Rabbit Vagus Research Articles

Partial Recovery of Respiratory Function and Diaphragm Reinnervation following Unilateral Vagus Nerve to Phrenic Nerve Anastomosis in Rabbits

Respiratory dysfunction is the leading cause of mortality following upper cervical spinal cord injury (SCI). Reinnervation of the paralyzed diaphragm via an anastomosis between phrenic nerve and a donor nerve is a potential strategy to mitigate ventilatory deficits. In this study, anastomosis of vagus nerve (VN) to phrenic nerve (PN) in rabbits was performed to assess the potential capacity of the VN to compensate for lost PN inputs. At first, we compared spontaneous discharge pattern, nerve thickness and number of motor fibers between these nerves. The PN exhibited a highly rhythmic discharge while the VN exhibited a variable frequency discharge pattern. The rabbit VN had fewer motor axons (105.3±12.1 vs. 268.1±15.4). Nerve conduction and respiratory function were measured 20 weeks after left PN transection with or without left VN-PN anastomosis. Compared to rabbits subjected to unilateral phrenicotomy without VN-PN anastomosis, diaphragm muscle action potential (AP) amplitude was improved by 292%, distal latency by 695%, peak inspiratory flow (PIF) by 22.6%, peak expiratory flow (PRF) by 36.4%, and tidal volume by 21.8% in the anastomosis group. However, PIF recovery was only 28.0%, PEF 28.2%, and tidal volume 31.2% of Control. Our results suggested that VN-PN anastomosis is a promising therapeutic strategy for partial restoration of diaphragm reinnervation, but further modification and improvements are necessary to realize the full potential of this technique.

Zacopride, a potent 5-HT3 antagonist.

The substituted benzamide derivative zacopride was found to antagonize competitively the effects of 5-hydroxytryptamine (5-HT) on the guinea-pig ileum, the rabbit vagus nerve and the von Bezold Jarisch reflex in the rat. The potency of zacopride was comparable with that of ICS 205-930 and it is concluded that zacopride possesses 5-HT3 receptor antagonizing properties.

Letter: The effect of local anaesthetics on the incorporation of 32P into the phosphoinositides of rabbit vagus nerve.

Letter: The effect of local anaesthetics on the incorporation of 32P into the phosphoinositides of rabbit vagus nerve.

Positively Active

The action of local anesthetics, containing a tertiary nitrogen, on mammalian nonmyelinated fibers of the rabbit's vagus nerve has been analyzed to determine whether the uncharged or the positively charged form of these compounds is responsible for their ability to block impulse conduction. The compounds studied were dibucaine, tetracaine, chlorpromazine, imipramine, and procaine. Impulse conduction was restored, in fibers in which it had been blocked by pretreatment with a local anesthetic, by increasing the pH of the perfusing solution from approximately 7.0 to 9.5; block was rapidly reestablished when the fibers were again perfused with the solution of pH approximately 7.0. From the way in which the size of the action potential varied with pH in nerve fibers pretreated with a local anesthetic. it has been concluded that the active form of the local anesthetic is the cation.

Axon–glia interactions modulate axonal excitability in mammalian unmyelinated nerves

A period of electrical activity in unmyelinated nerve fibers is followed by a post-tetanic hyperpolarization (PTH), generated by the hyperactivity of the electrogenic Na +–K + pump. In order to protect the membrane potential against these strong hyperpolarizations, different types of axonal inward currents are activated during the PTH. We investigated in the rabbit vagus nerve one of these currents, which was activated by carbamylcholine (CCh). We observed that the effect of CCh on the PTH amplitude could be blocked or reversed with scopolamine. Moreover, the PTH amplitude increased when scopolamine alone was added to the perfusate, indicating that an endogenous muscarinic agonist was liberated in the preparation during the period of electrical activity. This CCh-activated current was TEA but not Ba 2+ or Cs + sensitive. It has been shown previously that muscarinic acetylcholine receptors (mAChRs) in the rabbit vagus nerve are located on the axonal but not glial membrane and that Schwann cells express several types of purinergic receptors, which activation evoke Ca 2+ transients in Schwann cells. We hypothesise that during electrical activity axons release a transmitter, presumably ATP. This transmitter evoke in the neighbouring Schwann cells a Ca 2+-dependent liberation of a endogenous muscarinic agonist, which in turn activates a TEA-sensitive inward current in axons. We suggest that the major purpose of this mechanism is the control of the membrane potential during and after a period of intense electrical activity when the Na +–K + pump generates a robust PTH.

