O trabalho do Dr. Carvalho foi apoiado por uma bolsa de pesquisa do Instituto de Pesquisa de Saúde da Mulher e do Instituto Nacional de Saúde Infantil e Desenvolvimento Humano dos Institutos Nacionais de Saúde (5K12 HD043452). Angst Dr. receberam suprimentos (On-Q PainBuster Pós-Op Sistema de alívio da dor) e fundos (para os ensaios bioquímicos) de I-Flow (Lake Forest, Califórnia).

1. Protocolo de estudo <ul><li> Obter a aprovação do Conselho de Revisão Institucional e consentimento informado por escrito. </li><li> Selecione uma população adequada cirúrgico para condução do estudo. Esta metodologia tem sido somente aplicado em feridas cesarianas mas as feridas cirúrgicas seria mais apropriada. </li><li> Considerar os critérios de inclusão e exclusão de sua participação no estudo. Pacientes e procedimentos com altas taxas de infecção da ferida cirúrgica não pode ser apropriado. </li><li> Padronizar anestesia e pós-operatório para todos os pacientes, para evitar potenciais fatores de confusão. </li></ul> 2. Colecção Mediador nociceptiva e inflamatória Bioquímica <ul><li> Inserir o PainBuster ON-Q com ON-Q SilverSoaker (I-Flow, de Lake Forest, CA) para dentro da camada subcutânea imediatamente antes de fecho de feridas (camadas de tecido podem ser consideradas outras). O ON-Q PainBuster sistema proporciona o alívio da dor continuamente solução salina normal a uma taxa de 2 ml / h. O transportador pode ser penteINED ou comparado com medicamentos de interesse, tais como um anestésico local e não-esteróides anti-inflamatórios. </li><li> Incorporar uma torneira de três vias padrão para este sistema para permitir a aspiração de exsudado da ferida em pontos de tempo especificados. A infusão contínua impede que o cateter de coagulação e melhora a fiabilidade do sistema para a produção de amostras de exsudato. Se a aspiração de exsudado é difícil (a cerca de 5% dos casos), considere empurrando suavemente acima da ferida, alterando a posição do sujeito (por exemplo, o paciente sentado ou deitado eles se plana), utilizando uma descarga 0,5-1 ml de solução salina normal ou a retirar o centímetros cateter 1-2. </li><li> Em pontos de tempo especificados pelo protocolo (por exemplo, 1, 4, 6, 24, e 48 h após a cesariana), aspirar e retirar 1 ml de exsudado da ferida. Transferir o exsudado dentro de um copo de polietileno contendo 30 ul de inibidor de proteinase. Temos usado inibidor de protease completa (Roche Bioscience, Palo Alto, CA) para nossos propósitos. </li><li&gt; Nos mesmos intervalos de tempo especificados pelo protocolo do estudo, recolher 10 ml de sangue. Nós usamos um tubo de coleta de sangue contendo heparina de lítio, em nossa instituição é um tubo de vidro verde com tampo. Adicionar um adicional de 300 ul de inibidor de proteinase em amostras de sangue. </li><li> Dentro de 1 hora de recolha, colocar as amostras em gelo e centrifugar a 3000 rpm durante 10 min. </li><li> Remover sobrenadante soro e ferida e colocar num tubo de microcentrífuga de padrão e armazenar a -20 ° C. </li></ul> 3. Análise ensaio <ul><li> Uma vez que todo o exsudado o estudo e as amostras de soro são colhidas e armazenadas, descongelar e análise das amostras ao mesmo tempo. </li><li> Medimos as citocinas e quimiocinas, utilizando um imunoensaio multiplex. A técnica de multiplex de imuno-ensaio permite ensaio analitos a partir de numerosas pequenas amostras (50 ul) do volume. O multiplex produzir resultados comparáveis ​​com aqueles obtidos com ELISA. Temos utilizado o sistema de 17-Plex Bio-PlexTM (BioRad, HercEGRAS, CA) para nossa análise. Este ensaio é capaz de medir o factor de necrose tumoral α (TNFa), interferon α (INFα), interleucina 1β (IL-1β), interleucina 2 (IL-2), interleucina 4 (IL-4), interleucina 5 (IL-5 ), interleucina 6 (IL-6), interleucina 7 (IL-7), interleucina 8 (IL-8), interleucina (IL-10), interleucina 12 (IL-12), interleucina 13 (IL-13), interleucina 17 (IL-17), factor estimulante de colónias de granulócitos (G-CSF), de granulócitos-macrófagos factor estimulador de colónias (GM-CSF), proteína quimioatractiva de monócitos 1 (MCP-1) e uma proteína inflamatória de macrófagos (MIP-1β). Factor de crescimento do nervo foi medida com NGF anticorpo DY256 adicionando-o da placa de 17-plex com o kit de acoplamento de amina Bio-Plex. </li><li> Para garantir a análise de boa qualidade, cada medição deve ser feita em duplicata. As curvas padrão para cada analito são gerados usando os analitos de referência fornecidos pelo fabricante (em concentrações de 0,20, 0,78, 3,13, 12,5, 50, 200, 800, 3200 pg / ml, maisum padrão de zero para a solução salina normal). As curvas padrão são incluídos em cada ensaio e as concentrações de amostra são calculadas com Bio-Plex Manager. </li><li> A substância P e de prostaglandina E2 foram medidos utilizando kits de sensibilidade altamente ELISA. Temos usado Ensaio Designs, Inc. (Ann Arbor, Michigan) para esta análise. Realizar a análise em duplicado, de acordo com a especificação do fabricante. </li></ul>

