RESUMEN
Peripheral nerve injuries are debilitating, and current clinical management is limited to surgical intervention, which often leads to poor functional outcomes. Development of pharmacological interventions aimed at enhancing regeneration may improve this. One potential pharmacological target is the P2X purinergic receptor 7 (P2X7R) expressed in Schwann cells, which is known to play a role during the development of the peripheral nerves. Herein, we analysed differences in regeneration between genetically engineered P2X7 knockout mice and wild-type controls, using in vivo and ex vivo models of peripheral nerve regeneration. We have found that the speed of axonal regeneration is unaltered in P2X7 knockout mice, nevertheless regenerated P2X7 knockout nerves are morphologically different to wild-type nerves following transection and immediate repair. Indeed, the detailed morphometric analysis at 4 and 8 weeks after injury showed evidence of delayed remyelination in P2X7 knockout mice, compared to the wild-type controls. Furthermore, the Wallerian degeneration phase was unaltered between the two experimental groups. We also analysed gene expression changes in the dorsal root ganglia neurones as a result of the peripheral nerve injury, and found changes in pathways related to pain, inflammation and cell death. We conclude that P2X7 receptors in Schwann cells may be a putative pharmacological target to control cell fate following injury, thus enhancing nerve re-myelination.
Asunto(s)
Traumatismos de los Nervios Periféricos , Receptores Purinérgicos P2X7 , Animales , Axones , Ganglios Espinales , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Regeneración Nerviosa , Receptores Purinérgicos P2X7/genética , Células de Schwann , Nervio CiáticoRESUMEN
The development of neuropathy and of mood alterations is frequent after chemotherapy. These complications, independent from the antitumoral mechanism, are interconnected due to an overlapping in their processing pathways and a common neuroinflammatory condition. This study aims to verify whether in mice the treatment with the proteasome inhibitor bortezomib (BTZ), at a protocol capable of inducing painful neuropathy, is associated with anxiety, depression and supraspinal neuroinflammation. We also verify if the therapeutic treatment with the antagonist of the prokineticin (PK) system PC1, which is known to contrast pain and neuroinflammation, can prevent mood alterations. Mice were treated with BTZ (0.4 mg/kg three times/week for 4 weeks); mechanical allodynia and locomotor activity were evaluated over time while anxiety (dark light and marble burying test), depression (sucrose preference and swimming test) and supraspinal neuroinflammation were checked at the end of the protocol. BTZ treated neuropathic mice develop anxiety and depression. The presence of mood alterations is related to the presence of neuroinflammation and PK system activation in prefrontal cortex, hippocampus and hypothalamus with high levels of PK2 and PKR2 receptor, IL-6 and TNF-α, TLR4 and an upregulation of glial markers. PC1 treatment, counteracting pain, prevented the development of supraspinal inflammation and depression-like behavior in BTZ mice.
Asunto(s)
Afecto/efectos de los fármacos , Bortezomib/farmacología , Inhibidores de Proteasoma/farmacología , Factor de Crecimiento Endotelial Vascular Derivado de Glándula Endocrina/metabolismo , Animales , Ansiedad/tratamiento farmacológico , Ansiedad/metabolismo , Biomarcadores/metabolismo , Citocinas/metabolismo , Depresión/tratamiento farmacológico , Depresión/metabolismo , Modelos Animales de Enfermedad , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/metabolismo , Hipotálamo/efectos de los fármacos , Hipotálamo/metabolismo , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Locomoción/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Dolor/tratamiento farmacológico , Dolor/metabolismo , Enfermedades del Sistema Nervioso Periférico/tratamiento farmacológico , Enfermedades del Sistema Nervioso Periférico/metabolismo , Corteza Prefrontal/efectos de los fármacos , Corteza Prefrontal/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
BACKGROUND: DNA-RNA compounds have shown promising protection against cell oxidative stress. This study aimed to assess the cytotoxicity, protective, or preventive effect of different experimental formulations on oral epithelia's oxidative stress in vitro. METHODS: Reconstituted human oral epithelia (RHOE) were grown air-lifted in a continuous-flow bioreactor. Mouthwashes and gels containing DNA-RNA compounds and other bioactive molecules were tested on a model of oxidative stress generated by hydrogen peroxide treatment. Epithelia viability was evaluated using a biochemical MTT-based assay and confocal microscopy; structural and ultrastructural morphology was evaluated by light microscopy and TEM. RESULTS: DNA-RNA showed non-cytotoxic activity and effectively protected against oxidative stress, but did not help in its prevention. Gel formulations did not express adequate activity compared to the mouthwashes. Excipients played a fundamental role in enhancing or even decreasing the bioactive molecules' effect. CONCLUSION: A mouthwash formulation with hydrolyzed DNA-RNA effectively protected against oxidative stress without additional enhancement by other bioactive molecules. Active compounds, such as hyaluronic acid, ß-Glucan, allantoin, bisabolol, ruscogenin, and essential oils, showed a protective effect against oxidative stress, which was not synergistic with the one of DNA-RNA. Incorporation of surfactant agents showed a reduced, yet significant, cytotoxic effect.
