Morphometric study of the superior cervical and stellate ganglia of spontaneously hypertensive rats during the prehypertensive stage

To compare the functional state of the superior cervical (SCG) and stellate sympathetic ganglia (SG) of spontaneously hypertensive rats (SHR) with those of age-matched normotensive Wistar Kyoto rats (WKY), ganglion cell volume and area occupied by ganglion cells relative to each whole ganglionic area were morphometrically examined using the Texture Analyse System (TAS) in rats at 0, 10 and 30 days of age. The weight of each ganglion relative to animal weight was also measured. The ganglion cell volume and the relative area of ganglionic cells in both ganglia of SHR were significantly larger (P less than 0.05) than those of age-matched WKY at ages 0 and 10 days after birth. The relative ganglionic weights of SHR were significantly larger (P less than 0.01) compared with those of WKY at all ages examined, except for SG at 0 days after birth. These results show that the relative volume of sympathetic ganglion cells is greater in both SCG and SG of SHR than that of WKY, suggesting that hyperfunction of sympathetic ganglia occurs at the prehypertensive stage as a primary factor in the development of hypertension in SHR.     

Autoradiographic study of 3H-DOPA uptake by superior cervical and stellate ganglia of spontaneously hypertensive rats during the prehypertensive stage

The functional state of sympathetic ganglia in spontaneously hypertensive rats (SHR) was compared with that of ganglia in normotensive Wistar Kyoto rats (WKY) by examining catecholamine synthetic activity by light microscopic autoradiography 3H-L-dihydroxyphenyl alanine (3H-DOPA). The number of silver grains over the perikarya of ganglion cells in the superior cervical (SCG) and stellate ganglia (SG) of newborn, 10-day-old and 30-day-old animals was counted on photographic enlargements. There were significantly more silver grains over ganglion cells in SHR compared with those in age-matched WKY at almost all incorporation times at all ages examined in SCG, at all incorporation times in newborn rats, and at incorporation times of 15 and 60 min in SG of 10-day-old rats. The increased incorporation of the label by both sympathetic ganglia was more marked in newborn than in 30-day-old animals. These results indicate that catecholamine synthetic activity in these ganglion cells is increased in SHR from the newborn stage, suggesting that a congenital hyperfunction of sympathetic ganglia occurs in SHR.     

Autoradiographic study of 3H-lysine uptake by superior cervical and stellate ganglia in prehypertensive spontaneously hypertensive rats

In order to compare the functional state of sympathetic ganglia in spontaneously hypertensive (SHR) with those in normotensive Wistar Kyoto rats (WKY), protein synthetic activity was examined by light microscopic autoradiography with 3H-lysine. The number of silver grains over the cytoplasm of ganglion cells in the superior cervical and stellate ganglia of newborn and 30-day-old animals were counted on photographic enlargements. In both sympathetic ganglia there were significantly more silver grains over ganglion cells in SHR compared with age-matched WKY at 15, 60, and 120 min after injection of 3H-lysine. The increased incorporation of the label by both sympathetic ganglia was more marked in newborn than in 30-day-old animals. This result shows that protein synthetic activity in these ganglion cells is increased in SHR from the newborn stage. It is suggested that a congenital hyperfunction of sympathetic ganglia occurs in SHR.     

Enhanced ganglionic responses to substance P in spontaneously hypertensive rats

Intravenous injection of substance P (SP) increases blood pressure in normotensive rats by stimulating sympathetic ganglia. This study compared the effects of SP to increase renal nerve firing and blood pressure in normotensive and hypertensive rats treated with chlorisondamine. The increase in renal nerve firing was greatest in spontaneously hypertensive rats (SHR), intermediate in Wistar rats, and least in Wistar-Kyoto (WKY) rats. Blood pressure was increased more in SHR than in Wistar rats. Blood pressure was not increased in WKY rats. Responses to the ganglionic stimulant 1,1-dimethyl-4-phenylpiperazinium were the same in the three strains. These results suggest that there is a selective increase in the action of SP on sympathetic ganglia of SHR and that ganglion responsiveness to SP is correlated with its effect on blood pressure.     

