Neuronal injuries induced by perinatal hypoxic-ischemic insults are potentiated by prenatal exposure to lipopolysaccharide: animal model for perinatally acquired encephalopathy

We developed an original rat model for neonatal brain lesions whereby we explored the sequential effects of infectious and hypoxic-ischemic aggressions. We investigated the influence of combined exposure to prenatal infection with neonatal hypoxic-ischemic insult. Infectious effect was produced by administrating lipopolysaccharide (LPS) intraperitoneally to pregnant rats starting on embryonic day 17. Hypoxia-ischemia (H/I) was induced in the pups at postnatal day 1 (P1) by ligature of the right common carotid artery followed by exposure to hypoxia (8% O(2)) for 3.5 h. Animals were randomized into four groups: (1) control group: pups born to mothers subjected to intraperitoneal saline injection; (2) LPS group: pups exposed in utero to LPS; (3) H/I group: pups exposed to postnatal hypoxia after ligation of the right carotid artery, and (4) H/I plus LPS group: in utero exposure to LPS followed by postnatal hypoxia after ligation of the right carotid artery. Neuropathological findings in pups examined at P3 and P8 showed that groups 2, 3, and 4 presented a pattern of neuronal injury similar to those characterized as 'selective neuronal necrosis' within the context of human perinatal encephalopathy. Neuronal cellular injuries were particularly seen in the neocortex, mainly in parasagittal areas. The extent of neuronal cell injury in the brain of rats exposed to postnatal H/I was significantly increased by antenatal exposure to LPS. This animal model provides an experimental means to explore the respective roles of anoxic and infectious components in the pathogenesis of perinatal brain lesions and consequent cerebral palsy.     

Decreased expression of bcl-2 and bcl-x mRNA coincides with apoptosis following intracerebral administration of 3-nitropropionic acid

The mitochondrial toxin, 3-nitropropionic acid (3-NP), is an irreversible inhibitor of succinate dehydrogenase that induces apoptosis in vitro and in vivo. We injected 3-NP into the striatum of rats to examine the potential role of Bcl-2 or Bcl-x, proteins that can inhibit apoptosis, in brain injury due to 3-NP. Electrophoretic examination of striatal tissue indicated that 3-NP induced internucleosomal fragmentation typical of apoptosis. There was also histologic evidence of apoptosis based on staining by the terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL) method. Apoptosis was first observed 6 h after injection, was maximal at 1 day, and was still observed on day 7. Expression of bcl-2, bcl-x, and c-jun mRNA expression was evaluated 1, 3, 6, and 12 h and 1, 3, 5, and 7 days after injection using in situ hybridization. Both bcl-2 and bcl-x mRNA expression in the striatum decreased starting at 6 h and continued to 5 days after injection. This was in contrast to an apparent increase in c-jun expression. The similarity in the time course of apoptosis to that of suppression of bcl-2 and bcl-x mRNA suggests that changes in expression of these genes may contribute to apoptosis following 3-NP injection.     

大鼠脑出血后血肿周围组织线粒体功能变化的研究

目的:研究大鼠脑出血后脑组织线粒体功能的变化。方法:以50 μL鼠尾血注入大鼠尾状核制作大鼠脑出血模型,注血后分别于12 h及1、3、7 d处死大鼠,断头,取血肿周围脑组织,测定不同时相点血肿周围组织线粒体功能(琥珀酸脱氢酶活性、呼吸控制率)。结果:出血后12 h线粒体呼吸控制率及琥珀酸脱氢酶活性无明显变化,1 d后呼吸控制率较对照组下降,3 d时线粒体呼吸控制率及琥珀酸脱氢酶活性均较对照组下降,出血后7 d线粒体琥珀酸脱氢酶活性、呼吸控制率则较对照组明显下降(P<0.01)。结论:脑出血早期(尤其在12 h之内)血肿周围组织线粒体功能无明显下降,晚期则明显受损。     

