Ischemic preconditioning with non‐lethal ischemia can be protective against lethal forebrain ischemia. We hypothesized that aging may aggravate ischemic susceptibility and reduce brain plasticity ...against preconditioning. Magnetic resonance diffusion tensor imaging (DTI) is a sensitive tool to detect brain integrity and white matter architecture. This study used DTI and histopathology to investigate the effect of aging on ischemic preconditioning. In this study, adult and middle‐aged male Mongolian gerbils were subjected to non‐lethal 5‐min forebrain ischemia (ischemic preconditioning) or sham‐operation, followed by 3 days of reperfusion, and then lethal 15‐min forebrain ischemia. A 9.4‐Tesla MR imaging system was used to study DTI indices, namely fractional anisotropy (FA), mean diffusivity (MD), and intervoxel coherence (IC) in the hippocampal CA1 and dentate gyrus (DG) areas. In situ expressions of microtubule‐associated protein 2 (MAP2, dendritic marker protein) and apoptosis were also examined. The 5‐min ischemia did not cause dendritic and neuronal injury and any significant change in DTI indices and MAP2 in adult and middle‐aged gerbils. The 15‐min ischemia‐induced significant delayed neuronal apoptosis and early dendritic injury evidenced by DTI and MAP2 studies in both CA1 and DG areas with more severe injury in middle‐aged gerbils than adult gerbils. Ischemic preconditioning could improve neuronal apoptosis in CA1 area and dendritic integrity in both CA1 and DG areas with better improvement in adult gerbils than middle‐aged gerbils. This study thus suggests an age‐dependent protective effect of ischemic preconditioning against both neuronal apoptosis and dendritic injury in hippocampus after forebrain ischemia.
Ischemic preconditioning with nonlethal ischemia can be protective against lethal forebrain ischemia eventually in an age‐dependent manner. Magnetic resonance diffusion tensor imaging (DTI) is a sensitive tool to detect brain integrity and white matter architecture. The present study found delayed neuronal apoptosis and early dendritic injury evidenced by microtubule‐associated protein 2 (MAP2) and diffusion tensor imaging (DTI) indices after lethal forebrain ischemia is more severe in middle‐aged gerbils than adult gerbils. In the same way, preconditioning leads to better improvement in adult gerbils than middle‐aged gerbils. Our present study suggested an age‐dependent protective effect of ischemic preconditioning against both neuronal apoptosis and dendritic injury in hippocampus after forebrain ischemia.
Thrombolysis remains the only effective therapy to reverse acute ischaemic stroke. However, delayed treatment may cause serious complications including hemorrhagic transformation and reperfusion ...injury. The level of lipocalin‐2 (LCN2) is elevated in the plasma of ischaemic stroke patients, but its role in stroke is unknown. Here, we show that LCN2 was acutely induced in mice after ischaemic stroke and is an important mediator of reperfusion injury. Increased levels of LCN2 were observed in mouse serum as early as 1 hr after transient middle cerebral artery occlusion (tMCAO), reaching peak levels at 23 hrs. LCN2 was also detected in neutrophils infiltrating into the ipsilateral hemisphere, as well as a subset of astrocytes after tMCAO, but not in neurons and microglia. Stroke injury, neurological deficits and infiltration of immune cells were markedly diminished in LCN2 null mice after tMCAO, but not after permanent MCAO (pMCAO). In vitro, recombinant LCN2 protein induced apoptosis in primary cultured neurons in a dose‐dependent manner. Our results demonstrate that LCN2 is a neurotoxic factor secreted rapidly in response to cerebral ischaemia, suggesting its potential usage as an early stroke biomarker and a novel therapeutic target to reduce stroke‐reperfusion injury.
Oxidative stress is a key contributor to the pathogenesis of stroke-reperfusion injury. Neuroinflammatory peptides released after ischemic stroke mediate reperfusion injury. Previous studies, ...including ours, have shown that lipocalin-2 (LCN2) is secreted in response to cerebral ischemia to promote reperfusion injury. Genetic deletion of LCN2 significantly reduces brain injury after stroke, suggesting that LCN2 is a mediator of reperfusion injury and a potential therapeutic target. Immunotherapy has the potential to harness neuroinflammatory responses and provides neuroprotection against stroke. Here we report that LCN2 was induced on the inner surface of cerebral endothelial cells, neutrophils, and astrocytes that gatekeep the blood-brain barrier (BBB) after stroke. LCN2 monoclonal antibody (mAb) specifically targeted LCN2 in vitro and in vivo, attenuating the induction of LCN2 and pro-inflammatory mediators (iNOS, IL-6, CCL2, and CCL9) after stroke. Administration of LCN2 mAb at 4 h after stroke significantly reduced neurological deficits, cerebral infarction, edema, BBB leakage, and infiltration of neutrophils. The binding epitope of LCN2 mAb was mapped to the β3 and β4 strands, which are responsible for maintaining the integrity of LCN2 cup-shaped structure. These data indicate that LCN2 can be pharmacologically targeted using a specific mAb to reduce reperfusion injury after stroke.
