circulation after cardiac arrest (CA-ROSC). Inspired by the propensity of melanoma metastasis to the brain, B16F10 cell membranes are camouflaged on 2-methoxyestradiol (2ME2)-loaded reactive oxygen species (ROS)-triggered "Padlock" nanoparticles that are constructed by phenylboronic acid pinacol esters conjugated D-a-tocopheryl polyethylene glycol succinate (TPGS-PBAP). The biomimetic nanoparticles (BM@TP/2ME2) can be internalized, mainly mediated by the mutual recognition and interaction between CD44v6 expressed on B16F10 cell membranes and hyaluronic acid on cerebral vascular endothelial cells, and they responsively release 2ME2 by the oxidative stress microenvironment. Notably, BM@TP/2ME2 can scavenge excessive ROS to reestablish redox balance, reverse neuroinflammation, and restore autophagic flux in damaged neurons, eventually exerting a remarkable neuroprotective effect after CA-ROSC in vitro and in vivo. This biomimetic drug delivery system is a novel and promising strategy for the treatment of cerebral ischemia-reperfusion injury after CA-ROSC.

MCOLN1/TRPML1 in the lysosome: a promising target for autophagy modulation in diverse diseases.

MCOLN1/TRPML1 is a nonselective cationic channel specifically localized to the late endosome and lysosome. With its property of mediating the release of several divalent cations such as Ca2+, Zn2+ and Fe2+ from the lysosome to the cytosol, MCOLN1 plays a pivotal role in regulating a variety of cellular events including endocytosis, exocytosis, lysosomal biogenesis, lysosome reformation, and especially in Macroautophagy/autophagy. Autophagy is a highly conserved catabolic process that maintains cytoplasmic integrity by removing superfluous proteins and damaged organelles. Acting as the terminal compartments, lysosomes are crucial for the completion of the autophagy process. This review delves into the emerging role of MCOLN1 in controlling the autophagic process by regulating lysosomal ionic homeostasis, thereby governing the fundamental functions of lysosomes. Furthermore, this review summarizes the physiological relevance as well as molecular mechanisms through which MCOLN1 orchestrates autophagy, consequently influencing mitochondria turnover, cell apoptosis and migration. In addition, we have illustrated the implications of MCOLN1-regulated autophagy in the pathological process of cancer and myocardial ischemia-reperfusion (I/R) injury. In summary, given the involvement of MCOLN1-mediated autophagy in the pathogenesis of cancer and myocardial I/R injury, targeting MCOLN1 May provide clues for developing new therapeutic strategies for the treatment of these diseases. Exploring the regulation of MCOLN1-mediated autophagy in diverse diseases contexts will surely broaden our understanding of this pathway and offer its potential as a promising drug target.Abbreviation: CCCP:carbonyl cyanide3-chlorophenylhydrazone; CQ:chloroquine; HCQ: hydroxychloroquine;I/R: ischemia-reperfusion; MAP1LC3/LC3:microtubule associated protein 1 light chain 3; MCOLN1/TRPML1:mucolipin TRP cation channel 1; MLIV: mucolipidosis type IV; MTORC1:MTOR complex 1; ROS: reactive oxygenspecies; SQSTM1/p62: sequestosome 1.

cRGD-modified nanoparticles of multi-bioactive agent conjugate with pH-sensitive linkers and PD-L1 antagonist for integrative collaborative treatment of breast cancer.

Targeted co-delivery and co-release of multi-drugs is essential to have an integrative collaborative effect on treating cancer. It is valuable to use few drug carriers for multi-drug delivery. Herein, we develop cRGD-modified nanoparticles (cRGD-TDA) of a conjugate of doxorubicin as cytotoxic agent, adjudin as an anti-metastasis agent and D-α-tocopherol polyethylene glycol 1000 succinate (TPGS) as a reactive oxygen species inducer linked with pH-sensitive bonds, and then combine the nanoparticles with PD-L1 antagonist to treat 4T1 triple-negative breast cancer. cRGD-TDA NPs present tumor-targeted co-delivery and pH-sensitive co-release of triple agents. cRGD-TDA NPs combined with PD-L1 antagonist much more significantly inhibit tumor growth and metastasis than single-drug treatment, which is due to their integrative collaborative effect. It is found that TPGS elicits a powerful immunogenic cell death effect. Meanwhile, PD-L1 antagonist mitigates the immunosuppressive environment and has a synergistic effect with the cRGD-TDA NPs. The study provides a new strategy to treat refractory cancer integratively and collaboratively.

