Sham Ischemia Research Articles

Time course analysis of changes in neuronal loss, oxidative stress, and excitotoxicity in gerbil hippocampus following ischemia and reperfusion under hyperthermic conditions.

Oxidative stress and excitotoxicity are the major causes of neuronal death/loss in the brain following ischemia and reperfusion (IR). Hyperthermia is known to exacerbate ischemic neuronal damage; however, the underlying mechanisms remain unclear. This study investigated the mechanisms underlying neuronal damage caused by IR injury (IRI) under hyperthermic conditions in the gerbil hippocampal CA1 region. Gerbils were controlled at normothermia (37.5±0.2°C) or hyperthermia (39.5±0.2°C). After temperature control for 30 min, the animals received IRI (following 5 min of transient forebrain ischemia) or sham ischemia, and were subsequently sacrificed at 0, 3, 6, 12, 24, 48, and 120h after IRI. Neuronal death was examined using neuronal nuclear antigen immunohistochemistry and Fluoro-Jade B histofluorescence. Oxidative stress was analyzed by immunohistochemistry for 8-Hydroxy-2'-deoxyguanosine (8OHdG) and superoxide dismutase 2 (SOD2). Excitotoxicity was investigated by immunohistochemistry and western blotting for glutamate transporter 1 (GLT1). Immunohistochemical staining for glial fibrillary acidic proteins (GFAP) was performed to detect reactive astrogliosis. Loss of pyramidal neurons was detected earlier (48h post-IRI) in the hyperthermia-IRI group than in the normothermia-IRI group (120h post-IRI). Further, 8OHdG and SOD2 immunoreactivity in the hyperthermia-IRI group was significantly higher than that in the normothermia-IRI group. Changes in GLT1 immunoreactivity in both groups were biphasic, indicating that the immunoreactivity and protein levels were significantly lower in the hyperthermia-IRI group. GFAP immunoreactivity was enhanced following neuronal loss, indicating that the immunoreactivity was significantly higher in the hyperthermia-IRI group. Taken together, these results suggest that brain IR under hyperthermic conditions can aggravate neuronal damage in the hippocampal CA1 region through severe oxidative stress and excitotoxicity.

Regular transient limb ischemia improves endothelial function and inhibits endothelial cell apoptosis to prevent atherosclerosis in rabbit

AimsRegular transient limb ischemia (RTLI) can prevent atherosclerosis (AS) progression in hypercholesterolemic rabbits. This study aimed to investigate the minimum effective intensity and possible mechanisms of RTLI for preventing atherosclerosis.MethodsEighty rabbits were divided into eight groups: normal (N), high cholesterol (H), three RTLI [three RTLI cycles every other day (R3qod), three RTLI cycles daily (R3qd), and six RTLI cycles daily (R6qd), each cycle of RTLI included 5 min of limb ischemia followed by 5 min limb reperfusion], and three correlated sham RTLI [sham ischemia for 30 min once every other day (S3qod), sham ischemia for 30 min once daily (S3qd), and sham ischemia for 60 min once daily (S6qd)]. Rabbits in group N were kept normally, while the others were fed 1% cholesterol diet for 12 weeks. The RTLI and sham RTLI groups were received RTLI or sham RTLI procedure, respectively. The plaque area in the thoracic aorta was determined by oil red O staining, and quantifying the ratio of plaque area to intimal area (PA/IA). Endothelium-dependent and -independent relaxation were also determined. Endothelial cell were isolated from abdominal aorta of rabbits, and the apoptosis ratio was detected using flow cytometry.ResultsThe PA/IA and early apoptotic cell ratio was significantly lower as well as the endothelium-dependent relaxation response was higher in group R6qd than those in groups H and S6qd, while those in the R3qod group was not significantly different from those in groups H and S3qod, as well as those in the R3qd group showed no significant difference compared to those in groups H and S3qd.ConclusionsSix cycles of RTLI daily was the optimal effective intensity to prevent AS progression in rabbits. Endothelial function improvement and apoptosis inhibition might contribute to the anti-AS effects.

Effects of Cholinergic Receptor Activation and Magnetic Fields on Motor Behavior in Ischemic Gerbils

Introduction: Ischemic stroke stands as a leading global cause of death and disability, prompting the need for animal model experiments in stroke research and the protection of motor function. Recently, magnetic fields have gained significant interest in various biological contexts, showing promise in preserving neurons and reversing behavioral and morphological changes in stroke models. This study explores the potential synergy between static magnetic field and nAChR agonist administration in safeguarding motor behavior in ischemic gerbils. Objective: To determine whether the combined use of a static magnetic field and an agonist for nicotinic acetylcholine receptors (nAChR) can preserve motor behavior in ischemic gerbils.Methods: In this experimental study, 72 Mongolian gerbils were randomly allocated into nine groups (n=8): S, SISM, SINSM, ISM, INP, ISP, INSM, INNP, INSP, distributed according to surgical procedure and treatment. The animals were trained and evaluated on the Rotarod (RR) to assess motor performance.Results: The main finding was the preservation of motor behavior in the Sham Ischemia and Nicotine and Sham Magnetic Stimulation (SINSM) and Ischemia and Nicotine and South Pole Magnetic Field (INSP) groups, as evidenced by the results of the RR test.Conclusions: The findings are consistent with previous literature and provide insight into the mechanism of potentiation, as results showed that adding a nAChR agonist to the magnetic field preserved motor performance in the RR test of ischemic animals.

