Stage dependency in eliciting target-dependent enhanced neurite outgrowth from spinal cord explants in vitro

Explants of frog tadpole spinal cord exhibit varying degrees of enhanced neuritic outgrowth in tissue culture that is dependent on the presence of appropriately staged limb target tissue. Nerve fiber outgrowth from early larval spinal cords was greatly augmented in the presence of early larval limb tissue, but was unresponsive to older limb tissues. Spinal cord explants of midlarval animals, on the other hand, failed to respond significantly to limb tissues over a broad developmental range. Nerve-target growth interactions must be regarded as developmental time-dependent systems with consideration of the differentiative state of both components.     

Intrinsic properties inhibit axonal outgrowth from neonatal rat spinal cord explant

Lumbar spinal cord explants, harvested from neonatal rat pups aged between postnatal day 0 (P0) and P6, were cultured for a period of 48 hrs in the chemically defined medium R(12) on a poly-ethylene-imine (PEI) and on poly-D-lysin (PDL) coated surface. The outgrowth outside the explant was quantified. Lumbar explants from the same rat and embedded in a collagen matrix, and cortical explants from a P0 rat were used as controls. Statistical analysis demonstrated a clear relation between age-at-explantation and the number of neurites in the corona surrounding the explant. The number of outgrowing neurites decreased sharply with age-at-explantation. The average number of neurites per explant obeyed to the expression log (n) = -0.736x + 3.294 on PEI, and log (n) = -0.721x + 2.295 on PDL; x epsilon in [P0 - P6] (n, the number of neurites per explant; x, the age-at-explantation expressed in postnatal days). A similar observed age-related decrease of outgrowth has been described when culturing the lumbar explant inside a collagen matrix. The phenomenon appears to be an intrinsic property of the explant. We review growth inhibitory properties in different models and propose that the phenomenon occurs here at the interface explant-world.     

Synaptic profiles in the outgrowth from chick spinal cord in vitro

Summary Synaptic profiles have been identified in the outgrowth from chick embryo spinal cord maintained in vitro for short periods. Profiles corresponding to types that may be excitatory and inhibitory in the intact central nervous system have been found. Their presence outside expiants, and in occasional relation to glial cells, suggests that neurites themselves may possess a generalised capacity for synapse formation under appropriate circumstances, rather than be limited to specific targets.     

Conditioned medium derived from umbilical cord mesenchymal stem cells regenerates atrophied muscles

We investigated the regenerative effects and regulatory mechanisms of human umbilical cord mesenchymal stem cells (UC-MSCs)-derived conditioned medium (CM) in atrophied muscles using an in vivo model. To determine the appropriate harvest point of UC-CM, active factor content was analyzed in the secretome over time. A muscle atrophy model was induced in rats by hindlimb suspension (HS) for 2 weeks. Next, UC-CM was injected directly into the soleus muscle of both hind legs to assess its regenerative efficacy on atrophy-related factors after 1 week of HS. During HS, muscle mass and muscle fiber size were significantly reduced by over 2-fold relative to untreated controls. Lactate accumulation within the muscles was similarly increased. By contrast, all of the above analytical factors were significantly improved in HS-induced rats by UC-CM injection compared with saline injection. Furthermore, the expression levels of desmin and skeletal muscle actin were significantly elevated by UC-CM treatment. Importantly, UC-CM effectively suppressed expression of the atrophy-related ubiquitin E3-ligases, muscle ring finger 1 and muscle atrophy F-box by 2.3- and 2.1-fold, respectively. UC-CM exerted its actions by stimulating the phosphoinositol-3-kinase (PI3K)/Akt signaling cascade. These findings suggest that UC-CM provides an effective stimulus to recover muscle status and function in atrophied muscles.     

Conditioned serum-free medium from umbilical cord mesenchymal stem cells has anti-photoaging properties

Chronic exposure to solar radiation is the primary cause of photoaging and benign and malignant skin tumors. A conditioned serum-free medium (SFM) was prepared from umbilical cord mesenchymal stem cells (UC-MSCs) and its anti-photoaging effect, following chronic UV irradiation in vitro and in vivo, was evaluated. UC-MSC SFM had a stimulatory effect on human dermal fibroblast proliferation and reduced UVA-induced cell death. In addition, UC-MSC SFM blocked UVA inhibition of superoxide dismutase activity. Topical application of UC-MSC SFM to mouse skin prior to UV irradiation blocked the inhibition of superoxide dismutase and glutathione peroxidase activities, and prevented the upregulation of malonaldehyde. UC-MSC SFM thus protects against photoaging induced by UVA and UVB radiation and is a promising candidate for skin anti-photoaging treatments.     