<ol>
	<li>Carvalho, B., Clark, D. J., Angst, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+and+Systemic+Release+of+Cytokines,+Nerve+Growth+Factor,+Prostaglandin+E2,+and+Substance+P+in+Incisional+Wounds+and+Serum+Following+Cesarean+Delivery.">Local and Systemic Release of Cytokines, Nerve Growth Factor, Prostaglandin E2, and Substance P in Incisional Wounds and Serum Following Cesarean Delivery.</a> J. Pain. 9, 650-657 (2008).</li><li>Carvalho, B., Clark, D. J., Yeomans, D. C., Angst, M. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+epidural+clonidine+on+perioperative+cytokine+response,+postoperative+pain,+and+bowel+function+in+patients+undergoing+colorectal+surgery.">The effect of epidural clonidine on perioperative cytokine response, postoperative pain, and bowel function in patients undergoing colorectal surgery.</a> Anesth. Analg. 99, 502-509 (2004).</li><li>Buvanendran, A., Kroin, J. S., Berger, R. A., Hallab, N. J., Saha, C., Negrescu, C., Moric, M., Caicedo, M. S., Tuman, K. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+continuous+wound+catheters+delivering+local+anesthetic+for+postoperative+analgesia:+a+quantitative+and+qualitative+systematic+review+of+randomized+controlled+trials.">Efficacy of continuous wound catheters delivering local anesthetic for postoperative analgesia: a quantitative and qualitative systematic review of randomized controlled trials.</a> J. Am. Coll. Surg. 203, 914-932 (2006).</li><li>Eriksson, A. S., Sinclair, R., Cassuto, J., Thomsen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+lidocaine+on+leukocyte+function+in+the+surgical+wound.">Influence of lidocaine on leukocyte function in the surgical wound.</a> Anesthesiology. 77, 74-78 (1992).</li><li>Clark, J. D., Shi, X., Li, X., Qiao, Y., Liang, D., Angst, M. S., Yeomans, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphine+reduces+local+cytokine+expression+and+neutrophil+infiltration+after+incision.">Morphine reduces local cytokine expression and neutrophil infiltration after incision.</a> Mol. Pain. 3, 28 (2007).</li><li>Webb, S. T., Ghosh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intra-articular+bupivacaine:+potentially+chondrotoxic.">Intra-articular bupivacaine: potentially chondrotoxic.</a> Br. J. Anaesth. 102, 439-441 (2009).</li><li>Scott, N. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+infiltration+for+surgery.">Wound infiltration for surgery.</a> Anaesthesia. 65, 67 (2010).</li></ol>

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Esta metodologia descrita acima foi encontrado para ser suficientemente sensível para detectar bioquímicos mediadores inflamatórios e nociceptiva no exsudado da ferida. A amostragem em vários pontos de tempo também permitiu observar mudanças de tais mediadores com o tempo. 1,2 Não temos conhecimento de qualquer ensaio outra ferida cirúrgica humana que pode série medir o perfil de liberação de mediadores inflamatórios e nociceptivos em exsudato da ferida cirúrgica. Os resultados da nossa metodolog...

