Comparison between hypothermia and glutamate antagonism treatments on the immediate outcome of perinatal asphyxia.

This study investigated the influence of temperature or glutamate antagonism on the immediate outcome of perinatal asphyxia. Perinatal asphyxia was produced by water immersion of fetus-containing uterus horns removed by cesarean section from ready to deliver rats. The uterus horns were kept in a water bath for different time periods, before the pups were delivered and stimulated to breathe. After delivery, the pups were assessed for behavior and for systemic glutamate, aspartate, lactate and pyruvate levels measured with in vivo microdialysis, or ex vivo for energy-rich phosphates, including adenosine triphosphate (ATP), in brain, heart and kidney. In a series of experiments, asphyxia was initiated in a water bath at 37 degrees C, before the pup-containing uterus horns were moved for different time intervals to a 15 degrees C bath. In another series of experiments, the mothers were treated with N-methyl-D-aspartate (NMDA) antagonist, dizocilpine (MK-801), or alpha-amino-3-hydroxy-methylisoxazole-4-propionic acid (AMPA) antagonist,2,3-dihydroxy-6-nitro-7-sulfamoyl benzo(f) quinoxalin NBQX) 1 h before hysterectomy and asphyxia at 37 degrees C. The rate of survival rapidly decreased following exposure to more than 16 min of asphyxia, and no survival could be observed after 22 min of asphyxia. An LD50 was estimated to occur at approximately 19 min of asphyxia. The outcome was paralleled by a decrease in ATP in kidney, followed by a decrease in heart and brain. A maximal decrease in ATP was observed after 20 min of asphyxia in all tissues. Systemic microdialysis revealed that glutamate, aspartate and pyruvate levels were increased with a peak after 5 min of asphyxia. In contrast, lactate levels increased along with the length of the insult. Survival was increased when the pup-containing uterus horns were moved from a 37 degrees C to a 15 degrees C bath, at 15 min of asphyxia (the LD50 was thus increased to 30 min). If the shift occurred at 10 or 5 min of asphyxia, the LD50 increased to 80 or 110 min, respectively. The effect of glutamate antagonism was minor compared to hypothermia; the best effect (an increase in the LD50 to approximately 22 min) was observed after combining AMPA and NMDA antagonists.

Increased serum lipoprotein(a) levels in patients with early renal failure.

BACKGROUND: Elevated serum lipoprotein(a) levels have been found in patients with end-stage renal disease and in patients undergoing dialysis, suggesting that this lipoprotein contributes to the increased cardiovascular risk seen in these patients. It is not known whether lipoprotein(a) levels are elevated in the early phases of renal disease. OBJECTIVE: To evaluate levels of lipoprotein(a) and other lipids and the prevalence of atherosclerotic disease in patients with early renal failure. DESIGN: Cross-sectional study. SETTING: Hypertension clinic of a university medical center. PATIENTS: 257 patients with normal renal function and 160 patients with early impairment of renal function (creatinine clearance, 30 to 89 mL/min per 1.73 m2 of body surface area). MEASUREMENTS: Renal function was assessed by 24-hour creatinine clearance, proteinuria, and microalbuminuria. Cardiovascular disease status was also assessed. Serum lipoprotein(a), lipids, apolipoproteins, and apolipoprotein(a) isoforms were measured. RESULTS: Age, blood pressure, and serum lipoprotein(a) levels were greater in patients with early renal failure than in those with normal renal function and were independently associated with the presence of decreased creatinine clearance. Serum lipoprotein(a) and creatinine clearance were inversely correlated. The prevalence of coronary artery, cerebrovascular, and peripheral vascular disease was greater in patients with early renal failure than in those with normal renal function. The frequency distribution of apolipoprotein(a) isoforms was similar in patients with normal and those with impaired renal function. CONCLUSIONS: Serum lipoprotein(a) levels are elevated in patients with early impairment of renal function and are associated with greater prevalence of cardiovascular disease. An inverse correlation between serum lipoprotein(a) level and creatinine clearance and a frequency distribution of apolipoprotein(a) isoforms similar to that of normal patients point to decreased renal catabolism as a probable mechanism of lipoprotein(a) elevation in patients with early renal failure.