Effects of different ischemic pressures on bar velocity during the bench press exercise: A randomized crossover trial.

The main objective of this study was to evaluate the effects of different ischemic pressures applied during rest intervals on bar velocity during the bench press exercise. 10 resistance-trained males (age = 23.2 ± 2.7 years; body mass = 83.9 ± 9 kg; body height = 181 ± 5.2 cm; bench press 1 repetition maximum (1RM) = 125 ± 16.4 kg; training experience = 5.4 ± 3.4 years) participated in the study. During 4 experimental sessions, following a randomized crossover design, the subjects performed 5 sets of 3 repetitions of the bench press exercise with a load of 60% 1RM under conditions: with ischemia (50% or 80% of arterial occlusion pressure), with SHAM ischemia (20 mmHg) and without ischemia (control condition). For the ischemic conditions cuffs were applied before each set for 6.5 min and released 30 s before the start of the set as reperfusion (6.5 min ischemia + 0.5 min reperfusion). In the control condition, ischemia was not applied. The two-way repeated measures ANOVA showed no significant condition × set interaction for mean bar velocity (MV; p = 0.17) and peak bar velocity (PV; p = 0.66). There was also no main effect of condition for MV (p = 0.58) and PV (p = 0.61). The results indicate that ischemic or SHAM treatment (6.5 minutes ischemia or SHAM + 30 s reperfusion) does not affect mean and peak bar velocity during the bench press exercise regardless of the applied pressure.

Postischemic Infusion of Apigenin Reduces Seizure Burden in Preterm Fetal Sheep

Seizures are common in preterm newborns and are associated with poor neurodevelopmental outcomes. Current anticonvulsants have poor efficacy, and many have been associated with upregulation of apoptosis in the developing brain. Apigenin, a natural bioactive flavonoid, is a potent inhibitor of hyaluronidase and reduces seizures in adult animal models. However, its impact on perinatal seizures is unclear. In the present study, we examined the effect of apigenin and S3, a synthetic, selective hyaluronidase inhibitor, on seizures after cerebral ischemia in preterm fetal sheep at 0.7 gestation (98–99 days, term ~147 days). Fetuses received sham ischemia (n = 9) or ischemia induced by bilateral carotid occlusion for 25 min. Immediately after ischemia, fetuses received either a continuous infusion of vehicle (0.036% dimethyl sulfoxide, n = 8) or apigenin (50 µM, n = 6). In a pilot study, we also tested infusion of S3 (2 µM, n = 3). Fetuses were monitored continuously for 72 h after ischemia. Infusion of apigenin or S3 were both associated with reduced numbers of animals with seizures, total seizure time, and mean seizure burden. S3 was also associated with a reduction in the total number of seizures over the 72 h recovery period. In animals that developed seizures, apigenin was associated with earlier cessation of seizures. However, apigenin or S3 treatment did not alter recovery of electroencephalographic power or spectral edge frequency. These data support that targeting brain hyaluronidase activity with apigenin or S3 may be an effective strategy to reduce perinatal seizures following ischemia. Further studies are required to determine their effects on neurohistological outcomes.

Remote ischemic preconditioning protects against spinal cord ischemia–reperfusion injury in mice by activating NMDAR/AMPK/PGC-1α/SIRT3 signaling

BackgroundTo study the protective effects of delayed remote ischemic preconditioning (RIPC) against spinal cord ischemia–reperfusion injury (SCIRI) in mice and determine whether SIRT3 is involved in this protection and portrayed its upstream regulatory mechanisms.MethodsIn vivo, WT or SIRT3 global knockout (KO) mice were exposed to right upper and lower limbs RIPC or sham ischemia. After 24 h, the abdominal aorta was clamped for 20 min, then re-perfused for 3 days. The motor function of mice, number of Nissl bodies, apoptotic rate of neurons, and related indexes of oxidative stress in the spinal cord were measured to evaluate for neuroprotective effects. The expression and correlation of SIRT3 and NMDAR were detected by WB and immunofluorescence. In vitro, primary neurons were exacted and OGD/R was performed to simulate SCIRI in vivo. Neuronal damage was assessed by observing neuron morphology, detecting LDH release ratio, and flow cytometry to analyze the apoptosis. MnSOD and CAT enzyme activities, GSH and ROS level were also measured to assess neuronal antioxidant capacity. NMDAR-AMPK-PGC-1α signaling was detected by WB to portray upstream regulatory mechanisms of RIPC regulating SIRT3.ResultsCompared to the SCIRI mice without RIPC, mice with RIPC displayed improved motor function recovery, a reduced neuronal loss, and enhanced antioxidant capacity. To the contrary, the KO mice did not exhibit any effect of RIPC-induced neuroprotection. Similar results were observed in vitro. Further analyses with spinal cord tissues or primary neurons detected enhanced MnSOD and CAT activities, as well as increased GSH level but decreased MDA or ROS production in the RIPC + I/R mice or NMDA + OGD/R neurons. However, these changes were completely inhibited by the absence of SIRT3. Additionally, NMDAR-AMPK-PGC-1α signaling was activated to upregulate SIRT3 levels, which is essential for RIPC-mediated neuroprotection.ConclusionsRIPC enhances spinal cord ischemia tolerance in a SIRT3-dependent manner, and its induced elevated SIRT3 levels are mediated by the NMDAR-AMPK-PGC-1α signaling pathway. Combined therapy targeting SIRT3 is a promising direction for treating SCIRI.

