mTOR pathway inhibition attenuates skeletal muscle growth induced by stretching

The present study has aimed to verify the influence of calcineurin and mTOR pathways in skeletal muscle longitudinal growth induced by stretching. Male Wistar rats were treated with cyclosporin-A or rapamycin for 10 days. To promote muscle stretching, casts were positioned so as completely to dorsiflex the plantar-flexor muscles at the ankle in one hind limb during the last 4 days of treatment with either cyclosporin-A or rapamycin. Thereafter, we determined soleus length, weight, protein content, and phenotype. In addition, NFATc1, Raptor, S6K1, 4E-BP1, iNOS, and nNOS gene expression in the soleus were determined by real-time polymerase chain reaction. Soleus length, weight, and protein content were significantly reduced by rapamycin treatment in animals submitted to stretching (P<0.05). In contrast, cyclosporin-A treatment did not alter these parameters. In all cyclosporin-A treated groups, there was a significant reduction in NFATc1 expression (P<0.001). Similarly, a significant reduction was noted in Raptor (P<0.001) and S6K1 (P<0.01) expression in all rapamycin-treated groups. No alteration was observed in 4E-BP1 gene expression among rapamycin-treated groups. Stretching increased gene expression of both NOS isoforms in skeletal muscle. Rapamycin treatment did not interfere with NOS gene expression (P<0.05). Cyclosporin-A treatment did not impair muscle growth induced by stretching but instead caused a marked slow-to-fast fiber shift in the soleus; this was attenuated by stretching. The data presented herein indicate that mTOR pathway is involved in skeletal muscle longitudinal growth. We gratefully acknowledge the financial support given by FAPESP.     

Establishment and characterization of conditionally immortalized endothelial cell lines from the thoracic duct and inferior vena cava of tsA58/EGFP double-transgenic rats

The basic biology of blood vascular endothelial cells has been well documented. However, little is known about that of lymphatic endothelial cells, despite their importance under normal and pathological conditions. The lack of a lymphatic endothelial cell line has hampered progress in this field. The objective of this study has been to establish and characterize lymphatic and venous endothelial cell lines derived from newly developed tsA58/EGFP transgenic rats harboring the temperature-sensitive simian virus 40 (SV40) large T-antigen and enhanced green fluorescent protein (EGFP). Endothelial cells were isolated from the transgenic rats by intraluminal enzymatic digestion. The cloned cell lines were named TR-LE (temperature-sensitive rat lymphatic endothelial cells from thoracic duct) and TR-BE (temperature-sensitive rat blood-vessel endothelial cells from inferior vena cava), respectively, and cultured on fibronectin-coated dishes in HuMedia-EG2 supplemented with 20% fetal bovine serum and Endothelial Mitogen at a permissive temperature, 33°C. A temperature shift to 37°C resulted in a decrease in proliferation with degradation of the large T-antigen and cleavage of poly (ADP-ribose) polymerase. TR-LE cells expressed lymphatic endothelial markers VEGFR-3 (vascular endothelial growth factor receptor), LYVE-1 (a lymphatic endothelial receptor), Prox-1 (a homeobox gene product), and podoplanin (a glomerular podocyte membrane mucoprotein), together with endothelial markers CD31, Tie-2, and VEGFR-2, whereas TR-BE cells expressed CD31, Tie-2, and VEGFR-2, but no lymphatic endothelial markers. Thus, these conditionally immortalized and EGFP-expressing lymphatic and vascular endothelial cell lines might represent an important tool for the study of endothelial cell functions in vitro.M. Matsuo and K. Koizumi contributed equally to this work. This study was supported in part by Grants-in-Aid for the 21st Century COE Program and for CLUSTER (Cooperative Link of Unique Science and Technology for Economy Revitalization) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.     