Conditioned medium enhances the fusion capability of rat bone marrow mesenchymal stem cells and cardiomyocytes

Mesenchymal stem cells (MSCs) show accelerated regeneration potential when these cells experience hypoxic stress. This “preconditioning” has shown promising results with respect to cardio-protection as it stimulates endogenous mechanisms resulting in multiple cellular responses. The current study was carried out to analyze the effect of hypoxia on the expression of certain growth factors in rat MSCs and cardiomyocytes (CMs). Both cell types were cultured and assessed separately for their responsiveness to hypoxia by an optimized dose of 2,4,-dinitrophenol (DNP). These cells were allowed to propagate under normal condition for either 2 or 24 h and then analyzed for the expression of growth factors by RT-PCR. Variable patterns of expression were observed which indicate that their expression depends on the time of re-oxygenation and extent of hypoxia. To see whether the growth factors released during hypoxia affect the fusion of MSCs with CMs, we performed co-culture studies in normal and conditioned medium. The conditioned medium is defined as the medium in which CMs were grown for re-oxygenation till the specified time period of either 2 or 24 h after hypoxia induction. The results showed that the fusion efficiency of cells was increased when the conditioned medium was used as compared to that in the normal medium. This may be due to the presence of certain growth factors released by the cells under hypoxic condition that promote cell survival and enhance their fusion or regenerating ability. This study would serve as another attempt in designing a therapeutic strategy in which conditioned MSCs can be used for ischemic diseases and provide more specific therapy for cardiac regeneration.     

Lactoferrin enhances opioid-mediated analgesia via nitric oxide in the rat spinal cord

Lactoferrin (LF) is a multifunctional protein that is found in milk, neutrophils, and other biological fluids, and its receptors have also been identified in the central nervous system. Recently, we found that bovine milk-derived LF (BLF) produced analgesia via a mu-opioid receptor-mediated response in the spinal cord. However, the precise mechanism of this analgesic effect remains unclear. In this study, spinally applied BLF produced analgesia that was reversed by coadministration with a nitric oxide (NO) synthase inhibitor, NG-nitro-l-arginine methyl ester, during phases 1 and 2 in the formalin test. Spinal coadministration of a mu-opioid receptor agonist, morphine, with a subeffective dose of BLF produced a much more highly potentiated analgesia than that produced by morphine alone during phases 1 and 2 in the formalin test. This potentiated analgesia by morphine with BLF was reversed by a mu-opioid receptor antagonist, d-Phe-Cys-Tyr-d-Trp-Orn-Thr-NH2, or by NG-nitro-l-arginine methyl ester. In the tail-flick test, continuous spinal infusion of morphine via an osmotic minipump over 6 days resulted in development of tolerance by day 4, but no tolerance of BLF was observed throughout the experiment. These results suggest that BLF acts as an enhancer of the spinal opioidergic system via an NO-mediated mechanism.     

Conditioned medium from umbilical cord mesenchymal stem cells improves nasal mucosa damage by radiation

Objectives

To explore therapeutic effects of conditioned medium from human umbilical cord mesenchymal stem cells (hUC-MSCs) on nasal mucosa radiation damage both in vivo and in vitro.

Results

The mucus cilia clearance time (7 and 30 days), degree of mucosal edema (7, 30, 90 and 180 days), cilia coverage (180 days) of concentrated conditioned medium group improved compared with radiotherapy control group. The proliferation and migration abilities of irradiated and non-irradiated nasal epithelial cells significantly increased after culture in bronchial epithelial cell growth medium (BEGM) containing 10% conditioned medium of hUC-MSCs compared to cells cultured in BEGM alone.

Conclusions

Soluble factors secreted by hUC-MSCs may promote nasal epithelial cell proliferation and migration. Intranasal administration of hUC-MSC conditioned medium effectively repairs nasal mucosa radiation damage.