Depois de uma incisão cirúrgica, residente e migração de células inflamatórias liberam citocinas pró-e anti-inflamatórias, prostanóides, neuropéptidos e quimiocinas, que são importantes para a iniciação e manutenção da dor. 3,4 A capacidade de recolher-site marcadores bioquímicos específicos é essencial para determinar o relação entre os níveis no soro e da ferida cirúrgica, compreender a complexa relação entre a libertação de mediadores e dor cirúrgica, e para ser capaz de avaliar o efeito de ambos sistémica e administração da droga periférica sobre a bioquímica da ferida cirúrgica. Estudos têm-se centrado em medidas sistêmicas de mediadores bioquímicos e não considerou site-specific lançamento. 5,6 medições séricos de citocinas e quimiocinas podem não ser reflexo de eventos locais de feridas cirúrgicas bioquímicos e apenas indicam uma resposta sistémica global a lesão cirúrgica. Este artigo fornece uma descrição detalhada e visual único da metodologia que foi abetoSão referenciados em 2008 no Journal of Pain. 1 O objetivo deste artigo JoVE é fornecer uma descrição única e visualmente melhor dos métodos.

collecting and measuring wound exudate biochemical mediators in surgical wounds

Nós descrevemos uma metodologia pela qual somos capazes de recolher e medir bioquímicos mediadores inflamatórios e nociceptivos no local da ferida cirúrgica. Coleta de site-specific marcadores bioquímicos é importante para entender a relação entre os níveis de soro e ferida cirúrgica, determinar quaisquer associações entre a liberação do mediador, dor, uso de analgésicos e outros resultados de interesse, e avaliar o efeito da administração da droga sistêmica e periférica da ferida operatória bioquímica. Esta metodologia foi aplicada a mulheres saudáveis ​​submetidos a cesariana eletiva com raquianestesia. Nós medimos exsudato da ferida e mediadores séricos em intervalos de tempo iguais pontuações paciente da dor e consumo de analgésicos por até 48 horas pós-parto cesariana. Utilizando esta metodologia, foram capazes de detectar vários mediadores bioquímicos, incluindo factor de crescimento do nervo (NGF), a prostaglandina E2 (PG-E2) substância P, IL-1β, IL-2, IL-4, IL-6, IL-7, IL-8,IL-10, IL-12, IL-13, IL-17, TNFa, INFγ, G-CSF, GM-CSF, MCP-1 e MIP-1β. Estudos que aplicam este bioensaio ferida cirúrgica humano foi encontrado nenhum correlações entre ferida e concentrações séricas de citocinas ou seu perfil de liberação de tempo (J Dor 2008;. 9 (7) :650-7) 1 Nós também documentou a utilidade da técnica para identificar. drogas mediadas por alterações no conteúdo de citocinas ferida (Anesth Analg 2010; 111:1452-9). 2

Utilizando esta metodologia, foram capazes de detectar todos os mediadores bioquímicos descritos acima com a excepção de IL-5 no exsudado da ferida e plasma. 1,2 Algumas citocinas adicionais não foram detectados no plasma (IL-4, IL-17, TNFa, INFγ , GM-CSF). 1 curso O tempo de citocinas e quimiocinas, medidos em estudos relatados foram consistentes, atingindo um patamar dentro das primeiras horas que foi mantida durante o período de observação de 24 horas .. 1,2 No entanto, NGF au...

Watch this Scientific Journal Video about Collecting And Measuring Wound Exudate Biochemical Mediators In Surgical Wounds at JoVE.com

Collecting And Measuring Wound Exudate Biochemical Mediators In Surgical Wounds

Este artigo fornece uma descrição detalhada e visual de uma metodologia de coleta e medição bioquímicos mediadores inflamatórios e nociceptivos no local da ferida cirúrgica após cesariana. Este bioensaio humano tem sido usado para determinar a correlação entre a ferida e concentrações de citocinas no soro e de drogas mediadas mudanças na ferida citocinas, quimiocinas e neuropetides.

