WIN55,212-2 protects oligodendrocyte precursor cells in stroke penumbra following permanent focal cerebral ischemia in rats

Aim:

To explore whether the synthetic cannabinoid receptor agonist WIN55,212-2 could protect oligodendrocyte precursor cells (OPCs) in stroke penumbra, thereby providing neuroprotection following permanent focal cerebral ischemia in rats.

Methods:

Adult male SD rats were subjected to permanent middle cerebral artery occlusion (p-MCAO). The animals were administered WIN55,212-2 at 2 h, and sacrificed at 24 h after the ischemic insult. The infarct volumes and brain swelling were assessed. The expression of cannabinoid receptor type 1 (CB1) in the stroke penumbra was examined using Western blot assay. The pathological changes and proliferation of neural glial antigen 2-positive OPCs (NG2⁺ cells) in the stroke penumbra were studied using immunohistochemistry staining.

Results:

p-MCAO significantly increased the expression of CB1 within the stroke penumbra with the highest level appearing at 2 h following the ischemic insult. Administration of WIN55,212-2 (9 mg/kg, iv) significantly attenuated the brain swelling, and reduced the infarct volume as well as the number of tau-immunoreactive NG2⁺ cells (tau-1⁺/NG2⁺ cells) in the stroke penumbra. Moreover, WIN55,212-2 significantly promoted the proliferation of NG2⁺ cells in the stroke penumbra and in the ipsilateral subventricular zone at 24 h following the ischemic insult. Administration of the selective CB1 antagonist rimonabant (1 mg/kg, iv) partially blocked the effects caused by WIN55,212-2.

Conclusion:

Tau-1 is expressed in NG2⁺ cells following permanent focal cerebral ischemic injury. Treatment with WIN55,212-2 reduces the number of tau-1⁺/NG2⁺ cells and promotes NG2⁺ cell proliferation in the stroke penumbra, which are mediated partially via CB1 and may contribute to its neuroprotective effects.     

The edaravone and 3-n-butylphthalide ring-opening derivative 10b effectively attenuates cerebral ischemia injury in rats

Aim:

Compound 10b is a hybrid molecule of edaravone and a ring-opening derivative of 3-n-butylphthalide (NBP). The aim of this study was to examine the effects of compound 10b on brain damage in rats after focal cerebral ischemia.

Methods:

SD rats were subjected to 2-h-middle cerebral artery occlusion (MCAO). At the onset of reperfusion, the rats were orally treated with NBP (60 mg/kg), edaravone (3 mg/kg), NBP (60 mg/kg)+edaravone (3 mg/kg), or compound 10b (70, 140 mg/kg). The infarct volume, motor behavior deficits, brain water content, histopathological alterations, and activity of GSH, SOD, and MDA were analyzed 24 h after reperfusion. The levels of relevant proteins in the ipsilateral striatum were examined using immunoblotting.

Results:

Administration of compound 10b (70 or 140 mg/kg) significantly reduced the infarct volume and neurological deficits in MCAO rats. The neuroprotective effects of compound 10b were more pronounced compared to NBP, edaravone or NBP+edaravone. Furthermore, compound 10b significantly upregulated the protein levels of the cytoprotective molecules Bcl-2, HO-1, Nrf2, Trx, P-NF-κB p65, and IκB-α, while decreasing the expression of Bax, caspase 3, caspase 9, Txnip, NF-κB p65, and P-IκB-α.

Conclusion:

Oral administration of compound 10b effectively attenuates rat cerebral ischemia injury.     

Long-lasting neuroprotection and neurological improvement in stroke models with new, potent and brain permeable inhibitors of poly(ADP-ribose) polymerase

BACKGROUND AND PURPOSES

Thienyl-isoquinolone (TIQ-A) is a relatively potent PARP inhibitor able to reduce post-ischaemic neuronal death in vitro. Here we have studied, in different stroke models in vivo, the neuroprotective properties of DAMTIQ and HYDAMTIQ, two TIQ-A derivatives able to reach the brain and to inhibit PARP-1 and PARP-2.

EXPERIMENTAL APPROACH

Studies were carried out in (i) transient (2 h) middle cerebral artery occlusion (tMCAO), (ii) permanent MCAO (pMCAO) and (iii) electrocoagulation of the distal portion of MCA in conjunction with transient (90 min) bilateral carotid occlusion (focal cortical ischaemia).

