ドナーの動作と後肢の収穫<ol><li>手術手順については顕微器具の滅菌セットが利用されている。外科医は、外科的処置の開始前に変更する必要が滅菌手袋、を含む、適切な服装を着用してください。 </li><li>ラットは、ペントバルビタールの腹腔内注射（50mg/kg IP）で麻酔ですし、全体の手術を通して安定した動物の体の温度を容易に加熱された操作のフィールド上に仰臥位に置かれる。 </li><li>前の操作から提出された皮膚切開を3回の両方でそれぞれ、消毒剤やアルコールを消毒しています。乾燥から動物の角膜を防ぐために、眼は手術前に眼軟膏（Vidisic）で潤滑されています。 </li><li>皮膚切開は約鼠径靭帯のレベルで右脚の付け根を越え行われます。大腿血管はバイポーラ焼灼し、鈍的切開とフリー解剖され、深い枝は8から0絹と連結されています。 </li><li>ヘパリンの300μl（50 IU）は、抗凝固のための陰茎静脈を介して注入されています。 </li><li>大腿血管は鼠径靱帯のレベルで連結し、合字の遠位カットされています。 </li><li>後肢は、氷冷（4℃）HTK液（5ml）を5分かけて大腿動脈経由でフラッシュされます。 </li><li>すべての筋肉群を大幅にはさみで分割され、骨切り術は大腿骨の遠位第三のレベルで見た回転電機で行われます。 </li><li>収穫された四肢は、移植までの保存および低温貯蔵のための氷冷HTK液の20mlの中に沈めている。 </li></ol>受信者の動作と移植<ol><li>ラットでは、操作のフィールド上にペントバルビタールと置か仰臥位の腹腔内注射で麻酔です。 </li><li>皮膚切開は約鼠径靭帯のレベルで右脚の付け根を越え行われます。大腿血管はバイポーラ焼灼し、鈍的切開とフリー解剖されています。 </li><li>大腿血管は可能ですが、心窩部血管の離陸に近位のような遠位に連結される。血管クランプを動脈と静脈で鼠径靱帯のレベルに配置されます。 </li><li>ポリエチレンカフは、動脈を介してプルアップされる（カフの内径：0.912ミリメートル、カフ壁の厚さ：0.010ミリメートル、袖口の長さ：2.0 mm）と静脈（カフの内径：1.151ミリメートル、カフ壁の厚さ：0.010ミリメートル、カフの長ささ2.0mm）。血管の内腔の端は、血管拡張剤でプルアップされ、大腿動脈と静脈はカフ（約2mm）の全身にevertedと円周8から0絹糸結紮で固定されています。 </li><li>ドナーの手足用のスペースを確保するために受信者の手足は、筋肉群の鋭い解剖で削除され、骨切りは半ば大腿骨で行われます。 </li><li>ドナーの肢は、HTK液から除去し、移植は18ゲージの針を（長さ：1.5〜2.0センチメートル）を使用して大腿骨の骨接合術で開始されます髄内ロッドとして。 </li><li>腹側筋群は密接に4から0 VicrylまたはProlene中断縫合糸で近似されています。 </li><li>血管吻合はレシピエントの静脈と袖口の複雑な上にドナーの静脈と動脈を引いて実行されます。円周8から0絹糸結紮は再び吻合を確保するために配置されます。 </li><li>静脈クランプは、動脈クランプ続いてリリースされ、再灌流は、四肢遠位や足への色のリターンだけでなく、動脈を通る脈動流によって可視化されている。 </li><li>残りの背側の筋群は4から0 VicrylまたはProlene中断縫合糸で再近似されています。 </li><li>皮膚は4から0 vicrylまたはprolene中断縫合で閉じられます。 </li><li>レシピエントラットを加熱パッド上に配置され、回復するまで厳重に監視されています。 </li><li>レシピエント動物は、3つの移植後の日のために予防的抗生物質（セファゾリン、20mg /日、IM）とBuprenex（0.1 mg / kgを、SC）を受け取ります。 </li></ol>