Coleta e medição de mediadores bioquímicos feridas exsudato em feridas cirúrgicas

We describe a methodology by which we are able to collect and measure biochemical inflammatory and nociceptive mediators at the surgical wound site. ...

collecting-measuring-wound-exudate-biochemical-mediators-surgical

Research

JoVE Journal

Medicine

11.9K visualizações.  Stanford University School of Medicine. Este artigo fornece uma descrição detalhada e visual de uma metodologia de coleta e medição bioquímicos mediadores inflamatórios e nociceptivos no local da ferida cirúrgica após cesariana. Este bioensaio humano tem sido usado para determinar a correlação entre a ferida e concentrações de citocinas no soro e de drogas mediadas mudanças na ferida citocinas, quimiocinas e neuropetides.

Vídeo: Collecting And Measuring Wound Exudate Biochemical Mediators In Surgical Wounds

Biochemical Measurement of Neonatal Hypoxia

Neonatal hypoxia ischemia is characterized by inadequate blood perfusion of a tissue or a systemic lack of oxygen. This condition is thought to cause/exacerbate well documented neonatal disorders including neurological impairment 1-3. Decreased adenosine triphosphate production occurs due to a lack of oxidative phosphorylation. To compensate for this energy deprived state molecules containing high energy phosphate bonds are degraded 2. This leads to increased levels of adenosine which is subsequently degraded to inosine, hypoxanthine, xanthine, and finally to uric acid. The final two steps in this degradation process are performed by xanthine oxidoreductase. This enzyme exists in the form of xanthine dehydrogenase under normoxic conditions but is converted to xanthine oxidase (XO) under hypoxia-reperfusion circumstances 4, 5. Unlike xanthine dehydrogenase, XO generates hydrogen peroxide as a byproduct of purine degradation 4, 6. This hydrogen peroxide in combination with other reactive oxygen species (ROS) produced during hypoxia, oxidizes uric acid to form allantoin and reacts with lipid membranes to generate malondialdehyde (MDA) 7-9. Most mammals, humans exempted, possess the enzyme uricase, which converts uric acid to allantoin. In humans, however, allantoin can only be formed by ROS-mediated oxidation of uric acid. Because of this, allantoin is considered to be a marker of oxidative stress in humans, but not in the mammals that have uricase.
We describe methods employing high pressure liquid chromatography (HPLC) and gas chromatography mass spectrometry (GCMS) to measure biochemical markers of neonatal hypoxia ischemia. Human blood is used for most tests. Animal blood may also be used while recognizing the potential for uricase-generated allantoin. Purine metabolites were linked to hypoxia as early as 1963 and the reliability of hypoxanthine, xanthine, and uric acid as biochemical indicators of neonatal hypoxia was validated by several investigators 10-13. The HPLC method used for the quantification of purine compounds is fast, reliable, and reproducible. The GC/MS method used for the quantification of allantoin, a relatively new marker of oxidative stress, was adapted from Gruber et al 7. This method avoids certain artifacts and requires low volumes of sample. Methods used for synthesis of MMDA were described elsewhere 14, 15. GC/MS based quantification of MDA was adapted from Paroni et al. and Cighetti et al. 16, 17. Xanthine oxidase activity was measured by HPLC by quantifying the conversion of pterin to isoxanthopterin 18. This approach proved to be sufficiently sensitive and reproducible.

A method is described to measure biochemical markers of neonatal hypoxia-ischemia. The approach utilizes high pressure liquid chromatography (HPLC) and Gas Chromatography Mass Spectrometry (GC/MS).

Medição bioquímicos da Hipóxia Neonatal

Neonatal hypoxia ischemia is characterized by inadequate blood perfusion of a tissue or a systemic lack of oxygen. This condition is thought to ...

Medicina

Collecting Saliva and Measuring Salivary Cortisol and Alpha-amylase in Frail Community Residing Older Adults via Family Caregivers

Salivary measures have emerged in bio-behavioral research that are easy-to-collect, minimally invasive, and relatively inexpensive biologic markers of stress. This article we present the steps for collection and analysis of two salivary assays in research with frail, community residing older adults-salivary cortisol and salivary alpha amylase. The field of salivary bioscience is rapidly advancing and the purpose of this presentation is to provide an update on the developments for investigators interested in integrating these measures into research on aging. Strategies are presented for instructing family caregivers in collecting saliva in the home, and for conducting laboratory analyses of salivary analytes that have demonstrated feasibility, high compliance, and yield quality specimens. The protocol for sample collection includes: (1) consistent use of collection materials; (2) standardized methods that promote adherence and minimize subject burden; and (3) procedures for controlling certain confounding agents. We also provide strategies for laboratory analyses include: (1) saliva handling and processing; (2) salivary cortisol and salivary alpha amylase assay procedures; and (3) analytic considerations.