     

Imatinib enhances functional outcome after spinal cord injury

We investigated whether imatinib (Gleevec?, Novartis), a tyrosine kinase inhibitor, could improve functional outcome in experimental spinal cord injury. Rats subjected to contusion spinal cord injury were treated orally with imatinib for 5 days beginning 30 minutes after injury. We found that imatinib significantly enhanced blood-spinal cord-barrier integrity, hindlimb locomotor function, sensorimotor integration, and bladder function, as well as attenuated astrogliosis and deposition of chondroitin sulfate proteoglycans, and increased tissue preservation. These improvements were associated with enhanced vascular integrity and reduced inflammation. Our results show that imatinib improves recovery in spinal cord injury by preserving axons and other spinal cord tissue components. The rapid time course of these beneficial effects suggests that the effects of imatinib are neuroprotective rather than neurorestorative. The positive effects on experimental spinal cord injury, obtained by oral delivery of a clinically used drug, makes imatinib an interesting candidate drug for clinical trials in spinal cord injury.     

Kidney glomerular explants in serum-free media: role of individual medium components in cell outgrowth

Guinea pig glomeruli were grown for 22 days in a serum-free medium composed of Waymouth's MB 752/1 supplemented with sodium pyruvate, nonessential amino acids, and antibiotics. To this basic medium was added insulin, transferrin, selenium (Se), tri-iodothyronine, or fibronectin (FN) - either singly, or in various combinations - and sequential quantitative studies of the glomerular outgrowths were performed. Total cells in glomerular outgrowths, mitotic index, and glomerular attachment rate were determined and compared with values for glomerular outgrowths in media containing either no additions or all of the above components. FN was required for whole glomerular attachment, while transferrin plus FN was required for mitosis in glomerular cell outgrowths. Insulin and tri-iodothyronine slightly increased glomerular cell outgrowth by slightly increasing whole glomerular attachment, but had little effect on mitosis in glomerular outgrowths. The effect of Se was complex. Se did not affect whole glomerular attachment or mitosis in the presence of transferrin plus FN. However, in a medium containing transferrin, FN, and 3-amino-1,2,4-triazole (AT) (an inhibitor of catalase and glutathione peroxidase), Se increased total cell number but had little effect on the glomerular attachment rate or the mitotic index. Morphologic analysis of glomeruli early in culture suggested that Se may act by decreasing the amount of or delaying the time of cell death. In all of the media tested, total DNA was relatively constant over the course of 22 days, suggesting the possibility that glomerular cells cultured in a serum-free medium are part of a cell renewal system.     

Explant cultures of teleost spinal cord: source of neurite outgrowth

The source of neurite outgrowth in explant cultures of normal adult Apteronotus spinal cord was examined. Explants which contained the central region of spinal cord, including ependyma, showed neurite outgrowth in culture. Explants which did not contain ependyma showed no neurite outgrowth. It is concluded that the ependymal region is necessary for neurite outgrowth in these cultures of adult teleost spinal cord. In addition, our failure to observe axon outgrowth clearly attributable to fluorescently back-labeled electromotor neurons in these cultures suggests that the exuberant neurite outgrowth in vitro is most probably due to cells other than the electromotor neurons. This explant culture system provides a unique opportunity to study neuronal differentiation, regeneration, and neurogenesis in vitro.     

Adult rat spinal cord culture on an organosilane surface in a novel serum-free medium

Summary In this study, we have documented by morphological analysis, immunocytochemistry, and electrophysiology, the development of a culture system that promotes the growth and long-term survival of dissociated adult rat spinal cord neurons. This system comprises a patternable, nonbiological, cell growth-promoting organosilane substrate coated on a glass surface and an empirically derived novel serum-free medium, supplemented with specific growth factors (acidic fibroblast growth factor, heparin sulfate, neurotrophin-3, brain-derived neurotrophic factor, glial-derived neurotrophic factor, cardiotrophin-1, and vitronectin). Neurons were characterized by immunoreactivity for neurofilament 150, neuron-specific enolase, Islet-1 antibodies, electrophysiology, and the cultures were maintained for 4–6 wk. This culture system could be a useful tool for the study of adult mammalian spinal neurons in a functional in vitro system.     

Islet-neogenesis-associated protein enhances neurite outgrowth from DRG neurons

Islet-neogenesis-associated protein, INGAP, is a 175-amino-acid pancreatic acinar protein that stimulates pancreatic duct cell proliferation in vitro and islet neogenesis in vivo. To date, the mitogenic activity of INGAP has been identified only in nonneural tissues. The aim of this study was to examine the effects of a pentadecapeptide of INGAP (INGAP peptide), the biologically active portion of the native protein, in cultured dorsal root ganglia (DRG) explants from C57BL/6 mice. The present study provides evidence that INGAP peptide acts as a mitogen in the peripheral nervous system (PNS), and that it enhances neurite outgrowth from DRGs in vitro in a time- and dose-dependent manner. The neuritogenic action of INGAP peptide correlates with an increase in [(3)H]thymidine incorporation (P < 0.0001) and mitochondrial activity (P < 0.001). Results from these studies suggest that INGAP peptide promotes Schwann cell proliferation in the DRG which releases trophic factors that promote neurite outgrowth.     

