Glycine Intracerebroventricular Administration Disrupts Mitochondrial Energy Homeostasis in Cerebral Cortex and Striatum of Young Rats

High tissue levels of glycine (GLY) are the biochemical hallmark of nonketotic hyperglycinemia (NKH), an inherited metabolic disease clinically characterized by severe neurological symptoms and brain abnormalities. Considering that the mechanisms underlying the neuropathology of this disease are not fully established, the present work investigated the in vivo effects of intracerebroventricular administration of GLY on important parameters of energy metabolism in cerebral cortex and striatum from young rats. Our results show that GLY reduced CO₂ production using glucose as substrate and inhibited the activities of citrate synthase and isocitrate dehydrogenase in striatum, whereas no alterations of these parameters were verified in cerebral cortex 30 min after GLY injection. We also observed that GLY diminished the activities of complex IV in cerebral cortex and complex I–III in striatum at 30 min and inhibited complex I–III activity in striatum at 24 h after its injection. Furthermore, GLY reduced the activity of total and mitochondrial creatine kinase in both brain structures 30 min and 24 h after its administration. In contrast, the activity of Na⁺, K⁺-ATPase was not altered by GLY. Finally, the antioxidants N-acetylcysteine and creatine, and the NMDA receptor antagonist MK-801 attenuated or fully prevented the inhibitory effects of GLY on creatine kinase and respiratory complexes in cerebral cortex and striatum. Our data indicate that crucial pathways for energy production and intracellular energy transfer are severely compromised by GLY. It is proposed that bioenergetic impairment induced by GLY in vivo may contribute to the neurological dysfunction found in patients affected by NKH.     

Disruption of Energy Transfer and Redox Status by Sulfite in Hippocampus,Striatum, and Cerebellum of Developing Rats

Patients with sulfite oxidase (SO) deficiency present severe brain abnormalities, whose pathophysiology is not yet elucidated. We evaluated the effects of sulfite and thiosulfate, metabolites accumulated in SO deficiency, on creatine kinase (CK) activity, mitochondrial respiration and redox status in hippocampus, striatum and cerebellum of developing rats. Our in vitro results showed that sulfite and thiosulfate decreased CK activity, whereas sulfite also increased malondialdehyde (MDA) levels in all brain structures evaluated. Sulfite further diminished mitochondrial respiration and increased DCFH oxidation and hydrogen peroxide production in hippocampus. Sulfite-induced CK activity decrease was prevented by melatonin (MEL), resveratrol (RSV), and dithiothreitol while increase of MDA levels was prevented by MEL and RSV. Regarding the antioxidant system, sulfite increased glutathione concentrations in hippocampus and striatum. In addition, sulfite decreased the activities of glutathione peroxidase in all brain structures, of glutathione S-transferase in hippocampus and cerebellum, and of glutathione reductase in cerebellum. In vivo experiments performed with intrahippocampal administration of sulfite demonstrated that this metabolite increased superoxide dismutase activity without altering other biochemical parameters in rat hippocampus. Our data suggest that impairment of energy metabolism and redox status may be important pathomechanisms involved in brain damage observed in individuals with SO deficiency.     

Pristanic Acid Provokes Lipid,Protein, and DNA Oxidative Damage and Reduces the Antioxidant Defenses in Cerebellum of Young Rats

