Anoxia Group Research Articles

Melatonin attenuates developmental deficits and prevents hippocampal injuries in male and female rats subjected to neonatal anoxia.

Hypoxia in preterm infants is a clinical condition that has been associated with cognitive and behavioral disturbances for which treatment strategies are strongly required. Melatonin administration following brain insults has been considered a promising therapeutic strategy due to its antioxidant and anti-inflammatory effects. Not surprisingly, it has been extensively studied for preventing disturbances following brain injury. This study evaluated the effects of melatonin on developmental disturbances, memory disruption, and hippocampal cell loss induced by neonatal anoxia in rats. Neonatal Wistar rats were subjected to anoxia and subsequently treated with melatonin. Later, maturation of physical characteristics, ontogeny of reflexes, learning and memory in the Morris water maze (MWM), and estimates of the number of hippocampal neurons, were evaluated. Melatonin treatment attenuated (1) female anoxia-induced delay in superior incisor eruption, (2) female anoxia-induced vibrissae placement reflexes, and (3) male and female anoxia-induced hippocampal neuronal loss. Melatonin also promoted an increase (5) in swimming speeds in the MWM. In addition, PCA analysis showed positive associations between the acoustic startle, auditory canal open, and free fall righting parameters and negative associations between the male vehicle anoxia group and the male melatonin anoxia group. Therefore, melatonin treatment attenuates both anoxia-induced developmental deficits and hippocampal neuronal loss.

Effects of Stocking Density and Pre-Slaughter Handling on the Fillet Quality of Largemouth Bass (Micropterus salmoides): Implications for Fish Welfare.

There is currently insufficient acknowledgment of the relationship between fish welfare and ultimate fillet quality. The purpose of this study was to assess the impacts of pre-slaughter handling and stocking density as fish welfare markers on fillet quality of largemouth bass (Micropterus salmoides). Fish from three stocking densities of 35, 50, and 65 kg·m-3 were reared in a recirculating aquaculture system (RAS) for 12 weeks and received commercial feed. Ultimately, the fish were either stunned with percussion on the head (control group) or subjected to air exposure for 3 min (anoxia group) before stunning and subsequent collection of blood and fillet samples. Western blot analysis revealed the degradation of actin in both groups. Additionally, higher oxidation progress and lower hardness and pH were observed in anoxia compared to the control group. We observed higher hardness at 35 kg·m-3 in anoxia compared to 50 and 65 km-3. The initial hardness values at 35, 50, and 65 km-3 were 1073, 841, and 813 (g) respectively in the anoxia group. Furthermore, the anoxia and control groups had rigor mortis after 6 and 10 h, respectively. Cortisol and glucose levels, and oxidative enzymes activity were higher in anoxia than in the control group. In conclusion, oxidation induced by anoxia likely plays a crucial role as a promoter of the quality deterioration of largemouth bass fillets.

Pathophysiological aspects of neonatal anoxia and temporal expression of S100β in different brain regions.

The aim of this study was to investigate the temporal variations of S100β in the hippocampus, cerebellum and cerebral cortex of neonatal rats (Wistar strain) under anoxic conditions. Real-time PCR and western blotting techniques were used for gene expression and protein analysis. Animals were divided into two groups, a control group and an anoxic group, and further separated at different time points for analysis. After anoxia, S100β gene expression showed a significant peak in the hippocampus and cerebellum after 2 h, followed by a decline compared to the control group at other time points. The increased gene expression in these regions was also accompanied by an increase in S100β protein levels in the anoxia group, observable 4 h after injury. In contrast, S100β mRNA content in the cerebral cortex never exceeded control values at any time point. Similarly, the protein content of S100β in the cerebral cortex did not show statistically significant differences compared to control animals at any assessment time point. These results suggest that the production profile of S100β differs by brain region and developmental stage. The observed differences in vulnerability between the hippocampus, cerebellum and cerebral cortex may be attributed to their distinct developmental periods. The hippocampus and cerebellum, which develop earlier than the cerebral cortex, showed more pronounced effects in response to anoxia, which is supported by the gene expression and protein content in this study. This result reveals the brain region-dependent nature of S100β as a biomarker of brain injury.