A Comparison of Two Peripheral Nerve Blocks Combined With General Anesthesia in Elderly Patients Undergoing Arthroplasty for Hip Fractures: A Pilot Randomized Controlled Trial.

BACKGROUND: Combined anesthesia can be a promising option for hip surgery when neuraxial anesthesia is contraindicated. Lumbar and sacral plexus blocks, and femoral nerve and lateral femoral cutaneous (LFC) nerve blocks in combination with general anesthesia (GA) are commonly used in elderly patients undergoing arthroplasty for hip fracture surgery. However, no study has compared these two anesthetic strategies in the perioperative period. METHODS: A total of 41 elderly patients scheduled for arthroplasty for hip fracture surgery were randomized into group A (n = 20) and group B (n = 21). Group A received femoral nerve block, LFC nerve blocks, and GA, and group B received lumbar plexus block, sacral plexus block, and GA. Primary outcomes were incidences of hemodynamic events and changes in blood pressure (BP) and heart rate (HR). Secondary outcomes included time and drug consumption, infusion and bleeding volume, eyes opening time after surgery, and postoperative quality recovery rate. RESULTS: Compared with group B, group A showed a lower incidence of intraoperative hypotension (p < 0.001), higher BP [including mean arterial pressure (MAP), systolic BP (SBP), and diastolic BP (DBP)] following induction (IN), and higher HR from mid-surgery. Time required for nerve blockade (p < 0.001) and ephedrine consumption was significantly shorter in group A (p < 0.001), while sufentanil consumption was higher as compared to group B (p = 0.002). No significant differences in other intraoperative parameters and postoperative quality recovery rate were reported during the observation. CONCLUSION: Our pilot data indicate that compared with lumbar and sacral plexus blocks, femoral nerve and LFC nerve blocks may provide more stable intraoperative hemodynamics and a comparable postoperative recovery for elderly patients undergoing arthroplasty for hip fracture under GA. Further studies with a larger sample size are needed to derive stronger evidence.

Combining ultrasound with bio-indicators reveals progression of carotid stenosis.

BACKGROUND: Carotid artery stenosis (CAS) is one of the leading causes of ischemic stroke. However, knowledge of the changes in the plaque itself is lacking. Information about the ultrasound and clinical features of CAS will help elucidate the changes in prognostic and risk factors. METHODS: We evaluated 736 patients with carotid stenosis for an average 18-month follow-up. According to their degree of CAS stenosis, patients were allocated to one of three groups: regression (n=125), stable (n=443), or progression (n=168). An ordinal regression analysis was used to determine the risk factors for atherosclerosis progression. A logistic regression was subsequently applied to investigate the effects of CAS stenosis on cerebrovascular events after adjusting for various factors. RESULTS: The progression group had more male patients (P=0.02), hypoechoic plaque (P<0.01), high-risk high sensitivity C-reactive protein (hs-CRP) (P=0.02), ulcerative plaque (P=0.05), and hyperlipidemia (P=0.05) than the other two groups. There were no significant differences in residual ultrasound and clinical features among the three groups, including age, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), intima-media thickness (IMT), body mass index (BMI), diabetes mellitus (DM), hypertension (HTN), coronary heart disease (CHD), statin use, ulcerative plaque. The ordinal regression analysis identified hypoechoic plaque (OR, 1.53; 95% CI: 1.14-2.05; P<0.01) and high-risk hs-CRP (OR, 1.75; 95% CI: 1.17-2.61; P<0.01) as independent risk factors for CAS progression. Logistic regression analysis revealed that the stroke/transient ischemic attack adjusted odds ratio was 1.80 (95% CI: 1.03-3.13) in the progression group. CONCLUSIONS: High-risk hs-CRP and hypoechoic plaque are independently associated with CAS progression. The progression of carotid stenosis is associated with a high risk of cerebrovascular events.