Zellweger syndrome (ZS) and some peroxisomal diseases are severe inherited disorders mainly characterized by neurological symptoms and cerebellum abnormalities, whose pathogenesis is poorly understood. Biochemically, these diseases are mainly characterized by accumulation of pristanic acid (Prist) and other fatty acids in the brain and other tissues. In this work, we evaluated the in vitro influence of Prist on redox homeostasis by measuring lipid, protein, and DNA damage, as well as the antioxidant defenses and the activities of aconitase and α-ketoglutarate dehydrogenase in cerebellum of 30-day-old rats. The effect of Prist on DNA damage was also evaluated in blood of these animals. Some parameters were also evaluated in cerebellum from neonatal rats and in cerebellum neuronal cultures. Prist significantly increased malondialdehyde (MDA) levels and carbonyl formation and reduced sulfhydryl content and glutathione (GSH) concentrations in cerebellum of young rats. It also caused DNA strand damage in cerebellum and induced a high micronuclei frequency in blood. On the other hand, this fatty acid significantly reduced α-ketoglutarate dehydrogenase and aconitase activities in rat cerebellum. We also verified that Prist-induced increase of MDA levels was totally prevented by melatonin and attenuated by α-tocopherol but not by the nitric oxide synthase inhibitor N^ω-nitro-l-arginine methyl ester, indicating the involvement of reactive oxygen species in this effect. Cerebellum from neonate rats also showed marked alterations of redox homeostasis, including an increase of MDA levels and a decrease of sulfhydryl content and GSH concentrations elicited by Prist. Finally, Prist provoked an increase of dichlorofluorescein (DCFH) oxidation in cerebellum-cultivated neurons. Our present data indicate that Prist compromises redox homeostasis in rat cerebellum and blood and inhibits critical enzymes of the citric acid cycle that are susceptible to free radical attack. The present findings may contribute to clarify the pathogenesis of the cerebellar alterations observed in patients affected by ZS and some peroxisomal disorders in which Prist is accumulated.     

Sleep Disruption in Young Foster Children

In the current study, sleep actigraphy and parent-report measures were used to investigate differences in sleeping behavior among four groups of 3- to 7-year-olds (N = 79): children in regular foster care (n = 15); children receiving a therapeutic intervention in foster care (n = 17); low income community children (n = 18); and upper middle income community children (n = 29). The children in therapeutic foster care exhibited longer sleep latency and increased variability of sleep duration than the upper middle income community children. In addition, there was an indication of a treatment effect: the therapeutic foster care children slept longer than the regular foster care and low income community children and had earlier bedtimes, fell asleep earlier, and spent more time in bed than the regular foster care children. The results are discussed in terms of the effectiveness of early intervention for enhancing sleep in foster children.     

Disruption of Saccadic Adaptation with Repetitive Transcranial Magnetic Stimulation of the Posterior Cerebellum in Humans

Saccadic eye movements are driven by motor commands that are continuously modified so that errors created by eye muscle fatigue, injury, or—in humans—wearing spectacles can be corrected. It is possible to rapidly adapt saccades in the laboratory by introducing a discrepancy between the intended and actual saccadic target. Neurophysiological and lesion studies in the non-human primate as well as neuroimaging and patient studies in humans have demonstrated that the oculomotor vermis (lobules VI and VII of the posterior cerebellum) is critical for saccadic adaptation. We studied the effect of transiently disrupting the function of posterior cerebellum with repetitive transcranial magnetic stimulation (rTMS) on the ability of healthy human subjects to adapt saccadic eye movements. rTMS significantly impaired the adaptation of the amplitude of saccades, without modulating saccadic amplitude or variability in baseline conditions. Moreover, increasing the intensity of rTMS produced a larger impairment in the ability to adapt saccadic size. These results provide direct evidence for the role of the posterior cerebellum in man and further evidence that TMS can modulate cerebellar function.     

Mitochondrial Morphology and Intracellular Calcium Homeostasis in Cytochrome Oxidase-Deficient Human Fibroblasts

Mitochondrial encephalomyopathies arise from mutations in the mitochondrial or nuclear genome and result in defective energy metabolism. Investigation of cellular pathophysiology in these disorders has been limited to nonneuronal explant cultures such as fibroblasts and myoblasts. While investigating mitochondrial structure and function in fibroblasts obtained from control and cytochrome oxidase-deficient (COX) patients, we observed possible abnormalities by vital dye confocal microscopy. Most notable were swelling, reticulation (e.g., intricate fusion of mitochondria), and proliferation of mitochondria. However, a detailed quantitative comparison of mitochondrial morphology in age-, sex-, and passage-matched cultures revealed no significant differences between control and cytochrome oxidase-deficient fibroblasts, nor any differences with passage. In addition, COX fibroblasts exhibited no obvious impairment of intracellular calcium handling, measured by fura-2. These results indicate that cytochrome oxidase deficiency, at the level in these cultures, does not produce structural or ionic concentration alterations in fibroblasts. Future investigation of the pathophysiology of this respiratory chain disorder may require excitable tissue.     