Neonatal Anoxia Impacts Rats’ Maternal Behavior and Offspring Development due to Ultrasonic Vocalization’s Impairment

Hypoxic-ischemic encephalopathy is a severe clinical condition, among others, affecting the brain after offspring exposure to neonatal anoxia, which causes persistent sensorimotor and cognitive deficits. During peripartum, maternal behaviors are crucial for the healthy development of the offspring. In rats, the vocalization of newborns, around 40 kHz, corresponds to separation calls that encourage their mothers to retrieve them. Alterations in this pattern affect the maternal behavior addressed to the offspring. This study aimed to evaluate the maternal behavior of primiparous rats whose offspring were exposed to neonatal anoxia in P2 (postpartum day) during the lactation period, to assess mother-pup interactions through the pups’ vocalization from P3 to P18. It also intends to quantify eventual neuronal alterations in the mothers’ medial preoptic area after the last weaning (P21) through FOS protein expression. Anoxia offspring were found to reduce maternal behaviors toward them, increased frequency of separation calls in the male anoxia group, and reduced vocalization rate in the female anoxia group compared to their respective controls. Body weight gain reduction of males’ and females’ anoxia was observed. We concluded that anoxia exerts deleterious effects on the vocalization patterns of the pups, with sex differences that alter maternal behavior toward them. Impaired USV makes an additional negative impact on the already noxious effects of neonatal anoxia. Understanding those phenomena applies/contributes to guiding procedures and strategies to mitigate the deleterious outcomes and orient research concerning the complexity of neonatal anoxia events and the influence of maternal care quality concerning the pups, which should also be considered sex differences.

Anoxia at Birth Induces Behaviorally-Relevant Changes in alpha2-Noradrenergic Receptor Binding in the Adult Rat

Evidence suggests that Caesarean section birth in the rat, with or without an additional period of anoxia, results in long-term changes in brain catecholamine levels as well as reactivity to stress. The purpose of the present investigation was to determine whether Caesarean birth plus anoxia alters (alpha)2-noradrenergic receptor binding and sensitivity to the (alpha)2 receptor agonist, clonidine, in the Porsolt forced swim test. Sprague Dawley rat dams were decapitated and the uteruses were removed by Caesarean section. Pups were then delivered either immediately (Caesarean Only group), or were immersed in a saline bath for approximately 15 minutes (Caesarean plus Anoxia group) before delivery of the pups. A third group of animals born vaginally served as controls (Vaginally-born group). Four to five months postnatally, the expression of (alpha)2 receptors was measured by receptor autoradiography using [3H]-Rauwolscine binding. Receptor binding was increased in the area of the ventral hypothalamus and decreased in the CA1 region of the hippocampus in animals subjected to Caesarean plus Anoxia at birth. These animals also displayed a subsensitive response to the immobilizing effects of clonidine (100 micrograms/kg, i.p.) in the Porsolt forced swim test. Specifically these data show that Caesarean birth produces long-term changes in (alpha)2 receptor density and that, in animals subjected to Caesarean plus anoxia, these changes are reflected in a behavioral subsensitivity to the (alpha)2 agonist, clonidine. The findings reported here provide further experimental support for the hypothesis that birth complications may contribute to the pathophysiology of disorders such as schizophrenia that involve central catecholamine dysfunction.

