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The effects of 2-deoxy-D-glucose and sympathetic denervation of brown fat GDP binding in Sprague-Dawley rats.

Central administration of 2-deoxy-D-glucose (2-DG) decreases brown fat thermogenesis. This effect is suggested to be mediated via a central control mechanism. Our study was designed to determine the importance of the sympathetic nervous system in the response of brown fat to intraperitoneal (i.p.) injection of 2-DG. Unilateral denervation of interscapular brown adipose tissue (IBAT) was performed on male Sprague-Dawley rats (300 g body weight). Nine days after surgery, rats were injected i.p. with either saline vehicle (0.9% sodium chloride) or 2-DG (360 mg/kg wt) and then killed one hour later. Sympathetic denervation resulted in 50% decreases in total IBAT protein and in mitochondrial protein recovered. In the denervated lobes, mitochondrial GDP binding (expressed as nmol/mg mitochondrial protein and as total activity recovered) was decreased to 36% and 18%, respectively. Injection of 2-DG did not change mitochondrial protein content in either the innervated or denervated IBAT. In the innervated lobes, 2-DG significantly lowered GDP binding to 55% of that in saline-treated animals, whether expressed per mg mitochondrial protein or as total recovered activity. In contrast, 2-DG did not further decrease GDP binding in the denervated lobes. In conclusion, the effects of i.p. injection of 2-DG on brown fat thermogenesis (as evidenced by GDP binding) appear to be primarily mediated via the sympathetic nervous system.

Adipose Tissue, Brown↗

Effects of carotid body sympathetic denervation on ventilatory acclimatization to hypoxia in the goat.

Our objective was to test the hypothesis that diminishing sympathetic input to the carotid body (CB) during prolonged exposure to hypoxia results in increased CB afferent activity and increased ventilatory drive. Six awake goats were studied prior to and following sectioning of the efferent sympathetic input to the CB from the superior cervical ganglion. Ventilatory responses to acute and prolonged isocapnic hypoxia (PaO2 40 Torr) and drugs (norepinephrine and dopamine, 0.5, 1.0 and 5.0 micrograms.kg-1 min-1) were collected prior to the denervation. One week and 3-4 weeks following the sympathetic denervation, the animals were restudied following the above protocol. Ventilation was significantly lower following sympathetic denervation in normoxia and during the hypoxic exposure. However, the response to acute hypoxia and the time-course of ventilatory acclimatization to hypoxia was not altered by sympathetic denervation. All doses of norepinephrine and dopamine significantly inhibited VE in a dose-dependent manner. Sympathetic denervation did not significantly alter the response to the drug infusions. The sympathetic innervation to the CB does not appear to play a role in either the acute or prolonged ventilatory responses to hypoxia in the awake goat, but may affect overall ventilation.

Animals↗

Capsaicin induced afferent denervation and receptor-linked responses to CGRP in the rat.

We have examined the effect of neonatal capsaicin-induced destruction of primary afferent nerves in rats on the response to calcitonin gene-related peptide (CGRP) in vitro and in vivo. Denervation was confirmed by immunohistochemistry. Rat alpha-CGRP (rCGRP) activated adenylate cyclase in homogenates of rat spleen (basal activity 45 +/- 8.27, delta Vmax 75 +/- 16 pmol cyclic AMP min-1 protein; K(act) 2.04 +/- 0.44 nM). A single specific binding site for [125I]hCGRP was demonstrated in homogenates of spleen (Kd = 4.84 +/- 0.66 nM, Bmax = 1.43 +/- 0.35 pmol mg-1 protein). Neither adenylate cyclase activation nor binding site characteristics were affected by capsaicin-induced denervation. In addition, hypotensive responses to intravenous boluses of rCGRP were examined in anaesthetized rats. Neither the basal blood pressure nor the blood pressure fall in response to rCGRP were altered by neonatal denervation by capsaicin. In conclusion, there is no evidence of denervation hypersensitivity of receptor mediated responses to CGRP in vivo or in vitro, following capsaicin-induced denervation in the rat. This suggests that CGRP is unlikely to exert a sustained tonic influence on cardiovascular regulation in the rat.

