Effects of long-term phasic electrical stimulation on denervated soleus muscle: guinea-pig contrasted with rat

Guinea-pig soleus muscles were denervated and electrically stimulated for periods of 43 to 66 days. Stimuli were in 1 s bursts of 40 Hz pulses, repeated every 5 min. Other guinea-pigs were denervated for 82 days without stimulation and, in a third group, the soleus muscle was necrotized and allowed to regenerate without reinnervation for 13-15 days. Isometric and isotonic recordings were made in vivo. Denervated guinea-pig muscles were embedded in epoxy resin for light and electron microscopy. Chronic stimulation of denervated guinea-pig soleus had no effects on the prolonged twitch or on reduced maximal shortening velocity, maximal rate of rise of tension and tetanic force. This contrasts with the slow-to-fast conversion produced by denervation and denervation-stimulation of rat soleus. Loss of force was much greater in rat than guinea-pig after denervation, and chronic stimulation increased force in rat to the same level as in guinea-pig after denervation (with or without stimulation). Eighty-day denervated guinea-pig soleus did not reveal those morphological signs of fibre breakdown and regeneration which are prominent in denervated rat soleus muscles. Those changes in rat resembled aneurally regenerated muscles in several aspects, especially the increased incidence of fibres with internal myo-nuclei which did not appear in guinea-pig soleus after denervation. Aneurally regenerated guinea-pig soleus became fast like aneurally regenerated rat muscle. Our data are compatible with the hypothesis that slow-to-fast transformation of denervated rat soleus is not directly brought about by chronic stimulation but by de-novo formation of fast-contracting regenerated fibres. The persistence of fibrillation in guinea-pig but not rat after denervation may account for the species difference.     

Electrophysiological and pharmacological responses of chronically denervated lower esophageal sphincter of the opossum

BACKGROUND & AIMS: Achalasia is characterized by loss of myenteric neurons and incomplete relaxation of the lower esophageal sphincter (LES). The aim of this study was to develop an achalasia model in the opossum using the surfactant benzyldimethyltetradecylammonium chloride (BAC). This study further characterizes the achalasia model. METHODS: BAC or saline was injected circumferentially into the LES of 14 adult opossums. Eight months after injection, manometry, isolated muscle bath studies, electrical field stimulation, and histochemical analysis were performed. RESULTS: Manometrically, the LES of BAC-treated opossums showed higher pressures (38.7 +/- 12 mm Hg vs. 17 +/- 3.0 mm Hg) and reduced esophageal body contraction amplitudes (4.2 +/- 3 mm Hg vs. 27.4 +/- 12 mm Hg). Isolated muscle strips challenged with carbachol and sodium nitroprusside contracted and relaxed similarly to controls. Electrical field stimulation failed to induce relaxation in BAC-treated tissue but did induce contraction. Contractile responses were markedly reduced by tetrodotoxin and atropine in BAC-treated animals and controls. An altered nitric oxide system was shown by the lack of response to L-arginine and N omega-nitro-L-arginine. Histology showed loss of myenteric neurons and increased cholinergic nerve bundles. CONCLUSIONS: Loss of NO inhibitory myenteric neurons markedly reduces the relaxation of the LES, and histology and pharmacological responses suggest a proliferation of cholinergic nerves into the LES contributing to the static elevated pressures of the amyenteric LES.     

