AIMTo explore the effects of limb ischemic preconditioning (LIP) on cerebral ischemia/reperfusion injuries.

METHODSThirty six wistar rats, of which bilateral vertebral arteries were occluded permanently, were randomly divided into the following 6 groups: control group, cerebral ischemic group, limb ischemic group, LIP 0 d group (cerebral ischemia was given immediately after LIP), LIP 1 d group (cerebral ischemia was given 1 d after LIP) and LIP 2 d group (cerebral ischemia was given 2 d after LIP). Global cerebral ischemia was performed by four vessels occlusion in rats. LIP was performed by occluding the bilateral femoral arteries for 10 min 3 times in a interval of 10 min. The histological grade and pyramidal neuronal density in the CA1 hippocampus were measured to quantitate the degree of hippocampal injury under thionin staining.

RESULTSThe histological grade was increased and the pyramidal neuronal density was decreased in the CA1 hippocampus of the cerebral ischemic group (P < 0.01). The damage of the CA1 hippocampus in LIP 0 d group was significantly diminished, which represented by decreased histological grade and increased neuronal density compared with the cerebral ischemic group (P < 0.01). But the CA1 hippocampus still showed obvious injuries in the LIP 1 d and LIP 2 d group.

CONCLUSIONLIP performed immediately prior to cerebral ischemia could confer obvious protective effects on CA1 hippocampus against cerebral ischemia/reperfusion injuries. But LIP performed 1 d and 2 d prior to cerebral ischemia could not afford the protection against injuries induced by cerebral ischemia/reperfusion.

Animals ; Brain Ischemia ; prevention & control ; Extremities ; blood supply ; Hippocampus ; blood supply ; Ischemic Preconditioning ; methods ; Rats ; Rats, Wistar ; Reperfusion Injury ; prevention & control

OBJECTIVETo investigate the expression of vascular endothelial growth factor (VEGF) and microvessel density (MVD) in hippocampus of rats with aging.

METHODSParaffin sections of brain tissue of rats at the age of 3, 18, 24, 30 months were stained by immunohistochemistry, the expression of VEGF and MVD was quantitatively analyzed.

RESULTSInnunohistochemical staining showed that the VEGF-positive cells were mainly pyramidal neuron in hippocampus; the intensity of VEGF-positivity in neuron cells was decreased with the aging (P<0.05). The MVD in hippocampus was also decreased with the aging of rats (P<0.05).

CONCLUSIONIncreasing VEGF contents and improving blood circulation in brain tissue may prevent or treat vascular dementia and cerebrovascular diseases.

Aging ; Animals ; Capillaries ; pathology ; Hippocampus ; blood supply ; metabolism ; Male ; Rats ; Rats, Sprague-Dawley ; Vascular Endothelial Growth Factor A ; genetics ; metabolism

AIMTo study the effect of different intervals between occlusions of vertebral arteries and bilateral common carotid arteries on the Pulsinelli 4-vessel occlusion global cerebral ischemic model, and the features of ischemia of the brainstem and hippocampus induced by occulusion of bilateral common carotid arteries under the condition of occlusion of unilateral vertebral artery.

METHODSEighty four adult male Wistar rats were divided into 4 groups randomly: control group, bilateral vertebral artery occluding group, global brain ischemic insult group, and unilateral vertebral artery occluding plus bilateral common carotid arteries occluding group. In the global brain ischemic insult group, rats were further divided into 24 h, 48 h, and 72 h interval subgroups according to the interval between the occlusion of the vertebral arteries and bilateral common carotid arteries. The responses including enlarging of pupils and the light reflex during the brain ischemia were observed. The duration of right reflex disappearing, the general state, and the delayed neuronal death (DND) of pyramidal neurons in the CA1 hippocampus of the rats after the brain ischemia were also observed.

RESULTSAmong the global brain ischemic insult group, both the responses and DND were more severe in 72 h interval subgroup than those in 24 h and 48 h interval subgroups. There was no significant difference between 24 h and 48 h interval subgroups. When the bilateral common carotid arteries were occluded under the condition of occlusion of unilateral vertebral artery, severe DND was observed in the CA1 hippocampus ipsilateral to the occluding vertebral artery, but no significant DND was observed in the contralateral CA1 hippocampus.

CONCLUSIONThe results suggested that the prior occlusion of the bilateral vertebral arteries during producing Pulsinelli 4-vessel occlusion global cerebral ischemic model might be a cerebral ischemic preconditioning that could protect to some extent pyramidal neurons of the hippocampus against severe ischemic insult induced by occlusion of bilateral common carotid arteries within 48 h. Moreover, There is ipsilateral predominance of blood perfusion from one side of vertebral artery to the brainstem and hippocampus, although there was Willis artery circle in rats.

