This work was supported by the Ronald Peter Griggs Memorial Fellowship in ALS Research (to KP) and a Neuromuscular Research Partnership Program grant from the CIHR, the ALS Society of Canada, and Muscular Dystrophy Canada (JNM-69682) to CK and FMR. Additional support was also provided by the ALS Society of America (57969). We would like to thank Dr. R. Keith Humphries from the Terry Fox Laboratory/Department of Pathology, BC Cancer Agency, Vancouver, Canada and the staff at the Simon Fraser University Animal Care Unit.

NOTA: Ética Declaração: Este protocolo foi revisto e aprovado pelo Comitê de Cuidados Animal da Simon Fraser University (UACC; permitir números 1037K-12 e 1060K-03) e está em conformidade com a Canadian Council on Animal Care, o Guia do NIH para Cuidados e Uso de Animais de Laboratório, e da Directiva do Conselho CEE. 1. Considerações Especiais NOTA: O bussulfano é citotóxica. Manusear e eliminar de acordo com a ficha de segurança e diretrizes institucionais. <ol><li> Executar todas as técnicas de forma asséptica numa campânula de fluxo laminar. </li><li> Esterilizar instrumentos antes da sua utilização por envolvimento em um material de embalagem adequado, tal como um pacote de casca, e autoclavagem. </li><li> Embora o risco de infecção é baixo comparado com o condicionamento de busulfano a irradiação, apenas tratar os animais numa câmara de fluxo laminar para as primeiras 2 semanas após o transplante. </li></ol> 2. Condicionamento deOs ratinhos receptores <ol><li> Diluir busulfan, a 3 mg / ml com solução salina tamponada com fosfato estéril (PBS). NOTA: É importante fazer uma nova diluição de busulfan imediatamente antes de usar cada dia da injeção. </li><li> Administrar 20 mg / kg de busulfano diluída para murganhos receptores por via de injecção IP diariamente. </li><li> Repita injecções IP diárias de 20 mg / kg de busulfano até uma dose total de 60-100 mg / kg foi administrada (isto é, para um 80 mg / kg dose total, administrar 20 mg / kg de busulfano durante 4 dias consecutivos). </li></ol> 3. Isolamento de Doadores de Medula Óssea Cells NOTA: Este protocolo tem sido utilizado com sucesso para isolar e preparar a partir de células de MO até 5 ratinhos dadores. Normalmente, o rendimento das células por rato é de aproximadamente 30-40000000 BMDCs, o que é suficiente para transplantar 16/12 ratos destinatário. Se forem necessários mais ratos doadores o protocolo pode precisar de ser ajustado em conformidade. <ol><li> Após o último dia de busulfan condicionado euthanize um rato doador GFP (1-6 meses de idade), utilizando CO 2 (ou de qualquer outro procedimento de eutanásia aceitos na instituição). Para evitar complicações de enxerto utilizar doadores singeneicas que são do mesmo sexo, como os destinatários. </li><li> Spray de rato com etanol 70%. Elevador pele nas tesouras cirúrgicas abdómen e utilizando-se a incisão através da pele a partir da cavidade abdominal para cima da perna para o tornozelo. Segurando o pé, firmeza da pele a partir do tornozelo para o quadril expondo o tecido perna. </li><li> Apare músculo e tecido adiposo do fêmur para expor a articulação do quadril. </li><li> Enquanto puxa suavemente no pé para estender a perna, pressione a tesoura contra a articulação do quadril. Corte logo acima da cabeça do