The effect of denervated muscle and Schwann cells on axon collateral sprouting

The effects of denervated muscle and Schwann cells on collateral sprouting from peripheral nerve were studied in the peroneal and tibial nerves of 48 Sprague-Dawley rats. Three groups were prepared. In group MSW (muscle-Schwann cell-window), the peroneal nerves were transected 3 mm below the sciatic bifurcation. The proximal stumps were sealed in a blocked tube to prevent regeneration and the distal stumps were implanted into denervated muscle cells that were wrapped around the ipsilateral tibial nerve, which had a window of perineurium resected. Schwann cells from the ipsilateral sural nerve were implanted into the muscle. Group MS (muscle-Schwann cell) was similar to group MSW, except that the tibial nerve perineurium was kept intact. In group MW (muscle-window), the muscle was prepared without Schwann cells and the tibial nerve perineurium was windowed. S-100 immunostain was used to identify the Schwann cells surviving 1 week after transplantation. After 16 weeks of regeneration, horseradish peroxidase tracer was used to label motor neurons and sensory neurons reinnervating the peroneal nerve. Myelinated axons of the reinnervated peroneal nerves were quantified with the Bioquant OS/2 computer system (R&M Biometrics, Nashville, TN). A mean of 169 motor neurons in group MSW, 64 in group MW, and 26 in group MS reinnervated the peroneal nerve. In the dorsal root ganglion, the mean number of labeled sensory neurons was 1,283 in group MSW, 947 in group MS, and 615 in group MW. The mean number of myelinated axons in the reinnervated peroneal nerve was 1,659 in group MSW, 359 in group MS, and 348 in group MW. Reinnervated anterolateral compartment muscles in group MSW were significantly heavier than those in group MS or MW. This study demonstrates that the transplantation of denervated muscle and Schwann cells promotes motor and sensory nerve collateral sprouting through a perineurial window.     

Electron microscopic study of long-term denervated rat skeletal muscle

This article reports a secondary analysis of past therapy outcome meta-analysis. Fifteen meta-analysis provided effect sizes from 56 studies in previous reviews that met 1 of 3 increasingly stringent levels of criteria for clinical representativeness. The effect sizes were synthesized and compared with results from the original meta-analyses. Effect sizes from more clinically representative studies are the same size at all 3 criteria levels as in past meta-analyses. Almost no studies exist that meet the most stringent level of criteria. Results are interpreted cautiously because of controversy about what criteria best capture the notion of clinical representativeness, because so few experiments have tested therapy in clinical conditions, and because other models for exploring the generalizability of therapy outcome research to clinical conditions might yield different results.     

Immunolocalization of leukemia inhibitory factor in normal and denervated human muscle

The cytokine leukemia inhibitory factor (LIF) stimulates myoblast proliferation in vitro and vivo and is neurotrophic for motor neurons. In experimentally reinnervated muscle, exogenous LIF application increases muscle mass through myofiber hypertrophy. The goal of this study was to evaluate possible sources of endogenous LIF in human muscle, and whether LIF immunoreactivity (-IR) was detectable in specific myofiber types and/or re-expressed in human denervated muscle. Our study shows that LIF-IR is constitutively detectable in type I myofibers of normal human muscle. In acute and chronically denervated and reinnervated human muscle, LIF-IR is found in all type I myofibers and in addition in some atrophic and almost all angulated atrophic type II myofibers.     

The mechanism of the excitatory action of catecholamines and histamine on the smooth muscle of guinea-pig ureter

1. The ionic mechanism of the excitatory action of catecholamines and histamine on the smooth muscle cells of guinea-pig ureter was studied with the double sucrose-gap method. 2. In normal conditions adrenaline and noradrenaline in a concentration of 10(-5) g/ml., and histamine in a concentration of 10(-6) g/ml., prolonged the duration of the plateau of the action potential and increased the amplitude and duration of the phasic contraction. Sometimes these changes were accompanied by a slight depolarization of the muscle membrane and by a small increase (with noradrenaline) or decrease (with histamine) of the membrane resistance. The amplitude and duration of the fast spike component of the action potential were not changed. 3. Isoprenaline in a concentration of 10(-5) g/ml. either caused no change or it decreased the duration of the plateau, reduced the amplitude of contractions and reduced excitability. 4. Tetraethyl ammonium (TEA; 5 mM), which blocks the delayed outward K current, did not prevent the increase in the duration of the plateau nor the increase of the amplitude and duration of the contractions by noradrenaline and histamine. 5. In Na-free or in K-free solution or in the presence of ouabain, i.e. in conditions in which the Na-gradient across the membrane was reduced, noradrenaline and histamine were unable to increase the duration of the plateau and the amplitude and duration of the contraction. 6. In the presence of Mn2+ (2 mM) which suppressed the spike component of tha action potential and the phasic contraction, theeffects of noradrenaline and histamine were almost abolished. 7. The results suggest a dual ionic mechanism of the alpha-action of catecholamines and of the action of histamine on the smooth muscle of ureter: (1) these drugs affect the passive ionic permeability of the membrane in a manner that results in depolarization; (2) they specifically activate the potential-dependent conductance of the slow Na channels, thereby increasing the plateau duration. The increased amplitude and duration of the contraction is the result of their primary effect on the plateau of the action potential.     

