Time course of sympathetic denervation of the rat ovary after freezing its nerve supply

Brief freezing of the ovarian vascular pedicle in rats reduced ovarian noradrenaline concentration, measured with HPLC, by 67% (P less than 0.01). Freezing of the ovarian suspensory ligament caused a 22% reduction (P less than 0.01) and 33% reduction (P less than 0.05) in 2 experiments. The numbers of adrenergic nerve terminals detected by fluorescence microscopy after these procedures were similarly reduced (P less than 0.01). Freezing of both the pedicle and the ligament produced complete sympathetic denervation in about 50% of the ovaries. From Days 2 to 10 after operation no noradrenaline or nerve terminals were detected in 14 out of 27 ovaries. Nerve terminals were also eliminated from the oviduct. Reinnervation of the ovary began between Days 12 and 30. It is concluded that the adrenergic innervation of the ovary is predominantly through nerves that accompany the vascular supply to the ovary and the ovarian suspensory ligament. Freezing of these routes is a simple and relatively atraumatic means of denervating the ovary for experimental studies.     

Blood flow in the ovary and oviduct of rats after sympathetic denervation

The effect of sympathetic denervation on blood flow in the ovary and oviduct was studied in rats undergoing oestrous cycles or at Day 14 of pregnancy. The ovary and oviduct on one side were denervated by briefly freezing the ovarian vascular pedicle and the ovarian suspensory ligament. Blood flow was measured using 15 microns 57Co-labelled microspheres while the rats were under barbiturate anaesthesia. In cyclic rats denervation raised blood flow to the oviduct by 90% the next day (P less than 0.01) and 39% at 4-10 days (0.05 less than P less than 0.1). Blood flow to the ovary was not affected. Denervation on Day 13 of pregnancy raised blood flow in the oviduct 5-fold at Day 14 (P less than 0.01) and denervation on Day 7 raised blood flow 3-fold on Day 14 (P less than 0.05). Blood flow to the luteal and non-luteal components of the ovary was not affected. Sham-operation did not affect blood flow in the oviduct or ovary. It is concluded that sympathetic nerves exert tonic vasoconstrictor control on the vasculature of the oviduct but not on that of the ovary, and that these nerves do not regulate the major changes in blood flow that occur in ovaries in various physiological states.     

Pregnancy and parturition in rats after sympathetic denervation of the ovary, oviduct and utero-tubal junction

The ovarian vascular pedicle and ovarian suspensory ligament were briefly frozen to destroy the nerves. Examination of sections from the ovary, oviduct and utero-tubal junction by fluorescence histochemistry showed that they were usually devoid of adrenergic nerves. Measurement of noradrenaline in segments of the uterine horn by high-performance liquid chromatography showed that the transmitter was eliminated from the upper third but not the middle or lower thirds of the uterine horn. Unilateral or bilateral denervations at metoestrus in cyclic rats did not affect either the number of ovulations or the numbers or spacing of conceptuses at Day 7 of pregnancy. Bilateral denervations on Days 4, 7 or 11 of pregnancy did not affect ovarian weights or numbers of conceptuses observed 1 week later. Plasma progesterone concentrations were at least as high in the denervated groups as in the sham-operated control groups. After unilateral or bilateral denervations at Day 15, pregnancy continued normally and birth of normal young occurred, without apparent problems, at the same time as for sham-operated rats. The mothers tended their young and allowed them to suck. It is concluded that the adrenergic innervation of the ovary in the rat is not required for its normal function during pregnancy; that of the oviduct and mesosalpinx is not required for ovum pick-up or for transport of oocytes, spermatozoa or early embryos; and that of the utero-tubal junction is not required for uterine motility involved in embryo spacing and parturition.     

Ovulation in the isolated perfused rat ovary as documented by intravital microscopy

Surface cell changes at the apices of preovulatory follicles and ovulations were documented in isolated perfused ovaries from immature rats treated with pregnant mare serum gonadotropin (20 IU) and 48 h later with human chorionic gonadotropin (hCG) (10 IU). A video camera coupled to an inverted microscope and a video recorder captured the preovulatory and ovulatory events at a cellular level. At around 8 h post-hCG, the follicular apex changed from a smooth and optically homogeneous appearance into a rough surface with bleb formation and extrusions of single cells through minute perforations (early stigma formation). At approximately 10 h, a sticky material formed a basketlike structure with trapped cells (late stigma formation). At 12 to 15 h, ovulation took place at a constant speed and with no contractions of the follicular wall. This indicates that ovulation can occur with no visible circumfollicular muscular activity. Furthermore, the observations of a leakage of cells over an extended period of time indicates that the follicular wall is partly digested several hours before ovulation occurs.     