Animals ; Brain Ischemia ; prevention & control ; Hippocampus ; blood supply ; Ischemic Preconditioning ; methods ; Male ; Rats ; Rats, Wistar ; Vertebral Artery ; pathology

OBJECTIVETo investigate morphological changes of capillary in aging brain and explore the role of vascular factor in brain aging.

METHODSTwenty-eight brains of individuals (mean age 65 years) who died without clinical or pathological involvement of nervous system and 6 brains of Alzheimer's disease (AD) patients (mean age 83 years) were obtained at autopsy. Sections from frontal lobe, occipital lobe, striatum and hippocampus of normal subjects and sections from hippocampus of AD patients were used for hematoxylin eosin (HE), lox fast blue (LFB), toluidine blue stains and ulex europaeus agglutinin (UEA) immunostaining. After observations of morphological changes of neuron and capillary, computer-aid image analysis was performed to quantify numerical density and area density of neuron and capillary in frontal lobe, occipital lobe, putamen, CA3 sector of normal subjects and CA3 sector of AD patients. Numerical ratio and area ratio of neuron and capillary were then calculated. Correlations between neuron/capillary ratio and age were estimated using Pearson's correlation test. Difference of neuron/capillary ratio in CA3 sectors between AD patients and advanced aged normal subjects (> 75 years) was analyzed with Student's t-test.

RESULTSSeveral pathological microvascular changes, including increased tortuosity, looping, bundling, stringing, and effacement of endothelia were seen in aged subjects and more prevalent in AD patients. Numerical ratio and area ratio of neuron and capillary of frontal lobe, occipital lobe and putamen significantly increased with age in normal aging subjects.

CONCLUSIONSMorphological changes and relative decrease in number and capacity of capillary in aging brain may reduce cerebral blood flow and metabolism, and consequently result in functional impairment of aging brain. Vascular factors may play an important role in the development of brain aging.

Adult ; Aged ; Aged, 80 and over ; Aging ; Alzheimer Disease ; etiology ; pathology ; Capillaries ; anatomy & histology ; pathology ; Cell Count ; Cerebral Cortex ; blood supply ; pathology ; Female ; Frontal Lobe ; blood supply ; pathology ; Hippocampus ; blood supply ; pathology ; Humans ; Image Processing, Computer-Assisted ; Male ; Middle Aged ; Neurons ; pathology ; Occipital Lobe ; blood supply ; pathology

To investigate the effects of time interval and cumulative dosage of repetitive mild cellular hypoxia on shape of neurodegeneration and neuroprotection in mice, population spike amplitude (PSA) was measured during hypoxia and posthypoxic recovery in hippocampal slices from untreated control and mice pretreated in vivo with a single or repeatedly intraperitoneal injection of 3-nitropropionate (3-NP). Posthypoxic recovery of PSA was dose-dependent in single pretreated slices, with maximal recovery on pretreatment attained with 20 mg/kg 3-NP (82 +/- 32%, P < 0.01). Upon 5 and 9 treatments with 20 mg/kg 3-NP (dosage interval 3 days), PSA recovered to (38 +/- 9)% with the difference being not significant vs control group and (72 +/- 45)% with the difference being significant (P < 0.05 to control, P < 0.05 to 5 treatments), respectively. In contrast, with 2 days time interval, recovery after 5 and 9 treatments was (30 +/- 25)% and (16 +/- 14)%, respectively (without significant difference from control). Continued neuroprotection was also observed upon increase of dosage interval to 4 and 5 days. It was suggested that repetitive chemical hypoxia is a model for neurodegenerative disease and continued neuroprotection depending on time interval between repetitive hypoxic episodes rather than cumulative dosage. At appropriate time intervals increased neuronal hypoxic tolerance could be induced with number of hypoxic episodes.
Adaptation, Physiological ; Animals ; Cell Hypoxia ; Disease Models, Animal ; Hippocampus ; blood supply ; cytology ; Huntington Disease ; physiopathology ; Ischemic Preconditioning ; Male ; Mice ; Neuronal Plasticity ; physiology ; Nitro Compounds ; Propionates ; Time Factors

