Immunohistochemistry and double immunofluorescent labeling techniques combined with confocal laser scanning microscope analysis were used to investigate the characteristic spatial induction profile of nestin following a transient middle cerebral artery occlusion in adult rat brain. The results showed that nestin was induced in ischemic core at 1 day after reperfusion. In addition to ischemic core, the expression of nestin increased in peri-ischemic I, II and III regions at 3 days and 1 week, then it decreased and narrowed along the rim of ischemic core 2 weeks after reperfusion. Double immunofluorescent labeling showed that nestin positive cells were mostly co-stained with GFAP,a astrocyte marker, in peri-ischemic I region 3 days after reperfusion. At 2 weeks, however nestin cells showed a long process and the cells double stained with nestin and NSE,a neuonal specific marker,increased in the ischemic brain. The results suggest that cerebral ischemia induces nestin expression in damaged neurons which might favor the neuroprotection against ischemic damage.
Animals ; Brain ; metabolism ; pathology ; Brain Ischemia ; metabolism ; pathology ; Immunohistochemistry ; Infarction, Middle Cerebral Artery ; metabolism ; pathology ; Nestin ; metabolism ; Neurons ; metabolism ; Rats

AIMTo observe the effect of nonselective nitro oxide synthase inhibitor N(G)-nitro-L-arginine(L-NA) on mitochondria injury in focal cerebral ischemia rats.

METHODSThe rats were randomly divided into sham, ischemia and L-NA treatment group. The model of focal cerebral ischemia was prepared with thread embolism in rats. L-NA was administrated respectively at 2 h, 6 h, 12 h after middle cerebral artery occlusion (MCAO). Rats were killed and the mitochondria of cerebral tissue were isolated by differential centrifugation after L-NA treatment for 3 days. The swelling and the activity of mitochondria, and the activities of ATPase, SOD, GSH-Px in mitochondria and the contents of NO, MDA in mitochondria were measured. Ultrastructure changes of neuronal mitochondria were examined by electronic microscope in ischemia and L-NA treatment group.

RESULTSThe swelling of mitochondria was markedly increased and the activity of mitochondria was decreased, and the contents of mitochondria NO and MDA were markedly increased, the activity of ATPase, SOD and GSH-Px in mitochondria were decreased significantly after MCAO. Compared with ischemia group, the contents of NO were decreased after ischemia 2h, 6h, 12h administered by L-NA, and the swelling of mitochondria was decreased and the activity of mitochondria was increased, and the activities of ATPase, SOD, GSH-Px in mitochondria were enhanced and the contents of MDA in mitochondria were decreased after ischemia 12 h administered by L-NA. The neuronal cytoplasm and the mitochondria swelled, the cristae were disrupted, dissolved or disappeared in MCAO rats. Administration of L-NA could reduce these changes induced by cerebral ischemia in rats.

CONCLUSIONIt could be concluded that L-NA could beneficially inhibit NO production. But it could't protect brain against damage in ischemia acute stage. It could improve mitochondria energy pump, ameliorate oxidative injury and increase the activities of mitochondria during postischemia, and then could effectively protect brain against damage induced by focal cerebral ischemia.

Animals ; Arginine ; pharmacology ; Brain ; metabolism ; Brain Ischemia ; metabolism ; pathology ; Male ; Mitochondria ; metabolism ; pathology ; Rats ; Rats, Wistar

Mitochondria play a key role in various cell processes including ATP production, Ca homeostasis, reactive oxygen species (ROS) generation, and apoptosis. The selective removal of impaired mitochondria by autophagosome is known as mitophagy. Cerebral ischemia is a common form of stroke caused by insufficient blood supply to the brain. Emerging evidence suggests that mitophagy plays important roles in the pathophysiological process of cerebral ischemia. This review focuses on the relationship between ischemic brain injury and mitophagy. Based on the latest research, it describes how the signaling pathways of mitophagy appear to be involved in cerebral ischemia.
Animals ; Brain Ischemia ; metabolism ; pathology ; Humans ; Mitochondrial Degradation ; Reactive Oxygen Species ; metabolism ; Stroke ; metabolism ; pathology

AIMTo make approach to the relationship between the changes of free zinc and ischemic neuronal damage in hippocampus after forebrain ischemia/reperfusion.

METHODSThe models of forebrain ischemia/reperfusion were established in rats. The contents of free Zn2+ were measured by TSQ fluorescence method. The Zn2+ chelator (CaEDTA) was injected into lateral ventricles in order to evaluate the effect of free Zn2+ on ischemic neuronal damage.

RESULTS(1) Zn2+ fluorescence in the hilus of dentate gyrus, CA3 region and the stratum radiatum and stratum oriens of CA1 decreased slightly at forty-eight hours after reperfusion. From seventy-two hours to ninety-six hour after reperfusion, the decreased fluorescence gradually returned to the normal level, but some fluorescence dots were found in pyramidal neurons of CA1 and the hilus of dentate gyrus. Seven days after reperfusion, all the changes of the fluorescence almost recovered. (2) The cell membrane-impermeable Zn2+ chelator CaEDTA could reduce the intracellular concentration of free Zn2+ and reduced neuronal damage after forebrain ischemia/reperfusion.