The balance between AIM2-associated inflammation and autophagy: the role of CHMP2A in brain injury after cardiac arrest.

BACKGROUND: Activation of the absent in melanoma 2 (AIM2) inflammasome and impaired autophagosome clearance in neurons contribute significantly to cardiac arrest and return of spontaneous circulation (CA-ROSC) injury, while the mechanism by which the AIM2 inflammasome is regulated and relationship between the processes remain poorly understood. Recently, charged multivesicular body protein 2A (CHMP2A), a subunit of endosomal sorting complex required for transport (ESCRT), was shown to regulate phagophore closure, and its depletion led to the accumulation of autophagosomes and induced cell death. Here, we investigated whether CHMP2A-mediated autophagy was an underlying mechanism of AIM2-associated inflammation after CA-ROSC and explored the potential link between the AIM2 inflammasome and autophagy under ischemic conditions. METHODS: AIM2 inflammasome activation and autophagic flux in the cortex were assessed in the CA-ROSC rat model. We injected LV-Vector or LV-CHMP2A virus into the motor cortex with stereotaxic coordinates and divided the rats into four groups: Sham, CA, CA+LV-Vector, and CA+LV-CHMP2A. Neurologic deficit scores (NDSs), balance beam tests, histopathological injury of the brain, and expression of the AIM2 inflammasome and proinflammatory cytokines were analyzed. RESULTS: AIM2 inflammasome activation and increased interleukin 1 beta (IL-1ß) and IL-18 release were concurrent with reduced levels of CHMP2A-induced autophagy in CA-ROSC rat neurons. In addition, silencing CHMP2A resulted in autophagosome accumulation and decreased autophagic degradation of the AIM2 inflammasome. In parallel, a reduction in AIM2 contributed to autophagy activation and mitigated oxygen-glucose deprivation and reperfusion (OGD-Rep)-induced inflammation. Notably, CHMP2A overexpression in the cortex hindered neuroinflammation, protected against ischemic brain damage, and improved neurologic outcomes after CA. CONCLUSIONS: Our results support a potential link between autophagy and AIM2 signaling, and targeting CHMP2A may provide new insights into neuroinflammation in the early phase during CA-ROSC.

Blood flow changes in the forearm arteries after ultrasound-guided costoclavicular brachial plexus blocks: a prospective observational study.

BACKGROUND: An increase in blood flow in the forearm arteries has been reported after brachial plexus block (BPB). However, few studies have quantitatively analysed the blood flow of the forearm arteries after BPB or have studied only partial haemodynamic parameters. The purpose of the present study was to comprehensively assess blood flow changes in the distal radial artery (RA) and ulnar artery (UA) after BPB performed via a new costoclavicular space (CCS) approach using colour Doppler ultrasound. METHODS: Thirty patients who underwent amputated finger replantation and received ultrasound-guided costoclavicular BPB were included in the study. The haemodynamic parameters of the RA and UA were recorded before the block and 10 min, 20 min, and 30 min after the block using colour Doppler ultrasound to determine the peak systolic velocity (PSV), end-diastolic velocity (EDV), mean velocity (Vmean), pulsatility index (PI), resistance index (RI) and area. The volumetric flow rate (VFR) was calculated using the formula Q = area×Vmean. The aforementioned parameters were compared not only before and after the BPB but also between the RA and UA. RESULTS: Compared with those of the respective baselines, there was a significant increase in the PSV, EDV, Vmean, area, and VFR and a significant decrease in the PI and RI of the RA and UA 10 min, 20 min, and 30 min post-block. The increase 30 min post-block in EDV (258.68 % in the RA, 279.63 % in the UA) was the most notable, followed by that in the Vmean (183.36 % in the RA, 235.24 % in the UA), and the PSV (139.11 % in the RA, 153.15 % in the UA) changed minimally. The Vmean and VFR of the RA were significantly greater than those of the UA before the BPB; however, there was no significant difference in the VFR between the RA and UA after the BPB. CONCLUSIONS: A costoclavicular BPB can increase blood flow in the forearm arteries. The RA had a higher volumetric flow rate than the UA before the BPB; however, the potential blood supply capacity of the UA was similar to that of the RA after a BPB. TRIAL REGISTRATION: This study was registered at the Chinese Clinical Trial Registry (http://www.chictr.org.cn/searchproj.aspx, clinical trial number: ChiCTR 1900023796, date of registration: June 12, 2019).

