Comparison of innervated and noninnervated free flaps in oral reconstruction.

Thirteen patients who had undergone ablative surgery for advanced squamous cell carcinoma in which more than half of the tongue had been resected underwent reconstruction in which the cutaneous nerve of a free flap was anastomosed to the stump of the transected lingual nerve. Eight of the patients underwent reconstruction with an innervated anterolateral thigh flap and five patients underwent reconstruction with an innervated rectus abdominis musculocutaneous flap. Sensory recovery of the flap at least 6 months postoperatively was compared in these 13 patients and in 16 additional patients who received noninnervated versions of the same flaps for the same defect. The degree of sensory recovery of innervated thigh flaps was significantly greater than that of noninnervated ones in all modalities and that of innervated rectus abdominis flaps was also greater than that of noninnervated flaps, except for hot and cold perception. These results indicate that sensory regrowth occurs in most areas through the surgically created pathways. However, results of Semmes-Weinstein testing showed that recovery did not reach the level of protective sensation in either type of innervated flap. Although these findings must be followed by additional objective and functional tests and the need for sensory reeducation should be considered, this simple operative procedure can improve postoperative intraoral function and should be attempted whenever possible after ablative surgery.     

Acetylcholine sensitivity of innervated and noninnervated Xenopus muscle cells in culture

The chemosensitivity of Xenopus muscle cells grown in culture to iontophoretically applied acetylcholine (ACh) in the presence or absence of neurons was examined. Muscle cells grown without nerve cells are sensitive to ACh over their entire surface (2.4 mV/pC) with occasional spots of high chemosensitivity (“hot spots”). In cultures containing neural tube cells, the ACh sensitivity of muscle cells increased by approximately 50% regardless of the presence of nerve contacts or functional synapses. A similar increase in the ACh sensitivity was observed in muscle cells cultured in medium conditioned by neural tube cells. The ACh sensitivity of the extrajunctional region in functionally innervated muscle cells was not different from that of noninnervated cells growing in the same cultures. However, the chemosensitivity at the junctional region was about fivefold higher than that of the extrajunctional area. This increase in junctional chemosensitivity may well account for the increase in miniature endplate potential amplitude which has previously been reported to occur during nerve-induced ACh receptor accumulation.     

Distribution of fiber types in embryonic chick limb muscles innervated by foreign motoneurons

The differentiation of distinct myotube fiber types in chick limb muscle development is coincident with innervation. The role of motoneurons in influencing fiber type differentiation was analyzed by causing chick hind limb muscles to be innervated by inappropriate motoneurons and then examining experimental muscles for changes in the distribution of myosin ATPase fiber types. Motoneuron innervation of limb muscles was altered by performing either limb shifts, limb reversals, or large spinal cord reversals on early neural tube or limb bud stage chick embryos. The distribution of fiber types was then analyzed in muscles from stage 36 (E10) to stage 45 (E20) embryos after processing hind limb sections for myosin ATPase histochemistry. In the majority of experimental muscles examined (267/312), the distribution of myosin ATPase fiber types was unaltered. In the remaining experimental muscles (14%), alterations in the distribution of myosin ATPase fiber types occurred, indicating that in some cases, foreign innervation may alter the developmental program of differentiating myotubes. The results suggest that myotubes differentiate myosin ATPase staining characteristics according to an intrinsic program and that these differentiating myotubes are selectively innervated by motoneurons of the appropriate type under most conditions including normal development. Under exceptional circumstances of motoneuron-muscle fiber type mismatch, embryonic motoneurons can alter fiber type expression.     

The effect of oxidized isoprenaline on the chick embryonic heart

Intra-amnial administration of isoprenaline (IPRO) to chick embryos induces a number of myocardial lesions. The purpose of the present study was to investigate whether similar changes may also be induced after injection of spontaneously oxidized isoprenaline and commercially obtained adrenochrome. Cardiotoxicity of these substances has been demonstrated in adult animals. IPRO, oxidized IPRO, or adrenochrome were administered intra-amnially to 10-day-old chick embryos at doses of 0.1, 1.0, 10.0, and 100.0 mg X kg-1. Parallel experimental groups received propranolol at a dose of 1 mg X kg-1, 15 s before injection of IPRO or oxidized IPRO. The cAMP level in the heart was determined by radioimmunoassay 2 and 30 min after administration of IPRO, oxidized IPRO, or adrenochrome at a single dose of 10.0 mg X kg-1. It has been found that in embryos the effect of IPRO and oxidized IPRO is dose dependent. The rise in mortality and development of cardiomegaly together with increased hydration and disturbances of the development of coronary vascularization were highly significant starting from the dose of 10 mg X kg-1. Furthermore, both drugs significantly increased cAMP levels in the embryonic heart. On the other hand, the administration of adrenochrome was without any effect. The changes induced by IPRO were prevented by the administration of the beta-blocking agent propranolol; the lesions induced by spontaneously oxidized IPRO were, however, prevented only partially.     

