No Evidence of Neurogenesis in Adult Rat Sympathetic Ganglia Following Guanethidine-Induced Neuronal Loss.

The potential for neurogenesis in the cranial (superior) cervical ganglia (SCG) of the sympathetic nervous system was evaluated. Eleven consecutive daily doses of guanethidine (100 mg/kg/d) were administered intraperitoneally to rats in order to destroy postganglionic sympathetic neurons in SCG. Following the last dose, animals were allowed to recover 1, 3, or 6 months. Right and left SCG from guanethidine-treated and age-matched, vehicle-treated control rats were harvested for histopathologic, morphometric, and stereologic evaluations. Both morphometric and stereologic evaluations confirmed neuron loss following guanethidine treatment. Morphometric analysis revealed a 50% to 60% lower number of tyrosine hydroxylase (TH)-positive neurons per unit area of SCG at both 3 and 6 months of recovery, compared to ganglia of age-matched controls, with no evidence of restoration of neuron density between 3 and 6 months. Reductions in TH-positive neurons following guanethidine treatment were corroborated by unbiased stereology of total hematoxylin and eosin-stained neuron numbers in SCG. Stereologic analyses revealed that total neuron counts were lower by 37% at 3 months of recovery when compared to age-matched vehicle controls, again with no obvious restoration between 3 and 6 months. Thus, no evidence was found that postganglionic neurons of the sympathetic nervous system in the adult rat have a neurogenic capacity.

Physiological approaches to assess diminished sympathetic activity in the conscious rat.

The purpose of this study was to evaluate functional measures of diminished sympathetic activity after postganglionic neuronal loss in the conscious rat. To produce variable degrees of sympathetic postganglionic neuronal loss, adult rats were treated daily with toxic doses of guanethidine (100mg/kg) for either 5days or 11days, followed by a recovery period of at least 18days. Heart rate, blood pressure, cardiac baroreflex responsiveness, urinalysis (for catecholamine metabolite, 3-methoxy-4-hydroxyphenylethylenglycol; MHPG), and pupillometry were performed during the recovery period. At the end of the recovery period stereology of superior cervical ganglia (SCG) was performed to determine the degree of neuronal loss. Total number of SCG neurons was correlated to physiological outcomes using regression analysis. Whereas guanethidine treatment for 11days caused significant reduction in the number of neurons (15,646±1460 vs. 31,958±1588), guanethidine treatment for 5days caused variable levels of neuronal depletion (26,009±3518). Regression analysis showed that only changes in urinary MHPG levels and systolic blood pressure significantly correlated with reduction of SCG neurons (r2=0.45 and 0.19, both p<0.05). Although cardiac baroreflex-induced reflex tachycardia (345.7±19.6 vs. 449.7±20.3) and pupil/iris ratio (0.50±0.03% vs. 0.61±0.02%) were significantly attenuated in the 11-day guanethidine treated rats there was no significant relationship between these measurements and the number of remaining SCG neurons after treatment (p>0.05). These data suggest that basal systolic blood pressure and urinary MHPG levels predict drug-induced depletion of sympathetic activity in vivo.

Neurophysiological assessment of sympathetic cardiovascular activity after loss of postganglionic neurons in the anesthetized rat.

The goal of this study was to determine the degree of sympathetic postganglionic neuronal loss required to impair cardiovascular-related sympathetic activity. To produce neuronal loss separate groups of rats were treated daily with guanethidine for either 5days or 11days, followed by a recovery period. Sympathetic activity was measured by renal sympathetic nerve activity (RSNA). Stereology of thoracic (T13) ganglia was performed to determine neuronal loss. Despite loss of more than two thirds of neurons in T13 ganglia in both treated groups no effect on resting blood pressure (BP) or heart rate (HR) was detected. Basal RSNA in rats treated for 5days (0.61±0.10µV∗s) and 11days (0.37±0.08µV∗s) was significantly less than vehicle-treated rats (0.99±0.13µV∗s, p<0.05). Increases in RSNA by baroreceptor unloading were significantly lower in 5-day (1.09±0.19µV∗s) and 11-day treated rats (0.59±0.11µV∗s) compared with vehicle-treated rats (1.82±0.19µV∗s, p<0.05). Increases in RSNA to chemoreceptor stimulation were significantly lower in 5-day treated rats (1.54±0.25µV∗s) compared with vehicle-treated rats (2.69±0.23µV∗s, p<0.05). Increases in RSNA in 11-day treated rats were significantly lower (0.75±0.15µV∗s, p<0.05) compared with both vehicle-treated and 5-day treated rats. A positive correlation of neurons to sympathetic responsiveness but not basal activity was detected. These data suggest that diminished capacity for reflex sympathetic responsiveness rather than basal activity alone must be assessed for complete detection of neurophysiological cardiovascular impairment.