3-Nitropropionic acid neurotoxicity in hippocampal slice cultures: developmental and regional vulnerability and dependency on glucose

We investigated whether neurotoxic effects of the mitochondrial toxin 3-nitropropionic acid (3-NP) in hippocampal slice cultures are dependent on glucose levels in the culture medium and whether such effects occur via apoptosis or necrosis. In addition, 3-NP toxicity was investigated at two developmental stages of the cultures, prepared from rat brain at postnatal day 5-7 and grown in Neurobasal medium for 1 or 3 weeks. Cultures were exposed to 3-NP in the presence of high (25 mM), normal (5 mM), or low (3 mM) glucose for 48 h, followed by 48 h incubation in medium without 3-NP. Cellular propidium iodide (PI) uptake and lactate dehydrogenase (LDH) efflux into the medium revealed time- and dose-dependent cell death by 3-NP, with EC(50) values of about 60 microM in high or normal glucose. Regional vulnerability, as assessed by PI uptake and MAP2 immunostaining, in 3-week-old cultures was as follows: CA1 > CA3 > fascia dentata. In low glucose much lower concentrations of 3-NP (25 microM) triggered neurotoxicity. One-week-old cultures were less susceptible to 3-NP toxicity than 3-week-old cultures, but the dentate granule cells were relatively more affected in the immature cultures. We found no evidence for apoptotic cell death by 3-NP in 3-week-old cultures, but in 1-week-old cultures the putative apoptotic marker c-JUN/AP1 and nuclear fragmentation (Hoechst) were significantly increased in the dentate granule cells.     

Effects of early chronic diazepam treatment on incorporation of glucose and β-hydroxybutyrate into cerebral amino acids: Relation to undernutrition

The effects of early chronic diazepam (DZP) exposure on blood glucose and ketone body concentration and glucose and β-hydroxybutyrate (βHB) utilization for regional cerebral amino acid biosynthesis were studied in suckling rats. The animals were treated from postnatal day 2 (P2) to 21 (P21) by a daily subcutaneous injection of 10 mg/kg DZP or of the dissolution vehicle and studied at P5, P10, P14 and P21, together with an additional group of food-restricted rats obtained by an increase in litter size. DZP treatment induced a 9–26% decrease in body and brain weight. Undernutrition decreased body weight by 20–24% at all ages whereas brain weight was relatively spared. DZP and N-desmethyldiazepam concentrations decreased with age and were cleared from brain and plasma by 6–8 hrs after the injection. DZP decreased plasma glucose concentrations by 6–12% at P5, P14 and P21, whereas undernutrition did not change plasma glucose concentrations, except for a 7% decrease at P14. DZP treatment had no consequences on circulating concentrations of both ketone bodies while undernutrition increased their concentration by 45–362% at all ages. The conversion of [¹⁴C]glucose into cerebral amino acids was reduced by DZP at P5 and P10. The cerebral concentration of neurotransmitter amino acids was not affected by DZP treatment which only increased the amount of neutral amino acids mainly in the cerebellum at P5 and P10. After [U-¹⁴C]glucose injection, specific radioactivities of cerebral amino acids were mostly decreased by DZP from P5 to P14 and significantly increased at P21. With [3-¹⁴C]βHB as a precursor, specific radioactivities of neurotransmitter amino acids were increased by DZP. In conclusion, P5 and P10 rats appear to be most sensitive to DZP effects whereas some tolerance to the drug seems to develop by P21. The lack of effects of DZP on blood ketone body concentrations compared to food restriction as well as the relative sparing of brain weight in undernourished rats confirms that the cerebral metabolic consequences of early DZP exposure on brain energy metabolism are mostly direct effects not mediated by sedation-induced undernutrition.     