Effect of ketamine on mitochondrial function of rat neurons subjected to anoxia

Objective To investigate the effect of ketamine on the mitochondrial function of rat neurons subjected to anoxia. Methods Primarily cultured rat hippocampal neurons were seeded in culture dishes(35 mm in diameter)at the density of 5×105-1×106 cells/ml, and divided into 3 groups(n=11 each)using a random number table: control group, anoxia group and ketamine group.The neurons were exposed to 90% N2 plus 10% CO2 50 ml/min for 5 min in anoxia group.In ketamine group, ketamine was added to the culture medium with the final concentration of 20 μmol/L at 1 h before anoxia, and then the neurons were exposed to 90% N2 plus 10% CO2 50 ml/min for 5 min.After the end of treatment in each group, the dead neurons were detected using trypan blue staining, the ATP content was determined by ATP bioluminescence assay, and mitochondrial membrane potential was measured by rhodamine 123 staining. Results Compared with control group, the mortality rate of hippocampal neurons was significantly increased, and the ATP content and mitochondrial membrane potential were significantly decreased in anoxia group and ketamine group(P<0.05). Compared with anoxia group, the mortality rate of hippocampal neurons was significantly decreased, and the ATP content and mitochondrial membrane potential were significantly increased in ketamine group(P<0.05). Conclusion The mechanism by which ketamine ameliorates anoxia-induced damage to rat neurons is related to improved mitochondrial function. Key words: Ketamine; Hypoxia, brain; Neurons; Mitochondria

Negative regulation of exogeneous polyl-4-hydroxylase domain proteins on hypoxia-inducible factor pathway in human RPE cells

Background Anti-VEGF drugs are generally applied in the treatment of ocular neovascular diseases.However, the therapy effect is unsatisfactory in some patients.Studing the effect of hypoxia-inducible factor-1 (HIF-1), a upstream regulatory gene of VEGF, and its limiting enzyme prolyl-4-hydroxylase domain proteins (PHDs) is of important clinical significance. Objective This study was to investigate the negtive regulation of exogeneous PHDs on HIF-1 pathway in human RPE cells. Methods pFLAG-PHD1, pFLAG-PHD2 and pFLAG-PHD3 plasmids were constructed by extracting RNA from Hela cell line and coloning PHD1, PHD2 and PHD3 using reverse transcription PCR with restriction enzyme.The plasmids were identified by gene sequencing.ARPE-19 cells were cultured at 21% O2 (normoxia group), 1% O2 (hypoxia group), or in hypoxia-mimicking agents (CoCl2, anoxia group), respectively, and then were transfected with plasmids encoding FLAG-tagged PHD1, PHD2, PHD3 and pFLAG-CMV2 transfected cells served as blank control.The expressional intensities of PHD1, PHD2 and PHD3 in the cells were detected and compared among different groups by using Western blot assay.The transcriptional activity of HIF-1 in the cells was evaluated with dual luciferase reporter assay. Results Western blot assay showed that PHD1, PHD2 and PHD3 all were expressed in ARPE-19 cells in the normoxia group, hypoxia group and anoxia group.The expression was strong in PHD2 protein and was weak in PHD3 protein, a statistically significant difference was found between PHD2 protein expression and PHD1 or PHD3 expressions (all at P<0.05). Endogenous HIF-1 activity was elevated in pFLAG-CMX transfected cells in the hypoxia group and anoxia group than that in the normoxia group.Compared with pFLAG-CMX transfected cells, no obvious change was seen in the endogenous HIF-1 activity in the normoxia group, however, HIF-1 activity was declined in the hypoxia group and anoxia group after pFLAG-PHD1, pFLAG-PHD2 or pFLAG-PHD3 transfection.Under the same oxygen environment, HIF-1 activity was lower in the pFLAG-PHD2 transfected cells than that in the pFLAG-PHD1 or pFLAG-PHD3 transfected cells (both at P<0.05). Conclusions PHDs play a negative regulation to HIF-1 activating pathway in human RPE cells, especially in hypoxia and anoxia cells.Among PHDs proteins, PHD2 presents the strongest inhibition on HIF-1 activating pathway. Key words: Cell hypoxia; Vascular endothelial growth factors; Gene expression regulation; Hypoxia-inducible factor-1; Procollagen-proline dioxygenase/metabolism; Humans; Transcription factors; Epithelial cells, retinal