Mite-control activities of active constituents isolated from Pelargonium graveolens against house dust mites.

The mite-control activities of materials obtained from Pelargonium graveolens oil against Dermatophagoides farinae and D. pteronyssinus were examined using an impregnated fabric disk bioassay and were compared with those shown by commercial benzyl benzoate and N,N-diethylm- toluamide (DEET). Purification of the biologically active constituents from P. graveolens oil was done by silica gel chromatography and high performance liquid chromatography. The structures of the active components were analyzed by EI/MS, (1)H-NMR, (13)C-NMR, (1)H-(13)C COSYNMR, and DEPT-NMR spectra, and were identified as geraniol (C(10)H(18)O, MW 154.25, trans-3,7-dimethyl-2,6- octadien-1-ol) and beta-citronellol (C(10)H(20)O, MW 156.27, 3,7-dimethyl-6-octen-1-ol). Based on the LD50 values, the most toxic compound was geraniol (0.26 microg/cm(2)), followed by beta-citronellol (0.28 microg/cm(2)), benzyl benzoate (10.03 microg/ cm(2)), and DEET (37.12 microg/cm(2)) against D. farinae. In the case of D. pteronyssinus, geraniol (0.28 microg/cm(2)) was the most toxic, followed by beta-citronellol (0.29 microg/cm(2)), benzyl benzoate (9.58 microg/cm(2)), and DEET (18.23 microg/cm(2)). These results suggest that D. farinae and D. pteronyssinus may be controlled more effectively by the application of geraniol and beta-citronellol than benzyl benzoate and DEET. Furthermore, geraniol and beta-citronellol isolated from P. graveolens could be useful for managing populations of D. farinae and D. pteronyssinus.

Chemical sympathectomy further increases muscle protein degradation of acutely diabetic rats.

The present work investigated the role of the sympathetic nervous system (SNS) in the control of protein degradation in skeletal muscles from rats with streptozotocin (STZ)-induced diabetes. Diabetes (1, 3, and 5 days after STZ) induced a significant increase in the norepinephrine content of soleus and EDL muscles, but it did not affect plasma catecholamine levels. Chemical sympathectomy induced by guanethidine (100 mg/kg body weight, for 1 or 2 days) reduced muscle norepinephrine content to negligible levels (less than 5%), decreased plasma epinephrine concentration, and further increased the high rate of protein degradation in muscles from acutely diabetic rats. The rise in the rate of proteolysis (nmol.mg wet wt(-1).2h(-1)) in soleus from 1-day diabetic sympathectomized rats was associated with increased activities of lysosomal (0.127 +/- 0.008 vs. 0.086 +/- 0.013 in diabetic control) and ubiquitin (Ub)-proteasome-dependent proteolytic pathways (0.154 +/- 0.007 vs. 0.121 +/- 0.006 in diabetic control). Increases in Ca2+-dependent (0.180 +/- 0.007 vs. 0.121 +/- 0.011 in diabetic control) and Ub-proteasome-dependent proteolytic systems (0.092 +/- 0.003 vs. 0.060 +/- 0.002 in diabetic control) were observed in EDL from 1-day diabetic sympathectomized rats. The lower phosphorylation levels of AKT and Foxo3a in EDL muscles from 3-day diabetic rats were further decreased by sympathectomy. The data suggest that the SNS exerts acute inhibitory control of skeletal muscle proteolysis during the early stages of diabetes in rats, probably involving the AKT/Foxo signaling pathway.