<ol>
	<li>Shapiro, R. I., Cerra, F. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+for+reimplantation+and+transplantation+of+a+complex+organ:+the+rat+hind+limb.">A model for reimplantation and transplantation of a complex organ: the rat hind limb.</a> J Surg Res. 24 (6), 501-506 (1978).</li><li>Petruzzo, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+international+registry+on+hand+and+composite+tissue+transplantation.">The international registry on hand and composite tissue transplantation.</a> Transplantation. 86 (4), 487-492 (2008).</li><li>Schneeberger, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First+forearm+transplantation:+outcome+at+3+years.">First forearm transplantation: outcome at 3 years.</a> Am J Transplant. 7 (7), 1753-1762 (2007).</li></ol>

利害の衝突は宣言されません。

実験的な研究の十年は前と最近の過去に成功した人間の手の移植が有効になっている。 1978年に、シャピロら。よく、様々な病理学的、生理学的、および免疫学的な側面だけでなく、複合組織移植片の機能的な結果を研究に適したラットにおける同所性後肢移植モデルを、開発した。我々は、非縫合カフ技術を導入することにより、すなわち、血管吻合を手順の最も困難な部分を変更しまし?...

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orthotopic hind-limb transplantation in rats

複合組織の同種移植（CTA）は、手、前腕または数字の消失後に有効な治療選択肢を表しており、再建手術における新しい治療のエンティティとなっています。しかし、長期的に高用量の多剤免疫抑制療法は、より広範なアプリケーションをホールター重篤な副作用のリスクに耐える、移植片の生存を保証するために必要です。この分野でのさらなる進行は、基本的な免疫学および複合組織移植における虚血再灌流障害のよりよい理解に依存するかもしれません。 これまでに、ラットの同所後肢移植が再建移植（RT）の優先げっ歯類のモ​​デルをされている、しかし、それは血管、骨、筋肉と神経の再付着のために特別顕微スキルを必要とする過酷な手順です。 我々は、後肢の移植をラットに迅速で信頼性の高いアプローチを提供して、このモデルに血管カフ吻合技術を導入している。この手法では、簡素化と短縮外科手術をして正常に高い生存率と低い合併症率との操作を実行するための基本的な顕微経験を持つ外科医が可能になります。テクニックはよく免疫だけでなく、虚血再灌流障害（IRI）の研究に適しているように見える。

Watch this Scientific Journal Video about Orthotopic Hind-Limb Transplantation in Rats at JoVE.com

Orthotopic Hind-Limb Transplantation in Rats

ここでは、ゴールドスタンダードと思われる同所性ラット後肢移植の手順を、説明します。<em&gt; in vivoで</em複合組織の同種移植の研究のための&gt;モデル。

ラット同所性後肢移植

Composite tissue allotransplantation (CTA) now represents a valid therapeutic option after the loss of a hand, forearm or digits and has become a novel ...

orthotopic-hind-limb-transplantation-in-rats

Research

JoVE Journal

Immunology and Infection

11.6K ビュー.  Innsbruck Medical University. ここでは、ゴールドスタンダードと思われる同所性ラット後肢移植の手順を、説明します。 in vivoでモデル。

ビデオ: Orthotopic Hind-Limb Transplantation in Rats

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that distinguish acute and chronic inflammation in mouse models. With tissue damage or pathogen invasion, neutrophils are the first line of defense, playing a major role in mediating the acute inflammatory response. As the inflammatory reaction progresses, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages, which mediate chronic inflammation and promote tissue repair by removing tissue debris and producing anti-inflammatory cytokines. Intraperitoneal injection of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt) enables detection of acute inflammation largely mediated by tissue-infiltrating neutrophils. Luminol specifically reacts with the superoxide generated within the phagosomes of neutrophils since bioluminescence results from a myeloperoxidase (MPO) mediated reaction. Lucigenin (bis-N-methylacridinium nitrate) also reacts with superoxide in order to generate bioluminescence. However, lucigenin bioluminescence is independent of MPO and it solely relies on phagocyte NADPH oxidase (Phox) in macrophages during chronic inflammation. Together, luminol and lucigenin allow non-invasive visualization and longitudinal assessment of different phagocyte populations across both acute and chronic inflammatory phases. Given the important role of inflammation in a variety of human diseases, we believe this non-invasive imaging method can help investigate the differential roles of neutrophils and macrophages in a variety of pathological conditions.

