Percentage Of Apoptotic Neurons Research Articles

Downregulation of NHE1 expression attenuates apoptosis of primary hippocampal neurons of an epilepsy model through the calpain-1 pathway

ObjectiveNa(+)/H(+) exchanger isoform 1 (NHE1), a membrane protein that regulates intracellular pH, is abundantly expressed in brain tissues. It is associated with pathophysiologies in several brain diseases. The present study aimed to investigate the effects of NHE1 on the apoptosis of primary neurons of an epilepsy model. MethodsPrimary hippocampal neurons were cultured in an Mg2+-free medium to establish an epilepsy cell model. Designed shNHE1 lentivirus was used to silence NHE1 level in primary neurons. Nonselective pharmacological inhibitor MDL-28170 (20 μmol/L) was used to inhibit calpain-1 protein in neurons treated with Mg2+-free medium. The expression levels of NHE1 and calpain-1, intracellular Ca2+ (Ca2+i) and H+ (H+i) levels, and the expression levels of apoptosis-related proteins Bcl-2 and Bax were detected in neurons. TUNEL staining was performed to determine apoptosis in different groups. ResultsNHE1 expression was increased in primary neurons treated with an Mg2+-free medium, and it was correlated with increased expression of calpain-1 and cell apoptosis. Neurons from the in vitro epilepsy model showed significantly decreased Bcl-2 protein expression and significantly increased Bax protein expression. In the presence of LV-shNHE1 and the calpain-1 inhibitor MDL-28170, the changes in the expression of apoptosis-related proteins Bcl-2 and Bax were blocked in the epileptic model, and the percentage of apoptotic neurons among neurons from the in vitro epilepsy model was significantly decreased. The increase in calpain-1 expression was suppressed by LV-shNHE1; however, the inhibition of calpain-1 did not affect NHE1 expression. ConclusionThese results demonstrate that NHE1 participates in the promotion of neuronal apoptosis of epilepsy model in vitro through the calpain-1 pathway. Downregulation of NHE1 expression could exert a neuroprotective effect on epilepsy.

Apoptosis Neuron Nukleus Merah pada Tikus Model Penyakit Parkinson Akibat Induksi Parakuat Diklorida

Red nucleus plays a role in motoric control, so that disturbanced of neurons in this nucleus could be affected on motor impairment. Previous study reported that paraquat (PQ) could induced Parkinson disease, a neurodegeneratif disease that affected subtantia nigra pars compactca and clinically characterized by motoric disorders. Apoptosis is a programed cell death that physiologically normal for the body, but in large quantities will cause progresive cell death. This study aimed to observed apoptosis in rats RN neurons following by PQ injection. Ten male Wistar rats with the aged of 3 month, devided into two groups of five. Control group was given aquadest and treatment group was given PQ, injected intraperitoneally, dosage 7 mg/kg BW, twice a week for three weeks. At day 24th all rats were anesthetized using ketamin dosage 40 mg/kg BW and xylazine dosage 5 mg/kg BW, perfused intracardially using 4% paraformaldehyde and mesencephalons were collected. Mesencephalons were processed for histological preparations using the paraffin method and cut stereologically in coronal section, in 4 ?m thickness. Immunohistochemistry staining was done using caspase 3 antibody as a marker of apoptosis. Data were analyzed descriptively and quantitatively. The results in both groups showed RN neurons were large size and stelat or fusiform in shape. The percentage of apoptotic neurons in the RN of treatment group was significantly increased (80,4%±13,8%) compared to the control group (26,6%±18,32%) (P<0.01). In conclusion, following PQ exposure there is a significant increased of apoptosis neurons number in the RN of rat model of Parkinson disease.

Targeting CCL20 inhibits subarachnoid hemorrhage-related neuroinflammation in mice.

Recent evidence suggests that CC chemokine ligand 20 (CCL20) is upregulated after subarachnoid hemorrhage (SAH). Here, we investigated the functions of CCL20 in SAH injury and its underlying mechanisms of action. We found that CCL20 is upregulated in an SAH mouse model and in cultured primary microglia and neurons. CCL20-neutralizing antibody alleviated SAH-induced neurological deficits, decreased brain water content and neuronal apoptosis, and repressed microglial activation. We observed increased levels of CCL20, CC chemokine receptor 6 (CCR6), interleukin 1 beta (IL-1β), and tumor necrosis factor alpha (TNF-α), as well as of microglial activation in microglia treated with oxyhemoglobin (OxyHb). CCL20 or CCR6 knockdown reversed the effects of OxyHb on microglia. Conditioned medium from OxyHb-treated microglia induced neuronal apoptosis, while the percentage of apoptotic neurons in the conditioned medium from microglia transfected with CCL20 siRNA or CCR6 siRNA was decreased. We observed no decrease in OxyHb-induced apoptosis in CCL20-knockdown neurons. Conditioned medium from OxyHb-treated neurons led to microglial activation and induced CCR6, IL-1β and TNF-α expression, while CCL20 knockdown in neurons or CCR6 knockdown in microglia reversed those effects. Our results thus suggest CCL20 may be targeted to elicit therapeutic benefits after SAH injury.

