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Enhanced accumulation of granulation tissue in a wound healing model was observed in the presence of regenerating skeletal muscle. This study characterized the tissue accumulated in the normal wound healing and regeneration-adapted models. DNA, RNA, total protein, collagen, and noncollagen protein content, and protein:DNA and noncollagen protein:collagen ratios were determined using standard methods. DNA did not differ; but RNA increased and total protein decreased compared to early granulation tissue. Mean protein: DNA ratio was lower in regeneration-associated granulation tissue compared to age-matched normal and early granulation tissue. Collagen was reduced and noncollagen protein was increased in tissue surrounding regenerating muscle, yielding an elevated noncollagen:collagen ratio. The relative cellularity of granulation tissue was enhanced and collagen deposition potentially reduced, when accompanied by regeneration. Factors from a regeneration environment can interact with cells engaged in wound repair and potentially alter their behavior.

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BACKGROUND: Factors differentially regulating regeneration and repair as contrasting resolutions to injury are unknown. We adapted a common wound healing model to further characterize mammalian wound repair and regeneration microenvironments. METHODS: Polyvinyl alcohol sponges, sponges containing minced muscle, or blocks of minced muscle were implanted onto the backs of Fischer rats. Vascularization was assessed by infusion with india ink and progress of regeneration was evaluated histologically. RESULTS: Regeneration occurring within sponges was histologically similar to that of free muscle blocks; but, was initiated more slowly. Vascularization of minced muscle implanted in sponges was delayed slightly compared to implanted free muscle blocks. CONCLUSIONS: Regeneration of minced muscle in sponges parallels normal regeneration of free minced muscle. Incorporation into a wound repair model provides access to interstitial fluids conditioned by regenerating muscle and will lead to more detailed comparisons of the content and properties of repair and regeneration microenvironments.

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Dexamethasone induces a relative cardiomegaly in neonatal rats. Biochemical analyses, performed on hearts at seven days postpartum, disclosed that dexamethasone increased protein content without significantly altering DNA or RNA content. While dexamethasone decreased absolute actinomyosin and collagen content, the relative abundance of these proteins as a function of heart mass was increased and the ratio of muscle protein to collagen remained constant. In addition, dexamethasone increased glycogen but decreased fatty acid content. Thus, dexamethasone treatment of rats during the neonatal period appears to induce a relative hypertrophic cardiomyopathy complicated by fibrosis and metabolic derrangement of myocardial maturation.

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The ability of wound fluids to support events required for skeletal muscle regeneration was examined. Wound fluids were obtained from polyvinyl alcohol sponges 1, 3, 5, 10, and 15 days after implantation. Neonatal rat L8 myoblasts were used to test the ability of early wound fluids to promote myoblast proliferation and late wound fluids to promote myoblast differentiation-two characteristics deemed critical for effective skeletal muscle regeneration. Early wound fluids (1- and 3-day) stimulated DNA replication by myoblasts, as judged by tritiated thymidine uptake, up to ninefold (p < 0.05). Later wound fluids (5-, 10-, and 15-day) displayed decreasing ability to stimulate proliferation, with 15-day wound fluid failing to significantly stimulate proliferation. In contrast, myoblast differentiation, as judged by myotube fusion and creatine kinase activity, was progressively reduced by wound fluids of increasing age. In fact, late wound fluids (5, 10, and 15 days) reduced myotube fusion by 88% to 100% and depressed creatine kinase activity by 60% to 75% (p < 0.05). Thus, wound fluids from a repair environment appear to support myoblast proliferation early but suppress myoblast differentiation later. These characteristics suggest that the wound repair environment cannot fully support skeletal muscle regeneration.

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Dexamethasone-induced relative cardiomegaly in neonatal rats mimics hypertrophic cardiomyopathy seen in premature infants receiving dexamethasone for bronchopulmonary dysplasia. Dexamethasone reduced cellular density by approximately 20%. However, dose related increases in mean fiber diameter were seen and mitotic index was doubled at 12.5 micrograms dexamethasone/injection. In addition, dexamethasone induced collagen accumulation throughout the myocardium in an apparently dose dependent manner. These data suggest that dexamethasone promotes (i) myocardial hypertrophy and possibly hyperplasia and (ii) fibrosis and possibly fibroplasia. Further studies are required to determine relationships between disseminated collagen deposition and other suggested metabolic changes and the observed compensatory myocardial hypertrophy and hyperplasia.

