Expression of hypoxia inducible factor-1 alpha and ischemic erythropoietin tolerance in the brain of cerebral ischemic tolerance model rats

BACKGROUND: Hypoxia inducible factor-1 alpha (HIF-1 α) and erythropoietin(EPO), possessing neuroprotective effect in the cerebral ischemia, might play an important role in the formation of cerebral ischemic tolerance (IT). OBJECTIVE:To observe the neuroprotective effect of cerebral ischemic preconditioning(IPC) of rats, and the expression and mechanism of HIF-1α and target gene erythropoietin in the brain tissue following the formation of cerebral IT. DESIGN:A randomized and controlled observation. SETTING: Department of Neurology, the Affiliated Hospital of Medical College, Qingdao University. MATERIALS: Totally 84 enrolled adult healthy male Wistar rats of clean grade, weighing 250 to 300 g, were provided by the Animal Experimental Department, Tongji Medical College of Huazhong University of Science and Technology. Ready-to-use SABC reagent kit and rabbit anti-rat HIF-1α monoclonal antibody were purchased from Boshide Bioengineering Co.Ltd (Wuhan); Rabbit anti-rat EPO monoclonal antibody was purchased from Santa Cruz Company (USA). METHODS: This experiment was carried out in the Department of Anatomy, Medical College, Qingdao University during March 2005 to March 2006. ① The 84 rats were divided into 3 groups by a lot: IPC group (n =40), sham-operation group (n =40) and control group (n =4). In the IPC group, middle cerebral artery was occluded for 2 hours respectively on the 1st, 3rd, 7th, 14th and 21st days of the reperfusion following 10-minute preischemia was made using a modified middle cerebral artery second suture method from Zea-Longa. The rats were sacrificed 22 hours after reperfusion in the end of middle cerebral artery occlusion (MCAO). That was to say, after 10-minute preischemia, suture was exited to the external carotid artery and embedded subcutaneously. Middle cerebral artery was occluded again to form the second reperfusion at the set time point after reperfusion. Twenty-two hours later, rats were sacrificed; In the sham-operation group,the preischemia was substituted by sham-operation(only common carotid artery and crotch were exposed, and MCAO by suture was omitted), and the other procedures were the same as those in the IPC group. In the control group, rats were given sham-operation twice at an interval of one day, and they were sacrificed 24 hours after the second sham-operation. ② Brain tissue was taken from the rats in each group. Cerebral infarction area of each layer was measured with TTC staining, and total cerebral infarction volume (The total cerebral infarction area of each layer×interspace ) was calculated. After brain tissue was stained by haematoxylin-esoin (HE), the form of nerve cells was observed under an optical microscope, and the expressions of HIF-1α and EPO protein in the brain tissue were detected with immunohistochemical method. MAIN OUTCOME MEASURES: ①Cerebral infarction volume;②form of nerve cell; ③ the expression of HIF-1α and EPO protein in the brain tissue. RESULTS: Totally 84 rats were enrolled in the experiment. The dead rats were randomly supplied during the experiment, and finally 84 rats entered the stage of result analysis. ① Detection of cerebral infarction volume of rats in each group: Cerebral infarction volume in the IPC group was significantly smaller than that in the sham-operation group on the 1st, 3rd and 7th days after reperfusion respectively [(161.2±6.9) mm3 vs (219.9±11.2) mm3, (134.9±9.0) mm3 vs (218.6±13.0) mm3, (142.9±13.7) mm3 vs (221.3±14.2) mm3, t =8.924，10.587，7.947, P < 0.01]. ② Observation of nerve cell form of brain tissue: HE staining showed that the ischemic degree, range and cerebral edema degree of IPC group were significantly milder than those of sham-operation group. ③ The expressions of HIF-1α and EPO protein in cerebral cortex and hippocampus : The expression of HIF-1α of IPC group was significantly higher than that of sham-operation group on the 1st, 3rd and 7th days after reperfusion respectively (125.93±3.79 vs 117.65±5.60, 140.63±4.64 vs 119.33±4.26, 131.15±2.74 vs 107.60±3.89, t=2.449, 6.763，9.899，P < 0.05-0.01). The expression of EPO of IPC group was significantly higher than that of sham-operation group on the 3rd and 7th days after perfusion respectively (141.68±3.29 vs 126.33±4.51, 138.88±2.59 vs 125.58±6.18，t=5.499，3.970, P < 0.05). CONCLUSION: ① IPC can protect the never cells in rat brain and the best time to onset of cerebral IT induced by IPC is 1 to 7 days after reperfusion. ② Neuroprotective effect of cerebral IT might be related to the expression of HIF-1α and its target gene EPO.     