Therapeutic Hypothermia Attenuates Cortical Interneuron Loss after Cerebral Ischemia in Near-Term Fetal Sheep.

Therapeutic hypothermia significantly improves outcomes after neonatal hypoxic-ischemic (HI) encephalopathy but is only partially protective. There is evidence that cortical inhibitory interneuron circuits are particularly vulnerable to HI and that loss of interneurons may be an important contributor to long-term neurological dysfunction in these infants. In the present study, we examined the hypothesis that the duration of hypothermia has differential effects on interneuron survival after HI. Near-term fetal sheep received sham ischemia or cerebral ischemia for 30 min, followed by cerebral hypothermia from 3 h after ischemia end and continued up to 48 h, 72 h, or 120 h recovery. Sheep were euthanized after 7 days for histology. Hypothermia up to 48 h recovery resulted in moderate neuroprotection of glutamate decarboxylase (GAD)+ and parvalbumin+ interneurons but did not improve survival of calbindin+ cells. Hypothermia up to 72 h recovery was associated with significantly increased survival of all three interneuron phenotypes compared with sham controls. By contrast, while hypothermia up to 120 h recovery did not further improve (or impair) GAD+ or parvalbumin+ neuronal survival compared with hypothermia up to 72 h, it was associated with decreased survival of calbindin+ interneurons. Finally, protection of parvalbumin+ and GAD+ interneurons, but not calbindin+ interneurons, with hypothermia was associated with improved recovery of electroencephalographic (EEG) power and frequency by day 7 after HI. The present study demonstrates differential effects of increasing the duration of hypothermia on interneuron survival after HI in near-term fetal sheep. These findings may contribute to the apparent preclinical and clinical lack of benefit of very prolonged hypothermia.

Lack of Neuroprotection with a Single Intravenous Infusion of Human Amnion Epithelial Cells after Severe Hypoxia-Ischemia in Near-Term Fetal Sheep.

Background: Hypoxic–ischemic encephalopathy (HIE) around the time of birth results from loss of oxygen (hypoxia) and blood supply (ischemia). Exogenous infusion of multi-potential cells, including human amnion epithelial cells (hAECs), can reduce hypoxic–ischemic (HI) brain injury. However, there are few data on treatment of severe HI in large animal paradigms at term. The aim of the current study was to determine whether infusion of hAECs early after injury may reduce brain damage after ischemia in near-term fetal sheep. Methods: Chronically instrumented fetal sheep (0.85 gestation) received 30 min of global cerebral ischemia followed by intravenous infusion of hAECs from 2 h after the end of ischemia (ischemia-hAEC, n = 6) or saline (ischemia-vehicle, n = 7). Sham control animals received sham ischemia with vehicle infusion (sham control, n = 8). Results: Ischemia was associated with significant suppression of EEG power and spectral edge frequency until the end of the experiment and a secondary rise in cortical impedance from 24 to 72 h, which were not attenuated by hAEC administration. Ischemia was associated with loss of neurons in the cortex, thalamus, striatum and hippocampus, loss of white matter oligodendrocytes and increased microglial numbers in the white matter, which were not affected by hAEC infusion. Conclusions: A single intravenous administration of hAECs did not reduce electrographic or histological brain damage after 30 min of global cerebral ischemia in near-term fetal sheep.

Effect of ischemic compression performed by family caregivers on myofascial pain syndrome and the care burden of the families of patients: a multicenter open-label randomized comparative study.

