Protective effects of polydeoxyribonucleotides on cartilage degradation in experimental cultures

The capacity of cartilage self‐regeneration is considered to be limited. Joint injuries often evolve in the development of chronic wounds on the cartilage surface. Such lesions are associated with articular cartilage degeneration and osteoarthritis. Re‐establishing a correct micro/macro‐environment into damaged joints could stop or prevent the degenerative processes. This study investigated the effect of polydeoxyribonucleotides (PDRNs) on cartilage degradation in vitro and on cartilage extracted cells. The activities of matrix metalloproteinases 2 and 9 were measured in PDRN‐treated cells and in controls at days 0 and 30 of culture. Human nasal cartilage explants were cultured, and the degree of proteoglycan degradation was assessed by measuring the amount of glycosaminoglycans released into the culture medium. The PDRN properties compared with controls were tested on cartilage tissues to evaluate deposition of extracellular matrix. Chondrocytes treated with PDRNs showed a physiological deposition of extracellular matrix (aggrecan and type II collagen: Western blot, IFA, fluorescence activated cell sorting, Alcian blue and safranin O staining). PDRNs were able to inhibit proteoglycan degradation in cartilage explants. The activities of matrix metalloproteinases 2 and 9 were reduced in all PDRN‐treated samples. Our results indicate that PDRNs are suitable for a long‐term cultivation of in vitro cartilage and have therapeutic effects on chondrocytes by protecting cartilage. Copyright © 2012 John Wiley & Sons, Ltd.     

Toxic effects of resazurin on cell cultures

Resazurin, introduced as a cell viability indicator under the trade name alamarBlue^®, is generally regarded as nontoxic when used according to manufacturer’s suggested shorter-term incubation time specifications. However, problems arise when exposure times are extended to longer-term cultures on the order of days. To assess the effect of resazurin over longer incubation times, MCF7 (HTB-22), MCF10A (CRL-10317), 3T3-L1 (CL-173), and D1 (CRL-12424) cultures were tested with varying amounts of resazurin over 4- and 8-day periods. MCF7, 3T3-L1, and D1 cells cultured for 8 days with 20 % alamarBlue^® had significantly less cell survivability. Specifically, levels of metabolic activity, deoxyribonucleic acid (DNA) concentration, and glucose consumption of the cell lines cultured for 8 days in medium with 20 % alamarBlue^® were significantly lower (p < 0.05) than metabolic activity, DNA concentration, and glucose consumption of MCF7 cells cultured for 8 days in medium with no alamarBlue^®. MCF7, 3T3-L1, and D1 cells used less glucose at concentrations as low as 5 %. Data also suggests the toxic effects are more pronounced in the cancerous cell line as compared to the noncancerous cells.     

Protective effect of radicicol against LPS/IFN-gamma-induced neuronal cell death in rat cortical neuron-glia cultures

We investigated the protective activity of radicicol, an antifungal antibiotic, against inflammation-induced neurotoxicity in neuron-glia cultures. Radicicol potently prevented the loss of neuronal cell bodies and neurites from LPS/IFN-gamma-induced neurotoxicity in rat cortical neuron-glia cultures with an EC(50) value of 0.09 microM. Radicicol inhibited the LPS/IFN-gamma-induced expression of inducible nitric oxide synthase (iNOS) and production of nitric oxide (NO) in microglia. Additionally, radicicol decreased the LPS/IFN-gamma-induced release of tumor necrosis factor-alpha (TNF-alpha) in the cultures. The inhibitory potency of radicicol against the production of NO and TNF-alpha was well correlated with the protection of neurons. These results suggest that the protective effect of radicicol against LPS/IFN-gamma-induced neuronal cell death in neuron-glia cultures is mediated via the inhibition of TNF-alpha release, as well as the suppression of iNOS expression in microglia.     

