Impact of Perinatal Systemic Hypoxic–Ischemic Injury on the Brain of Male Offspring Rats: An Improved Model of Neonatal Hypoxic–Ischemic Encephalopathy in Early Preterm Newborns

In this study, we attempted to design a model using Sprague-Dawley rats to better reproduce perinatal systemic hypoxic-ischemic encephalopathy (HIE) in early preterm newborns. On day 21 of gestation, the uterus of pregnant rats were exposed and the blood supply to the fetuses of neonatal HIE groups were thoroughly abscised by hemostatic clamp for 5, 10 or 15 min. Thereafter, fetuses were moved from the uterus and manually stimulated to initiate breathing in an incubator at 37 °C for 1 hr in air. We showed that survival rates of offspring rats were decreased with longer hypoxic time. TUNEL staining showed that apoptotic cells were significant increased in the brains of offspring rats from the 10 min and 15 min HIE groups as compared to the offspring rats in the control group at postnatal day (PND) 1, but there was no statistical difference between the offspring rats in the 5 min HIE and control groups. The perinatal hypoxic treatment resulted in decreased neurons and increased cleaved caspase-3 protein levels in the offspring rats from all HIE groups at PND 1. Platform crossing times and the percentage of the time spent in the target quadrant of Morris Water Maze test were significantly reduced in the offspring rats of all HIE groups at PND 30, which were associated with decreased brain-derived neurotrophic factor levels and neuronal cells in the hippocampus of offspring rats at PND 35. These data demonstrated that perinatal ischemic injury led to the death of neuronal cells and long-lasting impairment of memory. This model reproduced hypoxic ischemic encephalopathy in early preterm newborns and may be appropriate for investigating therapeutic interventions.     

Dynamic Changes of Cerebral-Specific Proteins in Full-Term Newborns with Hypoxic–Ischemic Encephalopathy

The aim of this study was to observe the dynamic changes of serum brain-derived neurotrophic factor (BDNF), S-100B, and Tau proteins levels in full-term newborns with hypoxic–ischemic encephalopathy (HIE) and to discuss their significance in brain damage. Serum samples of 28 full-term newborns diagnosed with HIE and 20 controls were obtained in the first 24 h of life. Another serum samples were also taken, respectively, at 3 and 7 days of life in HIE group. The concentrations of BDNF, S-100B, and Tau proteins were measured by the enzyme-linked immunosorbent assay method. Mean concentrations of BDNF, S-100B, and Tau proteins among different time period and in different grades of HIE group were calculated and compared. Compared with the control group, serum BDNF and proteins S-100B levels in HIE group were significantly elevated in 24 h after birth (P < 0.05) and their concentrations were also significantly higher among patients with mod-severe HIE compared to those with mild HIE at 24 h and 7 days after asphyxia (P < 0.05). Regardless of whether mod-severe HIE or mild HIE, there were no significant difference of serum BDNF and proteins S-100B among the three different time periods. There was no difference in Tau protein levels between HIE group and control group, also no difference between mod-severe HIE group and mild HIE group. BDNF and proteins S-100B are up-regulated early in asphyxia neonates with HIE; and the released amount of BDNF and proteins S-100B from nerve center system correlate with the extent of encephalopathy.     

The Protective Mechanism for the Blood–Brain Barrier Induced by Aminoguanidine in Surgical Brain Injury in Rats

This study was performed to investigate the mechanism of blood–brain barrier (BBB) permeability change, which was induced by aminoguanidine (AG) after surgical brain injury (SBI) in rats. Compared to control group, AG (150 mg/kg, i.p.) significantly reduced Evans blue extravasation into brain tissue at 24 h after surgical resection, it also induced a 32% decrease of malondialdehyde (MDA) values and a 1.1-fold increase of the glutathione (GSH) levels at 12 h after injury. The expression of inducible nitric oxide synthase (iNOS) reached the peak value at 24 h after SBI, which was significantly attenuated after AG treatment. In addition, ZO-1 protein was up-regulated by AG (150 mg/kg) treatment at 24 h after SBI. Our results indicated that AG could protect the BBB after SBI, which could be correlated with antioxidative property, the down-regulation of iNOS and up-regulation of tight junction protein expression.     

