The study was supported by the Swedish Research Council, the Swedish Rheumatism Asociation, Karolinska Institute Foundations, Stockholm County Council, The Wallenberg Foundation, and the GV 80 Years' Foundation for research. The authors would also like to thank Hannah Aucott for proofreading the manuscript.

모든 동물 실험은 관리 및 카롤린스카 연구소, 스톡홀름에서 현지 윤리위원회의 승인을 동물의 사용 지침에 따라 수행 하였다. 지역 윤리위원회는 동물 보호에 관한 유럽 연합 지침을 따른다. 주 : 수술 (6 시간 내지 1 대) 및 LPS의 레벨 (15 밀리그램 / kg 대 2 밀리그램 / kg)를 주입 한 후 원래의 프로토콜에서의 주요 변화는 복구 시간이다. 그렇지 않으면, 수술 자체에 관련된 다른 설정이 변경되지 않았습니다. 1. 자극에 대한 자료를 준비 <ol><li> 컴퓨터와 자극 전극 (도 1A)에 연결된 데이터 수집 시스템 켜기. </li><li> 인정 인 프로그램을 시작합니다. </li><li> 5 밀리그램 / 1X PBS에 mL를 분취하여 농도의 LPS의 스톡 용액을 준비하고 -20 ℃에서 보관. 실험의 날, 나누어지는을 해동하고 에이드를 준비중량에 따라, 동물에 약 100 μL를 주입하기 위해 LPS (0.5 ㎎ / ㎖)의 quate 샘플. </li></ol> 2. 마취에 대한 동물 준비 <ol><li> C57BL / 6 마우스를 사용합니다. , 12 시간 빛 / 어둠주기와 기후의 조건을 유지 그들에게 표준 설치류 식사를 공급하고, 그들에게 임의 량의 물을 제공합니다. </li><li> 실험의 날에 약 25g 무게 쥐에 수술을 수행합니다. 참고 : 염증 반응이 유발되는 경우,이 정기 검진과 동물 임상 반응의 관찰을 수행하는 것이 특히 중요하다. 동물의 조건이 윤리적 기준을 충족하지 않는 경우 CO 2 흡입 조기 안락사가 필요합니다. </li><li> 마취 기계를 설정합니다. <ol><li> 튜브가 제대로 연결되어 있고 손상되지 않았는지 확인합니다. 통풍 영역이 제대로 작동하는지 확인합니다. 이소 플루 란 병에 키 필터를 연결하고 VAPO을 채우기이소 플루 란 충분한 양 rizer. </li><li> 가스 공급 (공기와 산소)를 열고 병 실험에 대한 충분한 가스를 포함하고 있는지 확인하십시오. 삼원 커넥터는 유도 챔버 또는 마스크에 이소 플루 란 흐름을 전송할 수있다. </li></ol></li><li> 유도 챔버에 삼원 커넥터를 돌립니다. 홈 케이지에서 하나의 마우스를 선택하고 챔버로 동물을 삽입합니다. 1.0 L / 분의 산소 및 1.0 L / 분으로 공기 유동 조절기를 조정. 5 % - 4로 이소 플루 란 농도를 조정합니다. </li><li> 마취의 원하는 레벨에 도달하면, 마취 원하는 레벨에 도달하면, 외과 영역을 면도하고, 마스크 챔버로부터 동물을 이동. 마스크 흐름에 세 방향 커넥터를 돌립니다. 0.25 L / 분의 산소 및 0.25 L / 분으로 공기 유동 조절기를 조정. <ol><li> 2.5 % - 1.5로 이소 플루 란 농도를 조정합니다. 