We demonstrate: (1) procedures for collection of salivary samples in cognitive impaired older adults by family caregivers in the home setting, (2) procedures for measuring stress activity via salivary cortisol and alpha amylase, and (3) representative profiles. Protocols that allow researchers to study stress-linked processes advance our understanding of biological sensitivity and susceptibility.

Salivary measures have emerged in bio-behavioral research  that are easy-to-collect, minimally invasive, and relatively inexpensive  biologic markers of ...

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease and injury regional estimates, World Health Organization, 2004). Surgeries that often compromise the quality of life are required to correct heart defects, reminding us of the importance of finding the causes of CHD. Mutant mouse models and live imaging technology have become essential tools to study the etiology of this disease. Although advanced methods allow live imaging of abnormal hearts in embryos, the physiological and hemodynamic states of the latter are often compromised due to surgical and/or lengthy procedures. Noninvasive ultrasound imaging, however, can be used without surgically exposing the embryos, thereby maintaining their physiology. Herein, we use simple M-mode ultrasound to assess heart rates of embryos at E18.5 in utero. The detection of abnormal heart rates is indeed a good indicator of dysfunction of the heart and thus constitutes a first step in the identification of developmental defects that may lead to heart failure.

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Ultra-high frequency ultrasound is a powerful live imaging tool to examine cardiac abnormalities in small animals. Its noninvasiveness allows maintaining the physiologic state of embryos. Herein, we show the use of M-mode ultrasound to measure the heart rates of embryos at E18.5 in utero.

Na medição utero da frequência cardíaca em rato por não invasiva em modo M Ecocardiografia

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease ...

Demonstration of the Rat Ischemic Skin Wound Model

The propensity for chronic wounds in humans increases with ageing, disease conditions such as diabetes and impaired cardiovascular function, and unrelieved pressure due to immobility. Animal models have been developed that attempt to mimic these conditions for the purpose of furthering our understanding of the complexity of chronic wounds. The model described herein is a rat ischemic skin flap model that permits a prolonged reduction of blood flow resulting in wounds that become ischemic and resemble a chronic wound phenotype (reduced vascularization, increased inflammation and delayed wound closure). It consists of a bipedicled dorsal flap with 2 ischemic wounds placed centrally and 2 non-ischemic wounds lateral to the flap as controls. A novel addition to this ischemic skin flap model is the placement of a silicone sheet beneath the flap that functions as a barrier and a splint to prevent revascularization and reduce contraction as the wounds heal. Despite the debate of using rats for wound healing studies due to their quite distinct anatomic and physiologic differences compared to humans (i.e., the presence of a panniculus carnosus muscle, short life-span, increased number of hair follicles, and their ability to heal infected wounds) the modifications employed in this model make it a valuable alternative to previously developed ischemic skin flap models.

The rat, due to its size, availability, and rather docile behavior, has been utilized as a research model for many years. The goal of this protocol is to utilize the rat as an ischemic skin wound healing model to provide valuable insight into the pathophysiology of chronic wounds.

Demonstração da Wound modelo de pele de rato Isquêmico

The propensity for chronic wounds in humans increases with ageing, disease conditions such as diabetes and impaired cardiovascular function, and ...

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an otherwise dynamic system, and successive phases are easily overlooked or misinterpreted. Live-cell imaging and time-lapse microscopy, in which living cells can be followed for hours or even days in a more or less continuous fashion, are therefore very informative. The protocol described here allows for the investigation of the fate of chemotherapeutic nanoparticles after the delivery of doxorubicin (dox) in living cells. Dox is an intercalating agent that must be released from its nanocarrier to become biologically active. In spite of its clinical registration for more than two decades, its uptake, breakdown, and drug release are still not fully understood. This article explores the hypothesis that lipid-based nanoparticles are taken up by the tumor cells and are slowly degraded. Released dox is then translocated to the nucleus. To prevent fixation artifacts, live-cell imaging and time-lapse microscopy, described in this experimental procedure, can be applied.

