Isabella Zini

Detection of free radicals during brain ischemia and reperfusion by spin trapping and microdialysis

Extracellular free radicals were detected in rat striatal perfusate samples by intracerebral microdialysis coupled to the spin trapping technique. Five Sprague-Dawley rats were subjected to 30 min of global ischemia followed by reperfusion; throughout the experimental period the intrastriatal dialysing probe was perfused with Ringers solution containing the spin trap agent pyridyl-N-oxide-t-butylnitrone (100 mM) together with the iron chelating agent diethylentriaminepentacetic acid (100 microM). A radical adduct occurred during ischemia and early reperfusion, but not in basal conditions; the spin adduct was characterized as a carbon centered radical, consistent with the presence of an oxidative attack on membrane lipids. The direct evidence of the formation of free radicals supports the hypothesis that free radicals play a role in the pathogenesis of the histological damage during brain ischemia.

Morphometrical and microdensitometrical studies on phenylethanolamine-N-methyltransferase- and neuropeptide Y-immunoreactive neurons in the rostral medulla oblongata of the adult and old male rat.

In the present paper the neuronal systems of the medulla oblongata containing phenylethanolamine-N-methyltransferase- and neuropeptide Y-like immunoreactivity have been characterized in adult (3-month-old) and old (24-month-old) male rats. The phenylethanolamine-N-methyltransferase and neuropeptide Y-immunoreactive neurons have been visualized by means of immunocytochemistry (peroxidase-antiperoxidase technique) and analysed in a quantitative fashion by means of morphometrical (phenylethanolamine-N-methyltransferase- and neuropeptide Y-immunoreactive cell groups) and microdensitometrical (phenylethanolamine-N-methyltransferase-immunoreactive cell groups) approaches developed on the IBAS II image analyser (Zeiss-Kontron). During aging there is (a) a reduction in the area covered by the phenylethanolamine-N-methyltransferase-immunoreactive neuropil for both the C1 and C2 adrenaline cell groups; (b) a reduction in the area covered by the phenylethanolamine-N-methyltransferase-immunoreactive cell bodies, which is highly significant only for the C2 cell group; (c) a decrease in the area covered by the phenylethanolamine-N-methyltransferase-positive cell cluster for both C1 and C2 cell groups; (d) a decrease in the degree of phenylethanolamine-N-methyltransferase immunoreactivity present in the C1 and C2 cell groups; (e) a decay of neuropeptide Y immunoreactivity in the C1 and C2 groups, while the C3 group is unaffected by aging as evaluated by number of phenylethanolamine-N-methyltransferase- and neuropeptide Y-immunoreactive cell body profiles. These results indicate heterogeneities in the responses of the adrenaline-neuropeptide Y cell groups to the aging process. The possible functional consequences of aging-induced changes in the cardiovascular adrenergic neurons are discussed, especially in relation to development of hypertension.

Aspects of neural plasticity in the central nervous system-I. Computer-assisted image analysis methods.

Microdensitometric and morphometric techniques have been developed to quantitatively characterize cell groups and terminal populations of transmitter-identified neuronal systems. Various microdensitometric methods implemented on the image analyzer or on the scanning microdensitometer were introduced and compared. On this basis a technique to assess the half-life of dopamine stores determined by quantitative immunocytochemistry has been developed. The problem of relative and absolute quantification of microdensitometric analysis of immunocytochemical preparations is discussed here. A method has been developed for the study, both in 2- and 3-dimensions, of the overall features of the profile distribution in a defined neuroanatomical area. An approach to determine the degree of uniformity of a certain profile distribution is also proposed. Furthermore, methods for the evaluation of the codistribution of two or more different types of profiles and to characterize the morphometric features of patches of profiles in a certain region are presented. All these quantitative morphological approaches were tested in relevant preparations of the central nervous system.

Diethyldithiocarbamate, a superoxide dismutase inhibitor, counteracts the maturation of ischemic-like lesions caused by endothelin-1 intrastriatal injection.

