Delta neutrophil index: A reliable marker to differentiate perforated appendicitis from non-perforated appendicitis in the elderly.

BACKGROUND: Delta neutrophil index (DNI) is a new inflammatory marker and the present study aimed to evaluate the predictive value of the DNI for the presence of a perforation in elderly with acute appendicitis. METHODS: This retrospective observational study was conducted on 108 consecutive elderly patients (≥65 years old) with acute appendicitis treated over a 24-month period. RESULTS: Sixty-nine of the 108 patients (median, IQR: 72, 67-77 years) were allocated to the perforated appendicitis group (63.9%) and 39 to the non-perforated appendicitis group (36.1%). WBC, neutrophil-to-lymphocyte ratio, platelet-to-lymphocyte ratio and DNI were significantly higher in the perforated group. In multiple logistic regression analyses, initial DNI was the only independent marker that can significantly predict the presence of perforation in multiple regression [odds ratio 9.38, 95% confidence interval (2.51-35.00), P=.001]. Receiver operator characteristic curve analysis showed that DNI is a good predictor for the presence of appendiceal perforation at an optimal cut-off for DNI being 1.4% (sensitivity 67.7%, specificity 90.0%, AUC 0.807). CONCLUSION: Clinicians can reliably differentiate acute perforated appendicitis from non-perforated appendicitis by DNI level of 1.4 or more in elderly patients.

Neuroprotection of Ischemic Preconditioning is Mediated by Anti-inflammatory, Not Pro-inflammatory, Cytokines in the Gerbil Hippocampus Induced by a Subsequent Lethal Transient Cerebral Ischemia.

Ischemic preconditioning (IPC) induced by sublethal transient cerebral ischemia could reduce neuronal damage/death following a subsequent lethal transient cerebral ischemia. We, in this study, compared expressions of interleukin (IL)-2 and tumor necrosis factor (TNF)-α as pro-inflammatory cytokines, and IL-4 and IL-13 as anti-inflammatory cytokines in the gerbil hippocampal CA1 region between animals with lethal ischemia and ones with IPC followed by lethal ischemia. In the animals with lethal ischemia, pyramidal neurons in the stratum pyramidale (SP) of the hippocampal CA1 region were dead at 5 days post-ischemia; however, IPC protected the CA1 pyramidal neurons from lethal ischemic injury. Expressions of all cytokines were significantly decreased in the SP after lethal ischemia and hardly detected in the SP at 5 days post-ischemia because the CA1 pyramidal neurons were dead. IPC increased expressions of anti-inflammatory cytokines (IL-4 and IL-13) in the stratum pyramidale of the CA1 region following no lethal ischemia (sham-operation), and the increased expressions of IL-4 and IL-13 by IPC were continuously maintained is the SP of the CA1 region after lethal ischemia. However, pro-inflammatory cytokines (IL-2 and TNF-α) in the SP of the CA1 region were similar those in the sham-operated animals with IPC, and the IL-4 and IL-13 expressions in the SP were maintained after lethal ischemia. In conclusion, this study shows that anti-inflammatory cytokines significantly increased and longer maintained by IPC and this might be closely associated with neuroprotection after lethal transient cerebral ischemia.

Time-course changes in immunoreactivities of glucokinase and glucokinase regulatory protein in the gerbil hippocampus following transient cerebral ischemia.

Glucose is a main energy source for normal brain functions. Glucokinase (GK) plays an important role in glucose metabolism as a glucose sensor, and GK activity is modulated by glucokinase regulatory protein (GKRP). In this study, we examined the changes of GK and GKRP immunoreactivities in the gerbil hippocampus after 5 min of transient global cerebral ischemia. In the sham-operated-group, GK and GKRP immunoreactivities were easily detected in the pyramidal neurons of the stratum pyramidale of the hippocampus. GK and GKRP immunoreactivities in the pyramidal neurons were distinctively decreased in the hippocampal CA1 region (CA), not CA2/3, 3 days after ischemia-reperfusion (I-R). Five days after I-R, GK and GKRP immunoreactivities were hardly detected in the CA1, not CA2/3, pyramidal neurons; however, at this point in time, GK and GKRP immunoreactivities were newly expressed in astrocytes, not microglia, in the ischemic CA1. In brief, GK and GKRP immunoreactivities are changed in pyramidal neurons and newly expressed in astrocytes in the ischemic CA1 after transient cerebral ischemia. These indicate that changes of GK and GKRP expression may be related to the ischemia-induced neuronal damage/death.