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

Live-cell imaging is a powerful tool to visualize dynamic processes. The examination of fixed cells provides only static pictures, which can lead to misinterpretation and confusion about the process. This work presents a method to study uptake, drug release, and intracellular localization of liposomal nanoparticles in living cells.

Usando em Vitro viver-pilha de imagem para explorar Chemotherapeutics entregues por nanopartículas de lipídios-baseado

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an ...

Collecting Hair Samples for Hair Cortisol Analysis in African Americans

The hormone cortisol is typically assessed in saliva, serum, or urine samples. More recently, cortisol has been successfully extracted from hair, including humans. The advantage of hair cortisol concentration is that it reflects a retrospective representation of hypothalamic-pituitary-adrenal (HPA) axis function over time, much like hemoglobin A1C represents glycemic control. However, obtaining hair samples can be challenging, due to the cultural beliefs and hair care practices of minority participants. For example, African Americans may be reluctant to provide samples. Additionally, few researchers are trained to collect hair samples from African Americans. The purpose of this paper is to present a culturally informed protocol to help researchers obtain hair samples from African Americans. To illustrate the representative results of this protocol implementation, de-identified data from African Americans that participated in a community-based study on chronic stress are provided. Hair practice preferences are assessed. The participants are made comfortable by showing pictures of hair samples prior to cutting their hair. The single strain twist and gently pull method is used to collect approximately 30 - 50 strands of hair from the posterior vertex region of the scalp. This protocol will significantly improve collection of hair samples from African Americans.

Hair cortisol concentration analysis provides an alternative to traditional measures of cortisol; however, to collect hair samples from African Americans, scientists need to be culturally informed and competent. The purpose of this protocol is to demonstrate a culturally informed technique to collect hair samples for cortisol analysis from African Americans.

Coletar amostras de cabelo para cabelo análise de Cortisol em americanos africanos

The hormone cortisol is typically assessed in saliva, serum, or urine samples. More recently, cortisol has been successfully extracted from hair, ...

Protocol to Create Chronic Wounds in Diabetic Mice

Chronic wounds develop as a result of defective regulation in one or more complex cellular and molecular processes involved in proper healing. They impact ~6.5M people and cost ~$40B/year in the US alone. Although a significant effort has been invested in understanding how chronic wounds develop in humans, fundamental questions remain unanswered. Recently, we developed a novel mouse model for diabetic chronic wounds that have many characteristics of human chronic wounds. Using db/db-/- mice, we can generate chronic wounds by inducing high levels of oxidative stress (OS) in the wound tissue immediately after wounding, using a one-time treatment with inhibitors specific to the antioxidant enzymes catalase and glutathione peroxidase. These wounds have high levels of OS, develop biofilm naturally, become fully chronic within 20 days after treatment and can remain open more for more than 60 days. This novel model has many features of diabetic chronic wounds in humans and therefore can contribute significantly to advancing fundamental understanding of how wounds become chronic. This is a major breakthrough because chronic wounds in humans cause significant pain and distress to patients and result in amputation if unresolved. Moreover, these wounds are very expensive and time-consuming to treat, and lead to significant loss of personal income to patients. Advancements in this field of study through the use of our chronic wound model can significantly improve health care for millions who suffer under this debilitating condition. In this protocol, we describe in great detail the procedure to cause acute wounds to become chronic, which has not been done before.

Chronic wounds are developed from acute wounds on a diabetic mouse model by inducing high levels of oxidative stress after a full-thickness cutaneous wound. The wound is treated with inhibitors for catalase and glutathione peroxidase, resulting in impaired healing and biofilm development by bacteria present in the skin microbiome.

Protocolo para criar feridas crônicas em camundongos diabéticos

Chronic wounds develop as a result of defective regulation in one or more complex cellular and molecular processes involved in proper healing. They impact ...

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.

This protocol describes a method to inflict an abrasion to the ocular surface of the mouse, and to follow the wound healing process thereafter. The protocol takes advantage of an ocular burr to partially remove the surface epithelium of the eye in anaesthetized mice.

Cicatrização de feridas abrasão epitelial da córnea com Burr Ocular como um modelo para córnea

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...