Use of Naïve Bayes Classifier to Assess the Effects of Antipsychotic Agents on Brain Electrical Activity Parameters in Rats

Journal of Evolutionary Biochemistry and Physiology - Research and development of novel methods to determine the effects of antipsychotic agents is an important challenge for experimental...     

Protective Effects of Notoginsenoside R1 via Regulation of the PI3K-Akt-mTOR/JNK Pathway in Neonatal Cerebral Hypoxic–Ischemic Brain Injury

Notoginsenoside R1 (NGR1) is a predominant phytoestrogen extracted from Panax notoginseng that has recently been reported to play important roles in the treatment of cardiac dysfunction, diabetic kidney disease, and acute liver failure. Studies have suggested that NGR1 may be a viable treatment of hypoxic-ischemic brain damage (HIBD) in neonates by reducing endoplasmic reticulum stress via estrogen receptors (ERs). However, whether NGR1 has other neuroprotective mechanisms or long-term neuroprotective effects is unclear. In this study, oxygen-glucose deprivation/reoxygenation (OGD/R) in primary cortical neurons and unilateral ligation of the common carotid artery (CCL) in 7-day-old postnatal Sprague Dawley (SD) rats followed by exposure to a hypoxic environment were used to mimic an HIBD episode. We assessed the efficacy of NGR1 by measuring neuronal damage with MTT assay and assessed brain injury by TTC staining and brain water content detection 24–48 h after OGD/HIE. Simultaneously, we measured the long-term neurophysiological effects using the beam walking test (5 weeks after HI) and Morris water maze test 5–6 weeks after HI. Expression of PI3K-Akt-mTOR/JNK (24 h after HI or OGD/R) proteins was detected by Western blotting after stimulation with HI, NGR1, LY294002 (PI3K inhibitor), 740Y-P (PI3K agonist), or ICI 182780(estrogen receptors inhibitor). The results indicated that NGR1 exerted neuroprotective effects by inhibiting neuronal apoptosis and promoting cell survival via the PI3K-Akt-mTOR/JNK signaling pathways by targeting ER in neonatal hypoxic–ischemic injury.     

Histamine-Mediated Regulation of Electrical Activity during the Bladder–Urethra Interaction in Rats

The effect of histamine on background spontaneous electrical activity of the interrelated bladder and urethra was studied in rats. The basic characteristics of pacemaker activity (action potential amplitude, average peak rise rate, peak rise time, peak half-width, rhythmogenicity frequency) were analyzed both in normal conditions and upon exposure to histamine (10^–4 mol/L). A comparison of the action potential parameters in the above organs demonstrated far lower values of the amplitude (by 34.19%; p ≤ 0.001), peak rise rate (by 30.39%; p ≤ 0.01) and peak rise time (by 18%; p ≤ 0.01) at a reduced rhythmogenicity frequency. Histamine evoked a considerable increase in the amplitude and its rise rate in the bladder (by 50.59 and 56.36%, respectively; p ≤ 0.001) and rhythmogenicity frequency (by 18%; p ≤ 0.01) at a constancy of the remaining two parameters. In the urethra, no obvious changes were detected in the action potential parameters. Morphohistochemical analysis also supported the involvement of histamine in the activation of rhythmogenicity only in the bladder. Thus, histamine is not implicated in the genesis of tonic contractions in the urethra, in contrast to its activating effect on the bladder.     

Effects of Pyrazinamide on Tryptophan–Niacin Conversion in Rats

In the course of our screening for a new anti-tumor substance, the bisabolane sesquiterpenoid endoperoxide, 3,6-epidioxy-1,10-bisaboladiene (EDBD), was isolated from the edible wild-plant, Cacalia delphiniifolia. EDBD showed cytotoxicity toward human chronic myelogenous leukemia K562 and human prostate carcinoma LNCaP cell lines with IC₅₀ values of 9.1 μM and 23.4 μM, respectively. DNA fragmentation and condensation of chromatin, the hallmarks of apoptosis, appeared in K562 cells after an 18-h treatment with EDBD. α-Curcumene, a bisabolane sesquiterpene that lacks the endoperoxide moiety of EDBD, also showed cytotoxicity toward both K562 and LNCaP cell lines at over a 10-times higher dose than that of EDBD. The results indicate the importance of the endoperoxide structure within EDBD to its anti-tumor activity in vitro.     