fêmur tomando cuidado para não cortar o próprio fêmur. </li><li> Para ajudar a manter a esterilidade, segure a perna pelo pé e limpe qualquer tecido restante dos ossos, esfregando a superfície do osso com tecidos autoclavada. </li><li> Separe o fêmur e da tíbia, cortando através da articulação do joelho ecoloque o fêmur em uma placa de cultura contendo PBS estéril. Incubar em gelo. </li><li> Retire e elimine a fíbula por corte nos pontos em que a fíbula se conecta à tíbia. Coloque a tíbia do prato de cultura com o fémur, e incubar em gelo. </li><li> Repita os passos de 3,2-3,7 para a outra perna, e se necessário, dos ratinhos dadores adicionais. Após a remoção dos ossos, esterilizar os instrumentos cirúrgicos com um esterilizador quente talão ou usar um novo conjunto de ferramentas estéreis para as etapas subseqüentes. </li><li> Para os fêmures, segure o fêmur com uma pinça e usando uma tesoura cirúrgica cuidadosamente &quot;shave&quot; as extremidades distais da osso. Remover o mínimo do osso como necessário para expor a cavidade BM. </li><li> Encha uma seringa com 3 ml de PBS estéril e anexar uma agulha G 23. Suportou cuidadosamente a agulha na cavidade BM e lave o BM em um prato de cultura estéril. Certifique-se de raspar a cavidade medular com a ponta da agulha para assegurar a remoção de todas as células desejadas. Após a extracção,assegurar que o BM vermelho já não é visível e o osso agora parece branca. </li><li> Repita os passos 3,9-3,10 para fêmures posteriores, reunindo todo o BM no mesmo prato de cultura. </li><li> Para as tíbias, segure a tíbia com uma pinça e cuidadosamente &quot;shave&quot; o final, onde a tíbia foi anexado ao joelho para expor a cavidade BM. Faça um segundo corte ao longo do osso onde o BM vermelho visível termina. </li><li> Encha uma seringa com 3 ml de PBS estéril e anexar uma agulha G 25. Inserir a agulha suavemente para dentro da cavidade BM (a partir da extremidade que foi ligado ao joelho) e lave o BM para o prato de cultura contendo o BM a partir dos fémures. Raspe as paredes da cavidade BM com a agulha para remover todas as células. Após a extracção, garantir que o BM vermelho não é mais visível e o osso agora aparece branco. </li><li> Repita os passos de 3,12-3,13 para tíbias subsequente, reunindo todo o BM no mesmo prato de cultura. </li><li> Delicadamente, triturar o BM com uma ponteira de 1 ml para dissociar a cvaras. </li><li> Passar a suspensão de células através de um filtro de carrinho 40 uM em um tubo de centrífuga de 50 mL estéril. Lave o prato com PBS para obter todas as células restantes e transferir este através do filtro no tubo de centrífuga. </li><li> Centrifugar durante 5 minutos a 450 xg, a 4 ° C. Retire e elimine o sobrenadante. </li><li> Re-suspender o sedimento em 3 ml de tampão de lise de eritrócitos. Incubar em gelo durante 8,5 min. </li><li> Adicionar ~ 30 ml de PBS estéril para saciar o tampão de lise. </li><li> Centrifugar durante 5 minutos a 450 xg, a 4 ° C. Retire e elimine o sobrenadante. </li><li> Re-suspender o sedimento em 1 ml de PBS estéril. Mantenha no gelo. </li><li> Tomar uma pequena aliquota do homogeneizado de células e dilui-se com PBS (1: 100 para 1 de ratinho; 1: 200 durante 2 murganhos, etc.) para quantificar o número de células utilizando um