Sympathectomy alters bone architecture in adult growing rats.

Sympathetic nervous system (SNS) fibres and alpha- and beta-receptors are present in bone, indicating that the SNS may participate in bone metabolism. The importance of these observations is controversial because stimulation or inhibition of the SNS has had various effects upon both anabolic and catabolic activity in this tissue. In this study we evaluated the effects of pharmacological sympathectomy, using chronic treatment of maturing male rats with 40 mg of guanethidine/kg i.p., upon various parameters in bone. Double labelling with tetracycline injection was also performed 20 and 2 days before sacrifice. Bone mass, mineral content, density and histomorphometric characteristics in different skeletal regions were determined. Bone metabolic markers included urinary deoxypyridinoline and serum osteocalcin measurements. Guanethidine significantly reduced the accretion of lumbar vertebral bone and of mineral content and density, compared to controls. Femoral bone mineral content and density were also significantly reduced, compared to controls. Histomorphometric analyses indicated these effects were related to a reduction of cortical bone and mineral apposition rate at femoral diaphysials level. Both markers of bone metabolism were reduced in controls as they approached maturity. Guanethidine significantly decreased serum osteocalcin compared to controls, while urinary deoxypyridinoline was unchanged. These data indicate that guanethidine-induced sympathectomy caused a negative balance of bone metabolism, leading to decreased mass by regulating deposition rather than resorption during modeling and remodeling of bone.

Paeoniflorin reverses guanethidine-induced hypotension via activation of central adenosine A1 receptors in Wistar rats.

1. Intravenous injection of paeoniflorin, a glycoside purified from the root of Paeonia lactiflora, reversed guanethidine-induced hypotension in Wistar rats. 2. Pretreatment with the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine inhibited this effect of paeoniflorin in a dose-dependent manner. 3. The action of paeoniflorin was not modified by 8-(p-sulfophenyl)theophylline, the polar antagonist of the adenosine A1 receptor, which is not able to enter the central nervous system. 4. We conclude that paeoniflorin can reverse guanethidine-induced hypotension via activation of adenosine A1 receptors in the brain of Wistar rats.

Simulatory effect of porcine insulin on noradrenaline secretion in guinea-pig ileum myenteric nerve terminals.

1. The effect of insulin on the release of noradrenaline (NA) from nerve terminals was investigated in isolated ileal synaptosomes of guinea-pig. Release was determined as the amount of NA, quantified by h.p.l.c.-electrochemical detection, from samples incubated with insulin minus that in parallel blanks treated with some volume of vehicle. 2. Porcine insulin stimulated the secretion of NA in a concentration-dependent manner from 0.01 i.u. ml-1, while the value of lactate dehydrogenase in the incubated medium was not influenced by insulin. 3. The presence of insulin receptors in this preparation was illustrated by immunoblotting with insulin receptor monoclonal antibodies. 4. The release of NA by insulin was reduced by guanethidine and bretylium and it was markedly lowered in the samples obtained from guinea-pigs that had received an intraperitoneal injection of DSP-4, the noradrenergic neurotoxin. 5. Tetrodotoxin attenuated the action of insulin at concentrations sufficient to block sodium channels. The depolarizing effect of insulin on the membrane potential was also illustrated by a concentration-dependent increase in the fluorescence of bisoxonol, a potential-sensitive dye. 6. The action of insulin was attenuated by removal of calcium chloride from the bathing medium. The induction of calcium ion influx by insulin into the synaptosomes is supported by the inhibitory effects of the calcium channel blockers omega-conotoxin GVIA (for the N-type channels) and nifedipine (for the L-type channels). 7. These findings suggest that insulin can stimulate NA release from noradrenergic terminals via activation of calcium influx.

Anti-asialo GM1 antibodies prevents guanethidine-induced sympathectomy in athymic rats.