수술 procedur을 시작하기 전에 반사 제어 및 호흡에 의한 마취의 수준을 확인이자형. </li></ol></li><li> 접착 테이프를 사용하여 작업 벤치에 마우스의 다리를 고정합니다. 동물의 코는 여전히 신중하게 마스크에 위치되어 있는지 확인합니다. </li></ol> 미주 신경 3. 수술 및 자극 <ol><li> 70 % 에탄올로 수술 영역을 소독. </li><li> 메스를 사용하여 조심스럽게 목 (약 1 ~ 1.5 cm의 절개)의 수준에서 피부를 절개. 참고 : 프로토콜에서, 수술 절차가 SHAM-운영하는 동물 여기를 종료합니다. 사실, 단지 금속 도구 신경을 터치하면 이미 어느 정도를 촉진하는 것이 밝혀졌다. LPS의 소량을 사용하는 경우 따라서,보다 정확한 수술 제어 동물을 얻기 위해,이 단계에서 수술을 중지합니다. </li><li> 현미경 (12.5X 목적)의 도움으로, 해부 집게를 사용 경동맥에서 좌측 미주 신경을 분리. 먼저 피부와 지방이 층을 제거하여 흉쇄 근육을 찾은 다음에 그것을 철회신경과 동맥 모두 뒤에 집게를 넣어 주문. 주 : 신경과 혈관이 서로 밀접하게 부착되어, 상기 다음 공정이 매우 까다 롭다. 따라서, 선박을 절감하고 동물을 죽이는 것은 매우 쉽습니다. 그러나, 신경 및 동맥 사이에 매우 신중하게 집게를 배치하여, 그들은 결국 분리, 그리고 신경을 분리하는 것이 가능하다. </li><li> 미주 신경 아래의 전극 (도 1b)를 놓는다. 바늘 전극은 매우 긴, 그래서 신경이 자극 동안 약간 이동하더라도, 그것은 항상 전극과 접촉 할 것이다. </li><li> (사이토 카인 수준에 대한 판독을위한, 즉, 하향 조절 효과를 측정하기 위해) 주사기의 도움 LPS의 복강 내 (IP) 주입 (2 밀리그램 / kg)을 수행한다. </li><li> 자극을 시작하기 전에 5 분을 기다립니다. </li><li> 인정 인 프로그램의 시작 버튼을 눌러 5 V 및 1 Hz에서 5 분 동안 미주 신경을 자극한다. </li><li> 레전극을 이동 외과 용 봉합 실을 가진 동물의 상처 봉합. </li><li> 순서 치유를 개선하고 감염으로부터 보호하기에 상처에 없음 스팅 배리어 필름 (NSBF)를 스프레이. </li></ol> 동물의 4. 복구 <ol><li> 수술 후, 각성 및 복구를 위해 다시 집 케이지에 동물을 이동합니다. 체온을 유지하기 위해 적외선 빛에 따라, 전체 의식이 회복 될 때까지 동물을 모니터링해야합니다. </li><li> 동물이 분석을 위해 희생하기 전에 6 시간 동안 새장에 복구 할 수 있습니다. 주 : 미주 신경 자극의 효과는 매우 고속이고 또한 (48 시간까지) 지속되는 긴 것으로 도시되어 있으므로 복구 시간은 연구의 필요에 따라 실험에 의해 설정 될 수있다. </li></ol> 추가 분석을 위해 희생 5. <ol><li> 공동 2 관리 장치에 연결된 장에 동물을 놓는다. </li><li> 5에서 장치를 설정CO 2 흡입라는 우수한 성능을 나타내었다주기. </li><li> 안락사가 완료되면, 관심의 장기를 수집하고 직접 추가 분석을 위해 드라이 아이스에 그들을 동결 (예를 들어, 마우스 TH1을 사용하여 비장 추출물에서 사이토 카인 수준의 측정 / TH2 9 플렉스 분석) 3. </li></ol>