Delayed neuronal death following perinatal asphyxia in rat.

The consequences of perinatal asphyxia on the rat brain were studied 80 min to 8 days after birth with hematoxylin-eosin and in situ DNA double-strand-breaks labeling histochemistry. Asphyxia was induced by immersing fetus-containing uterus horns, removed from ready-to-deliver Sprague-Dawley rats, in a water bath at 37 degrees C for various time periods (0-22 min). Spontaneous- and cesarean-delivered pups were used as controls. Perinatal asphyxia led to a decrease in the rate of survival, depending upon the length of the insult. No gross morphological changes could be seen in the brain of either control or asphyctic pups at any of the studied time points after delivery. However, in all groups, nuclear chromatin fragmentation, corresponding to in situ detection of DNA fragmentation, was observed at different stages. Nuclear fragmentation in control pups showed a specific distribution that appeared to be related to brain maturation, thus indicating programmed cell death. A progressive and delayed increase in nuclear fragmentation was found in asphyctic pups, which was dependent upon the length of the perinatal insult. The most evident effect was seen in frontal cortex, striatum, and cerebellum at postnatal day 8, although changes were also found in ventral-posterior thalamus, at days 1 and 2. Thus, nuclear chromatin fragmentation in asphyctic pups indicates a delayed post-asphyctic neuronal death. The absence of signs of inflammation or necrosis suggests that delayed neuronal cell death following perinatal asphyxia is an active, apoptosis-like phenomenon.

Decrease of brain protein kinase C, protein kinase A, and cyclin-dependent kinase correlating with pH precedes neuronal death in neonatal asphyxia.

BACKGROUND: Acidosis, energy depletion, overstimulation by excitatory amino acids, and free radical-mediated reactions are the major, current concepts for the explanation of damage and death resulting from asphyxia. Impaired protein phosphorylation by protein kinase C represents another mechanism incriminated in cell death. METHODS: We used a nonsophisticated perinatal asphyxia model to study brain (frontal cortex) pH, ATP, protein kinases PKC, PKA, and cyclin-dependent kinase. We used o-tyrosine, a marker for hydroxyl radical attack, and LPO 586, a spectrophotometric assay, to study lipid peroxidation products. The antioxidant enzymes catalase, superoxide dismutase, and glutathione peroxidase were used in the frontal cortex. In addition, a cell death ELISA and histology to evaluate cell death were performed. RESULTS: Brain pH and protein kinases were decreasing with the length of the asphyctic periods, and energy depletion was shown by a drop of ATP levels, whereas no evidence for the involvement of free radical-mediated mechanisms was obtained. Cell death was shown by the cell death ELISA as early as 10 minutes after the asphyctic period, and histologically, cell death could be revealed but not before day 8 after asphyxia. CONCLUSION: Acidosis and/or impaired protein kinases, but not free radical mechanisms, may play a role in the pathobiochemistry of cell death in neonatal asphyxia of the rat.

Effect of perinatal asphyxia on systemic and intracerebral pH and glycolysis metabolism in the rat.