KEY RESULTS

In male rats with tMCAO, HYDAMTIQ (0.1–10 mg·kg⁻¹) injected i.p. three times, starting 4 h after MCAO, reduced infarct volumes by up to 70%, reduced the loss of body weight by up to 60% and attenuated the neurological impairment by up to 40%. In age-matched female rats, HYDAMTIQ also reduced brain damage. Protection, however, was less pronounced than in the male rats. In animals with pMCAO, HYDAMTIQ administered 30 min after MCAO reduced infarct volumes by approximately 40%. In animals with focal cortical ischaemia, HYDAMTIQ treatment decreased post-ischaemic accumulation of PAR (the product of PARP activity) and the presence of OX42-positive inflammatory cells in the ischaemic cortex. It also reduced sensorimotor deficits for up to 90 days after MCAO.

CONCLUSION AND IMPLICATIONS

Our results show that HYDAMTIQ is a potent PARP inhibitor that conferred robust neuroprotection and long-lasting improvement of post-stroke neurological deficits.     

Ginsenoside Rd promotes neurogenesis in rat brain after transient focal cerebral ischemia via activation of PI3K/Akt pathway

Aim:

To investigate the effects of ginsenoside Rd (Rd) on neurogenesis in rat brain after ischemia/reperfusion injury (IRI).

Methods:

Male SD rats were subjected to transient middle cerebral artery occlusion (MCAO) followed by reperfusion. The rats were injected with Rd (1, 2.5, and 5 mg·kg⁻¹·d⁻¹, ip) from d 1 to d 3 after MCAO, and with BrdU (50 mg·kg⁻¹·d⁻¹, ip) from d 3 to d 6, then sacrificed on 7 d. The infarct size and neurological scores were assessed. Neurogenesis in the brains was detected by BrdU, DCX, Nestin, and GFAP immunohistochemistry staining. PC12 cells subjected to OGD/reperfusion were used as an in vitro model of brain ischemia. VEGF and BDNF levels were assessed with ELISA, and Akt and ERK phosphorylation was measured using Western blotting.

Results:

Rd administration dose-dependently decreased the infarct size and neurological scores in the rats with IRI. The high dose of Rd 5 (mg·kg⁻¹·d⁻¹) significantly increased Akt phosphorylation in ipsilateral hemisphere, and markedly increased the number of BrdU/DCX and Nestin/GFAP double-positive cells in ischemic area, which was partially blocked by co-administration of the PI3 kinase inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. Treatment with Rd (25, 50, and 100 μmol/L) during reperfusion significantly increased the expression of VEGF and BDNF in PC12 cells with IRI. Furthermore, treatment with Rd dose-dependently increased the phosphorylation of Akt and ERK, and significantly decreased PC12 cell apoptosis, which were blocked by co-application of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002.

Conclusion:

Rd not only attenuates ischemia/reperfusion injury in rat brain, but also promotes neurogenesis via increasing VEGF and BDNF expression and activating the PI3K/Akt and ERK1/2 pathways.     

The inhibitor of 20-HETE synthesis, TS-011, improves cerebral microcirculatory autoregulation impaired by middle cerebral artery occlusion in mice

BACKGROUND AND PURPOSE

20-Hydroxyeicosatetraenoic acid is a potent vasoconstrictor that contributes to cerebral ischaemia. An inhibitor of 20-Hydroxyeicosatetraenoic acid synthesis, TS-011, reduces infarct volume and improves neurological deficits in animal stroke models. However, little is known about how TS-011 affects the microvessels in ischaemic brain. Here, we investigated the effect of TS-011 on microvessels after cerebral ischaemia.

EXPERIMENTAL APPROACH

TS-011 (0.3 mg·kg⁻¹) or a vehicle was infused intravenously for 1 h every 6 h in a mouse model of stroke, induced by transient occlusion of the middle cerebral artery occlusion following photothrombosis. The cerebral blood flow velocity and the vascular perfusion area of the peri-infarct microvessels were measured using in vivo two-photon imaging.