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저자는 공개 아무것도 없어.

2000 년대 초반의 발견 이후, CAP의 메커니즘을 철저하게 연구되고있다. 우리는 지금 경로의 좋은 사진을 가지고 있고, 특히, 대상 기관, 비장, 매우 효율적인 팀으로 NE, 메모리 T 세포, 이크, 대 식세포 작업은 염증 매개 2 다운 조절하는 곳. 우리는 또한 최근에 분명히 CAP 3의 활성화 후 비장에서 아세틸 콜린의 방출을위한 필수 구성 요소, 특히 마우스의 기능 프로...

선천성 면역은 유기체의 넓은 범위에서 감염과 질병에 대한 방어의 즉각적인 첫 번째 라인을 제공합니다. 그것은 위협을 제거하기 위해 차 면역 반응을 시작 할뿐 아니라 활성화하고 병원균 고유의 방식으로 차 면역 반응을 수행하는 후천성 면역 교육에 중추적 인 역할을한다. 염증은 다시 감염 부위에 다른 면역 세포를 유치 할 수있는 능력을 가지고 같은 발적, 부종, 통증, 기능의 손실 및 발열과 같은 염증의 추기경 표지판을 유도하는 사이토 카인과 케모카인의 과다에 의해 조율한다 . 기간과 염증의 강도는 여러 가지 요인에 따라 달라집니다,하지만 염증을 해결하고 항상성을 회복하는 만성 염증성 질환의 발병을 방지하기 위해 중요한 단계입니다. 신경 과학 및 면역학 분야의 최근 발전 infl을 제어하는 ​​엄청난 치료 가능성과 특정 신경 메커니즘을 풀어 한중추 신경계 및 주변의 ammation. 이들 메커니즘 중 하나는 자율 신경계 (4, 5)에 의해 구동되는 염증성 반사적으로 알려진 콜린성 소염 통로 (CAP)이다. 현재 염증 매개체는 중추 신경계 염증의 상태에 관한 신호를 감각 신경을 활성화하고 전송할 것으로 생각된다. 반사 응답은 원심성 미주 신경을 통해 작동된다. 캡의 해부학 적 세부 사항에 대한 광범위한 연구는 두 개의 신경으로 구성 부교감 - 교감 모델, 미주 신경 및 비장 신경 각각 6을 공개했다. 카프에서 활성화 콜린성 원심성 미주 신경은 아직 탐구하는기구에 의해 비장 아드레날린 신경의 활성화의 결과로, 복강 신경절-장간막에서 끝난다. 따라서 활성화 된 비장 신경, 내측으로 알려져흰색 펄프의 면역 세포에 근접, 한계 영역 및 CAP (7), (8)의 비장, 원금과 필수 기관의 빨간 펄프에서 되어진. 비장 신경 말단에서 노르 에피네프린 (NE)의 비장 T 림프구상에서 발현 해당 β 2 아드레날린 수용체에 결합한다. 이것은 차례로함으로써 사이토 카인 생성 및 염증이 제한 식세포 α7 니코틴 성 아세틸 콜린 수용체 (α7nACh)를 활성화 콜린 아세틸 트랜스퍼 라제 (하는 ChAT) - 매개 성 아세틸 콜린 (ACH) 방출을 유도한다. 결과적으로, 신경계는 말초 조직에 염증을 조절하는 지역 면역 항상성을 복원 할 수 있는지 지금은 분명하다. 통로의 이름에서 알 수 있듯이, 아세틸 콜린 시스템이 신경 면역 조절 경로의 기능에 중앙 중요하다. 흥미롭게도, 메커니즘의 활성화에 관여캡 주변부 및 중추 신경계에서 다른 것처럼 보인다. 비장의 니코틴 수용체의 중요성 (α7nAChR)가 이전 9 입증되었지만, 무스 카린 수용체 (mAChR)는 경로 (10), (11)의 중앙 활성화를위한 필수입니다. 치명적인 뮤린 패혈증 동안 최근 중앙에 작용하는 M1 무스 카린 성 작용제의 말초 투여가 현저히 억제 혈청 비장 종양 괴사 인자 α (TNFα), (12) 신호 그대로 미주 신경 및 신경 비장 필요한 액션. 우리는 또한 프로스타글란딘 E이 결핍 된 마우스 (PGE 2) 미주 신경 자극에 응답 할 수 없었다 혈청과 비장 3 사이토 카인의 LPS 유도 방출을 다운 조절하지 않았다는 것을 최근에 보여 주었다. 따라서 CAP은 주요 아세틸 콜린의 pathw 이외의 다른 시스템에 의해 규제 될 수있다찬성. 미주 신경 때문에 체내에서의 방황 물론 같은 지명되었다, 간, 폐, 비장, 신장 및 창자 (13)를 포함한 주요 기관을 지배하는. 이 큰 신경 분포 및 미주 신경의 매우 강력한 면역 억제 효과를 고려하여, 캡의 치료 가능성은 염증 상태의 넓은 범위를 커버 할 수있다. 어떤 약물은 신체에 추가되지으로 미주 신경은 전기적 (또는 기계적) 통상적 인 처리의 전압 및 주파수와 반대로 제어하여, 활성화 될 수있다. 시험은 만성 염증 (14) 치료에 VNS의 임상 적 의의를 테스트하기 위해, 예를 들어, 류마티스 환자에서 현재 진행되고 있습니다. 전부, 신경 - 면역 통신 및 염증의 조절은 기존의 치료에 대한 가능한 대안 치료를 제공 할 것이다 조사 중이다. 따라서, 미주 신경의 분석 stimulation 개의 서로 다른 분포하는 장기 효과하지만 만성 염증의 동물 모델에서 잠재적 인 치료 행동의도 특성은, 확실히 통찰력을 제공하고 새로운 잠재적 인 치료 목표에 대한 희망 것이다. 트레이시 동료 (4)에 의해 개발 된 기존 방법 때문에 (LPS의 치사량) 염증 반응의 자극을 넘어선 및 CAP 활성화 및 판독 사이에 너무 짧은 시간 범위에 조사 우리 필드 전치 수 없었다. 본 논문에서는, 원래의 프로토콜에 대한 변경을 제시한다 사이토킨 수준의 두 가지 방법을 비교하여 표적 기관 (비장)에 대향하는 새로운 관찰을 강조.