Asunto(s)
Mucosa Bucal/metabolismo , Antisépticos Bucales/farmacología , Estrés Oxidativo/efectos de los fármacos , Reactores Biológicos/microbiología , ADN/farmacología , Epitelio/efectos de los fármacos , Epitelio/metabolismo , Geles/farmacología , Ingeniería Genética/métodos , Humanos , Mucosa Bucal/efectos de los fármacos , Antisépticos Bucales/metabolismo , ARN/farmacologíaRESUMEN
BACKGROUND: Neuropathy is a dose-limiting side effect of many chemotherapeutics, including bortezomib. The mechanisms underlying this condition are not fully elucidated even if a contribution of neuroinflammation was suggested. Here, we investigated the role of a chemokine family, the prokineticins (PKs), in the development of bortezomib-induced peripheral neuropathy (BIPN), and we used a PK receptor antagonist to counteract the development and progression of the pathology. METHODS: Neuropathy was induced in male C57BL/6J mice by using a protocol capable to induce a detectable neuropathic phenotype limiting systemic side effects. The presence of allodynia (both mechanical and thermal) and thermal hyperalgesia was monitored over time. Mice were sacrificed at two different time points: 14 and 28 days after the first bortezomib (BTZ) injection. At these times, PK system activation (PK2 and PK-Rs), macrophage and glial activation markers, and cytokine production were evaluated in the main station involved in pain transmission (sciatic nerve, DRG, and spinal cord), and the effect of a PK receptors antagonist (PC1) on the same behavioral and biochemical parameters was assessed. Structural damage of DRG during BTZ treatment and an eventual protective effect of PC1 were also evaluated. RESULTS: BTZ induces in mice a dose-related allodynia and hyperalgesia and a progressive structural damage to the DRG. We observed a precocious increase of macrophage activation markers and unbalance of pro- and anti-inflammatory cytokines in sciatic nerve and DRG together with an upregulation of GFAP in the spinal cord. At higher BTZ cumulative dose PK2 and PK receptors are upregulated in the PNS and in the spinal cord. The therapeutic treatment with the PK-R antagonist PC1 counteracts the development of allodynia and hyperalgesia, ameliorates the structural damage in the PNS, decreases the levels of activated macrophage markers, and prevents full neuroimmune activation in the spinal cord. CONCLUSIONS: PK system may be a strategical pharmacological target to counteract BTZ-induced peripheral neuropathy. Blocking PK2 activity reduces progressive BTZ toxicity in the DRG, reducing neuroinflammation and structural damage to DRG, and it may prevent spinal cord sensitization.
Asunto(s)
Antineoplásicos/toxicidad , Bortezomib/toxicidad , Hormonas Gastrointestinales/metabolismo , Neuropéptidos/metabolismo , Enfermedades del Sistema Nervioso Periférico/inducido químicamente , Enfermedades del Sistema Nervioso Periférico/metabolismo , Animales , Modelos Animales de Enfermedad , Hiperalgesia/inducido químicamente , Hiperalgesia/metabolismo , Inflamación/inducido químicamente , Inflamación/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Péptidos/metabolismoRESUMEN
We aimed at analyzing the effect of the 3D-arrangement on the expression of some genes and proteins which play a key role in pancreatic adenocarcinoma (PDAC) progression in HPAF-II, HPAC and PL45 PDAC cells cultured in either 2D-monolayers or 3D-spheroids. Cytokeratins 7, 8, 18, 19 were differently expressed in 3D-spheroids compared to 2D-monolayers. Syndecan 1 was upregulated in HPAF-II and PL45 3D-spheroids, and downregulated in HPAC. Heparanase mRNA levels were almost unchanged in HPAF-II, and increased in HPAC and PL45 3D-spheroids. Hyaluronan synthase (HAS) 2 and 3 mRNA increased in all 3D-spheroids compared to 2D-monolayers. CD44 and CD44s were expressed to a lower extent in HPAF-II and HPAC 3D-spheroids. By contrast, the CD44s/v3 and the CD44s/v6 ratio increased in HPAC and PL45 3D-spheroids, compared to 2D-monolayers. The expression of MMP-7 was strongly upregulated in 3D-spheroids. STAT3 was similarly expressed 3D-spheroids or 2D-monolayers, while pSTAT3 was almost undetectable in 2D-monolayers and strongly upregulated in 3D-spheroids. These results suggest that 3D-spheroids represent a cell culture model that allows the characterization of PDAC cell phenotype, adding new information that contributes to a better understanding of the biology and behavior of PDAC cells.
Asunto(s)
Adenocarcinoma , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas/patología , Esferoides Celulares/patología , Adenocarcinoma/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Línea Celular Tumoral , Humanos , Neoplasias Pancreáticas/metabolismo , Fenotipo , Factor de Transcripción STAT3/metabolismo , Neoplasias PancreáticasRESUMEN
Melanoma is the most severe type of skin cancer. Its unique and heterogeneous metabolism, relying on both glycolysis and oxidative phosphorylation, allows it to adapt to disparate conditions. Mitochondrial function is strictly interconnected with mitochondrial dynamics and both are fundamental in tumour progression and metastasis. The malignant phenotype of melanoma is also regulated by the expression levels of the enzyme acid sphingomyelinase (A-SMase). By modulating at transcriptional level A-SMase in the melanoma cell line B16-F1 cells, we assessed the effect of enzyme downregulation on mitochondrial dynamics and function. Our results demonstrate that A-SMase influences mitochondrial morphology by affecting the expression of mitofusin 1 and OPA1. The enhanced expression of the two mitochondrial fusion proteins, observed when A-SMase is expressed at low levels, correlates with the increase of mitochondrial function via the stimulation of the genes PGC-1alpha and TFAM, two genes that preside over mitochondrial biogenesis. Thus, the reduction of A-SMase expression, observed in malignant melanomas, may determine their metastatic behaviour through the stimulation of mitochondrial fusion, activity and biogenesis, conferring a metabolic advantage to melanoma cells.
Asunto(s)
Regulación hacia Abajo , Melanoma Experimental/enzimología , Melanoma Experimental/metabolismo , Dinámicas Mitocondriales , Esfingomielina Fosfodiesterasa/metabolismo , Animales , Modelos Animales de Enfermedad , Femenino , GTP Fosfohidrolasas/metabolismo , Melanoma Experimental/ultraestructura , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Biogénesis de Organelos , Oxidación-ReducciónRESUMEN
To verify the hypothesis that by enmeshing lubricants, microvilli reduce the coefficient of kinetic friction (µ) of pleural mesothelium, µ was measured during reciprocating sliding of rabbit's visceral against parietal pleura before and after addition of hyaluronan, and related to the morphological features of the microvillar network. Because no relation was found between µ or µ changes after hyaluronan and microvillar characteristics, the latter are not determinants of the frictional forces which oppose sliding of normal mesothelial surfaces under physiological conditions, nor of the effects of hyaluronan. Addition of hyaluronan increased µ slightly but significantly in normal specimens, probably by altering the physiological mix of lubricants, but decreased µ of damaged mesothelia, suggesting protective, anti-abrasion properties. Indeed, while sliding of an injured against a normal pleura heavily damaged the latter and increased µ when Ringer was interposed between the surfaces, both effects were limited or prevented when hyaluronan was interposed between the injured and normal pleura before onset of sliding.