Cerebral hypoxic preconditioning (CHP), which was induced by repetitive sub-lethal hypoxic insult, is an endogenous protection of neuron against subsequent severe hypoxic injury. Although a number of possible induction pathways have been investigated, such as neuroactive cytokines, activation of glutamate receptors, the ATP-sensitive potassium channel, nitric oxide and oxidative stress, the exact mechanism underlying CHP-induced protection remains unclear. It is interesting that all the above-mentioned mechanisms are involved in the activation of protein kinases C (PKC). Recently we reported that the level of PKCs membrane translocation was significantly increased in the brain of hypoxic preconditioned mice. In order to explore the role of conventional protein kinases C (cPKC) in the development of cerebral hypoxic preconditioning, biochemical techniques of SDS-PAGE and Western bolt were applied to observe the effects of repetitive hypoxic exposure (H1-H4) on the level of cPKCalpha and gamma membrane translocation in the cortex and hippocampus of mice. Experiments were carried out in accordance with the National Institutes of Health guide for the care and use of laboratory animals. The hypoxic preconditioned mice model was adapted with minor modification from our previous report. In brief, healthy adult BALB/C mice weighing 18-20 g of either sex were randomly divided into 5 groups: control group (H0), hypoxic control group (H1, hypoxic exposure once ), hypoxic preconditioned group (H2-H4, repetitive hypoxic exposure for 2-4 times respectively). The first sign of gasping breath was taken as the end of each hypoxic exposure, and then the mice were kept in normal control condition for a 30-min interval to recover before the following hypoxic insult. We found that the level of cPKCgamma membrane translocation was increased significantly (*P<0.05, n=6) with the increase of the hypoxic exposure times in both hippocampus (H0: 100% vs H1 approximately H4: 119.2%+/-7.0% *, 139.3% +/-7.4%*, 134.2% +/-8.95%*, 184.0% +/-10.8%*) and cortex (H0: 100% vs H1-H4: 129.7% +/-13.8%, 143.3% +/-13.9%*, 204.0% +/-12.1%*, 229.5% +/-14.6%*) of mice. But there were no significant changes in cPKCalpha membrane translocation in cortex and hippocampi of hypoxic preconditioned mice. These results suggest that cPKCgamma plays an important role in the development of cerebral hypoxic preconditioning. The changes in some other forms of novel and atypical PKCs are still under investigation.
Animals ; Brain ; blood supply ; metabolism ; physiology ; Cell Membrane ; metabolism ; Female ; Hippocampus ; metabolism ; Ischemic Preconditioning ; Male ; Mice ; Mice, Inbred BALB C ; Protein Kinase C ; metabolism ; Protein Transport

The purpose of this study was to investigate the effects of limb ischemic preconditioning (LIP) on apoptosis of pyramidal neurons in the CA1 hippocampus induced by global cerebral ischemia-reperfusion in rats. Forty-six rats whose bilateral vertebral arteries were occluded permanently were assigned to one of four groups: sham group, limb ischemia group, cerebral ischemia group and LIP group. LIP was performed by occluding the bilateral femoral arteries for 10 min 3 times in an interval of 10 min. Global cerebral ischemia was underwent by occluding the bilateral common carotid arteries for 8 min immediately after LIP. Assays for apoptosis of the hippocampal neurons were biologically and morphologically performed using gel electrophoresis, TUNEL and AO/EB staining. Characteristic DNA ladder was clearly visualized with gel electrophoresis in the hippocampus in cerebral ischemia group, but not in LIP group. The number of TUNEL-positive cells in the CA1 hippocampus was significantly reduced by LIP from 69.8+/-12 (cerebral ischemia group) to 17.8+/-5.8 (P<0.01). AO/EB staining also showed a reduction of apoptosis in LIP group compared with cerebral ischemia group. These results suggest that LIP can inhibit hippocampal neuronal apoptosis induced by cerebral ischemia-reperfusion, which contributes to the protection against the delayed neuronal death induced by cerebral ischemic insult.
Animals ; Apoptosis ; physiology ; Brain Ischemia ; physiopathology ; Hippocampus ; pathology ; Ischemic Preconditioning ; methods ; Lower Extremity ; blood supply ; Male ; Neurons ; pathology ; Pyramidal Cells ; pathology ; Rats ; Rats, Wistar ; Reperfusion Injury ; prevention & control

Under global cerebral ischemia, the effect of different brain temperature on cerebral ischemic injury was studied. Male Sprague-Dawley rats were divided into normothermic (37-38°C) ischemia, mild hypothermic (31-32°C) ischemia, hyperthermic (41-42°C) ischemia and sham-operated groups. Global cerebral ischemia was established using the Pulsinelli four-vessel occlusion model and brain temperature was maintained at defined level for 60 min after 20-min ischemia. The expression of c-fos protein and the levels of malondialdehyde (MDA) and lactate in brain regions were detected by immunochemistry and spectrophotometrical methods, respectively. C-fos positive neurons were found in the hippocampus and cerebral cortex after cerebral ischemia reperfusion. Mild hypothermia increased the expression of c-fos protein in both areas, whereas hyperthermia decreased the expression of c-fos protein in the hippocampus at 24 h reperfusion, and the cerebral cortex at 48 h reperfusion when compared to normothermic conditions. In normothermic, mild hypothermic and hyperthermic ischemia groups, the levels of MDA and lactate in brain tissue were increased at 24, 48 and 72 h reperfusion following 20-min ischemia as compared with the sham-operated group (P<0.01). The levels of MDA and lactate in mild hypothermic group were significantly lower than those in normothermic group (P<0.01). It is suggested that brain temperature influences the translation of the immunoreactive protein product of c-fos after global cerebral ischemia, and MDA and lactate are also affected by hypothermia and hyperthermia.
Animals ; Body Temperature ; Brain ; blood supply ; metabolism ; physiopathology ; Brain Ischemia ; metabolism ; physiopathology ; Cerebral Cortex ; blood supply ; metabolism ; physiopathology ; Hippocampus ; blood supply ; metabolism ; physiopathology ; Immunochemistry ; Lactic Acid ; metabolism ; Male ; Malondialdehyde ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Proto-Oncogene Proteins c-fos ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; metabolism ; physiopathology ; Spectrophotometry ; Temperature ; Time Factors ; Tumor Suppressor Protein p53 ; metabolism