Ultrastructural changes in the rat pineal gland after sympathetic denervation. Quantitative study

Ultrastructural changes in the rat pineal gland were studied quantitatively 7 and 60 days after the sympathetic denervation by bilateral excission or decentralization of superior cervical ganglia. The surface occupied by pineal parenchymal cells decreased in rats of experimental groups with respect to the control group. Furthermore, profile areas of the cytoplasm, nucleus and nucleolus of the pinealocytes were also diminished. Cytoplasmic lipid droplets in the pinealocytes were markedly decreased in number and size in experimental rats. As demonstrated by the Kruskal-Wallis H test, statistically significant differences were found between rats of the control and operated groups. Rats treated by superior cervical ganglionectomy or decentralization showed morphological changes indicating a hypofunctional pineal gland, although differences were found between both groups.     

Gastric erosions following sympathetic denervation in the maternally deprived rat.

The adenosine triphosphate (ATP) content of rat mast cells was studied during and after histamine release induced by compound 48/80. The almost identical time course of ATP decrease from mast cells treated with either glycolytic or respiratory inhibitors seems to indicate that the ATP depletion was largely related to the histamine release process and not to an uncoupling of the oxidative phosphorylation. These results support the view that histamine release induced by compound 48/80 is an energy-requiring process. The ATP content of the cells was not, however, restored within the two hours of observation. The cause of the prolonged decrease in the ATP level has been discussed.     

Afferent renal denervation impairs baroreflex control of efferent renal sympathetic nerve activity

Increasing efferent renal sympathetic nerve activity (ERSNA) increases afferent renal nerve activity (ARNA), which decreases ERSNA to prevent sodium retention. High-sodium diet enhances ARNA, suggesting an important role for ARNA in suppressing ERSNA during excess sodium intake. Mean arterial pressure (MAP) is elevated in afferent renal denervated by dorsal rhizotomy (DRX) rats fed high-sodium diet. We examined whether the increased MAP in DRX is due to impaired arterial baroreflex function. In DRX and sham DRX rats fed high-sodium diet, arterial baroreflex function was determined in conscious rats by intravenous nitroprusside and phenylephrine or calculation of transfer function gain from arterial pressure to ERSNA (spontaneous baroreflex sensitivity). Increasing MAP did not suppress ERSNA to the same extent in DRX as in sham DRX, -60 +/- 4 vs. -77 +/- 6%. Maximum gain, -4.22 +/- 0.45 vs. -6.04 +/- 0.90% DeltaERSNA/mmHg, and the maximum value of instantaneous gain, -4.19 +/- 0.45 vs. -6.04 +/- 0.81% DeltaERSNA/mmHg, were less in DRX than in sham DRX. Likewise, transfer function gain was lower in DRX than in sham DRX, 3.9 +/- 0.2 vs. 6.1 +/- 0.5 NU/mmHg. Air jet stress produced greater increases in ERSNA in DRX than in sham DRX, 35,000 +/- 4,900 vs. 20,900 +/- 3,410%.s (area under the curve). Likewise, the ERSNA responses to thermal cutaneous stimulation were greater in DRX than in sham DRX. These studies suggest impaired arterial baroreflex suppression of ERSNA in DRX fed high-sodium diet. There were no differences in arterial baroreflex function in DRX and sham DRX fed normal-sodium diet. Impaired arterial baroreflex function contributes to increased ERSNA, which would eventually lead to sodium retention and increased MAP in DRX rats fed high-sodium diet.     

Alkaline ribonuclease activity is increased in rat sympathetic ganglia after nerve injury

Ribonuclease activity at pH 7.1 (alkaline ribonuclease) was determined in homogenates of rat superior cervical ganglion up to 5 days after postganglionic nerve injury under optimal conditions of assay. Measurements were performed in the presence and absence of the sulfhydryl blocking agent, N-ethylmaleimide, to assess the proportion of alkaline ribonuclease apparently bound to endogenous inhibitor. Total ribonuclease activity per ganglion was stimulated 1.3 fold by 1 day after injury and remained elevated over the 5 day period. Free ribonuclease activity accounted for about 60% of the observed increase in total activity at day 1, but had returned to control level by day 3. At day 3 the entire 90% increase in total activity was attributable to ribonuclease bound to endogenous inhibitor (i.e. latent activity). These changes are occurring at times after nerve injury when marked alterations in RNA turnover have been observed, implicating alkaline ribonucleases in the control of RNA metabolism during nerve regeneration.     

