Ischemic Preconditioning Mediates Neuroprotection against Ischemia in Mouse Hippocampal CA1 Neurons by Inducing Autophagy

<div><p>The hippocampal CA1 region is sensitive to hypoxic and ischemic injury but can be protected by ischemic preconditioning (IPC). However, the mechanism through which IPC protects hippocampal CA1 neurons is still under investigation. Additionally, the role of autophagy in determining the fate of hippocampal neurons is unclear. Here, we examined whether IPC induced autophagy to alleviate hippocampal CA1 neuronal death in vitro and in vivo with oxygen glucose deprivation (OGD) and bilateral carotid artery occlusion (BCCAO) models. Survival of hippocampal neurons increased from 51.5% ± 6.3% in the non-IPC group (55 min of OGD) to 77.3% ± 7.9% in the IPC group (15 min of OGD, followed by 55 min of OGD 24 h later). The number of hippocampal CA1 layer neurons increased from 182 ± 26 cells/mm² in the non-IPC group (20 min of BCCAO) to 278 ± 55 cells/mm² in the IPC group (1 min × 3 BCCAO, followed by 20 min of BCCAO 24 h later). Akt phosphorylation and microtubule-associated protein light chain 3 (LC3)-II/LC3-I expression were increased in the preconditioning group. Moreover, the protective effects of IPC were abolished only by inhibiting the activity of autophagy, but not by blocking the activation of Akt in vitro. Using in vivo experiments, we found that LC3 expression was upregulated, accompanied by an increase in neuronal survival in hippocampal CA1 neurons in the preconditioning group. The neuroprotective effects of IPC on hippocampal CA1 neurons were completely inhibited by treatment with 3-MA. In contrast, hippocampal CA3 neurons did not show changes in autophagic activity or beneficial effects of IPC. These data suggested that IPC may attenuate ischemic injury in hippocampal CA1 neurons through induction of Akt-independent autophagy.</p></div

Protective effects of IPC-induced autophagy against lethal BCCAO in the hippocampal CA1 layer.

<p>(A) Mice were exposed to control (I, II) or 1 min × 3 sublethal BCCAO followed by 20 min lethal BCCAO 24 h later. The distribution of LC3 was then quantified in hippocampal CA1 neurons by immunohistochemistry at 1 (III, IV), 3 (V, VI), 6 (VII,VIII), and 24 h (IX,X). (B) Mice were exposed to 20 min lethal BCCAO alone, or 1 min × 3 sublethal BCCAO followed by 20 min lethal BCCAO 24 h later. Western blot analysis of LC3 and β-actin expression in mice hippocampus 6 h after lethal BCCAO. (C)Mice were exposed to 1 min × 3 sublethal BCCAO, IPC plus 3-MA, or IPC plus saline, followed by 20 min lethal BCCAO 24 h later. The distribution of LC3 was then quantified in hippocampal area (a, e, i, m, q), and CA1 neurons (b, c, f, g, j, k, n, o, r, s) and CA3 neurons (d, h, l, p, t) 6 h later. Immunohistochemistry showed a strong increase in LC3 expression at 6 h after the lethal BCCAO in the preconditioning group (j, k) and after pretreatment with IPC and saline (r, s) compared with that of the control (b, c) and lethal BCCAO alone (f, g). However, the increase in LC3 expression was inhibited following pretreatment with a combination of IPC and 3-MA (n, o). High-magnification views revealed the presence of numerous LC3-positive dots in hippocampal CA1 neurons only in the preconditioning subgroup (k) and following pretreatment with IPC and saline (s), but not in the control (c) or following 20 min of lethal BCCAO (g) or pretreatment with IPC and 3-MA (o). No changes in LC3 expression were detected in the CA3 layer (d, h, l, p, t). Original magnification, 2× (I, III, V, VII, IX,a, e, i, m, and q), and 10× (II, IV, VI, VIII, X, b, d, f, h, j, l, n, p, r and t) and 100× (c, g, k, o, and s). Error bars denote SDs. *<i>P</i> < 0.01 compared with the control group.</p