Targeting Gray Rami Communicantes in Selective Chemical Lumbar Sympathectomy

Chemical lumbar sympathectomy (CLS) is a commonly used, minimally invasive procedure for the treatment of conditions including ischemic diseases of the lower extremities, hyperhidrosis, etc. It is commonly practiced to position the puncture needle tip in front of the anterior fascia of the psoas major muscle and inject the inactivating agent around the sympathetic trunk, which is defined as conventional CLS. Although relatively rare, ureteropelvic damage is the most frequently reported complication of conventional CLS and can cause serious harm to patients. We found that injecting the inactivating agent behind the anterior fascia, which only targets gray rami communicantes, helped achieve therapeutic efficacy in&#160;vasodilation, sweat reduction, and pain relief comparable to conventional CLS, and serious complications were largely reduced. We define this procedure as selective CLS. Here, we present a protocol of selective CLS. The precise needle tract and accurate evaluation of the spreading of the contrast agent are critical to ensure that the drug is injected behind the anterior fascia of the psoas major muscle. The needle tip is at approximately one-third the dividing line of the vertebral body in the lateral view of a lumbar X-ray. The contrast is mainly confined around the needle tip and spreads outward and downward along the psoas muscle fibers. In this way, the anterior fascia provides a natural barrier for the ureteropelvic area, and the psoas major muscle provides a natural barrier for the lumbar nerve root. There are several highlights of this article, including 1) a detailed description of the selective CLS procedures, 2) an explanation of the anatomical basis for the implementation of selective CLS, and 3) an explanation of the differences between selective and conventional CLS.

We present a protocol for selective chemical lumbar sympathectomy (CLS), which inactivates only gray rami communicantes and not the sympathetic trunk. Selective CLS can help achieve therapeutic efficacy in vasodilation, sweat reduction, and pain relief that are comparable to conventional CLS, and serious complications, particularly ureteropelvic damages, can be reduced.

Direcionamento de Rami cinza Communicantes em simpatectomia lombar química seletiva

Chemical lumbar sympathectomy (CLS) is a commonly used, minimally invasive procedure for the treatment of conditions including ischemic diseases of the ...

Application of Lucilia sericata Larvae in Debridement of Pressure Wounds in Outpatient Settings

Biological therapy using Lucilia sericata larvae has numerous advocates worldwide, yet it is still fairly unknown and not commonly applied in daily practice because of the limited awareness and insufficient experience of medical and nursing personnel. There are case reports suggesting that maggot therapy can be applied and supported by lay caregivers, provided they are supervised and informed by physicians/nurses. The foregoing observation suggests that the method should be considered for implementation by a wider group of caregivers if accepted and meticulously supervised by trained and experienced medical staff. The concerns related to the therapeutic use of maggots in certain regions seem understandable, but are not supported by scientific facts. It should be noted that many therapeutic agents (including brood) used in medicine are of natural origin, and are associated with low production costs and high possibilities of implementation in the course of therapy. By analyzing the literature and using our own clinical and research experience, we have come to conclusions related to using larvae therapy, as a quick and safe method providing cleaning and revitalization in the process of treating wounds of various etiologies, especially pressure ulcers. In the current study, medical-grade Lucilia sericata maggots were applied to remove necrotic tissue from deep pressure sores. The treatment is mostly accepted by both caregivers and patients. In most cases, it is conducted by trained and experienced medical personnel in home and outpatient settings. Over the course of the conducted analyzes involving the collected specimens, there were no statistically significant relationships (p&gt; 0.05) confirmed between the wound surface successfully cleared by brood and variables, such as time from wound formation, location, surface size, and the depth of damage to the tissue structure. The lack of statistical dependence may result from the small size of the studied group. Based on the current findings, we have formulated the following conclusions: Maggot Debridement Therapy (MDT) is a fast and effective method enabling the preparation of the wound bed. The use of MDT in outpatient settings is safe and acceptable for patients and their caregivers.

Application of Lucilia sericata Larvae in Debridement of Pressure Wounds in Outpatient Settings

Maggots of Lucilia sericata were used in the debridement of deep pressure sores in outpatient settings for the treatment of pressure sores involving the complete skin thickness. The technique presented in the article does not pose risks for the patient.

Aplicação de Lucilia sericata larvas em desbridamento de feridas de pressão em ambientes ambulatoriais

Biological therapy using Lucilia sericata larvae has numerous advocates worldwide, yet it is still fairly unknown and not commonly applied in daily ...