Ischemic compression is a manual therapy technique for myofascial pain. This study aimed to verify the effect of ischemic compression performed by family caregivers on myofascial pain syndrome (MPS) in patients and on the family's care burden. This multicenter, open-label, randomized, comparative study included patients with myofascial pain and their family caregivers who were randomized into the following groups: ischemia compression (performed by a family caregiver), sham ischemia compression, or untreated control. The effectiveness and safety of ischemic compression and the burden on family caregivers were evaluated. The primary endpoint was the rate of 50% or more improvement in the patient's mean numerical rating scale pain score in the previous 24 hours, 14 days after starting the intervention. The secondary endpoint was the rate of change in the family caregivers' reaction assessments. A total of 75 patients and caregivers (70 patients with cancer and family caregivers) who received home medical care were enrolled at three facilities. The study completion rate was 94.7%, and there were no adverse events. The rate of 50% or more improvement in the numerical rating scale score was 64.0% in the ischemic compression group, 16.0% in the sham ischemic compression group, and 4.0% in the control group (P<0.001). Caregivers' self-esteem was significantly lower in the ischemic compression and sham ischemic compression groups than in the control group. However, there was no significant difference between the two groups (P=0.370). Ischemic compression for myofascial pain in patients performed by family caregivers can increase the analgesic effect in patients and self-esteem in family caregivers. The University Hospital Medical Information Network Clinical Trials Registry (approval number: UMIN000036605).

Effects of Bone Marrow Mesenchymal Stem Cells on Neurological Function, Transforming Growth Factor Beta 1 and Nogo-A Expression in Stroke Rats

To investigate BMSCs’ effect on neurological function, TGF-β1 and Nogo-A expression in stroke rats. Rats were assigned into sham operation group, ischemia group (MACO rat model) and BMSCs group (BMSCs transplantation) followed by analysis of neurological function, brain pathological changes, cerebral infarction volume, TGF-β1 and Nogo-A level by Western blot. Compared with sham operation group, the score of rats was significantly elevated in ischemic group and decreased in BMSCs group (P &lt;0.05). Compared with sham-operated group, ischemic group showed significantly increased cerebral infarction area (P &lt;0.05) and BMSCs group had a significant decreased water level and brain infarct volume (P &lt; 0.05). Compared with sham-operated group, ischemic group had more edema in the nerve cells with serious vacuole, uneven cytoplasm staining and reduced number of neurons, which were all significantly improved in BMSCs group. Compared to sham group, ischemic group showed significantly reduced TGF-β1 and increased Nogo-A level (P &lt;0.05), which were all reversed in BMSCs group (P &lt;0.05). BMSCs transplantation can significantly improve the nerve function of stroke rats, promote TGF-β1 secretion and inhibit Nogo-A expression.

Changes in Cyclin D1, cdk4, and Their Associated Molecules in Ischemic Pyramidal Neurons in Gerbil Hippocampus after Transient Ischemia and Neuroprotective Effects of Ischemic Preconditioning by Keeping the Molecules in the Ischemic Neurons.

Simple SummaryCyclin D1 and cyclin-dependent kinase 4 (cdk4) is implicated in neuronal death induced by various pathological conditions. Ischemic preconditioning (IPC) confers neuroprotective effect, but underlying mechanisms have been poorly addressed. In this study, IPC protected pyramidal neurons (cells) in gerbil hippocampus after transient ischemia. Additionally, IPC controlled expressions of cyclin D1, cdk4, phosphorylated retinoblastoma (p-Rb), and E2 promoter binding factor 1 (E2F1). In particular, the expression of p16INK4a was not different by IPC. These findings indicate that cyclin D1/cdk4-related signals may play important roles in events in neurons related to damage/death following ischemic insults. Especially, the preservation of p16INK4a by IPC may be crucial in attenuating neuronal death/damage or protecting neurons after brain ischemic insults.Inadequate activation of cell cycle proteins including cyclin D1 and cdk4 is involved in neuronal cell death induced by diverse pathological stresses, including transient global brain ischemia. The neuroprotective effect of ischemic preconditioning is well-established, but the underlying mechanism is still unknown. In this study, we examined changes in cyclin D1, cdk4, and related molecules in cells or neurons located in Cornu Ammonis 1 (CA1) of gerbil hippocampus after transient ischemia for 5 min (ischemia and reperfusion) and investigated the effects of IPC on these molecules after ischemia. Four groups were used in this study as follows: sham group, ischemia group, IPC plus (+) sham group, and IPC+ischemia group. IPC was developed by inducing 2-min ischemia at 24 h before 5-min ischemia (real ischemia). Most pyramidal cells located in CA1 of the ischemia group died five days after ischemia. CA1 pyramidal cells in the IPC+ischemia group were protected. In the ischemia group, the expressions of cyclin D1, cdk4, phosphorylated retinoblastoma (p-Rb), and E2F1 (a transcription factor regulated by p-Rb) were significantly altered in the pyramidal cells with time after ischemia; in the IPC+ischemia group, they were controlled at the level shown in the sham group. In particular, the expression of p16INK4a (an endogenous cdk inhibitor) in the ischemia group was reversely altered in the pyramidal cells; in the IPC+TI group, the expression of p16INK4a was not different from that shown in the sham group. Our current results indicate that cyclin D1/cdk4-related signals may have important roles in events in neurons related to damage/death following ischemia and reperfusion. In particular, the preservation of p16INK4a by IPC may be crucial in attenuating neuronal death/damage or protecting neurons after brain ischemic insults.