Transient Receptor Potential Canonical 5-Scramblase Signaling Complex Mediates Neuronal Phosphatidylserine Externalization and Apoptosis.

Phospholipid scramblase 1 (PLSCR1), a lipid-binding and Ca2+-sensitive protein located on plasma membranes, is critically involved in phosphatidylserine (PS) externalization, an important process in cell apoptosis. Transient receptor potential canonical 5 (TRPC5), is a nonselective Ca2+ channel in neurons that interacts with many downstream molecules, participating in diverse physiological functions including temperature or mechanical sensation. The interaction between TRPC5 and PLSCR1 has never been reported. Here, we showed that PLSCR1 interacts with TRPC5 through their C-termini in HEK293 cells and mouse cortical neurons. Formation of TRPC5-PLSCR1 complex stimulates PS externalization and promotes cell apoptosis in HEK293 cells and mouse cerebral neurons. Furthermore, in vivo studies showed that PS externalization in cortical neurons induced by artificial cerebral ischemia-reperfusion was reduced in TRPC5 knockout mice compared to wild-type mice, and that the percentage of apoptotic neurons was also lower in TRPC5 knockout mice than in wild-type mice. Collectively, the present study suggested that TRPC5-PLSCR1 is a signaling complex mediating PS externalization and apoptosis in neurons and that TRPC5 plays a pathological role in cerebral-ischemia reperfusion injury.

Morroniside protects against cerebral ischemia/reperfusion injury by inhibiting neuron apoptosis and MMP2/9 expression.

The aim of the present study was to investigate the effect of morroniside against matrix metalloproteinase (MMP)2/9 and focal cerebral ischemia/reperfusion (I/R) injury in rats. A rat model of focal cerebral I/R injury rats was established and rats were administered with 30, 90 or 270 mg/kg/day morroniside for 7 days. The expression of MMP2/9 and neuronal apoptosis were assessed. In addition, the expression of active caspase-3, B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax) were measured. The results revealed that MMP2 and MMP9 expression was upregulated and the percentage of apoptotic neurons was increased in rats with focal cerebral I/R injury compared with the control. However, treatment with morroniside significantly inhibited I/R-induced MMP2/9 expression and neuron apoptosis compared with the untreated I/R injury group. Morroniside administration also decreased the expression of active caspase-3 and increased the Bcl-2/Bax ratio compared with untreated rats with focal cerebral I/R injury. The inhibitory effect of morroniside on MMP2/9 expression and neuron apoptosis was dose dependent. In summary, the results of the present study suggest that morroniside is able to protect against cerebral I/R injury in the brain and may have potential as a therapeutic treatment for patients who have suffered a stroke.

Heme oxygenase-1 dependant pathway contributes to protection by tetramethylpyrazine against chronic hypoxic injury on medulla oblongata in rats

Tetramethylpyrazine (TMP), one of the active ingredients of the Chinese herb Lingusticum Wallichii (Chuan Xiong) has been proved to protect the medulla oblongata from chronic hypoxia injury. However, the underlying mechanism remains unclear. The purpose of this study was to determine whether the protective effects of TMP are associated with the heme oxygenase-1 (HO-1) dependant pathway in adult rats. The morphological changes of neurons in the hypoglossal nucleus (12N), the nucleus ambiguus (Amb), the nucleus tractus solitarius (NTS), and the pre-Bötzinger complex (pre-BötC) were investigated by Nissl staining; the malondialdehyde (MDA) content and superoxide dismutase (SOD) activity were measured to evaluate the anti-oxidant effect; some apoptosis parameters, Bax mRNA and Bcl-2 mRNA, were tested; and the double immunochemistry staining of active caspase-3/NeuN was performed. Meanwhile, the HO-1 protein expression and heme oxygenase (HO) activity were examined. Tin-protoporphyrin (SnPP), a potent inhibitor of HO, was used to further confirm the effect of HO-1. We found that TMP ameliorated the neuron loss in 12N, Amb and NTS, the decrease in SOD activity and the increase in MDA content, the decrease in Bcl-2 mRNA of medulla oblongata (P<0.05), and the increase in percentage of apoptotic neurons in Amb (P<0.05) induced by chronic hypoxia. Co-administration with SnPP abolished the beneficial effects above of TMP to some extent (P<0.05). Moreover, TMP significantly increased HO activity and HO-1 protein expression, which was most likely enhanced in the neurons (P<0.05), and co-administration of SnPP reduced these up-regulated effects (P<0.05). This study demonstrated that HO-1 dependant pathway may be involved in the protective action of TMP against chronic hypoxic damage on medulla oblongata in the rats.