Adenylyl Cyclases↗

Prolonged expression of Fos-related antigens, Jun B and TrkB in dopamine-denervated striatal neurons.

Previous studies have demonstrated that striatal dopamine-denervation leads to a long-term increase in Fos-related antigen(s) (FRA's) in striatal neurons. Because Fos-family proteins bind to DNA by dimerizing to Jun-family proteins we investigated the expression of Jun B protein 2 weeks and 1 month after striatal dopamine-denervation, produced by medial forebrain bundle transection. We also investigated the effects of this lesion on TrkB-immunoreactivity in the striatum. FRA's (as previously reported) and Jun B were expressed in striatal neurons following dopamine-denervation, and in addition, there was an increase in expression of TrkB in the striatum on the dopamine-denervated side. These results show that striatal dopamine depletion leads to a long-term up-regulation of FRA's and Jun B in the striatum, and this may be related to other biochemical changes previously reported to occur in striatal neurons (e.g.: D2-dopamine receptor up-regulation) after dopamine depletion. In addition, FRA and Jun B expression may induce increased production of TrkB after dopamine-denervation.

Animals↗

Parasympathetic denervation increases responses to VIP in isolated rat parotid acini.

Vasoactive intestinal peptide (VIP) is a putative neurotransmitter found in the salivary glands of many species, including the rat parotid gland. Parasympathetic denervation has been reported to deplete VIP in the rat parotid gland and to lead to supersensitivity to this peptide in vivo. We have compared the effects of VIP on acini isolated from parasympathetically denervated and unoperated parotid glands to examine possible supersensitivity to the peptide in vitro. VIP normally produced responses similar to those obtained with a low concentration of the beta adrenergic agonist isoproterenol (ISO), but strikingly different from the effects obtained with the muscarinic agonist carbachol (CARB). In parotid membrane preparations, VIP stimulated adenylate cyclase activity. Dissociated acini treated with VIP showed increases in cAMP accumulation and amylase release which were potentiated by forskolin and also by inhibition of phosphodiesterase. After parasympathetic denervation, maximal effects of VIP on adenylate cyclase, cAMP accumulation and amylase release in intact cells were increased two- to five-fold over contralateral control (or unoperated) parotid responses. The increase in adenylate cyclase-mediated responses after denervation was specific to VIP; there was no increased response nor increased sensitivity of any of these responses to ISO. Specific [125I]VIP binding to parotid acini increased two-fold per gland and three-fold per mg of protein after denervation; this probably explains the observed increases in the response to VIP.

4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone↗

Increased vascular sensitivity and connexin43 expression after sympathetic denervation.

OBJECTIVE: Following denervation, arteries demonstrate a heightened sensitivity to alpha-adrenergic agonists and increased oscillatory contractions that may partly result from increased gap junction expression. Hence, we wanted to study the effect of sympathetic denervation on connexin43 (Cx43) expression and agonist-induced contractility in the vascular smooth muscle (VSM). METHODS: Effects of denervation with reserpine (3 mg/kg/day, i.p.) or topical 5% phenol-glycerol on VSM contractions and expression of the gap junction Cx43 mRNA by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting for Cx43 protein were examined. Wistar-Kyoto (WKY) rat tail arteries were exposed to norepinephrine (NE) (10(-9)-10(-5) M). Reactivity was also examined in the carotid arteries and thoracic aortas from Cx43 heterozygote deficient (KO) mice. RESULTS: The concentration for NE-induced contraction was lower in reserpine- and phenol-treated vessels than controls (p<0.05). NE-induced oscillatory activity (OA) was seen in 5/5 reserpine- and 5/8 phenol-treated vessels vs. 0/12 controls (p<0.05). Spontaneous OA was observed more frequently in carotid and aortic rings from WT than Cx43 KO rings. Cumulative OA in response to alpha-adrenergic stimulation was significantly greater in WT carotid (429+/-101 vs. 128+/-7 mN s, p<0.05) and aortic rings (337+/-85 vs. 134+/-11 mN s, p<0.05) than in Cx43 KO rings. Following denervation, RT-PCR showed significantly increased levels of Cx43 mRNA (p<0.05). Western blot analysis revealed near doubling of Cx43 protein (p<0.05). CONCLUSION: We conclude that sympathetic denervation results in increased expression of Cx43, which in turn, contributes to increased spontaneous and agonist-induced OA in VSM.