OBJECTIVETo investigate the effect of acupuncture serum on Ca2+ content in the cultured nervous cells of hippocampus after ischemia-reperfusion, so as to probe into humoral factors in acupuncture treatment.

METHODSThe neurons of the hippocampus from the new born rats were cultured for 9-11 days. Fluorescein-molecular probe Fluo-3 AM was used for staining of intracellular Ca2+. Fluorescent levels in the nervous cells cultured with the serum of the normal rats or the rats given electroacupuncture at "Baihui" (GV 20), "Zusanli" (ST 36), "Quchi" (LI 11) and "Sanyinjiao" (SP 6) for 2 weeks were determined by using a laser confocal microscope.

RESULTSAfter the normal serum was added, the intracellular Ca2+ fluorescent levels increased to 697 +/- 113 from 461 +/- 96, while after acupuncture serum was added, the Ca2+ fluorescent levels decreased to 584 +/- 103 from 673 +/- 108, indicating that after addition of acupuncture serum, the increased intracellular Ca2+ content could be decreased.

CONCLUSIONThere are some active substances in acupuncture serum which can obviously decrease intracellular Ca2+ content after ischemia-reperfusion, so as to provide a direct evidence for role of humoral factor in acupuncture treatment.

Acupuncture Therapy ; Animals ; Blood Proteins ; pharmacology ; Brain Ischemia ; metabolism ; therapy ; Calcium ; metabolism ; Cells, Cultured ; Hippocampus ; blood supply ; drug effects ; metabolism ; Humans ; Neurons ; drug effects ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; metabolism ; prevention & control ; therapy ; Serum ; chemistry

OBJECTIVEThere was consanguineous relationship between caspase-3 and early damage after hypoxia and ischemia. Caspase-3 plays a key role in the process of apoptosis in neuron. Magnesium sulfate could protect neuron from injuries, but the mechanism was not clear. The study was to investigate the expression of caspase-3 mRNA in the hippocampus of seven-day-old hypoxic-ischemic rats and the possible mechanism of neural protection with magnesium sulfate.

METHODSThe model of seven-day-old hypoxia-ischemia rats was established. The rats were divided randomly into 6 groups as follows: (1) normal control (n = 4); (2) sham surgery control (n = 4); (3) hypoxia-ischemia (n = 4); (4) sodium chloride injection with hypoxia-ischemia (n = 4); (5) magnesium sulfate pre-injection with hypoxia-ischemia (n = 4); (6)magnesium sulfate post-injection with hypoxia-ischemia (n = 4). The therapy groups received a bolus injection of 500 mg/kg magnesium sulfate intraperitoneally 0.5 hour before or after hypoxia-ischemia. Semi-quantitative RT-PCR was used to measure caspase-3 mRNA expression in the hippocampus 24 hours after hypoxia-ischemia.

RESULTSThe expression of caspase-3 mRNA was significantly increased in the hippocampus of the hypoxia-ischemia pups (1.88 +/- 0.36 vs 0.97 +/- 0.46, P < 0.05). The expression of caspase-3 mRNA in rats with magnesium sulfate pre-injection and post-injection decreased significantly (1.54 +/- 0.49, 1.65 +/- 0.48 vs 1.88 +/- 0.36, P < 0.05).

CONCLUSIONCaspase-3 was activated in the hippocampus of the seven-day-old rats 24 hours after hypoxia-ischemia. The suppression of the expression of caspase-3 mRNA in the hippocampus was probably related to the protective effect of magnesium sulfate on the brain injury of hypoxia-ischemia.

Animals ; Animals, Newborn ; Caspase 3 ; Caspases ; genetics ; Female ; Gene Expression Regulation, Enzymologic ; Hippocampus ; blood supply ; metabolism ; Hypoxia-Ischemia, Brain ; physiopathology ; prevention & control ; Magnesium Sulfate ; therapeutic use ; Male ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction