Nutritional Management of Short Bowel Syndrome.

Short bowel syndrome (SBS) of infancy is a cause of prolonged morbidity with intolerance to enteral feeding, specialized nutritional needs, and partial/total dependence on parenteral nutrition. These infants can benefit from individualized nutritional strategies to support and enhance the process of intestinal adaptation. Early introduction of enteral feeds during the period of intestinal adaptation is crucial, even though the enteral feedings may need to be supplemented with an effective, safe, and nutritionally adequate parenteral nutritional regimen. Newer generation intravenous lipid emulsions can be effective in preventing and treating intestinal failure-associated liver disease. Prevention of infection(s), pharmaceutical interventions to enhance bowel motility and prevent/mitigate bacteria overgrowth, and specialized multidisciplinary care to minimize the injury to other organs such as the liver, kidneys, and the brain can assist in nutritional rehabilitation and lower the morbidity in SBS.

Small Proportion of Low-Birth-Weight Infants With Ostomy and Intestinal Failure Due to Short-Bowel Syndrome Achieve Enteral Autonomy Prior to Reanastomosis.

BACKGROUND: It is challenging to provide optimum nutrition in low-birth-weight (LBW) infants with short-bowel syndrome (SBS) and ostomy. This study aims to evaluate the clinical course of LBW infants with SBS and ostomy in response to enteral feeds, recognize characteristics associated with achievement of enteral autonomy prior to reanastomosis, and evaluate associated short-term outcomes. METHODS: A retrospective analysis of 52 LBW neonates with intestinal failure (IF) caused by SBS and ostomy treated in a neonatal intensive care unit from 2012 to 2018 was performed. Clinical characteristics and short-term outcomes were studied in relation to the location of the ostomy and the success with enteral feeding achieved prior to reanastomosis. RESULTS: Of the 52 infants with SBS, jejunostomy, ileostomy, and colostomy were present in 9, 40, and 3 infants, respectively. Fourteen (26.92%) infants achieved enteral autonomy transiently, and 7 (13.46%) sustained until reanastomosis. All 9 infants with jejunostomy were parenteral nutrition dependent, compared with 22 with ileostomy and none with colostomy (P = 0.002). Infants who achieved enteral autonomy showed lower incidence of cholestasis (P = 0.038) and better growth velocity (P = 0.02) prior to reanastomosis. CONCLUSIONS: A minority of LBW infants with SBS and ostomy achieved enteral autonomy prior to reanastomosis. Distal ostomy (ileostomy and colostomy), reduced cholestasis, and better growth were associated with achievement of enteral autonomy. Our report highlights the challenges in establishing enteral autonomy in LBW infants with IF and ostomy, and the feasibility of that approach in a minority of patients, with tangible benefits.

Prolactin prevents mitochondrial dysfunction induced by glutamate excitotoxicity in hippocampal neurons.

Prolactin (PRL) is a pleiotropic hormone secreted by several cells and tissues in the body, such as mammary glands, T-lymphocytes, hypothalamus, among others. This hormone possess neuroprotective properties against glutamate-excitotoxicity through the activation of NF-kB, suggesting it could exert an antioxidant action. However, the role of PRL on the antioxidant defense during glutamate-induced excitotoxicity is not clear to date. Therefore, in the present study, we have evaluated the effect of PRL on SOD activity and protein content of both of its isoforms (Mn2+-SOD and Cu2+/Zn2+-SOD), as well as, its action on mitochondrial activity in primary culture of hippocampal neurons of rats. Additionally, we have evaluated the possible antioxidant effect of PRL through the determination of lipid peroxidation products (LPO), measured as malondialdehyde (MDA). Results show that PRL enhances the activity and the protein content of Mn2+-SOD and Cu2+/Zn2+-SOD in neurons exposed to glutamate-induced excitotoxicity. Moreover, our results demonstrate that PRL prevents mitochondrial dysfunction induced by glutamate and significantly decreases the levels of LPO products. To our knowledge, this is the first time that a potential antioxidant effect of PRL has been described in hippocampal neurons exposed to glutamate excitotoxicity, opening questions of its potentiality for therapeutics.

Nutritional considerations in the care of conjoined twins.

