VEGF overexpression in the astroglial cells of rat brainstem following ozone exposure.

Ozone, a major photochemical pollutant, produces rapid damages in the pulmonary airway tract and in the central nervous system. This study focused on the neural mechanisms underlying the adaptive responses to an acute ozone exposure. Vascular endothelial growth factor (VEGF) is a factor associated with cellular recovery following brain injury. The aim of this study was to assess and localize the cellular expression of VEGF, since the central respiratory areas show a neuroplasticity in response to ozone. Adult rats were subjected to 0.5ppm ozone for 3h and then recovered for further 3h. The expression of VEGF was evaluated by immunocytochemistry in the central respiratory areas, i.e., the nucleus tractus solitarius (NTS) and the ventrolateral medulla (VLM). The data show a VEGF overexpression at the end of ozone exposure, which persisted during the 3-h recovery. Interestingly, using confocal analysis the bulk of VEGF labeling was observed in astroglial cell bodies and branches, while neuronal labeling was hardly noticed. Moreover, VEGF colocalized with IL-6 and TNFalpha in astrocytes closely apposed to blood vessel walls. The vasculature area was markedly increased (+58%) during post-ozone recovery. The data show that an acute ozone exposure affects primarily glial cells in the central nervous system. The VEGF up-regulation which persists after ozone exposure may contribute to brain repair and consecutive functional adaptations.

Prenatal nicotine alters maturation of breathing and neural circuits regulating respiratory control.

While perinatal nicotine effects on ventilation have been widely investigated, the prenatal impact of nicotine treatment during gestation on both breathing and neural circuits involved in respiratory control remains unknown. We examined the effects of nicotine, from embryonic day 5 (E5) to E20, on baseline ventilation, the two hypoxic ventilatory response components and in vivo tyrosine hydroxylase (TH) activity in carotid bodies and brainstem areas, assessed at postnatal day 7 (P7), P11 and P21. In pups prenatally exposed to nicotine, baseline ventilation and hypoxic ventilatory response were increased at P7 (+48%) and P11 (+46%), with increased tidal volume (p<0.05). Hypoxia blunted frequency response at P7 and revealed unstable ventilation at P11. In carotid bodies, TH activity increased by 20% at P7 and decreased by 48% at P11 (p<0.05). In most brainstem areas it was reduced by 20-33% until P11. Changes were resolved by P21. Prenatal nicotine led to postnatal ventilatory sequelae, partly resulting from impaired maturation of peripheral chemoreceptors and brainstem integrative sites.

Long-term prenatal hypoxia alters maturation of brain catecholaminergic systems and motor behavior in rats.

In order to determine the influence of long-term prenatal hypoxia on the maturation of the brain catecholaminergic structures involved in motor and cognitive functions, pregnant rats were subjected to hypoxia (10% O2) from the 5th to 20th day of gestation. The in vivo activity of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, was assessed, by accumulation of L-DOPA after i.p. administration of NSD-1015, in the motor cortex areas, the hippocampus, and the striatum at birth and at the 3rd, 7th, 14th, 21st, and 68th postnatal days. The motor reactivity to novelty and the circadian motor activity were measured at the 21st and 68th postnatal days. Exposure to prenatal hypoxia strongly altered the developmental pattern of in vivo TH activity in restricted noradrenergic terminals of the brain. In the 21-day-old prenatal hypoxic rats, the TH activity was reduced by 80% in the motor cortex areas and by 43% in the hippocampus, compared to control rats, while no differences could be detected in the striatum. Compared to control rats, the prenatal hypoxic pups exhibited a higher motor reactivity to novelty and a nocturnal motor hypoactivity at the 21st postnatal day. The neurochemical and behavioral alterations were no longer observed at the 68th postnatal day. The altered in vivo TH activity in the young rats might be part of the neural mechanisms contributing to the motor behavioral impairments induced by prenatal hypoxia. Long-term prenatal hypoxia could be linked to the development of psychopathologies that can be detected in infancy.

Central and peripheral changes in catecholamine biosynthesis and turnover in rats after a short period of ozone exposure.

