Comparative study on cerebrovascular injuries by three chlorophenoxyacetic acids (2,4-D, 2,4,5-T and MCPA).

At toxic doses in rats, 2,4-dichlorophenoxyacetic acid (2,4-D) and 2-methyl-4-chlorophenoxyacetic acid (MCPA) caused injuries in blood vessels in the brain. 2. 2,4-D caused extravasations of the circulating Evans blue-albumin complex also in the spinal cord. 3. By contrast, potency in damaging cerebral vessels was almost non-existent with 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). 4. None of the three chlorophenoxyacetic acids caused such cerebrovascular injuries in mice, guinea pigs, Syrian hamsters, rabbits and chickens.

Cytochemical demonstration of adenylate cyclase activity with cerium.

Cerium was applied for the ultrastructural, cytochemical localization of adenylate cyclase (EC 4.6.1.1.). The enzyme activity was stimulated with norepinephrine, prenalterol and cholera toxin in the brown fat cells of newborn rats. The final reaction product was observed in the plasmalemmas of the stimulated adipocytes. The precipitate was finely crystalline, easily visible in the electron microscope and in the X-ray microprobe analysis it yielded cerium and phosphate peaks, respectively. The use of cerium offers a new tool valid for the cytochemical localization of adenylate cyclase enzyme related to the membrane receptors.

Dithiocarbamate fungicides decrease histochemical reactivity of cholinesterases in the gut wall of the rat.

The toxicity of dithiocarbamate fungicides is poorly known, especially in the autonomic nervous system. Male Wistar rats were given tetraethylthiuramdisulfide (DSF), maneb, and zineb as a suspension in saline i.p. 200 mg/kg daily for 4 d. Rats received a similar dose of ethylenethiourea (ETU) as a solution in saline i.p., and the control rats received a corresponding volume of saline i.p. After decapitation, pieces of ileum were taken, and the enzyme histochemical reactivity for the acetylcholinesterase (AChE) and for the nonspecific cholinesterase (nsChE) in the ilea of the treated and control animals was studied. The reaction intensities in the samples were estimated by eye using a scale from 0 (no reaction product) to 3+ (maximal reaction). DSF was used as an internal standard. Controls showed the maximal reaction for the AChE and for the nsChE. Maneb, ETU and zineb showed consistent decrease in the reactivity for the AChE (+ +) and for the nsChE (+ +). The decrease by DSF of the reactions for both the AChE (+) as well as for the nsChE(+) was even more pronounced indicating a possible nerve damage to the cholinergic innervation of rat intestine. Kinetic differences may explain the divergent effects between the agents studied.

Neurotoxic effects of disulfiram on autonomic nervous system in rat.

Two groups of male Wistar rats weighing about 140 (WI) and 200 g (WII) and a group of Sprague-Dawley (S.D.) rats (140 g) received oral disulfiram 220-580 mg/kg (DSF) daily for one or three weeks. Isolated ilea of both control and treated rats showed similar responses to acetylcholine, but the responses to 5-hydroxytryptamine (5-HT) were decreased after one and three weeks' treatment in the WI and SD rats. Pretreatment with reserpine intensified this effect in treated WI rats. A distinct decrease in the histochemical reactivity for the acetylcholinesterase and the non-specific cholinesterase was observed in the nerve plexuses of the gut wall indicating a DSF-induced nerve damage. Autonomic (cholinergic) nerves seemed thus to be affected by DSF. The two rats strains studied did not differ in their responses to 5-HT.

Endothelial surface sulfhydryl-groups in blood-brain barrier transport of nutrients.

The sulfhydryl-inhibitors p-chloromercuribenzoic acid (PCMB) and p-chloromercuribenzene sulfonic acid (PCMB-S) were used to study the mechanism of nutrient transport across the blood-brain barrier (BBB) in the rabbit. The vascular endothelium of the left hemisphere was exposed to these agents at 10(-4) M concentration for 35 sec by intracarotid perfusion. The non-perfused right hemisphere served as intact control. 3H-methyl-O-glucose, 14C-cycloleucine and L-DOPA were used as tracers for carrier-mediated nutrient transport, and 3H-norepinephrine, 14C-sucrose and horseradish peroxidase were given to demonstrate abnormal extravasation. Assays of symmetrical samples from left and right hemispheres showed that PCMB-S reduced the uptake of cycloleucine and methylglucose in the perfused hemisphere down to 14.1 +/- 2.3% and 33.2 +/- 2.2%, respectively, without deranging the barrier to the other tracers. PCMB also diminished the uptake of the nutrient tracers, but simultaneously induced extravasation of the normally barred tracers, an effect similar to that induced by mercuric ions in corresponding earlier studies. Our findings indicate that sulfhydryl-groups located on the surface of the luminal membrane of the brain endothelial cells, where they are accessible to PCMB-S binding, are essential for the blood-brain barrier transport of important nutrients.

Synthesis and storage of hormonal granules in cultured cells of the rat pituitary pars intermedia.

Tissue from the pars intermedia of the pituitary from new-born and adult rats was maintained in tissue culture for periods up to 14 days. Both new-born and adult rat pars intermedia cells showed active production of secretory granules during tissue culture, and also showed formaldehyde-chloral induced fluorescence. Immunohistochemically, beta-endorphin could be demonstrated in cultured pars intermedia cells. In contrast to the appearance of the pars intermedia cells in vivo, the granules in the cultured cells were most often seen along the cell membrane, and the cytoplasm was often dominated by clear vacuoles slightly larger than the granules. No exocytotic figures were observed in the cultured cells. The mechanism of hormonal release from the organ, if release does occur, thus remains obscure. In conclusion, cultured cells from the rat pituitary pars intermedia continue their production of hormonal granules in tissue culture, and thus form a valuable tool for further investigations on the complex regulation of hormonal secretion form the organ.

Increase in the penetration of tracer compounds into the rat brain during 2-methyl-4-chlorophenoxyacetic acid (MCPA) intoxication.

The penetration of different intravenous tracer molecules such as 14C-labelled 2-methyl-4-chlorophenoxyacetic acid (14C-MCPA), 14C-p-aminobenzoic acid (14C-PABA), 14C-sucrose, 14C-antipyrine and iodinated (125I) human albumin (125I-HA) into the brain and cerebrospinal fluid (CSF) was studied in MCPA-intoxicated and control rats. Toxic subcutaneous doses of sodium salt of MCPA (200-500 mg/kg) increased highly the brain/plasma and CSF/plasma ratios of 14C-MCPA and 14C-PABA, as compared to the muscle/plasma ratio. Probenecid (200 mg/kg) did not affect the cerebral MCPA concentration in the intoxicated animals. The tissue/plasma ratios of 14C-sucrose, 14C-antipyrine and 125I-HA were also increased in the brain and CSF of intoxicated animals, but the increases were less pronounced than those of 14C-MCPA or 14C-PABA. The results indicate that MCPA intoxication caused a selective damage of the blood-brain barrier in the brain areas studied.

Ultrastructural changes in the neurohypophysis of the aged male rat.

The neurohypophyses (neural lobes of the pituitary) of young adult (3-6 months) and aged (12-30 months) male rats were studied by means of electron microscopy. Stereomorphometric analyses were performed to determine the size, number and relative volume of the hormone-containing vesicles. The principal observations included: 1) the conspicuous depletion in aged rats of the granular hormone-containing vesicles from the axon terminals and the Herring bodies, with a decrease in the relative volume fraction of the vesicles from 4.8 Vv% in the control animals to 1.1 Vv% in the aged rats; 2) a change in the size-distribution of the hormone vesicles; 3) an increase in the extracellular space around the nerve terminals, axons and capillaries; and 4) lipid accumulations and signs of activation in the pituicytes. The possible physiological significance of the findings is discussed in the light of several regulatory functions known to be altered during the process of ageing.

Effect of norepinephrine, prenalterol and metoprolol on the histochemical localization of adenylate cyclase activity in the brown adipose tissue of newborn rat.

Norepinephrine-stimulated adenylate cyclase enzyme (EC 4.6.1.1.) was histochemically demonstrated by electron microscope in the plasmalemmas of the newborn rat adipocytes. The precipitating agent in the histochemical reaction was cobalt salt. Metoprolol, a selective beta 1-blocker given in vivo abolished almost totally the reaction products from the outer surfaces of the plasmalemmas. In vitro treatment did not yield constantly reproducible results. Prenalterol (H 133/22-CGP 7760/B Hässle) a selective beta-agonist given in vivo stimulated enzyme activity in the plasmalemmas. The reaction, however, showed different localization patterns and enhanced amounts of reaction products in the membranes. It is proposed that the histochemical demonstration of amine-stimulated adenylate cyclase activity in the adipocyte plasmalemmas corresponds to the hormonal receptor sites. The question of the sensitivity of the receptors is discussed.

Vacuolated neurons in the hypogastric ganglion of the rat.

The vacuolated neurons (VN) of the main hypogastric ganglion of the male rat were studied using the formaldehyde-induced fluorescence (FIF) method for the histochemical demonstration of catecholamines. Microspectrofluorimetry was performed to identify the fluorophores and to quantify the FIF. The thiocholine method (Koelle-Gomori) was used to demonstrate acetylcholinesterase activity. The fine structure of the VN was studied using glutaraldehyde/OsO4 fixation. (1) In the untreated adult male rat VN represent only a small population of the total number of hypogastric neurons (0.8--1.2%). The vacuoles are similar to those of the VN from the corresponding female ganglion. (2) The VN are considered to be adrenergic due to the nature of their fluorophore, indicating a primary catecholamine. (3) The first VN appear in the hypogastric ganglia at the age of 7 weeks. After testosterone administration to young rats, VN are found at the age of 4 weeks. (4) The basic fine structure of the VN is similar to that of other ordinary neurons of the hypogastric ganglia. (5) The content of the vacuoles could not be identified. (6) Indications of degeneration were not observed in the VN. (7) The VN are interpreted as being a functional stage of the "short" adrenergic neurons, which are under the control of steroid hormones. (8) Fifteen months after castration, no VN could be found in the hypogastric ganglia, while their number was normal in the corresponding control animals.

Light and electron microscopic histochemistry of the monoamines in the human foetal sympathetic ganglion in culture.

Sympathetic ganglia of 13 to 19-week-old human foetuses were cultured in small pieces with and without nerve growth factor for up to 5 weeks in vitro. The cultures were studied using phase-contrast, fluorescence and electron microscopy. Monoamines were demonstrated with the formaldehyde-induced fluorescence method, with and without pretreatment of the cultures with catecholamines or monoamine oxidase inhibitor. In the long-term cultures, primitive sympathetic cells, sympathicoblasts of types I and II, and young sympathetic neurons showed a fine structure identical to that described earlier in vivo. There were virtually no satellite or Schwann cells in the cultures. The neurons showed a considerable capacity to grow new nerve fibres in culture, even without nerve growth factor. Nerve terminals with accumulations of synaptic vesicles were regularly observed, occasionally in synapse-like contact with other nervous structures. Large granular vesicles were regularly found in the sympathicoblasts after glutaraldehyde-osmium tetroxide fixation. After permanganate fixation, dense-cored vesicles typical of adrenergic neurons were not seen, either in the perikarya, or in the processes, although it was possible to demonstrate specific fluorescence. No small intensity fluorescent (SIF) cells were observed. Variable formaldehyde-induced fluorescence was observed in the nerve cell perikarya and nerve fibres. The intensity of the fluorescence increased after treatment of the cultures with monoamine oxidase inhibitor and after incubation with catecholamines.

Effects of ageing on the histochemically demonstrable catecholamines and acetylcholinesterase of human sympathetic ganglia.

The sympathetic ganglia of adult and aged humans were obtained during vascular, gynaecological and urological surgery, and studied using the formaldehyde-induced fluorescence (FIF) method for histochemical demonstration of catecholamines. Microspectrofluorimetry was applied to characterize the emission spectra of the flurophores. The sympathetic ganglia contained two types of cells exhibiting FIF: the adrenergic neurons and the small, intensely fluorescent (SIF) cells. The adrenergic neurons contained pigment granules exhibiting autofluorescence with emission maximum at 530---540 nm. The number of these granules increased with age while the FIF of the neurons decreased. In the oldest patients, most of the neurons in the sympathetic ganglia showed only pigment fluorescence but not FIF. The decrease of the perikaryonal catecholamine stores is interpreted as a degenerative change leading to impaired function of the neurons. The patterns of AChE did not change with age. The SIF cells were sparse and were mostly observed near the ganglia, forming paraganglia of varying size. Because of the rarity of the SIF cells, the effect of ageing could not be judged reliably.

Ultrastructural demonstration of amine granules in the adrenal medullary cells of the rat using acid permanganate fixation.

Potassium permanganate fixative is usually employed at pH 7.0. At this pH the amines in the granules of the adrenal medullary cells do not react with permanganate. When the pH was adjusted to 5.0, electron dense precipitates were seen in the amine granules of part of the medullary cells, probably noradrenalin containing cells.