The effects of perinatal asphyxia on systemic and brain pH and glycolysis metabolism were studied in the rat. Perinatal asphyxia was induced by immersing pup-containing uterus horns, obtained by cesarean section from rats within the last day of gestation, in a water bath at 37 degrees C for various periods of time (0-23 min). Subcutaneous levels of pyruvate (Pyr), lactate (Lact), glutamate (Glu), and aspartate (Asp) were monitored with microdialysis 40-80 min after delivery. In parallel experiments, the pups were sacrificed 40 min after delivery and the heart and brain were removed for measuring pH. Brain (striatum) Pyr, Lact, Glu, and Asp levels were also analyzed. A decrease in the rate of survival was first observed following asphyctic periods longer than 16 min, and no survival could be observed after 22 min of asphyxia. In control (cesarean-delivered) pups, heart and brain pH were 7.36 +/- 0.01 (N = 8) and 7.30 +/- 0.01 (N = 8), respectively. Significant decreases in pH were first observed following 5-6 and 10-11 min of asphyxia, in heart and brain, respectively. In both regions pH decreased along with the length of asphyxia, but a decrease below 7 was only observed in the brain, following asphyctic periods longer than 16 min. A significant increase in subcutaneous Lact levels was first observed following 2-3 min of asphyxia, with a maximum after 20-21 min of asphyxia. In the brain, the increase in Lact levels was delayed compared to that observed in subcutaneous tissue. Pyr and Asp levels increased in subcutaneous tissue following perinatal asphyxia and decreased in brain tissue following > 15 min of asphyxia. Glu levels were increased subcutaneously by moderate (5-16 min) asphyctic periods, but, in the brain, were only transiently increased by 10-11 min of asphyxia. Thus, changes in systemic pH, glycolysis, and excitatory amino acid metabolism are observed following shorter asphyctic periods than are changes in the brain. In particular, increases in subcutaneous Lact levels precede: (i) a decrease in brain pH, (ii) an increase in brain Lact levels, (iii) a decrease in the rate of survival, and, probably, (iv) brain damage. It is suggested that monitoring Lact levels by subcutaneous microdialysis is a useful method for predicting the outcome produced by hypoxic-ischemic insults.

Effect of acetyl-L-carnitine on hyperactivity and spatial memory deficits of rats exposed to neonatal anoxia.

The effect of acetyl-L-carnitine (ALC) on behavioral deficits following neonatal anoxia (N2 100% for 25 min at 30 h after birth) was studied in the rat. Transient hyperactivity at P20-P45 postnatal days and permanent spatial memory deficits were shown by anoxic rats. A chronic ALC treatment (50 mg/kg per die injected intraperitoneally from P2, after anoxia, to P60) significantly reduced the transient increase in sniffing, rearing and locomotory activity of anoxic rats, but, mostly, ameliorated the spatial memory performances in a maze at P30-P40 and in a water maze at P50-P60. No behavioral changes were seen in ALC-treated animals that received sham-exposure at birth. On the basis of these results, the use of ALC for the treatment of perinatal asphyctic insults in children is suggested.

Short- and long-term effects of perinatal asphyxia on monoamine, amino acid and glycolysis product levels measured in the basal ganglia of the rat.

The effects of perinatal asphyxia on levels of dopamine (DA) and its metabolites, amino acids and glycolysis products, measured in tissue samples from substantia nigra (SN), striatum, ventral tegmental area (VTA), and nucleus accumbens (Acb), were studied 80 min to 8 days after birth with high performance liquid chromatography (HPLC). Furthermore, extracellular levels of DA, amino acids and glycolysis products were measured with in vivo microdialysis in the striatum 40-140 min and 4 weeks after birth. Asphyxia was induced by immersing foetus-containing uterus horns, removed from ready-to-deliver Sprague-Dawley rats, in a water bath at 37 degrees C for various time periods (0-22 min). Spontaneous- and caesarean-delivered pups were used as controls. Perinatal asphyxia led to a decrease in the rate of survival, depending upon the length of the insult. In parallel, lactate (LACT) levels were increased with the length of the insult in all examined brain regions, monitored ex vivo or in vivo immediately after birth. DA, glutamate (GLU) and aspartate (ASP) levels were also increased, mainly in tissue samples taken from the mesencephalon. Only minor changes were observed in tissue samples taken from the telencephalon. However, in experiments with in vivo microdialysis, DA and GLU levels were increased following 20-21 and 21-22 min of perinatal asphyxia, but the effect of K+ depolarisation on extracellular DA and ASP levels was strongly diminished. DA and metabolites increased with development in SN and striatum, with no clear differences between control and asphyctic rats. However, 8 days after birth, it was found that DA levels were increased, alternatively decreased in mesencephalic and telencephalic regions following 20-21 and 21-22 min of perinatal asphyxia, periods associated with 60% and 90% of perinatal mortality, respectively. Furthermore, in microdialysis experiments performed 4 weeks after birth, extracellular DA and its metabolites levels were also increased, alternatively decreased in rats exposed to a 20-21 and 21-22 min perinatal asphyctic insult. In this last group, GLU and ASP levels were also decreased. Furthermore, the effect of K+ depolarisation on DA and ASP levels was strongly decreased in both asphyctic groups. Thus, perinatal asphyxia produces short- and long-term consequences in general metabolism, and induces region-specific changes in several neurotransmitter systems, mainly affecting meso-telencephalic DA systems.

