이 연구는 NIH / NIDDK에서 부여 1K01DK079019A2 (PT)에 의해 지원되었다.

1. 마우스 접종에 대한 박테리아 준비 다음은 무균 조건 하에서 수행되어야합니다. <ol><li> 모자와 하나 17 × 100 밀리미터 폴리 프로필렌 튜브를 타고 신선한 Luria 국물 (LB) 미디어 3 ML를 추가합니다. autoclaved 팁을 사용하면, 스트레인 CP1 2의 일부 냉동 박테리아 글리세롤 주식을 가지고 LB 미디어를 포함하는 관에 문화의 작은 수량을 전송할 수 있습니다. 산소는 여전히 튜브를 입력 할 수 있도록 튜브 캡을 교체 - 문화 에어로빅 조건 하에서 성장해야합니다. 37 번 장소 튜브 ° 하루 아침에 220 rpm으로 흔들어 보육의 C. </li><li> 그 다음날, 3 ML의 LB 미디어와 함께 새로운 튜브에 밤 문화의 5 μl를 전송하고 37 ° C.에서 보육 밤의 정적 조건 하에서 성장 </li><li> 날 AA 50 ML 튜브에 3 전송 문화의 40 μl는 40 ML의 LB 미디어 각을 포함. 37 ° C 정적 보육의 하룻밤의 장소. </li><li> 켜, 4 ° C.에 원심 분리기를 설정일단 원심 분리기 최종 온도에 도달, 40 ML Nalgene의 원심 분리기 튜브에 각각 40 ML 문화를 전송할 수 있습니다. </li><li> 필요에 따라 LB 미디어와 함께 볼륨을 조절 - 원심 분리기의 균형을 튜브를 무게. 20 분에 6,000 rpm으로 튜브를 봐. </li><li> 조심스럽게 표면에 뜨는 제거하고 부드럽게 40 ML 멸균 PBS에서 박테리아 펠렛을 일시 중지합니다. </li><li> PBS와 단계 1.5를 반복합니다. </li><li> 표면에 뜨는을 대기음, 500 μl 멸균 PBS에서 박테리아를 일시 중지합니다. 1.5 ML의 microfuge 관에 솔루션을 전송합니다. 정지 10 μl를 타고 얼음처럼 차가운 PBS의 990 μl에 희석. 접종에 필요한 볼륨을 확인하려면 OD 420의 NM을 읽어 분광 광도계를 사용합니다. 의 적절한 볼륨을 확인하려면 얼음처럼 차가운 PBS가 펠릿을 중지 할 수 : OD 420의 나노 번호 (ML)를 타고 박테리아 펠렛 [예의 예상 볼륨을 뺍니다. OD 420 nm 정도 = 0.545, 펠렛 약 0.075 ML. 0.545-0.075 = 0.470]. </li><li> 10 박테리아 정지 μl와 디를 제거990 μl 얼음처럼 차가운 PBS의 류트. OD 420의 나노를 참조하십시오. 목표는 1.000의 희석 정지 ± 0.010을위한 OD 420의 nm의 값을 달성하는 것입니다. 귀하의 희석의 수가이 목표를 초과하면, 정지에 더 많은 볼륨을 추가하고 또 다른 읽기를. 수 아래 인 경우 0.990는 원하는 반응을 얻을 때까지 중지 및 반복 OD 420의 나노 읽기 스핀 다운. </li></ol> 2. 동물 감염 마우스 (5-7주 세, 그룹 당 10)가 잭슨 연구소 (바 하버, ME)에서 구입했다. <ol><li> 마우스는 Plexiglas 챔버에서 isoflurane 1~4%의 흡입에 의한 anesthetized하고 휴식하고있는 때까지 모니터링한다. </li><li> 한 번에 하나의 마우스는 유리 해밀턴 주사기 (1.5-2.0 cm의 길이)의 수정 된 30 G 바늘에 부착 된 폴리에틸렌 (PE10) 카테터와 catheterization에 의해 박테리아의 기지에 이루어집니다. 카테터는 바늘의 허브에 삽입됩니다. 