Lipocalin‐2 (LCN2) has been implicated in promoting apoptosis and neuroinflammation in neurological disorders; however, its role in neural transplantation remains unknown. In this study, we cultured ...and differentiated Lund human mesencephalic (LUHMES) cells into human dopaminergic‐like neurons and found that LCN2 mRNA was progressively induced in mouse brain after the intrastriatal transplantation of human dopaminergic‐like neurons. The induction of LCN2 protein was detected in a subset of astrocytes and neutrophils infiltrating the core of the engrafted sites, but not in neurons and microglia. LCN2‐immunoreactive astrocytes within the engrafted sites expressed lower levels of A1 and A2 astrocytic markers. Recruitment of microglia, neutrophils, and monocytes after transplantation was attenuated in LCN2 deficiency mice. The expression of M2 microglial markers was significantly elevated and survival of engrafted neurons was markedly improved after transplantation in LCN2 deficiency mice. Brain type organic cation transporter (BOCT), the cell surface receptor for LCN2, was induced in dopaminergic‐like neurons after differentiation, and treatment with recombinant LCN2 protein directly induced apoptosis in dopaminergic‐like neurons in a dose‐dependent manner. Our results, therefore, suggested that LCN2 is a neurotoxic factor for the engrafted neurons and a modulator of neuroinflammation. LCN2 inhibition may be useful in reducing rejection after neural transplantation.
Transient global cerebral ischemia (tGCI) resulting from cardiac arrest causes selective neurodegeneration in hippocampal CA1 neurons. Although the effect is clear, the underlying mechanisms ...directing this process remain unclear. Previous studies have shown that phosphorylation of Erk1/2 promotes cell survival in response to tGCI. DUSP6 (also named MKP3) serves as a cytosolic phosphatase that dephosphorylates Erk1/2, but the role of DUSP6 in tGCI has not been characterized. We found that DUSP6 was specifically induced in the cytoplasm of hippocampal CA1 neurons 4 to 24 h after tGCI. DUSP6-deficient mice showed normal spatial memory acquisition and retention in the Barnes maze. Impairment of spatial memory acquisition and retention after tGCI was attenuated in DUSP6-deficient mice. Neurodegeneration after tGCI, revealed by Fluoro-Jade C and H&E staining, was reduced in the hippocampus of DUSP6-deficient mice and DUSP6 deficiency enhanced the phosphorylation and nuclear translocation of Erk1/2 in the hippocampal CA1 region. These data support the role of DUSP6 as a negative regulator of Erk1/2 signaling and indicate the potential of DUSP6 inhibition as a novel therapeutic strategy to treat neurodegeneration after tGCI.
The ubiquitous nature of protein phosphorylation makes it challenging to map kinase-substrate relationships, which is a necessary step toward defining signaling network architecture. To trace the ...activity of individual kinases, we developed a semisynthetic reaction scheme, which results in the affinity tagging of substrates of the kinase in question. First, a kinase, engineered to use a bio-orthogonal ATPgammaS analog, catalyzes thiophosphorylation of its direct substrates. Second, alkylation of thiophosphorylated serine, threonine or tyrosine residues creates an epitope for thiophosphate ester-specific antibodies. We demonstrated the generality of semisynthetic epitope construction with 13 diverse kinases: JNK1, p38alpha MAPK, Erk1, Erk2, Akt1, PKCdelta, PKCepsilon, Cdk1/cyclinB, CK1, Cdc5, GSK3beta, Src and Abl. Application of this approach, in cells isolated from a mouse that expressed endogenous levels of an analog-specific (AS) kinase (Erk2), allowed purification of a direct Erk2 substrate.
Stroke is the most common fatal neurological disease in the United States. The majority of strokes (88%) result from blockage of blood vessels in the brain (ischemic stroke). Since most ischemic ...strokes (~80%) occur in the territory of middle cerebral artery (MCA), many animal stroke models that have been developed have focused on this artery. The intraluminal monofilament model of middle cerebral artery occlusion (MCAO) involves the insertion of a surgical filament into the external carotid artery and threading it forward into the internal carotid artery (ICA) until the tip occludes the origin of the MCA, resulting in a cessation of blood flow and subsequent brain infarction in the MCA territory. The technique can be used to model permanent or transient occlusion. If the suture is removed after a certain interval (30 min, 1 h, or 2 h), reperfusion is achieved (transient MCAO); if the filament is left in place (24 h) the procedure is suitable as a model of permanent MCAO. This technique does not require craniectomy, a neurosurgical procedure to remove a portion of skull, which may affect intracranial pressure and temperature. It has become the most frequently used method to mimic permanent and transient focal cerebral ischemia in rats and mice. To evaluate the extent of cerebral infarction, we stain brain slices with 2,3,5-triphenyltetrazolium chloride (TTC) to identify ischemic brain tissue. In this video, we demonstrate the MCAO method and the determination of infarct size by TTC staining.