Effect of ketamine on aquaporin-4 expression and neuronal apoptosis in brain tissues following brain injury in rats

INTRODUCTIONAquaporin (AQP), found recently, is a group of water permeability-re-lated cell membrane transport protein. Among them, AQP-4 is mostnoticeable. To suppress AQP-4 expression can relieve the formationof brain edema, and then reduce the death ra…     

Mild hypobaric hypoxic postconditioning increases the expression of HIF-1α and erythropoietin in the CA1 field of the hippocampus of rats that survive after severe hypoxia

Using immunohistochemistry, we studied the expression of the alpha regulatory subunit of the hypoxia-inducible factor (HIF-1α) and the product of its target gene, which encodes the protective cytokine erythropoietin in the hippocampal CA1 field of rats in response to damaging severe hypoxia and severe hypoxia followed by three sessions of postconditioning with mild hypobaric hypoxia. We found that the immunoreactivity to the proteins studied in the hippocampus of rats was reduced in response to severe hypoxia. Hypoxic postconditioning sessions of mild hypobaric hypoxia (360 mmHg, 2 h, three times at intervals of 24 h) up regulated the expression of HIF-1α and erythropoietin in hippocampal CA1 neurons of rats that survived after severe hypoxia. Our results indicate that postconditioning led to compensation of hypoxia-induced neuron damage in the brain, which actively involved HIF-1α and erythropoietin.     

Alteration of GSK-3β in the hippocampus and other brain structures after chronic paraquat administration in rats

Systemic exposure of rodents to the herbicide paraquat (PQ) was suggested to reproduce pathological features of Parkinson's disease. Our recent data showed that long-term PQ administration influenced levels of glycogen synthase kinase 3β (GSK-3β) and its active form phosphorylated on tyrosine 216 in the nigrostriatal system, which may be related to its vulnerability to PQ toxicity. The aim of this study was to analyse selectivity of the toxic effect of PQ after its systemic administration in rats. PQ was administered for 37 weeks and the protein level of total GSK-3β and its active GSK-3β (pY216) form in subcellular fractions of hippocampus, brain cortex and cerebellum was examined. Our data indicated that the long-term administration of PQ significantly decreased the level of both GSK-3β forms in nuclear and cytosolic fractions of hippocampus in rats. In the brain cortex and cerebellum PQ decreased the level of both forms of GSK-3β in the nuclear fraction but increased their levels in mitochondria and in some cases also in the cytosol. The results of the present study indicate that PQ influenced levels and activation of GSK-3β in different brain structures, which may contribute to its toxicity, but on the other hand may suggest development of adaptive, protective mechanisms.     

Effect of Ganoderma lucidum spore on expression of insulin-like growth factor-1, nuclear factor-kappa B, and neuronal apoptosis in the epileptic rat brain*★

