RESUMEN
OBJECTIVE: To describe and standardize a protocol that overcomes the technical limitations of Western blot (WB) analysis in the quantification of the neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) following nociceptive stimuli in rat skin. DESIGN: Male Wistar rats (Rattus norvegicus albinus) weighing 250 to 350 g were used in this study. Elements of WB analysis were adapted by using specific manipulation of samples, repeated cycles of freezing and thawing, more thorough maceration, and a more potent homogenizer; increasing lytic reagents; promoting greater inhibition of protease activity; and using polyvinylidene fluoride membranes as transfer means for skin-specific protein. Other changes were also made to adapt the WB analysis to a rat model. SETTING: University research center. MAIN OUTCOME MEASURE: Western blot analysis adapted to a rat model. RESULTS: This research design has proven effective in collecting and preparing skin samples to quantify SP and CGRP using WB analysis in rat skin. CONCLUSION: This study described a research design that uses WB analysis as a reproducible, technically accessible, and cost-effective method for the quantification of SP and CGRP in rat skin that overcomes technical biases.
Asunto(s)
Western Blotting , Péptido Relacionado con Gen de Calcitonina/metabolismo , Dolor Nociceptivo/metabolismo , Sustancia P/metabolismo , Animales , Modelos Animales de Enfermedad , Masculino , Dolor Nociceptivo/etiología , Ratas , Ratas Wistar , Proyectos de InvestigaciónRESUMEN
Low-level laser therapy (LLLT) has been used with the aim of improving vascular perfusion of the skin and musculocutaneous flaps. This study evaluated the effect of LLLT on transverse rectus abdominis musculocutaneous flap (TRAM) viability, vascular angiogenesis, and VEGF release. Eighty-four Wistar rats were randomly divided into seven groups with 12 rats in each group. Group 1 received sham laser treatment; group 2, 3 J/cm(2) at 1 point; group 3, 3 J/cm(2) at 24 points; group 4, 72 J/cm(2) at 1 point; group 5, 6 J/cm(2) at 1 point; group 6, 6 J/cm(2) at 24 points; and group 7, 144 J/cm(2) at 1 point. All experimental groups underwent LLLT immediately after the TRAM operation and on the following 2 days; thus, animals underwent 3 days of treatment. The percentage of skin flap necrosis area was calculated on the fourth postoperative day using the paper template method, and two skin samples were collected using a 1-cm(2) punch to evaluate alpha smooth muscle actin (1A4) and VEGF levels in blood vessels. Significant differences were found in necrosis percentage, and higher values were seen in group 1 than in the other groups. Statistically significant differences were not found among groups 3 to 7 (p<0.292). Groups 5 and 7 showed significantly higher VEGF levels compared to other groups. Groups 3 and 5 had an increase in levels of blood vessels compared to other groups. LLLT at energy densities of 6 to 144 J/cm(2) was efficient to increase angiogenesis and VEGF levels and promote viability in TRAM flaps in rats.
Asunto(s)
Terapia por Luz de Baja Intensidad/métodos , Recto del Abdomen/efectos de la radiación , Recto del Abdomen/trasplante , Colgajos Quirúrgicos , Actinas/metabolismo , Animales , Masculino , Necrosis , Neovascularización Fisiológica/efectos de la radiación , Ratas , Ratas Wistar , Recto del Abdomen/irrigación sanguínea , Trasplante de Piel , Colgajos Quirúrgicos/irrigación sanguínea , Colgajos Quirúrgicos/patología , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
PURPOSE: To investigate the subcutaneous injection of carbon dioxide (CO2) on neuropeptides Calcitonin Gene-Related Peptide (CGRP) and Substance P (SP) secretion in rat skin. METHODS: Fifty-six Wistar-EPM rats were distributed in two groups: one for CGRP analysis, the other for SP analysis. Each group was subdivided into four subgroups: control (Cont), control with needle (ContNd), CO2 injection (CO2Inj) and atmospheric air injection (AirInj) - with seven animals each. Sample analyses of partial skin were conducted by Western Blotting (WB). RESULTS: In SP group, there was a decrease in the amount of neuropeptides in subgroups CO2Inj and AirInj. Similarly, in CGRP group, there was a decrease in the amount of pro-CGRP neuropeptides (15 kDa) in subgroups CO2Inj and AirInj; Nevertheless, there was no decrease in the amount of CGRP (5 kDa) in any subgroups. CONCLUSION: Subcutaneous injection of CO2 and atmospheric air decreased the amount of Substance P and pro-Calcitonin Gene-Related Peptide (15 kDa) neuropeptides in rat skin.
Asunto(s)
Péptido Relacionado con Gen de Calcitonina/efectos de los fármacos , Dióxido de Carbono/administración & dosificación , Piel/efectos de los fármacos , Sustancia P/efectos de los fármacos , Animales , Western Blotting , Péptido Relacionado con Gen de Calcitonina/metabolismo , Inyecciones Subcutáneas , Masculino , Distribución Aleatoria , Ratas Wistar , Reproducibilidad de los Resultados , Piel/metabolismo , Sustancia P/metabolismo , Factores de TiempoRESUMEN
To investigate the subcutaneous injection of carbon dioxide (CO2) on neuropeptides Calcitonin Gene-Related Peptide (CGRP) and Substance P (SP) secretion in rat skin. Fifty-six Wistar-EPM rats were distributed in two groups: one for CGRP analysis, the other for SP analysis. Each group was subdivided into four subgroups: control (Cont), control with needle (ContNd), CO2 injection (CO2Inj) and atmospheric air injection (AirInj) - with seven animals each. Sample analyses of partial skin were conducted by Western Blotting (WB). RESULTS: In SP group, there was a decrease in the amount of neuropeptides in subgroups CO2Inj and AirInj. Similarly, in CGRP group, there was a decrease in the amount of pro-CGRP neuropeptides (15 kDa) in subgroups CO2Inj and AirInj; Nevertheless, there was no decrease in the amount of CGRP (5 kDa) in any subgroups. Subcutaneous injection of CO2 and atmospheric air decreased the amount of Substance P and pro-Calcitonin Gene-Related Peptide (15 kDa) neuropeptides in rat skin.