Бензиловый эфир креатина эффективен для профилактики и лечения неврологических и когнитивных нарушений при фокальной ишемии головного мозга у крыс

Введение. Ишемический инсульт головного мозга является одной из главных причин преждевременных смертей во всем мире, вызванные им когнитивные и функциональные нарушения ведут к инвалидизации. Для клинического использования в настоящее время одобрено лишь одно средство - рекомбинантный активатор плазминогена, который обеспечивает восстановление (реканализацию) мозгового кровотока. Средства эффективной нейропротекции, обеспечивающие непосредственную защиту нейронов от ишемии (предотвращение апоптоза, сохранение функциональной активности), несмотря на огромное число исследований, по-прежнему не найдены. Поиск новых подходов к профилактике и лечению острых нарушений мозгового кровообращения по-прежнему остается одной из наиболее актуальных проблем современной медицины. Цель исследования - изучение влияния бензилового эфира креатина, нового синтетического соединения, обладающего способностью проникать через гематоэнцефалический барьер и осуществлять нейропротекторное действие на неврологические и когнитивные нарушения, вызванные фокальной ишемией головного мозга у крыс линии Спрэг-Доули. Методика. Фокальную ишемию головного мозга (ФИМ) индуцировали путем хронической окклюзии левой средней мозговой артерии (СМА) в соответствии с модификацией методики A. Tamura и соавт., 1981. Использовали комбинированный наркоз: Золетил/Домитор (внутрибрюшинно (в/б) по 50 мг/кг и 0.2 мг/кг, соответственно), температуру тела поддерживали постоянной на уровне 38±0.5 oC. Окклюзию СМА производили путем биполярной высокочастотной электрокоагуляции (аппарат ЭХВЧ-25-11-С, «Медия», Россия) наиболее проксимального участка артерии, начиная с 0.5 мм от места ее отхождения от Виллизиева круга и на протяжении 2-3 мм. Процедура имитации ишемии (ложная операция) была аналогична вышеизложенной, но без электрокоагуляции СМА. Оценку степени неврологических нарушений проводили с использованием неврологической шкалы Гарсии и модифицированной шкалы тяжести неврологических нарушений (ОТНН) последовательно за 1 сут до и через 1, 3 и 7 сут после моделирования ФИМ. Для выявления когнитивных нарушений использовали водный лабиринт Морриса. Стандартный тест исследования процессов обучения и памяти применяли в версии для оценки пространственной референтной памяти, заключающейся в поиске животным скрытой под водой платформы при ориентации только по приметам окружающей обстановки. Бензиловый эфир креатина вводили внутрибрюшинно трижды - за 3, 2 и 1 ч до фокальной ишемии при профилактическом применении, либо через 1, 2 и 3 ч после моделирования ишемии при лечебном применении. Результаты. Получены приоритетные данные о том, что бензиловый эфир креатина при профилактическом введении эффективно предупреждает развитие неврологических нарушений и ослабляет когнитивные нарушения после фокальной ишемии мозга; при лечебном применении препарат ослабляет тяжесть неврологических нарушений и снижает выраженность когнитивных расстройств. Заключение. Результаты исследования позволяют рекомендовать данное производное креатина (после оценки возможной токсичности) для проведения первой фазы клинических испытаний в качестве препарата противоишемического действия. Background. Ischemic stroke is one of the main causes for premature death worldwide. Cognitive and functional disorders induced by stroke result in disability. Currently, only one agent has been approved for the clinical use, recombinant plasminogen activator, which provides recovery (recanalization) of cerebral blood flow. Despite numerous studies, an effective treatment to protect directly neurons from ischemia (preventing apoptosis, maintaining functional activity) has not been found so far. Thus, search for new approaches to prevention and treatment of acute cerebrovascular accidents remains one of the most pressing challenges of modern medicine. The aim of this work was to study the effect of creatine benzyl ester, a new synthetic compound capable for penetrating the blood-brain barrier to provide neuroprotection, on neurological and cognitive disorders induced by focal cerebral ischemia in Sprague-Dawley rats. Methods. Focal cerebral ischemia was induced by chronic occlusion of the left middle cerebral artery (MCA) by a A. Tamura et al. (1981) modified method. Combined intraperitoneal (i.p.) anesthesia with Zoletil/Domitor (50 mg/kg and 0.2 mg/kg, respectively) was used. Body temperature was maintained at 38±0.5 oC. MCA occlusion was performed by bipolar high-frequency electrocoagulation (EKVCh-25-11-C, Media, Russia) of the most proximal part of the artery, from 0.5 mm from the place where it originates from the circle of Willis and over 2-3 mm. The sham ischemia procedure was similar to that described above but without MCA electrocoagulation. Severity of neurological disorders was assessed with the Garcia neurological scale and a modified scale for severity of neurological disorders (OTTN) one day before and at 1, 3, and 7 days following ischemia. Cognitive impairment was assessed with the Morris water maze, a standard test for learning and memory. This test was used in a version for studying spatial reference memory, which consists of finding a platform hidden under the water by the animal guided only by surrounding marks. Creatine benzyl ester was injected i.p. three times, 3, 2 and 1 h before focal ischemia for prevention or 1, 2 and 3 h after ischemia for treatment. Results. The study produced priority data showing that preventive administration of creatine benzyl ester effectively abolished the development of neurological disorders and alleviated the cognitive impairment after focal cerebral ischemia whereas the therapeutic treatment restricted the severity of neurological and cognitive disorders. Conclusion. The results of the study supported recommendation of this creatine derivative (after assessing potential toxicity) for the first phase of clinical trials as an anti-ischemic drug.