Nerve injury affects the capillary supply in rat slow and fast muscles differently

The goal of this study was to determine the acute effects of permanent denervation on the length density of the capillary network in rat slow soleus (SOL) and fast extensor digitorum longus (EDL) muscles and the effect of short-lasting reinnervation in slow muscle only. Denervation was performed by cutting the sciatic nerve. Both muscles were excised 2 weeks later. Reinnervation was studied 4 weeks after nerve crush in SOL muscle only. Capillaries and muscle fibres were visualised by triple immunofluorescent staining with antibodies against CD31 and laminin and with fluorescein-labelled Griffonia (Bandeira) simplicifolia lectin. A recently developed stereological approach allowing the estimation of the length of capillaries adjacent to each individual fibre (Lcap/Lfib) was employed. Three-dimensional virtual test grids were applied to stacks of optical images captured with a confocal microscope and their intersections with capillaries and muscle fibres were counted. Interrelationships among capillaries and muscle fibres were demonstrated with maximum intensity projection of the acquired stacks of optical images. The course of capillaries in EDL seemed to be parallel to the fibre axes, whereas in SOL, their preferential direction deviated from the fibre axes and formed more cross-connections among neighbouring capillaries. Lcap/Lfib was clearly reduced in denervated SOL but remained unchanged in EDL, although the muscle fibres significantly atrophied in both muscle types. When soleus muscle was reinnervated, capillary length per unit fibre length was completely restored. The physiological background for the different responses of the capillary network in slow and fast muscle is discussed. This study was supported by the Slovenian Research Agency and the Ministry of Education, Youth and Sport of the Czech Republic (KONTAKT grant no. 19/2005).     

Multicellular recordings of cultured brainstem neurons in microelectrode arrays

Several vital systemic functions are controlled by the brainstem, which has been studied in a variety of experimental preparations and by various techniques, including in-vitro electrophysiological preparations. Although these in-vitro approaches have greatly advanced the understanding of brainstem neurons, most recording methods with microelectrodes and patch pipettes are invasive. To take advantage of in-vitro approaches but avoid their potential problems, we have studied brainstem neurons in microelectrode arrays (MEA). Neurons were isolated from the medulla oblongata and cultured in DMEM. Extracellular recordings were performed with no evident perturbations to the cellular environment. Neurons started firing after 24–48 h in culture, reached stable activity in 3–4 weeks, and retained this activity for at least 3 months. From their firing patterns, these neurons could be divided into tonic and bursting units. The latter could be further divided into regular and irregular bursters based on their burst intervals. Cells were stimulated or inhibited by exposure to 10% CO₂. The stimulatory effect of CO₂, though smaller, was still seen after selective ablation of serotonergic neurons or with low Ca⁺⁺ and high Mg⁺⁺ in the extracellular medium. Similar treatments had no significant effect on CO₂-inhibited units. The abundance of units with respect to their firing patterns and CO₂ responses, together with the long-term stable non-invasive recordings with no evident perturbation to cellular environments, suggests that MEA represent another promising in-vitro approach for studying brainstem neurons.This work was supported by NIH grant (HL058410, HL067890).     

Characterization of human islet-like structures generated from pancreatic precursor cells in culture

This study addresses the characterization of human islet-like structures generated from a newly discovered sparse population of precursor cells (Petropavlovskaia and Rosenberg, 2002) in the human pancreas. These cells may be progenitor cells capable of producing pancreatic cells suitable for the treatment of type 1 diabetes. The cells were cultured successfully in non-adherent stationary cultures and yielded, as an important first step, a 1.9-fold expansion in a serum-free medium developed specifically for this cell type. This expanded population grew as pancreatic cell aggregates, which were analyzed for islet-like characteristics. Specifically, through RT-PCR analyses and functionality assays, we show that cells within the population expressed all four of the endocrine hormone genes and proteins (insulin, glucagon, somatostatin, pancreatic polypeptide). As well, the expanded pancreatic precursor cell population exhibited glucose responsiveness although the produced cells appeared to be still primitive in nature.     