BACKGROUND：It has been reported that Ganoderma lucidum spore powder, a very well known Chinese traditional medicine, can affect immunoregulation, free radical scavenging, and anti-hypoxia responses. OBJECTIVE： To investigate the effect of Ganoderma lucidum spore powder on expression of insulin-like growth factor-1 （IGF-1）, nuclear factor-κB （NF-κB） and neuronal apoptosis in rats with pentylenetetrazol （PTZ）-induced epilepsy. DESIGN, TIME AND SETTING： A cellular and molecular biology experiment with randomized controlled study design was performed at the Central Laboratory of Basic Medical College of Jiamusi University from June to August 2005. MATERIALS： Thirty healthy, adult, male, Wistar rats were selected and randomly divided into 3 groups （10 rats per group）： control, epilepsy model, and Ganoderma lucidum spore powder. A sub-eclampsia PTZ dose （35 mg/kg） was intraperitoneally injected to induce epilepsy in the latter two groups. Wild Ganoderma lucidum spore powder （30 g/L） was provided by the wild Ganoderma lucidum plant nursery at Jiamusi, China. Immunohistochemical detection and terminal deoxynucleotidyl transferase-mediate dUTP nick end-labeling （TUNEL） kits were purchased from Wuhan Boster Biological Technology Co., Ltd., China. METHODS： Ganoderma lucidum spore powder was intragastrically administered at a dose of 10.0 mL/kg, once a day for 28 days. In the epilepsy and control groups, an equivalent volume of normal saline was intragastrically administered. MAIN OUTCOME MEASURES： Immunoreactivity for IGF-1 and NF-κB/P65 were detected by immunohistochemical staining. Neuronal apoptosis was detected using TUNEL methods. RESULTS： The hippocampus and cerebral cortex of rats with PTZ-induced epilepsy exhibited a higher number of apoptotic cells at high magnification （×400）, compared with the control group. Expression of IGF-1 and NF-κB were higher in the epilepsy group, compared with the control group （P 〈 0.01）. In Ganoderma lucidum spore-treated rats,     

Caspase-3 mRNA expression in rats with apoptosis of retinal photoreceptor cells

BACKGROUND: Apoptosis of retinal photoreceptor cells is a commonly pathological characteristic of various eye diseases, while caspase-3 is an important regulating gene and plays a key role in apoptosis. OBJECTIVE: To measure the expression of caspase-3 mRNA in rats with apoptosis of retinal photoreceptor cells by using real-time fluorescent quantitative polymerase chain reaction and compare with those of the normal rats. DESIGN: Randomized controlled animal study. SETTING: Zhongshan Ophthalmological Center of Sun Yat-sen University. MATERIALS: A total of 36 female SD rats of 50 days old and clean grade and weighing (150±10) g were provided by Experimental Animal Center of Northern Area of Sun Yat-sen University. All rats were randomly divided into normal control group (n =6) and N-methyl-N-nitrosourea (MNU) group (n =30), and they were observed at 12 hours, 1, 2, 3 and 5 days after model establishment, with 6 rats at each time point. METHODS: The experiment was carried out at Zhongshan Ophthalmological Center, Key Laboratory of Ophthalmology by State Ministry of Education from March to December 2004. Rats in the normal control group were intraperitoneally injected with saline and rats in the MNU group were intraperitoneally injected with 40 mg/kg MNU. And then, retinal photoreceptor injured models were established. At 12 hours, 1, 2, 3 and 5 days after model establishment, the rats were sacrificed for enucleating right eyeballs, isolating retina immediately and extracting total RNA. The expression of caspase-3 mRNA in retina was measured with real-time fluorescent quantitative polymerase chain reaction. MAIN OUTCOME MEASURES: Expression of caspase-3 mRNA in retina of rats in the two groups. RESULTS: A total of 36 SD rats were involved in the final analysis. The expressions of caspase-3 mRNA in the rat retina of both groups at the five time points (12 hours, 1, 2, 3 and 5 days) after model establishment were 1.52×105, 18.35×105, 25.14×105, 29.25×105, 13.72×105 and 12.24×105, respectively. The expression of caspase-3 mRNA in the MNU group increased after 12 hours of intraperitoneal injection, and rose to the top on the 2nd day, which was 19 times as many as that of the normal control group. Then, it decreased gradually and was still 8 times as many as that of the normal control group on the 5th day. CONCLUSION: The expression of caspase-3 mRNA is related to apoptosis of retinal photoreceptor cells, while caspase-3 plays an important role in occurrence and development of apoptosis of retinal photoreceptor cells.     