hemocitómetro. </li><li> Dilui-se a suspensão de células a 8 x 10 6 células / ml e incuba-se em gelo até injecção. </li></ol> 4. Transplante de Medula Óssea <ol><li> Fill uma seringa de 0,5 ml (com uma agulha de insulina 28 L) com 300 uL de suspensão diluída de células para cada ratinho a ser injectado. Certifique-se de remover todas as bolhas de ar presentes na seringa. </li><li> Coloque a gaiola do rato contendo os ratinhos receptores condicionados numa almofada de aquecimento e permita que as veias da cauda para dilatar. </li><li> Tome um rato destinatário e lugar no dispositivo de retenção. Limpe a cauda com gaze cirúrgica embebido com álcool 70%. </li><li> Segurar firmemente a cauda e com o bisel para cima, inserir a agulha suavemente numa das veias da cauda laterais e injectar a suspensão de células. </li><li> Segurar gaze cirúrgica no local da injecção até o sangramento pára antes de introduzir o rato em uma gaiola limpa novo. </li></ol> 5. estimativa da extensão da BM quimerismo NOTA: Os níveis de quimerismo são tipicamente variável no sangue periférico em uma semana pós-TMO, e, como tal, os níveis de quimerismo são normalmente estimada pelo menos 2 semanas de pós-TMO. Níveis de chimerism deve atingir&gt; 80% GFP + células no sangue periférico dentro de 3-4 semanas pós-TMO. <ol><li> Preparar tampão de FACS (2 mM de EDTA + 2% de soro fetal de bovino em PBS). NOTA: tampão FACS pode ser armazenado a 4 ° C durante várias semanas. </li><li> Posicione o mouse em um tubo de restrição e raspar a pele na perna posterior para expor a veia safena lateral. </li><li> Coat fina da perna com vaselina e perfuram a veia safena com uma agulha 25 G. </li><li> Recolhe aproximadamente 50 ul de sangue num tubo capilar com heparina revestidos. Adicionar o sangue para um tubo de micro-centrifugação contendo 1 ml de tampão de FACS. Misturar por inversão do tubo e armazenar em gelo. </li><li> Segure gaze cirúrgica no local da injeção, até o sangramento parar antes de retornar mouse de volta para a jaula. </li><li> Centrifuga-se as amostras de sangue durante 5 minutos a 900 x g. Retire e elimine o sobrenadante. </li><li> Re-suspender o sedimento em 500 mL de tampão de lise de eritrócitos. Incubar em gelo durante 8,5 min. </li><li> Adicionar 1 mL de tampão de FACS para extinguir o tampão de lise. </li><li> Centrifuga-se as amostras durante 5 min a 900 x g. Retire e elimine o sobrenadante. </li><li> Executar um segundo passo de lise em 500 uL de tampão de lise de eritrócitos. Incubar em gelo durante 8,5 min. </li><li> Adicionar 1 mL de tampão de FACS para extinguir o tampão de lise. </li><li> Centrifuga-se as amostras durante 5 min a 900 x g. Retire e elimine o sobrenadante. Verifique se o pellet é agora branco. NOTA: Se o pellet ainda aparece de vermelho, uma lise adicional pode ser necessária antes de prosseguir para a próxima etapa. </li><li> Re-suspender o sedimento em 1 ml de tampão de FACS. Centrifuga-se as amostras durante 5 min a 900 x g. Retire e elimine o sobrenadante. </li><li> Re-suspender o sedimento em 300 ul de tampão de FACS e quantificar as células GFP + utilizando citometria de fluxo (NOTA: Nesta altura desejada qualquer imunocoloração das células pode ser realizada, usando técnicas convencionais, antes da análise por citometria de fluxo). </li></ol>