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Plasma atrial natriuretic factor (ANF) levels are markedly altered in diabetic rodents suggesting that insulin might be a modulator of ANF release. Effects of insulin (25 mU/ml) on basal (nonstimulated) immunoreactive (ir)-ANF release by isolated neonatal pig hearts paced at 150 beats/min and perfused with no change in atrial stretch were examined. Release of ir-ANF decreased with age from 1-3 days postpartum (146.0 +/- 38.4 to 62.2 +/- 20.5 fmol/g/min). Insulin stimulated myocardial glucose utilization and lactate production approximately 2-fold, but uniformly decreased ir-ANF release by approximately 25%, regardless of age. Correlations between ir-ANF release and myocardial metabolism suggest independent influences of insulin on these two events. In addition, it appears that this model might be reasonably exploited to investigate episodic ANF release and its regulation during ontogeny and in a variety of physiological states.

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Traditional signal transduction pathways appear to be operating during amphibian forelimb regeneration, as in other developing systems. A consistent picture appears to be developing that suggests that growth factors promoting proliferation during regeneration act through cAMP-independent mechanisms while factors that modulate differentiation and morphogenesis include signaling pathways in which cAMP participates. Although our understanding of these pathways is far from complete, it is becoming increasingly evident that further characterization of signal transduction events will facilitate (a) identifying major signaling substances, (b) determining the cellular events in regeneration that they modulate, and (c) defining the mechanisms through which they act. The present study demonstrates that PPtases, and in particular PTPases, can be studied in regenerating newt limbs. More importantly, this investigation demonstrates that there are progressive and significant increases in PPtase activity during regeneration. Moreover, the observed patterns of PPtase activity during regeneration conform to an emerging picture that ligands acting through receptors possessing intrinsic tyrosine kinase activity promote proliferation within the regeneration blastema. However, the substrates used in this study precluded acquiring insights about modulating effects of serine/threonine kinases (e.g., PKA and PKC). Nevertheless, these data suggest that activation of PPtases contribute to the orchestration of the diverse cellular activities required to regenerate a vertebrate appendage.

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Effects of s.c. administration of dexamethasone (1-25 micrograms/rat pup/d) during the first 5 d postpartum on survival rates, body weight, heart weight, heart-to-body weight ratios, and heart dimensions were determined. Dexamethasone decreased survival, body weight, and heart weight, but increased heart-to-body weight ratios and myocardial percentage of dry weight (0.44 to 0.52-1.06 and 20 to 23%, respectively; p less than 0.05) by 7 d postpartum. By 21 d postpartum, differences in absolute body weight and heart weight between control and experimental animals were reduced; however, myocardial percentage of dry weight and heart-to-body weight ratios were indistinguishable. Microscopic analysis of 7-d-old hearts disclosed that interventricular septum, left ventricular free wall thicknesses, and left ventricle chamber diameter were reduced (0.93 to 0.76-0.85 mm, 1.19-1.34 to 0.92-1.07 mm, and 1.35-1.40 to 0.89-1.23 mm, respectively; p less than 0.05), whereas right ventricular free wall thickness was unaffected and right ventricle chamber diameter was increased (0.29-0.31 to 0.42-0.46 mm) by dexamethasone. Thus, dexamethasone reduced survival and retarded growth of neonatal rats. Retardation of heart growth was less severe, producing a transient relative cardiomegaly characterized by 1) increased myocardial percentage of dry weight, 2) increased left ventricular free wall-to-chamber ratio, and 3) retention of absolute right ventricular free wall thickness. This relative cardiomegaly appeared to be resolved by 21 d postpartum, as reflected in myocardial percentage of dry weight and heart-to-body weight ratios.(ABSTRACT TRUNCATED AT 250 WORDS)

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The potential induction of cardiac effects by high-dose dexamethasone therapy was evaluated prospectively in 13 respirator-dependent infants with bronchopulmonary dysplasia by means of two-dimensional and M-mode echocardiography. The initial divided dose of dexamethasone was 500 micrograms/kg per day, tapered progressively for as long as 6 weeks. Evaluations were made before treatment and at 3, 7, 14, 21, 28, 35, and 42 days after the start of dexamethasone therapy. This regimen was associated with a significant (p less than 0.01) increase in thickness of the interventricular septum (2.60 +/- 0.09 to 4.00 +/- 0.16 mm), diastolic left ventricular free wall (2.80 +/- 0.13 to 4.06 +/- 0.20 mm), and diastolic right ventricular free wall (1.55 +/- 0.08 to 2.02 +/- 0.12 mm). In addition, seven dexamethasone-treated infants but no control infants had systolic anterior motion of the mitral valve (p less than 0.001). These effects were transient, reached their maximal degree by the third week of treatment, and approached pretreatment conditions by the sixth week of treatment. Ejection fraction was not affected; heart rate and mean arterial pressure were transiently increased during dexamethasone therapy. We conclude that a transient absolute myocardial hypertrophy is associated with dexamethasone therapy in infants with bronchopulmonary dysplasia. The mechanism or mechanisms through which this hypertrophy arises and the cardiopulmonary implications are unclear.