Effect of encapsulation in liposomes on toxic effect of bupivacaine on spinal cord in rats

Objective To study the toxic effect of bupivacaine encapsulated in liposomes on spinal cord in rats.Methods One hundred and eight SD rats (200-225 g) in which intrathecal (IT) catheter was successfully implanted without complications were randomly divided into 6 groups ( n =18 each):control group (group C) ; liposome group (group L) ; 0.5％ and 1.0％ bupivacaine groups (groups B1 and B2 ) and 0.5％ and 1.0％ bupivacaine encapsulated in liposomes groups (groups LB1 and LB2 ).In groups L,B1,B2,LB1 and LB2,liposome,0.5 ％ bupivacaine,1.0 ％ bupivacaine,0.5 ％ liposomal bypivacaine and 1.0 ％ liposomal bupivacaine 20 μl were injected IT respectively once a day for 7 consecutive days,while in group C nothing was injected IT.Pain threshold was measured by mechanical stimulation of the plantar surface of hindpaw.Motor function of the hindlimbs was also assessed.The animals were sacrificed at 8 day after IT injection.The lumbar segment of the spinal cord was removed for microscopic examination,detection of neuronal apoptosis (by flow cytometry) and Fos protein expression (by immuno-histochemistry).Results 1.0％ bupivacaine IT significantly increased the percentage of apoptotic neurons in group B2 as compared with control group.0.5％ and 1.0％ bupivacaine IT significantly increased the number Fos protein positive cells in group B1 and B2 as compared with group C.1.0％ bupivacaine IT induced severe histologic damage including shrinkage of nucleus and vacuole formation in mitochondria.Encapsulation of bupivacaine in liposomes significantly attenuated bupivacaine-induced increase in apoptosis and Fos protein expression and histologic damage in group LB2 as compared with group B2.Conclusion The encapsulation in liposomes can decrease the neurotoxicity of 1.0 ％ bupivacaine administered IT in rats. Key words: Bupivacaine; Liposomes; Neurotoxicity Syndromes; Spinal Cord

GnRH analogue attenuated apoptosis of rat hippocampal neuron after ischemia–reperfusion injury

The expression and new functions of reproductive hormones in organs beyond hypothalamus-pituitary-gonad axis have been reported. So far, there is no report about the protective effects of GnRH analogue to hippocampal neurons suffering from ischemia-reperfusion injury. Middle cerebral artery occlusion model together with TUNEL staining were made in vivo and oxygen-glucose deprivation model together with double staining of Annexin V/PI with flow cytometer were made in vitro to observe the anti-apoptotic effects of GnRH analogue to hippocampal neurons after ischemia-reperfusion injury. The results found that the number of TUNEL positive pyramidal neurons in CA1 region in GnRH analogue experiment group was less than that in control group in vivo; the percentage of apoptotic neurons in GnRH analogue experiment group was less than that in control group in vitro. These findings suggested that pretreatment with certain concentration of GnRH analogue could attenuate apoptosis of hippocampal neurons. GnRH analogue has the protective effects to neurons.

Antioxidants prevent ethanol-associated apoptosis in fetal rhombencephalic neurons