The effect of graded hypoxia on the embryonic chick heart

Embryonic ventricular function in the chick was measured in response to graded levels of hypoxia. Myocardial contractility, as measured by cinephotoanalysis and expressed as shortening fraction, was significantly depressed after 1 hour of moderate hypoxia (6% O2) and after 5 hours of milder (16% O2 and 11% O2) levels of hypoxia (P less than .05). Microscopy confirmed associated myocyte damage with cell death noted after 5 hours of moderate hypoxic stress. Heart rate change was not related to the severity of hypoxia. The greatest level of tachycardia was noted with conditions of mildest hypoxia (16% O2). The data confirm that cardiac contractility, as measured by shortening fraction, is depressed on exposure to hypoxia, with impairment of function related to the severity of the hypoxic conditions.     

Glycosaminoglycan synthesis by the early embryonic chick heart

Glycosaminoglycans of embryonic chick hearts labeled in situ were characterized by means of labeled precursor incorporation, electrophoretic mobility, sensitivity to testicular hyaluronidase, elution characteristics from CPC-cellulose columns, and hexosamine content. During the initial period of overt cardiac muscle differentiation (approximately stage 10) chondroitin sulfates are not detectable but an undersulfated component is present. Chondroitin sulfate synthesis appears shortly after overt muscle differentiation. Hyaluronate is present both during and after overt myocardial differentiation. Although epimerization of ³H-glucosamine-derived labeled UDP-N-acetyl-d-glucosamine occurs (determined by recovery of incorporated labeled galactosamine), label does not appear in chondroitin sulfate. ³H-Glucosamine is thus a relatively specific precursor for unsulfated glycosaminoglycans, a fact that we exploited in demonstrating their distribution radioautographically. Glycosaminoglycan synthesis was also examined in hearts labeled (a) in isolated organ culture, (b) in situ but exposed directly to the medium by removal of the splanchnopleure. In both cases fully sulfated chondroitin sulfate and chondroitin are not synthesized. Hearts make only hyaluronate and undersulfated chondroitin sulfate.     

Neuregulin stimulates DNA synthesis in embryonic chick heart cells.

Neuregulins are a family of growth factors that have been shown to promote the growth or differentiation of various cell types. Recently, targeted mutations of the genes for neuregulins or their putative receptors by homologous recombination resulted in embryonic lethality characterized by cardiac malformation. Here we investigate a role for neuregulin in the growth of cultured chick heart cells. Neuregulin induced the tyrosine phosphorylation of a 185-kDa protein in cultured heart cells, and it also stimulated an increase in [(3)H]thymidine incorporation and BrDU labeling in the cell cultures. Immunocytochemistry revealed that the increased DNA synthesis was primarily in mesenchymal cells and not detected in myocytes or endocardial cells. These data suggest that neuregulin may function as a paracrine signal in mesenchymal-endothelial interactions during cardiac development.     

Repolarization currents in embryonic chick atrial heart cell aggregates.

Outward membrane currents in aggregates of atrial cells prepared from 7-12-d-old chick embryonic hearts were measured with the two microelectrode voltage-clamp technique. Two outward current components, Ix1 and Ix2, were found in the plateau potential range of the action potential. The Ix1 component is activated between -50 and -20 mV; the Ix2 component is activated between -15 and +20 mV. The Ix1 component inwardly rectifies, whereas Ix2 has an approximately linear current-voltage relation. These preparations lack a time-dependent pacemaker current component, even though they beat spontaneously with an interbeat interval of approximately 1 s. A mathematical model of electrical activity is described based on our measurements of time-dependent outward current, and measurements in the literature of inward current components.     