Age-dependent changes of the autonomic neurons expressing vanilloid TRPV1 receptors

Expression of vanilloid receptors in sympathetic and afferent ganglionic neurons was studied in rats of different ages (newborn, 10-day old, 20-day old, 30-day old, 60-, 180-day old) using immunohistochemical methods. The results obtained indicate that the majority of the afferent neurons in the nodose ganglion of vagus nerve (GNVN) and in the spinal ganglia (SG) were TRPV1-positive from birth onwards. The percentage of neurons containing TRPVT receptors in SG slightly increased with age up to 30 days postnatally. In the GNVN, the percentage of TRPV1-positive neurons was higher in comparison with the SG in all age groups. The vast majority of the sympathetic neurons were TRPV1-positive from birth onwards, and the percentage of TRPV1-immunoreactive neurons substantially decreased during further development. In 20-day old and older animals, we observed only few TRPV1-immunoreactive neurons in sympathetic ganglia. Finally, the percentage of neurons containing these types of neurons, become similar to adult animals to the end of the first month of life.     

Expression of phenylethanolamine N-methyltransferase in rat sympathetic ganglia and extra-adrenal chromaffin tissue

To determine whether similar mechanisms regulate adrenergic phenotypic expression in different cellular populations, the superior cervical sympathetic ganglion (SCG) and extra-adrenal chromaffin tissue were studied in the fetal and neonatal rat; results were compared to those previously obtained with the adrenal medulla. Phenylethanolamine N-methyltransferase (PNMT), the enzyme which converts norepinephrine to epinephrine, was used as an index of adrenergic expression. PNMT catalytic activity was initially detectable in the SCG of normal, untreated fetuses at 17.0 days of gestation (E17.0), and increased three- to fourfold until postnatal day 2. Thereafter activity decreased precipitously, and was undetectable 2 weeks after birth. Immunohistochemical studies, using specific antisera to PNMT, were employed to localize the enzyme. Immunoreactivity (PNMT-IR) was undetectable in sympathetic ganglia of control animals, suggesting that this method is less sensitive than the catalytic assay. Following glucocorticoid treatment, cells heavily stained for PNMT-IR were observed in paravertebral sympathetic ganglia, including the SCG, and in the organ of Zuckerkandl. In the SCG, PNMT-IR was present in small cells presumed to be small, intensely fluorescent (SIF) cells and was never observed in principal ganglion neurons. The increase in PNMT-IR after steroid treatment was strikingly age dependent: initiation of treatment at progressively older ages during the first week of life resulted in fewer and fewer PNMT-IR cells. No response was apparent after 1 week. Moreover, treatment of pregnant rats was associated with appearance of PNMT-IR at E18.5, but not at E16.5. After treatment from days 0 to 6 of life, PNMT-IR gradually disappeared. However, retreatment on days 24–30 caused the reappearance of PNMT-IR, suggesting that exposure to steroids at birth causes (a) an immediate increase in PNMT-IR and (b) responsiveness to steroids during adulthood. Consequently, the disappearance of PNMT-IR after exposure to steroids at birth, is not simply due to death of SIF cells. We conclude that proximity to the adrenal cortex is not necessary for initial expression of PNMT. More generally, the expression of PNMT by ganglion SIF cells parallels that in adrenal chromaffin cells since initial expression was not dependent on high local concentrations of glucocorticoids, whereas subsequent development did require high levels of the hormones. Our observations suggest that similar mechanisms regulate expression and development of the adrenergic phenotype in adrenal and sympathetic ganglia.     

Retrograde Transport of Endogenous Nerve Growth Factor in Superior Cervical Ganglion of Adult Rats

The concentration of naturally synthesized nerve growth factor (NGF) was measured in various tissues of adult rats, using a highly sensitive two-site enzyme immunoassay. The highest concentration was found in the superior cervical sympathetic ganglion (SCG). Transection of the postganglionic external carotid nerve (ECN) reduced the ganglionic level of NGF more than did section of the internal carotid nerve (ICN). When both the preganglionic nerve and the ECN were cut, the ganglionic NGF level decreased even more. On the other hand, when the preganglionic nerve and the ICN were both sectioned, leaving the ECN intact, endogenous NGF content in the SCG was significantly enhanced 3-9 h after operation. Bilateral extirpation of submaxillary gland produced a rapid decrease in ganglionic NGF 3-6 h after operation, and even unilateral removal of one salivary gland caused a decrease in both ganglia, which was however much greater in the ipsi- than in the contralateral ganglion. Removal of the eyeballs caused a much smaller reduction in ganglionic NGF than did removal of the glands. These results suggest that the endogenous NGF that accumulates in the SCG is mostly synthesized in the submaxillary gland rather than in the iris, and that it is transported to the SCG, mostly via the ipsilateral ECN.     