The psychosocial outcomes of anoxic brain injury following cardiac arrest

Aim of the studyThis exploratory study aimed to investigate the psychosocial outcomes for cardiac arrest survivors and explore if there is a greater impact on psychosocial outcome for individuals experiencing anoxic brain injury as a result of the cardiac arrest. MethodsSelf-report measures were used to compare the quality of life, social functioning and symptoms of anxiety, depression and post-traumatic stress of individuals with and without anoxic brain injury. Secondary measures of subjective memory and executive difficulties were also used. Fifty-six participants (27 with anoxia, 29 without anoxia) took part in the study between six months and four years after experiencing cardiac arrest. ResultsA MANOVA identified a significant difference between the two groups, with the anoxia group reporting more psychosocial difficulties. They reported more social functioning difficulties and more anxiety, depression and post-traumatic stress symptoms. There was, however, no significant difference in self-reported quality of life between the two groups. ConclusionAs the first known study to compare psychosocial outcomes for cardiac arrest survivors experiencing anoxic brain injury with those without anoxia, the current results suggest that cardiac arrest survivors with subsequent acquired brain injury experience more psychosocial difficulties. This could be due to a combination of neuropsychological, social and psychological factors.

A modified rat model of neonatal anoxia: Development and evaluation by pulseoximetry, arterial gasometry and Fos immunoreactivity

Neonatal anoxia is a worldwide clinical problem that has serious and lasting consequences. The diversity of models does not allow complete reproducibility, so a standardized model is needed. In this study, we developed a rat model of neonatal anoxia that utilizes a semi-hermetic system suitable for oxygen deprivation. The validity of this model was confirmed using pulse oximetry, arterial gasometry, observation of skin color and behavior and analysis of Fos immunoreactivity in brain regions that function in respiratory control. For these experiments, 87 male albino neonate rats ( Rattus norvegicus, lineage Wistar) aged approximate 30 postnatal hours were divided into anoxia and control groups. The pups were kept in an euthanasia polycarbonate chamber at 36 ± 1 °C, with continuous 100% nitrogen gas flow at 3 L/min and 101.7 kPa for 25 min. The peripheral arterial oxygen saturation of the anoxia group decreased 75% from its initial value. Decreased pH and partial pressure of oxygen and increased partial pressure of carbon dioxide were observed in this group, indicating metabolic acidosis, hypoxia and hypercapnia, respectively. Analysis of neuronal activation showed Fos immunoreactivity in the solitary tract nucleus, the lateral reticular nucleus and the area postrema, confirming that those conditions activated areas related to respiratory control in the nervous system. Therefore, the proposed model of neonatal anoxia allows standardization and precise control of the anoxic condition, which should be of great value in indentifying both the mechanisms underlying neonatal anoxia and novel therapeutic strategies to combat or prevent this widespread public health problem.

The change of F-actin and actin binding protein in intstinal epithelial cells under the ischemia and anoxia injury

Objective To investigate the relationship between the apoptosis and the change of F-actin and actin binding protein in intstinal epithelial cells (IEC) under the ischemia and anoxia injury, and the effect of calcium channel antagonist. Methods Primary culture of rat IECs and ischemia and an-oxia injury model in vitro were established. IECs were divided into 8 groups : group A ( control group), group B (ischemia group) ,group C (anoxia group) ,group D (ischemia and anoxia group) ,group A1 ( A +2 mg/L Verapamil) ,group B1 (B +2 mg/L Verapamil) ,group C1 (C +2 mg/L Verapamil) ,group D1 ( D + 2 mg/L Verapamil). Apoptosis rate (AR) was determined by flow cytometry. F-actin, talin and α-ac-tin were observed by LSCM. Results The AR in groups B, C and D was significantly higher than in group A (P < 0.05). The AR in groups B1, C1 and D1 was lower than in groups B. C and D ( P < 0.05 ). The form and arragement of F-actin, talin and α-actin in groups B, C and D were changed with the decrease of fluorescence intensity compared with group A (P < 0.01 ), the most significant in group D. The change in groups B1, C1 and D1 was reduced and the fluorescence intensity was increased as compared with the cor-responding injury groups (P < 0.05). Conclusion The ischemia and anoxia injury results in the break-down of F-actin and actin binding protein, which is related to the apotosis of IEC. Calcium channel antago-nist can decrease AR and reduce the change of these cytoskeleton. Key words: Intestinal epithelial ceils ; Ischemia; F-actin