<table><tbody><tr><td>Computer</td><td>Toshiba</td><td>-</td><td>Any computer is actually compatible</td></tr><tr><td>MP-150 data acquisition system</td><td>Biopac Systems</td><td>MP150WSW</td><td></td></tr><tr><td>Acknowledge software</td><td>Biopac Systems</td><td></td><td></td></tr><tr><td>Mice C57Bl/6</td><td>Charles River</td><td></td><td></td></tr><tr><td>Anesthetic machine</td><td>Simtec Engineering</td><td></td><td></td></tr><tr><td>Medical oxygen bottle</td><td>AGA</td><td>107563</td><td></td></tr><tr><td>Medical air bottle&amp;nbsp;</td><td>AGA</td><td>108639</td><td></td></tr><tr><td>Vetflurane (1,000 mg/g)</td><td>Virbac</td><td>137317</td><td></td></tr><tr><td>LPS</td><td>Sigma-Aldrich</td><td>L2630</td><td></td></tr><tr><td>Saline</td><td>Merck Millipore</td><td>1024060080</td><td></td></tr><tr><td>PBS 10x</td><td>Sigma-Aldrich</td><td>P5493</td><td>Diluted 10 times for used concentration</td></tr><tr><td>Syringe (1 ml)</td><td>BD Plastipak</td><td>303172</td><td></td></tr><tr><td>Needles 23 G</td><td>KD-FINE</td><td>900284</td><td>0.6 x 30 mm (blue)</td></tr><tr><td>Microdissecting forceps (curved)</td><td>Sigma-Aldrich</td><td>F4142</td><td></td></tr><tr><td>Dissecting scissors</td><td>Sigma-Aldrich</td><td>Z265969</td><td></td></tr><tr><td>Surgical suture 4-0</td><td>Ethicon</td><td>G667G</td><td></td></tr><tr><td>Euthanasia unit</td><td>Euthanex Smartbox</td><td>EA-32000</td><td></td></tr><tr><td>Cavilon No Sting Barrier Film</td><td>3M Health Care</td><td>3346N</td><td></td></tr><tr><td>TH1/TH2 9-Plex assay, ultrasensitive kit</td><td>MesoScale Discovery</td><td>K15013C-1</td><td></td></tr><tr><td>Stimulating electrode device</td><td>Biopac Systems</td><td>STIMSOC</td><td></td></tr><tr><td>Aesculap Isis shaver</td><td>Agnthos</td><td>GT420</td><td></td></tr><tr><td>R70</td><td></td><td></td><td>Rodent diet from Lantmannen, Stockholm, Sweden</td></tr></tbody></table>

increased recovery time and decreased lps administration to study the vagus nerve stimulation mechanisms in limited inflammatory responses