White matter injury in the neonatal hypoxic-ischemic brain and potential therapies targeting microglia.

Neonatal hypoxic-ischemic (H-I) injury, which mainly causes neuronal damage and white matter injury (WMI), is among the predominant causes of infant morbidity (cerebral palsy, cognitive and persistent motor disabilities) and mortality. Disruptions to the oxygen and blood supply in the perinatal brain affect the cerebral microenvironment and may affect microglial activation, excitotoxicity, and oxidative stress. Microglia are significantly associated with axonal damage and myelinating oligodendrocytes, which are major pathological components of WMI. However, the effects of H-I injury on microglial functions and underlying transformation mechanisms remain poorly understood. The historical perception that these cells are major risk factors for ischemic stroke has been questioned due to our improved understanding of the diversity of microglial phenotypes and their alterable functions, which exacerbate or attenuate injuries in different regions in response to environmental instability. Unfortunately, although therapeutic hypothermia is an efficient treatment, death and disability remain the prognosis for a large proportion of neonates with H-I injury. Hence, novel neuroprotective therapies to treat WMI following H-I injury are urgently needed. Here, we review microglial mechanisms that might occur in the developing brain due to neonatal H-I injury and discuss whether microglia function as a double-edged sword in WMI. Then, we emphasize microglial heterogeneity, notably at the single-cell level, and sex-specific effects on the etiology of neurological diseases. Finally, we discuss current knowledge of strategies aiming to improve microglia modulation and remyelination following neonatal H-I injury. Overall, microglia-targeted therapy might provide novel and valuable insights into the treatment of neonatal H-I insult.

Increased PINK1/Parkin-mediated mitophagy explains the improved brain protective effects of slow rewarming following hypothermia after cardiac arrest in rats.

Cerebral ischemia-reperfusion (I/R) after cardiac arrest (CA) induces mitochondrial dysfunction, and the timely removal of damaged mitochondria by mitophagy is reported to protect against cerebral I/R injury. Therapeutic hypothermia (TH) has become an important component of postresuscitation care for patients who return to spontaneous circulation after CA. Previous studies have shown that TH can activate mitophagy and can contribute a protective effect; however, the optimal rewarming rate and underlying mechanism of rewarming following TH remain largely unexplained. Here, we investigated the effects of different rewarming rates and whether mitophagy is involved in rewarming. After 5 min of asphyxial CA following 4 h of cooling, Sprague-Dawley rats were randomized into the normothermia, hypothermia, slow rewarming (0.5 °C/h) and fast rewarming (4 °C/h) groups. The hypothermia group was kept cool until tissue harvest, the rewarming duration for the slow rewarming group and fast rewarming group was 6 h and 45 min, respectively. We found that slowly rewarmed rats had better survival at 72 h than normothermic rats and fast-rewarmed rats (70%, 25.71%, and 50%, respectively) and higher neurological deficit scores (NDSs), in which the medians were 57.33, 26, and 28.83, respectively. In addition, we explored the underlying mechanism during this process and found that PINK1/Parkin-mediated mitophagy was activated during hypothermia in the slow rewarming group but was inhibited in the fast rewarming group. Further inhibition of mitophagy in the slowly rewarmed rats resulted in severe apoptosis and decreased the mean NDS from 58.39 to 33.11, indicating the protective role of mitophagy. Moreover, the fast rewarming group exhibited deficiencies in PINK1 expression and mitophagy activity and marked accumulation of reactive oxygen species (ROS). Overall, our results highlighted a neuroprotective role of PINK1/Parkin-mediated mitophagy during slow rewarming after hypothermia.