We investigated in rat the effects of ozone exposure (0.7 ppm) for 5 h on the catecholamine biosynthesis and turnover in sympathetic efferents and various brain areas. For this purpose, the activity of tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, was assessed in superior cervical ganglia and in two major noradrenergic cell groups, A2 and A6 (locus coeruleus). Tyrosine hydroxylase activity was estimated in vivo by measuring the accumulation of l-dihydroxyphenylalanine after pharmacological blockade of L-aromatic acid decarboxylases by NSD-1015 (100 mg/kg i.p.). The catecholamine turnover rate was measured after inhibition of tyrosine hydroxylase by alpha-methyl-para-tyrosine (AMPT, 250 mg/kg, i.p., 2.5 h) in peripheral sympathetic target organ (heart and lungs) as well as in some brain catecholamine terminal areas (cerebral cortex, hypothalamus and striatum). Ozone caused differential effects according to the structure. Catecholamine biosynthesis was stimulated in superior cervical ganglia (+44%, P < 0.05) and caudal A2 subset (+126%, P < 0.01), whereas catecholamine turnover was increased in heart (+183%, P < 0.01) and cortex (+22%, P < 0.05). On the other hand, catecholamine turnover was inhibited in lungs (-53%, P < 0.05) and striatum (-24%, P < 0.05). A brief exposure to ozone, at a concentration chosen to mimic pollution level encountered in urban areas, can modulate catecholamine biosynthesis and utilization rate in the sympathetic and central neurones.

Gestational hypoxia induces white matter damage in neonatal rats: a new model of periventricular leukomalacia.

In the premature infant, periventricular leukomalacia, usually related to hypoxicischemic white matter damage, is the main cause of neurological impairment. We hypothesized that protracted prenatal hypoxia might induce white matter damage during the perinatal period. Pregnant Sprague-Dawley rats were placed in a chamber supplied with hypoxic gas (10% O2-90% N2) from embryonic day 5 (E5) to E20. Neonatal rat brains were investigated by histology, immunocytochemistry, western blotting, in situ hybridization, DNA fragmentation analysis, and in vivo magnetic resonance imaging (MRI). Body weight of pups subjected to prenatal hypoxia was 10 to 30% lower from P0 to P14 than in controls. Specific white matter cysts were detected between P0 and P7 in pups subjected to prenatal hypoxia, in addition to abnormal extra-cellular matrix, increased lipid peroxidation, white matter cell death detected by TUNEL, and increased activated macrophage counts in white matter. Subsequently, gliotic scars and delayed myelination primarily involving immature oligodendrocytes were seen. In vivo MRI with T1, T2, and diffusion sequences disclosed similar findings immediately after birth, showing strong correlations with histological abnormalities. We speculate that protracted prenatal hypoxia in rat induces white matter damage occurring through local inflammatory response and oxidative stress linked to re-oxygenation during the perinatal period.

Sympathetic and brain monoaminergic regulation of energy balance in obesity-resistant rats (Lou/C).

In contrast to the Wistar rat, the Lou/C rat does not develop obesity with age. To determine the role of sympathetic output and brain monoamines in the different energy balance of Lou/C rats, the monoamine contents and activity of rate-limiting enzymes in catecholamine and serotonin biosynthesis were assessed in brain structures involved in energy balance regulation, i.e., brainstem noradrenergic (A6, A5, A2) and serotonergic cell groups (dorsal raphe, and median raphe), and two hypothalamic nuclei (ventromedial nucleus and paraventricular nucleus). In vivo tyrosine hydroxylase activity and noradrenaline content were measured in sympathetic target organs storing fuel substrates, the liver, white adipose and brown adipose tissues in the Lou/C rat and compared to the Wistar rat. In Lou/C rats, indirect calorimetric measurements showed a higher energy expenditure despite a reduced food intake. The Lou/C rat displayed selective monoamine features. The catecholaminergic activity was higher in the white adipose tissue and interscapular brown adipose tissue but lower in the liver and adrenal gland of Lou/C rats. The noradrenergic activity in A2, A6 and ventromedial nucleus, and the serotonergic pattern in A6, dorsal raphe and median raphe were lower in Lou/C. The metabolic particularities of Lou/C rats are associated with (i) a selectively enhanced sympathetic activity restricted to the white adipose tissue and brown adipose tissue, (ii) a reduced noradrenergic activity in selective brainstem and hypothalamic areas, which control the energy expenditure and food intake.

Prenatal hypoxia impairs circadian synchronisation and response of the biological clock to light in adult rats.

The aim of this study was to test the hypothesis that prenatal hypoxia in rats might lead to consistent changes in the entrainment of the circadian clock by light. Pregnant female rats were placed in a chamber provided with hypoxic gas (10 % O2--90 % N2) at gestational day 5 and returned to normoxia before delivery. Once adult, rats born to hypoxic mothers had significant alterations in their circadian rhythm of locomotor activity (recorded in freely accessible running wheels). Under a regular 12/12 light/dark (LD) cycle, they showed a phase advance of their rhythm of activity (mean phase advance of 87 min) and were less active than control rats. After an abrupt 6 h phase delay in the LD cycle, rats from the prenatal hypoxic group (PNH) took significantly more time to resynchronise to the new LD cycle compared to controls (+53 %; 6.0 +/- 1.5 vs. 9.2 +/- 0.5 days respectively). Under constant darkness, PNH and control rats had a similar period of activity (24.27 +/- 0.20 vs. 24.40 +/- 0.13) but the response of PNH rats to a light pulse in the early subjective night was less marked than that of control rats (101 +/- 9 vs. 158 +/- 13 min). When submitted to acute restraint stress, PNH rats had a prolonged secretion of corticosterone compared to controls. These results indicate that prenatal hypoxia is a factor that has long lasting consequences for the functional output of the biological clock and the hormonal response to stress.

Long-lasting adverse effects of prenatal hypoxia on developing autonomic nervous system and cardiovascular parameters in rats.

To determine whether prenatal hypoxia increases the risk of developing cardiovascular disorders as an adult and, if so, the identity of the cell mechanisms involved in such dysfunction, we evaluated the sympathoadrenal system and central areas related to cardiovascular events during development and the cardiovascular parameters in adults. Pregnant rats were exposed to hypoxia (10% oxygen) from embryonic day (E) 5 to E20 and the offspring studied at 1, 3, 9 and 12 weeks of age for neurochemistry and at 12 weeks of age for cardiovascular analysis. In the 1-, 3- and 9-week-old offspring, the levels and utilization of catecholamines were reduced in sympathetic ganglia, in target organs, in adrenals and in the rostral part of the A2 cell group in the nucleus tractus solitarius, but were increased in the locus coeruleus. In the 12-week-old adult offspring, the lowered autonomic nervous activity was restricted to cardiac-related structures, i.e. the stellate ganglion, heart and adrenals. In adult rats, prenatal hypoxia did not affect the cardiac parameters under resting conditions but increased blood pressure and the variability of blood pressure and heart rate under stress conditions. The altered metabolic activity of the sympathoadrenal system and related central areas during development and at adulthood for most structures might be part of the potential mechanisms contributing to cardiovascular disorders in adults.

Long-term prenatal hypoxia alters maturation of adrenal medulla in rat.

Catecholamine release from the adrenal medulla glands plays a vital role in postnatal adaptation. A number of pathologic situations are characterized by oxygen deficiency. The objective of the present study was to determine the influence of long-term prenatal hypoxia on maturation of the adrenal medulla. Pregnant rats were subjected to hypoxia (10% O2) from the fifth to the 20th d of gestation. The offspring were examined on the 19th d of gestation (E19), the day of birth (P0), and at postnatal (P) day of life P3, P7, P14, P21, and P68. The catecholamine content and activity of tyrosine hydroxylase (TH) in vivo were assayed by HPLC with electrochemical detection. Cellular expression of TH and phenylethanolamine N-methyl transferase was evaluated by protein immunohistochemistry and in situ hybridization of the corresponding mRNA species. Exposure to prenatal hypoxia reduced the epinephrine content of the adrenal medulla on E19, P0, P3, and P7 while increasing the norepinephrine content on E19, P0, and P14. Furthermore, the peak epinephrine to norepinephrine ratio appearing between P7 and P10 in the normoxic offspring was absent in the hypoxic offspring. The in vivo TH activity was increased on P3 and P14 and decreased on P68. The percentage of chromaffin cells in the medulla expressing TH and phenylethanolamine N-methyl transferase was lowered on E19, P0, and P7. TH and phenylethanolamine N-methyl transferase mRNA levels were reduced on P7. Clearly prenatal hypoxia results in major changes in adrenal catecholamine stores and synthesis during the perinatal period, which persist into adulthood. The capacity to cope with postnatal stress might be disturbed as a consequence of prenatal hypoxia.