Neonatal hypoxia suppresses oligodendrocyte Nogo-A and increases axonal sprouting in a rodent model for human prematurity

Premature human infants frequently suffer from periventricular leukomalacia (PVL) characterized by the loss of central myelinated tracts in the brain [Neuropathology, 22 (2002) 193]. Rodent chronic sublethal hypoxia (CSH) from P3 to 33 (postnatal day 3-33) provides a model for PVL characterized by cerebral ventriculomegaly and reductions in cerebral white matter volume [Brain Res. Dev. Brain Res. 111 (1998) 197; Proc. Natl. Acad. Sci. USA 100 (2003) 11718]. Here, we demonstrate that mice exposed to CSH from P3 to P33 followed by normoxia from P33 to P75 continue to exhibit a locomotor hyperactivity that resembles behavioral changes observed in some human children with very low birth weights. Because periventricular white matter is specifically lost in PVL, we examined the expression of oligodendrocyte proteins. Hypoxic rearing dramatically decreases the level of the axon outgrowth inhibitor Nogo-A in oligodendrocytes of CNS white matter at P12. The Nogo-A decrease exceeds the moderate decrease in another myelin protein, myelin associated glycoprotein (MAG). Although myelin protein expression returns to normal by maturity (P75), persistent abnormalities in axonal trajectories are detectable. Anterograde axonal tracing from motor cortex demonstrates ectopic corticofugal fibers in the corticospinal tract (CST), corpus callosum, and caudate nucleus of adult animals reared in CSH. Thus, hypoxia-induced reduction in myelin-derived axon outgrowth inhibitors appears to contribute axonal misconnection to the pathology of very low birth weight infants.     

Intranasal administration of IGF-1 attenuates hypoxic-ischemic brain injury in neonatal rats

To determine whether intranasal administration (iN) of recombinant human insulin-like growth factor-1 (rhIGF-1) provides neuroprotection to the neonatal rat brain following cerebral hypoxia-ischemia (HI), two doses of rhIGF-1 (50 μg at a 1 h interval) were infused into the right naris of postnatal day 7 (P7) rat pups with or without a prior HI insult (right common carotid artery ligation, followed by an exposure to 8% oxygen for 2 h). Our result showed that rhIGF-1 administered via iN was successfully delivered into the brain 30 min after the second dose. In the following studies rhIGF-1 was administered to P7 rat pups at 0, 1 or 2 h after HI at the dose described above. Pups in the control group received cerebral HI and vehicle treatment. Pups that underwent sham operation and vehicle treatment served as the sham group. Brain pathological changes were evaluated 2 and 15 days after HI. Our results showed that rhIGF-1 treatment up to 1 h after cerebral HI effectively reduced brain injury as compared to that in the vehicle-treated rats. Moreover, rhIGF-1 treatment improved neurobehavioral performance (tested on P5–P21) in juvenile rats subjected to HI. Our results further showed that rhIGF-1 inhibited apoptotic cell death, possibly through activating the Akt signal transduction pathway. rhIGF-1 enhanced proliferation of neuronal and oligodendroglial progenitors after cerebral HI as well. These data suggest that iN administration of IGF-1 has the potential to be used for clinical treatment.     

A strong epileptogenic effect of mechanical injury can be reduced in the dysplastic rat brain. Long-term consequences of early prenatal gamma-irradiation