마우스 plac입니다에드는 마취상의 등쪽 표면에 코 콘을 사용하여 유지. </li><li> 마우스의 음경은 음경 요도의 입구의 윤활에 사용되는 부드러운 압력과 면봉에 윤활제의 자유 양에 의해 압출입니다. </li><li> 10 μl 인산염은 버퍼 1 개 10 8 박테리아를 포함하는 염분은 catheterization 다음 anesthetized 마우스의 요도에 소개되어 있습니다. </li><li> 세균 첨부 파일을 허용하고 즉각적인 배뇨을 방지하기 위해 세균 소개 한 후 마우스가 Plexiglas 챔버에서 15 분 동안 anesthetized 상태로 유지됩니다. </li><li> 마우스들은 케이지에 다시 배치하고 24 시간 모니터링하고 있습니다. </li></ol> 3. 동작 테스트 마우스는 사전 감염 (기준)과 일 3, 7, 14, 21, 감염 후 28 테스트되었습니다. 라고 hyperalgesia와 촉각 allodynia은 복부 11,12과 발바닥 R에 적용 폰 프라이의 필라멘트를 사용하여 테스트되었습니다뒷다리 앞발로 땅을 차다 13 egion. 테스트는 시간의 하루 고정에 perfomed 된 표준 방법론과 모든 동물의 한 실험 테스트는 고용했다. 그룹의 눈이 멀어 테스트는 동물 모델에서 동작 기반의 통증 테스트의 한계를 방지하기 위해 활용되었다. 0.04의 힘, 0.16, 0.4, 1.0 및 4.0 g (Stoelting, USA)와 다섯 개인 폰 프라이의 필라멘트가 탈퇴 응답의 복부 및 주파수에 적용되었다가 계산되었습니다. <ol><li> 새 환경 순응의 30 분 기간 후, 마우스는 스테인레스 스틸 와이어 그리드 바닥 - 집에서 만든 개인 Plexiglas 챔버 (6 × 10 x12 cm)에 배치됩니다. </li><li> 라고 hyperalgesia와 촉각 allodynia은 다섯 폰 프라이의 필라멘트를 사용하여 테스트되었습니다. 각 필라멘트는 총 10 회에 5 초의 상호 자극 간격으로 1-2 초에 대해 적용하고, 머리카락이 힘의 오름차순으로 테스트되었습니다. </li><li> 각 필라멘트는 전립선의 일반적인 주변에 낮은 복부 지역에 적용되고주의 desensitization을 피하​​거나 효과 &quot;를 맺어야&quot;이 지역 내의 다른 지역을 자극하는 촬영되었습니다. 필라멘트는 10 배의 총 stimulations 사이에 최소 5 초 간격으로 1-2 초에 대해 힘의 순서를 증가에 적용되었습니다. </li><li> 행동의 세 가지 유형은 필라멘트 자극에 긍정적 인 반응으로 간주됩니다 : 복부 1) 날카로운 철회, 2) 즉시 핥아 또는 필라멘트 자극의 지역을 긁적, 3) 점프. </li><li> 응답 주파수는 긍정적 인 반응의 비율 (10 중에 10 = 50 % 예를 들어 5 응답)로 계산 및 데이터가 응답 주파수의 평균 비율 ± SE로보고되었다 </li><li> 25 개 이상의 긍정적 총 기본 응답과 동물은 연구에서 제외됩니다. </li><li> 촉각 allodynia는 0.04의 힘, 0.16, 0.4, 1.0 및 4.0 g과 폰 프라이의 필라멘트를 사용하여 뒷다리 손 안에 발바닥 지역에서 테스트되었습니다. 중간 50 % 탈퇴 임계 값 (5)이 위, 아래를 사용하여 평가 한필라멘트가 약간 구부러 때까지 테스트가 0.04 g 필라멘트로 시작 방법은 뒷다리 손 안에 발바닥 표면에 수직 적용. 필라멘트는 긍정적 인 반응이 관찰 될 때까지 오름차순으로 테스트되었습니다. 필라멘트에 대한 긍정적 인 반응은 테스트 손 안에 앞발로 땅을 차다이나 핥아의 날카로운 철회 중 하나로 정의되었다. 긍정적 인 반응이 기록되었을 때 다음 약한 필라멘트가 적용되고, 부정적인 응답이 관찰 된 경우, 다음 강한 필라멘트가 적용되었습니다. </li><li> 설명 실험과 방법론은 검토 및 승인 노스 웨스턴 대학 동물 의료 의해위원회를 사용하고 있습니다. </li></ol>