Comparison of muscle mass preservation in denervated muscle and transplanted muscle flaps after motor and sensory reinnervation and neurotization

The gracilis muscle model was used either as a denervated muscle in situ or as a transplanted flap in 273 rats to compare the trophic effects of muscle reinnervation and neurotization using sensory and motor nerves. The average gracilis muscle flap weighed 626 +/- 94 mg at the time of the initial procedure. Experimental muscles were examined 6 months following the procedure. In denervated, nontransplanted muscles, both motor nerve reinnervation and neurotization resulted in significantly preserved muscle mass, averaging 570 +/- 69 and 521 +/- 116 mg, respectively, compared with the denervated control average of 178 +/- 22 mg (p < 0.05). Sensory nerve reinnervation and neurotization produced much smaller trophic effects (p > 0.05). In transplanted gracilis free flaps, however, only direct reinnervation with motor or sensory nerves resulted in improved bulk preservation, with average weights of 313 +/- 83 and 327 +/- 91 mg compared with the control average of 201 +/- 76 mg (p < 0.05). Neither sensory nor motor neurotization was significantly effective in the free-flap model (p > 0.05). These data suggest that transplantation may alter the response of muscle to reinnervation.     

Measurement of intercellular electrical coupling in guinea-pig detrusor smooth muscle

Ropivacaine, a new long-acting local anesthetic, is currently being investigated for the treatment of ulcerative colitis. In view of the increased incidence of dysplasia and neoplasia associated with ulcerative colitis, it is important that the medical treatment of these patients does not stimulate cell proliferation further. This study was performed to evaluate the effect of ropivacaine on the proliferation of human colon adenocarcinoma cells (HT-29 and Caco-2) in vitro. A serum-induced proliferation assay of human colon adenocarcinoma cells was used. Ropivacaine inhibited the growth of HT-29 and Caco-2 cells in a dose-dependent manner. Fifty percent inhibition of growth was found at a ropivacaine concentration of 250 microM when the HT-29 cells were cultured in 1% fetal calf serum and of 550 microM when the HT-29 cells were cultured in 10% serum. The effective concentrations are within the range of the therapeutic concentrations obtained in the colon of patients treated rectally with ropivacaine. Lidocaine, hydrocortisone, and 5-aminosalicylic acid were found to be less potent than ropivacaine in inhibiting proliferation. Ropivacaine caused a dose-dependent membrane depolarization that appeared to correlate with the inhibited cell proliferation, whereas the effect was not related to inhibition of leukotriene B4 or prostaglandin E2. In conclusion, the antiproliferative activity of ropivacaine, combined with previously reported anti-inflammatory activities, makes this drug an interesting new alternative for the local treatment of ulcerative colitis.     

Muscarinic receptor subtypes controlling the cationic current in guinea-pig ileal smooth muscle

1. The effects of muscarinic antagonists on cationic current evoked by activating muscarinic receptors with the stable agonist carbachol were studied by use of patch-clamp recording techniques in guinea-pig single ileal smooth muscle cells. 2. Ascending concentrations of carbachol (3-300 microM) activated the cationic conductance in a concentration-dependent manner with conductance at a maximally effective carbachol concentration (Gmax) of 27.4+/-1.4 nS and a mean -log EC50 of 5.12+/-0.03 (mean+/-s.e.mean) (n=114). 3. Muscarinic antagonists with higher affinity for the M2 receptor, methoctramine, himbacine and tripitramine, produced a parallel shift of the carbachol concentration-effect curve to the right in a concentration-dependent manner with pA2 values of 8.1, 8.0 and 9.1, respectively. 4. All M3 selective muscarinic antagonists tested, 4-DAMP, p-F-HHSiD and zamifenacin, reduced the maximal response in a concentration-dependent and non-competitive manner. This effect could be observed even at concentrations which did not produce any increase in the EC50 for carbachol. At higher concentrations M3 antagonists shifted the agonist curve to the right, increasing the EC50, and depressed the maximum conductance response. Atropine, a non-selective antagonist, produced both reduction in Gmax (M3 effect) and significant increase in the EC50 (M2 effect) in the same concentration range. 5. The depression of the conductance by 4-DAMP, zamifenacin and atropine could not be explained by channel block as cationic current evoked by adding GTPgammaS to the pipette (without application of carbachol) was unaffected. 6. The results support the hypothesis that carbachol activates M2 muscarinic receptors so initiating the opening of cationic channels which cause depolarization; this effect is potentiated by an unknown mechanism when carbachol activates M3 receptors. As an increasing fraction of M3 receptors are blocked by an antagonist, the effects on cationic current of an increasing proportion of activated M2 receptors are disabled.     