Development of GABA and calcium binding proteins immunoreactivity in the rat hippocampus following neonatal anoxia.

The consequences of neonatal anoxia (N2 100% for 25 min at 30 h after birth) on the rat hippocampus were studied 7-60 days postnatally with immunocytochemistry for gamma-aminobutyric acid (GABA), parvalbumin (PV) and calbindin-D28k (CB). In both sham-treated and anoxic rats, GABA immunoreactivity presented a mature expression since early stages, while PV and CB immunoreactivity showed a major postnatal development. In anoxic animals, a significant reduction in the number of hippocampal GABA-immunoreactive neurons was observed at all time-points analysed, a transitory effect on PV immunoreactivity was seen at P7 and P21, while no modifications in the number of CB-immunoreactive neurons could be found. Thus, selective vulnerability of GABA-containing neurons and relative resistance of neurons in which PV or CB immunoreactivity is present or is expressed later, occur in the hippocampus after neonatal anoxia. The role of calcium binding proteins (CBP) in nerve cell protection is discussed.

Changes in open field behavior, spatial memory, and hippocampal parvalbumin immunoreactivity following enrichment in rats exposed to neonatal anoxia.

Behavioral and neurochemical changes following enriched housing were studied in Wistar rats neonatally exposed to anoxia (100% N2 for 25 min at approximately 30 h after birth) or to sham treatment. Neonatal anoxia provoked transient hyperactivity during the P25-P40 period, and spatial memory disturbances persisting into adult life. Enriched housing, from P21, at weaning, to P60, improved behavior in open field and spatial memory abilities in a water maze, reducing the deficits that followed neonatal anoxia. Changes in the expression of the calcium binding protein parvalbumin were present in the CA1, CA3, and DG regions of the hippocampus in both sham-treated and anoxic rats exposed to enrichment. The present findings give further support to the evidence of a positive effect of enriched housing on behavior and learning of normal and lesioned animals, which is sustained by modifications in the neuronal activity, and suggest that modifications in the environment can be useful to counteract the development of some neurological disturbances that follow neonatal insults, e.g., perinatal asphyxia.

Superior cervical ganglion regenerating axons through peripheral nerve grafts and reversal of behavioral deficits in hemiparkinsonian rats.

The superior cervical ganglion (SCG) has been grafted to the brain of adult rats in an attempt to reverse the parkinsonian syndrome that follows destruction of central dopamine systems. However, the main limitation to this approach is the massive cell death that occurs in the grafted SCG after direct transplantation into the brain. In adult rats, 6-hydroxydopamine (6-OHDA) was stereotactically injected into the right substantia nigra (SN). One month later, dopamine denervation was assessed using the apomorphine-induced rotational test. In rats with a positive test, an autologous peripheral nerve (PN) graft was tunneled from the right cervical region to the ipsilateral parietal cortex. One end of PN graft was sutured to the transected postganglionic branch of the SCG and the other end was inserted into a surgically created cortical cavity. The apomorphine test was repeated at 3 days and again at 1, 3, and 5 months after surgery. The brain, SCG, and PN graft were studied under light and electron microscopy and with the tyrosine hydroxylase immunohistochemical and horseradish peroxidase tracing methods. Three days after grafting, there were no significant differences on the apomorphine test as compared to the preoperative test. Conversely, 1,3, and 5 months after grafting, the number of rotations was reduced by 69% (+/-20.2), 66.6% (+/-17.1), and 72.5% (+/-11.3), respectively. Control rats that received a free PN graft to the brain and underwent section of the postganglionic branch of the SCG did not show significant changes on the apomorphine test after surgery. Histological examination revealed that the PN graft was mostly reinnervated by amyelinic axons of small caliber. Approximately 40% of the SCG neuronal population that normally projects to the postganglionic branch survived axotomy and regenerated the transected axons into the PN graft. Axons arising from the SCG elongated the whole length of the graft, crossed the graft-brain interface and extended into brain regions adjacent to the denervated striatum up to 2037 micrometer from the graft insertion site. This work shows that the ingrowth of catecholamine-regenerating axons from the SCG to dopamine-depleted brain parenchyma significantly reduces behavioral abnormalities in hemiparkinsonian rats. This effect cannot be ascribed either to the brain cavitation or to the PN tissue placement in the brain.