Inhibition of the I(h) current in isolated peripheral nerve: a novel mode of peripheral antinociception?

Although the alpha2-adrenergic agonist clonidine has been shown to promote peripheral antinociception, its mechanism of action has not yet been clearly elucidated. By the use of the sucrose-gap method, we have shown that in C fibers of the rabbit vagus nerve, clonidine at micromolar concentrations enhances activity-dependent hyperpolarizations generated by the Na+-K+ pump during and after repetitive stimulation. Similar results were obtained with 10 microM of ZD 7288, a specific blocker of the hyperpolarization-activated cation current (I(h)) and with 2 mM of Ba2+ that blocks the inwardly rectifying potassium current (I(KIR)). Furthermore, clonidine had no added effect on the ZD 7288-induced response, whereas it produced a marked enhancement of Ba2+induced response. From these results, it can be concluded that clonidine enhances activity-dependent hyperpolarization by inhibiting the current I(h). We propose that clonidine, by increasing the threshold for initiating the action potential, induces a slowing or block of conduction and that this mechanism is the origin of the clonidine-induced antinociception. Finally, this study suggests a novel role for inwardly rectifying hyperpolarization-activated conductances in peripherally mediated antinociception.

Inhibition of the Ih current in isolated peripheral nerve: A novel mode of peripheral antinociception?

Although the alpha2-adrenergic agonist clonidine has been shown to promote peripheral antinociception, its mechanism of action has not yet been clearly elucidated. By the use of the sucrose-gap method, we have shown that in C fibers of the rabbit vagus nerve, clonidine at micromolar concentrations enhances activity-dependent hyperpolarizations generated by the Na+-K+ pump during and after repetitive stimulation. Similar results were obtained with 10 microM of ZD 7288, a specific blocker of the hyperpolarization-activated cation current (I(h)) and with 2 mM of Ba2+ that blocks the inwardly rectifying potassium current (I(KIR)). Furthermore, clonidine had no added effect on the ZD 7288-induced response, whereas it produced a marked enhancement of Ba2+induced response. From these results, it can be concluded that clonidine enhances activity-dependent hyperpolarization by inhibiting the current I(h). We propose that clonidine, by increasing the threshold for initiating the action potential, induces a slowing or block of conduction and that this mechanism is the origin of the clonidine-induced antinociception. Finally, this study suggests a novel role for inwardly rectifying hyperpolarization-activated conductances in peripherally mediated antinociception.

Mechanism of antinociceptive action of clonidine in nonmyelinated nerve fibres

Despite a large body of clinical evidence in favour of a local anesthetic effect of clonidine, the underlying mechanism has not yet been elucidated. In this study we have used the sucrose-gap method to measure the effects of clonidine on the electrophysiological properties of nonmyelinated nerve fibers in the rabbit vagus nerve. The results showed that clonidine enhanced the hyperpolarizing and reduced the depolarizing afterpotential that follow compound action potentials during electrical activity. We showed that summation of these afterpotentials shifts the membrane potential toward more negative values, thus creating a region of low safety conduction, where the local circuit currents might fail to depolarize the axonal membrane to the threshold value needed to open voltage-dependent Na + channels. Yohimbine did not reverse the inhibitory effects of clonidine on impulse propagation, indicating that the observed effects of clonidine relies on mechanisms not mediated by α 2-adrenoceptors.

Nitric oxide synthase in damaged sensory neuron

NADPH diaphorase colocalized with nitric oxide synthase was studied in damaged protoneurons of the nodose ganglion of rabbit vagus nerve. The number and activity of nitric oxide synthase-expressing neurons increased. Nitric oxide is involved in neuron remodeling.

The Neurotoxicity of Drugs Given Intrathecally (Spinal)

The Neurotoxicity of Drugs Given Intrathecally (Spinal)

Lactate is released and taken up by isolated rabbit vagus nerve during aerobic metabolism.