Protective effects of cynaroside on oxidative stress in retinal pigment epithelial cells

Cynaroside is a flavonoid compound proved to possess antioxidant activity, but its protective effect on age‐related macular degeneration still remains unclear. In this study, the protective effects of cynaroside on oxidative stress and apoptosis in retinal pigment epithelial (RPE) cells induced by hydrogen peroxide (H₂O₂) were investigated. Results showed that cynaroside effectively attenuated the decrease of cell activity induced by H₂O₂. The total reactive oxygen species can be remitted by decreasing malondialdehyde level, as well as increasing glutathione level, and superoxide dismutase and catalase activities. In addition, Western blot analysis indicated that cynaroside protected ARPE‐19 cells from apoptosis through downregulation of caspase‐3 protein activation which was controlled by the upstream proteins Bcl‐2 and Bax. It was finally proved that cynaroside could enhance the antioxidant and antiapoptotic ability in ARPE‐19 cells by promoting the expression of p‐Akt.     

Protective effect of tetramethylpyrazine on absolute ethanol-induced renal toxicity in mice

Acute administration of absolute ethanol (10 ml/kg) per os to fasted mice produced extensive renal failure as measured by a rise in blood urea nitrogen and creatinine. Pretreatment with oral administration of tetramethylpyrazine (TMP) prevented such failure. The maximal effect against absolute ethanol-induced renal failure could be observed 1 h after TMP administration. In order to further investigate the renal protective mechanism of TMP, experiments on lipid peroxidation and superoxide scavenging activity were conducted. Renal homogenates made from mice treated with ethanol showed that TMP pretreatment had an antioxidant effect. Mice in acute renal failure had higher malonic dialdehyde concentrations than those pretreated with TMP. The renal protective mechanism of TMP was attributed, in part, to its prominent superoxide scavenging effect, which protects the kidney from superoxide-induced renal damage.     

Protective effect of hypothermia during ischemia in neural cell cultures

Hypothermia offers protection from the effects of ischemia in small animals. We have recently shown that similar to small animals, hypothermia may also be protective in an astrocytic model of simulated ischemia in cell culture. This study was designed to look at the protective effects of hypothermia in cultures of cerebellar granular (glutamatergic) and cortical (GABAergic) neurons. We used LDH release into the medium as an indicator for neuron damage. Experiments were all done in sister cultures, in groups of six cultures at two temperatures (37 and 32 degrees Celsius). The duration of ischemia was three hours in cerebellar granular neuronal cell cultures and six hours in cortical neurons. LDH release was measured immediately after the insult. Hypothermia protected both granular and cortical neurons. In granular cells, LDH release was 62+/–18 at 32 degrees and 212+/–15 at 37 degrees (p=0.02). Cortical neurons showed LDH release of 15+/–2 at 32 degrees and 32+/–2 at 37 degrees (p=0.005). Our study suggests that similar to astrocytes, the protective effects of hypothermia are evident in neuronal cell cultures from the cerebellum and the cerebral cortex. Cell culture systems should prove useful techniques in understanding mechanisms of hypothermic protection during simulated ischemia in neurons from different sites.     

Neuroprotective effects of interleukin-6 on NMDA-induced rat retinal damage

This study shows that interleukin-6 (IL-6) combined with soluble interleukin-6 receptors (sIL-6R) modulates N-methyl-D-aspartate (NMDA)-induced retinal damage. Eyes pretreated with a combined injection of IL-6 and sIL-6R had NMDA administered into the vitreous cavity. Morphometric analysis and retrograde labeling analysis found that pretreatment with either IL-6 or sIL-6R alone did not bring about any neuroprotective effect. However, pretreatment with a combined administration of IL-6 and sIL-6R induced a significant neuroprotective effect against NMDA-induced retinal damage. Apoptotic changes in the retina were assessed by the TUNEL method. The results indicated that pretreatment with IL-6 combined with sIL-6R prevents NMDA-induced apoptosis. Western blotting studies demonstrated upregulation of gp130 expression in the NMDA-injected retina. Present studies suggest that IL-6 combined with sIL-6R provides a neuroprotective effect on NMDA-induced retinal damage.     