实验性脑积水幼鼠脑脊液中一氧化氮及谷氨酸浓度的变化

目的探讨实验性脑积水幼鼠脑脊液中一氧化氮（NO）和谷氨酸（Glu）浓度的动态变化。方法SD幼鼠40只,随机平分为A、B、C实验组和对照组共4组,用显微外科技术向枕大池内注入25%白陶土混悬液,分别在注射后第2（A组）、3（B组）、4周（C组）后行MRI检测,鉴定脑积水形成情况;之后24h内取脑脊液,用硝酸还原酶法测NO浓度、高效液相色谱仪检测Glu浓度。对照组幼鼠于枕大池内注入生理盐水,用同样方法检测。结果实验组21只幼鼠成功诱发脑积水。与对照组相比,实验组脑脊液中NO和Glu浓度显著增加（P〈0.01）。结论脑脊液中高水平的NO和Glu可能参与了实验性脑积水幼鼠的病理损伤过程。     

Intrastriatal Quinolinic Acid Administration Impairs Redox Homeostasis and Induces Inflammatory Changes: Prevention by Kynurenic Acid

Neurotoxicity Research - Kynurenic acid (KYNA) and quinolinic acid (QUIN) are metabolites formed in the degradation of tryptophan (Trp). QUIN is a selective NMDA receptor antagonist and may exert...     

Organization of Host Afferents to Cerebellar Grafts Implanted into Kainate Lesioned Cerebellum in Adult Rats

This paper examines the organization of host afferents within cerebellar grafts implanted into kainic acid lesioned cerebellum. Our selection of a cerebellum, a prime example of a 'point-to-point' system, permits precise determination of the degree and the specificity of host-graft interactions. One month after a cerebellar injection of kainic acid, the lesion produced can be divided into two concentric regions: (i) a central necrotic zone, totally depleted of neurons (zone 1), and (ii) a peripheral zone which lacks all Purkinje cells but preserves its cortical lamination (zone 2). Two months after the implantation of solid pieces of embryonic cerebellum, the graft has evolved into a minicerebellar structure, occupying most of zone 1. The grafted minicerebellum consists of a highly convoluted trilaminated cortex with a core containing deep nuclear neurons. Purkinje cells are positioned between the molecular and granular layer with their short and irregular dendrites branching within the former. Donor foetal Purkinje cells migrate into the contiguous portion of the molecular layer of the host zone 2. These embryonic neurons set up within the upper three-quarters of the host molecular layer, and develop monoplanar dendritic trees that span the whole width of the layer. The organization of host-graft interactions was studied by autoradiography of anterogradely transported tritiated leucine, injected in the host bulbar region containing the caudal half of the inferior olivary complex (origin of all vermal climbing fibres) and the dorsally adjacent paramedian reticular nucleus (origin of a few mossy fibres). Numerous labelled fibres cross the host-graft interface from the white matter of the host cerebellum, and provide innervation to the minicerebellar structure. The vast majority of these labelled axons terminate in the molecular layer, forming axonal arborizations that follow the shape of the Purkinje cell dendrites. The labelled climbing fibres are organized into uneven sagittally aligned strips, which mimic that of olivocerebellar projections in control rats. Only a small proportion of host labelled fibres end in the donor granular layer, forming typical mossy fibre rosettes. The latter are present in the region of the graft close to the host-graft interface. In addition, labelled axons are observed climbing over the dendritic trees of grafted Purkinje cells that have invaded a portion of the host molecular layer of zone 2. In all regions containing grafted Purkinje cells and labelled climbing fibres, the density of the innervation is close to normal with practically all Purkinje cells receiving a climbing fibre. The extensive integration of the grafted cells into the deficient neuronal networks of the host clearly illustrates the positive neurotropic effect exerted by immature cerebellar neurons on adult extracerebellar afferent fibres. The hodological integration, allowing a possible restoration of the impaired cerebellar circuitry, takes place respecting the specificity and topographic distribution which characterize the 'point-to-point' arrangement of normal cerebellar circuitry.     