To determine if lactate is produced during aerobic metabolism in peripheral nerve, we incubated pieces of rabbit vagus nerve in oxygenated solution containing D-[U-14C]glucose while stimulating electrically. After 30 min, nearly all the radioactivity in metabolites in the nerve was in lactate, glucose 6-phosphate, glutamate, and aspartate. Much lactate was released to the bath: 8.2 pmol (microg dry wt)(-1) from the exogenous glucose and 14.2 pmol (microg dry wt)(-1) from endogenous substrates. Lactate release was not increased when bath PO2 was decreased, indicating that it did not come from anoxic tissue. When the bath contained [U-14C]lactate at a total concentration of 2.13 mM and 1 mM glucose, 14C was incorporated in CO2 and glutamate. The initial rate of formation of CO2 from bath lactate was more rapid than its formation from bath glucose. The results are most readily explained by the hypothesis that has been proposed for brain tissue in which glial cells supply lactate to neurons.

Axonal and glial currents activated during the post-tetanic hyperpolarization in non-myelinated nerve.

Changes in membrane potential and potassium concentration in the extracellular space ([K+]e) of rabbit vagus nerve were measured simultaneously during electrical activity and during the period of recovery using a modified sucrose-gap method and potassium-sensitive microelectrodes. After stimulation for 15 s at 15 Hz the main activity-induced increase in [K+]e reached 16.9 mM. This increase in [K+]e was paralleled by a depolarization of the preparation. The period of activity was followed by a post-tetanic hyperpolarization (PTH) lasting tens of seconds, generated by the axonal electrogenic Na+-K+ pump and to a lesser extent by the pump of the surrounding Schwann cells. The amplitude of the PTH dramatically increased in experiments in which inward currents were blocked by removal of Cl– or after application of Cs+ or Ba2+, indicating that under normal conditions the current generated by the Na+-K+ pump is strongly short-circuited. A pharmacological and kinetic study showed that these currents are: (1) the hyperpolarization-activated current I h, and (2) the inwardly rectifying I KIR current. The results show that the latter originates from Schwann cells. Our data indicate that in non-myelinated nerves there is a subtle association of inward ionic channels which (1) helps the cell to maintain an optimal membrane potential after a period of activity, and (2) contributes to the removal of excess K+ from the extracellular space.

Neurophysiologic actions and neurological consequences of veratridine on the rat sciatic nerve.

The quest for a drug that would provide analgesia with minimal motor deficiency, through the selective inhibition of impulses in small-diameter fibers, was brightened by a previous report of veratridine's C-fiber-selective actions on the isolated rabbit vagus nerve. The goal of the present research was to demonstrate the same actions on rat sciatic nerve in vitro and to observe the functionally differential blockade in the rat in vivo. Sciatic nerves were removed from rats, mounted in a recording chamber, wherein a 1-cm length of the ensheathed nerve was superfused with the plant alkaloid veratridine (2 microM) in bicarbonate-buffered Liley's solution, and the compound action potential (CAP) was stimulated supramaximally to give A- and C-fiber elevations. Onset, steady-state, and recovery from veratridine effects were assayed for a range of stimulus frequencies. Open-field behavior and quantitative neurological assessments of proprioception, motor function, and nociception were tested in 15 trained rats after injection near the sciatic nerve of 0.1 ml veratridine at 0.5, 0.7, and 1.0 mM each plus epinephrine (1:200,000). Veratridine inhibited the C-fiber component of the CAP in a frequency-dependent manner. At 0.1 Hz the CAP was 65% of the control amplitude, 50% at 0.5 Hz, and 40% at 5 Hz. A-fiber elevations were unattenuated at stimulus frequencies as high as 50 Hz. Steady-state inhibition was reached 5 min after drug administration, and recovery from the effects was 30% complete by 15 min of drug washout. Proprioception, measured as a "hopping" or "placing" reaction, was inhibited dose dependently by maximum degree and for durations of, respectively, 0.5 mM, 61%, 180 min; 0.7 mM, 100%, 360 min; and 1 mM, 100%, 420 min. Extensor postural thrust, as a measure of motor function, was inhibited by and for 0.5 mM, 77%, 240 min; 0.7 mM, 99%, 390 min; and 1 mM, 100%, 420 min. Analgesia, as a prolonged withdrawal latency to a noxious thermal stimulus, had the following profile: 0.5 mM, 10%, 30 min; 0.7 mM, 52%, 150 min; and 1 mM, 66%, 150 min. Despite the fact that veratridine gave a C-fiber preferential blockade in the isolated sciatic nerve, heightened analgesia over motor block was not achieved in vivo. Indeed, just the opposite occurred. If preferential C-fiber blockade also occurs in vivo, then its traditionally expected correlation with analgesia must be re-examined.