Steroid synthesis in rat brain cell cultures

Primary cultures derived from neonatal rat forebrains were established and cultured for several weeks. They grow entirely as glial cultures composed of oligodendrocytes and astrocytes. Glial cells undergo maturation and differentiation in culture. This was shown by measuring the oligodendroglial enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), a specific marker for expression of oligodendrocyte differentiation. CNPase activity increased from days 10-21 of culture. Both cell types were characterized by indirect immunofluorescence staining using monoclonal antibodies to galactocerebroside (Gal C) and myelin basic protein (MBP) for oligodendrocytes, and glial fibrillary acidic protein (GFAP) for astrocytes. Using the above criteria, we measured about 60% oligodendrocytes and 40% astrocytes after 3 weeks of culture. Oligodendrocytes, expressing Gal C and MBP, were highly immunoreactive to monospecific polyclonal antibodies to the cytochrome P-450scc, enzyme involved in the synthesis of pregnenolone from cholesterol. After incubation of glial cultures with [3H]mevalonolactone in the presence of mevinoline and trilostane, biosynthesis of [3H]cholesterol, [3H]pregnenolone (P) and [3H]pregn-5-ene-3 beta, 20 alpha-diol (20-OHP) was demonstrated. Steroid biosynthesis was related to oligodendroglial differentiation, as the initial and rapid rate of increase in CNPase activity was found to occur at the same time as the onset of steroid synthesis. Both reached a maximum at 3 weeks of culture and remained stable for several weeks. Steroid synthesis was increased by dibutyryl cAMP (0.2 mM), as well as by dexamethasone (10 nM). When aminoglutethimide, a potent inhibitor of cytochrome P-450scc, was added during the incubation of cells with [3H]mevalonolactone, [3H]cholesterol accumulated in the cells. After the release of aminoglutethimide blockade, [3H]20-OHP was the major steroid produced and released in the culture medium. The demonstration of de novo steroid biosynthesis and of the cholesterol side-chain cleavage cytochrome P-450 in normal rat glial cells brings additional support to the concept of "neurosteroids".     

Environmental enrichment effects on development of retinal ganglion cell dendritic stratification require retinal BDNF

A well-known developmental event of retinal maturation is the progressive segregation of retinal ganglion cell (RGC) dendrites into a and b sublaminae of the inner plexiform layer (IPL), a morphological rearrangement crucial for the emergence of the ON and OFF pathways. The factors regulating this process are not known, although electrical activity has been demonstrated to play a role. Here we report that Environmental Enrichment (EE) accelerates the developmental segregation of RGC dendrites and prevents the effects exerted on it by dark rearing (DR). Development of RGC stratification was analyzed in a line of transgenic mice expressing plasma-membrane marker green fluorescent protein (GFP) under the control of Thy-1 promoter; we visualized the a and b sublaminae of the IPL by using an antibody selectively directed against a specific marker of cholinergic neurons. EE precociously increases Brain Derived Neurotrophic Factor (BDNF) in the retina, in parallel with the precocious segregation of RGC dendrites; in addition, EE counteracts retinal BDNF reduction in DR retinas and promotes a normal segregation of RGC dendrites. Blocking retinal BDNF by means of antisense oligos blocks EE effects on the maturation of RGC dendritic stratification. Thus, EE affects the development of RGC dendritic segregation and retinal BDNF is required for this effect to take place, suggesting that BDNF could play an important role in the emergence of the ON and OFF pathways.     

Characterization of photoreceptor cell differentiation in the rat retinal cell culture

Photoreceptor cell differentiation in the rat retina was studied in vivo and in vitro, using an immunohistochemical method to demonstrate opsin-like immunoreactivity. Cells in a dissociated monolayer culture expressed some properties characteristic of rat rod cells developing in vivo, including a ciliary structure and opsin-like immunoreactivity. Immunoblot analysis revealed that cultured retinal cells synthesize a polypeptide with the same molecular weight as that synthesized by the intact retina. Although the outer segment (OS) was not present in the culture, immunoreactive cells possessed a ciliary structure. Opsin-like immunoreactivity was found on the plasma membrane, including the cilia. The neuritic extensions were also intensely stained. In mature rod cells of the intact rat retina, opsin was detected only on the OS but, during development, it was found both in the somatic region of the rod cells and on the differentiating OS. During maturation of rod cells opsin immunoreactivity seemed to shift to the OS from other locations. However, some "displaced" photoreceptor cells, found in the inner nuclear layer and extending fibers bipolarly, retained immunoreactivity throughout their structure. The absence of polarized distribution of opsin in these cells is considered to be due to an abnormal environment, which may also be the case with cultured retinal cells. The present culture conditions will offer a useful model system to understand the cellular mechanism of the hereditary retinal dystrophy of rodent animals in which photoreceptor cells selectively degenerate.     