不同日龄大鼠颈上交感神经节神经元电转染方法

目的 建立一种高效电转染不同日龄大鼠颈上交感神经节(superior cervical sympathetic ganglion,SCG)神经元细胞的方法.提高转染后细胞的成活率、转染效率和干扰效率.方法 用传统的及经改良的神经元培养液分别培养电转染后的7日龄、14日龄和40日龄SD大鼠SCG细胞,24 h后用台盼蓝染色方法观察并计算细胞成活率;通过改变质粒DNA和siRNA与转染液比例,优化转染条件,于转染24h后在共聚焦显微镜下观察并计算转染效率或干扰效率.结果 改良培养液可使14日龄以上SD大鼠SCG细胞转染后成活率达到75％以上,明显高于传统培养液转染后的成活率(P＜0.01),且结果稳定,细胞状态良好,能够满足后续实验研究的要求;优化转染条件后,DNA 的转染率及siRNA的干扰率显著提高,当DNA与转染液比例为1∶100(μg∶μL)时,细胞转染率最高;当siRNA与转染液比例为1∶50(μg∶ μL)时干扰率最高.结论 通过改良神经元培养液及优化转染条件,成功提高了电转染后细胞的成活率、转染效率和干扰效率,利用电转染方法可成功转染不同日龄SD大鼠SCG神经元.     

Regeneration of Cholinesterases in the Stellate and Normal and Denervated Superior Cervical Ganglia of the Cat Following Inactivation by Sarin

Abstract: Experiments were designed to test the hypothesis that ganglionic butyrylcholinesterase (BuChE) is derived from acetylcholinesterase (AChE). At 5 to 8 days following preganglionic denervation of the right superior cervical ganglion (SCG), cats were given sarin, 2.0 μmol/kg, i.v. At intervals of 1 h and 1, 2, 3, 6, 11, and 22 days later, they were killed, and the AChE and BuChE contents of both SCG and both stellate ganglia (StG) were assayed. The regeneration of AChE in the normal ganglia occurred in two phases: an initial rapid phase, to 25-40% of control activity in 1 day, and a slow phase, to approximately 70% of control activity in 22 days. BuChE reached approximately 85% of control activity in normal SCG and StG at 22 days. In the denervated SCG, AChE activity reached a maximum of approximately 17% of normal at 1 day, the value prior to the administration of sarin, and did not increase appreciably above this subsequently. BuChE activity in the denervated SCG reached approximately 50% of normal ganglia at 22 days. At each interval, its activity approached 55% of that of the contralateral normal SCG, the value found in the denervated SCG prior to the administration of sarin. Hence, the regeneration of BuChE appears to be independent of the presence of AChE in the neuropil. The origin of ganglionic BuChE remains obscure.     

Enkephalin-like immunoreactivity in ganglionic cells in the larynx and superior cervical ganglion of the rat.

The distribution of enkephalin-like immunoreactivity (ENK-LI) in the larynx, the superior cervical ganglion (SCG) and the nodose ganglion of adult rats was examined in the present study. A substantial number of the local acetylcholinesterase (AChE)-positive, presumably parasympathetic, ganglionic cells in the larynx displayed ENK-LI. These cells also exhibited neuropeptide Y (NPY)- and vasoactive intestinal polypeptide (VIP)-LI. Varicose nerve fibers showing ENK-LI were observed close to the acini and ducts of the glands, in the perichondrium and in the lamina propria. The varicosities exhibiting ENK-LI frequently displayed NPY- and VIP-LI. The ENK-LI was detected in a subpopulation of AChE-positive nerve fibers in the laryngeal tissue. In the SCG, only a small number of the ganglionic cells displayed ENK-LI. These cells, in contrast to other ganglionic cells of the SCG, did not show NPY-LI. None of the ganglionic cells of the nodose ganglion exhibited ENK-LI. Sympathectomy and vagotomy affected neither the number nor the distribution of fibers showing ENK-LI in the larynx. In conclusion, ENK appears to be present together with NPY and VIP in the parasympathetic innervation of the larynx and in a very limited number of the ganglionic cells of a sympathetic ganglion, the SCG, of the adult rat.     