Conjoined twins represent an interesting nutritional challenge as nutrient delivery and absorption is greatly affected by anatomy and, therefore, unique to each twin pair. Nutritional support is essential to optimize growth and development in the neonatal period; however, very little data exists on the topic in this population. Conjoined twins require individualized nutritional assessment that focuses on the interaction between the metabolic rate, nutrient uptake, and nutrient delivery of each twin in the dyad. This report describes one center's experience with monitoring growth, establishing nutrient requirements, and determining substrate utilization in three sets of conjoined twins.

Sustained metabolic inhibition induces an increase in the content and phosphorylation of the NR2B subunit of N-methyl-D-aspartate receptors and a decrease in glutamate transport in the rat hippocampus in vivo.

The concentration of glutamate is regulated to ensure neurotransmission with a high temporal and local resolution. It is removed from the extracellular medium by high-affinity transporters, dependent on the maintenance of the Na(+) gradient through the activity of Na(+),K(+)-ATPases. Failure of glutamate clearance can lead to neuronal damage, named excitotoxic damage, due to the prolonged activation of glutamate receptors. Severe impairment of glycolytic metabolism during ischemia and hypoglycemia, leads to glutamate transport dysfunction inducing the elevation of extracellular glutamate and aspartate, and neuronal damage. Altered glucose metabolism has also been associated with some neurodegenerative diseases such as Alzheimer's and Huntington's, and a role of excitotoxicity in the neuropathology of these disorders has been raised. Alterations in glutamate transporters and N-methyl-D-aspartate (NMDA) receptors have been observed in these patients, suggesting altered glutamatergic neurotransmission. We hypothesize that inhibition of glucose metabolism might induce changes in glutamatergic neurotransmission rendering neurons more vulnerable to excitotoxicity. We have previously reported that sustained glycolysis impairment in vivo induced by inhibition of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), facilitates glutamate-mediated neuronal damage. We have now investigated whether this facilitating effect involves altered glutamate uptake, and/or NMDA receptors in the rat hippocampus in vivo. Results indicate that metabolic inhibition leads to the progressive elevation of extracellular glutamate and aspartate levels in the hippocampus, which correlates with decreased content of the GLT-1 glutamate transporter and diminished glutamate uptake. In addition, we observed increased Tyr(1472) phosphorylation and protein content of the NR2B subunit of the NMDA receptor. Results suggest that moderate sustained glycolysis inhibition alters glutamatergic neurotransmission.

Exacerbation of excitotoxic neuronal death induced during mitochondrial inhibition in vivo: relation to energy imbalance or ATP depletion?

During the past two decades a close relationship between the energy state of the cell and glutamate neurotoxicity has been suggested. We have previously shown that increasing the extracellular concentration of glutamate does not cause neuronal death unless a deficit in energy metabolism occurs. The mechanisms of glutamate-induced neuronal death have been extensively studied in vitro and it has been associated with a rapid and severe decrease in ATP levels, accompanied with mitochondrial dysfunction. In this study we aimed to investigate the time course of the changes in energy metabolites during glutamate-induced neuronal death, in the presence of a moderate inhibition of mitochondrial metabolism in the rat striatum in vivo. We also aimed to study whether or not, as reported in vitro, changes in ATP levels are related to the extension of neuronal death. Results show that glutamate-induced lesions are exacerbated when rats are previously treated with a subtoxic dose of the mitochondrial toxin 3-nitropropionic acid (3-NP). However, changes in nucleotide levels were similar in rats injected with glutamate alone and in rats injected with glutamate and previously treated with 3-NP. In spite of the presence of an extensive striatal lesion, nucleotide levels were recovered in 3-NP-treated rats 24 h after glutamate injection. Results show that 3-NP pre-treatment induced an imbalance in nucleotide levels that predisposed cells to glutamate toxicity; however it did not influence the bioenergetic changes induced by glutamate alone. Enhancement of glutamate neurotoxicity in 3-NP pre-treated rats is more related to a sustained nucleotide imbalance than just to a rapid decrease in ATP levels.

The anion channel blocker, 4,4'-dinitrostilbene-2,2'-disulfonic acid prevents neuronal death and excitatory amino acid release during glycolysis inhibition in the hippocampus in vivo.