Effect of daptomycin on primary rat muscle cell cultures in vitro

Daptomycin is a lipopeptide antibiotic that has strong bactericidal activity against Gram-positive bacteria and that was previously reported to exhibit minor side effects on skeletal muscle. This study was designed to further characterize the effect of daptomycin on skeletal muscle through the use of primary cultures of muscles from rats. Our investigations demonstrated that daptomycin has a concentration-dependent and time-dependent effect on the plasma membrane of primary cultures of differentiated, spontaneously contracting rat myotubes. No effects were evident in non-differentiated myoblasts or other mononucleated cells present in cultures even at the highest daptomycin concentrations tested (6,000 μg/mL). In cultures treated with daptomycin at a concentration of 2,000 μg/mL, plasma membrane damage was observed in ∼20–30% of differentiated myotubes; no myotube damage was detected at concentrations of 1,000 μg/mL and below. A transient loss of spontaneous myotube contractions was evident at 750 μg/mL, while at 2,000 μg/mL and above, a permanent loss of spontaneous contractility was observed. These results suggest that the putative targets for daptomycin effects on skeletal muscle are structures on the plasma membrane of highly differentiated myotubes.     

Opposing effects by glucocorticoid and bone morphogenetic protein-2 in fetal rat bone cell cultures

Glucocorticoid in excess produces bone loss in vivo. Consistent with this, it reduces the stimulatory effect of transforming growth factor β (TGF-β) on collagen synthesis in osteoblast-enriched cultures in vitro, where it also suppresses TGF-β binding to its type I receptors. Analogous studies with bone morphogenetic protein-2 (BMP-2) show directly opposite results. These findings prompted us to assess the effect of glucocorticoid on BMP-2 activity in cultured bone cells, and whether either agent had a dominant influence on TGF-β binding or function. BMP-2 activity was retained in part in osteoblast-enriched cultures pre-treated or co-treated with cortisol, and was fully evident when glucocorticoid exposure followed BMP-2 treatment. In addition, BMP-2 suppressed the effects of cortisol on TGF-β activity, on TGF-β binding, and on gene promoter activity directed by a glucocorticoid sensitive transfection construct. While BMP-2 also alters the function of less-differentiated bone cells, it only minimally prevented cortisol activity in these cultures. Our studies indicate that BMP-2 can oppose certain effects by cortisol on differentiated osteoblasts, and may reveal useful ways to diminish glucocorticoid-dependent bone wasting. J. Cell. Biochem. 67:528–540, 1997. © 1997 Wiley-Liss, Inc.     

Protective effects of autophagy against blue light-induced retinal degeneration in aged mice

The aim of this study was to explore the role of autophagy in response to blue light damage in aged mice and in human retinal pigmented epithelium(hRPE) cells. Blue light damage to the retina was induced in 10-month-old(10 mo) C57 mice and hRPE cells. Flash electroretinography was used to assess retinal function. Retinal structure changes were observed by electron microscopy. Western blot was conducted to determine the expression levels of the following proteins: cleaved caspase-3, p38 mitogen-activated protein kinases, protein kinase R-like endoplasmic reticulum kinase(PERK), autophagy marker light chain 3(LC3), P62, and Beclin-1. On day 1 after light damage to the 10 mo mice, retinal function was changed. The latent periods of awave and b-wave were delayed, and amplitude was reduced. The electron microscopy results revealed mitochondria damage in the retinal pigmented epithelium and a disorganized photoreceptor outer segment(OS). PERK, LC3, and Beclin-1 were upregulated, whereas P62 was not. On day 5 after the blue light damage, restoration of electroretinography and OS was observed.PERK, LC3, and Beclin-1 were downregulated, whereas P62 was not. Protein changes in vitro were consistent with in vivo. The present study provided structural and functional evidence that autophagy plays an important role in the response to blue lightinduced retinal damage.     

Optimal trypsin concentration for rat heart cell cultures

Three concentrations (0.5, 1 and 2 per 1000, w/v) of a single batch of trypsin were compared regarding their influence on cultured cardiac cell of newborn rats. All three allowed to obtain cardiac cells in good conditions, as evidence by beating frequencies and [16-14C]-palmitate beta-oxidation. However the 0.5 per 1000 concentration appeared to be the optimal one, inducing a smaller loss of cells during the first two days in culture.