Nitric oxide synthase expression in the cerebral cortex of patients with epilepsy.

PURPOSE: Nitric oxide (NO), a short-lived radical synthesized from L-arginine by activation of the enzyme nitric oxide synthase (NOS), has been implicated in the pathophysiology of epilepsy by some investigators. However, the current data about NO and NOS in epilepsy are controversial and are derived only from animal models of epilepsy. In this study we investigated possible changes in NOS expression in the cerebral cortex of patients with epilepsy. METHODS: Qualitative and quantitative parameters of the immunolabeling pattern of the neuronal, endothelial, and inducible isoforms of NOS were analyzed in biopsy material obtained from patients with short and long seizure history and from patients without epilepsy. RESULTS: The comparative study showed that in the cerebral cortex of patients with epilepsy, particularly in those with a long seizure history, the number and labeling intensity of NOS-positive neurons increased, and that a subpopulation of nonpyramidal GABAergic neurons (type II NOS neurons) was responsible for this phenomenon. CONCLUSIONS: The fact that NOS upregulation is more evident in patients with a long seizure history suggests that this is a consequence of seizures, acting probably as an adaptative response to the sustained release of excitatory amino acids.

Effect of hypertension on the angiotensin II fibres arriving at the posterior lobe of the hypophysis of the rat. An immunohistochemical study.

We studied immunohistochemically the posterior lobe of the hypophysis (PL) of 15-week-old spontaneously hypertensive rats (SHR) and of matched normotensive Wistar Kyoto rats (WKY), by using our own polyclonal antibody raised in mice against Angiotensin II (mouse-antiangiotensin II, MAAII). The blood pressure, water intake and volume of the PL were also recorded. The SHR rats were hypertensive, drank more water and showed a clear hypertrophy of their hypophysial PL. Also the PL of the SHR animals showed an increase in the immunoreactivity to the anti-angiotensin II antibody in the fibres arriving at the PL, with respect to the PL of WKY rats. This increase is compatible with the hyperactivity of the brain RAS, depletion of vasopressin content in the PL and increase in plasmatic levels of vasopressin described in SHR rats with respect to normotensive animals, as angiotensin II could locally stimulate vasopressin release to plasma from the neurohypophysis.

Effect of hypertension and captopril treatment on the vasopressin in the rat median eminence and posterior lobe of the hypophysis. An immunohistochemical study.

The present study analyses the effects of hypertension and/or its oral treatment with captopril (angiotensine-converting enzyme inhibitor) on the rat median eminence (ME) and the posterior lobe of the hypophysis (PL). After an immunohistochemical reaction using an antibody against arginine-vasopressin, we compared by densitometry the amount of vasopressin immunoreactive material (vasopressin-ir) of these centers in 4 groups of animals: control Wistar Kyoto rats (WKY), spontaneously hypertensive rats (SHR), WKY rats treated with captopril (WKY-T) and SHR rats also treated with the same drug (SHR-T). Captopril was administrated at a dosage of 0.1 mg/ml in the drinking water from the 8th to the 15th weeks. We have found that the rats showing the lowest level of vasopressin-ir, in both ME and PL, were those from the SHR group, the concentration increasing after oral captopril treatment (SHR-T), although without reaching the values of WKY rats. Then, ACE inhibition by captopril influences vasopressin content in brain areas where the hormone is concentrated before being released, which supports the hypothesis that suggests a central modulatory effect of ACE inhibitors, contributing to their therapeutic action on hypertension.

Changes in the secretory activity of the subcommissural organ of spontaneously hypertensive rats.

The subcommissural organ (SCO) is a glandular circumventricular organ secreting glycoproteins into the cerebrospinal fluid. The SCO of 15-week-old spontaneously hypertensive rats (SHR) and of matched normotensive Wistar-Kyoto rats (WKY) was studied immunocytochemically by using an antibody against the glycoproteins secreted by the SCO. The blood pressure, water intake and volume of brain ventricles of SHR and WKY rats were also recorded. The SHR were hypertensive, drank more water and did not display dilatation of the brain ventricles. The SCO of the SHR rats showed a drastic decrease of the immunoreactive material stored in the rough endoplasmic reticulum whereas the amount of immunoreactive apical secretory granules did not vary with respect to the SCO of WKY rats. These changes are compatible with an increased secretory activity of the SCO of the SHR rats. It is suggested that the changes in the SCO of SHR rats, and their hypertensive state, are interrelated phenomena.

Effects of alcohol and aging on the cingular (area 24) and frontal (area 6) cortical areas of the mouse.