Inflammation is a local response to infection and tissue damage mediated by activated macrophages, monocytes, and other immune cells that release cytokines and other mediators of inflammation. For a long time, humoral and cellular mechanisms have been studied for their role in regulating the immune response, but recent advances in the field of immunology and neuroscience have also unraveled specific neural mechanisms with interesting therapeutic potential. The so-called cholinergic anti-inflammatory pathway (CAP) has been described to control innate immune responses and inflammation in a very potent manner. In the early 2000s, Tracey and collaborators developed a technique that stimulates the vagus nerve and mimics the effect of the pathway. The methodology is based on the electrical stimulation of the vagus nerve at low voltage and frequency, in order to avoid any side effects of overstimulation, such as deregulation of heart rate variability. Electrical devices for stimulation are now available, making it easy to set up the methodology in the laboratory. The goal of this research was to investigate the potential involvement of prostaglandins in the CAP. Unfortunately, based on earlier attempts, we failed to use the original protocol, as the induced inflammatory response either was too high or was not suitable for enzymatic metabolism properties. The different settings of the original surgery protocol remained mostly unchanged, but the conditions regarding inflammatory induction and the time point before sacrifice were improved to fit our purposes (i.e., to investigate the involvement of the CAP in more limited inflammatory responses). 
The modified version of the original protocol, presented here, includes a longer time range between vagus nerve stimulation and analysis, which is associated with a lower induction of inflammatory responses. Additionally, while decreasing the level of lipopolysaccharides (LPS) to inject, we also came across new observations regarding mechanistic properties in the spleen.

TNFα 및 인터루킨-1β (IL-1β)의 수준 LPS의 용량을 수술 후 시간 경과 증가 및 감소 후 이전에 나타낸 바와 같이, 원래의 프로토콜을 사용 VNS는 TNFα의 수준 및 IL-1β (360.0 ± 40.21 PG / 밀리그램 (39.7 ± 10.8 PG / VNS에서 ㎎, p &lt;0.001 대 SHAM에서 169.3 ± 24.9 PG / ㎎) 감소 LPS를 복강 내 주사 (15 ㎎ / ㎏) (도 2A) 후의 비장 VNS에서 191.7...

Watch this Scientific Journal Video about Increased Recovery Time and Decreased LPS Administration to Study the Vagus Nerve Stimulation Mechanisms in Limited Inflammatory Responses at JoVE.com

Increased Recovery Time and Decreased LPS Administration to Study the Vagus Nerve Stimulation Mechanisms in Limited Inflammatory Responses

Vagus nerve stimulation has proven to have a strong efficacy for decreasing peripheral inflammation. Here, we present a modified vagus nerve stimulation protocol that allows for further examinations of the cholinergic anti-inflammatory mechanisms in limited inflammatory responses.

복구 시간 증가 및 제한 염증 반응에서 미주 신경 자극 메커니즘을 연구에 LPS 관리를 감소

Inflammation is a local response to infection and tissue damage mediated by activated macrophages, monocytes, and other immune cells that release ...

increased-recovery-time-decreased-lps-administration-to-study-vagus

Research

JoVE Journal

Immunology and Infection

9.8K 조회수.  Karolinska University Hospital. Vagus nerve stimulation has proven to have a strong efficacy for decreasing peripheral inflammation. Here, we present a modified vagus nerve stimulation protocol that allows for further examinations of the cholinergic anti-inflammatory mechanisms in limited inflammatory responses.