Effects of excess K+ on carbachol-induced contractions in the guinea-pig tracheal muscle

1. In smooth muscles isolated from the guinea-pig trachea, the effects of dihydropyridines, nifedipine and nicardipine on contractions produced by carbachol (Cch) were studied in normal (6 mM) and excess K+ concentration (60 mM). The tonic contraction produced by 1 microM Cch was highly dependent on the external Ca2+ concentration ([Ca2+]0) and was not significantly affected by cyclopiazonic acid or thapsigargin, Ca2+ uptake inhibitor. 2. [Ca2+]0-tension curves were steeper in the presence of 1 microM Cch (the Hill coefficient: 2.5) than in the presence of 60 mM K+ (Hill coefficient: 1.6) and their ED50 of Ca2+ was 0.16 and 0.39 mM, respectively. An increase of K+ to 60 mM in the presence of 1 microM Cch shifted the curve to the left roughly in parallel (ED50: 0.12 mM, Hill coefficient: 2.3). 3. [Ca2+]0-tension curve in the presence of 1 microM Cch was shifted to the right in parallel by nifedipine (1 microM). This was markedly potentiated by 60 mM K+ (the increase in ED50 of Ca2+ being 3 times at 6 mM and 15 times at 60 mM K+). No tension was evoked by Ca2+ up to 2.5 mM in 60 mM K+ solution containing 1 microM nifedipine but no Cch. 4. In the absence of nifedipine, Cch-induced contractions were potentiated by 60 mM K+, whereas in the presence of nifedipine, Cch-induced contractions were markedly inhibited by 60 mM K+. These mechanical changes were accompanied by an increase or a decrease in intracellular Ca2+. 5. A hypothesis is presented to explain the results which suggests that the kinetics of Ca2+ influx though a single type of pathway is modulated by membrane potential and receptor activation and that the susceptibility of the pathway to dihydropyridine blockade is closely related to the Ca2+ influx kinetics with receptor activation reducing and membrane depolarization increasing the susceptibility.     

Pharmacology of neurotransmission to the smooth muscle of the rat and the guinea-pig prostate glands

The advantages of customized Laboratory Information Management's Systems (LIMS) are their focus on the special aspects of their users' needs. Differences in the research and development or production chain in the individual organizations lead to an increase of interest in customized systems. Usually, also for customized systems, the core software is commercially available. The individual application modules as the Customized part of the LIMS are the most critical elements within the validation process. The topic of this paper is to give an example of the validation of a customized analytical LIMS. Validation of complex computerized systems guarantees the intended use and is therefore an unavoidable requirement of authorities. The audit of the supplier of the individual programmed modules, the user requirement specifications and the acceptance testing and results, respectively, on the software are of special interest within a customized LIMS. The hardware suitability and the principal processing routines are also a very important part of the whole validation process, but they will not be discussed in detail in this paper.     

A pharmacological evaluation of the pull-up test for muscle relaxation in rats

The pull-up test for muscle relaxation is described and validated. At testing, rats were evaluated for their ability to recover ('pull-up') from a fully inverted head-down position. Control animals rapidly regained position (median: 1 s). Known muscle relaxants increased latency to pull-up compared to controls. The test proved sensitive to the effects of barbiturates and benzodiazepines which produced graded dose-response functions. In general, results in the pull-up test corresponded with known potencies, with weaker muscle relaxants such as clobazam and oxazepam being less active. The test was relatively insensitive to non-benzodiazepine compounds (e.g., haloperidol, etomidate, morphine, fentanyl and risperidone) producing cataleptic, catatonic, neuroleptic, analgesic, sedative or hypnotic effects. In terms of ED50 values for barbiturates and benzodiazepines, the pull-up test correlated significantly with ED50s from the rotarod test, the antipentylenetetrazol test, ataxia in rats and muscle relaxation in cats. It was concluded that the pull-up test was relatively specific for muscle relaxation and provided a simple alternative to more time-consuming or equipment-intensive tests.     