A COMPARISON OF THE NEURAL REGULATION OF TYROSINE HYDROXYLASE ACTIVITY IN SYMPATHETIc GANGLIA OF ADULT MICE AND RATS

Surgical decentralization of the superior cervical ganglion (SCG) in rats and mice led to a fall in ganglionic tyrosine hydroxylase (T-OH) activity, and a loss of more than 90 per cent of the preganglionic neurone marker, choline acetyl transferase. T-OH activity was reduced by more than 50 per cent in mice SCG ten days after surgery, but fell by only 25 per cent in rat SCG after 21 days. The surgical procedure did not cause obvious histo-logical damage or loss of SCG cells in either species. Both T-OH and choline acetyl transferase activities in rat and mouse SCG recovered to normal three months after surgery. Reserpine treatment was more effective in rats in causing increased ganglionic T-OH activity than in mice. Neither decentralization nor reserpine treatment caused any changes in DOPA-decarboxylase or monoamine oxidase activities in rat SCG. These results demonstrate that T-OH activity in SCG is subject to trans-synaptic regulation in both rats and mice; this regulation does not apply to DOPA-decarboxylase or monoamine oxidase. Differences in basal sympathetic tone may explain the different results obtained in mice and rats.     

Effects of Denervation and Axotomy on Nervous System-Specific Protein, Ornithine Decarboxylase, and Other Enzyme Activities in the Superior Cervical Sympathetic Ganglion of the Rat

The time courses of changes of three enolase isozymes (alpha alpha, alpha gamma, and gamma gamma), S-100 protein, 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), ornithine decarboxylase (ODC), beta-galactosidase, and glucose-6-phosphate dehydrogenase (G6PDH) were examined from 1 to 14 days after cutting of the preganglionic nerve (denervation) or the postganglionic nerve (axotomy) of the superior cervical sympathetic ganglion (SCG) of the rat. The wet weight and protein content in the axotomized SCG increased continuously, to nearly twice those of the denervated SCG for 1-2 weeks after the operations. Among enolase isozymes in the SCG, neuron-specific gamma gamma-enolase decreased rapidly after denervation and stayed at a low level for 2 weeks, whereas the isozyme remained almost unchanged after axotomy. On the contrary, ganglionic alpha alpha-enolase and the alpha gamma-hybrid form increased remarkably to reach a maximum at the second day after axotomy, and remained above control for 1 to 2 weeks; these two enolase isozymes showed little change after denervation. Denervation caused a much larger increase than did axotomy in the ganglionic S-100 protein, an astrocyte-specific protein, during the first week after the operation, while the protein content decreased after 2 weeks of either denervation or axotomy. CNPase, a myelin-associated enzyme, rose suddenly 2 days after axotomy, and remained at a rather high level compared with the denervated ganglion, which showed little variation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased pressor responses to pressor agents in spontaneously hypertensive rats.

Pressor reactivity to a variety of pressor agents after partial ganglionic blockade induced with hexamethonium was investigated in intact, in spinalized, and in chemically sympathectomized, spontaneously hypertensive rats (SHR). Responses of unanaesthetized 6-month-old SHR to noradrenaline, phenylephrine, and angiotensin after hexamethonium administration (32 mg/kg) markedly exceeded those of unanaesthetized, age-matched normotensive Wistar-Kyoto rats (WKR). Responses of anaesthetized SHR to noradrenaline after hexamethonium administration (16 mg/kg) were also increased at the hypertensive stages but not at the prehypertensive stages, when compared with those of anaesthetized normotensive Wistar rats of respective ages. In spinalized and chemically sympathectomized preparations after hexamethonium administration (16 mg/kg), noradrenaline produced equal increases in blood pressure in 6-month-old SHR and WKR. It is suggested that the functional sympathetic nervous system is important for the hyperreactivity of intact SHR.     