Neuronal death associated with cerebral ischemia and hypoglycemia is related to increased release of excitatory amino acids (EAA) and energy failure. The intrahippocampal administration of the glycolysis inhibitor, iodoacetate (IOA), induces the accumulation of EAA and neuronal death. We have investigated by microdialysis the role of exocytosis, glutamate transporters and volume-sensitive organic anion channel (VSOAC) on IOA-induced EAA release. Results show that the early component of EAA release is inhibited by riluzole, a voltage-dependent sodium channel blocker, and by the VSOAC blocker, tamoxifen, while the early and late components are blocked by the glutamate transport inhibitors, L-trans-pyrrolidine 2,4-dicarboxylate (PDC) and DL-threo-beta-benzyloxyaspartate (DL-TBOA); and by the VSOAC blocker 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS). Riluzole, DL-TBOA and tamoxifen did not prevent IOA-induced neuronal death, while PDC and DNDS did. The VSOAC blockers 5-nitro-2-(3-phenylpropyl-amino) benzoic acid (NPPB) and phloretin had no effect either on EAA efflux or neuronal damage. Results suggest that acute inhibition of glycolytic metabolism promotes the accumulation of EAA by exocytosis, impairment or reverse action of glutamate transporters and activation of a DNDS-sensitive mechanism. The latest is substantially involved in the triggering of neuronal death. To our knowledge, this is the first study to show protection of neuronal death by DNDS in an in vivo model of neuronal damage, associated with deficient energy metabolism and EAA release, two conditions involved in some pathological states such as ischemia and hypoglycemia.

Differential effects of the substrate inhibitor l-trans-pyrrolidine-2,4-dicarboxylate (PDC) and the non-substrate inhibitor DL-threo-beta-benzyloxyaspartate (DL-TBOA) of glutamate transporters on neuronal damage and extracellular amino acid levels in rat brain in vivo.

The extracellular concentration of glutamate is highly regulated by transporter proteins, due to its neurotoxic properties. Dysfunction or reverse activation of these transporters is related to the extracellular accumulation of excitatory amino acids and neuronal damage associated with ischemia and hypoglycemia. We have investigated by microdialysis the effects of the substrate and the non-substrate inhibitors of glutamate transporters, l-trans-2,4-pyrrolidine dicarboxylate (PDC) and DL-threo-beta-benzyloxyaspartate (DL-TBOA), respectively, on the extracellular levels of amino acids in the rat hippocampus in vivo. In addition, we have studied the effect of both inhibitors on neuronal damage after direct administration into the hippocampus and striatum. Electroencephalographic activity was recorded after the intrahippocampal infusion of DL-TBOA or PDC. Microdialysis administration of 500 microM DL-TBOA into the hippocampus increased 3.4- and nine-fold the extracellular levels of aspartate and glutamate, respectively. Upon stereotaxic administration it induced neuronal damage dose-dependently in CA1 and dentate gyrus, and convulsive behavior. Electroencephalographic recording showed the appearance of limbic seizures in the hippocampus after DL-TBOA infusion. In the striatum it also induced dose-dependent neuronal damage. These effects were prevented by the i.p. administration of the glutamate receptor antagonists (+)-5-methyl-10,11-dihydroxy-5H-dibenzo(a,d)cyclohepten-5,10-iminemaleate and 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)-quinoxaline. In contrast to dl-TBOA, PDC (500 microM) induced a more discrete elevation of excitatory amino acids levels (2.6- and three-fold in aspartate and glutamate, respectively), no neuronal damage or behavioral changes, and no alterations in electroencephalographic activity. The differential results obtained with DL-TBOA and PDC might be attributed to their distinct effects on the extracellular concentration of amino acids. Results are relevant to the understanding of the role of glutamate transporters in amino acid removal or release and the induction of excitotoxic cell death.

2,3-Dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline protects against both AMPA and kainate-induced lesions in rat striatum in vivo.