It is well known that ethanol damages the developing nervous system by augmenting apoptosis. Previously, this laboratory reported that ethanol augments apoptosis in fetal rhombencephalic neurons, and that the increased apoptosis is associated with reduced activity of the phosphatidylinositol 3-kinase pathway and downstream expression of pro-survival genes. Other laboratories have shown that another mechanism by which ethanol induces apoptosis in developing neurons is through the generation of reactive oxygen species (ROS) and the associated oxidative stress. The present study used an in vitro model to investigate the potential neuroprotective effects of several antioxidants against ethanol-associated apoptosis in fetal rhombencephalic neurons. The investigated antioxidants included three phenolics: (−)-epigallocatechin-3-gallate (EGCG), a flavanoid polyphenol found in green tea; curcumin, found in tumeric; and resveratrol (3,5,4′-trihydroxystilbene), a component of red wine. Additional antioxidants, including melatonin, a naturally occurring indole, and α-lipoic acid, a naturally occurring dithiol, were also investigated. These studies demonstrated that a 24-hour treatment of fetal rhombencephalic neurons with 75 mM ethanol caused a 3-fold increase in the percentage of apoptotic neurons. However, co-treatment of these cultures with any of the five different antioxidants prevented ethanol-associated apoptosis. Antioxidant treatment did not alter the extent of apoptosis in control neurons, i.e., those cultured in the absence of ethanol. These studies showed that several classes of antioxidants can exert neuroprotection against ethanol-associated apoptosis in fetal rhombencephalic neurons.

Antiapoptotic effects of the peptidergic drug cerebrolysin on primary cultures of embryonic chick cortical neurons.

Cerebrolysin (EBEWE Arzneimittel, Austria, Europe) is a widely used drug relieving the symptoms of a variety of neurological disorders, particularly of neurodegenerative dementia of the Alzheimer's type. It consists of approximately 25% of low molecular weight peptides (<10k DA) and a mixture of approximately 75% free amino acids, this being based on the total nitrogen content. In this study we used a low serum (2% serum supplement) cell stress in-vitro model to assess drug effectiveness on neuronal viability and programmed cell death (PCD). In this in-vitro model the type of cell death was previously shown to be primarly apoptotic, which was verified by DNA-laddering and TUNEL-staining. For evaluation of neuronal viability a MTT-reduction assay was performed after 4 DIV and 8 DIV and the percentage of apoptotic neurons was determined by bis-benzimide staining of nuclear chromatin. To differentiate between possible effects of the free amino acids and the peptide fraction of Cerebrolysin an artificial amino acid mixture (AA-mix) was used as a control. Cerebrolysin, the AA-mix and 10% foetal calf serum (FCS) caused a similar increase in viability after 4 DIV, whereas the effects of the growth factors BDNF and FGF-2 were less pronounced. After 8 DIV Cerebrolysin, but not the AA-mix, was able to ameliorate neuronal viability, which could reflect a neuro-protective effect or an increased activity of the mitochondrial dehydrogenase measured in a MTT-reduction assay. The percentage of cells showing apoptotic chromatin changes was significantly reduced (p < 0.01) in cultures treated with Cerebrolysin, whereas the AA-mix failed to decrease the percentage of cells showing apoptotic chromatin changes. These findings ascertain an anti-apoptotic effect of the peptide fraction of Cerebrolysin and reveal a transient viability promoting effect of the amino acid fraction, which is most likely due to improved nutritional supply.

Neuroprotective effects of NV-31, a bilobalide-derived compound: evidence for an antioxidative mechanism

In previous studies we have already shown that the extract of Ginkgo biloba, and some of its constituents, such as ginkgolide B and bilobalide, protected cultured neurons against apoptotic and excitotoxic damage and reduced the infarct volume after focal cerebral ischemia in mice and rats. In this work, we determined the neuroprotective and antioxidative effects of 4-hydroxy-4- tert-butyl-2,3,5,6-tetrahydrothiopyran-1-oxide (NV-31), a stable compound which was synthesized to mimic the pharmacological activity profile of bilobalide. In pure neuronal cultures from chick embryo telencephalon, damage was induced by serum deprivation (24 h) and exposure to staurosporine (200 nM, 24 h) which caused an increase in the percentage of apoptotic neurons from 14 (controls) to 30 and 55%, respectively. NV-31 (1–100 nM) protected dose-dependently chick neurons against both serum deprivation- and staurosporine-induced apoptosis. Similarly, NV-31 (100 nM) reduced staurosporine (300 nM, 24 h)-induced neuronal damage in mixed cultures of neurons and astrocytes from neonatal rat hippocampus. The cellular ROS content increased 6-fold 4 h after serum deprivation as well as 4 h after the exposure to staurosporine and this increase was reduced by 50% in the presence of 10 and 100 nM NV-31, respectively. In mice, a treatment with 10 and 20 mg/kg NV-31 60 min before and immediately after focal cerebral ischemia, respectively, significantly reduced the infarct area compared with vehicle-treated animals. In the present study, we show that NV-31 promotes neuronal survival and we suggest that its antioxidative property contributes to the mechanism of neuroprotection.