11Beta-Hydroxysteroid Dehydrogenase Messenger Ribonucleic Acid Expression, Bioactivity and Immunoreactivity in Rat Cerebellum

11β-Hydroxysteroid dehydrogenase (11β-OHSD) metabolizes corticosterone to inactive 11-dehydrocorticosterone and thus protects non-specific mineralocorticoid receptors from exposure to corticosterone in the kidney in vivo. Clearly, 11β-OHSD might also regulate corticosterone access to glucocorticoid receptors. We have investigated cerebellum, a tissue with high glucocorticoid receptor, but very low mineralocorticoid receptor levels and have shown marked 11β-OHSD bioactivity with similar co-substrate requirements and inhibition kinetics to the renal enzyme. 11β-OHSD messenger ribonucleic acid was expressed in cerebellum and was localized in Purkinje and granule cells. This distribution was confirmed immunohistochemically. Thus, we provide evidence for 11β-OHSD in cerebellum and suggest that it may regulate the access of corticosterone to glucocorticoid receptors in addition to mineralocorticoid receptors.     

Disruption of Cortical Development as a Consequence of Repetitive Pilocarpine-induced Status Epilepticus in Rats

Summary:  Purpose: The aim of the present study was to observe possible cortical abnormalities after repetitive pilocarpine-induced status epilepticus (SE) in rats during development.
Methods: Wistar rats received intraperitoneal injection of pilocarpine hydrochloride 2% (380 mg/kg) at P7, P8, and P9. All experimental rats displayed SE after pilocarpine injections. Rats were killed at P10 and P35, and immunocytochemistry procedures were performed on 50-μm vibratome sections, by using antibodies against nonphosphorylated neurofilament (SMI-311), parvalbumin (PV), calbindin (CB), calretinin (CR), and glutamate decarboxylase (GAD-65). Selected sections were used for the TUNEL method and double-labeling experiments, with different mixtures of the same markers.
Results: The major findings of the present work were (a) altered intracortical circuitry development; (b) anticipation of PV immunoreactivity in neocortical interneurons; (c) increased GAD-65 immunoreactivity; and (d) reduced neocortical apoptotic process.
Conclusions: From these results, we suggest that previously healthy brain, without genetic abnormalities, might develop an "acquired" disruption of cortical development whose evolution reproduces some characteristics of the childhood epilepsies associated with cognitive impairment.     

Activity-dependent Decay of Early LTP Revealed by Dual EPSP Recording in Hippocampal Slices from Young Rats

The early maintenance of long-term potentiation (LTP) was studied in the CA1 region of hippocampal slices from 12- to 18-day-old rats in a low-magnesium solution (0.1 mM). The α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptor-mediated components of the field excitatory postsynaptic potential were estimated in parallel using early and late measurements of the composite potential. At the normal test stimulus frequency of 0.1 Hz, LTP was seen initially as a predominant increase in the AMPA component, but converted, via a substantial decay of this component and a gradual growth of the NMDA component, into nearly equal changes of the two components. Interrupting the test stimulation for 10 min, changing the test stimulus frequency to 1/60 Hz after LTP induction, or using a test stimulus frequency of 1/60 Hz during the entire experiment significantly reduced the decay of the potentiation of the AMPA component while enhancing the potentiation of the NMDA one. The ratio between the magnitudes of the two excitatory postsynaptic potential (EPSP) components showed a decaying time course that was independent of the manipulations used. Application of the NMDA antagonist D(-)-2-amino-5-phosphonopentanoic acid (50μM) after LTP induction stabilized the LTP of the AMPA component until washout was started. On the other hand, the phosphatase inhibitor okadaic acid (1 μM) resulted in decay of the potentiation of both EPSP components back to around baseline and altered the time course of the ratio between the components. Our results show that the early maintenance of LTP is controlled in an activity-dependent and NMDA-dependent manner. This process accelerates the decay of LTP of both AMPA and NMDA components in parallel, suggesting that it is similar to homosynaptic long-term depression, although it operates at the normal test stimulus frequency. The data support a scenario in which LTP ensues as a selective AMPA receptor modification and subsequently converts to another modification, possibly a presynaptic one.     