Comparison of neurogenesis in the dentate gyrus between the adult and aged gerbil following transient global cerebral ischemia.

In the present study, we compared differences in cell proliferation, neuroblast differentiation and neuronal maturation in the hippocampal dentate gyrus (DG) between the adult and aged gerbil induced by 5 min of transient global cerebral ischemia using Ki-67 and BrdU (markers for cell proliferation), doublecortin (DCX, a marker for neuroblast differentiation) and neuronal nuclei (NeuN, a marker for mature neuron). The number of Ki-67-immunoreactive (⁺) cells in the DG of both the groups peaked 7 days after ischemia/reperfusion (I/R). However, the number in the aged DG was 40.6 ± 1.8% of that in the adult DG. Thereafter, the number decreased with time. After ischemic damage, DCX immunoreactivity and its protein level in the adult and aged DG peaked at 10 and 15 days post-ischemia, respectively. However, DCX immunoreactivity and its protein levels in the aged DG were much lower than those in the adult. DCX immunoreactivity and its protein level in the aged DG were 11.1 ± 0.6% and 34.4 ± 2.1% of the adult DG, respectively. In addition, the number of Ki-67⁺ cells and DCX immunoreactivity in both groups were similar to those in the sham at 60 days postischemia. At 30 days post-ischemia, the number of BrdU⁺ cells and BrdU⁺/NeuN⁺ cells in the adult-group were much higher (281.2 ± 23.4% and 126.4 ± 7.4%, respectively) than the aged-group (35.6 ± 6.8% and 79.5 ± 6.1%, respectively). These results suggest that the ability of neurogenesis in the ischemic aged DG is much lower than that in the ischemic adult DG.

Na+/HCO3- cotransporter immunoreactivity changes in neurons and expresses in astrocytes in the gerbil hippocampal CA1 region after ischemia/reperfusion.

The maintenance of intracellular pH is important in neuronal function. Na(+)/HCO(3)(-) cotransporter (NBC), a bicarbonate-dependent acid-base transport protein, may contribute to cellular acid-base homeostasis in pathophysiological processes. We examined the alterations of NBC immunoreactivity and its protein levels in the hippocampal CA1 region after transient cerebral ischemia in gerbils. In the sham-operated group, moderate NBC immunoreactivity was detected in CA1 pyramidal neurons, and, 12 h after I/R, the immunoreactivity in the pyramidal neurons was markedly increased over controls. Three days after I/R, NBC immunoreactivity nearly disappeared in the CA1 pyramidal neurons. However, NBC immunoreactivity was detected in the non-pyramidal neurons of the ischemic CA1 region at 3 days after I/R. From double immunofluorescence study with glial markers, NBC immunoreactivity was detected in astrocytes, not in microglia, at 4 days after I/R. NBC protein level in the CA1 region was significantly increased at 12 h post-ischemia and significantly decreased at 2 days post-ischemia. Thereafter, NBC protein level was again increased and returned to the level of the sham-operated group at 4 days post-ischemia. On the other hand, treatment with 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS), an inorganic anion exchanger blocker including Cl-bicarbonate exchanger, protected CA1 pyramidal neurons from I/R injury at 4 days post-ischemia. These results indicate that changes in NBC expressions may play an important role in neuronal damage and astrocytosis induced by transient cerebral ischemia.

Rufinamide pretreatment attenuates ischemia-reperfusion injury in the gerbil hippocampus.