Animals↗

Predicting acute denervation in carpal tunnel syndrome.

OBJECTIVE: To determine which nerve conduction parameters can predict the presence of acute denervation in carpal tunnel syndrome. SETTING: The electrodiagnostic laboratories of a hospital and a county hospital district. DESIGN: A retrospective review. PATIENTS: A total of 1,590 consecutive cases from January 1992 to June 1996, diagnosed as having median neuropathy at the wrist. MAIN OUTCOME MEASURES: Evidence of acute denervation on needle electromyography of the abductor pollicis brevis and its relationship to patient age, gender, and parameters obtained from nerve conduction studies, including median sensory latency and amplitude, and median motor latency and amplitude. RESULTS: Logistic regression analysis identified gender, median motor latency, and median motor amplitude (all p < or = .008) as contributing to the prediction of denervation. Needle examination of the cases with a median motor amplitude <7 mV detected 95.3% (141/148) of all cases with denervation and could have spared 52% (708/1,362) of the population from a needle examination of the abductor pollicis brevis. CONCLUSION: The median motor amplitude can predict the presence of acute denervation in the thenar muscles in median neuropathy at the wrist and possibly eliminate a painful needle examination of the median-innervated thenar muscles in over 50% of the cases.

Acute Disease↗

The glutamate and carbachol effects on the early post-denervation depolarization in rat diaphragm are directed towards furosemide-sensitive chloride transport.

The membrane potentials of denervated muscle fibres of the rat diaphragm kept in a tissue culture medium are depolarized by about 8-10 mV (10-12%) within 3 h after denervation. This early post-denervation depolarization (EPD) is substantially reduced (2-3 mV) when muscle strips are bathed with 1 mM L-glutamate (GLU) which is found in motor nerve endings, or with 5 x 10(-8) M carbachol (CCh), which mimics the effect of nonquantally released acetylcholine (ACh). The hyperpolarizing effects of GLU and CCh on EPD are not influenced by ouabain, an active sodium transport inhibitor, but are absent when Cl- transport is augmented by increased osmolarity (500 mosmol/l) produced by addition of sucrose or NaCl. The EPD and the effect of hyperosmolarity are effectively prevented by the Cl- transport inhibitor furosemide (1 x 10(-4) M) or by a chloride-free bathing medium. It is suggested that the post-denervation cessation of nonquantal ACh release, and probably also GLU release, from nerve endings leads to the activation of the furosemide-sensitive Cl- transport in the sarcolemma, which is responsible for the early post-denervation depolarization.

Animals↗

Denervation supersensitivity to phenylephrine in guinea-pig vas deferens in vivo and in vitro: functional studies on alpha1-adrenoceptors.

The objective was to estimate alterations in adrenergic receptor sites of guinea pig vas deferens, in vivo and in vitro, induced by chronic denervation. The denervation process induced an increased sensitivity (3-fold at the EC50 level) without alteration in the maximum response to phenylephrine in vitro. The sensitivity alteration was characterized by the decrease in the dissociation constant of phenylephrine for alpha-adrenoceptor [K(A): normal tissue 3.50 (0.75-16.21) x 10(-5) and denervated tissue 0.43 (0.11-1.67) x 10(-5) M, p < 0.05] without changing the dissociation constant of prazosin. A decrease in pD'2 value for phenylephrine-phenoxybenzamine, probably due to a qualitative rather than a quantitative alteration in the alpha-adrenoceptor, was also shown in vitro [pD'2: normal tissue (8.2776+/-0.0402) and denervated tissue (8.0051+/-0.0442), p < 0.05]. No change in sensitivity and maximum response to phenylephrine was observed in vivo after denervation, although an increased resistance of vas deferens to phenoxybenzamine blockade has been evidenced in this condition.