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관심 없음 충돌이 선언 없습니다.

UPEC와 마우스 전립선의 감염은 세균 prostatitis, CP / CPPS의 pathogenesis 또는 만성 염증에 predisposing 이벤트로 참여 할 수 있습니다 이벤트의 생체 모델링에서 할 수 있습니다. 방법은 여성 비뇨기의 요로 감염이 7,14에 UPEC 모델에 문학의 큰 몸에 세균 준비 및 떨어 뜨림 조 추첨에 대해 설명했다. 모델은 잠재적 인 백신 후보와 면역 변조의 메커니즘을 테스트 pathogenesis을 공부에 대한 폭 넓...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> 문화 튜브 </td><td> BD 팔콘 </td><td> 352059 </td></tr><tr><td> LB 국물 밀러 </td><td> EMD </td><td> EM1.10285.0500 </td></tr><tr><td> Nalgene의 원심 분리기 튜브 </td><td> 열 </td><td> 3118-0050 </td></tr><tr><td> Isoflurane </td><td> 버틀러 Schein </td><td> NDC 11695-6776-1 </td></tr><tr><td> 카테 테르 바늘 30g의 </td><td> 해밀턴 </td><td> 91,030 </td></tr><tr><td> PE10 튜브 </td><td> BD intramedic </td><td> 427400 </td></tr><tr><td> 본 프라이의 필라멘트 </td><td> Stoelting </td><td> 58025-31 </td></tr></tbody></table>

measurement of tactile allodynia in a murine model of bacterial prostatitis

Uropathogenic 대장균 (UPEC)는 요로 감염 및 세균 prostatitis 1 중요한 역할을 병원균입니다. 우리는 최근에 UPEC는 만성 골반 통증 2, 만성 prostatitis / 만성 골반 통증 증후군 (CP / CPPS) 3,4의 기능의 개시에 중요한 역할을하는 것으로 나타났습니다. 임상 관련 UPEC하여 전립선의 감염은 조직 손상과 autoimmunity 5 메커니즘의 개시를 포함 할 수 있습니다 메커니즘을 통해 만성 통증을 시작하고 설정할 수 있습니다. 전립선에 UPEC의 pathogenesis를 이해하는 데 도전 조작에 전립선의 상대적 어려움입니다. 우리는이를 통해 전립선의 오름차순 감염을 수립 남성 생쥐에 UPEC의 임상 부담이 제공하는 이전에 설명 intraurethral 감염 방법 6을 활용. 여기, 우리는 학사 학위를 표준화에 대한 우리의 프로토콜을 설명뿐만 아니라 박테리아의 기지에 anesthetized 남성 쥐를 catheterizing 절차 등 cterial inoculum 7. CP / CPPS는 주로 촉각 allodynia (4)의 존재에 의해 특징입니다. 동작 테스트는 언급 내장 통증 8-10으로 인한 피부 hyperalgesia의 개념을 기반으로했다. 내장 조직에 짜증 초점은 일반적으로 비 고통스러운 자극 (allodynia)에 대한 과장된 반응을 허용하는 피부 통증 임계 값을 감소시킵니다. 기본 내장 고통의 강도를 증가하는 경향이 이상 반응의 피부 결과 정상적인 힘의 응용 프로그램입니다. 우리는 UPEC 박테리아와 하나의 감염에 대한 응답으로 시간이 지남에 촉각 allodynia를 정할 폰 프라이 섬유를 활용 NOD / ShiLtJ 마우스에 방법을 설명합니다.