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.

急性及び慢性炎症を区別するために生体内イメージング法における

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that ...

免疫・感染学

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

長期的な慢性<em&gt;緑膿菌</emマウスにおける&gt;気道感染

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

Complete Thymectomy in Adult Rats with Non-invasive Endotracheal Intubation

Thymectomy in neonatal rodents is an established and reliable procedure for immunological studies. However, in adult rats, complications of hemorrhage and pneumothorax from pleural disruption can result in a significant mortality rate. This protocol is a simple method of rat thymectomy that utilizes a mini-sternotomy and endotracheal intubation. Intubation is accomplished with a non-invasive and easily reproducible method and allows for positive pressure ventilation to prevent pneumothorax and a controlled airway that allows sufficient time for careful thymus dissection to minimize pleural disruption. A 1.5 cm sternal incision decreases contact with mediastinal vessels and pleura, while still providing full visualization of the thymus. Following exposure of the mediastinum, the thymus is removed by blunt dissection under magnification. The pleural space is then sealed by suture closure of the pre-tracheal muscles followed by the application of surgical glue. The thorax is then closed by suture closure of the sternum, followed by suture closure of the skin. All thymectomies were complete as evidenced by immunohistochemical (IHC) staining of mediastinal tissue, and absence of na&#239;ve T-cells by flow cytometry, and the procedure had a 96% survival rate. This method is suitable when complete thymectomy with minimal complications is desired for further immunological studies in athymic adult rats.

Rodent thymectomy is a valuable technique in immunological research. Here, a protocol for complete thymectomy in adult rats using a mini-sternotomy along with non-invasive intubation and positive pressure ventilation to minimize perioperative morbidity and mortality is described.

非侵襲的気管挿管と成体ラットにおける完全な胸腺摘出

Thymectomy in neonatal rodents is an established and reliable procedure for immunological studies. However, in adult rats, complications of hemorrhage and ...

Murine Hind Limb Long Bone Dissection and Bone Marrow Isolation

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, biomechanics, and stem cell biology. Despite the importance of the bone in healthy and pathologic states, however, it is a largely under-researched organ due to lack of specialized knowledge of bone dissection and bone marrow isolation. Mice are a common model organism to study effects on bone and bone marrow, necessitating a standardized and efficient method for long bone dissection and bone marrow isolation for processing of large experimental cohorts. We describe a straightforward dissection procedure for the removal of the femur and tibia that is suitable for downstream applications, including but not limited to histomorphologic analysis and strength testing. In addition, we outline a rapid procedure for isolation of bone marrow from the long bones via centrifugation with limited handling time, ideal for cell sorting, primary cell culture, or DNA, RNA, and protein extraction. The protocol is streamlined for rapid processing of samples to limit experimental error, and is standardized to minimize user-to-user variability.

Here we present a protocol for the dissection of hind limb long bones (femurs and tibiae) from the laboratory mouse. We further describe a rapid technique for bone marrow isolation from these bones that utilizes centrifugation for removal of bone marrow from the bone marrow space.

マウス後肢長骨解剖や骨髄の単離

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, ...

Murine Full-thickness Skin Transplantation

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited application to humans, skin transplantation in mice has been widely employed for transplantation research. The procedure is easy to learn and perform, and it does not require delicate microsurgical techniques nor extensive training. Moreover, graft rejection in this model occurs in a very reproducible immunological reaction and is easily monitored by direct inspection and palpation. In addition, secondary skin transplantation with donor-matched or third-party skin grafts can be performed on more complex transplant models as an alternative and uncomplicated method to assess donor-specific tolerance. The complications are low and are in general limited to anesthesia overdose or respiratory distress after the procedure. Graft failure, on the other hand, occurs commonly as a result of poor preparation of the graft, incorrect positioning in the graft bed, or inappropriate placement of the bandage. In this article, we present a protocol for full-thickness skin transplantation in mice and describe the important steps necessary for a successful procedure.