Mutation in the promoter of gene severely causes downregulation of the neo-Y copy

Dual-reporter assay of promoter activities of . Standard error among triplicates was shown on the bar.<p><b>Copyright information:</b></p><p>Taken from "Neo-sex chromosomes in the black muntjac recapitulate incipient evolution of mammalian sex chromosomes"</p><p>http://genomebiology.com/2008/9/6/R98</p><p>Genome Biology 2008;9(6):R98-R98.</p><p>Published online 14 Jun 2008</p><p>PMCID:PMC2481430.</p><p></p

Determination of the lethal OGD time and suitable IPC intervention in hippocampal neurons.

<p>(A) Time-dependent effects of hypoxia on neuronal death. Hippocampal neurons were exposed to OGD conditions for 0, 5, 10, 15, 30, or 55 min, followed by quantification of cell survival with MTT assays 24 h later. (B) Different OGD times conferred protection against the deleterious effects of 55 min of OGD. Hippocampal neurons were exposed to sublethal (0–30 min) OGD followed by 55 min of lethal OGD 24 h later. Values are given as percentages compared with cell survival in neurons maintained under normoxic conditions. Error bars denote SDs. *<i>P</i> < 0.01 compared with cells maintained under OGD for 0–30 min. ^<b>$</b><i>P</i> < 0.01 compared with cells maintained under normoxic conditions. ^#<i>P</i> < 0.05 compared with cells maintained under 55 min of OGD.</p

IPC-induces autophagy conferred neuroprotection independent of Akt phosphorylation.

<p>(A) Representative image of western blot analysis for Akt phosphorylated at serine 473 (pAkt), total Akt (tAkt), and β-actin in lysates from hippocampal neurons 6 h after exposure to normoxic conditions, 15 min of OGD alone, 55 min of OGD alone, or preconditioning. (B) Mean survival in hippocampal neurons exposed to 55 min of OGD; pretreated with IPC alone, 10 nM rapamycin alone, a combination of IPC and 10 nM GDC-0068, or a combination of 10 nM rapamycin and 10 nM GDC-0068 24 h before 55 min of OGD; or pretreated with 10 nM GDC-0068 alone under normoxic conditions. (C) Representative images of Nissl staining of the hippocampus. Mice were exposed to 1 min × 3 sublethal BCCAO or with a combination of IPC and intraventricular injection of wortmannin followed by 20 min of lethal BCCAO 24 h later. Cell survival was quantified by Nissl staining of the hippocampal CA1 and CA3 layers 24 h later. Values are given as the mean number of neurons. Error bars denote SDs. *<i>P</i> < 0.05 compared with other groups. **<i>P</i> < 0.05 compared with cells under normoxic conditions. ^#<i>P</i> < 0.05 compared with neurons maintained under 55 min of OGD. ^<b>☆</b><i>P</i> > 0.05 compared with the control. ^{<b>☆☆</b>}<i>P</i> < 0.05 compared with the control.</p

Autophagic activation induced by IPC protected hippocampal neurons.

<p>(A) Representative image of western blot analysis of LC3 and β-actin expression in lysates from hippocampal neurons at different times (0, 1, 3, or 24 h) after exposure to lethal OGD in the preconditioning group. (B) Western blot analysis of LC3 and β-actin expression in hippocampal neurons 3 h after exposure to normoxic conditions, 55 min of OGD alone, or 15 min of OGD followed by 55 min of OGD 24 h later. (C) Mean survival in hippocampal neurons exposed to 55 min of OGD; pretreated with IPC alone, 10 nM rapamycin alone, or IPC plus 100 nM 3-MA 24 h before, or pretreated with 100 nM 3-MA alone or 10 nM rapamycin alone under normoxic conditions. Error bars denote SDs. ^$<i>P</i> < 0.05 compared with the other groups. *<i>P</i> < 0.05 compared with cells under normoxic conditions. **<i>P</i> < 0.05 compared with neurons maintained under 55 min of OGD. ^#<i>P</i> < 0.05 compared with cells in the preconditioning group. ^☆<i>P</i> > 0.05 compared with cells maintained under normoxic conditions.</p