<html> 우리는 세균 감염시 만성 통증의 존재에 대한 NOD / ShiLtj와 C57BL/6J 수컷 쥐를 조사했다. 남성 마우스 (5~7주 이전)은 요도 (그림 1)에 생리 나 세균과 배우는되었습니다. 생리 - 배우는 C57BL/6J 마우스와 UPEC에 감염된 C57BL/6J 마우스의 폰 프라이의 필라멘트와 골반 지역의 기계적 자극이 실험의 28 일 코스 (그림 1) 동안 변경되지 않은 응답 주파수하게되었습니다. 반면, UPEC에...

Watch this Scientific Journal Video about Measurement of Tactile Allodynia in a Murine Model of Bacterial Prostatitis at JoVE.com

Measurement of Tactile Allodynia in a Murine Model of Bacterial Prostatitis

전립선의 감염은 만성 prostatitis의 골반 통증을 중재에 기여 요소가 될 수 있습니다. 우리는 표준화 된 박테리아 inoculum, 시간이 지남에 따라 마우스에 촉각 allodynia을 측정하는 남성 마우스 및 방법론의 요도에 박테리아의 기지의 준비에 대한 절차를 설명합니다.

세균 Prostatitis의 손쥐 모델에 촉각 Allodynia 측정

Uropathogenic Escherichia coli (UPEC) are pathogens that play an important role in urinary tract infections and bacterial prostatitis1. We have recently ...

measurement-tactile-allodynia-murine-model-bacterial

Research

JoVE Journal

Immunology and Infection

15.7K 조회수.  Northwestern University Feinberg School of Medicine. 전립선의 감염은 만성 prostatitis의 골반 통증을 중재에 기여 요소가 될 수 있습니다. 우리는 표준화 된 박테리아 inoculum, 시간이 지남에 따라 마우스에 촉각 allodynia을 측정하는 남성 마우스 및 방법론의 요도에 박테리아의 기지의 준비에 대한 절차를 설명합니다.

비디오: Measurement of Tactile Allodynia in a Murine Model of Bacterial Prostatitis

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

세균성 접착 연구에 전단 응력을 소개합니다

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

연구

면역학 및 감염병학

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

이식 - 대 - 호스트 질병의 유도 및<em&gt; 생체 내</emMHC 검색 손쥐 모델을 사용&gt; T 셀 모니터링

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

세대와의 멀티 표현형 높은 콘텐츠 상영<em&gt; Coxiella burnetii의</em&gt; 트랜스포존 돌연변이

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

그룹 B의 쥐 모델<em&gt; 연쇄상 구균</em&gt; 질 식민

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various pathologies such as cancer, atherosclerosis and diabetes. We have previously shown that dysfunction of the Nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/ Kelch-like erythroid cell-derived protein 1 (Keap1) signaling pathway leads to extreme ROS imbalance during cutaneous wound healing in diabetes. Since ROS levels are an important indicator of progression of wound healing, specific and accurate quantification techniques are valuable. Several in vitro assays to measure ROS in cells and tissues have been described; however, they only provide a single cumulative measurement per sample. More recently, the development of protein-based indicators and imaging modalities have allowed for unique spatiotemporal analyses. L-012 (C13H8ClN4NaO2) is a luminol derivative that can be used for both in vivo and in vitro chemiluminescent detection of ROS generated by NAPDH oxidase. L-012 emits a stronger signal than other fluorescent probes and has been shown to be both sensitive and reliable for detecting ROS. The time lapse applicability of L-012-facilitated imaging provides valuable information about inflammatory processes while reducing the need for sacrifice and overall reducing the number of study animals. Here, we describe a protocol utilizing L-012-facilitated in vivo imaging to quantify oxidative stress in a model of excisional wound healing using diabetic mice with locally dysfunctional Nrf2/Keap1.