Brachially innervated ectopic hindlimbs in the chick embryo. I. Limb motility and motor system anatomy during the development of embryonic behavior

The functional status of brachially innervated hindlimbs, produced by transplanting hindlimb buds of chick embryos in place of forelimb buds, was quantified by analyzing the number and temporal distribution of spontaneous limb movements. Brachially innervated hindlimbs exhibited normal motility until E10 but thereafter became significantly less active than normal limbs and the limb movements were more randomly distributed. Contrary to the findings with axolotls and frogs, functional interaction between brachial motoneurons and hindlimb muscles cannot be sustained in the chick embryo. Dysfunction is first detectable at E10 and progresses to near total immobility by E20 and is associated with joint ankylosis and muscular atrophy. Although brachially innervated hindlimbs were virtually immobile by the time of hatching (E21), they produced strong movements in response to electrical stimulation of their spinal nerves, suggesting a central rather than peripheral defect in the motor system. The extent of motoneuron death in the brachial spinal cord was not significantly altered by the substitution of the forelimb bud with the hindlimb bud, but the timing of motoneuron loss was appropriate for the lumbar rather than brachial spinal cord, indicating that the rate of motoneuron death was dictated by the limb. Measurements of nuclear area indicated that motoneuron size was normal during the motoneuron death period (E6-E10) but the nuclei of motoneurons innervating grafted hindlimbs subsequently became significantly larger than those of normal brachial motoneurons. Although the muscle mass of the grafted hindlimb at E18 was significantly less than that of the normal hindlimb (and similar to that of a normal forelimb), electronmicroscopic examination of the grafted hindlimbs and brachial spinal cords of E20 embryos revealed normal myofiber and neuromuscular junction ultrastructure and a small increase in the number of axosomatic synapses on cross-sections of motoneurons innervating grafted hindlimbs compared to motoneurons innervating normal forelimbs. The anatomical data indicate that, rather than being associated with degenerative changes, the motor system of the brachial hindlimb of late-stage embryos is intact, but inactive. © 1993 John Wiley & Sons, Inc.     

Effects of taurine on the electrical and mechanical activities of embryonic chick heart

The effect of taurine (2-aminoethanesulphonic acid) on myocardial slow action potentials (APs) and accompanying contractions was examined in isolated perfused chick hearts and reaggregated cultured cells. Isoproterenol (ISO), histamine (HIS), or tetraethylammonium (TEA) induced slow APs and contractions in hearts whose fast Na+ channels had been inactivated by elevated K+. Taurine (10 mM) not only failed to induce slow APs, but actually decreased ISO (10(-8) M), HIS (10(-4) M), or TEA (10 mM) induced slow APs and contractions transiently (about 30s-2 min after the addition of taurine). The properties of the slow APs recovered to control levels by 7-13 min after the addition of the taurine; at this time, there was an increase in developed tension of the contraction accompanying the slow APs. These results suggest that the positive inotropic action of taurine is not mediated through an increase in the slow inward Ca2+ current. However, the transient depression of Ca2+-dependent slow APs by taurine probably explains the transient negative inotropic effect of taurine.     

Fatty acid synthesis in chick embryonic heart and liver during development.

Fatty acid synthesis by subcellular fractions of heart and liver of chick embryos at varying stages of development has been studied. Fatty acid synthetase activity is associated with the embryonic heart at early stages of development, as suggested by substrate requirement, Schmidt decarboxylation of synthesized fatty acids and gas liquid chromatographic identification of the products as palmitic and stearic acids. The fatty acid synthetase activity decreases in heart cytosol with age of the embryo and is absent in the newly hatched chick and in older chicken. The acetyl CoA carboxylase activity is negligible in embryonic and adult chicken heart. The fatty acid synthetase activity in liver is low, but measurable during the entire embryonic development. The activity increases by about three-fold on hatching and thereafter in fed, newly hatched chicks by about 35-fold, over the basal embryonic activity. The acetyl and malonyl transacylase activities in the heart and liver cytosols during development followed closely the fatty acid synthetase activities in heart and liver, respectively. A non-coordinate induction of fatty acid synthetase and acetyl CoA carboxylase activities in liver was observed during development. The microsomal chain elongation in liver and heart followed the pattern of fatty acid synthetase activity in liver and heart, respectively. The mitochondrial chain elongation in embryonic heart is initially low and increases with age; while this activity in liver is higher in early stages of embryonic development than in the older embryos and the chicks. Measurement of lipogenesis from acetate-1-¹⁴C by liver and heart slices from chick embryos and newly hatched chicks support the conclusions reached in the studies with the subcellular fractions. The results obtained indicate that the major system of fatty acid synthesis in embryonic and adult heart is the mitochondrial chain elongation. In embryonic liver, fatty acid synthesis proceeds by chain elongation, while the de novo system is the major contributor to the lipogenic capacity of the liver after hatching.     