비디오: Increased Recovery Time and Decreased LPS Administration to Study the Vagus Nerve Stimulation Mechanisms in Limited Inflammatory Responses

Vagus Nerve Stimulation as a Tool to Induce Plasticity in Pathways Relevant for Extinction Learning

Extinction describes the process of attenuating behavioral responses to neutral stimuli when they no longer provide the reinforcement that has been maintaining the behavior. There is close correspondence between fear and human anxiety, and therefore studies of extinction learning might provide insight into the biological nature of anxiety-related disorders such as post-traumatic stress disorder, and they might help to develop strategies to treat them. Preclinical research aims to aid extinction learning and to induce targeted plasticity in extinction circuits to consolidate the newly formed memory. Vagus nerve stimulation (VNS) is a powerful approach that provides tight temporal and circuit-specific release of neurotransmitters, resulting in modulation of neuronal networks engaged in an ongoing task. VNS enhances memory consolidation in both rats and humans, and pairing VNS with exposure to conditioned cues enhances the consolidation of extinction learning in rats. Here, we provide a detailed protocol for the preparation of custom-made parts and the surgical procedures required for VNS in rats. Using this protocol we show how VNS can facilitate the extinction of conditioned fear responses in an auditory fear conditioning task. In addition, we provide evidence that VNS modulates synaptic plasticity in the pathway between the infralimbic (IL) medial prefrontal cortex and the basolateral complex of the amygdala (BLA), which is involved in the expression and modulation of extinction memory.

Vagus nerve stimulation (VNS) has emerged as a tool to induce targeted synaptic plasticity in the forebrain to modify a range of behaviors. This protocol describes how to implement VNS to facilitate the consolidation of fear extinction memory.

도구로 미주 신경 자극은 멸종 학습을위한 관련 진학 가소성을 유도

Extinction describes the process of attenuating behavioral responses to neutral stimuli when they no longer provide the reinforcement that has been ...

연구

행동분석학

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

골수 이식으로 마우스 대장염에서 면역 및 비 면역 세포에서 유전자 발현의 기능 역할을 차별화

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

면역학 및 감염병학

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

연쇄상 구균 폐렴으로 비강 내 식민지 화 중 염증 반응의 특성화

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

A Method of Nodose Ganglia Injection in Sprague-Dawley Rat

Afferent signaling via the vagus nerve transmits important general visceral information to the central nervous system from many diverse receptors located in the organs of the abdomen and thorax. The vagus nerve communicates information from stimuli such as heart rate, blood pressure, bronchopulmonary irritation, and gastrointestinal distension to the nucleus of solitary tract of the medulla. The cell bodies of the vagus nerve are located in the nodose and petrosal ganglia, of which the majority are located in the former. The nodose ganglia contain a wealth of receptors for amino acids, monoamines, neuropeptides, and other neurochemicals that can modify afferent vagus nerve activity. Modifying vagal afferents through systemic peripheral drug treatments targeted at the receptors on nodose ganglia has the potential of treating diseases such as sleep apnea, gastroesophageal reflux disease, or chronic cough. The protocol here describes a method of injection neurochemicals directly into the nodose ganglion. Injecting neurochemicals directly into the nodose ganglia allows study of effects solely on cell bodies that modulate afferent nerve activity, and prevents the complication of involving the central nervous system as seen in systemic neurochemical treatment. Using readily available and inexpensive equipment, intranodose ganglia injections are easily done in anesthetized Sprague-Dawley rats.

Afferent vagal signaling transmits important information to central nervous system from receptors located in organs of the abdomen and thorax. The nodose ganglia of vagus nerves contain many types of receptors that modulate vagal activity. This protocol describes a method of local injections of neurochemicals into the nodose ganglia.

스프 라그 - 돌리 쥐에 Nodose 신경절 주입하는 방법

Afferent signaling via the vagus nerve transmits important general visceral information to the central nervous system from many diverse receptors located ...