Dendrogram constructed using noncoding sequences of neo-Y and neo-X fragments

A total of 35.1-kilobase noncoding sequences of neo-Y and neo-X alleles in the black muntjac and the orthologous sequences in the Indian muntjac as the outgroup were used to construct the dentrogram. 'BM' stands for 'male black muntjac'; 'IM' stands for 'Indian muntjac'; '1'and '2' represent the two male black muntjac individuals separately. Tree constructed by neighbor-joining method. Branch lengths calculated using Kimura's two-parameter method are shown above the corresponding branches. Tree constructed by maximum likelihood method. Branch lengths calculated by baseml in PAML package using 'HKY85' method were shown above the corresponding branches.<p><b>Copyright information:</b></p><p>Taken from "Neo-sex chromosomes in the black muntjac recapitulate incipient evolution of mammalian sex chromosomes"</p><p>http://genomebiology.com/2008/9/6/R98</p><p>Genome Biology 2008;9(6):R98-R98.</p><p>Published online 14 Jun 2008</p><p>PMCID:PMC2481430.</p><p></p

Determination of the lethal BCCAO time and the effects of IPC against lethal BCCAO in hippocampal CA1 neurons.

<p>(A) The effects of different types of BCCAO on survival of neurons in the hippocampal CA1 layer. Mice were exposed to several patterns of BCCAO followed by quantification of cell survival with Nissl staining 24 h later. (B) Different patterns of BCCAO protected CA1 neurons against 20 min of BCCAO. Mice were exposed to several patterns of sublethal BCCAO followed by 20 min lethal BCCAO 24 h later, and quantification of cell survival was carried out using Nissl staining 24 h later. (C) Comparison of the vulnerability of the CA1 and CA3 regions of the hippocampus to preconditioning. Values are given as the mean number of neurons. Error bars denote SDs. *<i>P</i> < 0.01 compared with the control group. ^#<i>P</i> < 0.05 compared with other experimental groups.</p

Association between Elevated Hemoglobin A1c Levels and the Outcomes of Patients with Small-Artery Occlusion: A Hospital-Based Study

<div><p>Introduction</p><p>Abnormal glucose metabolism is an independent risk factor for poor outcome following acute ischemic stroke. However, the relationship between initial hemoglobin A1c level and functional outcome (defined by modified Rankin Scale scores) following small-artery occlusion, a subtype of ischemic stroke, is unknown. The aim of the present study was to evaluate this association among patients diagnosed with small-artery occlusion.</p><p>Materials and Methods</p><p>Data on 793 patients diagnosed with small-artery occlusion from October 25, 2012 to June 30, 2015 were collected from the stroke registry of the Department of Neurorehabilitation of HuanHu Hospital. Hemoglobin A1c values at admission were classified into three groups according to tertiles (<5.9,5.9to<6.7, and<b>≥</b>6.7). We used receiver operating characteristics curves to investigate the predictive value of hemoglobin A1c and examined the relationship between hemoglobin A1c levels at admission and modified Rankin Scale scores using univariate and multivariate analyses.</p><p>Results</p><p>The area under the curve was 0.570 (95%CI, 0.509–0.631; <i>P</i> = 0.023). Patients in the highest HbA1c stratification (<b>≥</b>6.7) had a significantly higher risk of an unfavorable outcome than patients in the lowest stratification (<5.9; adjusted odds ratio, 2.099; 95%CI, 1.160–3.798; <i>P</i> = 0.014). However, a significant association was not seen in the middle stratification (5.9 to <6.7; <i>P</i> = 0.115).</p><p>Conclusions</p><p>Elevated hemoglobin A1c level on admission was adversely associated with functional outcomes 3 months after stroke onset among patients presenting with small-artery occlusion.</p></div

Baseline demographics and clinical characteristics according to HbA1c tertiles.

<p>Baseline demographics and clinical characteristics according to HbA1c tertiles.</p