Experimental Evidence that In Vivo Intracerebral Administration of L-2-Hydroxyglutaric Acid to Neonatal Rats Provokes Disruption of Redox Status and Histopathological Abnormalities in the Brain

Tissue accumulation of L-2-hydroxyglutaric acid (L-2-HG) is the biochemical hallmark of L-2-hydroxyglutaric aciduria (L-2-HGA), a rare neurometabolic inherited disease characterized by neurological symptoms and brain white matter abnormalities whose pathogenesis is not yet well established. L-2-HG was intracerebrally administered to rat pups at postnatal day 1 (P1) to induce a rise of L-2-HG levels in the central nervous system (CNS). Thereafter, we investigated whether L-2-HG in vivo administration could disturb redox homeostasis and induce brain histopathological alterations in the cerebral cortex and striatum of neonatal rats. L-2-HG markedly induced the generation of reactive oxygen species (increase of 2′,7′-dichloroflurescein-DCFH-oxidation), lipid peroxidation (increase of malondialdehyde concentrations), and protein oxidation (increase of carbonyl formation and decrease of sulfhydryl content), besides decreasing the antioxidant defenses (reduced glutathione-GSH) and sulfhydryl content in the cerebral cortex. Alterations of the activities of various antioxidant enzymes were also observed in the cerebral cortex and striatum following L-2-HG administration. Furthermore, L-2-HG-induced lipid peroxidation and GSH decrease in the cerebral cortex were prevented by the antioxidant melatonin and by the classical antagonist of NMDA glutamate receptor MK-801, suggesting the involvement of reactive species and of overstimulation of NMDA receptor in these effects. Finally, L-2-HG provoked significant vacuolation and edema particularly in the cerebral cortex with less intense alterations in the striatum that were possibly associated with the unbalanced redox homeostasis caused by this metabolite. Taken together, it is presumed that these pathomechanisms may underlie the neurological symptoms and brain abnormalities observed in the affected patients.     

5-ALA介导的光动力治疗鼠C6胶质瘤的实验研究

目的研究以5-氨基乙酰丙酸（5-ALA）作为光敏剂的光动力治疗（PDT）对大鼠C6胶质瘤移植瘤的效果。方法通过皮下接种C6胶质瘤细胞建立大鼠移植胶质瘤模型。将24只荷瘤大鼠平均随机分成3组：A组按剂量20mg／kg在瘤内注射5-ALA，2h后在肿瘤局部行激光照射；B组行单纯激光照射，不予任何光敏剂；C组为荷瘤空白对照组，不给任何治疗。PDT后不同时间，测量肿瘤体积并观察其组织学变化。结果治疗后第4、8和14天A组肿瘤体积与B、C组相比明显缩小（P〈0．05），组织学上可见大量肿瘤细胞坏死。结论5-ALA作为光敏剂介导的PDT能使大鼠C6胶质瘤移植瘤组织坏死，生长速度明显减慢，有望成为胶质瘤治疗的新途径。     

Effect of Naloxone on the Secretion of Corticosterone Induced by Gamma-Hydroxybutyric Acid in Male Rats