The effect of EGF and the comitogen, norepinephrine, on the proliferative responses of fresh and cryopreserved rat and mouse hepatocytes

The effect of cryopreservation on the proliferative response of fresh and cryopreserved (CP) rat and mouse hepatocytes was studied. Of the parameters measured, incorporation of 3H-thymidine and bromodeoxyuridine (BdrU) incorporation were the most sensitive and LDH content was the least sensitive. The optimal seeding density for epidermal growth factor (EGF)-stimulated proliferative response in fresh rat and mouse hepatocytes was 1.8 x 10(4) cells/cm2 and 2.1 x 10(4) cells/cm2, respectively. 3H-thymidine incorporation by fresh rat and mouse hepatocytes was maximal in cultures treated with 10 and 5 ng/ml EGF, respectively. The cell attachment of fresh rat hepatocytes after 48 h was higher (68%) than CP (42%), therefore, the CP hepatocyte seeding density was increased to 7.1 x 10(4) cells/cm2 so that the cell number after 48 h was the same as fresh hepatocytes. Using the adjusted seeding density, the 3H-thymidine and BdrU incorporation into fresh and CP rat hepatocytes was equivalent. The attachment efficiencies of fresh and CP mouse hepatocytes were the same, therefore, no adjustment was needed. The proliferative response (3H-thymidine incorporation and DNA content) to EGF was the same in fresh and CP mouse hepatocytes. The comitogen, norepinephrine (NE), increased the proliferative response to EGF to the same extent in both fresh and CP rat hepatocytes. In summary, cryopreserved rat and mouse hepatocytes retain their ability to proliferate in culture. Adjustment and monitoring of the seeding density is of high importance, especially with rat hepatocytes, which lose some attachment capacity after cryopreservation. The secondary mitogenic effect of NE is also retained by cryopreserved rat hepatocytes, suggesting that these cells retain alpha1-receptor function.     

Expression of osteoblast marker genes in rat calvarial osteoblast-like cells, and effects of the endocrine disrupters diphenylolpropane, benzophenone-3, resveratrol and silymarin

Compelling evidence indicates that some endocrine disrupters (EDs), acting as selective estrogen-receptor modulators, interfere with osteoblast differentiation and function. Hence, we investigated whether four EDs [bisphenol-A (BSP), benzophenone-3 (BP3), resveratrol and silymarin] affect differentiation and growth of rat calvarial osteoblast-like (ROB) cells in primary in vitro culture. ROB cells were cultured for up 30 days in a medium supplemented with fetal calf serum (FCS), and conventional RT-PCR detected the expression of collagen-1alpha and osteonectin mRNAs through the entire culture period. Real time-PCR demonstrated that at days 2 and 7 of culture the expressions of collagen-1alpha and osteonectin were very low, and underwent a 192- and a 334-fold increase, respectively, at day 21 of culture. In contrast, osteocalcin expression remained unchanged from days 2 to 21 of culture. EIA showed that ROB cells secreted sizeable amounts of osteocalcin and osteopontin between days 13 and 15 of culture. EDs were added at day 13 of culture at concentrations ranging from 10(-10) to 10(-6) M, being the culture medium deprived of FCS, and their effects were tested 48 h later. None of EDs was found to affect osteocalcin and osteopontin secretion from ROB cells, suggesting that their effects were tested at a relatively earlier stage of culture, when ROB cell differentiation into osteoblats is not fully accomplished, and/or the presence of estrogens contained in FCS is needed for EDs to exert their osteoblast-differentiation modulating action. BSP and BP3, but not resveratrol and silymarin, decreased proliferative activity of cultured ROB cells, a cytotoxic effect conceivably independent of their estrogen-receptor modulating activity.     

Echinostoma caproni: intestinal pathology in the golden hamster, a highly compatible host, and the Wistar rat, a less compatible host

The histopathological changes induced by Echinostoma caproni (Trematoda: Echinostomatidae) in a high (golden hamster) and a low compatible host (rat) were compared at 15 and 30 days post-infection. Infection of rats was characterized by a progressive increase in erosion of villi and elevated numbers of goblet cells, which could be related to the early expulsion of the parasite in a host of low compatibility. In contrast to rats, the number of goblet cell in E. caproni-infected hamsters was low, but increased numbers of neutrophils and mesenteric inflammatory cells were observed. This indicated that local inflammatory responses in hamsters were greater than in rats. An immunohistochemical study using polyclonal IgG anti-E. caproni excretory-secretory antigens demonstrated a greater level of passage of E. caproni antigens through the intestinal mucosa in hamsters than in rats, probably in relation to the greater inflammatory response. Our results indicate the fact that early inflammatory responses could be important for the establishment of E. caproni chronic infections in highly compatible hosts.