In the present work we have tested the neuroprotective effect of 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (NBQX) on the excitotoxic damage induced by the injection of several glutamate receptor agonists into the rat striatum. NBQX was co-injected with each of the agonists studied (1 microliter) in the striatum and damage was assessed by the determination of both glutamate decarboxylase and choline acetyltransferase activities in striatal homogenates, five days after the lesion. Additionally, animals were transcardially perfused with 0.9% saline/4% paraformaldehyde and brain coronal sections were stained with Cresyl Violet for histological analysis. Our results show that NBQX (25 nmol) did not protect against the damage induced by the intrastriatal injection of 200 nmol quinolinic acid monitored by either choline acetyltransferase or glutamate decarboxylase activity. In contrast, the same concentration of NBQX partially protected against 200 nmol N-methyl-D-aspartate induced damage; this protection was more notable as detected by changes in choline acetyltransferase activity. When non-N-methyl-D-aspartate receptor agonists were used as excitotoxins, coinjection of NBQX (25 nmol) resulted in a notable protection against both alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA, 40 nmol) and kainate (10 nmol) induced neurodegeneration. At this concentration, protection was slightly better in AMPA-injected animals (71% protection averaged from choline acetyltransferase and glutamate decarboxylase enzyme activities) as compared to kainate-injected animals (47.5% protection). When a higher concentration of NBQX was tested (40 nmol) the protection against kainate improved to 65% while that against AMPA remained constant (64% protection).(ABSTRACT TRUNCATED AT 250 WORDS)

Neurotoxicity of glutamate uptake inhibition in vivo: correlation with succinate dehydrogenase activity and prevention by energy substrates.

Impairment of glutamate uptake or the reverse action of its transporters has been suggested as the mechanism responsible for the increased glutamate extracellular levels associated with ischemic neuronal damage. In previous studies we have shown that glutamate uptake inhibition by L-trans-pyrrolidine-2,4-dicarboxylate (PDC) in the rat striatum and hippocampus in vivo does not induce neuronal death despite the notable increase in the extracellular levels of glutamate and aspartate. However, PDC intracerebral administration leads to neuronal death in rats chronically injected with the mitochondrial toxin 3-nitropropionic acid (3-NP), an inhibitor of succinate dehydrogenase (SDH). In the present study we have determined the time course of inhibition of SDH activity in the striatum of rats acutely injected with a single dose of 3-NP (20 mg/kg), and studied its relation to PDC neurotoxicity. PDC induced larger lesions when administered during maximum inhibition of SDH activity while smaller lesions were found when it was injected during recovery of enzyme activity. We also studied the neuroprotective effect of different energy substrates such as creatine, pyruvate, and the ketone bodies beta-hydroxybutyrate and acetoacetate in this experimental model. Our results show partial protection with all compounds except for beta-hydroxybutyrate that showed no protection, while MK-801 completely prevented PDC-induced neuronal damage. We believe that the present results might be of relevance for the understanding of the mechanisms responsible for ischemic neuronal death and its prevention.

Acetoacetate protects hippocampal neurons against glutamate-mediated neuronal damage during glycolysis inhibition.

Glucose is the main substrate that fulfills energy brain demands. However, in some circumstances, such as diabetes, starvation, during the suckling period and the ketogenic diet, brain uses the ketone bodies, acetoacetate and beta-hydroxybutyrate, as energy sources. Ketone body utilization in brain depends directly on its blood concentration, which is normally very low, but increases substantially during the conditions mentioned above. Glutamate neurotoxicity has been implicated in neurodegeneration associated with brain ischemia, hypoglycemia and cerebral trauma, conditions related to energy failure, and to elevation of glutamate extracellular levels in brain. In recent years substantial evidence favoring a close relation between glutamate neurotoxic potentiality and cellular energy levels, has been compiled. We have previously demonstrated that accumulation of extracellular glutamate after inhibition of its transporters, induces neuronal death in vivo during energy impairment induced by glycolysis inhibition. In the present study we have assessed the protective potentiality of the ketone body, acetoacetate, against glutamate-mediated neuronal damage in the hippocampus of rats chronically treated with the glycolysis inhibitor, iodoacetate, and in hippocampal cultured neurons exposed to a toxic concentration of iodoacetate. Results show that acetoacetate efficiently protects against glutamate neurotoxicity both in vivo and in vitro probably by a mechanism involving its role as an energy substrate.

A comparative analysis of the neuroprotective properties of competitive and uncompetitive N-methyl-D-aspartate receptor antagonists in vivo: implications for the process of excitotoxic degeneration and its therapy.