OBJECTIVES: Rufinamide, a voltage-gated sodium channel (VGSC) blocker, is widely used for the clinical treatment of seizures associated with Lennox-Gastaut syndrome. Previous studies have demonstrated that VGSC blockers have neuroprotective properties against ischemic damage following experimental cerebral ischemia. However, protective effects of rufinamide against cerebral ischemic insults have not been addressed. Therefore, in the present study, we firstly examined neuroprotective effects of rufinamide using a gerbil model of transient global cerebral ischemia. METHODS: Gerbils were established by the occlusion of common carotid arteries for 5 min. The gerbils were divided into vehicle-treated sham-operated group, vehicle-treated ischemia-operated group, 50 and 100 mg/kg rufinamide-treated sham-operated groups, and 50 and 100 mg/kg rufinamide-treated ischemia-operated groups. Rufinamide was administrated intraperitoneally once daily for 3 days before ischemic surgery. To examine neuroprotective effects of rufinamide, we carried out cresyl violet staining, neuronal nuclear antigen immunohistochemistry and Fluoro-Jade B histofluorescence staining. In addition, we examined gliosis using immunohistochemistry for glial fibrillary acidic protein (a marker for astrocytes) and ionized calcium-binding adapter molecule 1 (a marker for microglia). RESULTS: We found that pre-treatment with 100 mg/kg of rufinamide effectively protected pyramidal neurons in the hippocampal cornus ammonis 1 (CA1) area after transient global cerebral ischemia. In addition, pre-treatment with 100 mg/kg of rufinamide significantly attenuated activations of astrocytes and microglia in the ischemic CA1 area. DISCUSSION: These findings suggest that rufinamide can display neuroprotective effect against cerebral ischemic insults and that its neuroprotective effect may involve the attenuation of ischemia-induced glial activation.

Effects of ischemic preconditioning on PDGF-BB expression in the gerbil hippocampal CA1 region following transient cerebral ischemia.

Ischemic preconditioning (IPC) is induced by exposure to brief durations of transient ischemia, which results in ischemic tolerance to a subsequent longer or lethal period of ischemia. In the present study, the effects of IPC (2 min of transient cerebral ischemia) were examined on immunoreactivity of platelet­derived growth factor (PDGF)­BB and on neuroprotection in the gerbil hippocampal CA1 region following lethal transient cerebral ischemia (LTCI; 5 min of transient cerebral ischemia). IPC was subjected to a 2­min sublethal ischemia and a LTCI was given 5­min transient ischemia. The animals in all of the groups were given recovery times of 1, 2 and 5 days and change in PDGF­BB immunoreactivity was examined as was the neuronal damage/death in the hippocampus induced by LTCI. LTCI induced a significant loss of pyramidal neurons in the hippocampal CA1 region 5 days after LTCI, and significantly decreased PDGF­BB immunoreactivity in the CA1 pyramidal neurons from day 1 after LTCI. Conversely, IPC effectively protected the CA1 pyramidal neurons from LTCI and increased PDGF­BB immunoreactivity in the CA1 pyramidal neurons post­LTCI. In conclusion, the results demonstrated that LTCI significantly altered PDGF­BB immunoreactivity in pyramidal neurons in the hippocampal CA1 region, whereas IPC increased the immunoreactivity. These findings indicated that PDGF­BB may be associated with IPC­mediated neuroprotection.

Effects of Paraquat Ban on Herbicide Poisoning-Related Mortality.

PURPOSE: In Korea, registration of paraquat-containing herbicides was canceled in November 2011, and sales thereof were completely banned in November 2012. We evaluated the effect of the paraquat ban on the epidemiology and mortality of herbicide-induced poisoning. MATERIALS AND METHODS: This retrospective study analyzed patients treated for herbicide poisoning at 17 emergency departments in South Korea between January 2010 and December 2014. The overall and paraquat mortality rates were compared pre- and post-ban. Factors associated with herbicide mortality were evaluated using logistic analysis. To determine if there were any changes in the mortality rates before and after the paraquat sales ban and the time point of any such significant changes in mortality, R software, version 3.0.3 (package, bcp) was used to perform a Bayesian change point analysis. RESULTS: We enrolled 2257 patients treated for herbicide poisoning (paraquat=46.8%). The overall and paraquat poisoning mortality rates were 40.6% and 73.0%, respectively. The decreased paraquat poisoning mortality rate (before, 75% vs. after, 67%, p=0.014) might be associated with increased intentionality. The multivariable logistic analysis revealed the paraquat ban as an independent predictor that decreased herbicide poisoning mortality (p=0.035). There were two major change points in herbicide mortality rates, approximately 3 months after the initial paraquat ban and 1 year after complete sales ban. CONCLUSION: This study suggests that the paraquat ban decreased intentional herbicide ingestion and contributed to lowering herbicide poisoning-associated mortality. The change point analysis suggests a certain timeframe was required for the manifestation of regulatory measures outcomes.