신경과학

Injections of Lipopolysaccharide into Mice to Mimic Entrance of Microbial-derived Products After Intestinal Barrier Breach

The intestinal epithelial barrier separates the host from the microbiota that is normally tolerated or ignored. The breach of this barrier results in the entrance of bacteria or bacteria-derived products into the host, accessing the host circulation and inner organs leading to the uncontrolled inflammation as observed in patients with inflammatory bowel disease (IBD), that are characterized by an increased intestinal epithelial permeability.
To mimic the entrance of bacterial-derived compounds into the host, an endotoxemia model has been adopted in which lipopolysaccharide (LPS), a component of the outer cell wall of Gram-negative bacteria, were injected into mice. In this study, a sublethal dose of LPS was intraperitoneally injected and the mice were subsequently monitored for 8 h using a disease score. Furthermore, the expression levels of the inflammatory cytokines Il6, Il1b, and Tnfa were analyzed in the spleen, liver and colon by qPCR at different time points post LPS injection. This model could be useful for the studies involving investigation of immune responses after the invasion of microorganisms or bacterial-derived products caused by a barrier breach of body surfaces.

Here a protocol to mimic the entrance of bacterial-derived compounds after intestinal barrier breach is presented. A low sublethal dose of lipopolysaccharide was injected systemically into mice, which were monitored for 24 h post-injection. The expression of pro-inflammatory cytokines was determined at several time points in spleen, liver, and colon.

장 방 벽 위반 후 미생물 파생 제품의 입구를 모방 하는 쥐로 Lipopolysaccharide의 주사

The intestinal epithelial barrier separates the host from the microbiota that is normally tolerated or ignored. The breach of this barrier results in the ...

Laboratory Administration of Transcutaneous Auricular Vagus Nerve Stimulation (taVNS): Technique, Targeting, and Considerations

Non-invasive vagus nerve stimulation (VNS) may be administered via a novel, emerging neuromodulatory technique known as transcutaneous auricular vagus nerve stimulation (taVNS). Unlike cervically-implanted VNS, taVNS is an inexpensive and non-surgical method used to modulate the vagus system. taVNS is appealing as it allows for rapid translation of basic VNS research and serves as a safe, inexpensive, and portable neurostimulation system for the future treatment of central and peripheral disease. The background and rationale for taVNS is described, along with electrical and parametric considerations, proper ear targeting and attachment of stimulation electrodes, individual dosing via determination of perception threshold (PT), and safe administration of taVNS.

Laboratory Administration of Transcutaneous Auricular Vagus Nerve Stimulation taVNS: Technique, Targeting, and Considerations

A methodological description of the technique, potential targets, and proper administration of transcutaneous auricular vagus nerve stimulation (taVNS) on the human ear is described.

실험실 관리의 경 피 성 귀의 미주 신경 자극 (taVNS): 기술, 타겟팅, 및 고려 사항

Non-invasive vagus nerve stimulation (VNS) may be administered via a novel, emerging neuromodulatory technique known as transcutaneous auricular vagus ...

Vagus Nerve Stimulation As an Adjunctive Neurostimulation Tool in Treatment-resistant Depression

Vagus nerve stimulation (VNS) is an approved neurostimulation therapy. The purpose of the method is to treat patients with therapy-resistant depression (TRD). VNS exhibits antidepressive and stabilizing effects. This method is particularly useful as a long-term treatment, in which up to two-thirds of patients respond. The vagus nerve stimulator is positioned on the left vagus nerve during a surgical procedure and is activated telemetrically by a wand connected to a handheld computerized device. The treating physician can perform various adjustments of the vagus nerve stimulator during in-office visits (e.g., by modifying stimulation intensity or stimulation frequency) to achieve maximum therapeutic effects with low side effects. Set-up of the device usually takes several months. Typical side effects include wound infection, temporary salivation, coughing, paralysis of the vocal cords, bradycardia, or even asystole. The patient can stop the VNS by placing a magnet over the generator. The current protocol describes delivery of the specific stimulation tool and methods for adjusting the tuning parameters to achieve the best remission rates in patients with TRD.

Vagus nerve stimulation (VNS) has been shown to be effective as an adjunct treatment for treatment-resistant depression (TRD). VNS leads to antidepressive and antisuicidal effects and quality of life improvements. This protocol offers a step-by-step guide to managing and adjusting a vagus nerve stimulator for efficient treatment of TRD.

Vagus nerve stimulation (VNS) is an approved neurostimulation therapy. The purpose of the method is to treat patients with therapy-resistant depression ...