Injection of the N-methyl-D-aspartate receptor agonist, quinolinic acid, into the rat striatum in vivo results in the degeneration of cholinergic and GABAergic neurons, as determined seven days later using the marker enzymes, choline acetyltransferase and glutamate decarboxylase, respectively. Such damage was dose-dependently prevented by CGP 37849 or MK-801 (competitive and uncompetitive N-methyl-D-aspartate receptor antagonists, respectively) administered systemically or intrastriatally at the same time as quinolinic acid. The neuroprotective activity of CGP 37849 was associated with the D-enantiomer, CGP 40116 (ED50 7.5 mg/kg i.p.), which was approximately 1.5-fold and 3.5-fold more potent than the related compounds, D-CPPene and CGS 19755, respectively. CGP 37849 was a weaker neuroprotectant than MK-801 (ED50 0.8 mg/kg i.p) when administered systemically, but was dramatically more potent following coinjection with quinolinic acid (ED50's 0.2 and 117 nmol, respectively). When injected intrastriatally 0.5-2 h post-quinolinic acid, CGP 37849 was protective over the entire period studied, whereas MK-801 was less effective at all post-quinolinic acid injection times. The finding that CGP 37849 is neuroprotective when administered intrastriatally 1-2 h post-quinolinic acid supports the hypothesis that a period exists following excitotoxic insult in which neurons are not committed to die, and can be rescued by blockade of ongoing N-methyl-D-aspartate receptor activation. Competition studies indicated that, when coinjected with 100-400 nmol quinolinic acid into the striatum, CGP 37849 exhibited kinetics predicted of a competitive N-methyl-D-aspartate receptor antagonist (declining neuroprotective potency with increasing doses of agonist), whereas MK-801 displayed a complex picture, with weak protective activity at low doses of quinolinic acid. Following systemic administration, neither antagonist was markedly affected by the dose of excitotoxin. When given i.p. at up to 6 h post-quinolinic acid, CGP 37849 and MK-801 showed essentially identical profiles of post-insult protection; degeneration of cholinergic neurons was reduced significantly throughout the entire post-insult period, whereas GABAergic neurons were protected only when drugs were administered 2 h or earlier post-quinolinic acid. The data indicate that competitive and uncompetitive N-methyl-D-aspartate receptor antagonists are effective neuroprotectants in vivo, and that parameters such as drug lipophilicity or mechanism of action at the receptor do not impinge upon their properties as systemically active cerebroprotectants.

Neuronal death and neurotrophin gene expression: long-lasting stimulation of neurotrophin-3 messenger RNA in the degenerating CA1 and CA4 pyramidal cell layers.

Neurotrophin-3 has been characterized as the product of a gene cloned by homology with nerve growth factor and brain-derived neurotrophic factor. Recombinant neurotrophin-3, like nerve growth factor and brain-derived neurotrophic factor, has been shown to enhance survival and differentiation of specific neuronal populations in vitro. However, little is known about its function and regulation in vivo. Both brain-derived neurotrophic factor and nerve growth factor messenger RNAs increased in adult rat brain, in a wide range of excitatory paradigms. In contrast, neurotrophin-3 messenger RNA decreased in some of them. Neurotrophin-3 is the most highly expressed neutrophic factor in immature areas of the central nervous system. However, no stimulation of its expression in the mature central nervous system, either in physiological or pathological conditions, has been described to date. This behaviour suggests that neurotrophin-3 could be involved in biological roles different from the prototypes nerve growth factor and brain-derived neurotrophic factor. Excitatory amino acid receptor-mediated neurotoxicity (excitotoxicity) is believed to contribute to neuronal loss in a wide range of neurodegenerative conditions (for a review, see Ref. 17). Moreover, locally increased levels of the endogenous excitotoxin quinolinic acid may be involved in the natural development of neurodegenerative diseases. The unilateral intrahippocampal injection of 120 nmol of quinolinic acid induced seizures together with local neurodegeneration in specific cell layers. In situ hybridization histochemistry was used to analyse the spatiotemporal pattern of expression of neurotrophin-3. As in other excitotoxic paradigms, neurotrophin-3 messenger RNA clearly decreased, nearly disappearing, in the contralateral hippocampus.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration of quinolinic acid in the rat hippocampus induces expression of c-fos and NGFI-A.