Variation of magnetic resonance spectroscopy of lactate, creatinine and N-acetylaspartate in newborn after intrauterine fetal anoxia

目的 通过对活体脑组织的代谢监测,了解脑组织早期缺氧的损伤情况.方法 采用新生儿行为神经评分(neonatal behviors neurological assessment,NBNA)对12例有宫内窘迫的新生儿及对照组新生儿进行评估,采用磁共振波谱扫描观察脑组织乳酸(lactate,Lac)、肌苷(creatinine,Cr)、N-乙酰天冬氨酸(N-acetylaspartate,NAA)等改变. 结果窘迫组新生儿NBNA评分(35.3±1.37)明显低于对照组(38.8±0.75)(t=3.25,P＜0.05).两组间磁共振波谱Cr峰值比较差异无统计学意义(P＞0.05),窘迫组基底核及颞叶皮质中Lac峰值及Lac/Cr比值明显高于对照组(P＜0.01),两组间NAA峰值及NAA/Cr比值比较差异无统计学意义(P＞0.05).窘迫组NBNA评分分值与基底核Lac/Cr比值呈负相关(r=-0.547,P＜0.05).结论基底核等磁共振波谱Lac峰值及Lac/Cr比值可能在新生儿脑组织缺氧后早期发生变化。

Effect of hypoxia on infiltration and migration of lung cancer cells and expression of MMP-2 and TIMP-2

It has been known that different degrees of hypoxia can produce different effects on tumor. The study is to investigate the effect of hypoxia on the infiltration and migration of lung adenocarcinoma cells. Lung adenocarcinoma cells were exposed to normoxic (air, 5%O₂), hypoxic (1%O₂, 5%CO₂, 94%N₂) or anoxic (95%N₂, 5%CO₂) condition for 48 hours. The migration ability of the cells was assayed by wound healing methods. The infiltration ability was assayed by HABM-HEM model. The cells exposed to hypoxia were planted subcutaneously in nude mice, and the growth of cells and the rate of metastasis to lymph node or lung were observed. The levels of matrix metalloproteinase 2 (MMP-2) and tissue inhibitor of metalloproteinase II (TIMP-2) in culture media were assayed by ELISA. Comparing with normoxia group, the infiltration, migration and metastasis of hypoxia group were increased significantly (P < 0.05) , as well as the level of MMP-2 (P < 0.01), and the TIMP-2 level was remarkably decreased (P < 0.05) . In anoxia group, the levels of MMP-2 and TIMP-2 were both significantly decreased (P < 0.01). Moderate hypoxia can up-regulate the expression of MMP-2, down-regulate the expression of TIMP-2, and increase the infiltration and migration of lung cancer cells. But serious hypoxia can decrease the expression of MMP-2 and TIMP-2, and inhibit the proliferation, infiltration and migration of lung cancer cells.