Murine full-thickness skin transplantation is a well-established model to study rejection in an alloimmune setting. Here, we provide a tutorial of each step involved in performing a BALB/c--&#62;C57BL/6 full-thickness skin transplant.

マウス全層皮膚移植

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited ...

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms requires reliable models. Here, we describe models of tolerance to cardiac allograft in rat, induced by blockade of costimulation signals or by upregulation of immunoregulatory molecules through gene transfer. Each of these models allowed in vivo generation of regulatory cells such as regulatory T cells (Tregs), regulatory B cells (Bregs) or regulatory myeloid cells (RegMCs). In this manuscript, we describe two complementary protocols that have been used to identify and define in vitro and in vivo regulatory cell activity to determine their responsibility in tolerance induction and maintenance. First, an in vitro suppressive assay allowed rapid identification of cells with suppressive capacity on effector immune responses in a dose dependent manner, and can be used for further analysis such as cytokine measurement or cytotoxicity. Second, the adoptive transfer of cells from a tolerant treated recipient to a newly irradiated grafted recipient, highlighted the tolerogenic properties of these cells in controlling graft directed immune responses and/or converting new regulatory cells (termed infectious tolerance). These methods are not restricted to cells with known phenotypic markers and can be extended to any cell population. Furthermore, donor directed allospecificity of regulatory cells (an important goal in the field) can be assessed by using third party donor cells or graft either in vitro or in vivo. Finally, to determine the specific tolerogenic capacity of these regulatory cells, we provide protocols to assess the humoral anti-donor antibody responses and the capacity of the recipient to develop humoral responses against new or former known antigens. The models of tolerance described can be used to further characterize regulatory cells, to identify new biomarkers, and immunoregulatory molecules, and are adaptable to other transplantation models or autoimmune diseases in rodent or human.

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

Here, we present a protocol to induce tolerance in transplantation, and assess in vitro and in vivo the suppressive capacity of distinct cell subsets from the recipient and the immune status of the recipient toward donor or exogenous antigens.

体外と体内の T, B, 骨髄細胞の抑制活性と移植から体液性応答の評価

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms ...

An Immunological Model for Heterotopic Heart and Cardiac Muscle Cell Transplantation in Rats

Heterotopic heart transplantation in rats has been a commonly used model for diverse immunological studies for more than 50 years. Several modifications have been reported since the first description in 1964. After 30 years of performing heterotopic heart transplantation in rats, we have developed a simplified surgical approach, which can be easily taught and performed without further surgical training or background.
After dissection of the ascending aorta and the pulmonary artery and ligation of superior and inferior caval and pulmonary veins, the donor heart is harvested and subsequently perfused with ice-cold saline solution supplemented with heparin. After clamping and incising the recipient abdominal vessels, the donor ascending aorta and pulmonary artery are anastomosed to the recipient abdominal aorta and inferior vena cava, respectively, using continuous running sutures.
Depending on different donor-recipient combinations, this model allows analyses of either acute or chronic rejection of allografts. The immunological significance of this model is further enhanced by a novel approach of in-ear injection of vital cardiac muscle cells and subsequent analysis of draining cervical lymphatic tissue.

We describe a model of heterotopic abdominal heart transplantation in rats, implying modifications of current strategies, which lead to a simplified surgical approach. Additionally, we describe a novel rejection model by in-ear injection of vital cardiac muscle cells, allowing further transplant immunological analyses in rats.

ラットにおける異所性心臓と心筋細胞移植のための免疫学的モデル

Heterotopic heart transplantation in rats has been a commonly used model for diverse immunological studies for more than 50 years. Several modifications ...

Optimization of the Cuff Technique for Murine Heart Transplantation

Murine cardiac transplantation has been performed for more than 40 years. With advancements in microsurgery, certain new techniques have been used to improve surgical efficiency. In our lab, we have optimized the cuff technique with two major steps. First, we used the inner tube technique to insert a temporary inner tube into the external jugular vein and carotid artery blood vessel to facilitate eversion of the vessel over the cuff. Second, we performed complete heterotopic cardiac transplantation through the collaboration of two experienced surgeons. These modifications effectively reduced the operation time to 25 minutes, with a success rate of 95%. In this report, we describe these procedures in detail and provide a supplemental video. We believe that this report on the improved cuff technique will offer practical guidance for murine heterotopic heart transplantation and will enhance the utility of this mouse model for basic research.