Effects of the Incidence Density of Fever (IDF) on Patients Resuscitated From In-Hospital Cardiac Arrest: A Mediation Analysis.

Objective: The aim of this research was to study the factors contributing to the survival rate of in-hospital cardiac arrest (IHCA) and to determine whether the incidence density of fever (IDF) acts as a mediator. Methods: Data from patients with IHCA who survived more than 48 h were collected from 2011 to 2017. IDF was defined as the fever duration divided by the hospitalization duration, prolonged fever was defined as fever lasting for more than 5 days, and early fever was defined as an initial onset within the first 2 days of IHCA. Possible clinical variables associated with IDF were examined by linear regression, and possible clinical variables associated with survival rate were examined by univariate and multivariate analyses. IDF was investigated as a mediator of the indirect effects of the risk factors on survival. Results: In our retrospective study, the median IDF was 0, with an interquartile range from 0 to 0.42. Prolonged fever was noted in 16% (97/605) of the total, and early fever was noted in 17.2% (104/605) of the total. Linear regression results showed that positive chest X-ray, central venous catheter and Glasgow Coma Score (GCS) ≤ 8 were related to IDF. The IDF (OR: 0.36, 95% CI, 0.13-0.97, P = 0.04), prolonged fever (adjusted OR = 0.13, 95% CI, 0.06-0.29, P < 0.001), positive chest X-ray (OR: 0.67, 95% CI, 0.46-0.98, P = 0.04), central venous catheter placement (OR: 0.54, 95% CI, 0.34-0.89, P = 0.01), and endotracheal intubation (OR: 0.47, 95% CI, 0.33-0.69, P < 0.001) were also related to the negative outcome of hospital discharge after adjustment. Additionally, positive chest X-ray had a 19% effect on survival outcome through IDF as a mediator, and the indirect effect of central venous catheter mediated by IDF accounted for 10% of the total. Conclusions: A higher IDF, prolonged fever, a positive chest X-ray, the use of a central venous catheter and endotracheal intubation reduced the survival rate of these patients, and the detrimental impacts of a positive chest X-ray and the use of a central venous catheter on survival outcomes were partially mediated by IDF.

Inhibition of neuronal ferroptosis in the acute phase of intracerebral hemorrhage shows long-term cerebroprotective effects.

Intracerebral hemorrhage (ICH) is a devastating subtype of stroke because it has few viable therapeutic options to intervene against primary or second brain injury. Recently, evidence has suggested that ferroptosis, a nonapoptotic form of cell death, is involved in ICH. In this study, we examined whether ICH-induced neuron death is partly ferroptotic in humans and assessed its temporal and spatial characteristics in mice. Furthermore, the ferroptosis inhibitor ferrostatin-1 (Fer-1) was used to examine the role of ferroptosis after ICH. Fold changes in ferroptosis-related gene expression, intracellular iron levels, malondialdehyde (MDA) levels, and both protein levels and cellular localization of cyclooxygenase-2 (COX-2) were measured to monitor ferroptosis. Transmission electron microscopy (TEM) was also performed to examine the ultrastructure of cells after ICH. We found that the expression level of prostaglandin-endoperoxide synthase (PTGS2) was increased in both in vitro and in vivo ICH models; by comparison, expression level of RPL8 was increased in human brain tissue. In mice, iron and MDA levels were significantly increased 3 h after ICH; COX-2 levels were increased at 12 h after ICH and peaked at 3 days after ICH; COX-2 colocalized with NeuN (a neuronal biomarker); and TEM showed that shrunken mitochondria were found at 3 h, 3 days, and 7 days after ICH. Moreover, ICH-induced neurological deficits, memory impairment and brain atrophy were reduced by Fer-1 treatment. Our results demonstrated that neuronal ferroptosis occurs during the acute phase of ICH in brain areas distant from the hematoma and that inhibition of ferroptosis by Fer-1 exerted a long-term cerebroprotective effect.