The effects of a focal lesion induced by endothelin-1 (ET-1, 0.8 microgram/0.8 microliter) on superoxide dismutase (SOD) were studied in the neostriatum of male rats. SOD activity was analyzed at several time intervals (5, 20, 60 min, 4, 24 h and 7 days) after the lesion. No significant changes were observed early after the injection, but SOD activity started to rise significantly at the 60-min time interval reaching a peak 24 h after the injection. In a second experiment the volume of ET-1-induced lesion was evaluated following treatments which induce variations of SOD activity. ET-1 caused a large lesion (9.20 +/- 1.32 mm3) in the neostriatum 24 h after the injection that was 3-fold greater than that observed 1 h after. Rats treated with the SOD inhibitor diethyldithiocarbamate showed a lesion equivalent to that observed 1 h after ET-1 injection, suggesting that SOD may be involved in the maturation of ET-1-induced neuronal damage.

Studies on neuropeptide Y-catecholamine interactions in the hypothalamus and in the forebrain of the male rat. Relationship to neuroendocrine function.

Neuropeptide Y-catecholamine interactions have been analyzed within the hypothalamus and in the forebrain of male rats by means of immunocytochemistry in combination with morphometry, quantitative histofluorimetry on catecholamine fluorescence in discrete catecholamine nerve terminal systems, biochemical analysis of catecholamines as well as by studies on serum levels of adenohypophyseal hormones vasopressin, adrenocortical hormones and angiotensin II using radioimmunoassay determinations. (1) Morphologic and morphometrical evidence indicates the existence of separate populations of neuropeptide Y and tyrosine hydroxylase immunoreactive nerve cell bodies in the parvo- and magnocellular components of the arcuate nucleus respectively. In addition, a significant codistribution of NPY immunoreactive nerve terminals and tyrosine hydroxylase immunoreactive nerve cell bodies were demonstrated in the ventrolateral part of the magnocellular component of the arcuate nucleus. (2) Immunocytochemical studies on the distribution of tyrosine hydroxylase, phenyl ethanolamine-N-methyltransferase and neuropeptide Y immunoreactive nerve terminal networks in the peri- and paraventricular hypothalamic nucleus indicated that these types of immunoreactive nerve terminals densely innervate the medial and anterior parvocellular part of the paraventricular hypothalamic nucleus and anterior periventricular hypothalamic nucleus. From studies on the pattern of terminal distribution results have been obtained compatible with the view that neuropeptide Y or a neuropeptide Y related peptide can be a comodulator in noradrenaline and adrenaline nerve terminal networks of these regions. (3) Acute intraventricular injections of neuropeptide Y (1.25 nmol) do not change dopamine and noradrenaline levels in any hypothalamic and telencephalic dopamine and noradrenaline nerve terminal system analyzed with the exception of the anteromedial frontal cortex, in which area a significant increase in the dopamine levels was observed as revealed biochemically. (4) By means of the tyrosine hydroxylase inhibition method it was possible to show that acute intraventricular injection of NPY (1.25 nmol) increased dopamine utilization in the medial and lateral palisade zone of the median eminence and in the anteromedial frontal cortex and reduced noradrenaline utilization in the parvocellular part of the paraventricular hypothalamic nucleus, while dopamine utilization was not influenced in the nucleus caudatus putamen, nucleus accumbens or in the tuberculum olfactorium. (5) In the intraventricular experiments reported above neuropeptide Y (1.25 nmol, 1 h) reduced the serum levels of thyreotropin stimulating hormone, prolactin and luteinizing hormone and increased serum corticosterone, adrenocorticotrophin, vasopressin, angiotensin II and aldosterone levels. The presence of the tyrosine hydroxylase inhibitor by itself, increased corticosterone, adrenocorticotrophin and aldosterone serum levels and reduced serum luteinizing hormone levels. Neuropeptide Y together with the tyrosine hydroxylase inhibitor further enhanced the adrenocorticotrophin, angiotensin II and aldosterone serum levels seen with the inhibitor, but could no longer produce its excitatory and inhibitory effects on serum corticosterone and luteinizing hormone levels, respectively. Vasopressin serum levels were increased to the same extent in the absence or presence of tyrosine hydroxylase inhibition. The present morphological, neurochemical and functional studies indicate that neuropeptide Y given intraventricularly inhibit the secretion of prolactin, luteinizing and thyreotropin stimulating hormones probably by activation mainly of neuropeptide Y receptors located in the somadendritic region of the arcuate DA cell bodies, leading to increased activity in inhibitory tubero-infundibular dopamine neurons. In addition, it is suggested that the ability of neuropeptide Y to increase adrenocorticotrophin and corticosterone secretion is at least in part related to its ability to reduce noradrenaline turnover in the parvocellular part of the paraventricular hypothalamic nucleus, rich in corticotrophin releasing factor immunoreactive nerve cell bodies. It is speculated that neuropeptide Y as a comodulator in the noradrenaline nerve terminals in this area may enhance the excitatory actions of noradrenaline on the corticotrophin releasing factor immunoreactive nerve cells. Such an action will lead to increases of corticotrophin releasing factor neuronal activity and of adrenocorticotrophin hormone secretion producing a feedback response, which may reduce noradrenaline turnover exclusively in this nucleus as was observed in the present experiments. The increase in aldosterone may be induced by the increased adrenocorticotrophin serum levels but the increase in vasopressin secretion and in angiotensin II serum levels may be secondary to the hypotensive activity of neuropeptide Y. Finally, it is suggested that neuropeptide Y mechanisms can increase dopamine synthesis and release in the anteromedial frontal cortex. Thus, neuropeptide Y mechanisms may participate in the control of cortical functions at least partly by regulating the cortical dopamine neurotransmission.