We have studied the effect of intrahippocampal administration of quinolinic acid (QUIN) on the temporal expression of mRNAs encoding the immediate early genes (IEGs) c-fos and NGFI-A, by in situ hybridization histochemistry. After administration of QUIN to the left hippocampus, expression of mRNA of both IEGs was transiently stimulated. Maximal expression was found between 1 and 3 h. mRNA of both IEGs was simultaneously expressed in the ipsilateral and contralateral sides in the granule cell layer of the dentate gyrus, the pyramidal cell layer of the CA1 and CA3 fields as well as in the cortex. After pretreatment with the non-competitive NMDA antagonist MK-801 (2 mg/kg i.p. -30 min) the increased expression of both IEGs was partially prevented in the hippocampus and completely in the cortex. No inhibition was observed after treatment with the AMPA antagonist NBQX (30 mg/kg i.p. -15, -5 and +10 min). Additional delayed expression of both IEGs was observed in the ipsilateral hippocampus. This expression was related to cell damage. Twelve h after QUIN administration, c-fos and NGFI-A mRNAs were present in the dentate gyrus. After 4 days, only c-fos mRNA was observed in the dentate gyrus and CA1 field while no NGFI-A mRNA was detected. The present results show that the effect of QUIN is mediated by NMDA and not by AMPA receptors.

Differential regulation of the expression of nerve growth factor, brain-derived neurotrophic factor and neurotrophin-3 mRNAs in adult rat brain after intrahippocampal injection of quinolinic acid.

Intrahippocampal injection of the endogenous excitotoxin quinolinic acid (QUIN) induces seizures together with local, delayed neurodegeneration in specific cell layers. In situ hybridization histochemistry was used to study the spatio-temporal pattern of expression of neurotrophins (NTFs) after this treatment. As in other excitatory paradigms, nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) mRNA levels increased dramatically and transiently in dentate gyrus after the administration of 120 nmol of QUIN to the left hippocampus. BDNF, but not NGF, mRNA also increased in the hippocampal pyramidal cell layer, mainly in the CA1 field. Neurotrophin-3 (NT3) mRNA levels decreased in dentate gyrus, practically disappeared around 12 h after the insult and returned to basal levels four days later. A very different pattern of expression of NTFs was found locally: (a) upregulation of NGF and BDNF mRNAs expression was prevented in a spherical region of 1-2 mm diameter around the injection site, (b) a delayed increase in NT3 mRNA levels, beginning at 12 h and lasting for at least 4 days after the administration of QUIN, was found in the same region, in cell layers showing neurodegeneration. Pretreatment with the non-competitive NMDA antagonist MK-801 (2 mg/kg, 30 min before the insult), partially blocked the increase in both BDNF and NGF mRNAs, as well as the decrease in NT3, in the contralateral hippocampus. However, this treatment did not prevent the QUIN-induced local downregulation of NGF and BDNF. Treatment with the AMPA/kainate antagonist NBQX (30 mg/kg, 15 and 5 min before, and 10 min after the insult) did not influence the effect of QUIN upon NGF or BDNF mRNA levels, although it partially prevented the hippocampal contralateral decrease in NT3 mRNA. In conclusion, the present study strongly supports previous work concerning different regulation of BDNF/NGF respect to NT3 in seizure inducing paradigms. Moreover, the different and to some extent opposite regulation of NTFs in the hippocampal region contiguous to the injection site, respect to the remaining hippocampus, suggests a differential regulation of NTFs in QUIN-induced neurodegenerative and seizural processes. Finally, our pharmacological data, (i) show that the upregulation of NGF and BDNF mRNAs, indirectly induced by QUIN, is not mediated by AMPA receptors, and (ii) suggest other effects for QUIN, apart from the stimulation of NMDA receptors.

Evaluation of quinolinic acid induced excitotoxic neurodegeneration in rat striatum by quantitative magnetic resonance imaging in vivo.

Excitotoxic neurodegeneration in the rat striatum was induced by direct injection of quinolinic acid. The degree of damage was evaluated in vivo 1 day later by quantitative magnetic resonance imaging (MRI) and 7 days later in the same animals by measuring the activities of the neuronal marker enzymes choline acetyltransferase and glutamic acid decarboxylase. Striatal damage assessed using the two approaches was highly correlated. Moreover the cerebroprotective efficacy of the N-methyl-D-aspartate receptor antagonist CGP 40116 was indistinguishable based on all analytical parameters. MRI, however, was more reproducible than the enzymatic methods and was faster and simpler for routine analyses of excitotoxic damage and cerebroprotection in vivo.