The requirement of extracellular signal-related protein kinase pathway in the activation of hypoxia inducible factor 1α in the developing rat brain after hypoxia–ischemia

Hypoxia inducible factor 1α (HIF-1α) is a key regulator of cellular oxygen homeostasis. However, the regulation of HIF-1α in neonates with hypoxia–ischemia (HI) is not clear. Under normoxic conditions, the extracellular signal-related protein kinase (ERK) pathway has been shown to be involved in the activation of HIF-1α in cell lines. Therefore, we hypothesized that the ERK pathway is involved in the activation of HIF-1α and its target genes in the developing rat brain following HI. To test this hypothesis, we set up an HI model by ligating the right common carotid artery followed by hypoxia using postnatal day 10 rats. Rat brains from HI and sham controls were collected to detect the expression of HIF-1α, its target gene, vascular endothelial growth factor (VEGF), and ERK using immunohistochemistry, Western blot analysis, and RT-PCR. We found that the expression of HIF-1α protein was significantly upregulated at 4 h and peaked at 8 h after HI compared with sham controls. Accordingly, VEGF was similarly upregulated. However, the expression of total ERK (Erk1/2) had no obvious changes. Even though the phosphorylated form of ERK, p-Erk1/2, was upregulated and peaked at 4 h after HI, it is earlier than that seen in HIF-1α expression. Furthermore, the induction of HIF-1α protein, but not its mRNA, could be significantly inhibited by Erk1/2 pathway specific inhibitor, U0126. Our findings suggest that Erk1/2 pathway is involved in the regulation of HIF-1α and VEGF in the developing rat brain after HI. The Erk1/2 pathway may work as a potential target for therapeutic intervention in neonates with HI. L. Li and Y. Xiong contributed equally to this report. This work was supported by grants from the National Natural Science Foundation of China (No. 30570623 and No. 30770748 to Dezhi Mu), China Medical Board of New York (00-722 to Dezhi Mu), Ministry of Education of China (2006331-11-7, 20070610092), and Bureau of Scientific Technology of Sichuan Province (07JY029-067).     

Pilocarpine-induced epilepsy alters the expression and daily variation of the nuclear receptor RORα in the hippocampus of rats

It is widely known that there is an increase in the inflammatory responses and oxidative stress in temporal lobe epilepsy (TLE). Further, the seizures follow a circadian rhythmicity. Retinoic acid receptor-related orphan receptor alpha (RORα) is related to anti-inflammatory and antioxidant enzyme expression and is part of the machinery of the biological clock and circadian rhythms. However, the participation of RORα in this neurological disorder has not been studied. The aim of this study was to evaluate the RORα mRNA and protein content profiles in the hippocampus of rats submitted to a pilocarpine-induced epilepsy model at different time points throughout the 24-h light–dark cycle analyzing the influence of the circadian rhythm in the expression pattern during the acute, silent, and chronic phases of the experimental model. Real-time PCR and immunohistochemistry results showed that RORα mRNA and protein expressions were globally reduced in both acute and silent phases of the pilocarpine model. However, 60 days after the pilocarpine-induced status epilepticus (chronic phase), the mRNA expression was similar to the control except for the time point 3 h after the lights were turned off, and no differences were found in immunohistochemistry. Our results indicate that the status epilepticus induced by pilocarpine is able to change the expression and daily variation of RORα in the rat hippocampal area during the acute and silent phases. These findings enhance our understanding of the circadian pattern present in seizures as well as facilitate strategies for the treatment of seizures.     

Tumor necrosis factor-α expression in muscles of polymyositis and dermatomyositis

We evaluated the expression of tumor necrosis factor-α (TNF-α) mRNA in muscle biopsy specimens from patients with polymyositis (PM) and dermatomyositis (DM) to clarify its role in the pathogenesis of PM and DM. We performed non-radioactive in situ hybridization studies for TNF-α combined with immunohistochemistry for cell type-specific markers on muscles from ten PM and five DM patients. TNF-α-positive infiltrating cells present in the endomysium and perimysium were found in all PM and DM muscles. The frequency of TNF-α-positive cells against total infiltrating cells was similar among PM and DM (27.1 ± 7.4% in PM and 28.5 ± 13.6% in DM). However, TNF-α/CD8-positive lymphocytes and TNF-α-positive macrophages invading the non-necrotic muscle fiber were observed only in PM but not in DM. TNF-α was more highly expressed in PM and DM than was previously thought, and it was suggested that TNF-α plays a role in muscle fiber degeneration in PM. Received: 9 June 1999 / Revised, accepted: 25 October 1999     

The dynamics of HIF-1α expression in the rat brain at different stages of experimental posttraumatic stress disorder and its correction with moderate hypoxia