The prognostic value of the grey-to-white matter ratio in cardiac arrest patients treated with extracorporeal membrane oxygenation.

AIM: The grey-to-white matter ratio (GWR) is a reliable predictor of the neurological outcome of out-of-hospital cardiac arrest (OHCA). However, the reliability in patients receiving extracorporeal membrane oxygenation-assisted cardiopulmonary resuscitation (ECPR) remains unknown. We evaluated the utility of the GWR in predicting neurological outcomes in ECPR-treated patients. METHODS: This single-centre retrospective study was conducted from July 2009 to January 2014. Patients who received ECPR for OHCA were classified into two groups: Cerebral performance category(CPC) 1-2 was defined as good, CPC 3-5 as poor outcome. Four GWR (GWR-AV[average], GWR-CO[cortex], GWR-BG[basal ganglia], and GWR-SI [simplified])were evaluated and compared between the groups. RESULTS: Of 38 patients who received ECPR for OHCA, 30 patients were enrolled. Five (16.7%) had a good outcome and 25(83.3%) a poor outcome. All GWR were significantly higher in the good outcome group than in the poor outcome group. ROC curve analysis produced the following areas under the curve: GWR-AV=0.920 (95% CI 0.761 to 0.987), GWR-BG=0.872 (95%CI 0.699 to 0.965), GWR-CO=0.952 (95% CI 0.806 to 0.997), and GWR-SI=0.848(95% CI 0.670 to 0.962). The cut-off value with 100% specificity for the prediction of the poor outcome was 1.23 for GWR-AV (sensitivity: 76%), 1.24 for GWR-BG (sensitivity: 88.0%), 1.22 for GWR-CO (sensitivity: 64%), and 1.21 for GWR-SI (sensitivity: 76%). CONCLUSIONS: In ECPR, GWR of patients with poor outcome was significantly lower than that of patients with good outcome.

Refractory ventricular fibrillation treated with esmolol.

AIMS: This study aimed to evaluate the effects of esmolol treatment for patients with refractory ventricular fibrillation (RVF) in out-of-hospital cardiac arrest (OHCA). METHODS: This single-centre retrospective pre-post study evaluated patients who were treated between January 2012 and December 2015. Some patients had received esmolol (loading dose: 500µg/kg, infusion: 0-100µg/kg/min) for RVF (≥3 defibrillation attempts), after obtaining consent from the patient's guardian. RESULTS: Twenty-five patients did not receive esmolol (the control group), and 16 patients received esmolol. Sustained return of spontaneous circulation (ROSC) was significantly more common in the esmolol group, compared to the control group (56% vs. 16%, p=0.007). Survival and good neurological outcomes at 30 days, 3 months and at 6 months were >2-fold better in the esmolol group, compared to the control group, although these increases were not statistically significant. CONCLUSIONS: The findings of our study suggest that administration of esmolol may increase the rate of sustained ROSC and ICU survival among patients with RVF in OHCA. Further larger-scale, prospective studies are necessary to determine the effect of esmolol for RVF in OHCA.

Long-term observation of neuronal degeneration and microgliosis in the gerbil dentate gyrus after transient cerebral ischemia.

Ischemic insults in the central nervous system evoke activation of microglia. In this study, we investigated long-term changes of neuronal damage and microglial activation in the gerbil dentate gyrus for 60 days after transient cerebral ischemia using immunohistochemistry and western blot. Neuronal damage or death was hardly found in the dentate gyrus after transient ischemia using cresyl violet staining and NeuN immunohistochemistry; however, neuronal degeneration was detected in the polymorphic layer of the dentate gyrus using Fluoro-Jade (F-J) B staining. F-J B-positive cells were significantly increased after ischemia-reperfusion (I-R) and peaked at 3 days post-ischemia, thereafter, F-J B-positive cells were decreased in a time-dependent manner and shown until 30 days post-ischemia; no F-J B-positive cells were observed 60 days after I-R. On the other hand, Iba-1-immunoreactive microglia were hypertrophied after I-R, and numbers of Iba-1-immunoreactive microglia were significantly increased along with the neuronal degeneration and highest 7 days after I-R, thereafter, numbers of Iba-1-immunoreactive microglia were decreased with time, although microglia activation lasted up to 60 days after I-R. In addition, Iba-1 protein level in the dentate gyrus after I-R was changed like immunohistochemical change. Our results, in brief, indicate that transient ischemia-induced neuronal degeneration in the dentate gyrus is maintained for about 30 days after I-R and that microglial activation lasts up to, at least, 60 days after I-R in the gerbil dentate gyrus after transient cerebral ischemia.