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The authors have nothing to disclose.

Busulfano condicionado permite a geração de alto nível BM quimerismo em ratinhos com células hematopoiéticas, que são prontamente detectáveis. Este condicionamento pode ser realizado sem a necessidade de instalações de irradiação. Além disso, a mielossupressão com as doses de bussulfano descrito neste protocolo é bem tolerado, minimizando os efeitos secundários tóxicos causados ​​por doses mieloablativas de irradiação, e, portanto, pode ser uma técnica mais apropriada para gerar BM quimerismo em j...

Recently, there has been considerable interest in the roles of microglia and bone marrow (BM)-derived cells (BMDCs) in the central nervous system (CNS), both during disease as well as with normal aging. Microglia, the resident immune cells of the CNS, are now known to develop in the CNS following the entry of primitive myeloid progenitors during embryogenesis1. Microglia retain aspects of their myelomonocytic lineage well into adult life. While evidence suggests that the contribution of BMDCs to the microglial pool is minimal in healthy adult animals1, the role BMDCs play in the progression of various neurodegenerative diseases remains unclear. This uncertainty is compounded by the fact that it is difficult to distinguish BMDCs that accumulate within the CNS from endogenous resident microglia, as no universally accepted discriminating immunohistochemical marker has been identified. In order to monitor BMDCs in vivo, we employ a BM transplantation (BMT) protocol whereby endogenous BM cells of a recipient mouse are replaced with those of a donor mouse ubiquitously expressing green fluorescent protein (GFP) under control of the &#946;-actin promoter. This protocol permits the determination of both the localization and morphology of GFP+ BMDCs within the recipient mouse using immunohistochemistry, and facilitates further characterization of these cells using fluorescence-activated cell sorting (FACS) followed by subsequent biochemical assays. 
For a successful BMT, recipient BM cells need to be ablated (termed myeloablation) in order to generate niche space within the recipient BM to allow for donor cell engraftment. Often, myeloablation is achieved using total body gamma irradiation which induces double strand breaks in DNA that leads to cell death, particularly in actively dividing cells such as hematopoietic progenitor cells2,3. The irradiation protocols are done to induce sufficient BM cell death such that animal lethality results if the recipient animal does not achieve adequate donor cell engraftment from BMT (so called &#8216;lethal irradiation&#8217;). However, irradiation requires a specialized facility and equipment, along with veterinary and animal husbandry resources not available to researchers at all institutions. Furthermore, myeloablative irradiation can cause potentially lethal damage to other tissues, and due to immunosuppresion, irradiated animals are more susceptible to secondary infections3. As such, reduced intensity conditioning (so called &#8216;RIC&#8217; protocols) regimens have been developed that are intended to minimize potentially toxic side effects of the conditioning protocol in patients, particularly for use in at risk populations such as children and the elderly4. 
Some RIC protocols rely upon chemotherapeutics such as busulfan in order to condition the BM compartment. Busulfan is a bifunctional DNA alkylating agent often used clinically as an alternative to irradiation5,6. Notably, busulfan can be safely administered to mice by intraperitoneal (IP) injection and does not require the specialized facilities and equipment necessary to irradiate mice. Busulfan conditioning has been used extensively in our lab7, as well as in several other recent publications8&#8211;11. When doses of 60-100 mg/kg are employed, high degrees of stable chimerism (>80% GFP+ cells) can be established in the peripheral blood and BM7. Importantly, at these doses myeloablation is not complete and as such mice are able to survive without receiving support BM cells (K. Peake, J. Manning, C. Lewis, and C. Krieger, unpublished observations). Moreover, with myelosuppressive busulfan conditioning there is rapid reconstitution of peripheral blood myelomonocytic cells by donor cells. However, the replacement of peripheral blood lymphocytes by donor cells is slower, highlighting the lack of immunosuppression that occurs with 60-100 mg/kg doses of busulfan compared to total body irradiation7. 
We have successfully used busulfan-induced chimerism to investigate BMDCs in wild-type mice, as well as in mouse models of the neurodegenerative disorders amyotrophic lateral sclerosis (ALS)7 and Alzheimer disease. It has been observed that under certain conditions significant numbers of GFP+ BMDCs accumulate within the CNS7,12. Importantly, like myeloablative conditioning with irradiation, busulfan conditioning followed by BMT is sufficient to allow for GFP+ BMDC accumulation within the CNS in both wild-type mice, and mice with neurological disorders7&#8211;11. As a major obstacle in treating neurodegenerative disorders is the ability to get therapeutic molecules from the circulation into the CNS where they can exert beneficial effects, this raises the possibility that BMDCs could be engineered to express therapeutic molecules, and subsequently used as a vehicle to deliver therapeutics to the CNS of busulfan conditioned recipients, a mechanism we are actively investigating. Although we have found that myeloablative conditioning with irradiation leads to greater accumulation of GFP+ BMDCs in the CNS compared to busulfan conditioning7, irradiation may have considerable toxicity in patients with neurodegenerative disorders and in murine models of CNS disease.
While we, and many others, have used BMT models to study BMDCs in neurodegenerative disorders, the ability to largely replace the BM compartment of a recipient mouse with a distinctly identifiable population of BM cells is an invaluable tool for studying various aspects of the hematopoietic system. This includes a wide array of research topics such as hematopoietic lineage development, leukemia, organ transplantation, graft-versus-host disease, and immunobiology.

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		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Busulfex</td>
			<td style="width:224px;">Otsuka Canada Pharmaceutical Inc.</td>
			<td style="width:119px;">DIN 02240602</td>
			<td>Dilute busulfan to 3 mg/mL with sterile PBS just prior to use. Busulfan is cytotoxic. Handle and dispose according to the MSDS and institutional guidelines.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ACK Lysing Buffer</td>
			<td style="width:224px;">Life Technologies</td>
			<td style="width:119px;">A10492-01</td>
			<td>Other lysis buffer could be substituted but incubation times may need to be adjusted accordingly</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal Bovine Serum</td>
			<td style="width:224px;">Life Technologies</td>
			<td>12483-020</td>
			<td></td>
		</tr>
	</tbody>
</table>

busulfan as a myelosuppressive agent for generating stable high-level bone marrow chimerism in mice