A study on thestunning phenomenon of nerve cells after transient anoxia-serum deprivation/reperfusion

：Objective To identifywhether there is nerve cells stunning after transient anoxia-serum deprivation/reperfusionand to explore the probable underlying mechanism of the phenomenon.Methods The PC12 cellswere randomly divided into a control group and a anoxia group, which were divided into 3subgroups respectively, according to the time of reperfusion after 15 min of anoxia-serumdeprivation. The content of adenosine triphosphate, mitochondria membrane potential andcell activity were determined at different time points after transient anoxia-serumdeprivation/reperfusion.Results The content of adenosine triphosphate(1.05±0.34),mitochondria membrane potential (41.39±1.242) and cell activity after 15 min ofanoxia-serum deprivation plus 1h of reperfusion in the anoxia group were significantlylower than those in the control group(P ＜0.05), and recovered to level of the control group after6h.Conclusion There was lower energy status after transient of anoxia-serumdeprivation/reperfusion, and the lower energy status can recover to the normal level.There might be nerve cell stunning. This phenomenon probably results from lower succinatedehydrogenase activity in the respiratory chain of the cells and the changes ofmitochondria membrane potential. Key words: Anoxia; Serum deprivation; Reperfusion; Nerve cell stunning; Energymetabolism

Social function changes in children and adolescents with acquired brain injury during inpatient rehabilitation.

To examine changes in social function capabilities between and within groups of children with six subtypes of acquired brain injury during inpatient rehabilitation. The Pediatric Evaluation of Disability Inventory (PEDI) Social Functional Skills Scale was administered to 139 children with acquired brain injury (mean age=9.28 years; SD = 5.27). Scaled summary score changes from hospital admission to discharge for the total group and six diagnostic sub-groups were calculated. One-way ANOVAs were performed for the total group and six sub-groups to determine between group differences in amount of change. Within-group changes were examined by paired t-tests, effect sizes and the proportion of children who made a clinically meaningful change (> or = 10 points). Children with traumatic brain injury demonstrated the greatest amount of change during inpatient rehabilitation, but only significantly greater than children with seizures. Scores were significantly higher at discharge for the total group (p < 0.001) and all sub-groups except for the anoxia group. Forty-five per cent of all children made clinically meaningful changes. Children with acquired brain injury made significant changes in social functioning from admission to discharge. Varying degrees of change existed for diagnostic sub-groups. Further research is needed to examine clinically meaningful changes and in which areas of social functioning children are changing during inpatient rehabilitation.

Birth Insult Alters Dopamine-Mediated Behavior in a Precocial Species, the Guinea Pig: Implications for Schizophrenia

Schizophrenia is associated with increased birth complications, suggesting that birth complications might alter CNS dopaminergic activity later in life. In rats, Caesarean section (C-section) birth can produce long term changes in dopaminergic biochemistry and behavior. However rat brain is somewhat immature compared to human brain at birth. The current study tested if mild birth complications also alter dopamine-mediated function in a species with a more mature CNS at birth, the guinea pig. As adults, guinea pigs born by C-section showed increased amphetamine-induced locomotion and disruption of prepulse inhibition (PPI) of acoustic startle, compared to vaginally born controls. Guinea pigs born by C-section with 1 min of added global anoxia showed reduced amphetamine-induced locomotion and disrupted PPI, while a C-section plus 2 min anoxia group showed no change in amphetamine-induced locomotion but increased amphetamine-induced startle. No group differences in effects of amphetamine or apomorphine on PPI were observed. Taken with previous findings, these results indicate that mild birth complications can cause long term changes in dopamine-mediated behavior in both guinea pig and rat, two species spanning the level of human brain maturity at birth.