Connexin Hemichannel Mimetic Peptide Attenuates Cortical Interneuron Loss and Perineuronal Net Disruption Following Cerebral Ischemia in Near-Term Fetal Sheep.

Perinatal hypoxia-ischemia is associated with disruption of cortical gamma-aminobutyric acid (GABA)ergic interneurons and their surrounding perineuronal nets, which may contribute to persisting neurological deficits. Blockade of connexin43 hemichannels using a mimetic peptide can alleviate seizures and injury after hypoxia-ischemia. In this study, we tested the hypothesis that connexin43 hemichannel blockade improves the integrity of cortical interneurons and perineuronal nets. Term-equivalent fetal sheep received 30 min of bilateral carotid artery occlusion, recovery for 90 min, followed by a 25-h intracerebroventricular infusion of vehicle or a mimetic peptide that blocks connexin hemichannels or by a sham ischemia + vehicle infusion. Brain tissues were stained for interneuronal markers or perineuronal nets. Cerebral ischemia was associated with loss of cortical interneurons and perineuronal nets. The mimetic peptide infusion reduced loss of glutamic acid decarboxylase-, calretinin-, and parvalbumin-expressing interneurons and perineuronal nets. The interneuron and perineuronal net densities were negatively correlated with total seizure burden after ischemia. These data suggest that the opening of connexin43 hemichannels after perinatal hypoxia-ischemia causes loss of cortical interneurons and perineuronal nets and that this exacerbates seizures. Connexin43 hemichannel blockade may be an effective strategy to attenuate seizures and may improve long-term neurological outcomes after perinatal hypoxia-ischemia.

Propofol improves intestinal ischemia-reperfusion injury in rats through NF-κB pathway.

The aim of this study was to investigate the effects of propofol on intestinal ischemia-reperfusion injury in rats through the nuclear factor-kappa B (NF-κB) pathway. A total of 24 Sprague-Dawley rats were selected and randomly divided into three groups, including sham operation group, ischemia group and propofol group. Rats in sham operation group were only treated with isolation of superior mesenteric artery, which was clipped for 1 h and reperfused for 2 h in ischemia group. Meanwhile, propofol (60 mg/kg) was injected into the femoral vein 1 h before ischemia in propofol group. TUNEL assay was performed to detect cell apoptosis of intestinal tissues. Real-time quantitative polymerase chain reaction (RT-qPCR) was conducted to measure the expression levels of malondialdehyde (MDA), superoxide dismutase (SOD), myeloperoxidase (MPO), caspase-3 and B-cell lymphoma-2 (Bcl-2) in rats of each group. Western blotting was utilized to detect the protein expression levels of NF-κB pathway related molecules, such as myeloid differential protein-88 (MyD88), v-rel avian reticuloendotheliosis viral oncogene homolog A (RelA) and NF-κB. Furthermore, changes in plasma cytokine levels were determined via enzyme-linked immunosorbent assay (ELISA). The number of apoptotic cells in ischemia group was remarkably higher than that in sham operation group (p<0.05). However, it decreased notably in propofol group compared with ischemia group (p<0.05). In comparison with sham operation group, significantly up-regulated expression of caspase-3 and down-regulated expression of Bcl-2 were observed in the intestinal tissues of rats in ischemia group (p<0.05). Caspase-3 was lowly expressed, while Bcl-2 was highly expressed in the intestinal tissues of rats in propofol group compared with ischemia group (p<0.05). In addition, no statistically significant differences were observed in the expression level of SOD among sham operation group, ischemia group and propofol group (p>0.05). The expression levels of MDA and MPO were overtly higher in the intestinal tissues of rats in ischemia group than those in sham operation group and propofol group (p<0.05). Besides, the protein expression levels of MyD88, RelA and NF-κB in the intestinal tissues of rats in ischemia group were remarkably higher than those in sham operation group and propofol group (p<0.05). The activity of the NF-κB pathway in the intestinal tissues of rats in propofol group significantly declined compared with ischemia group (p<0.05). Moreover, compared with sham operation group, plasma levels of TNF-α, interleukin (IL)-2, IL-6 and IL-4 increased significantly in rats of ischemia group (p<0.05). However, they were markedly lower in propofol group than those in ischemia group (p<0.05). Propofol protects rats from intestinal ischemia-reperfusion injury through the NF-κB pathway.