The dynamics of the expression of the HIF factor-1 α-subunit, which is related to products of early genes, has been studied in the neocortex, the hippocampus, and the hypothalamus of rats during the development of posttraumatic stress disorder (PTSD) in a stress–restress model and using triple moderate hypobaric hypoxia (MH₃), which prevents the formation of this anxiety pathology. The immunohistochemical method has shown that after pathogenic traumatic stress (TS), during the primary (“hidden”) period of modeled PTSD formation, the level of HIF-1α expression did not change significantly; however, after restress it rapidly increased in all regions of the brain. An increased expression of this factor remained in animals with experimental PTSD for up to 10 days after restress. Exposure to MH₃ before TS or before restress compensated for these disturbances: fully in the hippocampus and partly in the neocortex; it inhibited the prolonged over-induction of the HIF-1α, which may be one of the mechanisms that mediate an anxiolytic effect of hypoxia. Along with this, preconditioning with MH₃ significantly decreased the content of HIF-1α after TS, thus preventing activation of the HIF-1 factor during the hidden period, which is likely associated with the formation of pathological reactivity to restress. These facts indicate the pathogenetic role of HIF-1 in certain periods of experimental PTSD and the correcting effect of hypoxic preconditioning.     

Changes of learning and memory ability associated with neuronal nitric oxide synthase in brain tissues of rats with acute alcoholism

INTRODUCTION The discovery of nitric oxide was the greatest achievement of neuro- biology in late 20th century. The discoverers were awarded the Nobel Prize in Physiology and Medicine. Publications on all aspects of nitric oxide run into thousands. Nevert…     

Cell apoptosis in perihematomal brain regions and expression of Caspase-3 protein in patients with hypertensive intracerebral hemorrhage

INTRODUCTION Hypertensive intracerebral hemorrhage (HICH) has high mortality and morbidity rates. But effective therapeutic methods are still needed at present. Recent evidences from laboratory models suggest that cell death in the perihematomal region ma…     

Effect of cyclovirobuxine D on human growth-associated protein 43 and neurocan expression in ischemic brain tissue of stroke-prone renovascular hypertensive rats*★

BACKGROUND： Experimental data indicate that human growth-associated protein 43 mRNA expression coincides with axonal growth during nerve ganglion development; while neurocan, secreted from astrocytes, can inhibit sprouting and elongation of the axonal growth cone. OBJECTIVE： To verify regulatory effects of cyclovirobuxine D （CVB-D） extracted from Chinese box branchlet on human growth-associated protein 43 （GAP-43）, and neurocan expression in brain tissue of stroke-prone renovascular hypertensive （RHRSP） rats, at different time points after cerebral ischemia/reperfusion. DESIGN： Randomized grouping design and controlled animal study. SETTING： This study was performed at the Center of Guangdong Hospital of Traditional Chinese Medicine （a national key laboratory） from March 2003 to September 2006. MATERIALS： 100 healthy male Sprague-Dawley rats, aged 2 3 months and weighing 90-120 g, were selected for this study. CVB-D was provided by Nanjing Xiaoying Pharmaceutical Factory （Batch number： 307701）. METHODS： The initial tip of renal arteries was clamped bilaterally for 10 weeks to establish the RHRSP model. 100 RHRSP rats were randomly divided into 4 groups： naive group （n = 10）, sham surgery group （n = 10）, CVB-D group （n = 40）, and lesion group （n = 40）. Rats in the naive group did not undergo any treatment, and cervical vessels of rats in the sham surgery group were exposed, but not blocked. The right middle cerebral artery of rats in the CVB-D group and lesion group were occluded to establish cerebral ischemia. Rats in the CVB-D group were intraperitoneally injected with CVB-D （6.48 mg/kg） every day and with saline （1.5 mL/injection） twice a day. Rats in the lesion group were intraperitoneally injected with saline （2 mL/injection）. MAIN OUTCOME MEASURES： Immunohistochemistry was applied to detect GAP-43 and neurocan expression in the ischemic penumbra region of CVB-D and lesion brains at 2 hours post-cerebral ischemia and at 1, 7, 14, and 30 days po