Global cerebral ischemia that accompanies cardiac arrest is a major cause of morbidity and mortality. Protein Kinase C epsilon (PKCε) is a member of the novel PKC subfamily and plays a vital role in ...ischemic preconditioning. Pharmacological activation of PKCε before cerebral ischemia confers neuroprotection. The role of endogenous PKCε after cerebral ischemia remains elusive. Here we used male PKCε‐null mice to assess the effects of PKCε deficiency on neurodegeneration after transient global cerebral ischemia (tGCI). We found that the cerebral vasculature, blood flow, and the expression of other PKC isozymes were not altered in the PKCε‐null mice. Spatial learning and memory was impaired after tGCI, but the impairment was attenuated in male PKCε‐null mice as compared to male wild‐type controls. A significant reduction in Fluoro‐Jade C labeling and mitochondrial release of cytochrome C in the hippocampus was found in male PKCε‐null mice after tGCI. Male PKCε‐null mice expressed increased levels of PKCδ in the mitochondria, which may prevent the translocation of PKCδ from the cytosol to the mitochondria after tGCI. Our results demonstrate the neuroprotective effects of PKCε deficiency on neurodegeneration after tGCI, and suggest that reduced mitochondrial translocation of PKCδ may contribute to the neuroprotective action in male PKCε‐null mice.
Neural cell death after transient global cerebral ischemia (tGCI) was attenuated in the hippocampus of mice lacking PKCε. Representative images of cresyl violet‐stained brain slices from Prkce+/+ and Prkce−/− mice 3 days after tGCI. Arrowheads indicate degenerative neurons with pyknotic and shrunken nuclei in the enlarged CA1 region of a Prkce+/+ brain slice (lower panels).
TRIM28/KAP1/TIF1β is a crucial epigenetic modifier. Genetic ablation of
is embryonic lethal, although RNAi-mediated knockdown in somatic cells yields viable cells. Reduction in TRIM28 abundance at ...the cellular or organismal level results in polyphenism. Posttranslational modifications such as phosphorylation and sumoylation have been shown to regulate TRIM28 activity. Moreover, several lysine residues of TRIM28 are subject to acetylation, but how acetylation of TRIM28 affects its functions remains poorly understood. Here, we report that, compared with wild-type TRIM28, the acetylation-mimic mutant TRIM28-K304Q has an altered interaction with Krüppel-associated box zinc-finger proteins (KRAB-ZNFs). The
-K304Q knock-in cells were created in K562 erythroleukemia cells by CRISPR-Cas9 (Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein nuclease 9) gene editing method. Transcriptome analysis revealed that
-K304Q and
knockout K562 cells had similar global gene expression profiles, yet the profiles differed considerably from wild-type K562 cells. The expression levels of embryonic-related globin gene and a platelet cell marker integrin-beta 3 were increased in
-K304Q mutant cells, indicating the induction of differentiation. In addition to the differentiation-related genes, many zinc-finger-proteins genes and imprinting genes were activated in
-K304Q cells; they were inhibited by wild-type TRIM28 via binding with KRAB-ZNFs. These results suggest that acetylation/deacetylation of K304 in TRIM28 constitutes a switch for regulating its interaction with KRAB-ZNFs and alters the gene regulation as demonstrated by the acetylation mimic TRIM28-K304Q.
Hemorrhagic transformation (HT) is a serious complication after endovascular thrombectomy (EVT) for patients with acute ischemic stroke (AIS). We analyzed the plasma levels of MMP-9 before and after ...EVT and assessed the temporal changes of MMP-9 that may be associated with, and therefore predict, HT after EVT.
We enrolled 30 AIS patients who received EVT, and 16 (53.3%) developed HT. The levels of MMP-9 in plasma collected from the arteries of AIS patients before and immediately after EVT were measured using ELISA. The percent change in MMP-9 after EVT (after/before) was calculated and compared between patients with and without HT.
The median age of the AIS patients was 70 years, and 13 patients (43.3%) were men. The median National Institutes of Health Stroke Scale (NIHSS) scores of patients with HT were 18 on admission and 18 after EVT. The median NIHSS scores of patients without HT were 17 on admission and 11 after EVT. Patients with HT demonstrated significantly greater percentage increases in arterial MMP-9 levels after EVT.
Patients with AIS who developed HT had significantly increased arterial MMP-9 levels after EVT, suggesting that the upregulation of MMP-9 following EVT could serve as a predictive biomarker for HT.
PDF