The roles of oxidative stress and Beclin-1 in the autophagosome clearance impairment triggered by cardiac arrest.

During cardiac arrest and return of spontaneous circulation (CA-ROSC), autophagosome clearance in the cortex is progressively impaired, but the role of reactive oxygen species (ROS) in this process and the mechanism underlying the autophagy impairment remain unknown. In this study, we investigated the impacts of ROS on the autophagy-lysosome pathway after CA-ROSC in rats. Cortices from CA-ROSC rats revealed accumulation of LC3, p62 and ubiquitin, indicating impaired autophagic flux. Furthermore, impairment of autophagic flux was related to lysosomal lesion, as indicated by decreased cathepsin D and lysosomal-associated membrane protein 2 (LAMP2) levels after CA-ROSC. In vitro, the resulting ROS generation blocked autophagosome processing and caused accumulation of LC3-II, ubiquitin, and p62, leading to mitochondrial dysfunction and cell death; this outcome was alleviated by cyclosporine A (CsA) pretreatment. Interestingly, ischemia/reperfusion injury was connected with ROS-mediated Beclin-1 upregulation and a reduction in LAMP2, which is a pivotal protein in the autophagy-lysosome pathway. Recovery of the LAMP2 levels and partial Beclin-1 silencing restored the autophagic flux and reduced cell death after CA-ROSC. Taken together, our data indicate that CA-ROSC injury impairs autophagosome clearance partially through a ROS-induced decline in LAMP2 and increase in Beclin-1, leading to increased neuronal cell death.

Angiopep-2 modified PEGylated 2-methoxyestradiol micelles to treat the PC12 cells with oxygen-glucose deprivation/reoxygenation.

2-Methoxyestradiol (2ME2), as a microtubule and hypoxia-inducible factor-1 (HIF-1) inhibitor, can be used to treat cerebral ischemia-reperfusion (I/R) injury. However, its poor water solubility compromises its efficacy as a neuroprotectant. Herein, we synthesized PEGylated 2ME2 and angiopep-2 capped PEGylated 2ME2 and fabricated angiopep-2 modified PEGylated 2ME2 micelles containing free 2ME2 (ANG-PEG-2ME2/2ME2) via emulsion-solvent evaporation method. The effect of the micelles on ischemia-reoxygenation injury was evaluated by oxygen-glucose deprivation/reoxygenation (OGD/R) models with different degrees of PC12 cell damage. In comparison with free 2ME2, the micelles significantly increased the cell viability, inhibited reactive oxygen species (ROS) generation and apoptosis for PC12 cells with 0.5 and 4 h OGD followed by 24 h reoxygenation. Taken together, the angiopep-2 modified 2ME2-loaded micelles could effectively reduce the injury of PC12 cells induced by OGD/R.

[Application of targeted temperature management in the treatment of cardiac arrest in adult patients].

OBJECTIVE: Cardiac arrest (CA) is a fatal condition with low resuscitation rate and high mortality rate. Most of the survivors have neurological sequelae affecting their quality of life. Targeted temperature management (TTM) has been suggested by a number of studies to increase the survival rate and improve neurological outcome of CA. It is highly recommended by the International Liaison Committee on Resuscitation (ILCOR) for unconscious patients after resuscitation. In this review, we discuss the pathological mechanism of brain injury in CA and applications of TTM in adults with CA, with the aim of providing valuable information for clinical application.

bFGF plays a neuroprotective role by suppressing excessive autophagy and apoptosis after transient global cerebral ischemia in rats.