The effect of mannitol upon cochlear dysfunction induced by transient local anoxia

Transient local anoxia of the cochlea was induced by pressing the labyrinthine artery, and compound action potential (CAP) or endocochlear potential (EP) was measured before and after transient local anoxia ranging from 5 to 60 min using 106 albino guinea pigs. The complete interruption of the cochlear blood flow by this procedure and its full restoration after releasing the pressure on the artery was confirmed by a laser-Doppler flowmeter. The anoxia of less than 10 min induced no post-anoxic cochlear dysfunction, whereas the anoxia of a longer duration induced an irreversible dysfunction of the cochlea. It was evident that the post-anoxic recovery of the CAP threshold was worse as the anoxia period was prolonged, and CAP was almost completely abolished after 60-min anoxia. In animals which were administered mannitol intravenously just after the restoration of the cochlear blood circulation, the recovery of the CAP threshold was significantly better than that in the control animals, when the animals were subjected to local anoxia of 15- to 30-min duration. No beneficial effect, however, was observed in the 60-min anoxia group. In conclusion, local anoxia of 10 min or longer caused cochlear dysfunction, which was partially but significantly alleviated by mannitol.

Sparing by Rasagiline (Tvp-1012) of Cholinergic Functions and Behavior in the Postnatal Anoxia Rat

Rasagiline (N-propargyl-1(R)aminoindan) is a selective and potent MAO-B inhibitor currently under development as the mesylate salt (TVP-1012) for the treatment of various neurologic disorders. Preliminary work in adult and senescent rats, either normal or hypoxia-lesioned, showed that chronic rasagiline treatment improved performance in memory and learning tasks, suggesting some beneficial effect on central cholinergic function. We have now used the postnatal anoxia-lesioned rat as a model of cholinergic dysfunction. In the neonatal rat, anoxia strongly affects the cholinergic system, which has not yet reached full maturation at this state of life. Rasagiline mesylate was administered from day 1 to completion of the study (day 60), first through nursing mother milk until weaning (day 21), then in drinking water, at the rate of 0.5 mg/kg/day. Drug access to the CNS was verified by analysis of MAO activity in brain (at 21 days). Treatment improved the juvenile hyperactivity syndrome associated with anoxia (at day 28). It improved performance in the passive avoidance test to normal control level (at day 40). It improved spatial memory performance in the Morris water maze to normal control level (at day 50). The untreated anoxia group failed in these tasks and was significantly inferior to either the normal control and rasagiline-treated anoxia groups. Determination of ChAT activity in the caudate and hippocampus of rats from each of these groups gave the following results (pmol ACh/mg protein/min). Caudate: normal control, 588 ± 56; anoxia, 398 ± 54; rasagiline-treated anoxia, 536 ± 35. Hippocampus: normal control, 380 ± 31; anoxia, 275 ± 47; rasagiline-treated anoxia, 325 ± 35. Results are mean ± SD from each of seven to nine different donors in a group. Thus, improvement in memory and learning tasks of the rasagiline-treated anoxia group finds correspondence in the activity of the cholinergic marker ChAT in two brain regions that have prominent cholinergic innervation.

Age dependence of tolerance to anoxia and changes in cytosolic calcium in rabbit renal proximal tubules.