Combination therapy of the granulocyte colony stimulating factor and intravenous lipid emulsion protect the hippocampus after global ischemia in rat: focusing on CA1 region

Brain stroke is one of the causes of human death and disability worldwide. Global ischemia results in the accumulation of free radicals in the neurons. It leads to histologically brain damage. The CA1 region of the hippocampus is a sensitive area for free radicals. This study investigated the combined therapy of the Granulocyte colony stimulating factor (G-CSF) and the Intravenous lipid emulsion (ILE). These neuroprotective agents play a role in the regeneration of neurons. They improve the learning ability and memory in rats induced global ischemia. We divided 35 rats into five groups. The groups were sham group, ischemia group, G-CSF group, ILE group, and G-CSF plus ILE group. Ischemia was induced by occlusion of the bilateral common carotid about 10min. The drugs applied on days 1, 3 and 7. The treated groups received subcutaneous injection of 20μg/kgG-CSF and intravenous injection of 5ml/kg ILE. After two weeks, the memory and learning ability of the rats was evaluated by the shuttle box. Hematoxylin and Eosin and Nissl and TUNEL stainings were used to determine the necrosis, normal and apoptotic cells. The combined therapy increased normal cells compared to the ischemia group. They decreased the number of necrotic and apoptosis cells in other groups. The combined group improved the passive avoidance test compared to the other groups. The combination therapy of G-CSF plus ILE is more effective than each alone.

Absence of renal hypoxia in the subacute phase of severe renal ischemia-reperfusion injury.

Tissue hypoxia has been proposed as an important event in renal ischemia-reperfusion injury (IRI), particularly during the period of ischemia and in the immediate hours following reperfusion. However, little is known about renal oxygenation during the subacute phase of IRI. We employed four different methods to assess the temporal and spatial changes in tissue oxygenation during the subacute phase (24 h and 5 days after reperfusion) of a severe form of renal IRI in rats. We hypothesized that the kidney is hypoxic 24 h and 5 days after an hour of bilateral renal ischemia, driven by a disturbed balance between renal oxygen delivery (Do2) and oxygen consumption (V̇o2). Renal Do2 was not significantly reduced in the subacute phase of IRI. In contrast, renal V̇o2 was 55% less 24 h after reperfusion and 49% less 5 days after reperfusion than after sham ischemia. Inner medullary tissue Po2, measured by radiotelemetry, was 25 ± 12% (mean ± SE) greater 24 h after ischemia than after sham ischemia. By 5 days after reperfusion, tissue Po2 was similar to that in rats subjected to sham ischemia. Tissue Po2 measured by Clark electrode was consistently greater 24 h, but not 5 days, after ischemia than after sham ischemia. Cellular hypoxia, assessed by pimonidazole adduct immunohistochemistry, was largely absent at both time points, and tissue levels of hypoxia-inducible factors were downregulated following renal ischemia. Thus, in this model of severe IRI, tissue hypoxia does not appear to be an obligatory event during the subacute phase, likely because of the markedly reduced oxygen consumption.

U0126 protects hippocampal CA1 neurons against forebrain ischemia-induced apoptosis via the ERK1/2 signaling pathway and NMDA receptors

Objective:Cerebral ischemia can trigger the ERK1/2 signaling cascade that enables the brain to adapt to ischemic injury. However, the mechanism of ERK1/2 in ischemic brain injury remains unclear. The aim of this study was to examine the roles of the ERK1/2 signaling pathway and NMDA receptors in the apoptosis of CA1 pyramidal neurons after ischemia/reperfusion (I/R).Methods:Male Wistar rats were subjected to a sham or transient forebrain ischemia procedure. Animals received the intracerebroventricular injection of U0126 (5 μl, 0.2 μg/μl) or vehicle 30 min before ischemia. Homogenates of the hippocampal CA1 field were obtained from sham-operated and ischemic rats 6, 12 or 48 h after ischemia/reperfusion (n = 6 per group) and then subjected to Western blotting analysis and TUNEL staining. Caspase-3 activity was assayed with a colorimetric assay kit.Results:We found that the phosphorylation level of ERK1/2 is increased in the CA1 region following transient I/R. Blocking the ERK1/2 signaling pathway by administration U0126 attenuated apoptotic neuronal cell death via inhibition of NMDA receptors.Conclusion:These findings suggest a novel mechanism by which the ERK1/2 signaling pathway affects the post-I/R apoptosis of CA1 pyramidal neurons, which will provide a therapeutic target for the treatment of stroke.

Stat5-dependent cardioprotection in late remote ischaemia preconditioning.