An exposure of rats to gamma-radiation at different stages of prenatal development produces brain dysplasias of different degree displaying also different susceptibility to pilocarpine-induced seizures. Following irradiation on prenatal day 13 (E13), the susceptibility is minimal and significantly lower even in relation to non-irradiated rats [Setkowicz, Z., Janeczko, K., 2003. Long-term changes in susceptibility to pilocarpine-induced status epilepticus following neocortical injuries in the rat at different developmental stages. Epilepsy Res. 53, 216-224]. On the other hand, the rat brain injured on postnatal day 30 presents very high susceptibility to seizures in the same pilocarpine model of epilepsy [Setkowicz, Z., Kluk, K., Janeczko, K., 2003. Long-term changes in postnatal susceptibility to pilocarpine-induced seizures in rats exposed to gamma radiation at different stages of prenatal development. Epilepsia 44, 1267-1273]. It could, therefore, be hypothesised that the congenital brain dysplasia produced by irradiation on E13 would minimize the highly increased susceptibility to seizures observed in the injured brain. Wistar rats were exposed to gamma-rays on E13 and they received a mechanical brain injury on postnatal day 30 (P30). On postnatal day 60, pilocarpine was injected to evoke status epilepticus. During a 6-h period following the injection, motor manifestations of seizure activity were recorded and rated. Seven days after pilocarpine injection, the animals were sacrificed and their brains were fixed. Pilocarpine injections in non-irradiated rats with brains injured on P30 evoked seizures of very high intensity and extremely high mortality in relation to non-injured controls. This high susceptibility to seizures following the brain injury was considerably decreased in rats irradiated on E13. The data provide evidence that the brain dysplasia in the rat acquired at this stage of prenatal development can significantly reduce the increased susceptibility to seizures evoked by the postnatal brain injury.     

Maternal deprivation in the early versus late postnatal period differentially affects growth and stress-induced corticosterone responses in adolescent rats

Periodic maternal deprivation (MD) in the early postnatal period leads to permanently altered responsibility of the hypothalamus-pituitary-adrenocortical (HPA) axis to various types of stress. However, no reports appear to have described the effect of periodic MD under different conditions on growth of the developing rat and responsibility of the HPA axis to immobilization stress in adolescent rats. Furthermore, although body weight changes are known to affect stress responsibility, their relationship under periodic MD is not clear. The present study therefore used 4 different types of periodic MD: for 12 h/day from postnatal day (P)1 to P6 (12E group); for 3 h/day from P1 to P6 (3E group); for 12 h/day from P16 to P21 (12L group); and for 3 h/day from P16 to P21 (3L group). Mean body weights were less in the 3E and 12E groups than in the control group until at least 9 weeks old, although body weight gain in the 3L and 12L groups was only transiently affected. Stress-induced corticosterone levels in the 3E and 12E groups did not return to basal levels until at least 330 min after the termination of stress, while temporal variations of stress-induced corticosterone levels did not differ significantly between the 3L, 12L and control groups. Periodic MD in the first postnatal week affected growth of developing rats and responsibility of the HPA axis to immobilization stress in adolescent rats, and the extent of this modification was larger with MD for 12 h/day than with MD for 3 h/day. Conversely, periodic MD from P16 to P21 had little effect. Periodic MD in the postnatal period induces long-term effects on growth and stress responsibility of the HPA axis. Furthermore, a critical age of the pup at the time of MD exists as well as a critical length of MD for inducing these effects.     

Influence of premature rupture of membrane on the cerebral blood flow in low-birth-weight infant after the delivery

Cerebral white matter injury, usually called periventricular leukomalacia (PVL), is the most common form of injury to preterm infants that is associated with adverse motor and cognitive outcomes. Intrauterine infection may be an important etiological factor in PVL, and premature rupture of the membranes (PROM) can be identified antepartum. In order to investigate the pathophysiology of cerebral white matter injury induced by PROM, the cerebral blood flow (CBF) of the internal carotid artery and the vertebral artery was measured by neck ultrasonography. The CBF was determined in 84 low-birth-weight infants with gestational ages ranging from 24 to 35 weeks, including 71 infants without PROM and 13 infants with PROM. The mean blood flow velocity and diameter of each vessel were measured on postnatal days 0–70. The intravascular flow volume was determined by calculating the mean blood flow velocity and the cross-sectional area. The mean blood pressures were recorded, and the ejection fraction was determined. The total cerebral blood flow (CBF) was significantly lower in infants with PROM than in infants without PROM from day 10 to day 70. The ejection fraction was significantly higher in infants with PROM than in infants without PROM on days 0, 5, 10, 21, and 42. There was no difference in the mean blood pressure between infants with PROM and infants without PROM. The results of the present study suggest that PROM may decrease cerebral blood flow after the birth.