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Influence of the immune system on epimorphic regeneration of amphibian limbs has been suggested but not proved. The present investigation explored this hypothesis by examining the effects of x-irradiation on forelimb regeneration and rejection of skin allografts. Two kRad x-irradiation was provided either to a single limb or as whole-body irradiation to intact newts (with 1 limb shielded). Complete suppression of regeneration was observed when limbs to be amputated were irradiated directly. In addition, irradiated limbs displayed severe and protracted inflammation, with total resorption of the affected limbs in 85% of the cases. Moreover, delays in both the rate of forelimb regeneration and allograft rejection were found in animals receiving whole-body irradiation. However, in these cases neither forelimb regeneration nor allograft rejection were suppressed. These observations diffuse the challenge raised by irradiation studies to the notion of possible immunological influence on epimorphic regeneration. Moreover, the delays observed in both regeneration rate and allograft rejection following whole-body irradiation are consistent with possible interaction between the immune system and the regenerating limb. Nevertheless, confirmation that such interaction occurs and is integral to epimorphic regeneration must await further investigations.

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Palmitate oxidation and the effect of palmitate on glucose and lactate utilization were investigated in isolated, perfused, fetal (0.9 gestation), and neonatal (2 day old) pig hearts. Hearts were perfused under working conditions, developing a mean aortic pressure of 50-55 mmHg, paced at 180 beats/min for 30 min, with Krebs-Henseleit buffer containing 3% albumin, glucose (5 mM), and insulin (100 microU/ml). Palmitate (1 mM) and lactate (5 mM), either individually or in combination, were added to the perfusion buffer. Palmitate oxidation was assessed from 14CO2 production from [U-14C]-palmitate, glucose uptake as 3H2O production from D-[2-3H]-glucose, and lactate metabolism from changes in buffer lactate content. After perfusion, ATP, creatine phosphate, triglycerides, and glycogen were measured. Substantial palmitate oxidation was observed at both ages but was greater in neonatal hearts. Nevertheless, palmitate inhibited lactate utilization and glucose uptake similarly in fetal and neonatal hearts. Lactate also reduced palmitate uptake and oxidation by 40-60% in both fetal and neonatal hearts. During perfusions with palmitate, tissue concentrations of triglycerides increased approximately threefold in fetal hearts and were unaffected by lactate. Thus both palmitate and lactate can act as major energy substrates for the immature heart. Both substrates significantly (P less than 0.01) suppress glucose utilization, and each has suppressive effects on the other's metabolism.

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The transition from preferential use of carbohydrates to fatty acids for energy production by neonatal mammalian myocardium might be hormonally influenced. In neonatal pigs (between 1 and 4 days of age), plasma glucose, lactate, and glucagon concentrations remained constant at adult levels, while plasma triglycerides and insulin content remained stable at substantially lower levels. Insulin, whether administered in vivo or during perfusion, increased myocardial glucose utilization [GU] and lactate production [LP]. Stimulation of GU and LP by insulin exposure in vivo persisted for at least 1 h during perfusions after insulin was removed. Exogenous lactate (0.5 mM) diminished insulin-stimulated GU and LP; and was used as a substrate at 1.25 mM. Thus, insulin augmented glycolytic activity of neonatal piglet myocardium; however, exogenous lactate, within the physiological range, altered the nature of the myocardium's response by, as yet, undetermined mechanisms. Therefore, the transition from carbohydrate to fatty acid utilization for energy production by the neonatal myocardium does not appear to result from decreased carbohydrate metabolism or reduced myocardial responsiveness to insulin.

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The role of lactate as an energy substrate in fetal (0.9 gestation) and newborn (2 day old) hearts was investigated in isolated, perfused hearts. Perfusions were performed with Krebs-Henseleit buffer supplemented with glucose (5 mM) in combination with varying concentrations of lactate. Isolated working heart perfusions, in which the heart ejects the buffer at controlled pressure, were carried out with glucose (5 mM) alone and with glucose (5 mM) and lactate (5 mM) combined. With glucose as sole substrate, lactate was produced by the heart and glucose uptake accounted for approximately two-thirds of oxygen consumption. When both glucose and lactate were provided, lactate accounted for more than 80% of oxygen consumption and profoundly suppressed glucose uptake. Further investigations using retrograde perfusion through the aorta demonstrated that lactate uptake was consistently observed when exogenous lactate concentrations exceeded 1.25 mM. Glucose uptake was suppressed with lactate concentrations as low as 0.5 mM and progressive suppression occurred with increasing lactate concentrations. Fetal and newborn pig hearts utilize lactate as a primary substrate for energy production when lactate concentrations are in the physiological range.