The effect of intraperitoneally administered γ-hydroxybutyrate on the hypothalamo-hypophysial-adrenocortical axis was studied. A significant increase in plasma corticosterone was induced by the administration of γ-hydroxybutyrate (100 mg/kg). The lack of antagonism of this action of γ-hydroxybutyrate by picrotoxin or bicuculline pretreatments (GABAergic antagonists), suggests independence from the GABAergic system. Neither dopaminergic nor serotoninergic systems intervened in the γ-hydroxybutyrate-stimulating effect on the hypothalamo-hypophysial-adrenocortical axis. Both corticosterone release and synthesis in adrenocortical cells was not significantly affected by γ-hydroxybutyrate, suggesting that this is possibly due to a central action of γ-hydroxybutyrate. The increase in corticosterone levels was probably mediated by an opioid receptor, since a strong similarity existed between the effects of γ-hydroxybutyrate and those of morphine. Moreover, the results show that the opioid-receptor involvement was demonstrated by the naloxone-sensitivity of the γ-hydroxybutyrate effects.     

Carnosic Acid Affords Mitochondrial Protection in Chlorpyrifos-Treated Sh-Sy5y Cells

Carnosic acid (CA; C₂₀H₂₈O₄) is a phenolic diterpene found in rosemary (Rosmarinus officinalis L.) and exhibits protective properties, e.g., antioxidant, anti-inflammatory, antitumor, and antimicrobial activities. In this context, CA has been viewed as a neuroprotective agent due to its ability in rescuing neuronal cells from pro-oxidant and pro-apoptotic challenges. In the present work, we found that CA pretreatment at 1 µM for 12 h suppressed the mitochondria-related pro-oxidant and mitochondria-dependent pro-apoptotic effects of chlorpyrifos (CPF) in human neuroblastoma SH-SY5Y cells. CA prevented mitochondrial membrane potential disruption and decreased the levels of oxidative stress markers in mitochondrial membranes obtained from cells exposed to CPF. CA also inhibited cytochrome c release and activation of the caspases-9 and -3, as well as decreased DNA fragmentation, in CPF-treated cells. CA upregulated the content of glutathione (GSH) in mitochondria by a mechanism involving the activation of the phosphoinositide-3-kinase (PI3K)/Akt/nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway, since inhibition of PI3K/Akt or silencing of Nrf2 using siRNA strategy abolished the protection exerted by CA in SH-SY5Y cells. Therefore, CA protected mitochondria of SH-SY5Y cells through the activation of the PI3K/Akt/Nrf2 axis, causing upregulation of the mitochondrial GSH content and consequent antioxidant and anti-apoptotic effects.     

Activation of Neuronal Nitric Oxide Synthase in Cerebellum of Chronic Hepatic Encephalopathy Rats is Associated with Up-regulation of NADPH-Producing Pathway

Cerebellum-associated functions get affected during mild hepatic encephalopathy (MHE) in patients with chronic liver failure (CLF). Involvement of nitrosative and antioxidant factors in the pathogenesis of chronic hepatic encephalopathy is an evolving concept and needs to be defined in a true CLF animal model. This article describes profiles of NADPH-dependent neuronal nitric oxide synthase (nNOS) and those of glutathione peroxidase and glutathione reductase (GR) vis-a-vis regulation of NADPH-producing pathway in the cerebellum of CLF rats induced by administration of thioacetamide (100 mg kg⁻¹ b.w., i.p.) up to 10 days and confirming MHE on Morris water maze tests. Significant increases in the expression of nNOS protein and nitric oxide (NOx) level coincided with a similar increment in NADPH–diaphorase activity in the cerebellum of CLF rats. Glutathione peroxidase and GR utilize NADPH to regenerate reduced glutathione (GSH) in the cells. Both these enzymes and GSH level were found to be static and thus suggested efficient turnover of GSH in the cerebellum of MHE rats. Relative levels of glucose-6-phosphate dehydrogenase (G6PD) vs. phosphofructokinase 2 (PFK2) determine the rate of pentose phosphate pathway (PPP) responsible to synthesize NADPH. The cerebellum of CLF rats showed overactivation of G6PD with a significant decline in the expression of PFK2 and thus suggested activation of PPP in the cerebellum during MHE. It is concluded that concordant activations of PPP and nNOS in cerebellum of MHE rats could be associated with the implication of NOx in the pathogenesis of MHE.