Bone marrow transplantation (BMT) is often used to replace the bone marrow (BM) compartment of recipient mice with BM cells expressing a distinct biomarker isolated from donor mice. This technique allows for identification of donor-derived hematopoietic cells within the recipient mice, and can be used to isolate and characterize donor cells using various biochemical techniques. BMT typically relies on myeloablative conditioning with total body irradiation to generate niche space within the BM compartment of recipient mice for donor cell engraftment. The protocol we describe here uses myelosuppressive conditioning with the chemotherapeutic agent busulfan. Unlike irradiation, which requires the use of specialized facilities, busulfan conditioning is performed using intraperitoneal injections of 20 mg/kg busulfan until a total dose of 60-100 mg/kg has been administered. Moreover, myeloablative irradiation can have toxic side effects and requires successful engraftment of donor cells for survival of recipient mice. In contrast, busulfan conditioning using these doses is generally well tolerated and mice survive without donor cell support. Donor BM cells are isolated from the femurs and tibiae of mice ubiquitously expressing green fluorescent protein (GFP), and injected into the lateral tail vein of conditioned recipient mice. BM chimerism is estimated by quantifying the number of GFP+ cells within the peripheral blood following BMT. Levels of chimerism&nbsp;>80% are typically observed in the peripheral blood 3-4 weeks post-transplant and remain established for at least 1 year. As with irradiation, conditioning with busulfan and BMT allows for the accumulation of donor BM-derived cells within the central nervous system (CNS), particularly in mouse models of neurodegeneration. This busulfan-mediated CNS accumulation may be more physiological than total body irradiation, as the busulfan treatment is less toxic and CNS inflammation appears to be less extensive. We hypothesize that these cells can be genetically engineered to deliver therapeutics to the CNS.

A administração de 60-100 mg / kg de busulfano é geralmente bem tolerada pelos ratinhos. No entanto, os animais normalmente perdem peso durante a fase de condicionamento (~ 5-10%) e, assim, a dieta pode ter de ser complementada com guloseimas como irradiados sementes de girassol para promover a alimentação. Os níveis de quimerismo de células de&gt; 80% para GFP + no sangue periférico e BM deve ser consistentemente alcançável usando 60-100 mg / kg de busulfano 7. Nosso laboratório tem utilizado com ...

Watch this Scientific Journal Video about Busulfan as a Myelosuppressive Agent for Generating Stable High-level Bone Marrow Chimerism in Mice at JoVE.com

Busulfan as a Myelosuppressive Agent for Generating Stable High-level Bone Marrow Chimerism in Mice

We describe a protocol whereby busulfan conditioning permits the bone marrow of a recipient mouse to be replaced with bone marrow cells from donor mice ubiquitously expressing green fluorescent protein, in the absence of irradiation. This technique is useful to study bone marrow cell accumulation in the central nervous system.

Busulfan como Agente mielosupressores para a geração estável de alto nível de Medula Óssea quimerismo em Ratos

Bone marrow transplantation (BMT) is often used to replace the bone marrow (BM) compartment of recipient mice with BM cells expressing a distinct ...

busulfan-as-myelosuppressive-agent-for-generating-stable-high-level

Research

JoVE Journal

Medicine

13.1K visualizações.  Simon Fraser University. We describe a protocol whereby busulfan conditioning permits the bone marrow of a recipient mouse to be replaced with bone marrow cells from donor mice ubiquitously expressing green fluorescent protein, in the absence of irradiation. This technique is useful to study bone marrow cell accumulation in the central nervous system.

Vídeo: Busulfan as a Myelosuppressive Agent for Generating Stable High-level Bone Marrow Chimerism in Mice

Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are attractive druggable therapeutic targets. Here we describe an efficient four-step strategy for pre-clinical evaluation of tyrosine kinase inhibitors (TKIs) in the treatment of acute leukemia. Initially, western blot analysis is used to confirm target inhibition in cultured leukemia cells. Functional activity is then evaluated using clonogenic assays in methylcellulose or soft agar cultures. Experimental compounds that demonstrate activity in cell culture assays are evaluated in vivo using NOD-SCID-gamma (NSG) mice transplanted orthotopically with human leukemia cell lines. Initial in vivo pharmacodynamic studies evaluate target inhibition in leukemic blasts isolated from the bone marrow. This approach is used to determine the dose and schedule of administration required for effective target inhibition. Subsequent studies evaluate the efficacy of the TKIs in vivo using luciferase expressing leukemia cells, thereby allowing for non-invasive bioluminescent monitoring of leukemia burden and assessment of therapeutic response using an in vivo bioluminescence imaging system. This strategy has been effective for evaluation of TKIs in vitro and in vivo and can be applied for identification of molecularly-targeted agents with therapeutic potential or for direct comparison and prioritization of multiple compounds.