Guanethidine sulphate induces destruction of peripheral sympathetic neurons and infiltration of mononuclear cells in rat sympathetic ganglia. The effect of guanethidine is believed to be an autoimmune reaction. In order to determine the effect of anti-asialo GM1, an antibody that binds to the glycolipid asialo GM1 expressed on rodent natural killer cells, athymic Lewis rats received guanethidine 40 mg/kg i.p. daily from day 1 to 14 and anti-asialo GM1 i.p. 1 mg/rat on day -2, 0, 2, 6, and 10 in the study period. Saline and anti-asialo GM1 were given alone in the same doses as control. The number of neurons in the sympathetic ganglia were counted and the ganglionic volume determined. The presence of natural killer cells in the ganglia were determined by immunohistochemical methods. Our results shows that anti-asialo GM1 can prevent guanethidine-induced reduction of sympathetic neurons, but not prevent the initiation of an immunological reaction in the ganglia. Natural killer cells could only be identified in ganglia following guanethidine treatment alone. It is concluded that anti-asialo GM1 treatment can prevent the guanethidine-induced sympathectomy by eliminating the natural killer cells from the ganglia.

Innervation of the rat thymus gland.

Current views from different laboratories on the innervation of the thymus gland are reviewed with particular reference to the rat. Noradrenergic nerve profiles of the sympathetic nervous system have been demonstrated in the subcapsular cortex, at the corticomedullary junction and in the cortex itself, and extremely sparsely in the medulla. By following beta-adrenergic receptor development in postnatal rats, it has been shown that there is a marked increase in density and morphological organization of the receptor in the medulla with the maturation of thymocyte function (monitored by measuring the proliferation response to concanavalin A) and a sexual dimorphism during the ontogeny of the receptor. Chemical sympathectomy of adult rats with 6-hydroxydopamine (6-OHDA) or guanethidine resulted in a loss of thymus weight, decreased cellularity, and increased apoptosis but a rise in the numbers of proliferating cells in the cortex. By contrast, proliferation of peripheral T cells was reduced after the use of 6-OHDA. Chemical sympathectomy also demonstrated that there were at least three nerve nets in the gland: noradrenergic neural profiles that were destroyed with both 6-OHDA and guanethidine, vasoactive intestinal polypeptide (VIP)-positive profiles that persisted, and AChE- and CGRP-positive profiles and cells that also persisted but had a different distribution to VIP-positive fibers. Some functional correlates of thymic innervation are discussed although the subject now needs to be further researched.

Differential susceptibility of prevertebral and paravertebral sympathetic ganglia to experimental injury.

To investigate the response of selected sympathetic ganglia to experimental injury, neonatal rat pups were treated with either 6-hydroxydopamine (6-OHDA), guanethidine, or antiserum to nerve growth factor (anti-NGF). When examined at one month of age, each of the treatments resulted in a significantly greater loss of neurons and tyrosine hydroxylase activity in paravertebral (superior cervical and stellate) versus prevertebral (superior mesenteric and celiac) sympathetic ganglia. Guanethidine treatment produced the largest differential in neuron loss and tyrosine hydroxylase activity between pre- and paravertebral ganglia. Histologically, the acute phase of guanethidine-induced injury in the superior cervical, paravertebral, ganglia was characterized by a prominent mononuclear cell infiltrate and extensive neuronal degeneration. Minimal histopathologic changes were seen in the superior mesenteric, prevertebral, ganglia of the same animals. Immunolocalization of tyrosine hydroxylase and neuropeptide Y (NPY) in guanethidine-treated animals showed a preferential loss of sympathetic innervation of the extramural mesenteric vasculature with relative sparing of the noradrenergic innervation of Auerbach's myenteric plexus. Differences in the susceptibility of sympathetic ganglia to various insults may underlie the selective and heterogeneous involvement of sympathetic ganglia in clinical and experimental situations.

Thrombogenic effects of xenobiotics.