Effect of Acupuncture on Hypothalamic�CPituitary�CAdrenal System in Maternal Separation Rats

The maternal separation (MS) animal model has been widely used to study early life stress and several psychiatric conditions such as depression and anxiety. In this study, we investigated the effect of acupuncture on anxiety-related behaviors and hypothalamic-pituitary-adrenal (HPA) system in MS-induced early life stress of Sprague-Dawley rat pups (14-21 postnatal days). For determining anxiety-related behaviors, the elevated plus-maze test was performed. The effects of acupuncture on the activation of stress were measured by assessing plasma levels of corticosterone (CORT) and adrenocorticotropin hormone (ACTH). The hypothalamic immunoreactivity (IR) of arginine vasopressin (AVP) was also examined. Acupuncture was conducted at acupoint HT7, which is used to treat mental disorders in Oriental medicine, for seven consecutive days. Acupuncture significantly decreased the frequencies of open arm entries and the amount of time spent in the open arms in MS rats. In addition, acupuncture reduced CORT and ACTH levels in plasma of MS rats, and AVP-IR in the hypothalamic paraventricular nucleus of MS rats. In conclusion, acupuncture reduced anxiety-related behaviors and modulated the HPA system. These findings suggest that acupuncture at HT7 may be useful as a therapeutic treatment in MS-induced early life stress.     

Role of the Ryanodine Receptor in Ischemic Brain Damage—Localized Reduction of Ryanodine Receptor Binding During Ischemia in Hippocampus CA1

1. The ryanodine receptor has recently been shown to play a pivotal role in the regulation of intracellular Ca²⁺ concentration via Ca²⁺-induced Ca²⁺ release (CICR). Effects of ischemia on CICR in the brain tissue, however, remain largely unknown since only a few reports have been published on this subject. In this paper we report on work in this area by our group and review related progress in this field.2. We examined alterations of ryanodine receptor binding and local cerebral blood flow (LCBF) at 15 min, 30 min, and 2 hr after occlusion of the right common carotid artery in the gerbil brain. A quantitative autoradiographic method permitted simultaneous measurement of these parameters in the same brain. The LCBF was significantly reduced in most of the cerebral regions on the occluded side during each time period of ischemia. In contrast, only in the hippocampus CA1 on the occluded side was a significant reduction in ryanodine binding found at 15 min, 30 min and 2 hr after the occlusion.3. These findings suggest that suppression of ryanodine binding in the hippocampus CA1 may be attributable to a regionally specific perturbation of CICR and that this perturbation may be closely associated with the pathophysiological mechanism that leads to the selective ischemic vulnerability of this region.4. Other recent studies have also reported an important role for ryanodine receptors in neuronal injury such as the delayed neuronal death in the hippocampus CA1. These data suggest that derangement of CICR is likely to be involved in acute neuronal necrosis as well as in delayed neuronal death in ischemia.5. Further studies on clarifying the role of CICR in ischemic brain damage are needed in order to develop new therapeutic strategies for stroke patients.     

Human Cerebral Microvessel Endothelial Cell Culture as a Model System to Study the Blood–Brain Interface in Ischemic/Hypoxic Conditions

Summary 1. Cerebral ischemia and reperfusion induce several changes on the endothelial cells at the microcirculatory level.2. Vasogenic brain edema due to compromised blood–brain barrier, transformation of the endothelial cell surface from an anticoagulant to a procoagulant property are important factors in the pathogenesis of ischemic stroke.3. Release of prostaglandins, endothelin-1, complement proteins, and matrix metalloproteinase-9 by microvascular endothelial cells are other components in the complex mechanism of brain ischemia/hypoxia.4. Ultrastructural studies documented the opened paracellular avenues in the course of vasogenic edema in different experimental models.5. Tight junctions of endothelial cells have been characterized with freeze fracture electron microscopy, and the process of transvesiculation was analyzed using rapid freeze and freeze substitution procedure before electron microscopy studies.6. In endothelial cell-culture experiments, we used rodent and later human brains.7. Endothelial cells co-cultured with astroglia resulted in an elaborate tight junctional complex.8. This co-culture technique becomes the basis of in vitro blood–brain barrier studies. On endothelial cells of human brain origin, different regulatory factors found to be responsible for the complex mechanism of ischemic stroke.This paper is dedicated to the memory of F. Joó, the good friend and pioneer in endothelial cell research.This revised article was published online in May 2005 with a February 2005 cover date.     