We have studied the morphometric changes of the neurons of the cingular area 24 and frontal area 6 of the mouse, produced by age and/or chronic alcohol intake. The parameters analyzed were nuclear area of these cortical neurons and cellular density (cell/neuropil coefficient). We detected a decrease in the number of neurons with age in practically all layers of the control animals. In the animals that chronically ingested the alcoholic solution, we also detected a decrease in the number of neurons with age, but only in layer V of the frontal cortex and in layer VI of the cingular area 24. The comparison between the control and the alcoholic group showed that alcohol intake caused an increase in the nuclear area of the neurons in layer II-III of the frontal cortex at 180 days, while in the cingular cortex the increase in nuclear area of its neurons was significative at 180 days in layer II-III and at 35 and 180 days in layers V and VI. We think that these changes are the expression of the neuronal plasticity in both cortical areas in response to the alcohol exposure.

The effects of chronic administration of captopril on the mouse median eminence.

The effects of Captopril (an angiotensin-converting enzyme inhibitor) on the median eminence (ME) of the male albino mouse have been examined using morphometric and immunohistochemical procedures. We measured the nuclear area of the ependymocytes of the ME and of the glial cells of the reticular external zone of the ME. We also determined the cell/neuropil coefficient (CNC), which expresses the relation between cellular area and neuropil of the ME, and the global volume of the ME in each animal. For the immunohistochemical study we used rabbit antiarginine-vasopressin, and compared the results in the different groups of mice. We detected an increased in the immunoreactive material (arginine-vasopressin, A-V) and an increase in the global volume of the organ and also an increase of the neuropil of the ME after the longest exposure to the drug. These alterations could be related to the inhibition of the brain angiotensin II by captopril and the accumulation of vasopressin in the fibrous tract that runs from the paraventricular nucleus (PVN) to the neurohypophysis.

Effects of chronic alcohol exposure on the morphometric development of medial preoptic area neurons of the male mouse.

We have performed a karyometric study of the medial preoptic area of male mice from mothers that ingested chronically a solution of 20% of ethanol added to the drinking water. Pups then were exposed prenatally to alcohol. After parturition, pups were also exposed to alcohol, first through their mother's milk and after weaning by direct ingestion of the same solution of 20% of alcohol until the day of sacrifice. Animals were sacrificed at the 25th, 35th, 45th, 55th and 100th day and the results compared with those obtained in another group of control animals, sacrificed at the same ages. Chronic alcohol exposure reduces the studied nuclear sizes (perimeter, area and maximum diameter) in adult animals of 100 days of life, but does not produce significative changes in nuclear sizes of younger animals. However, nuclear shape, another of the nuclear parameters analysed, did show significative alterations in relation with the puberal age. These morphometric effects could be due to the reduction of plasmatic testosterone levels produced by alcohol and/or to a direct toxic effect of the alcohol on central nervous system neurons.

Alcohol intake effects on the dorsal vagal complex of the rat: a cellular morphometric study.

We have analyzed the morphometric effects on the dorsal vagal complex (DVC) of the rat of alcohol exposure and/or hypoproteic diet intake during 8 weeks. In the area postrema (AP), alcohol treatment (combined with normal isoproteic or hypoproteic diet) caused a significant decrease in karyometric parameters. In the dorsal motor nucleus of the vagus (DMV) and nucleus tractus solitari (NTS), the alcohol isoproteic intake (AI) produced an increase in neuron size (expressed by an increase in the neuronal nuclear area and the cell/neuropil coefficient). The hypoproteic diets produced a reduction in the global volume of each structure of the DVC which was accompanied by a decrease in global brain volume. These results indicate that after 8 weeks of treatment, alcohol is the main cause of the morphometric alteration found in the DVC, while variations in the amount of protein intake appear to produce global effects on the whole brain.

Postnatal development of the Ammon's horn (CA1 and CA3 fields). A karyometric and topographic study.

We have performed a karyometric study of the pyramidal neurons of CA1 and CA3 fields of the Ammon's horn, in male mice aged from the 5th to the 190th postnatal day. Nuclear sizes were measured with the aid of a Magiscan Analysis System, used in an interactive form, in both superficial and deep layers of the stratum pyramidal in those fields. The measurements were made at three different topographic levels: rostral; intermediate; and caudal, to detect any possible difference related to the topography of the neuron in the same field. We have found that both CA1 and CA3 fields are correlated in the postnatal development of their nuclear pyramidal sizes and that all topographic levels of the hippocampus reach their highest karyometric sizes at the 10th-15th postnatal day. Caudal levels show higher karyometric values than the other levels and some differences between neurons of the superficial and deep layers of both fields are also described here and analysed in relation to the different ontogenetic gradients of these cells.