의학

Preparation of Peripheral Nerve Stimulation Electrodes for Chronic Implantation in Rats

Peripheral nerve cuff electrodes have long been used in the neurosciences and related fields for stimulation of, for example, vagus or sciatic nerves. Several recent studies have demonstrated the effectiveness of chronic VNS in enhancing central nervous system plasticity to improve motor rehabilitation, extinction learning, and sensory discrimination. Construction of chronically implantable devices for use in such studies is challenging due to rats&#8217; small size, and typical protocols require extensive training of personnel and time-consuming microfabrication methods. Alternatively, commercially available implantable cuff electrodes can be purchased at a significantly higher cost. In this protocol, we present a simple, low-cost method for construction of small, chronically implantable peripheral nerve cuff electrodes for use in rats. We validate the short and long-term reliability of our cuff electrodes by demonstrating that VNS in ketamine/xylazine anesthetized rats produces decreases in breathing rate consistent with activation of the Hering-Breuer reflex, both at the time of implantation and up to 10 weeks after device implantation. We further demonstrate the suitability of the cuff electrodes for use in chronic stimulation studies by pairing VNS with skilled lever press performance to induce motor cortical map plasticity.

Existing approaches for constructing chronically implantable peripheral nerve cuff electrodes for use in small rodents often require specialized equipment and/or highly trained personnel. In this protocol we demonstrate a simple, low-cost approach for fabricating chronically implantable cuff electrodes, and demonstrate their effectiveness for vagus nerve stimulation (VNS) in rats.

쥐에서 만성 이식을 위한 말초 신경 자극 전극의 준비

Peripheral nerve cuff electrodes have long been used in the neurosciences and related fields for stimulation of, for example, vagus or sciatic nerves. ...

Transcutaneous Auricular Vagus Nerve Stimulation에 의한 신경조절 측정

Understanding the Effects of Non&#45;Invasive Transauricular Vagus Nerve Stimulation on EEG and HRV

Several studies have demonstrated promising results of transcutaneous auricular vagus nerve stimulation (taVNS) in treating various disorders; however, no mechanistic studies have investigated this technique's neural network and autonomic nervous system effects. This study aims to describe how taVNS can affect EEG metrics, HRV, and pain levels. Healthy subjects were randomly allocated into two groups: the active taVNS group and the sham taVNS group. Electroencephalography (EEG) and Heart Rate Variability (HRV) were recorded at baseline, 30 min, and after 60 min of 30 Hz, 200-250 &#181;s taVNS, or sham stimulation, and the differences between the metrics were calculated. Regarding vagal projections, some studies have demonstrated the role of the vagus nerve in modulating brain activity, the autonomic system, and pain pathways. However, more data is still needed to understand the mechanisms of taVNS on these systems. In this context, this study presents methods to provide data for a deeper discussion about the physiological impacts of this technique, which can help future therapeutic investigations in various conditions.

저자 스포트라이트: 경귓바퀴 미주 신경 자극의 효과 탐구

This protocol provides information on how to apply transcutaneous auricular vagus nerve stimulation (taVNS) in a clinical trial, including potential biomarkers such as EEG metrics and heart rate variability (HRV) to measure the effect of this treatment on the autonomic nervous system.

비침습적 경귓바퀴 미주 신경 자극이 EEG 및 HRV에 미치는 영향 이해

Several studies have demonstrated promising results of transcutaneous auricular vagus nerve stimulation (taVNS) in treating various disorders; however, no ...

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실험실 관리의 경 피 성 귀의 미주 신경 자극 (taVNS): 기술, 타겟팅, 및 고려 사항

Vagus Nerve Stimulation As an Adjunctive Neurostimulation Tool in Treatment-resistant Depression

쥐에서 만성 이식을 위한 말초 신경 자극 전극의 준비

비침습적 경귓바퀴 미주 신경 자극이 EEG 및 HRV에 미치는 영향 이해

비침습적 경귓바퀴 미주 신경 자극이 EEG 및 HRV에 미치는 영향 이해