Transient global cerebral ischemia (tGCI) is a cerebrovascular disorder that can cause apoptotic neuronal damage and functional deficits. Basic fibroblast growth factor (bFGF) was reported to be highly expressed in the central nervous system (CNS) and to exert neuroprotective effects against different CNS diseases. However, the effects of bFGF on tGCI have not been studied intensively. This study was conducted to investigate the effect of bFGF and its underlying mechanism in an animal model of tGCI. After intracerebroventricular (i.c.v.) injection of bFGF, functional improvement was observed, and the number of viable neurons increased in the ischemia-vulnerable hippocampal CA1 region. Apoptosis was induced after tGCI and could be attenuated by bFGF treatment via inhibition of p53 mitochondrial translocation. In addition, autophagy was activated during this process, and bFGF could inhibit activation of autophagy through the mTOR pathway. Rapamycin, an activator of autophagy, was utilized to explore the relationship among bFGF, apoptosis, and autophagy. Apoptosis deteriorated after rapamycin treatment, which indicated that excessive autophagy could contribute to the apoptosis process. In conclusion, these results demonstrate that bFGF could exert neuroprotective effects in the hippocampal CA1 region by suppressing excessive autophagy via the mTOR pathway and inhibiting apoptosis by preventing p53 mitochondrial translocation. Furthermore, our results suggest that bFGF may be a promising therapeutic agent to for treating tGCI in response to major adverse events, including cardiac arrest, shock, extracorporeal circulation, traumatic hemorrhage, and asphyxiation.

Impaired autophagosome clearance contributes to neuronal death in a piglet model of neonatal hypoxic-ischemic encephalopathy.

To examine the temporal relationship of cortical autophagic flux with delayed neuronal cell death after hypoxia-ischemia (HI) in neonatal piglets. HI was produced with 45-min hypoxia and 7-min airway occlusion in 3-5-day-old piglets. Markers of autophagic, lysosomal and cell death signaling were studied via immunohistochemistry, immunoblotting, and histochemistry in piglet brains. In vitro, autophagy was impaired in cultured mouse cortical neurons treated with chloroquine with or without rapamycin for 1 d in the presence of Z-VAD-fmk, cyclosporine A, or vehicle control, and cell viability was assessed with the MTT assay. In vivo, neuronal cell death of sensorimotor cortex was delayed by 1-2 days after HI, whereas LC3-II, Beclin-1, PI3KC3, ATG12-ATG-5, and p-ULK1 increased by 1.5-6 h. Autophagosomes accumulated in cortical neurons by 1 d owing to enhanced autophagy and later to decreased autophagosome clearance, as indicated by LC3, Beclin-1, and p62 accumulation. Autophagy flux impairment was attributable to lysosomal dysfunction, as indicated by low lysosomal-associated membrane protein 2, cathepsin B, and cathepsin D levels at 1 d. Ubiquitin levels increased at 1 d. Autophagosome and p62 accumulated predominantly in neurons at 1 d, with p62 puncta occurring in affected cells. Beclin-1 colocalized with markers of caspase-dependent and caspase-independent apoptosis and necrosis in neurons. In vitro, mouse neonatal cortical neurons treated with rapamycin and chloroquine showed increased autophagosomes, but not autolysosomes, and increased cell death that was attenuated by cyclosporine A. Neonatal HI initially increases autophagy but later impairs autophagosome clearance, coinciding with delayed cortical neuronal death.

Preparation and evaluation of pH-responsive charge-convertible ternary complex FA-PEI-CCA/PEI/DNA with low cytotoxicity and efficient gene delivery.