Vitamin D3 (soltriol) nuclear receptors in abdominal scent gland and skin of Siberian hamster (Phodopus sungorus) localized by autoradiography and immunohistochemistry.

In vivo autoradiography with [3H]1,25-dihydroxycholecalciferol (vitamin D, soltriol) and immunostaining with antibodies to vitamin D receptor were applied to identify specific binding sites in the abdominal scent gland of male Siberian hamster (Phodopus sungorus). Nuclear concentration of radiolabeled hormone and receptor antibodies was observed in the corresponding cell types including basal cells of sebaceous glands, cells of the outer hair sheaths and hair bulbs, and also keratinocytes in the epidermis. Cells of the hair dermal papillae and fibroblasts of the dermis did not show nuclear labeling. There was good correspondence between the autoradiographic and immunohistochemical data. The results indicate the presence of receptors for vitamin D-soltriol and suggest a seasonal regulation of scent gland marking activities by this steroid hormone of sunlight in cooperation with the sex steroid testosterone.

The effects of chronic administration of captopril on the mouse subfornical organ and area postrema.

We have studied by morphometric procedures the chronic effect of captopril on the subfornical organ (SFO) and area postrema (AP) of the adult mouse. Oral administration of captopril does not produce any change in the size of individual nuclei of the ependymocytes and neurons in both centers. However, there are other quantitative effects of captopril on the global volume of the SFO and on the neuropil and vascular elements of both the SFO and AP which present a significant increase. It is suggested that this increase is due to metabolic processes at the level of both circumventricular organs.

Vitamin D (soltriol) target cells in the harderian gland of the Siberian hamster (Phodopus sungorus).

Harderian glands of female and male Siberian hamsters (Phodopus sungorus) were studied after a subcutaneous injection of 3H-dihydroxycholecalciferol (vitamin D, soltriol). Autoradiograms revealed the presence of nuclear concentration of the hormone in certain alveolar cells and in myoepithelial cells. The proportion of labeled cells varied between 5.5 and 19% of the total cell number, with an average of 9.5% in female and 12.4% in male hamsters. The data suggest that the functions of the Harderian gland could undergo seasonal changes under the control of vitamin D.

Brainstem projections to the medial preoptic region containing the luteinizing hormone-releasing hormone perikarya in the rat. An immunohistochemical and retrograde transport study.

The afferent projections to the anterior medial preoptic area (MPA) from the brainstem have been studied, in female Wistar rats, by retrograde tracing with horseradish peroxidase (HRP). The HRP was injected by iontophoresis into the preoptic region containing the luteinizing hormone-releasing hormone (LHRH) perikarya. The brain sections including the MPA were reacted with diaminobenzidine (DAB) to reveal the injection site; the LHRH cells were then immunohistochemically identified using DAB with ammonium nickel sulphate. When the injection site incorporated the LHRH cells, the brainstem sections were reacted with the DAB nickel solution to detect lysosomal HRP and then immunohistochemically processed to locate the adrenaline-synthesizing cells using DAB alone. The results confirm the brainstem projections to the MPA from the central grey matter, ventral tegmental area, subcoeruleus area, the dorsal raphe nucleus, the lateral parabrachial nucleus, the raphe pontis nucleus, the raphe obscurus nucleus, the region of the paragigantocellular nucleus and the nucleus of the solitary tract. Given the considerable evidence implicating the ascending adrenergic systems in the regulation of LHRH, we focused our attention on the afferents from the locus coeruleus, area postrema and the adrenaline-synthesizing cell groups (C1-3). The only cells which were retrogradely labelled and immunopositive for phenylethanolamine N-methyltransferase were found in C3.

Postnatal development of the dentate gyrus: a karyometric and topographic study.

We have analysed the postnatal development of the nuclear sizes of the granular cells of the dentate gyrus in 5- to 190-day-old male mice. The study was performed in three topographic levels: rostral, intermediate and caudal. Three subdivisions were analysed in each level: suprapyramidal blade, infrapyramidal blade and the transition between them, the angular zone. Additionally, each of these subdivisions was measured in its external and internal layer, separately. Three gradients of postnatal karyometric development can be described: external-to-internal, suprapyramidal-to-infrapyramidal, and caudal-to-rostral, indicating that the external, suprapyramidal and caudal cells show higher karyometric sizes than the other subdivisions. These gradients are related to the ontogenetic gradients of these neurons.