Characterization of apoptosis in cultured rat sympathetic neurons after nerve growth factor withdrawal

Sympathetic neurons depend on nerve growth factor (NGF) for their survival both in vivo and in vitro. In culture, the neurons die after NGF withdrawal by an autonomous cell death program but whether these neurons die by apoptosis is under debate. Using vital DNA stains and in situ nick translation, we show here that extensive chromatin condensation and DNA fragmentation occur before plasma membrane breakdown during the death of NGF-deprived rat sympathetic neurons in culture. Furthermore, kinetic analysis of chromatin condensation events within the cell population is consistent with a model which postulates that after NGF deprivation nearly all of the neurons die in this manner. Although the dying neurons display membrane blebbing, cell fragmentation into apoptotic bodies does not occur. Apoptotic events proceed rapidly at around the time neurons become committed to die, regardless of neuronal culture age. However the duration of NGF deprivation required to commit neurons to die, and the rate at which apoptosis occurs, increase with culture age. Thus, within the first week of culture, apoptosis is the predominant form of cell death in sympathetic neurons.     

The effects of sympathetic denervation on spontaneous ventricular defibrillation in the rat.

Ventricular tachycardia or ventricular fibrillation was electrically induced in 38 normal rats (group 1) and 24 sympathetically denervated rats (6-hydroxydopamine) (group 2). The time for spontaneous reversion to sinus rhythm was measured during (1) control, (2) isoproterenol, and (3) the combination of isoproterenol and phenylephrine. The time for spontaneous reversion was the same in both groups in the three states. The reversion time was prolonged threefold by isoproterenol, and restored to control values when phenylephrine was added to the infusion of isoproterenol. The tachycardia duration and the refractory period were inversely related: log10 (tachycardia duration) = 3.466-0.091 (refractory period). Ventricular tachycardia/fibrillation induction was examined as follows: (i) Ventricular tachycardia/fibrillation was induced in 100% of normal rats (group 1), but only 42% of the denervated rats (group 2, p less than 0.001); (ii) during isoproterenol, ventricular tachycardia/fibrillation was induced in 100% of rats of both groups; and (iii) when phenylephrine was added to isoproterenol, ventricular tachycardia/fibrillation was induced in 100% of group 1 rats versus 82% of group 2 rats, (p = NS). These observations suggest (1) the induction of ventricular tachycardia/fibrillation is highly dependent on intact sympathetic innervation, and (2) exogenous adrenergic agonists modulate the duration of ventricular fibrillation through their effects on ventricular refractory period, independent of sympathetic innervation.     

Recovery of baroreflex control of renal sympathetic nerve activity after spinal lesions in the rat

Spinal cord injury (SCI) has serious long-term consequences on sympathetic cardiovascular regulation. Orthostatic intolerance results from insufficient baroreflex regulation (BR) of sympathetic outflow to maintain proper blood pressure upon postural changes. Autonomic dysreflexia occurs due to insufficient inhibition of spinal sources of sympathetic activity. Both of these conditions result from the inability to control sympathetic activity caudal to SCI. It is well established that limited motor ability recovers after incomplete SCI. Therefore, the goal of this study was to determine whether recovery of BR occurs after chronic, left thoracic spinal cord hemisection at either T(3) or T(8). Baroreflex tests were performed in rats by measuring the reflex response of left (ipsilateral) renal sympathetic nerve activity to decreases and increases in arterial pressure produced by ramped infusions of sodium nitroprusside and phenylephrine, respectively. One week after a T(3) left hemisection, BR function was modestly impaired. However, 8 wk after a T(3) left hemisection, BR function was normal. One week after a T(8) left hemisection, BR function was significantly impaired, and 8 wk after a T(8) left hemisection, BR function was significantly improved. These results indicate that BR of renal sympathetic nerve activity in rats may partially recover after spinal cord hemisections, becoming normal by 8 wk after a T(3) lesion, but not after a T(8) lesion. The nature of the spinal cord and/or brain stem reorganization that mediates this recovery remains to be determined.     

Characteristics of contractile properties of the cat skeletal muscle after sympathetic denervation]

Twitching and tetanus were registered during stimulation in the cat desympathised m. semitendinosus, similar to those occurring in the motor units during thermoregulatory tone. The contractile responses were greater in the early desynpathisation phase. By the 14th day, the contractile responses became similar to those of the control.     