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

We describe a non-invasive in vivo imaging protocol that is streamlined and cost-effective, utilizing L-012, a chemiluminescent luminol-analog, to visualize and quantify reactive oxygen species (ROS) generated in a mouse excisional wound model.

Vivo에서 영상에는 Murine 반응성 산소 종의 상처 모델

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various ...

Isolation of Single Intracellular Bacterial Communities Generated from a Murine Model of Urinary Tract Infection for Downstream Single-cell Analysis

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected in the urinary tract. The protocol can be broadly divided into three sections: the infection, bladder epithelial cell harvesting, and mouth micropipetting to isolate individual infected epithelial cells. The isolated epithelial cell contains viable bacterial cells and is nearly free of contaminating extracellular bacteria, making it ideal for downstream single-cell analysis. The time taken from the start of infection to obtaining a single intracellular bacterial community is about 8 h. This protocol is inexpensive to deploy and uses widely available materials, and we anticipate that it can also be utilized in other infection models to isolate single infected cells from cell mixtures even if those infected cells are rare. However, due to a potential risk in mouth micropipetting, this procedure is not recommended for highly infectious agents.

This protocol describes a simple method of isolating single, infected-bladder epithelial cells from a murine model of urinary tract infection.

다운스트림 단일 세포 분석에 대 한 요로 감염의 Murine 모델에서 생성 된 단일 세포내 세균성 지역 사회의 절연

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected ...

A Precise Pathogen Delivery and Recovery System for Murine Models of Secondary Bacterial Pneumonia

Secondary bacterial pneumonias following influenza infections consistently rank within the top ten leading causes of death in the United States. To date, murine models of co-infection have been the primary tool developed to explore the pathologies of both the primary and secondary infections. Despite the prevalence of this model, considerable discrepancies regarding instillation procedures, dose volumes, and efficacies are prevalent among studies. Furthermore, these efforts have been largely incomplete in addressing how the pathogen may be directly influencing disease progression post-infection. Herein we provide a precise method of pathogen delivery, recovery, and analysis to be used in murine models of secondary bacterial pneumonia. We demonstrate that intratracheal instillation enables an efficient and accurate delivery of controlled volumes directly and evenly into the lower respiratory tract. Lungs can be excised to recover and quantify the pathogen burden. Following excision of the infected lungs, we describe a method to extract high quality pathogen RNA for subsequent transcriptional analysis. This procedure benefits from being a non-surgical method of delivery without the use of specialized laboratory equipment and provides a reproducible strategy to investigate pathogen contributions to secondary bacterial pneumonia.

Here, we present methods to improve secondary bacterial pneumonia studies by providing a non-invasive route of instillation into the lower respiratory tract followed by pathogen recovery and transcript analysis. These procedures are reproducible and can be performed without specialized equipment such as cannulas, guide wires, or fiber optic cables.

이차 세균성 폐렴의 뮤린 모델을 위한 정확한 병원균 전달 및 회복 시스템

Secondary bacterial pneumonias following influenza infections consistently rank within the top ten leading causes of death in the United States. To date, ...

Measurement of Pulse Propagation Velocity, Distensibility and Strain in an Abdominal Aortic Aneurysm Mouse Model

An abdominal aortic aneurysm (AAA) is defined as a localized dilation of the abdominal aorta that exceeds the maximal intraluminal diameter (MILD) by 1.5 times of its original size. Clinical and experimental studies have shown that small aneurysms may rupture, while a subpopulation of large aneurysms may remain stable. Thus, in addition to the measurement of intraluminal diameter of the aorta, knowledge of structural traits of the vessel wall may provide important information to assess the stability of the AAA. Aortic stiffening has recently emerged as a reliable tool to determine early changes in the vascular wall. Pulse propagation velocity (PPV) along with the distensibility and radial strain are highly useful ultrasound-based methods relevant for assessing aortic stiffness. The primary purpose of this protocol is to provide a comprehensive technique for the use of ultrasound imaging system to acquire images and analyze the structural and functional properties of the aorta as determined by MILD, PPV, distensibility and radial strain.