KEY RESULTS

The cerebral blood flow velocities in the peri-infarct microvessels decreased at 1 and 7 h after reperfusion, followed by an increase at 24 h after reperfusion in the vehicle-treated mice. We found that TS-011 significantly inhibited both the decrease and the increase in the blood flow velocities in the peri-infarct microvessels seen in the vehicle-treated mice after reperfusion. In addition, TS-011 significantly inhibited the reduction in the microvascular perfusion area after reperfusion, compared with the vehicle-treated group. Moreover, TS-011 significantly reduced the infarct volume by 40% at 72 h after middle cerebral artery occlusion.

CONCLUSIONS AND IMPLICATIONS

These findings demonstrated that infusion of TS-011 improved defects in the autoregulation of peri-infarct microcirculation and reduced the infarct volume. Our results could be relevant to the treatment of cerebral ischaemia.     

Combined prostaglandin E1 and lithium exert potent neuroprotection in a rat model of cerebral ischemia

Aim:

To examine the effects of a mixed formulation composed of prostaglandin E1 and lithium (PGE1+Li mixture) on brain damage after cerebral ischemia. The effects of the mixture on protein expression of heat shock proteins (HSPs), p53, and Bcl-2 were also determined.

Methods:

Brain ischemia was induced with a permanent middle cerebral artery occlusion (pMCAO) in rats. Rats were treated with a single intravenous administration of PGE1, lithium or a PGE1+Li mixture immediately after the ischemic insult. The infarct volume and motor behavior deficits were analyzed 24 h after the ischemic insult. The protein levels of HSP70, glucose-regulated protein 78 (GRP78), HSP60, Bcl-2, and p53 in the striatum of the ipsilateral hemisphere were examined using immunoblotting.

Results:

The mixture (PGE1 22.6 nmol/kg+Li 0.5 mmol/kg) reduced infarct volume and neurological deficits induced by focal cerebral ischemia. Moreover, the mixture had a greater neuroprotective effect against cerebral ischemia compared with PGE1 or lithium alone. The mixture was effective even if it was administered 3 h after ischemia. PGE1+Li also significantly upregulated cytoprotective HSP70, GRP78, HSP60, and Bcl-2 protein levels, while decreasing p53 expression.

Conclusion:

These results demonstrated a PGE1+Li mixture with a therapeutic window of up to 3 h for clinical treatment of cerebral ischemia. The PGE1+Li mixture potentially exerts a protective effect after stroke through the induction of HSPs and Bcl-2 proteins.     

The endoplasmic reticulum stress inhibitor salubrinal inhibits the activation of autophagy and neuroprotection induced by brain ischemic preconditioning

Aim:

To investigate whether endoplasmic reticulum (ER) stress participates in the neuroprotective effects of ischemic preconditioning (IPC)-induced neuroprotection and autophagy activation in rat brains.

Methods:

The right middle cerebral artery in SD rats was occluded for 10 min to induce focal cerebral IPC, and was occluded permanently 24 h later to induce permanent focal ischemia (PFI). ER stress inhibitor salubrinal (SAL) was injected via intracerebral ventricle infusion 10 min before the onset of IPC. Infarct volume and motor behavior deficits were examined after the ischemic insult. The protein levels of LC3, p62, HSP70, glucose-regulated protein 78 (GRP 78), p-eIF2α and caspase-12 in the ipsilateral cortex were analyzed using immunoblotting. LC3 expression pattern in the sections of ipsilateral cortex was observed with immunofluorescence.

Results:

Pretreatment with SAL (150 pmol) abolished the neuroprotective effects of IPC, as evidenced by the significant increases in mortality, infarct volume and motor deficits after PFI. At the molecular levels, pretreatment with SAL (150 pmol) significantly increased p-eIF2α level, and decreased GRP78 level after PFI, suggesting that SAL effectively inhibited ER stress in the cortex. Furthermore, the pretreatment with SAL blocked the IPC-induced upregulation of LC3-II and downregulation of p62 in the cortex, thus inhibiting the activation of autophagy. Moreover,SAL blocked the upregulation of HSP70, but significantly increased the cleaved caspase-12 level, thus promoting ER stress-dependent apoptotic signaling in the cortex.

Conclusion:

ER stress-induced autophagy might contribute to the neuroprotective effect of brain ischemic preconditioning.     