Dynorphin A inhibits nociceptin-converting enzyme from the rat spinal cord

Cysteine proteinase found in the spinal cord of rat, called nociceptin-converting enzyme (NCE), is competitively inhibited by dynorphin A and its fragment des-[Tyr(1)]-DYN A. This proteinase converts orphanin FQ/nociceptin (OFQ/N) to two major fragments: OFQ/N(1-11) and further OFQ/N(1-6) with analgesic properties. Dynorphin A at the concentration of 10 microM increases K(M) from 15.0 to 55.9 microM. The calculated K(i) for this interaction was estimated at 3.7 microM. This observation may suggest an interaction between opioid and nociceptive systems which may be affected by the balance between opioid and antiopioid systems. This balance between particular OFQ/N sequences that are derived from the same precursor and regulated by proteinases may play an important role in pain. Interestingly, dynorphin B does not reveal a similar action on the NCE.     

Innervation in cultures of fetal rodent skeletal muscle by organotypic explants of spinal cord from different animals

Summary Functional neuromuscular junctions formin vitro between spatially separated explants of fetal mammalian spinal cord and fetal skeletal muscle, even across species lines (rat and mouse). Differentiation and innervation occur when the muscle explant is oriented toward the ventral edge of the spinal cord cross-section, in the path of ventral-root nerve fibers. Arrival of these neurites enhances muscle development. This trophic influence is particularly apparent when cortisone is included in the nutrient fluid. Cross-striations begin to form toward the end of the first week of coupling, and acetylcholinesterase-positive loci appear by three weeks. In cultures maintained for 5–11 weeks, the more differentiated motor endplate structures show characteristic subneural infoldings, increased soleplate sarcoplasm, and terminal Schwann cells. Myelinated ventral-root fibers can be seen to bridge the gap between the cord and muscle explants, and to arborize and terminate on muscle fibers. Selective stimulation of ventral cord or ventral root can evoke widespread synchronized contractions of large numbers of fibers in the muscle expiant, demonstrating abundant formation of functional neuromuscular junctions between the coupled tissues.This study was supported by grants NS-06735, NS-06545 and NS-08770 from the National Institute of Neurological Diseases and Stroke, and the Nancy Louise Tryner Memorial Grant (No. 433) from the National Multiple Sclerosis Society.Kennedy Scholar at the Rose F. Kennedy Center for Research in Mental Retardation and Human Development (Albert Einstein College of Medicine).     

Conditioned medium from hypoxic cells protects cardiomyocytes against ischemia

The hypothesis of the present study is that cardiomyocytes subjected to prolonged ischemia, may release survival factors that will protect new cardiac cells from ischemic stress. We exposed neonatal rat cardiomyocyte primary cultures to hypoxia, collected the supernatant, treated intact cardiac cells by this posthypoxic supernatant, and exposed them to hypoxia. The results show cardioprotection of the treated cells compared with the untreated ones. We named the collected posthypoxic supernatant "conditioned medium" (CM), which acts in a dose-dependent manner to protect new cardiac cells from hypoxia: 100 or 75% of CM diluted in phosphate-buffered saline (PBS) protected cells as if they were not exposed to hypoxia (P < 0.001). When CM was removed from the cells before hypoxia, protection was not observed. CM also protected skeletal muscle cultures from hypoxia, but not cardiac cells against H(2)O(2)-induced cell damage. Finally, CM treatment protected the isolated heart in Langendorff set-up against ischemia. Smaller infarct size (9.9 ± 4.4% vs. 28.3 ± 8.5%, P < 0.05), better Rate Pressure Product (67 ± 11% vs. 48.6 ± 13.4%, P < 0.05) and better rate of contraction and relaxation were observed following ischemia and reperfusion (1341 ± 399 mmHg/s vs. 951 ± 349 mmHg/s, P < 0.05 and 1053 ± 347 mmHg/s vs. 736 ± 314 mmHg/s, P < 0.05). To conclude, there are factors that are released from the heart cells subjected to ischemia/hypoxia that protects cardiomyocytes from ischemic stress.