Adrenergic Agonists↗

Cholinergic denervation and sympathetic ingrowth result in persistent changes in hippocampal muscarinic receptors.

Our laboratory has been utilizing the model of hippocampal sympathetic ingrowth, which has been suggested to occur in Alzheimer's disease, to investigate the effects of cholinergic denervation and hippocampal rearrangements. After cholinergic denervation by medial septal lesions, peripheral sympathetic fibres originating from the superior cervical ganglia grow into the rat hippocampus. This hippocampal sympathetic ingrowth can be prevented by superior cervical ganglionectomy. We examined the long-term effects of these treatments on muscarinic receptors by comparing [3H]quinuclidinyl benzilate binding in rat dorsal hippocampus four and 12 weeks post lesion. Four groups of animals were employed, including controls (sham lesion+sham ganglionectomy), animals with ingrowth (medial septal lesion+ sham ganglionectomy), animals with cholinergic denervation alone (medial septal lesion+ ganglionectomy), and ganglionectomy alone (sham lesion+ganglionectomy) animals. In dorsal hippocampus four weeks post lesion, binding affinity was similar among all groups, while muscarinic receptor number was increased in ingrowth animals as compared to both the control (P<0.0002) and ganglionectomy animals (P<0.01). By 12 weeks, receptor affinity was significantly decreased in ingrowth (P<0.0001) and cholinergic denervation (P<0.0003) groups, and receptor number remained significantly elevated in ingrowth animals as compared to control (P<0.01), ganglionectomy (P<0.02) and cholinergic denervation (P<0.01) groups. The decrease in muscarinic receptor affinity may provide some insight into the ineffectiveness of cholinomimetic therapies in Alzheimer's disease, in that agonist efficacy would be reduced at the receptor.

Animals↗

Effects of myenteric denervation on gastric epithelial cells and gastric emptying.

The influence of myenteric denervation on gastric emptying and epithelial cell population was studied by chemical lesion of myenteric neurons of rat stomach with benzalkonium chloride. Three groups were evaluated: denervated, denervated with pyloroplasty, and control. After three months, the animals were submitted to functional (gastric emptying of liquids) and morphological (neuronal counting; mucosal area, height, and volume; mucous, chief, and parietal cell population; parietal cell area; and gastrin-G and somatostatin-D cell population) studies. After benzalkonium chloride treatment, there was significant delay in gastric emptying of liquids and an increase in mucosal area of all gastric portions, in antral mucosal volume, in parietal cell area, and gastrin-G cell and somatostatin-D cell populations. These changes did not occur when denervation was combined with pyloroplasty, indicating that after denervation, gastric distension and stasis are the major stimuli for parietal cell hypertrophy and G and D cell hyperplasia

Animals↗

Effect of denervation of pyloric antrum on the gastric secretory response to food.

The effect of selective denervation of the pyloric antrum on secretory response to three types of food was determined in 3 dogs with innervated and 4 with denervated stomach pouches. With the antrum in situ this procedure has surprisingly little effect on the total meal responses in innervated pouches, on the variation in response induced by altered buffering capacity of the meals, or on the effect of subsequent vagotomy. However, the antrum appears less sensitive to local stimuli after denervation, as evidenced by the reduced meal response from the denervated indicator pouch after denervation of the antrum.

Animals↗

Denervation retards but does not prevent toetip regeneration.