Asunto(s)
Epitelio/fisiología , Fricción , Pleura/fisiología , Respiración , Animales , Epitelio/efectos de los fármacos , Epitelio/ultraestructura , Ácido Hialurónico/administración & dosificación , Soluciones Isotónicas/administración & dosificación , Lubricantes/administración & dosificación , Microscopía Electrónica de Transmisión , Microvellosidades/efectos de los fármacos , Microvellosidades/fisiología , Microvellosidades/ultraestructura , Pleura/efectos de los fármacos , Pleura/ultraestructura , Conejos , Solución de RingerRESUMEN
AIM: To analyze the effect of three-dimensional (3D)-arrangement on the expression of epithelial-to-mesenchymal transition markers in pancreatic adenocarcinoma (PDAC) cells. METHODS: HPAF-II, HPAC, and PL45 PDAC cells were cultured in either 2D-monolayers or 3D-spheroids. Ultrastructure was analyzed by transmission electron microscopy. The expression of E-cadherin, ß-catenin, N-cadherin, collagen typeâ Iâ (COL-I), vimentin, α-smooth muscle actin (αSMA), and podoplanin was assayed by confocal microscopy in cells cultured on 12-mm diameter round coverslips and in 3D-spheroids. Gene expression for E-cadherin, Snail, Slug, Twist, Zeb1, and Zeb2 was quantified by real-time PCR. E-cadherin protein level and its electrophoretic pattern were studied by Western blot in cell lysates obtained from cells grown in 2D-monolayers and 3D-spheroids. RESULTS: The E-cadherin/ß-catenin complex was expressed in a similar way in plasma membrane cell boundaries in both 2D-monolayers and 3D-spheroids. E-cadherin increased in lysates obtained from 3D-spheroids, while cleavage fragments were more evident in 2D-monolayers. N-cadherin expression was observed in very few PDAC cells grown in 2D-monolayers, but was more evident in 3D-spheroids. Some cells expressing COL-I were observed in 3D-spheroids. Podoplanin, expressed in collectively migrating cells, and αSMA were similarly expressed in both experimental conditions. The concomitant maintenance of the E-cadherin/ß-catenin complex at cell boundaries supports the hypothesis of a collective migration for these cells, which is consistent with podoplanin expression. CONCLUSION: We show that a 3D-cell culture model could provide deeper insight into understanding the biology of PDAC and allow for the detection of marked differences in the phenotype of PDAC cells grown in 3D-spheroids.
Asunto(s)
Carcinoma Ductal Pancreático/patología , Transición Epitelial-Mesenquimal , Neoplasias Pancreáticas/patología , Antígenos CD , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Western Blotting , Cadherinas/genética , Cadherinas/metabolismo , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/ultraestructura , Línea Celular Tumoral , Forma de la Célula , Supervivencia Celular , Regulación Neoplásica de la Expresión Génica , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Microscopía Confocal , Microscopía Electrónica de Transmisión , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/ultraestructura , Fenotipo , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esferoides Celulares , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Polyamidoamines (PAAs) are a well-known family of synthetic biocompatible and biodegradable polymers, which can be prepared as soft hydrogels characterized by low interfacial tension and tunable elasticity. For the first time we report here on the in vivo performance of a PAA hydrogel implant as scaffold for tissue engineering. In particular, an amphoteric agmatine-deriving PAA hydrogel shaped as small tubing was obtained by radical polymerization of a soluble functional oligomeric precursor and used as conduit for nerve regeneration in a rat sciatic nerve cut model. The animals were analyzed at 30, 90, and 180 days post-surgery. PAA tubing proved to facilitate nerve regeneration. Good surgical outcomes were achieved with no signs of inflammation or neuroma. Moreover, nerve regeneration was morphologically sound and the quality of functional recovery satisfactory. In conclusion, PAA hydrogel scaffolds may represent a novel and promising material for peripheral nerve regeneration.