Neurofilament-like and glial fibrillary acidic protein-like immunoreactivities in rat and guinea-pig sympathetic ganglia in situ and after perturbation

Summary The presence of neurofilament (NF)-like and glial fibrillary acidic protein (GFAP)-like immunoreactivities was studied in sympathetic ganglia of adult rats and guinea pigs during normal conditions and after perturbation. In the superior cervical ganglion (SCG) of normal rats, many ganglion cells and nerve fibers show NF immunoreactivity. Some of these nerve fibers disappear after preganglionic decentralization of SCG; this indicates the presence of a mixture of preand postganglionic NF-positive nerves in the ganglion. Cuts in both preand postganglionic nerves result in a marked increase in GFAP immunoreactivity in SCG, whereas NF immunoreactivity increases in nerve cell bodies after preganglionic cuts. Only a few ganglion cells show NF immunoreactivity in the normal SCG of guinea pig. All intraganglionic NF-positive nerves are of preganglionic origin; decentralization abolishes NF immunoreactivity in these nerve fibers. The inferior mesenteric ganglion, the hypogastric nerves and colonic nerves in guinea pigs contain large numbers of strongly NF-immunoreactive nerve fibers.When the SCG of adult rat is grafted to the anterior eye chamber of adult rat recipients, both ganglionic cell bodies and nerve fibers, forming on the host iris from the grafted ganglion, are NF-positive. As only the perikarya of these neurons normally exhibit NF immunoreactivity, and the terminal iris arborizations are NF-negative, it appears that the grafting procedure causes NF immunoreactivity to become more widespread in growing SCG neurons.     

Sympathetic activation increases basilar arterial blood flow in normotensive but not hypertensive rats

The close apposition between sympathetic and parasympathetic nerve terminals in the adventitia of cerebral arteries provides morphological evidence that sympathetic nerve activation causes parasympathetic nitrergic vasodilation via a sympathetic-parasympathetic interaction mechanism. The decreased parasympathetic nerve terminals in basilar arteries (BA) of spontaneously hypertensive rat (SHR) and renovascular hypertensive rats (RHR) compared with Wistar-Kyoto rats (WKY), therefore, would diminish this axo-axonal interaction-mediated neurogenic vasodilation in hypertension. Increased basilar arterial blood flow (BABF) via axo-axonal interaction during sympathetic activation was, therefore, examined in anesthetized rats by laser-Doppler flowmetry. Electrical stimulation (ES) of sympathetic nerves originating in superior cervical ganglion (SCG) and topical nicotine (10-30 μM) onto BA of WKY significantly increased BABF. Both increases were inhibited by tetrodotoxin, 7-nitroindazole (neuronal nitric oxide synthase inhibitor), and ICI-118,551 (β(2)-adrenoceptor antagonist), but not by atenolol (β(1)-adrenoceptor antagonist). Topical norepinephrine onto BA also increased BABF, which was abolished by atenolol combined with 7-nitroindazole or ICI-118,551. Similar results were found in prehypertensive SHR. However, in adult SHR and RHR, ES of sympathetic nerves or topical nicotine caused minimum or no increase of BABF. It is concluded that excitation of sympathetic nerves to BA in WKY causes parasympathetic nitrergic vasodilation with increased BABF. This finding indicates an endowed functional neurogenic mechanism for increasing the BABF or brain stem blood flow in coping with increased local sympathetic activities in acutely stressful situations such as the "fight-or-flight response." This increased blood flow in defensive mechanism diminishes in genetic and nongenetic hypertensive rats due most likely to decreased parasympathetic nitrergic nerve terminals.     

Nerve stimulation induced overflow of neuropeptide Y and modulation by angiotensin II in spontaneously hypertensive rats