Toetips of mammals regrow after amputation by a process similar, but not identical, to that which occurs during regeneration of a newt limb. Nerve is needed as a mitotic stimulant for newt limb regeneration but the requirement for nerve during rodent digit-tip regeneration is not known. Nerve dependence in rats was tested by severing the sciatic nerve in one hindlimb, amputating digit-tips from the central digits of both hind feet, and comparing the amount of regrowth in innervated and denervated digits. Denervation delayed soft-tissue wound healing. However, denervation did not significantly affect bone regrowth when animals were examined at one month. Because we suspected delayed bone regrowth, we used a new method that we developed to follow bone growth at several time points in each animal. Termed visible bone fluorescence through nail, this technique used serial injections of fluorescent calcium-deposition markers and observation through the toenails to observe bone growth in living animals. Using this method it was possible to detect retarded bone regrowth in denervated digits. Thus, although denervation of rodent tips delayed both soft tissue healing and bone regrowth, it did not prevent ultimate restitution of the amputated part. This suggests that neurotrophic stimulation in the mammalian digit-tip is not identical to that documented during newt limb regeneration, and that growth stimulation may be provided by tissues other than nerve.

Amputation, Surgical↗

Sensory denervation modulates eIF-2 alpha kinase expression in the rabbit lacrimal gland.

PURPOSE: To investigate the hypothesis that sensory denervation of the rabbit lacrimal gland results in dysregulation of protein synthesis. We used differential display of mRNA to identify genes associated with protein synthesis and secretion that may be altered in this situation. METHODS: New Zealand white rabbits underwent unilateral sensory denervation by the ablation of the trigeminal ganglion. After 7 days, the denervated and contralateral control lacrimal glands were removed. The effects of denervation on gene expression were carried out using differential mRNA display. Northern and Western blot analyses were used to verify differential gene expression. RESULTS: Differential mRNA display identified the gene heme-regulated inhibitor eukaryotic initiation factor-2 alpha kinase (HRI eIF-2a kinase) in the lacrimal gland, the expression of which was reduced in the denervated lacrimal gland. The sequenced fragment from differential display showed 94% identity to rabbit HRI eIF-2a kinase. The decreased expression of HRI eIF-2a kinase was confirmed by Northern and Western blots, and measurement of HRI eIF-2a kinase phosphorylation activity in the lacrimal gland after ablation of sensory neurons showed that it was significantly decreased compared with that of normal and control lacrimal glands. CONCLUSIONS: The results suggest that loss of sensory innervation has a role in the lacrimal gland, contributing to the expression of HRI eIF-2a kinase, a pivotal negative regulator of protein synthesis. A reduction in control of protein synthesis may lead to the translation of repressed messages associated with cell stress responses.

Animals↗

Effects of nerve growth factor on the neurotization of denervated muscles.

Studies on surgical repair techniques of the peripheral nerve are still trying to improve the outcome. There are many studies on the effects of various neurotrophic factors on the transected peripheral nerve. Muscular neurotization, which is the direct implantation of the nerve to the target denervated skeletal muscle, is one of the techniques used when the primary repair of the peripheral nerves is not possible. The effects of nerve growth factor (NGF), which is one of the primary neurotrophic factors, on the reinnervation of denervated muscles by neurotization is investigated in this experimental study. The denervated soleus muscle was neurotized via peroneal nerve implantation (group 1), and NGF was administered to the neurotized muscle (group 2). All animals were evaluated at weeks 8, 10, and 12 using electromyography. Muscle contractility, muscle weight, and histological morphometric tests were performed at week 12. The experimental groups were compared with each other and normal control values. Electromyographically, group 2 (direct nerve implantation + NGF) demonstrated better reinnervation in all evaluations. The study of muscle weight showed that the muscle mass was 75% of the normal soleus muscle in group 1 and was 85% of the normal side in group 2 at the end of week 12. In group 1, the twitch force was 56% of the normal soleus muscle and was 71% in group 2. Tetanic force was 53% of the normal soleus muscle in group 1 and 68% in group 2. Histological morphometric studies revealed that there was a decrease in the density of the motor end plates in group 1, but there was no statistically significant difference between the normal soleus muscles and the NGF applied to group 2. The positive effects of NGF on the neurotization of denervated muscles seen in this study suggest that it may be useful for treating some difficult reconstructions caused by denervation.

Animals↗

Histological appearances of the nerves of the bladder in patients with denervation of the bladder after excision of the rectum.