Out-of-hospital cardiac arrest patients treated with cardiopulmonary resuscitation using extracorporeal membrane oxygenation: focus on survival rate and neurologic outcome.

BACKGROUND: Extracorporeal membrane oxygenation (ECMO) is a useful treatment for refractory out-of-hospital cardiac arrest (OHCA). However, little is known about the predictors of survival and neurologic outcome after ECMO. We analyzed our institution's experience with ECMO for refractory OHCA and evaluated the predictors of survival and neurologic outcome after ECMO. METHODS: This was a retrospective review of the medical records of 23 patients who were treated with ECMO due to OHCA that was unresponsive to conventional cardiopulmonary resuscitation, between January 2009 and January 2014. RESULTS: Our ECMO team was activated within 10 min for refractory OHCA, and the 30-day survival rate was 43.5 %. In a multivariate analysis that evaluated independent factors contributing to mortality, urine output ≤ 0.5 mL · kg(-1) · h(-1) (defined as oliguria) during the 24 h after ECMO was statistically significant (OR, 32.271; 95 % CI, 1.379-755.282; p = 0.031). Just after ECMO implantation, 6 of the 9 patients (66.7 %) who had normal findings on brain computed tomography (CT) survived with a cerebral performance category (CPC) of grade 1. However, only 3 of the 11 patients (27 %) who had evidence of hypoxic brain damage on initial brain CT survived (their CPC grade was 4). CONCLUSIONS: Based on our findings, the survival rate can be improved by rapid implantation of ECMO, and oliguria seen during the first 24 h after ECMO may be an independent predictor of mortality. Furthermore, findings on brain CT just after ECMO and subsequent images may represent an important predictor for neurologic outcome after ECMO.

Effect of ischemic preconditioning on the expression of c-myb in the CA1 region of the gerbil hippocampus after ischemia/reperfusion injury.

OBJECTIVES: In the present study, we investigated the effect of ischemic preconditioning (IPC) on c-myb immunoreactivity as well as neuronal damage/death after a subsequent lethal transient ischemia in gerbils. MATERIALS AND METHODS: IPC was subjected to a 2 min sublethal ischemia and a lethal transient ischemia was given 5 min transient ischemia. The animals in all of the groups were given recovery times of 1 day, 2 days and 5 days and we examined change in c-myb immunoreactivity as well as neuronal damage/death in the hippocampus induced by a lethal transient ischemia. RESULTS: A lethal transient ischemia induced a significant loss of cells in the stratum pyramidale (SP) of the hippocampal CA1 region at 5 days post-ischemia, and this insult showed that c-myb immunoreactivity in cells of the SP of the CA1 region was significantly decreased at 2 days post-ischemia and disappeared at 5 days post-ischemia. However, IPC effectively prevented the neuronal loss in the SP and showed that c-myb immunoreactivity was constitutively maintained in the SP after a lethal transient ischemia. CONCLUSION: Our results show that a lethal transient ischemia significantly decreased c-myb immunoreactivity in the SP of the CA1 region and that IPC well preserved c-myb immunoreactivity in the SP of the CA1 region. We suggest that the maintenance of c-myb might be related with IPC-mediated neuroprotection after a lethal ischemic insult.