Receptor tyrosine kinases are ectopically expressed in many cancers and have been identified as therapeutic targets in acute leukemia. This manuscript describes an efficient strategy for pre-clinical evaluation of tyrosine kinase inhibitors for the treatment of acute leukemia.

A avaliação pré-clínica da tirosina quinase inibidores para o tratamento de leucemia aguda

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are ...

Medicina

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

A Cultura de Tecidos modelo tridimensional para estudar primária da medula óssea humana e suas Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

Urinary Bladder Distention Evoked Visceromotor Responses as a Model for Bladder Pain in Mice

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized by increased urgency and frequency of urination, as well as nocturia and general pelvic pain, especially upon bladder filling or voiding. Despite years of research, the cause of IC/BPS remains elusive and treatment strategies are unable to provide complete relief to patients. In order to study nervous system contributions to the condition, many animal models have been developed to mimic the pain and symptoms associated with IC/BPS. One such murine model is urinary bladder distension (UBD). In this model, compressed air of a specific pressure is delivered to the bladder of a lightly anesthetized animal over a set period of time. Throughout the procedure, wires in the superior oblique abdominal muscles record electrical activity from the muscle. This activity is known as the visceromotor response (VMR) and is a reliable and reproducible measure of nociception. Here, we describe the steps necessary to perform this technique in mice including surgical manipulations, physiological recording, and data analysis. With the use of this model, the coordination between primary sensory neurons, spinal cord secondary afferents, and higher central nervous system areas involved in bladder pain can be unraveled. This basic science knowledge can then be clinically translated to treat patients suffering from IC/BPS.

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized in part by pelvic pain. In order to study nervous system contributions to the condition, a physiological model of urinary bladder pain is used in mice and rats.

Respostas Bexiga Distensão Evocado Visceromotor como um modelo para Bexiga dor em camundongos

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition ...

Femoral Bone Marrow Aspiration in Live Mice

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed step-by-step technical procedure for an analogous procedure in live mice which allows for serial characterization of cells present in the BM. This procedure facilitates studies aimed to detect the presence of exogenously administered cells within the BM of mice as would be done in xenograft studies for instance. Moreover, this procedure allows for the retrieval and characterization of cells enriched in the BM such as hematopoietic stem and progenitor cells (HSPCs) without sacrifice of mice. Given that the cellular composition of peripheral blood is not necessarily reflective of proportions and types of stem and progenitor cells present in the marrow, procedures which provide access to this compartment without requiring termination of the mice are very helpful. The use of femoral bone marrow aspiration is illustrated here for cytological analysis of marrow cells, flow cytometric characterization of the hematopoietic stem/progenitor compartment, and culture of sorted HSPCs obtained by femoral BM aspiration compared with conventional marrow harvest.

This protocol describes a procedure for serial sampling of femoral bone marrow (BM) without requiring the sacrifice of mice. This procedure facilitates longitudinal studies of the BM composition of mice over time and provides serial access to cells within the BM for ex vivo and transplantation studies.

Femoral Medula Óssea aspiração Live Ratos

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

Infusões artéria carótida para farmacocinéticas e farmacodinâmicas Análise de taxanos em Ratos

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

The study of breast cancer dormancy in the bone marrow is an exceptionally difficult undertaking due to the complexity of the interactions of dormant cells with their microenvironment, their rarity and the overwhelming excess of hematopoietic cells. Towards this end, we developed an in vitro 2D clonogenic model of dormancy of estrogen-sensitive breast cancer cells in the bone marrow. The model consists of a few key elements necessary for dormancy. These include 1) the use of estrogen sensitive breast cancer cells, which are the type likely to remain dormant for extended periods, 2) incubation of cells at clonogenic density, where the structural interaction of each cell is primarily with the substratum, 3) fibronectin, a key structural element of the marrow and 4) FGF-2, a growth factor abundantly synthesized by bone marrow stromal cells and heavily deposited in the extracellular matrix. Cells incubated with FGF-2 form dormant clones after 6 days, which consist of 12 or less cells that have a distinct flat appearance, are significantly larger and more spread out than growing cells and have large cytoplasm to nucleus ratios. In contrast, cells incubated without FGF-2 form primarily growing colonies consisting of >30 relatively small cells. Perturbations of the system with antibodies, inhibitors, peptides or nucleic acids on day 3 after incubation can significantly affect various phenotypic and molecular aspects of the dormant cells at 6 days and can be used to assess the roles of membrane-localized or intracellular molecules, factors or signaling pathways on the dormant state or survival of dormant cells. While recognizing the in vitro nature of the assay, it can function as a highly useful tool to glean significant information about the molecular mechanisms necessary for establishment and survival of dormant cells. This data can be used to generate hypotheses to be tested in vivo models.