Effect of Propofol Post-treatment on Blood–Brain Barrier Integrity and Cerebral Edema After Transient Cerebral Ischemia in Rats

Although propofol has been reported to offer neuroprotection against cerebral ischemia injury, its impact on cerebral edema following ischemia is not clear. The objective of this investigation is to evaluate the effects of propofol post-treatment on blood–brain barrier (BBB) integrity and cerebral edema after transient cerebral ischemia and its mechanism of action, focusing on modulation of aquaporins (AQPs), matrix metalloproteinases (MMPs), and hypoxia inducible factor (HIF)-1α. Cerebral ischemia was induced in male Sprague–Dawley rats (n = 78) by occlusion of the right middle cerebral artery for 1 h. For post-treatment with propofol, 1 mg kg^?1 min^?1 of propofol was administered for 1 h from the start of reperfusion. Nineteen rats undergoing sham surgery were also included in the investigation. Edema and BBB integrity were assessed by quantification of cerebral water content and extravasation of Evans blue, respectively, following 24 h of reperfusion. In addition, the expression of AQP-1, AQP-4, MMP-2, and MMP-9 was determined 24 h after reperfusion and the expression of HIF-1α was determined 8 h after reperfusion. Propofol post-treatment significantly reduced cerebral edema (P < 0.05) and BBB disruption (P < 0.05) compared with the saline-treated control. The expression of AQP-1, AQP-4, MMP-2, and MMP-9 at 24 h and of HIF-1α at 8 h following ischemia/reperfusion was significantly suppressed in the propofol post-treatment group (P < 0.05). Propofol post-treatment attenuated cerebral edema after transient cerebral ischemia, in association with reduced expression of AQP-1, AQP-4, MMP-2, and MMP-9. The decreased expression of AQPs and MMPs after propofol post-treatment might result from suppression of HIF-1α expression.     

Lifelong Consumption of Sodium Selenite: Gender Differences on Blood–Brain Barrier Permeability in Convulsive,Hypoglycemic Rats

The aim of this study was to compare the effects of hypoglycemia and induced convulsions on the blood-brain barrier permeability in rats with or without lifelong administration of sodium selenite. There is a significant decrease of the blood-brain barrier permeability in three brain regions of convulsive, hypoglycemic male rats treated with sodium selenite when compared to sex-matched untreated rats (p<0.05), but the decrease was not significant in female rats (p>0.05). The blood-brain barrier permeability of the left and right hemispheres of untreated, moderately hypoglycemic convulsive rats of both genders was better than their untreated counterparts (p<0.05). Our results suggest that moderate hypoglycemia and lifelong treatment with sodium selenite have a protective effect against blood-brain barrier permeability during convulsions and that the effects of sodium selenite are gender-dependent.     

The Influence of Oxygen on [NiFe]–Hydrogenase Cofactor Biosynthesis and How Ligation of Carbon Monoxide Precedes Cyanation

The class of [NiFe]–hydrogenases is characterized by a bimetallic cofactor comprising low–spin nickel and iron ions, the latter of which is modified with a single carbon monoxide (CO) and two cyanide (CN⁻) molecules. Generation of these ligands in vivo requires a complex maturation apparatus in which the HypC–HypD complex acts as a ‘construction site’ for the Fe–(CN)₂CO portion of the cofactor. The order of addition of the CO and CN^– ligands determines the ultimate structure and catalytic efficiency of the cofactor; however much debate surrounds the succession of events. Here, we present an FT–IR spectroscopic analysis of HypC–HypD isolated from a hydrogenase–competent wild–type strain of Escherichia coli. In contrast to previously reported samples, HypC–HypD showed spectral contributions indicative of an electron–rich Fe–CO cofactor, at the same time lacking any Fe–CN^– signatures. This immature iron site binds external CO and undergoes oxidative damage when in contact with O₂. Binding of CO protects the site against loss of spectral features associated with O₂ damage. Our findings strongly suggest that CO ligation precedes cyanation in vivo. Furthermore, the results provide a rationale for the deleterious effects of O₂ on in vivo cofactor biosynthesis.     