Effect of sympathetic denervation on the rate of protein synthesis in rat skeletal muscle

Rates of protein synthesis were investigated in skeletal muscles from rats submitted to chemical and surgical sympathectomy. Three models of sympathetic denervation were used: 1) treatment with guanethidine (100 mg.kg(-1).day(-1) sc); 2) lumbar sympathetic denervation (surgical excision of the second and third lumbar ganglia of the sympathetic chain, from which arises the postganglionic fibers to the skeletal muscles of rat hindlimb); and 3) adrenodemedullation. Protein synthesis was estimated in isolated soleus muscle by the rate of incorporation of [(14)C]tyrosine (0.1 mM, 0.05 microCi/ml) into total protein. Soleus isolated after 2 and 4 days of chemical sympathectomy or after 3 days of lumbar denervation showed a 17-20% statistically significant decrease in in vitro rates of protein synthesis. These effects were reverted by addition of 10(-5) M isoproterenol or epinephrine in vitro. Neither clenbuterol nor isoproterenol (10(-7), 10(-6), or 10(-5) M) in vitro affected the rate of protein synthesis in soleus from normal rats. On the other hand, clenbuterol or epinephrine (10(-5) M) increased by 20% the rate of protein synthesis in soleus muscles from adrenodemedullated rats and prevented its decrease in muscles from fasted rats. The data suggest that the sympathetic nervous system stimulates protein synthesis in oxidative muscles, probably through the activation of beta(2)-adrenoceptors, especially in situations of hormonal or nutritional deficiency.     

Effect of different fixation methods and partial chemical sympathetic denervation on cell size and 3H-leucine incorporation by rat sympathetic neurocytes

Dimensions of sympathetic nerve cells and of their nuclei were studied using various methods of fixation and embedding media, as well as autoradiographic indices of the protein synthesized system of neurocytes in normal rats, and in rats partially sympathectomized at an early age. On the ground of different changes in the average size of neurocytes seen under various methods of fixation, and of decreased labeling intensity of large neurocytes after the injection of the labeled protein biosynthesis precursor there is some reason to believe that large neurocytes may appear in the late reproductive age mainly due to the nerve cell cytoplasm swelling as a consequence of exhaustion of their growth resources.     

Reinnervation of the rat levator ani muscle after neonatal denervation

After axonal injury on postnatal day 14 (P14), but not P21, motoneurons in the spinal nucleus of the bulbocavernosus (SNB) do not display their normal response to circulating testosterone levels. This could result from a permanent disruption of communication between motoneurons and their testosterone-sensitive target muscles. We assessed the extent of reinnervation of one of these target muscles, the levator ani (LA) muscle, 5 months after the pudendal nerve was cut either on P14 or P21. The number of motoneurons innervating the LA in control and nerve cut animals was determined using retrograde labeling procedures. Functional recovery of the LA muscle was determined via the testing of its in situ contractile properties. Compared to control muscles, reinnervated LA muscles were smaller, had fewer muscle fibers, generated a lower maximum tetanic tension, and were more fatigable. In spite of the fact that fewer motoneurons reinnervated the LA muscle after nerve cut on P14 than on P21, there were no differences in the weight or contractile properties of the LA muscle between these two groups. These data suggest that motoneurons that survived injury on P14 innervated more muscle fibers than normal and exhibited a similar ability to functionally reinnervate the target muscle as those motoneurons that survived injury on P21.     

Glycoproteins of submaxillary saliva of the cat: differences in composition produced by sympathetic and parasympathetic nerve stimulation

Abstract— Saliva samples were obtained from the cannulated submaxillary ducts of the cat during stimulation of the peripheral cut end(s) of (1) the cervical sympathetic nerve, (2) the chordalingual (parasympathetic) nerve and (3) both nerves at the same time. In nine experiments the ratios of neuraminic acid to fucose and to hexosamine were consistently 2·5–4 times higher in saliva evoked by sympathetic nerve stimulation than in that produced by parasympathetic stimulation. This was not attributable to differences in the rate of synthesis of the carbohydrate of the glycoproteins or in salivary flow rate. The presence of glycolipids and blood glycoproteins was excluded. Saliva produced by stimulation of the sympathetic and parasympathetic nerves each showed a single, but different, peak after ultracentrifugation in 0·1 m -NaCl with 0·01 m -phosphate buffer (pH 7·4). The S_{20, w} of the former was 6·5 and of the latter, 39. Both peaks were demonstrable in saliva produced when both nerves were stimulated at the same time.