An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

We developed an in vitro model of dormancy in the bone marrow for estrogen-sensitive breast cancer cells. The goal of this protocol is to demonstrate use of the model for the study of the molecular and cellular biology of dormancy and for generation of hypotheses for subsequent testing in vivo.

Um<em&gt; In Vitro</em&gt; Dormência Modelo de câncer de mama estrogênio-sensíveis na medula óssea: Uma Ferramenta para Estudos Moleculares Mecanismo e Hipótese Geração

The study of breast cancer dormancy in the bone marrow is an exceptionally difficult undertaking due to the complexity of the interactions of dormant ...

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest improved outcome measures in both experimental and clinical studies. Although the BMMSC are implanted into the tendon lesion in large numbers (usually 10 - 20 million cells), only a relatively small number survive (&#60;10%) although these can persist for up to 5 months after implantation. This appears to be a common observation in other species where BMMSC have been implanted into other tissues and it is important to understand when this loss occurs, how many survive the initial implantation process and whether the cells are cleared into other organs. Tracking the fate of the cells can be achieved by radiolabeling the BMMSC prior to implantation which allows non-invasive in vivo imaging of cell location and quantification of cell numbers.
This protocol describes a cell labeling procedure that uses Technetium-99m (Tc-99m), and tracking of these cells following implantation into injured flexor tendons in horses. Tc-99m is a short-lived (t1/2 of 6.01 hr) isotope that emits gamma rays and can be internalized by cells in the presence of the lipophilic compound hexamethylpropyleneamine oxime (HMPAO). These properties make it ideal for use in nuclear medicine clinics for the diagnosis of many different diseases. The fate of the labeled cells can be followed in the short term (up to 36 hr) by gamma scintigraphy to quantify both the number of cells retained in the lesion and distribution of the cells into lungs, thyroid and other organs. This technique is adapted from the labeling of blood leukocytes and could be utilized to image implanted BMMSC in other organs.

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

This protocol describes the radiolabeling of equine mesenchymal stem cells and their implantation into tendon injuries in the horse in order to determine cell survival and tissue distribution using gamma scintigraphy.

In Vivo Imaging and Rastreamento de Tecnécio-99m Rotulado de Medula Óssea Células-Tronco Mesenquimais em Equine Tendinopatia

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest ...

Database-guided Flow-cytometry for Evaluation of Bone Marrow Myeloid Cell Maturation

A working group initiated within the French Cytometry Association (AFC) was developed in order to harmonize the application of multiparameter flow cytometry (MFC) for myeloid disease diagnosis in France. The protocol presented here was agreed-upon and applied between September 2013 and November 2015 in six French diagnostic laboratories (University Hospitals of Saint-Etienne, Grenoble, Clermont-Ferrand, Nice, and Lille and Institut Paoli-Calmettes in Marseille) and allowed the standardization of bone marrow sample preparation and data acquisition. Three maturation databases were developed for neutrophil, monocytic, and erythroid lineages with bone marrow from "healthy" donor individuals (individuals without any evidence of a hematopoietic disease). A robust method of analysis for each myeloid lineage should be applicable for routine diagnostic use. New cases can be analyzed in the same manner and compared against the usual databases. Thus, quantitative and qualitative phenotypic abnormalities can be identified and those above 2SD compared with data of normal bone marrow samples should be considered indicative of pathology. The major limitation is the higher variability between the data achieved using the monoclonal antibodies obtained with the methods based on hybridoma technologies and currently used in clinical diagnosis. Setting criteria for technical validation of the data acquired may help improve the utility of MFC for MDS diagnostics. The establishment of these criteria requires analysis against a database. The reduction of investigator subjectivity in data analysis is an important advantage of this method.

The MDS diagnosis is difficult in the absence of morphological criteria or non-informative cytogenetics. MFC could help refine the MDS diagnostic process. To become useful for clinical practice, the MFC analysis must be based on parameters with sufficient specificity and sensitivity, and data should be reproducible between different operators.