To study the protective effects of late remote ischaemic preconditioning (RIPC) against myocardial ischaemia/reperfusion (I/R) injury and determine whether Stat5 is involved in this protection by using cardiomyocyte-specific Stat5 knockout mice (Stat5-cKO). Mice were exposed to lower limb RIPC or sham ischaemia. After 24 h, the left anterior descending artery (LAD) was ligated for 30 min, then reperfused for 180 min. The myocardial infarct size (IS), apoptotic rate of cardiomyocytes, and serum myocardial enzymes were measured to evaluate for cardioprotective effects. Heart tissues were harvested to determine the cardiomyocytes' anti-apoptotic and survival signaling. When compared with the Stat5fl/fl mice without RIPC, Stat5fl/fl mice with RIPC (Stat5fl/fl+RIPC + I/R) displayed a decreased myocardial IS/LV (16 ± 1.5 vs. 30.1 ± 3.1%, P < 0.01; IS/ area at risk (AAR), 42.2 ± 3.5 vs. 69.2 ± 4.9%, P < 0.01), a reduced cardiomyocyte apoptotic rate (2.1 ± 0.37 vs. 5.5 ± 0.53%, P < 0.01), and lower creatine kinase (CK), lactate dehydrogenase (LDH), and creatine kinase-MB (CK-MB) levels. To the contrary, the Stat5-cKO mice (Stat5fl/fl; Tnnt2Cremice with Doxycycline treatment for 7 days) did not exhibit any effect of RIPC-induced cardioprotection. Activation of STAT5 protein was significantly higher in the Stat5fl/fl+RIPC + I/R group than in the Stat5fl/fl+I/R group, while there was no significant difference between the Stat5-cKO + RIPC + I/R and the Stat5-cKO + I/R group. Further analyses with heart tissues detected decreased protein expressions of cytochrome c (Cyt c) and cleaved Caspase-3 in the Stat5fl/fl+RIPC + I/R mice, along with increased anti-apoptotic molecules, including B-cell lymphoma-extra large (Bcl-xL) and B-cell lymphoma-2 (Bcl-2); such changes were not noted in the Stat5-cKO + RIPC + I/R mice. Additionally, RIPC increased cardiac hypoxia inducible factor-1 (HIF-1α) and interleukin-10 (IL10) protein levels and caused activation of AKT, phosphatidylinositol 3 kinase (PI3K), and vascular endothelial growth factor in the heart of the Stat5fl/fl mice. However, these changes were completely inhibited by the absence of Stat5. These results suggest that RIPC-induced late cardioprotection against myocardial I/R injury is Stat5-dependent and is correlated with the activation of anti-apoptotic signaling and cardiomyocyte-survival signaling.

Connexin hemichannel blockade improves survival of striatal GABA-ergic neurons after global cerebral ischaemia in term-equivalent fetal sheep

Basal ganglia injury at term remains a major cause of disability, such as cerebral palsy. In this study we tested the hypotheses that blockade of astrocytic connexin hemichannels with a mimetic peptide would improve survival of striatal phenotypic neurons after global cerebral ischaemia in term-equivalent fetal sheep, and that neuronal survival would be associated with electrophysiological recovery. Fetal sheep (0.85 gestation) were randomly assigned to receive a short or long (1 or 25 h) intracerebroventricular infusion of a mimetic peptide or vehicle, starting 90 minutes after 30 minutes of cerebral ischaemia. Sheep were killed 7 days after ischaemia. Cerebral ischaemia was associated with reduced numbers of calbindin-28k, calretinin, parvalbumin and GAD positive striatal neurons (P < 0.05 ischaemia + vehicle, n = 6 vs. sham ischaemia, n = 6) but not ChAT or nNOS positive neurons. Short infusion of peptide (n = 6) did not significantly improve survival of any striatal phenotype. Long infusion of peptide (n = 6) was associated with increased survival of calbindin-28k, calretinin, parvalbumin and GAD positive neurons (P < 0.05 vs. ischaemia + vehicle). Neurophysiological recovery was associated with improved survival of calbindin-28k, calretinin and parvalbumin positive striatal neurons (P < 0.05 for all). In conclusion, connexin hemichannel blockade after cerebral ischaemia in term-equivalent fetal sheep improves survival of striatal GABA-ergic neurons.

How long is sufficient for optimal neuroprotection with cerebral cooling after ischemia in fetal sheep?

The optimal duration of mild “therapeutic” hypothermia for neonates with hypoxic-ischemic encephalopathy is surprisingly unclear. This study assessed the relative efficacy of cooling for 48 h versus 72 h. Fetal sheep (0.85 gestation) received sham ischemia (n = 9) or 30 min global cerebral ischemia followed by normothermia (n = 8) or delayed hypothermia from 3 h to 48 h (n = 8) or 72 h (n = 8). Ischemia was associated with profound loss of electroencephalogram (EEG) power, neurons in the cortex and hippocampus, and oligodendrocytes and myelin basic protein expression in the white matter, with increased Iba-1-positive microglia and proliferation. Hypothermia for 48 h was associated with improved outcomes compared to normothermia, but a progressive deterioration of EEG power after rewarming compared to 72 h of hypothermia, with impaired neuronal survival and myelin basic protein, and more microglia in the white matter and cortex. These findings show that head cooling for 48 h is partially neuroprotective, but is inferior to cooling for 72 h after cerebral ischemia in fetal sheep. The close association between rewarming at 48 h, subsequent deterioration in EEG power and increased cortical inflammation strongly suggests that deleterious inflammation can be reactivated by premature rewarming.