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Chemical signals, whether in the form of hormones, neurosecretions (neurotransmitters and neuropeptides), or growth factors and chalones are used to communicate information to cells at all stages of their life cycle. These signals inform the cells when it is time to progress through developmental change, when to change the rates of various activities (e.g. metabolism or contraction), and, in some cases, even when it is time to die. Throughout all of these interactive exchanges, the signal molecule itself carries no intrinsic message of a universal nature. The chemical identity of the signal molecule has meaning only for those cells competent to receive the signal (i.e. does it possess an appropriate receptor?). Moreover, it is the nature of the cell receiving the signal (itself the product of innumerable previous encounters with signals from other cells) that dictates the specific response that a particular signal will evoke. The signal emitted by the communicating cell only informs the target cell that it is time to act in a manner consistent with that signal. The majority of the discussion has been from the perspective of vertebrate organisms. Moreover, of necessity, the discussion has been general and superficial. The primary objective of the preceding discussion has been to underscore major similarities and differences existing among hormones, neurosecretions (neurotransmitters and neuropeptides), and growth factors as information-bearing substances used in communication among vertebrate cells. It should be realized that similar means of communication are employed by multicellular invertebrates, by plants, and even by single-celled organisms such as the protists and bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)

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The possible role of leukocytes in regeneration was examined through the evaluation of quantitative changes in circulating blood cell counts during forelimb regeneration in adult newts. Leukocyte counts declined during the first 2 weeks, then returned to preamputational levels. Thrombocytes were also depressed (40-67%) throughout the progressive stages (9-30 days). In addition, lymphocytes were reduced, especially during the preblastemic phase (less than 15 days). In contrast, neutrophils were increased immediately following wound healing and during the differentiation stages, but were otherwise unchanged. These variations appear to reflect physiological changes occurring during regeneration and are consistent with a potential role for leukocytes in regeneration.

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Amphibian forelimb regeneration is under neural influence. Although the precise role of nerves is unknown, clear influence on macromolecular synthesis and on mitosis have been demonstrated. The hypothesis presented here proposes that neural input is directed primarily at influencing the decision of blastemal cells either to proliferate or to prepare to express differentiated phenotypes. This is considered to be accomplished by an interplay between the catecholamine neurotransmitters and neurotrophic peptides using cyclic nucleotides (cAMP) and Ca2+, respectively, as intracellular mediators. This coordination of proliferation-differentiation decisions in regenerating limbs is proposed to be primarily, but not exclusively, the function of nerves.

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The participation of leukocytes in regeneration was studied by determining changes in circulating leukocyte counts following putative immunological manipulations. Splenectomy failed to produce leukopenia during regeneration, although a 24-35% reduction in leukocytes occurred in otherwise intact newts. Bovine serum albumin and anti-lymphocyte serum produced initial lympho- and granulocytopenias, but blood counts soon returned to more normal levels. Lymphocytosis followed treatment with cobra venom factor, but marked lymphopenia occurred shortly thereafter. Regeneration occurred in all cases. These data failed to establish a clear correlation between the nature of quantitative changes in circulating leukocyte levels following these treatments and regenerative capacity.

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Cyclophosphamide (cytoxan), a clinical immunosuppressant, produced a marked, persistent leukopenia (greater than 40% reduction) in adult newts in contrast to a transient, milder leukopenia accompanying amputations without cytoxan treatments. In addition, cytoxan suppressed the formation of regeneration blastemata initially: however, blastema formation occurred if a second amputation was performed two or more weeks following the cessation of cytoxan treatments. The failure of the leukopenias of these latter cases to be corrected suggests that although a cytoxan-induced leukopenia is correlated with the absence of forelimb regeneration, this leukopenia is not, of itself, a sufficient condition to inhibit regeneration.

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The influence of thyroid hormones on neurogenesis suggested that these hormones might affect the maturation and development of neural centers which ultimately regulate thyroid function. Changes in the levels of the thyroid hormones, thyrotropin and the thyrotropin-releasing hormone in 'brain' and 'thyroid' fractions were, therefore, correlated at several developmental stages. All hormones showed increases from their initial detection through metamorphosis. The thyroid hormones were observed earliest in both fractions. These data are consistent with the notion that thyroid hormones influence the maturation of the particular neural regions whose later function is the regulation of the hypophyseal-thyroid axis.

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