Effect of Baicalin on Matrix Metalloproteinase-9 Expression and Blood–Brain Barrier Permeability Following Focal Cerebral Ischemia in Rats

Focal cerebral ischemia results in an increased expression of matrix metalloproteinase-9 (MMP-9), which induces vasogenic brain edema via disrupting the blood–brain barrier (BBB) integrity. Recent studies from our laboratory showed that baicalin reduces ischemic brain damage by inhibiting inflammatory reaction and neuronal apoptosis in a rat model of focal cerebral ischemia. In the present study, we first explored the effect of baicalin on the neuronal damage, brain edema and BBB permeability, then further investigated its potential mechanisms. Sprague–Dawley rats underwent permanent middle cerebral artery occlusion (MCAO). Baicalin was administrated by intraperitoneally injected twice at 2 and 12 h after the onset of MCAO. Neuronal damage, brain edema and BBB permeability were measured 24 h following MCAO. Expression of MMP-9 protein and mRNA were determined by western blot and RT–PCR, respectively. Expression of tight junction protein (TJP) occludin was detected by western blot. Neuronal damage, brain edema and BBB permeability were significantly reduced by baicalin administration following focal cerebral ischemia. Elevated expression of MMP-9 protein and mRNA were significantly down-regulated by baicalin administration. In addition, MCAO caused the decreased expression of occludin, which was significantly up-regulated by baicalin administration. Our study suggested that baicalin reduces MCAO-induced neuronal damage, brain edema and BBB permeability, which might be associated with the inhibition of MMP-9 expression and MMP-9-mediated occludin degradation.     

Single Administration of Tripeptide α-MSH(11–13) Attenuates Brain Damage by Reduced Inflammation and Apoptosis after Experimental Traumatic Brain Injury in Mice

Following traumatic brain injury (TBI) neuroinflammatory processes promote neuronal cell loss. Alpha-melanocyte-stimulating hormone (α-MSH) is a neuropeptide with immunomodulatory properties, which may offer neuroprotection. Due to short half-life and pigmentary side-effects of α-MSH, the C-terminal tripeptide α-MSH(11–13) may be an anti-inflammatory alternative. The present study investigated the mRNA concentrations of the precursor hormone proopiomelanocortin (POMC) and of melanocortin receptors 1 and 4 (MC1R/MC4R) in naive mice and 15 min, 6, 12, 24, and 48 h after controlled cortical impact (CCI). Regulation of POMC and MC4R expression did not change after trauma, while MC1R levels increased over time with a 3-fold maximum at 12 h compared to naive brain tissue. The effect of α-MSH(11–13) on secondary lesion volume determined in cresyl violet stained sections (intraperitoneal injection 30 min after insult of 1 mg/kg α-MSH(11–13) or 0.9% NaCl) showed a considerable smaller trauma in α-MSH(11–13) injected mice. The expression of the inflammatory markers TNF-α and IL-1β as well as the total amount of Iba-1 positive cells were not reduced. However, cell branch counting of Iba-1 positive cells revealed a reduced activation of microglia. Furthermore, tripeptide injection reduced neuronal apoptosis analyzed by cleaved caspase-3 and NeuN staining. Based on the results single α-MSH(11–13) administration offers a promising neuroprotective property by modulation of inflammation and prevention of apoptosis after traumatic brain injury.     

The Effect of Butylphthalide on the Brain Edema,Blood–Brain Barrier of Rats After Focal Cerebral Infarction and the Expression of Rho A