CONCLUSION(1) The synaptic vesicle Zn2+ released and then translocated into postsynaptic neurons after forebrain ischemia/reperfusion and played a role in ischemic neuronal damage. (2) The cell membrane-impermeable chelator CaEDTA could provide neuroprotection.

Animals ; Brain Ischemia ; metabolism ; pathology ; Hippocampus ; pathology ; Male ; Neurons ; metabolism ; Rats ; Rats, Wistar ; Reperfusion Injury ; metabolism ; pathology ; Zinc ; metabolism

Humans ; Ischemia ; metabolism ; pathology ; therapy ; Ischemic Postconditioning ; methods ; Myocardial Reperfusion Injury ; metabolism ; pathology ; therapy

OBJECTIVETo investigate the effect and mechanism of portal blood stasis on lung and renal injury induced by hepatic ischemia reperfusion.

METHODSA rabbit hepatic ischemia reperfusion injury model was established by hepatic portal occlusion and in situ hypothermic irrigation for 30 min. Twenty-four New Zealand white rabbits were employed and randomly divided into 3 groups equally by different dosage of portal blood stasis removal: group A5 (5 ml blood removal), group A10 (10 ml blood removal),and group B (no blood removal). Eight rabbits were served as controls with no hepatic portal occlusion and hypothermic irrigation. After reperfusion 4 h serum endotoxin content, tumor necrosis factor-alpha (TNF-alpha), urea nitrogen (BUN), and creatinine (Cr) were examined respectively, meantime lung and kidney tissues were sampled to determine the content of malondialdehyde (MDA), superoxide dismutase (SOD), the pathology, and wet to dry weight ratio, broncho-alveolar lavage fluid protein content in lung tissues.

RESULTSRemoving portal blood stasis ameliorated lung and renal injury as shown by decreasing the level of serum endotoxin, TNF-alpha, BUN, Cr, wet to dry weight ratio, broncho-alveolar lavage fluid protein content, MDA, SOD. TNF-alpha, Cr, broncho-alveolar lavage fluid protein content in lung tissues and MDA in kidney tissue in group A5 were significantly reduced compared with those in group B (P < 0.05), while in lung tissue in group A10 were also markedly reduced (P < 0.05). The activation of SOD in group A5 were significantly increased (P < 0.05).

CONCLUSIONSRemoval of portal blood stasis before the resume of splanchnic circulation may ameliorate the lung and renal injury induced by hepatic ischemia reperfusion. The possible mechanism may be that portal blood stasis removal reduces endotoxin absorption, and further decreases production of serum TNF-alpha.

Animals ; Disease Models, Animal ; Female ; Ischemia ; metabolism ; pathology ; Kidney ; metabolism ; pathology ; Liver ; blood supply ; Lung ; metabolism ; pathology ; Male ; Portal Vein ; pathology ; Rabbits ; Random Allocation ; Reperfusion Injury ; metabolism ; pathology

AIMTo study the expression of leptin receptor (OB-R) and neuronal damage following focal ischemia/reperfusion in rats.

METHODS20 adult male Wistar rats were divided into four groups randomly: sham-operated 24 h,72 h control group and ischemic/reperfusion 24 h, 72 h experiment group. Focal ischemia/reperfusion model was made with MCAO. Immunohistochemistry and immunoelectron microscope were used to observe the expression of OB-R of the cortex and neuronal damage.

RESULTSThe positive cells of OB-R were found in pyramidal cells of the parietal cortex, choroid plexus and blood vessel endothelium. Compared with sham-operated group, significant reduction of OB-R positive cells in the pyramidal cells was observed in the ischemia/reperfusion rats 24 hours after cerebral ischemia (P < 0.05). The positive cells of OB-R of sham-operated 72 h group reduced further (P < 0.01). Histochemistry and electron microscope showed neuronal damage in the core area of cerebral ischemia in the late period was more obvious than in the early period.

CONCLUSIONThe early and delayed ischemia/reperfusion neuronal damage were accompanied with reduction of OB-R expression. Thus, it is worth to study the effect of OB-R in cerebral ischemia.

Animals ; Brain Ischemia ; genetics ; metabolism ; pathology ; Cerebral Cortex ; metabolism ; pathology ; Disease Models, Animal ; Gene Expression ; Male ; Neurons ; pathology ; Rats ; Rats, Wistar ; Receptors, Leptin ; metabolism ; Reperfusion Injury ; metabolism ; pathology

OBJECTIVETo study the transport of glutamine and glucose, expression of their transporters and tissue morphology in intestinal hypoperfusion.

METHODSSprague-Dawley rats were randomized to receive 60 min of intestinal hypoperfusion (superior mesenteric artery clamp) or serve as normoxic controls (celiotomy only). At the same time, jejunal loops were randomized to receive in situ perfusion of mannitol,glucose,or glutamine.Intestinal brush border membrane vesicles (BBMV) were prepared by calcium precipitation. Sodium-dependent uptake of glucose and glutamine into BBMV were quantitated by rapid mixing and filtration. Histologic examination and immunohistochemistry were performed by pathologists blinded to the groups.