Activation of immediate-early response gene c-Fos protein in the rat paralimbic cortices after myocardial infarction.

c-Fos is a good biological marker for detecting the pathogenesis of central nervous system disorders. Few studies are reported on the change in myocardial infarction-induced c-Fos expression in the paralimbic regions. Thus, in this study, we investigated the changes in c-Fos expression in the rat cingulate and piriform cortices after myocardial infarction. Neuronal degeneration in cingulate and piriform cortices after myocardial infarction was detected using cresyl violet staining, NeuN immunohistochemistry and Fluoro-Jade B histofluorescence staining. c-Fos-immunoreactive cells were observed in cingulate and piriform cortices at 3 days after myocardial infarction and peaked at 7 and 14 days after myocardial infarction. But they were hardly observed at 56 days after myocardial infarction. The chronological change of c-Fos expression determined by western blot analysis was basically the same as that of c-Fos immunoreactivity. These results indicate that myocardial infarction can cause the chronological change of immediate-early response gene c-Fos protein expression, which might be associated with the neural activity induced by myocardial infarction.

The pre-ECMO simplified acute physiology score II as a predictor for mortality in patients with initiation ECMO support at the emergency department for acute circulatory and/or respiratory failure: a retrospective study.

BACKGROUND: In the emergency department (ED), extracorporeal membrane oxygenation (ECMO) can be used as a rescue treatment modality for patients with refractory circulatory and/or respiratory failure. Serious consideration must be given to the indication, and the PRESERVE and RESP scores for mortality have been investigated. However these scores were validated to predict survival in patients who received mainly veno-venous (VV) ECMO in the intensive care unit. The aim of the present study was to investigate the factors that predicted the outcomes for patients who received mixed mode (veno-arterial [VA] and VV) ECMO support in the ED. METHODS: This single center retrospective study included 65 patients who received ECMO support at the ED for circulatory or respiratory failure between January 2009 and December 2013. Pre-ECMO SAPS II and other variables were evaluated and compared for predicting mortality. RESULTS: Fifty-four percent of patients received ECMO-cardiopulmonary resuscitation (E-CPR), 31 % received VA and V-AV ECMO, and 15 % received VV ECMO. The 28-day and 60-month mortality rates were 52 % and 63 %. In the multivariate analysis, only the pre-ECMO Simplified Acute Physiology Score II (SAPS II) (odd ratio: 1.189, 95 % confidence interval: 1.032-1.370, p = 0.016) could predict the 28-day mortality. The area under the receiver operating characteristic curve and the optimal cutoff value for pre-ECMO SAPS II in predicting 28-day mortality was 0.852 (95 % CI: 0.753-0.951, p < 0.001) and 80 (sensitivity of 97.1 % and specificity of 71.0 %), respectively. Validation of the 80 cutoff value revealed a statistically significant difference for the 28-day and 60-month mortality rates in the overall, E-CPR, and VA groups (28-day: p < 0.001, p = 0.004, p = 0.005; 60-month: p < 0.001, p = 0.004, p = 0.020). In the Kaplan-Meier analysis, the 28-day and 60-month survival rates were lower among the patients with a pre-ECMO SAPS II of ≤ 80, compared to those with a score of > 80 (both, p < 0.001). CONCLUSION: The pre-ECMO SAPS II could be helpful for identifying patients with refractory acute circulatory and/or respiratory failure who will respond to ECMO support in the ED.

Efficacy of veno-venous extracorporeal membrane oxygenation in severe acute respiratory failure.

PURPOSE: The objective of this study was to evaluate our institutional experience with veno-venous (VV) extracorporeal membrane oxygenation (ECMO) in patients with severe acute respiratory failure (ARF). MATERIALS AND METHODS: From January 2007 to August 2013, 31 patients with severe ARF that was due to various causes and refractory to mechanical ventilation with conventional therapy were supported with VV ECMO. A partial pressure of arterial oxygen (PaO2)/inspired fraction of oxygen (FiO2) <100 mm Hg at an FiO2 of 1.0 or a pH <7.25 due to CO2 retention were set as criteria for VV ECMO. RESULTS: Overall, 68% of patients survived among those who had received VV ECMO with a mean PaO2/FiO2 of 56.8 mm Hg. Furthermore, in trauma patients, early use of ECMO had the best outcome with a 94% survival rate. CONCLUSION: VV ECMO is an excellent, life-saving treatment option in patients suffering from acute and life-threatening respiratory failure due to various causes, especially trauma, and early use of VV ECMO therapy improved outcomes in these patients.

Ischemic preconditioning-induced neuroprotection against transient cerebral ischemic damage via attenuating ubiquitin aggregation.