Endogenous neurosteroids modulate epileptogenesis in a model of temporal lobe epilepsy.

Neurosteroids modulate seizure susceptibility, but their role in the regulation of epileptogenesis is unknown. Status epilepticus (SE) induces temporal lobe epileptogenesis following a latent period in which glial cells are activated. Here, we found that P450scc, the rate-limiting enzyme in steroid synthesis, is upregulated in hippocampal glia during the latent period after pilocarpine-induced SE in rats. More prolonged SE was associated with greater P450scc expression and longer latencies to the development of seizures, suggesting that enhanced steroid synthesis retards epileptogenesis. The 5alpha-reductase inhibitor finasteride, which blocks neurosteroid synthesis, reduced the latent period, indicating that glia-derived neurosteroids may be antiepileptogenic.

Chapter 19 Aspects on the information handling by the central nervous system: focus on cotransmission in the aged rat brain

Publisher Summary This chapter discusses a study on a particular animal model—the aged brain of the rat—to evaluate the possible functional meaning of cotransmission. Aging is a progressive change in the morphological and biochemical features of the organism that takes place toward the end of adult life. Regional data on the effects of aging on two basic parameters of neural function—namely, Na + /K + ATPase, which is involved in the maintenance of membrane polarization, and protein phosphorylation, which is a biochemical system related to the intracellular decoding of extracellular signals—is reported. Cyclic adenosine monophosphate and Ca 2+ -induced protein phosphorylation was carried out. The studies were performed on crude mitochondrial membrane preparations obtained from dorsal cortex, striatum, limbic system, hippocampal formation, and spinal cord. It has been shown that the density of [ 3 H]ouabain binding sites shows a gradient with the highest density in the phylogenetically oldest regions of the central nervous system (CNS) and the lowest density in the most recent ones (cerebral cortex has the lowest value), while protein phosphorylation tends to have an opposite gradient.

Polyamines, Ornithine Decarboxylase, and Diamine Oxidase in the Substantia Nigra and Striatum of the Male Rat After Hemitransection