Because the surface of the cationic polymer gene complex is positively charged, it can result in problems such as poor blood stability and cytotoxicity. Therefore, reducing the positive charge of the cationic gene complex without affecting its transfection efficiency is crucial. To achieve this objective, a pH-responsive charge-convertible ternary complex was developed in this study. Modified plyethylenimine (PEI) with two different degrees of substitution of NH2 (plyethylenimine-1,2-cyclohexanedicarboxylic anhydride, PEI-CCA, and folic acid-plyethylenimine-1,2-cyclohexanedicarboxylic anhydride, FA-PEI-CCA) were first obtained by a chemical graft reaction. PEI-CCA and FA-PEI-CCA have significantly lower cytotoxicities and much better blood compatibilities than PEI does, and the former have an undifferentiated compression capability of DNA. The zeta potential values of the as-prepared ternary complexes (PEI-CCA/PEI/DNA and FA-PEI-CCA/PEI/DNA) were negative at pH 7.4 and positive at pH 6.5, with particle sizes of approximately 150nm. MTT assays demonstrated the significantly lower cytotoxicities of the ternary complexes compared to that of PEI/DNA. Moreover, the cytotoxicities of the ternary complexes were lower at pH 7.4 than pH 6.5. Transfection experiments in vitro revealed that the mean fluorescence intensities and transfection efficiencies of the ternary complexes were lower than for PEI/DNA at pH 7.4 but were almost the same at pH 6.5. The ternary complex with a FA group had significantly higher mean fluorescence intensity and transfection efficiency than did the ternary complex without it. In addition, the transfection experiment in 293T cells preliminarily validated the targeting function of the FA group of the ternary complex.

Cardiac arrest triggers hippocampal neuronal death through autophagic and apoptotic pathways.

The mechanism of neuronal death induced by ischemic injury remains unknown. We investigated whether autophagy and p53 signaling played a role in the apoptosis of hippocampal neurons following global cerebral ischemia-reperfusion (I/R) injury, in a rat model of 8-min asphyxial cardiac arrest (CA) and resuscitation. Increased autophagosome numbers, expression of lysosomal cathepsin B, cathepsin D, Beclin-1, and microtubule-associated protein light chain 3 (LC3) suggested autophagy in hippocampal cells. The expression of tumor suppressor protein 53 (p53) and its target genes: Bax, p53-upregulated modulator of apoptosis (PUMA), and damage-regulated autophagy modulator (DRAM) were upregulated following CA. The p53-specific inhibitor pifithrin-α (PFT-α) significantly reduced the expression of pro-apoptotic proteins (Bax and PUMA) and autophagic proteins (LC3-II and DRAM) that generally increase following CA. PFT-α also reduced hippocampal neuronal damage following CA. Similarly, 3-methyladenine (3-MA), which inhibits autophagy and bafilomycin A1 (BFA), which inhibits lysosomes, significantly inhibited hippocampal neuronal damage after CA. These results indicate that CA affects both autophagy and apoptosis, partially mediated by p53. Autophagy plays a significant role in hippocampal neuronal death induced by cerebral I/R following asphyxial-CA.

Protein Kinase C Isoforms Distinctly Regulate Propofol-induced Endothelium-dependent and Endothelium-independent Vasodilation.

Protein kinase C (PKC) isoforms improve endothelial nitric oxide synthase activity and contractile Ca sensitivity in blood vessels. These actions may have opposite effects on propofol-induced vasodilation. This study examines the hypothesis that propofol induces relaxation by enhancing the PKC-mediated nitric oxide synthesis in endothelium and/or inhibiting the PKC-regulated Ca sensitivity in vascular smooth muscle (VSM). Propofol (1-100 µM) induced greater relaxation in endothelium-intact rings compared with denuded rings, and this effect was antagonized by the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME). In contrast, treatment with the general PKC inhibitor GF-109203X augmented both the endothelium-dependent and endothelium-independent relaxation induced by propofol, and this enhancement was more profound in the intact rings at lower propofol concentrations. The enhancement was unaffected by L-NAME. Interestingly, calphostin C (an inhibitor of conventional and novel PKCs) and Gö-6976 (an inhibitor of conventional PKCs) had similar effects in augmenting propofol-induced relaxation in endothelium-denuded rings. Downregulation of novel isoforms not only reduced the norepinephrine-elicited contraction but also decreased the magnitude of propofol-induced relaxation. In vascular smooth muscle cells, propofol prevented norepinephrine-elicited phosphorylation of myosin light chain. Propofol can increase the PKC-mediated availability of nitric oxide but inhibit the novel PKC-regulated Ca-sensitization, which provides a novel explanation for the mechanism of propofol-induced vasodilation.