Spin-Lattice Relaxation Times of Water in Polarized and Depolarized Rabbit Vagus Nerves

Spin-lattice relaxation times (T 1) of intracellular water of the nonmyelinated fibers of rabbit cervical vagus nerve were measured using a paramagnetic shift reagent, s-FDF. Spin-lattice relaxation decay curves were composed of the fast (T 1,F) and slow (T 1,S) relaxation components. The mean values of T 1,F and T 1,S in the polarized nerve fibers at 25°C were 0.12 ± 0.04 and 0.61 ± 0.13 sec., respectively, and their fractions were 0.71 ± 0.07 and 0.29 ± 0.07, respectively (n = 21). On the other hand, those values in the depolarized nerve fibers were 0.16 ± 0.01 sec., 0.77 ± 0.17 sec., 0.77 ± 0.10 and 0.23 ± 0.10, respectively (n = 15). T 1,F and T 1,S for the depolarized nerve fibers were significantly elongated (P < 0.01).

Uptake of potassium by nonmyelinating Schwann cells induced by axonal activity.

1. The electrophysiological properties of the rabbit vagus nerve (membrane potential, compound action potentials, and afterpotentials) and potassium accumulation were measured simultaneously during low-frequency stimulation (LFS) (0.5 and 1 Hz) by using a modified sucrose-gap apparatus and potassium-sensitive microelectrodes (KSM). 2. During LFS at 0.5 and 1 Hz, the concentration of K+ in the extracellular space ([K+]c) increased in approximately 30 s to a maximal level that was 0.6 and 1.5 mM, respectively, above the resting concentration. Concomitantly the preparation developed an ouabain-sensitive hyperpolarization. 3. The compound action potential (CAP) was followed by a fast hyperpolarizing afterpotential (fHAP), a depolarizing afterpotential (DAP), and a slow hyperpolarizing afterpotential (sHAP). During LFS the characteristics of all these afterpotentials were profoundly modified. In parallel to the increase in [K+]e, the fHAP was decreased and the amplitude of the DAP was dramatically enhanced. Furthermore, the sHAP which had a duration of < 1 s when it followed a single CAP, turned into a ouabain-sensitive hyperpolarization (indicating that it was generated by the electrogenic Na(+)-K+ pump) that lasted several minutes. 4. The application of external Ba2+ produced a hyperpolarizing sag on the sHAP following a single isolated CAP. During LFS, Ba2+ enhanced the build-up of the DAP, raised the maximal level of [K+]e, and increased the activity-induced ouabain-sensitive hyperpolarization. 5. The increase by Ba2+ of the activity-induced hyperpolarization shifted the spikes from both myelinated and nonmyelinated fibers toward a more negative potential but did not increase their amplitude, indicating that this Ba(2+)-induced hyperpolarization originated from an extra-axonal source, presumably the Schwann cells. 6. It is proposed that the electrogenic activity of the Na(+)-K+ pump was enhanced in Schwann cells situated near active axons. This hyperpolarization was, however, not recorded in normal conditions because it was fully short-circuited by a K+ influx through Ba(2+)-sensitive channels. 7. Our results lead to the hypothesis that the Na(+)-K+ pump of the nonmyelinating Schwann cells is important in the mechanisms maintaining the homeostasis of K+ in the axonal microenvironment. They show that the Na(+)-K+ pump contributes to the K+ buffering not only by actively pumping K+ but also by generating a hyperpolarization that drives a passive K+ influx through Ba(2+)-sensitive K+ channels.

Changes in slow axonal transport of tubulin induced by local application of colchicine to rabbit vagus nerve.

The biochemical and morphological responses of the rabbit vagus nerve to local application of colchicine and to nerve crush were investigated. Fourteen days after the cervical vagus nerve had been crushed or subjected to local application of colchicine for 2 h, nodose ganglia of anaesthetized rabbits were either injected with [35S]methionine or [3H]leucine for studies of slow and fast axonal transport, respectively, or prepared for light microscopical examination. The radio-labelled proteins of the faster of the two slow transport groups (SCb; 25-30 mm day-1) were separated by one- or two-dimensional polyacrylamide gel electrophoresis and both radio-labelled tubulin and actin were quantified by densitometry from resulting fluorographs of gels. A relative increase in radio-labelled tubulin was found in SCb in the crushed and colchicine-treated nerves; this increase persisted for up to 50 days after nerve crush. Morphological changes in nerve cell bodies induced by colchicine were similar, but smaller in magnitude than those in crushed nerves. It is concluded that a temporary arrest of axonal transport produced by colchicine can lead to a redistribution of tubulin transport comparable with that found in regenerating nerve.