This manuscript describes a detailed protocol for using high frequency ultrasound imaging to measure luminal diameter, pulse propagation velocity, distensibility and radial strain on a mouse model of abdominal aortic aneurysm.

복부 대동맥류 마우스 모델에서 맥박 전파 속도, 감도 및 변형률 측정

An abdominal aortic aneurysm (AAA) is defined as a localized dilation of the abdominal aorta that exceeds the maximal intraluminal diameter (MILD) by 1.5 ...

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant advantage over other methods. To prepare these probes, azide derivatives of antibiotics are synthesized, then coupled with alkyne-fluorophores using azide-alkyne dipolar cycloaddition by click chemistry. Following purification, the antibiotic activity of the fluorescent antibiotic is tested by minimum inhibitory concentration assessment. In order to study bacterial accumulation, either spectrophotometry or flow cytometry may be used, allowing for much simpler analysis than methods relying on radioactive antibiotic derivatives. Furthermore, confocal microscopy can be used to examine localization within the bacteria, affording valuable information about mode of action and changes that occur in resistant species. The use of fluorescent antibiotic probes in the study of antimicrobial resistance is a powerful method with much potential for future expansion.

Fluorescently tagged antibiotics are powerful tools that can be used to study multiple aspects of antimicrobial resistance. This article describes the preparation of fluorescently tagged antibiotics and their application to studying antibiotic resistance in bacteria. Probes can be used to study mechanisms of bacterial resistance (e.g., efflux) by spectrophotometry, flow cytometry, and microscopy.

형광 항생제 프로브를 사용한 세균 저항성 시각화

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant ...

A Neonatal Imaging Model of Gram-Negative Bacterial Sepsis

Neonates are at an increased risk of bacterial sepsis due to the unique immune profile they display in the first months of life. We have established a protocol for studying the pathogenesis of E. coli O1:K1:H7, a serotype responsible for high mortality rates in neonates. Our method utilizes intravital imaging of neonatal pups at different time points during the progression of infection. This imaging, paralleled by measurement of bacteria in the blood, inflammatory profiling, and tissue histopathology, signifies a rigorous approach to understanding infection dynamics during sepsis. In the current report, we model two infectious inoculums for comparison of bacterial burdens and severity of disease. We find that subscapular infection leads to disseminated infection by 10 h post-infection. By 24 h, infection of luminescent E. coli was abundant in the blood, lungs, and other peripheral tissues. Expression of inflammatory cytokines in the lungs is significant at 24 h, and this is followed by cellular infiltration and evidence of tissue damage that increases with infectious dose. Intravital imaging does have some limitations. This includes a luminescent signal threshold and some complications that can arise with neonates during anesthesia. Despite some limitations, we find that our infection model offers an insight for understanding longitudinal infection dynamics during neonatal murine sepsis, that has not been thoroughly examined to date. We expect this model can also be adapted to study other critical bacterial infections during early life.

Infection of neonatal mice with bioluminescent E. coli O1:K1:H7 results in a septic infection with significant pulmonary inflammation and lung pathology. Here, we describe procedures to model and further study neonatal sepsis using longitudinal intravital imaging in parallel with enumeration of systemic bacterial burdens, inflammatory profiling, and lung histopathology.

그람 음성 세균 패혈증의 신생아 화상 진찰 모형

Neonates are at an increased risk of bacterial sepsis due to the unique immune profile they display in the first months of life. We have established a ...