Ubiquitin binds to short-lived proteins, and denatured proteins are produced by various forms of injuries. In the present study, we investigated the effect of ischemic preconditioning (IPC) on free ubiquitin and its mutant form (ubiquitin(+1)) in the gerbil hippocampus induced by transient cerebral ischemia. The animals were randomly assigned to 4 groups (sham-operated-group, ischemia-operated-group, IPC plus (+)-sham-operated-group, and IPC+ischemia-operated-group). IPC was induced by subjecting gerbils to a 2 min of ischemia followed by 1 day of recovery. A significant loss of neurons was observed in the stratum pyramidale (SP) of the hippocampal CA1 region (CA1) in the ischemia-operated-groups 5 days after ischemia-reperfusion (I-R). In all the IPC+ischemia-operated-groups, neurons in the SP were well protected. We found that strong ubiquitin immunoreactivity was detected in the SP in the sham-operated-group and the immunoreactivity was decreased with time after I-R. In all the IPC+ischemia-operated-groups, ubiquitin immunoreactivity in the SP was similar to that in the sham-operated group. Moderate ubiquitin(+1) immunoreactivity was detected in the SP of the sham-operated-group, and the immunoreactivity was markedly increased 2 days after I-R. Five days after I-R, ubiquitin(+1) immunoreactivity was very weak in the SP. In all the IPC+ischemia-operated-groups, ubiquitin(+1) immunoreactivity in the SP was slightly decreased with time after I-R. Western blot analysis showed that, in all the IPC+ischemia-ischemia-groups, the levels of ubiquitin and ubiquitin(+1) proteins were well maintained after I-R. In brief, our findings suggest that the inhibition of the depletion of free ubiquitin and the formation of ubiquitin(+1) may have an essential role in inducing cerebral ischemic tolerance by IPC.

Comparison of the immunoreactivities of NMDA receptors between the young and adult hippocampal CA1 region induced by experimentally transient cerebral ischemia.

Young gerbils are much more resistant to transient cerebral ischemia than the adult. In the present study, we observed that about 90% of CA1 pyramidal cells in the adult hippocampus died 4days post-ischemia; however, about 56% of them in the young hippocampus died at 7days post-ischemia. To compare excitotoxicity between them, we carried out immunoreactivities of NMDA receptor 1 (NMDAR1) and NMDAR2A/B in the hippocampal CA1 region (CA1) induced by 5min of transient cerebral ischemia in the young and adult gerbils. Their immunoreactivities and protein levels in the young sham-group were much lower than those in the adult sham-group. Four days after ischemia-reperfusion, they were significantly decreased in the adult ischemia-group; however, in the young ischemia-group, they were much higher than those in the adult. Seven days after ischemia-reperfusion, NMDAR1 immunoreactivity and its level in the young were much higher than those in the adult; NMDAR2A/B immunoreactivity and its level in the young were lower than in the adult. In brief, the immunoreactivities of NMDARs were not decreased in the ischemic CA1 region of the young 4days after transient cerebral ischemia. This finding indicates that longer maintenance of NMDARs may contribute to less and more delayed neuronal death/damage in the young CA1.

Neuroprotection of ebselen against ischemia/reperfusion injury involves GABA shunt enzymes.

Seleno-organic compound, ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one), is a substrate with radical-scavenging activity. In this study, we observed the neuroprotective effects of ebselen against ischemic damage and on GABA shunt enzymes such as glutamic acid decarboxylase 67 (GAD67), GABA transaminse (GABA-T) and succinic semialdehyde dehydrogenase (SSADH) in the hippocampal CA1 region after 5 min of transient forebrain ischemia in gerbils. For this, vehicle (physiological saline) or ebselen was administered 30 min before or after ischemia/reperfusion and sacrificed 4 days after ischemia/reperfusion. The administration of ebselen significantly reduced the neuronal death in the CA1 region induced by ischemia/reperfusion. In addition, treatment with ebselen markedly elevated GAD67, GABA-T and SSADH immunoreactivity and their protein levels compared to that in the vehicle-treated group, respectively. These results suggest that ebselen protects neurons from ischemic damage via control of the expressions of GABA shunt enzymes to enter the TCA cycle.