Isolation and characterization of TsTX-V, a new neurotoxin from tityus serrulatus scorpion venom which delays the inactivation of NA + channels

TsTX-V, a new neurotoxin from Tityus serrulatus scorpion venom able to induce a prolongation of the inactivation of Na + channels, ahs been purified to homogeneity. The venom was chromatographed on CM-cellulose-52 and 13 fractions were first collected. A subsequent stepwise elution chromatography of fraction XI afforded, among other toxins, highly purified TsTx-V, which showed a single band by PAGE, SDS-PAGE or isoelectric focusing, a distinctive amino acid composition, mol. wt. = 7230, p I = 8.0 and i.v. LD 50 = 94 ± 7 μg/kg in mice. TsTX-V induced a long lasting hypertension in anesthetized rats and prolonged the action potential of the B fibers of the rabbit vagus nerve at 0.03 μg/ml. At 0.3 μg/ml and higher concentrations it caused also a nerve depolarization. These effects on nerve membranes were irreversible and could be suppressed by tetrodotoxin (200 –500 nM). Nerve fibers depolarized by high extracellular K +(15–30mM) concentrations still displayed long duration action potentials after TsTX-V treatment. It is suggested that TsTX-V blocks the Na + channel inactivation system probably as an α-toxin.

Hyperpolarizing afterpotentials in C fibers and local anesthetic effects of clonidine and lidocaine.

Effects of clonidine and lidocaine on the hyperpolarizing after-potential (HAP) and frequency-dependent block in C fibers were examined on desheathed rabbit vagus nerves, using the sucrose gap technique. A single action potential (AP) was followed by a fast and a slow HAP. Clonidine, at concentrations from 0.05 to 50 mumol/l, decreased the fast HAP, while the AP amplitude was unchanged. At a 500 mumol/l concentration of clonidine, the fast HAP amplitude was similar to control, the slow HAP was increased, and the AP amplitude decreased. Lidocaine at 500 mumol/l delayed and broadened the HAP, making a distinction between fast and slow HAP impossible, and decreased and delayed the AP amplitude. In the presence of lidocaine (500 mumol/l), clonidine at concentrations from 0.05 to 500 mumol/l decreased the HAP amplitude, without modifying the lidocaine-induced shape of the HAP. The modifications of the HAP, however, do not contribute to the local anesthetic effects of clonidine, as the addition of clonidine (0.5 and 500 mumol/l) to Locke or lidocaine (500 mumol/l) solution does not enhance the frequency-dependent block (3 and 10 Hz) observed with either Locke or lidocaine solution alone.

Axonal transport and morphological changes following nerve compression An experimental study in the rabbit vagus nerve

Axonal transport and morphological changes were studied in the rabbit vagus nerve after the nerves had been subjected to compression at either 0, 50 or 200 mmHg for two hours. Slow axonally transported proteins, tubulin and actin, were radiolabelled with 35S-methionine two, seven or 14 days after the injury and the distribution of radiolabelled tubulin and actin within component b of slow transport was measured three days later by densitometric analysis of fluorographs of polyacrylamide gel. No significant differences were found in the distribution of tubulin two (50 and 200 mmHg) or seven (200 mmHg) days after injury, but at 14 days (200 mmHg) there was significantly increased radiolabelling of tubulin relative to actin in the nerve 60 to 70 mm from the nodose ganglion. Morphometric measurements of the nerve cell bodies two days after the compression injury at 200 mmHg revealed no significant changes. Previous work has shown that morphological changes, similar to those found after axotomy, were present in nerve cell bodies seven days after a compression injury. This, taken together with the present results, indicates that compression can induce both morphological and biochemical changes in the neurone. The altered axonal transport of tubulin associated with nerve injury follows a slower time course and does not precede the morphological changes. The findings may be of relevance when discussing the double crush syndrome.

The effect of nedocromil sodium on the isolated rabbit vagus nerve

Nedocromil sodium depolarized the isolated rabbit vagus nerve. The depolarization was blocked by DIDS (4,4'-diisothiocyanostilbcnc-2, 2'-disulphonic acid and did not occur when the nerve was bathed in solutions low in chloride. After depolarization the nerve was refractory to nedocromil sodium for an hour. It is suggested that nedocromil sodium can affect a chloride chanrcl.