RESULTSWhen compared with the control group, tissue lactate concentration of the hypoperfused group increased significantly (4.9+/-0.3 vs 3.1+/-0.2), especially in the glucose perfused groups (P<0.01). Transport and transporters of glucose in brush border, but not glutamine, decreased during hypoperfusion [(76+/-10) pmol d mg(-1) d 10 s(-1) vs (290+/-13)pmol d mg(-1) d 10 s(-1)]. Tissue structural damage was most severe in glucose perfused groups during hypoperfusion.

CONCLUSIONTransport and expression of transporters of glucose and glutamine in enteral nutrition are differently regulated under conditions of trauma and stress.

Animals ; Enteral Nutrition ; Glucose ; metabolism ; Glutamine ; metabolism ; Intestine, Small ; blood supply ; metabolism ; pathology ; Ischemia ; metabolism ; Male ; Rats ; Rats, Sprague-Dawley

Ischemia and reperfusion (I/R) injury is a major cause of hepatic failure after liver surgery, but no method could monitor or predict it real-time during surgery. We measured bioelectrical impedance (BEI) and cell viability to assess the usefulness of BEI during I/R in rat liver. A 70% partial liver ischemia model was used. BEI was measured at various frequencies. Adenosine triphosphate (ATP) content, and palmitic acid oxidation rate were measured, and histological changes were observed in order to quantify liver cell viability. BEI changed significantly during ischemia at low frequency. In the ischemia group, BEI increased gradually during 60 min of ischemia and had a tendency to plateau thereafter. The ATP content decreased below 20% of the baseline level. In the I/R group, BEI recovered to near baseline level. After 24 hr of reperfusion, the ATP contents decreased to below 50% in 30, 60 and 120 min of ischemia and the palmitic acid metabolic rates decreased to 91%, 78%, and 74%, respectively, compared with normal liver. BEI may be a good tool for monitoring I/R during liver surgery. The liver is relatively tolerant to ischemia, however after reperfusion, liver cells may be damaged depending upon the duration of ischemia.
Adenosine Triphosphate/metabolism ; Animals ; *Cell Survival ; Electric Impedance ; Energy Metabolism ; Ischemia/*metabolism ; Liver/*metabolism/pathology ; Male ; Palmitates/metabolism ; Rats ; Rats, Sprague-Dawley ; *Reperfusion ; Reperfusion Injury/metabolism/pathology

BACKGROUNDTherapeutic angiogenesis has been shown to promote blood vessel growth and improve tissue perfusion. Nerve growth factor (NGF) has been reported to play an important role in both physiological and pathological angiogenesis. This study aimed to investigate the effects of NGF on angiogenesis and skeletal muscle fiber remodeling in a murine model of hindlimb ischemia and study the relationship between NGF and vascular endothelial growth factor (VEGF) in angiogenesis.

METHODSTwenty-four mice were randomly allocated to normal control group (n = 6), blank control group (n = 6), VEGF gene transfection group (n = 6), and NGF gene transfection group (n = 6). The model of left hindlimb ischemia model was established by ligating the femoral artery. VEGF165plasmid (125 μg) and NGF plasmid (125 μg) was injected into the ischemic gastrocnemius of mice from VEGF group and NGF group, respectively. Left hindlimb function and ischemic damage were assessed with terminal points at 21th day postischemia induction. The gastrocnemius of four groups was tested by hematoxylin-eosin staining, proliferating cell nuclear antigen and CD34 immunohistochemistry staining, and myosin ATPase staining. NGF and VEGF protein expression was detected by enzyme-linked immunosorbent assay.

RESULTSOn the 21th day after surgery, the functional assessment score and skeletal muscle atrophy degree of VEGF group and NGF group were significantly lower than those of normal control group and blank control group. The endothelial cell proliferation index and the capillary density of VEGF group and NGF group were significantly increased compared with normal control group and blank control group (P < 0.05). The NGF and VEGF protein expression of NGF group showed a significant rise when compared with blank control group (P < 0.05). Similarly, the VEGF protein expression of VEGF group was significantly higher than that of blank control group (P < 0.05), but there was no significant difference of the NGF protein expression between VEGF group and blank control group (P > 0.05). The type I skeletal muscle fiber proportion in gastrocnemius of NGF group and VEGF group was significantly higher than that of blank control group (P < 0.05).

CONCLUSIONSNGF transfection can promote NGF and VEGF protein expression which not only can induce angiogenesis but also induce type I muscle fiber expression in ischemic limbs.

Animals ; Antigens, CD34 ; metabolism ; Female ; Hindlimb ; metabolism ; pathology ; Ischemia ; metabolism ; pathology ; Mice ; Muscle, Skeletal ; metabolism ; pathology ; Neovascularization, Physiologic ; genetics ; physiology ; Random Allocation ; Vascular Endothelial Growth Factor A ; genetics ; physiology