Human umbilical cord blood mesenchymal stem cell transplantation suppresses inflammatory responses and neuronal apoptosis during early stage of focal cerebral ischemia in rabbits

Aim： Human umbilical cord blood mesenchymal stem cells （hUCB-MSCs） have been shown to ameliorate cerebral ischemia in animal models. In this study we investigated the effects of hUCB-MSCs on inflammatory responses and neuronal apoptosis during the early stage of focal cerebral ischemia in rabbits. Methods： Focal cerebral ischemia was induced in male New Zealand rabbits by occlusion of MCA for 2 h. The blood samples were collected at different time points prior and during MCAO-reperfusion. The animals were euthanized 3 d after MCAO, and the protein levels of IL-113, IL-6, IL-IO, and TNF-a in the serum and peri-ischemic brain tissues were detected using Western blot and ELISA, respectively. Inflammatory cell infiltration, neuronal apoptosis and neuronal density were measured morphologically, hUCB-MSCs （5x106） were iv injected a few minutes after MCAO. Results： The serum levels of IL-113, IL-6, and TNF-~ were rapidly increased, and peaked at 2 h after the start of MCAO. hUCB-MSC transplantation markedly and progressively suppressed the ischemia-induced increases of serum IL-113, IL-6, and TNF-a levels within 6 h MCAO-reperfusion. Focal cerebral ischemia decreased the serum level of IL-IO, which was prevented by hUCB-MSC transplantation The expression of IL-113, IL-6, IL-IO, and TNF-a in the peri-ischemic brain tissues showed similar changes as in the serum, hUCB-MSC transplantation markedly suppressed the infiltration of inflammatory cells, and increased the neuronal density around the ischemic region. Furthermore, hUCB-MSC transplantation significantly decreased the percentage of apoptosis around the ischemic region. Conclusion： hUCB-MSCs transplantation suppresses inflammatory responses and neuronal apoptosis during the early stage focal cerebral ischemia in rabbits.     

DanHong injection dose-dependently varies amino acid metabolites and metabolic pathways in the treatment of rats with cerebral ischemia

Aim:

To determine how the relative amino acid contents and metabolic pathways regulate the pharmacological phenotypes in rats with cerebral ischemia after treatment with varying doses of DanHong injection (DHI).

Methods:

Adult male rats underwent middle cerebral artery occlusion (MCAO), and were injected with DHI (DH-1: 1 mL/kg; DH-2: 2.5 mL/kg; DH-3: 5 mL/kg, and DH-4: 10 mL/kg, iv) daily for 3 d. The neurological deficit score, body weights and infarct volume were assessed. Serum levels of 20 free amino acids were determined using HPLC, and the values were transformed through the quantitative analysis of the amino acids in the serum metabolic spectrum. Multivariate statistical analysis methods (PCA and PLS-DA) and web-based metabolomics tools (MetPa and MetaboAnalyst) were used to analyze the biological data sets for the amino acids.

Results:

Administration of DHI dose-dependently decreased cerebral infarct volume, and ameliorated neurological deficits. A total of 5, 6, 7 and 7 non-overlapping metabolites were identified in the DH-1, DH-2, DH-3, and DH-4 groups, respectively. Eight metabolites were shared between the DHI groups and the vehicle group. In addition, the serum levels of glutamic acid, aspartic acid and serine increased with increasing DHI dose. A total of 3, 2, 2 and 5 non-overlapping metabolic pathways were identified in the DH-1, DH-2, DH-3 and DH-4 groups, respectively, and glycine, serine, threonine and histidine metabolism were identified as overlapping pathways among the 4 dose groups.

Conclusion:

Overlapping and non-overlapping amino acid metabolites and metabolic pathways are associated with the dose-dependent neuroprotective effect of DHI.     

Effects of delayed puerarin treatment in long-term neurological outcomes of focal ischemic stroke in rats

Objective:

The present study aimed at investigate the therapeutic effects of delayed puerarin treatment in neurological outcomes after middle cerebral artery occlusion (MCAO) in rats.

Materials and Methods:

Male Wistar rats were subjected to MCAO for 120 min followed by reperfusion for 14 days. Puerarin (0, 50, 100, 200 mg/kg, intra-peritoneally) was administered at 24 h after stroke onset and repeated daily for 14 days. Neurological deficits were evaluated at 1, 4, 7, 14 days after stroke. Brain infarct volume and peri-infarct context vessel density were examined at 14 days after stroke.

Results:

Puerarin significantly improved neurological functions up to 14 days after stroke and decreased the infarct volume with doses of 50 mg/kg and 100 mg/kg compared with saline controls. Puerarin treatment also significantly increased peri-infarct context vessel density at 14 days after stroke.

Conclusions:

Delayed treatment of puerarin initiated at 24 h after stroke is beneficial with improved long-term neurological outcomes and reduced infarction volume in focal ischemic stroke in rats. Enhanced vascular remodeling by puerarin might at least partially contribute to its beneficial effects.KEY WORDS: Cerebrovascular remodeling, focal cerebral ischemia, long-term neurological outcomes, puerarin, rats     

Increased brain damage after ischaemic stroke in mice lacking the chemokine receptor CCR5

Background and purpose:

The chemokine receptor CCR5 is well known for its function in immune cells; however, it is also expressed in the brain, where its specific role remains to be elucidated. Because genetic factors may influence the risk of developing cerebral ischaemia or affect its clinical outcome, we have analysed the role of CCR5 in experimental stroke.

Experimental approach:

Permanent cerebral ischaemia was performed by occlusion of the middle cerebral artery in wild-type and CCR5-deficient mice. Locomotor behaviour, infarct size and histochemical alterations were analysed at different time points after occlusion.

Key results:

The cerebral vasculature was comparable in wild-type and CCR5-deficient mice. However, the size of the infarct and the motor deficits after occlusion were markedly increased in CCR5-deficient mice as compared with wild type. No differences between wild-type and CCR5-deficient mice were elicited by occlusion with respect to the morphology and abundance of astrocytes and microglia. Seven days after occlusion the majority of CCR5-deficient mice displayed neutrophil invasion in the infarct region, which was not observed in wild type. As compared with wild type, the infarct regions of CCR5-deficient mice were characterized by increased neuronal death.

Conclusions and implications:

Lack of CCR5 increased the severity of brain injury following occlusion of the middle cerebral artery. This is of particular interest with respect to the relatively frequent occurrence of CCR5 deficiency in the human population (1–2% of the Caucasian population) and the advent of CCR5 inhibitors as novel drugs.     

Acute metformin preconditioning confers neuroprotection against focal cerebral ischaemia by pre-activation of AMPK-dependent autophagy

BACKGROUND AND PURPOSE

Recent clinical trials report that metformin, an activator of AMP-activated protein kinase (AMPK) used to treat type 2 diabetes, significantly reduces the risk of stroke by actions that are independent of its glucose-lowering effects. However, the underlying molecular mechanisms are not known. Here, we tested the possibility that acute metformin preconditioning confers neuroprotection by pre-activation of AMPK-dependent autophagy in a rat model of permanent middle cerebral artery occlusion (pMCAO).

EXPERIMENTAL APPROACH

Male Sprague-Dawley rats were pretreated with either vehicle, an AMPK inhibitor, Compound C, or an autophagy inhibitor, 3-methyladenine, and were injected with a single dose of metformin (10 mg kg⁻¹, i.p.). Then, AMPK activity and autophagy biomarkers in the brain were assessed. At 24 h after metformin treatment, rats were subjected to pMCAO; infarct volume, neurological deficits and cell apoptosis were evaluated 24 and 96 h later.

KEY RESULTS

A single dose of metformin significantly activated AMPK and induced autophagy in the brain. The enhanced autophagic activity was inhibited by Compound C pretreatment. Furthermore, acute metformin preconditioning significantly reduced infarct volume, neurological deficits and cell apoptosis during a subsequent focal cerebral ischaemia. The neuroprotection mediated by metformin preconditioning was fully abolished by Compound C and partially inhibited by 3-methyladenine.

CONCLUSIONS AND IMPLICATIONS

These results provide the first evidence that acute metformin preconditioning induces autophagy by activation of brain AMPK, which confers neuroprotection against subsequent cerebral ischaemia. This suggests that metformin, a well-known hypoglycaemic drug, may have a practical clinical use for stroke prevention.     

Reduction of cerebral infarct volume by apocynin requires pretreatment and is absent in Nox2-deficient mice

Background and purpose:

Reactive oxygen species (ROS) derived from Nox2-containing reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity is reportedly detrimental in cerebrovascular disease. However, ROS generation by other Nox isoforms may have a physiological role. No Nox2-selective inhibitors have yet been identified, and thus it is unclear whether isoform non-selective Nox inhibitors would necessarily improve outcome after stroke. We assessed the effect of apocynin on cerebrovascular ROS production and also on outcome following cerebral ischaemia when administered either before ischaemia or after cerebral reperfusion. The involvement of Nox2-containing NADPH oxidase in the effects of apocynin was assessed using Nox2^−/− mice.

Experimental approach:

Transient cerebral ischaemia was induced by 0.5 h middle cerebral artery occlusion followed by 23.5 h reperfusion. Mice received apocynin (2.5 mg·kg⁻¹, i.p.) either 0.5 h before ischaemia or 1 h after reperfusion. In situ superoxide production after cerebral ischaemia-reperfusion was measured in brain sections of wild-type mice at 24 h using dihydroethidium fluorescence.

Key results:

Treatment with apocynin 0.5 h before ischaemia reduced total infarct volume, neurological impairment and mortality in wild-type but not Nox2^−/− mice. Conversely, treatment with apocynin 1 h after initiation of reperfusion had no protective effect. Cerebral ischaemia and reperfusion increased superoxide production in the brain at 24 h, and pretreatment but not posttreatment with apocynin reduced superoxide levels.

Conclusions and implications:

Apocynin improves outcome following stroke when administered before ischaemia in wild-type but not Nox2^−/− mice.     

Neutrophils contribute to intracerebral haemorrhages after treatment with recombinant tissue plasminogen activator following cerebral ischaemia

Background and purpose:

Polymorphonuclear neutrophils (PMNs) contribute to the vascular damage caused by transient cerebral ischaemia. Here we have evaluated the role of PMNs in intracerebral haemorrhage (ICH) induced in a model of thrombolysis with recombinant tissue plasminogen activator (t-PA) during the acute phase of cerebral ischaemia.

Experimental approach:

The middle cerebral artery (MCA) of male spontaneously hypertensive rats was occluded for 1 h followed by reperfusion and, 5 h later, infusion of thrombolytic products (generated in vitro by t-PA on autologous clots). Effects of pretreatment (before the MCA occlusion) with vinblastine (4 days before; 0.5 mg·kg⁻¹), monoclonal anti-neutrophil antibody (mAbRP3; 12 h, 0.3 mg·kg⁻¹) or saline on ICH, neutrophil infiltration, MCA vascular reactivity and brain infarct volume were assessed, 24 h after the beginning of reperfusion.

Key results:

Depletion of circulating neutrophils significantly reduced t-PA-induced ICH (vinblastine, 4.6 ± 1.0; mAbRP3, 5.2 ± 1.0 vs. saline, 10.8 ± 2.7 haemorrhages; P < 0.05). This depletion was associated with a decrease in cerebral infiltration by neutrophils and a decrease of endothelium-dependent, vascular dysfunction in isolated MCA, induced by the ischaemia/reperfusion and t-PA treatment. Brain infarct volume was significantly decreased after vinblastine treatment (159 ± 13 mm³ vs. 243 ± 16 mm³ with saline; P < 0.01) but not after depletion with mAbRP3 (221 ± 22 mm³).

Conclusions and implications:

Our results showed that pharmacological depletion of PMNs prevented t-PA-induced ICH, in parallel with a decrease in cerebral infiltration by PMNs and a decreased endothelial dysfunction in cerebral blood vessels.     

The regulation of N-terminal Huntingtin (Htt552) accumulation by Beclin1

Aim:

Huntingtin protein (Htt) was a neuropathological hallmark in human Huntington''s Disease. The study aimed to investigate whether the macroautophagy regulator, Beclin1, was involved in the degradation of Htt.

Methods:

PC12 cells and primary cultured brain neurons of rats were examined. pDC316 adenovirus shuttle plasmid was used to mediate the expression of wild-type Htt-18Q-552 or mutant Htt-100Q-552 in PC12 cells. The expression of the autophagy-related proteins LC3 II and Beclin1, as well as the lysosome-associated enzymes Cathepsin B and L was evaluated using Western blotting. The locations of Beclin1 and Htt were observed with immunofluorescence and confocal microscope.

Results:

Htt552 expression increased the expression of LC3 II, Beclin1, cathepsin B and L in autophagy/lysosomal degradation pathway. Treatment with the autophagy inhibitor 3-MA or the proteasome inhibitors lactacystin and MG-132 increased Htt552 levels in PC12 cells infected with Ad-Htt-18Q-552 or Ad-Htt-100Q-552. The proteasome inhibitor caused a higher accumulation of Htt552-18Q than Htt552-100Q, and the autophagy inhibitor resulted in a higher accumulation of Htt552-100Q than Htt552-18Q. Similar results were observed in primary cultured neurons infected with adenovirus. In Htt552-expressing cells, Beclin1 was redistributed from the nucleus to the cytoplasm. Htt siRNA prevented Beclin1 redistribution in starvation conditions. Blockade of Beclin1 nuclear export by leptomycin B or Beclin1 deficiency caused by RNA interference induced the formation of mHtt552 aggregates.

Conclusion:

Beclin1 regulates the accumulation of Htt via macroautophagy.     

Inhibition of inflammasome activation improves the impaired pattern of healing in genetically diabetic mice

Background and Purpose

Type 2 diabetes impairs the healing process because of an exaggerated and persistent inflammatory response, and an altered expression pattern of angiogenic molecules. We investigated the effects of inflammasome blockade in diabetes-related wound-healings defects, in genetically diabetic mice.

Experimental Approach

An incisional skin wound model was produced on the back of female diabetic C57BL/KsJ-m +/+ Lept^db mice (db⁺/db⁺) and their normal littermates (db⁺/m⁺). Animals were treated daily with two inflammasome blocking agents, BAY 11-7082 (20 mg·kg⁻¹ i.p.), or Brilliant Blue G (BBG, 45.5 mg·kg⁻¹ i.p.), or vehicle. Mice were killed on 3, 6 and 12 days after skin injury to measure expression of the NOD-like receptor NLRP3, caspase-1, VEGF, the inflammasome adapter protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and the chemokine CXCL12. Wound levels of IL-1β and IL-18 were also measured, along with histological assessments of wound tissue and the time to complete wound closure.

Key Results

During healing, the diabetic mice exhibited increased activation of NLRP3, caspase-1, ASC, IL-1β and IL-18. They also showed a reduced expression of VEGF and CXCL12.Treatment with BAY 11-7082 or BBG, to block activation of the inflammasome, decreased the levels of pro-inflammatory molecules. Histological evaluation indicated that inflammasome blockade improved the impaired healing pattern, at day 12 in diabetic mice, along with a decreased time to complete skin healing.

Conclusions and Implications

These data strongly suggest that activation of the NLRP3 inflammasome is one of the key contributors to the delayed healing of wounds in diabetic mice.     

Connexin 43 stabilizes astrocytes in a stroke-like milieu to facilitate neuronal recovery

Aim:

Connexin 43 (Cx43) is a member of connexin family mainly expressed in astrocytes, which forms gap junctions and hemichannels and maintains the normal shape and function of astrocytes. In this study we investigated the role of Cx43 in astrocytes in facilitating neuronal recovery during ischemic stroke.

Methods:

Primary culture of astrocytes or a mixed culture of astrocytes and cortical neurons was subjected to oxygen glucose deprivation and reperfusion (OGD/R). The expression of Cx43 and Ephrin-A4 in astrocytes was detected using immunocytochemical staining and Western blot assays. Intercellular Ca²⁺ concentration was determined with Fluo-4 AM fluorescent staining. Middle cerebral artery occlusion (MCAO) model rats were used for in vivo studies.

Results:

OGD/R treatment of cultured astrocytes caused a decrement of Cx43 expression and translocation of Cx43 from cell membrane to cytoplasm, accompanied by cell retraction. Furthermore, OGD/R increased intracellular Ca²⁺ concentration, activated CaMKII/CREB pathways and upregulated expression of Ephrin-A4 in the astrocytes. All these changes in OGD/R-treated astrocytes were alleviated by overexpression of Cx43. In the cortical neurons cultured with astrocytes, OGD/R inhibited the neurite growth, whereas overexpression of Cx43 or knockdown of Ephrin-A4 in astrocytes restored the neurite growth. In MCAO model rats, neuronal recovery was found to be correlated with the recuperation of Cx43 and Ephrin-A4 in astrocytes.

Conclusion:

Cx43 can stabilize astrocytes and facilitate the resistance to the deleterious effects of a stroke-like milieu and promote neuronal recovery.