The mechanisms by which xenobiotics may cause or promote thrombosis include vascular damage, induction of a hypercoagulable state and disturbances of blood flow. This paper discusses the methods available to detect various types of thrombogenic substances. Pathomorphological techniques are best suited to demonstrate thrombosis caused by localized vascular damage or generalized endothelial lesions. For the assessment of disseminated microcirculatory thrombosis, the consumption of platelets and clotting factors and the appearance of specific platelet proteins and fibrinogen and fibrin split products can be determined in the blood. Hypercoagulability which is defined as a perturbation of the hemostatic equilibrium resulting in a shift in the direction of thrombosis, is of particular importance in toxicology. Many in vitro, ex vivo and in vivo methods have been proposed to detect and to measure the ability of xenobiotics to induce a prethrombotic state. Their usefulness is demonstrated with several examples.

Dependence of the cytotoxic effect of guanethidine on the degree of sympathetic activity.

Young rats aged 15-29 days received a subcutaneous injection of guanethidine sulphate (5 mg/kg body weight) every day. Owing to damage to the postganglionic sympathetic neurones, on about the 60th day of life we observed a significant decrease in the noradrenaline concentration in these animals' hearts compared with the controls. If every guanethidine injection was followed immediately by intensive physical exercise, there was no drop in the heart noradrenaline concentration. Physical exercise of the same intensity performed a few hours before injecting guanethidine did not prevent the drop in the noradrenaline concentration in the heart. The results show that an exercise-induced increase in sympathetic activity, at a time when guanethidine is circulating in the blood and accumulating in the adrenergic neurones, inhibits the cytotoxic effect of guanethidine. Isolated physical exercise performed between the 15th and 29th day of life leads to an increase in the noradrenaline content of the heart of rats aged 60 days.

Influence of neuronal activity levels on the cytotoxic effects of guanethidine.

Reduction of activity levels in the noradrenergic innervation of the iris of the rat by decentralization of the superior cervical ganglion resulted in a marked increase in sensitivity of these fibers to damage by chronic guanethidine treatment (15 mg/kg/day) as shown by fluorescence histochemistry. Guanethidine also had an increased cytotoxic effect on the decentralized nerve cell bodies. These results suggest that the relative inactivity of male "short" noradrenergic neurons may contribute to their sensitivity to damage by guanethidine. The nature of the damage produced in other tissues by this dose of guanethidine indicates that levels of neuronal activity are not the only factor determining sensitivity to the cytotoxic effects of this drug.

[Hypotensive effect and toxicity of octadin and papaverine following their combined administration]. / Gipotensivnoe deistvie i tiksichnost' oktadina i papaverina pri ikh kombinirovannom vvedenii

A study on the hypotensive action of combined injections of papaverine and octadine (guanethidine) was carried out in acute experiments on 90 anaesthetized cats. It was shown that with combined injections of octadine (in doses exceeding by 22% ED100) and of papaverine (in doses inferior by 22% to ED100) the hypotensive action was potentiated. A change of the said proportions led to a diminished hypotensive effect. It was also found that the most effective combinations of the 2 drugs was the one consisting of 0.5 mg/kg of papaverine and 5 mg/kg of octadine (in a ratio of 1:10). By studying the toxicity of the 2 drugs and of their combinations on 350 albino mice it was ascertained that their toxicity diminished if the drugs were used together.

The acute and target organ toxicity of 1-methyl-3-keto-4-phenylquinuclidinium bromide (MA540) and guanethidine in the rat and dog.

The effects of acute and repeated increasing oral doses of 1-methyl-3-keto-4-phenylquinuclidinium bromide (MA540) and guanethidine in the rat and dog have been described. On repeated oral administration guanethidine produced histopathological changes in cervical ganglia of rats and dogs which were clearly dose-dependent and reproduced lesions reported in the literature. Repeated oral administration of MA540 resulted in no histopathological changes in either species. The maximum tolerated oral dose for guanethidine was estimated to be 515 mg/kg in the rat and 26 mg/kg in the dog compared with an estimated maximum tolerated oral dose for MA540 of 1750 mg/kg in the rat and 460 mg/kg in the dog. On the basis of these findings it is suggested that subchronic studies with MA540 in the rat and dog should provide evidence which would justify the use of MA540 in man at daily oral doses as high as 9 mg/kg.