The action of toxins derived from scorpion venom on the ileal smooth muscle preparation

The properties of the 37400 oncogenic strain of Agrobacterium tumefaciens are described. This strain was derived from the VI lysogenic strain originally isolated by Hamilton from a Zinnia elegans tumour. Strain 37400 has a number of properties which render it suitable for quantitative and genetic studies. It is cured of prophages and can serve as a universal sensitive indicator for a number of phages isolated from various lysogenic strains of Agrobacterium tumefaciens. Its good growth properties in synthetic media and at elevated temperatues enable the isolation of auxotrophic mutants and temperature sensitive phage mutants. Preliminary experiments show that strain 37400 will serve as suitable starting material for conjugation experiments under defined conditions.     

The influence of post-mortem conditions on contractile and relaxant responsiveness of guinea-pig isolated tracheal smooth muscle

Responsiveness to various contractile and relaxant agonists was assessed in tracheal preparations from guinea-pigs that had been incubated in situ at 4-37 degrees C for 0-168 h post-mortem. The potencies of histamine and acetylcholine were increased up to 168 h at 4 degrees C post-mortem and up to 24 h post-mortem at 22 degrees C. Histamine potency also increased with increasing post-mortem time at 37 degrees C. After 48 h at 22 degrees C and 8 h at 37 degrees C, responses to all spasmogens were abolished. Increases in histamine and acetylcholine potencies were similarly observed in tracheal tissue that had been removed at death and then incubated at 4 degrees C in oxygenated Krebs-bicarbonate solution for 0-168 h. The increased potency of these drugs may be explained by epithelial damage and/or loss of an epithelium-derived inhibitory factor (EpDIF). Both basal and spasmogen-stimulated increases in intracellular phosphoinositides fell with increasing time and ambient temperature post-mortem, despite the fact that contraction in response to these agonists could still be evoked. This suggests the selective failure of this signal transduction pathway and the maintenance of responsiveness via other mechanisms. The potencies and maximum effects of relaxant agonists remained unaltered in tracheal tissue with increasing time post-mortem, suggesting little change in the function of the appropriate receptor-signal transduction processes. This study has therefore demonstrated that at 4 degrees C. contractile and relaxant responses were preserved for up to 168 h post-mortem, although the modulatory influence of the epithelium on histamine and acetylcholine responses was rapidly lost.     

The angio-architecture of normal, hypertrophic and denervated muscle. A study in the rat, using micro-angiography and the scanning electron microscope

The IQ's of Jamaican boys aged 6-10 were associated significantly with the presence or absence of severe malnutrition in infancy, with height at time of IQ testing, and with a measure of the boys' social background. A multiple correlation coefficient of 0.674 was obtained between IQ and the three factors. Social background contributed 0.294 of the variance, height 0.112, and severe malnutrition 0.049. The two extreme groups of boys, i.e., those malnourished, small at follow-up, and with unfavorable social backgrounds and those not malnourished, tall at follow-up, and with favorable social backgrounds had average IQ's of 49.4 and 74.9, respectively (from Table 5). Only two of the boys in the most advantaged group had IQ scores that overlapped with the most disadvantaged group. Boys with severe malnutrition in infancy, but who are tall at follow-up and have a favorable social background have an average IQ 11 points higher than boys who did not experience severe malnutrition, but who are short at follow-up and have a unfavorable social background. The difference in IQ between boys who did and did not experience severe malnutrition in infancy varies under different conditions of height and social background when those are held constant for both groups. Under the most favorable conditions of being tall and having an advantageous social history the average IQ of the malnourtished boys in only 2 points lower than those not malnourished. Unde the most unfavorable conditions of short stature and a disadvantageous social background the IQ of the malnourished boys is 9 points lower than those not malnourished (Table 6 and Fig. 1).