Orientada para o banco de dados-citometria de fluxo para avaliação da maturação de células mieloides de medula óssea

A working group initiated within the French Cytometry Association (AFC) was developed in order to harmonize the application of multiparameter flow ...

Quantitative Mapping of Specific Ventilation in the Human Lung using Proton Magnetic Resonance Imaging and Oxygen as a Contrast Agent

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of the fresh gas entering a lung region divided by the region&#8217;s end-expiratory volume &#8213; in the human lung, using only inhaled oxygen as a contrast agent. Regional quantification of specific ventilation has the potential to help identify areas of pathologic lung function. Oxygen in solution in tissue shortens the tissue&#8217;s longitudinal relaxation time (T1), and thus a change in tissue oxygenation can be detected as a change in T1-weighted signal with an inversion recovery acquired image. Following an abrupt change between two concentrations of inspired oxygen, the rate at which lung tissue within a voxel equilibrates to a new steady-state reflects the rate at which resident gas is being replaced by inhaled gas. This rate is determined by specific ventilation. To elicit this sudden change in oxygenation, subjects alternately breathe 20-breath blocks of air (21% oxygen) and 100% oxygen while in the MRI scanner. A stepwise change in inspired oxygen fraction is achieved through use of a custom three-dimensional (3D)-printed flow bypass system with a manual switch during a short end-expiratory breath hold. To detect the corresponding change in T1, a global inversion pulse followed by a single shot fast spin echo sequence was used to acquire two-dimensional T1-weighted images in a 1.5&#160;T MRI scanner, using an eight-element torso coil. Both single slice and multi-slice imaging are possible, with slightly different imaging parameters. Quantification of specific ventilation is achieved by correlating the time-course of signal intensity for each lung voxel with a library of simulated responses to the air/oxygen stimulus. SVI estimations of specific ventilation heterogeneity have been validated against multiple breath washout and proved to accurately determine the heterogeneity of the specific ventilation distribution.

Specific ventilation imaging is a functional magnetic resonance imaging technique that allows for quantification of regional specific ventilation in the human lung, using inhaled oxygen as a contrast agent. Here, we present a protocol to collect and analyze specific ventilation imaging data.

Mapeamento quantitativo de ventilação específica no pulmão humano usando ressonância magnética de prótons e oxigênio como agente de contraste

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of ...

Flow Cytometry Analysis of Immune Cell Subsets within the Murine Spleen, Bone Marrow, Lymph Nodes and Synovial Tissue in an Osteoarthritis Model

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent evidence indicates a potential inflammatory component of the disease, with both T-cells and monocytes/macrophages potentially associated with the pathogenesis of OA. Further studies postulated an important role for subsets of both inflammatory cell lineages, such as Th1, Th2, Th17, and T-regulatory lymphocytes, and M1, M2, and synovium-tissue-resident macrophages. However, the interaction between the local synovial and systemic inflammatory cellular response and the structural changes in the joint is unknown. To fully understand how T-cells and monocytes/macrophages contribute towards OA, it is important to be able to quantitively identify these cells and their subsets simultaneously in synovial tissue, secondary lymphatic organs and systemically (the spleen and bone marrow). Nowadays, the different inflammatory cell subsets can be identified by a combination of cell-surface markers making multi-color flow cytometry a powerful technique in investigating these cellular processes. In this protocol, we describe detailed steps regarding the harvest of synovial tissue and secondary lymphatic organs as well as generation of single cell suspensions. Furthermore, we present both an extracellular staining assay to identify monocytes/macrophages and their subsets as well as an extra- and intra-cellular staining assay to identify T-cells and their subsets within the murine spleen, bone marrow, lymph nodes and synovial tissue. Each step of this protocol was optimized and tested, resulting in a highly reproducible assay that can be utilized for other surgical and non-surgical OA mouse models.

Here, we describe a detailed and reproducible flow cytometry protocol to identify monocyte/macrophage and T-cell subsets using both extra- and intracellular staining assays within the murine spleen, bone marrow, lymph nodes and synovial tissue, utilizing an established surgical model of murine osteoarthritis.

Análise de Citometria de Fluxo de Subconjuntos de Células Imunes dentro do Baço de Murina, Medula Óssea, Linfonodos e Tecido Sinovial em um Modelo de Osteoartrite