The sympathetic nervous system and renin-angiotensin system are both thought to contribute to the development and maintenance of hypertension in experimental models such as the spontaneously hypertensive rat (SHR). We demonstrated that periarterial nerve stimulation (NS) increased the perfusion pressure (PP) and neuropeptide Y (NPY) overflow from perfused mesenteric arterial beds of SHRs at 4-6, 10-12, and 18-20 wk of age, which correspond to prehypertensive, developing hypertensive, and maintained hypertensive stages, respectively, in the SHR. NS also increased PP and NPY overflow from mesenteric beds of Wistar-Kyoto (WKY) normotensive rats. NS-induced increases in PP and NPY were greater in vessels obtained from SHRs of all three ages compared with WKY rats. ANG II produced a greater increase in PP in preparations taken from SHRs than WKY rats. ANG II also resulted in a greater increase in basal NPY overflow from 10- to 12-wk-old and 18- to 20-wk-old SHRs than age-matched WKY rats. ANG II enhanced the NS-induced overflow of NPY from SHR preparations more than WKY controls at all ages studied. The enhancement of NS-induced NPY overflow by ANG II was blocked by the AT1 receptor antagonist EMD-66684 and the angiotensin type 2 receptor antagonist PD-123319. In contrast, ANG II greatly enhanced norepinephrine overflow in the presence of PD-123319. Both captopril and EMD-66684 decreased neurotransmitter overflow from SHR mesenteric beds; therefore, we conclude that an endogenous renin-angiotensin system is active in this preparation. It is concluded that the ANG II-induced enhancement of sympathetic nerve stimulation may contribute to the development and maintenance of hypertension in the SHR.     

Increased sympathetic innervation in the cerebral and mesenteric arteries of hypertensive rats

The density of catecholamine-containing nerve fibers was studied in the cerebral and mesenteric arteries from normotensive Wistar-Kyoto rats (WKY), spontaneously hypertensive rats (SHR), and stroke-prone SHR (SHRSP) in the growing (SHR, WKY) and adult (SHR, SHRSP, WKY) animals. Cerebral arteries from SHR showed an increased adrenergic innervation from day 1. The nerve plexuses reached an adult pattern earlier in SHR than in WKY. The arteries from adult SHR and SHRSP (22 weeks old) showed a markedly higher nerve density than WKY. There was a positive linear correlation between blood pressure and nerve density for four cerebral arteries. The mesenteric arteries were not innervated at birth. However, hyperinnervation of these arteries in the SHR was already present at 10 days of age as compared with WKY. Sympathectomy with anti-nerve growth factor and guanethidine caused a complete disappearance of fluorescent fibers in the mesenteric arteries from SHR and WKY, and in the cerebral arteries of WKY. The same procedure caused only partial denervation of the cerebral arteries from hypertensive animals. We postulate that the increase in nerve density in the cerebral arteries from the hypertensive rats may contribute to the development of arterial hypertrophy in chronic hypertension through the trophic effect of the sympathetic innervation on vascular structure.     

Kinin B1 and B2 receptor mRNA expression in the hypothalamus of spontaneously hypertensive rats

The central hypertensive effects induced by bradykinin are known to be mediated via B2 receptors, which are present constitutively in the brain. B, receptors are rapidly upregulated during inflammation, hyperalgesia, and experimental diabetes. The hypothalamus plays an important role in the regulation of cardiovascular homeostasis, and all components of kallikrein-kinin system have been identified in this area. Therefore, we analyzed the mRNA expression of B1 and B2 receptors in the hypothalamus of spontaneously hypertensive rats (SHR) by RT-PCR. Male SHR were studied at three different ages corresponding to the three phases in the development of hypertension: (i) 3-4 (prehypertensive), (ii) 7-8 (onset of hypertension), and (iii) 12-13 weeks (established hypertension) after birth, and compared with age-matched Wistar-Kyoto (WKY) rats. At all ages tested, B2 receptor mRNA levels in the hypothalamus of SHR were higher than age-matched WKY rats (p < 0.001). However, the B1 receptor mRNA levels were higher at the established phase of hypertension only. We conclude that B1 and B2 receptor mRNA are differentially expressed in the hypothalamus of SHR and may play different roles in the pathogenesis of hypertension: upregulation of B2 receptor mRNA from early age may participate in the pathogenesis of hypertension, whereas an upregulation of B1 receptor mRNA in the established phase of hypertension may reflect an epiphenomenon in essential hypertension.     

Increased number of sympathetic neurons in the superior cervical ganglia of rats of SHR and Wistar-Kyoto strains as compared with Wistar rats

With histological technique increased number of sympathetic neurons in cranial ganglia of SHR and Wistar-Kyoto rats in comparison with Wistar line has been shown. The resistance to hypobaric hypoxia (simulated altitude 12000m) was also enhanced in SHR and WKY rats.