The aim of this study was to explore the effect of butylphthalide on the brain edema, blood–brain barrier of rats of rats after focal cerebral infarction and the expression of Rho A. A total of 195 sprague–dawley male rats were randomly divided into control group, model group, and butylphthalide group (40 mg/kg, once a day, by gavage). The model was made by photochemical method. After surgery 3, 12, 24, 72, and 144 h, brain water content was done to see the effect of butylphthalide for the cerebral edema. Evans blue extravasation method was done to see the changes in blood–brain barrier immunohistochemistry, and Western blot was done to see the expression of Rho A around the infarction. Compared with the control group, the brain water content of model group and butylphthalide group rats was increased, the permeability of blood–brain barrier of model group and butylphthalide group rats was increased, and the Rho A protein of model group and butylphthalide group rats was increased. Compared with the model group, the brain water content of butylphthalide group rats was induced (73.67 ± 0.67 vs 74.14 ± 0.46; 74.89 ± 0.57 vs 75.61 ± 0.52; 77.49 ± 0.34 vs 79.33 ± 0.49; 76.31 ± 0.56 vs 78.01 ± 0.48; 72.36 ± 0.44 vs 73.12 ± 0.73; P < 0.05), the permeability of blood–brain barrier of butylphthalide group rats was induced (319.20 ± 8.11 vs 394.60 ± 6.19; 210.40 ± 9.56 vs 266.40 ± 7.99; 188.00 ± 9.22 vs 232.40 ± 7.89; 288.40 ± 7.86 vs 336.00 ± 6.71; 166.60 ± 6.23 vs 213.60 ± 13.79; P < 0.05), and the Rho A protein of butylphthalide group rats was decreased (western blot result: 1.2230 ± 0.0254 vs 1.3970 ± 0.0276; 1.5985 ± 0.0206 vs 2.0368 ± 0.0179; 1.4229 ± 0.0167 vs 1.7930 ± 0.0158;1.3126 ± 0.0236 vs 1.5471 ± 0.0158; P < 0.05). The butylphthalide could reduce the brain edema, protect the blood–brain barrier, and decrease the expression of Rho A around the infarction.     

Effects of Peroxidation Products of Linoleic Acid on Tryptophan–nicotinamide Metabolism in Rats

The flavin and pyridine nucleotide coenzymes are involved in the detoxication of autoxidation products of lipids. In tryptophan-nicotinamide metabolism, kynurenine 3-hydroxylase and N¹-methylnicotinamide (MNA) oxidase contain FAD as a coenzyme. So, the effects of dietary autoxidation products of linoleic acid on the metabolism of tryptophan-nicotinamide were investigated using rats. The administration of linoleic acid hydroperoxides or secondary products reduced the urinary excretion of xanthurenic acid, nicotinamide and its metabolites such as MNA, N¹-methyl-2-pyridone-5-carboxamide (2-Py), and N¹-methyl-4-pyridone-3-carboxamide (4-Py) as compared with the group administered saline or linoleic acid. Among the enzyme activities involved in the tryptophan-nicotinamide metabolism, the activity of NAD⁺ synthetase was decreased by the administration of linoleic acid hydroperoxides or secondary products. The activities of tryptophan oxygenase and 4-Py-forming MNA oxidase were also decreased by the administration of secondary products. These results indicate that the conversion of tryptophan to nicotinamide would be lower in the groups administered the hydroperoxides and secondary products than in saline and linoleic acid groups.     

Effects of Fractions 1–10 kDa from Brain of the Black Bear Ursus arctos on Rats and Mice

There were studied effects of administration into brain ventricle of Wistar rats of the 1–10 kDa fractions (BAF) isolated from the brain of the black bear Ursus arctos caught in winter at exit from the den after the provoked awakening. Injection of BAF at a doze of 0.1 mg decreased motor activity and produced a sleep-like state of rats. Phases of the slow-wave EEG activity, with an enhancement of - and suppression of - and -frequencies, alternated with -rhythm periods. An intranasal BAF injection to white breedless mice previously cooled to 17–19°C under conditions of hypoxia-hypercapnia decelerated the exit of the mice from the hypothermia state.     

Modulation of Paraoxonase 2 (PON2) in Mouse Brain by the Polyphenol Quercetin: A Mechanism of Neuroprotection?

Quercetin is a common flavonoid polyphenol which has been shown to exert neuroprotective actions in vitro and in vivo. Though quercetin has antioxidant properties, it has been suggested that neuroprotection may be ascribed to its ability of inducing the cell’s own defense mechanisms. The present study investigated whether quercetin could increase the levels of paraoxonase 2 (PON2), a mitochondrial enzyme expressed in brain cells, which has been shown to have potent antioxidant properties. PON2 protein, mRNA, and lactonase activity were highest in mouse striatal astrocytes. Quercetin increased PON2 levels, possibly by activating the JNK/AP-1 pathway. The increased PON2 levels induced by quercetin resulted in decreased oxidative stress and ensuing toxicity induced by two oxidants. The neuroprotective effect of quercetin was significantly diminished in cells from PON2 knockout mice. These findings suggest that induction of PON2 by quercetin represents an important mechanism by which this polyphenol may exert its neuroprotective action.