Calcium(Ca2+)-dependent processes mediate, in part, anoxic cell injury. These may account for the difference in sensitivity to anoxia between certain immature and mature renal cells. To address this question, we studied the effects of anoxia on cytosolic free Ca2+ concentration ([Ca2+]i), cell integrity, and transport functions in microdissected proximal convoluted tubules (PCT) of < 3-week-old (newborn) and > 12-week-old (adult) rabbits. Tubules were loaded with 10 microM fura-2 AM by incubation for 60 min at 37 degrees C, and then superfused with isosmotic saline solution gassed with either 95%O2-5%CO2 (control group) or 95%N2-5%CO2 (anoxia group) for 30 min. [Ca2+]i was measured ratiometrically; cell damage was assessed by nuclear binding of propidium iodide (PI). Anoxia resulted in a fourfold increase in [Ca2+]i in adult tubules (from resting values of 245 +/- 10 to 975 +/- 100 nM, P < 0.001), whereas in newborn tubules the rise was significantly less (from resting values of 137 +/- 5 to 165 +/- 5 nM, P < 0.001 between anoxic groups). Transient exposure to 100 mM potassium chloride, which depolarizes the PCT cells, induced increases in [Ca2+]i from baseline, to 920 +/- 90 nM in tubules from adult and to 396 +/- 16 nM in those from newborn rabbits (P < 0.001 between age groups). After exposure to ligands such as parathyroid hormone (PTH) and ATP, [Ca2+]i increased in both newborn and adult tubules, but to lower levels in newborn tubules. The response to PTH and ATP was transient in both age groups, [Ca2+]i returning to baseline levels after 2 min. Following anoxia, tubules from adult animals exhibited staining of all cell nuclei by 1 min exposure to PI, indicative of gross permeabilization of the cells. Nuclei of anoxic immatures tubules did not stain with PI. The sodium-dependent uptakes of a glucose analogue (14C-alpha-methyl-glucopyranoside) and phosphate (32Pi) were preserved in agarose-filled tubules of newborns after anoxia, whereas in those of adults recovery from anoxia was associated with drastic reduction in the uptake of these solutes. Overall, our results suggest that: (1) during anoxia, cell Ca2+ rises to critical levels in PCTs of adults compared with those of < 3-week-old animals, (2) Ca2+ influx occurs via a pathway activated by exposure to high [K+]o, presumably voltage-sensitive Ca2+ channels or reversal of Na(+)-Ca2+ exchange, (3) these pathways are either less active or less abundant in proximal tubules of newborn compared with adult rabbits, and (4) secondary active transport activity and cellular integrity are well preserved after anoxia in PCT cells of newborn but not of adult rabbits.

Immunohistochemically demonstrated increase of prostaglandin F2-alpha in neurons after reoxygenation in anoxic rats

Immunohistochemical staining for prostaglandin F2-alpha (PG F2α) was conducted to identify PG F2α synthesizing or binding sites in anoxic rat brains. Anoxia was produced in 22 rats to lower the arterial oxygen tension (PaO2) to 21 ± 4 mmHg by ventilation with a 95% nitrogen and 5% carbon dioxide gas mitxture. In 8 animals anoxia was continued for 30 sec, and in 14 rats for 3 min. Prior to decapitation, 5 animals in the 30-sec anoxia group and 8 rats in the 3-min anoxia group were reoxygenated fo 5 min, while the remaining 9 were not. Five-min reoxygenation returned the PaO2 to 106 ± 7. Three non-reoxygenated and 3 reoxygenated rats, all pretreated with indomethacin, and 5 normal rats served as controls. The brains were snap-frozen. The cryosections were stained by the indirect immunofluorescence method. PG F2α was noted mainly in pial vessels in all normal rats. All reoxygenated rats showed a positive reaction not only in blood vessels, but also in neurons, particularly hippocampal neurons and Purkinje cells. The staining of the above neurons was noted to be less in non-reoxygenated rats. The stronger staining was observed in rats reoxygenated after 3-min anoxia than 30 sec anoxia. The indomethacin-pretreated rats showed almost no increase in staining intensity. The above results indicate that reoxygenation after anoxia results in an increase of PG F2α in neurons of both cerebrum and cerebellum.

Behavioral differences in the developing rat following postnatal anoxia or postnatally injected AF-64A, a cholinergic neurotoxin

Rat pups were submitted postnatally to one of two procedures: a 25-min exposure to 100% nitrogen or an i.c.v. bilateral injection of AF-64A, 2 nmol contained in 1-μl saline. Throughout further development of either group, their performance in passive and active avoidance tests and in amphetamine-induced stereotype behavior was followed and compared. Both groups exhibited hyperactivity which persisted until 42 days of age in the anoxia group and beyond 120 days in the AF-64A group. Both groups were equally inferior to controls in the passive avoidance test, but only the anoxia group was inferior to controls in the active avoidance test. Amphetamine-induced stereotype behavior was much less pronounced in the anoxia group relative to AF-64A-treated rats or to controls. The results suggest that the lesion induced by the neurotoxin is more specific and less widespread than the one caused by anoxia.