Partial hemitransection at the mesodiencephalic junction in the rat increased striatal and nigral putrescine concentrations on the lesioned side for at least 168 h, with maximal increases between 24 and 48 h. Spermidine and spermine levels declined at 24 h in the striatum, rising above control values at 48 h and further at 168 h. In the substantia nigra, they remained unchanged for the first 48 h and then increased by 168 h. Cadaverine in the striatum also increased at 48 h. On the intact side putrescine increased but to a much lesser extent (at 48 h in the striatum and at 24 and 48 h in the substantia nigra). Ornithine decarboxylase and diamine oxidase activities showed maximal increases at 24 h in the striatum of the lesioned side, whereas in the substantia nigra ornithine decarboxylase attained a very high value as early as 4 h after the operation and diamine oxidase activity peaked at 48 h. The enzyme activities returned toward the basal values at 168 h. On the intact side, ornithine decarboxylase showed a small increase starting at 4 h and diamine oxidase was enhanced at 48 h. These results indicate that the stimulation of biosyn‐thetic and degradative enzymes of polyamine metabolism accompanied by marked and prolonged increases in putrescine may be essential events in the early phases of neuronal response to mechanical injury in the CNS.

Short- and Long-term Changes in Striatal Neurons and Astroglia After Transient Forebrain Ischemia in Rats

BACKGROUND AND PURPOSE The striatum is one of the regions most sensitive to transient forebrain ischemia. After 30-minute ischemia, areas of massive neuronal degeneration are clearly detectable a few hours after the insult and attain their maximal extension 24 hours after the insult. However, for most cellular and neurochemical parameters it is not known whether some recovery occurs at later times. We examined certain cell populations in the caudate putamen at different times after transient ischemia. METHODS Adult male Sprague-Dawley rats were subjected to 30-minute forebrain ischemia (four-vessel occlusion model). Six experimental groups were considered: control animals and ischemic animals killed 4 hours, 1 day, 7 days, 40 days, and 8 months after reperfusion. Three striatal cell populations were examined by means of immunocytochemistry coupled to computer-assisted image analysis: vulnerable medium spiny neurons, resistant aspiny neurons, and reactive astrocytes, labeled for their content of dopamine- and cAMP-regulated phosphoprotein mr32 (DARPP-32), somatostatin and neuropeptide Y, and glial fibrillary acidic protein, respectively. RESULTS (1) The area containing DARPP-32 immunoreactive neurons was markedly decreased (15% to 20% of control caudate putamen area) at 1 day after reperfusion and partially recovered at the following times (40% to 50% at 7 days and 50% to 60% at 40 days and 8 months after reperfusion). (2) The appearance of reactive astrocytes was precocious (4 hours to 1 day after ischemia) in the medial caudate putamen, the region in which DARPP-32 recovered within 40 days after ischemia, and late (7 to 40 days after ischemia) in the lateral caudate putamen, where no DARPP-32 recovery was detected. (3) Neuropeptide Y/somatostatin-containing neurons resisted the ischemic insult and could be detected in areas devoid of DARPP-32 immunoreactive neurons as long as 8 months after reperfusion. CONCLUSIONS The present results show a marked recovery of DARPP-32-positive neurons within 40 days after 30-minute forebrain ischemia in the medial, but not the lateral, caudate putamen. Medial caudate putamen also contains a high density of reactive astrocytes on the first day after ischemia, suggesting that astrocytic support has an important role in the spontaneous recovery of ischemic neurons.

Neuropeptide Y produces anxiolytic effects in spontaneously hypertensive rats

The sedative and anxiolytic effects of intracerebroventricular administration of neuropeptide Y (NPY) were studied in spontaneously hypertensive rats (SHR) and in normotensive Wistar-Kyoto (WKy) rats using the two-compartment exploratory test, and in the open-field test after habituation. In the two-compartment tests, NPY produced anxiolytic effects by increasing the exploratory activity in SHR at a dose (0.25 nmol) lower than the minimal effective dose in WKy rats (1.25 nmol). In SHR, anxiolytic effects were observed for the whole NPY dose range (0.25-5.0 nmol), whereas in normotensive WKy rats the highest dose (5.0 nmol) failed to increase exploratory activity. The open-field test showed reduced locomotor activity and rearings in WKy rats when injected with 5.0 nmol NPY. These effects were not observed in SHR. The absence of sedative effects and the higher sensitivity to the anxiolytic effects of NPY in SHR are suggestive of a genetically determined difference in central NPY systems involved in behavioral adaptation that may be relevant for the development of hypertension.