Short-term effects of perinatal asphyxia studied with Fos-immunocytochemistry and in vivo microdialysis in the rat.

In the present study, the short-term consequences of various perinatal asphyctic periods were studied at the peripheral and CNS levels in the rat. Perinatal asphyxia was induced in rat pups delivered by caesarean section within the last day of gestation, by placing the uterus horns including the fetuses in a water bath at 37 degrees C for various periods of time (0-23 min). Following asphyxia, the uterus horns were opened. The pups were then removed and stimulated to breathe. Subcutaneous levels of pyruvate (Pyr), lactate (Lact), glutamate (Glu), and aspartate (Asp) were monitored with microdialysis 40 min after delivery. In parallel experiments, the pups were sacrificed 80 min after delivery. The brains were removed, fixed, cut, and processed for Fos immunocytochemistry. The number of Fos-immunoreactive (IR) cells in different brain structures was counted under light microscopy. Subcutaneous levels of Pyr, Lact, Glu, and Asp increased following perinatal asphyxia, as compared to caesarean-delivered pups or to spontaneously delivered controls. A maximum increase in Pyr levels (approximately threefold) was observed with 2-3 min of asphyxia, while Lact levels increased along with the length of asphyxia. A maximum increase in Glu and Asp levels (approximately threefold) was observed with 10-11 min of asphyxia. Fos-IR nuclei were predominantly found in the piriform cortex, and in the cortical amygdaloid complex. In some cases, mainly in pups exposed to asphyxia, Fos-positive cells were also seen in other tele-diencephalic structures.

Effects of DM-9384, a pyrrolidone derivative, on ischemia-induced changes in the central monoamine systems.

Alterations in brain tissue levels of monoamines and monoamine metabolites were studied in gerbils 60 min after cerebral ischemia induced by 10 min carotid ligation after pretreatment with the antiischemic drug DM-9384 (1, 3, 10, 30 mg/kg, PO). The DA levels decreased in striatum after the ischemia, while cortical and hippocampal DA levels increased. The DOPAC levels increased in cortex, but were essentially unaffected in other regions. The HVA levels increased in all forebrain regions studied. NA levels decreased in hippocampus and superior colliculus, while a general increase in MHPG levels was seen. Decreases in 5-HT levels were seen in all forebrain regions except cortex. The 10 mg/kg and 30 mg/kg doses of DM-9384 counteracted the decrease in striatal 5-HT and hypothalamic MHPG/NA ratio, respectively. Thus pretreatment with DM-9384 exerted minor protective effects on the alterations induced in monoamine systems by transient forebrain ischemia.

Immunochemical detection of S-100 protein in non-nervous structures of the rabbit eye.

S-100 is a protein originally believed to be unique to the nervous system. We report here on its immunochemical detection in non-nervous structures of the rabbit eye, namely in the lens, cornea and iris, at a markedly higher concentration in the latter.

Immunochemical and immunocytochemical study of S-100 protein in rat adipocytes.

S-100 is a protein originally believed to be unique to the nervous system. We report here on the presence of S-100 in rat adipocytes, using immunohistochemical and immunochemical methods. We demonstrate that the protein in adipose tissue is present at a concentration comparable to that measured in the nervous tissue and is immunologically identical to brain S-100, indicating that the protein can no longer be regarded as being specific to the nervous system.