We introduce an inner tube approach to the cuff technique for mouse cervical heterotopic heart transplantation to help evert the vessel over the cuff. We found that cooperation between two experienced surgeons markedly shortens the operation time.

マウスの心臓移植のためのカフ技術の最適化

Murine cardiac transplantation has been performed for more than 40 years. With advancements in microsurgery, certain new techniques have been used to ...

Bone Marrow Transplantation Procedures in Mice to Study Clonal Hematopoiesis

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor cells (HSPCs). Mutations in driver genes, that confer cellular fitness, can lead to the development of expanding HSPC clones that increasingly give rise to progeny leukocytes harboring the somatic mutation. Because clonal hematopoiesis has been associated with heart disease, stroke, and mortality, the development of experimental systems that model these processes is key to understanding the mechanisms that underly this new risk factor. Bone marrow transplantation procedures involving myeloablative conditioning in mice, such as total-body irradiation (TBI), are commonly employed to study the role of immune cells in cardiovascular diseases. However, simultaneous damage to the bone marrow niche and other sites of interest, such as the heart and brain, is unavoidable with these procedures. Thus, our lab has developed two alternative methods to minimize or avoid possible side effects caused by TBI: 1) bone marrow transplantation with irradiation shielding and 2) adoptive BMT to non-conditioned mice. In shielded organs, the local environment is preserved allowing for the analysis of clonal hematopoiesis while the function of resident immune cells is unperturbed. In contrast, the adoptive BMT to non-conditioned mice has the additional advantage that both the local environments of the organs and the hematopoietic niche are preserved. Here, we compare three different hematopoietic cell reconstitution approaches and discuss their strengths and limitations for studies of clonal hematopoiesis in cardiovascular disease.

We describe three methods of bone marrow transplantation (BMT): BMT with total-body irradiation, BMT with shielded irradiation, and BMT method with no pre-conditioning (adoptive BMT) for the study of clonal hematopoiesis in mouse models.

クローン造造を研究するマウスにおける骨髄移植手順

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor ...

Purification and Expansion of Mouse Invariant Natural Killer T Cells for in vitro and in vivo Studies

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or microbial lipid antigens presented by the non-polymorphic MHC class I-related molecule CD1d. Preclinical and clinical studies support a role for iNKT cells in cancer, autoimmunity and infectious diseases. iNKT cells are very conserved throughout species and their investigation has been facilitated by mouse models, including CD1d-deficient or iNKT-deficient mice, and the possibility to unequivocally detect them in mice and men with CD1d tetramers or mAbs specific for the semi-invariant TCR. However, iNKT cells are rare and they need to be expanded to reach manageable numbers for any study. Because the generation of primary mouse iNKT cell line in vitro has proven difficult, we have set up a robust protocol to purify and expand splenic iNKT cells from the iV&#945;14-J&#945;18 transgenic mice (iV&#945;14Tg), in which iNKT cells are 30 times more frequent. We show here that primary splenic iV&#945;14Tg iNKT cells can be enriched through an immunomagnetic separation process, yielding about 95-98% pure iNKT cells. The purified iNKT cells are stimulated by anti-CD3/CD28 beads plus IL-2 and IL-7, resulting in 30-fold expansion by day +14 of the culture with 85-99% purity. The expanded iNKT cells can be easily genetically manipulated, providing an invaluable tool to dissect mechanisms of activation and function in vitro and, more importantly, also upon adoptive transfer in vivo.

We describe a rapid and robust protocol to enrich invariant natural killer T (iNKT) cells from mouse spleen and expand them in vitro to suitable numbers for in vitro and in vivo studies.

インビトロおよびインビボ研究のためのマウス不変性ナチュラルキラーT細胞の精製と拡大

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or ...