Two groups of patients who developed denervation of the bladder after excision of the rectum for carcinoma have been studied by histological examination of bladder biopsies. The groups were compared with a group of control patients of a similar age. One group with denervation of the bladder was studied soon after (7 weeks) and the other group in the long term (10 months) after operation. Bladder biopsies were stained for acetylcholinesterase, catecholamines and connective tissue. Tissue was also processed for electron microscopy. In control patients, the ratio of cholinesterase positive nerves to the number of smooth muscle nuclei (24:100) was significantly greater than in patients with denervation of the bladder studied soon after operation (2.5: 100; P less than 0.01). Appearances consistent with degenerate nerve terminals were observed on electron microscopy in these patients. No increase in the density of adrenergic nerves was observed in either group of patients with denervation of the bladder. In the long term, a greater density of cholinergic innervation was noted compared with patients studied soon after operation (P less than 0.05). In addition, nerve terminals, similar in appearance to those of control patients, were observed on electron microscopy. The findings in patients with denervation of the bladder studied in the long term after operation are consistent with partial regeneration of autonomic nerves. They suggest that, whilst histological examination of bladder biopsies may be useful soon after excision of the rectum, they may be less sensitive in the long term.

Adrenergic Fibers↗

Contractile effects of various vasoactive agents in small rat portal veins and hepatic arteries and the influence of sympathetic denervation on the noradrenaline response.

Contractile responses were studied in isolated tubal segments of branches of the rat portal vein (luminal diameter approximately 300 microns) and hepatic artery (luminal diameter approximately 200 microns). Portal veins were approximately three times more sensitive to noradrenaline (NA) than hepatic arteries. 5-hydroxytryptamine contracted hepatic arteries concentration-dependently, whereas it produced only weak and inconsistent contractions in portal veins. Vasopressin effectively contracted hepatic arteries, whereas it had no effect on portal veins. Both vessel types responded to prostaglandin F2 alpha with contractions, although the drug potency was relatively low (EC50 greater than 10(-5) mol l-1). Histamine and carbachol failed to induce (hepatic arteries) or caused only weak (portal veins) contractions. Microsurgical hepatic hilar denervation reduced the catecholamine content of the parenchyma to less than or equal to 25% of controls. In both portal veins and hepatic arteries, the denervation procedure increased the NA sensitivity by factors of 3.1 and 2.0, respectively. In non-denervated livers, cocaine produced a similar increase of the NA sensitivity, whereas the drug had no significant effect in vessels from denervated animals. Thus, there was a marked difference between rat portal veins and hepatic arteries in their responsiveness to several contractile agents. Furthermore, the results of the present study indicate that the adrenergic nerves in both vessel types can be adequately removed by the microsurgical denervation procedure used.

Animals↗

Extension and magnitude of denervation in skeletal muscle from ageing mice.

In this work we hypothesized that denervation in flexor digitorum brevis (FDB) muscle from ageing mice is more extensive than predicted by standard functional and structural assays used in the past. In addition, we asked whether denervation is a fully or partially developed process. Despite the reported alteration in skeletal muscle innervation, the quantification of the extension and magnitude of denervation in ageing rodents has remained elusive. To address these two questions we utilized a combination of electrophysiological and immunohistochemical assays directed to detecting the expression of tetrodotoxin (TTX)-resistant sodium channels (Na(v)1.5) in FDB muscles from young-adult and senescent mice. Sodium current density measured with the macropatch cell-attached technique did not show significant differences between FDB fibres from young and old mice. The TTX dose-response curve, using the whole cell voltage-clamp technique, showed three populations of fibres in senescent mice, one similar to fibres from young mice (TTX sensitive), another one similar to fibres from experimentally denervated muscle (TTX resistant), and a third group intermediate between these two. Partially and fully denervated fibres added up to approximately 50% of the total number of fibres tested, a number that concurs with the percentage of fibres positive for the Na(v)1.5 channel by specific immunostaining.

Aging↗