Alcohol effects on the morphometric development of the subfornical organ and area postrema of the albino mouse.

We have studied the development of the nuclear sizes of ependymocytes and neurons of two circumventricular organs of the male alcoholic mouse: the Subfornical Organ (SFO) and the Area Postrema (AP), comparing the results with a control group. The global volume of both centers was also studied. The results show that the SFO, a structure related to the control of fluid balance, responds to alcoholism with an increase of the global volume. This increase could be related to the variations of salt-water balance and/or blood pressure in chronic alcoholism. However, the size of cell nuclei in the SFO is not affected. In contrast, the AP responds to chronic alcoholism like other nervous centres, with a decrease of the nuclear size of its cells. The global volume of AP does not change.

Development of the median eminence in the male mouse. Karyometric effect of neonatal and prepuberal castration.

We have studied the karyometric development of the ependymocytes of the median eminence and ependyma adjacent of the arcuate nucleus in a control group of male albino mice from the 5th to the 160th postnatal day, and in other two experimental groups of animals that were castrated at two different days: the first and the 20th day of life. We have found differences in the spontaneous development of both ependymocytes showing the median eminence ependyma a more closely relationship to changes of gonadal hormone levels around puberty. In both zones, the response of the ependymocytes to neonatal castration was clearly more significative than that obtained after prepuberal castration, with lower values in the castrated animals than in the control mice. We suggest that this could be related to nervous and hormonal mechanisms.

Effect of propylthiouracil on liver cell development in the male albino mouse: protective effect against ethanol-induced alterations.

The aim of the present study is to analyze whether the addition of propylthiouracil reverts the influence of ethanol on the development of periportal and pericentral hepatocytes and their nuclei in male albino mice. Propylthiouracil-treated animals showed decreased cellular and nuclear areas when compared with the control animals, except for the 180-day-old animals, whose pericentral cells and nuclei were greater than those of the controls and exhibited fatty infiltration. Pericentral hepatocytes and nuclei of the ethanol-fed animals showed an increase of their sizes, especially in 180-day-old animals. In contrast, hepatocyte and nuclear sizes of the animals treated with both propylthiouracil and ethanol were similar to those of the control group, suggesting a protective effect of propylthiouracil against the ethanol-induced alterations.

Development of the subfornical organ and area postrema of the male albino mouse. Karyometric effect of neonatal and prepuberal castration.

We have studied the karyometric development of the nuclei of the ependymal cells and neurons of the subfornical organ and the area postrema in the male albino mouse from the 5th to the 190th postnatal day. We have found similar patterns of development in both although the area postrema showed more significant postnatal oscillations than those of the subfornical organ, suggesting a more intimate chronological relationship to gonadal development. We have furthermore analyzed the development in two experimental groups: in the one animals were castrated at birth, in the other, castration was made on the 20th postnatal day. We have found that neonatal castration produced a significant decrease of nuclear sizes; this was more evident in the subfornical organ than in the area postrema in earlier stages of development while the response was similar in both at peripuberal ages. The response to prepuberal castration was similar in both organs.

Effects of hypothyroidism on the karyometric development of pyramidal neurons of the hippocampus (CA1), area 6 and area 17 in the male mouse.

This study has examined the karyometric changes within pyramidal neurons of the hippocampus, motor area 6 and visual area 17 in a hypothyroid group of male mice treated with propylthiouracil, with or without interruption of treatment at the 35th postnatal day. Hypothyroidism resulted in decrease of nuclear size in the three areas before puberty and even after puberty in the hippocampus. Where the treatment was continued throughout the experimental period there was a progressive increase of nuclear size in both visual and motor areas.

Topographic organization of the karyometric response to neonatal castration of the male mouse in the paraventricular and ventromedial hypothalamic nuclei.

We have analysed the karyometric development of four topographic subdivisions of the paraventricular nucleus (rostral, medial, lateral and caudal) and three topographic subdivisions of the ventromedial nucleus (ventral, central and dorsal) of the hypothalamus in neonatally castrated male mice. Castration at birth produces a decrease of the nuclear sizes from the 10th to the 35th postnatal day, but after this age, no differences were detected in comparison with the sizes of the control male mice. Neonatal gonadal hormone deprivation also produces changes in the nuclear shape of the nuclei, that tend to be more spherical in both centres, as we could establish after the study of the form factor parameter (perimeter/area ratio). We have found differences in the response of both hypothalamic nuclei to neonatal castration and differences in the response of the topographic subdivisions of both of them.