Seizures and wet-dog shakes induced by 4-aminopyridine, and their potentiation by nifedipine.

The behavioral and electrographic effects of 4-aminopyridine (4-AP) administered i.p. or microinjected into the hippocampal CA1 region (i.h.) were studied in rats. The modification of such effects by the systemic administration of the Ca2+ antagonist dihydropyridine, nifedipine, was also studied. 4-AP i.p. (5 mg/kg) induced generalized tonic convulsions in 74% of the animals and death in 13%. Convulsions were characterized by electrical discharges of relatively short duration in all structures studied (frontal cortex, amygdala, dorsal hippocampus and dorsal raphe). Limbic seizures and frequent wet-dog shakes were observed when 4-AP was administered i.h. (2-4 nmol) and this behavior was correlated with hippocampal discharges, which rapidly propagated to the other structures. Pretreatment with nifedipine (7.5-50 mg/kg s.c.) markedly potentiated the effects of 4-AP. The percentage of rats that died during generalized convulsion after i.p. 4-AP increased to 56-87% and the frequency of wet-dog shakes increased after i.h. microinjection of 4-AP. Moreover, nifedipine-treated rats showed long-lasting (greater than 60 min) continuous discharges in all structures studied (status epilepticus). These results are discussed in the light of the possible participation of Ca2+ channels in the convulsant effect of 4-AP and its potentiation by nifedipine.

Selective vulnerability of brain regions to oxidative stress in a non-coma model of insulin-induced hypoglycemia.

Insulin-induced hypoglycemia causes the death of neurons in particular brain regions including the cerebral cortex, the striatum and the hippocampus, while the cerebellum and the brain stem are more resistant. The mechanisms underlying this selective vulnerability to hypoglycemic damage are unknown. In the present study we have analyzed the presence of lipoperoxidation products and nitrosilated protein residues in different rat brain regions during and after the induction of hypoglycemia. Insulin-injected hypoglycemic rats were sacrificed before the onset of the isoelectric period or infused with glucose to end hypoglycemia, and then sacrificed at different times. Increased lipoperoxidation levels were observed before the onset of the isoelectric period, while 3-nitrotyrosine (NT) residues in proteins and NT-positive cells were only observed after glucose reperfusion. These changes were found only in vulnerable brain regions, while none of them was evident in the cerebellum, suggesting a correlation between oxidative damage and vulnerability to hypoglycemic neuronal death in selective brain regions. Results suggest that a pro-oxidant state is promoted in certain brain regions during hypoglycemia and after the glucose reperfusion phase, which might result from the activation of several oxidative stress pathways and may be related to subsequent cell death.

Pathways involved in the generation of reactive oxygen and nitrogen species during glucose deprivation and its role on the death of cultured hippocampal neurons.

Oxidative stress has been suggested as a mechanism contributing to neuronal death induced by hypoglycemia, and an early production of reactive species (RS) during the hypoglycemic episode has been observed. However, the sources of reactive oxygen (ROS) and nitrogen (RNS) species have not been fully identified. In the present study we have examined the contribution of various enzymatic pathways to RS production and neuronal death induced by glucose deprivation (GD) in hippocampal cultures. We have observed a rapid increase in RS during GD, which depends on the activation of NMDA and non-NMDA receptors and on the influx of calcium from the extracellular space. Accordingly, intracellular calcium concentration [Ca(2+)](i) progressively increases more than 30-fold during the GD period. It was observed that superoxide production through the activation of the calcium-dependent enzymes, phospholipase A(2) (cPLA(2)) and xanthine oxidase (XaO), contributes to neuronal damage, while nitric oxide synthase (NOS) is apparently not involved. Inhibition of cPLA(2) decreased RS at early times of GD whereas inhibition of XaO diminished RS at more delayed times. The antioxidants trolox and ebselen also showed a protective effect against neuronal death and diminished RS generation. Inhibition of NADPH oxidase also contributed to the early generation of superoxide. Taking together, the present results suggest that the early activation of calcium-dependent ROS producing pathways is involved in neuronal death associated with glucose deprivation.