Staurosporine-induced apoptosis in cultured chick embryonic neurons is reduced by polyethylenimine of low molecular weight used as a coating substrate.

The survival of neurons largely depends on adhesion to extracellular matrix proteins. This study investigated the influence of polycationic macromolecules of different molecular weights used as coating substrates on apoptosis in primary cultures of chick embryonic neurons. Coating of the culture flasks with positively charged polyethylenimine (PEI) of 12, 32 and 1616 kDa led to different susceptibilities of the neurons to apoptosis induced by staurosporine and serum deprivation. In cultures grown as usual on polylysine (PL)-coated flasks, we found, after 24 h of incubation in medium with serum (controls), serum-free medium and staurosporine (200 nM)-containing serum-free medium, 15, 35 and 63% apoptotic neurons, respectively as evaluated by nuclear staining with Hoechst 33258. Using 12-kDa PEI as a coating substrate, only 11, 15 and 47% apoptotic neurons could be determined in controls, serum-deprived and staurosporine-treated cultures, respectively. No change in the percentage of apoptotic neurons was found after 24 h of serum deprivation or treatment with staurosporine in cultures grown on 32-kDa PEI compared with cultures grown on PL. However, in staurosporine-treated cultures grown on 1616-kDa PEI, the percentage of apoptotic neurons was even higher than in cultures grown on PL. Immunostaining using neurofilament (NF) antibodies revealed that the reduction of staurosporine-induced apoptosis using 12-kDa PEI instead of PL as a coating substrate was accompanied by a reduced disruption and aggregation of the neurofilaments. Thus, the usage of the newly synthesized 12-kDa PEI as a coating substrate enhanced neuronal resistance to apoptosis.

Inhibition of serum deprivation- and staurosporine-induced neuronal apoptosis by Ginkgo biloba extract and some of its constituents

Previous studies have already demonstrated that some constituents of an extract of Ginkgo biloba (EGb), such as ginkgolide B and bilobalide, protect cultured neurons from hypoxia- and glutamate-induced damage. This prompted us to investigate whether they were also able to inhibit neuronal apoptosis. We induced apoptosis in cultured chick embryonic neurons as well as in mixed cultures of neurons and astrocytes from neonatal rat hippocampus by serum deprivation and staurosporine. The increase in the percentage of apoptotic chick neurons from 12% in controls to 30% after 24 h of serum deprivation was reduced to control level by EGb (10 mg/l), ginkgolide B (10 μM), ginkgolide J (100 μM) and bilobalide (1 μM). After treatment with staurosporine (200 nM) for 24 h we observed 74% apoptotic chick neurons. This percentage of apoptotic neurons was reduced to 24%, 62% and 31% in the presence of EGb (100 mg/l), ginkgolide J (100 μM) and ginkgolide B (10 μM), respectively. Bilobalide (10 μM) decreased apoptotic damage induced by staurosporine treatment for 12 h nearly to the control level. In mixed neuronal/glial cultures, the extract of EGb (100 mg/l) and bilobalide (100 μM) rescued rat neurons from apoptosis caused by serum deprivation, whereas, bilobalide (100 μM) and ginkgolide B (100 μM) reduced staurosporine-induced apoptotic damage. Ginkgolide A revealed no anti-apoptotic effect in either serum-deprived or staurosporine-treated neurons. Our results suggest that EGb and some of its constituents possess anti-apoptotic capacity and that bilobalide is the most potent constituent.

Cultured neurons expressing phosphorylated tau are more resistant to apoptosis induced by NMDA or serum deprivation.

Apoptosis is a programmed cell death that occurs during the development of the nervous system and in neurodegenerative disorders. Tau protein is a cytoskeletal component that promotes microtubule polymerization and stabilization. Apoptosis was induced in primary neuronal cultures by a prolonged exposure (16 h) to the NMDA (N-methyl-D-aspartate 20 microM) or by serum deprivation. The percentages of apoptotic neurons expressing phosphorylated tau (AT8) immunoreactivity are comparable in control and NMDA-exposed cultures (7.5 +/- 1.9 and 6.9 +/- 1.9%, respectively). At the opposite, the percentage of apoptotic neurons expressing de-phosphorylated tau (tau 1) immunolabelings is dramatically increased in NMDA-treated cultures (X 2.3 of controls). Similar results were also observed 48 h after serum deprivation. These results demonstrate in vitro that under these conditions, resistant and sensitive cortical neurons to apoptosis can be partly differentiated according to their phosphorylated tau immunoreactivities.