Phase resetting of embryonic chick atrial heart cell aggregates. Experiment and theory.

The influence of brief duration current pulses on the spontaneous electrical activity of embryonic chick atrial heart cell aggregates was investigated experimentally and theoretically. A pulse could either delay or advance the time of the action potential subsequent to the pulse depending upon the time in the control cycle at which it was applied. The perturbed cycle length throughout the transition from delay to advance was a continuous function of the time of the pulse for small pulse amplitudes, but was discontinuous for larger pulse amplitudes. Similar results were obtained using a model of the ionic currents which underlie spontaneous activity in these preparations. The primary ion current components which contribute to phase resetting are the fast inward sodium ion current, INa, and the primary, potassium ion repolarization current, IX1. The origin of the discontinuity in phase resetting of the model can be elucidated by a detailed examination of the current-voltage trajectories in the region of the phase response curve where the discontinuity occurs.     

Morphologic study of ventricular trabeculation in the embryonic chick heart

This paper presents a morphologic study of ventricular trabeculation in chick embryo hearts between days 2 and 5 of incubation. Trabeculation appears to be the expression of three closely interrelated events: the formation of endocardial outgrowths that eventually invade the myocardium; the development of large intercellular spaces between the myocytes, and the decrease in thickness of the cardiac jelly. Endocardial cells present morphologic differences between trabeculated and nontrabeculated areas of the ventricular region. The elongation of the endocardial cells in the endocardial outgrowths and the presence of mitoses suggest that the endocardium grows out by means of an increase in cell number and by redistribution and elongation of the preexisting endocardial cells. The intercellular spaces of the myocardium appear filled with abundant extracellular material. It is suggested that the continuous synthesis of extracellular material by the myocytes may increase the hydrostatic pressure within the myocardium, inducing the formation and the enlargement of these intercellular spaces. The development and later rupture of endocardium-covered cords is described here. These cords are made up of a core of cardiac jelly material revested by endocardium. The cords may be engaged in the removal of substantial amounts of cardiac jelly during the formation of the trabeculae.     

Intramyocardial pressure measurements in the stage 18 embryonic chick heart

Intramyocardial pressure (IMP) and ventricular pressure (VP) were measured in the trabeculating heart of the stage 18 chick embryo (3 days of incubation). Pressure was measured at several locations across the ventricle using a fluid-filled servo-null system. Maximum systolic and minimum diastolic IMP tended to be greater in the dorsal wall than in the ventral wall, but transmural distributions of peak active (maximum minus minimum) IMP were similar in both walls. Peak active IMP near midwall was similar to peak active VP, but peak active IMP in the subepicardial and subendocardial layers was four to five times larger. These results suggest that the passive stiffness of the dorsal wall is greater than that of the ventral wall and that during contraction the inner and outer layers of both walls generate more contractile force and/or become less permeable to flow than the middle part of the wall. Measured pressures likely correspond to regional variations in wall stress that may influence morphogenesis and function in the embryonic heart.     

Different effect of propranolol and verapamil on isoprenaline-induced changes in the chick embryonic heart

Isoprenaline (IPRO) has been reported to cause pathological lesions of the embryonic heart. The purpose of the present study was to ascertain whether the development of IPRO-induced changes can be reduced--similarly as in adults--by beta blockade or calcium antagonists. IPRO was administered to 10-day-old chick embryos intraamnially (i.a.) in a dose of 2 X 10 mg.kg-1 per 48 h; propranolol (Inderal) and verapamil (Isoptin) were injected i.a. in a dose of 1.0 or 10.0 mg.kg-1 before each injection of IPRO. It was found that propranolol completely blocked the cardiac IPRO-induced changes, i.e. cardiomegaly, avascular areas and elevation of cAMP. On the other hand, verapamil was found to have no protective effect in any dose used. Furthermore, it increased the mortality of experimental embryos. This fact support the hypothesis that cardiac sensitivity to calcium antagonists may differ during prenatal development.