이 작품은 컨셉 교부금의 UC 발견 증명 의해 NIH (R00 EB007241)에 의해 지원되었다. 저자는 또한이 실험에서 그녀의 도움 재클린 허바드 감사드립니다.

1. 외과 준비 <ol><li> 6~8주 세 사이의 여성 CD 1 마우스는 우리 실험에 사용되었습니다. </li><li> 케타민과 xylazine 조합 (80 MG / kg ketamine/10 MG / kg xylazine)의 intraperitoneal 주사와 마우스를 마취. ~ 37 ° C.에 최적의 체온을 보장하기 위해 homeothermic 패드에서 마우스를 놓으십시오 지속적인 동물의 반사 신경 (예를 들면, 무딘 집게와 발을 곤란하게) 테스트에 의해 마취의 수준을 모니터링하고 필요한 경우 더 많은 마취를 삽입. </li><li> 인공 눈물의 연고와 함께 두 눈에 기름칠. 면도칼을 사용하여 두피에 머리카락을 제거하고 70 % 알코올 시험을 치루 패드를 사용하여 잔류 머리카락을 제거합니다. 두피 이상 Nair 헤어 제거 크림의 얇은 층을 적용하고 적용하려면 2 분 정도 기다리십시오. 부드럽게 생리를 사용하여 Nair와 나머지 머리는 면봉과 알코올 시험을 치루 패드를 moistened 닦아. 두피는 이제 완전히 사나이 답게해야합니다. </li><li> betadine 면봉 스틱을 사용하여 두피를 소독하고 7 청소0 % 에탄올 사립 패드. </li><li> ~ 37 최적의 체온을 보장하기 위해 수술 커튼에 동물을주의 깊게 포장 ° C와 두개골을 고정 할 수있는 stereotaxic 프레임에 동물을 탑재합니다. 살짝의 안정성을 보장하기 위해 두개골을 누릅니다. 사용 재료의 목록은 표 1에 제공됩니다. </li></ol> 2. 얇게 - 두개골 두피 창 준비 <ol><li> 눈 사이의 중간 선 지점에 절개를 시작합니다. 귀 사이의 중간 선 지점으로 caudally 계속합니다. 집게가있는 부분 피부를. </li><li> 해부 현미경으로 얇게 할 지역을 찾아 부드럽게 핀셋을 사용하여 근막을 제거합니다. 얇게 대뇌 피질의 창을 만들기 전에 멸균 면봉으로 두개골을 말린다. 우리의 실험에서, 우리는 4 만든 × 4mm는 두개의 창 ~ bregma에 뒤쪽과 측면 1mm를 얇게. </li><li> 빛 한눈에 모션 onl를 사용하여 수술 핸드 드릴에 드릴 비트 크기 0.75 mm를 둥근 카바이드 버를 사용하여 두개골을 썼음 시작Y. 두개골로 직접 압력을 가하지 마십시오. 멸균 생리와 면봉을 사용하여 뼈 먼지를 제거하고 두개골을 과열 방지를 위해 매 20-30 초를 드릴 중지합니다. 생리는 또한 두개골 전체의 열을 사라지고에 도움이됩니다. </li><li> 소형 뼈의 바깥 층이 완전히 제거되면 중간 작은 구멍이 많은 뼈 층은 이제 표시됩니다. 혈관이 작은 구멍이 많은 뼈 계층에서 더 명백만큼 ​​일부 약간의 출혈이있을 수 있습니다. 녹색 돌 버로 전환하여 작은 구멍이 많은 층 더 섬세하므로 특히주의를 사용하여 드릴 계속 진행합니다. 두개골 창 전체 반반 함을 만들 때 녹색 돌 버 적은 뼈 자료를 삭제합니다. 뼈 먼지를 제거하고 두개골을 냉각 가끔 드릴 중지합니다. </li><li> 마지막으로, 두개골이 더 투명하고 뇌에 vasculature 나면 이제 볼 수 있습니다, 연마 버를 사용하여 두개골을 연마 시작합니다. 이 수 두개골을 부드럽게하면서 숱이 더 정확.주세요 SKU의 두께를 확인부드럽게 포셉과에 눌러 붙인다. 두개골이 약간 유연하게 될 때 연마를 중지합니다. </li><li> 얇게 두개골 창이 완전히 부드럽고 반사 및 이미징 (그림 1)에 대한 준비가되어 있어야합니다. 뇌의 고도의 분산 조직의 특성으로 인해, 두개골은 최적 깊이 침투에 대해 최소 55 μm로 얇게해야합니다. 사용 재료의 목록은 표 1에 제공됩니다. </li></ol> 3. 광 결맞음 단층 촬영 영상 <ol><li> 수술이 완료되면 마취의 적절한 수준을 보장하고 필요한 경우 추가 마취를 관리하는 동물의 호흡 속도와 반사 신경을 확인합니다. stereotaxic 프레임에서 동물을 제거 동물 이미징 역 수술 커튼에 싸여 있으며, 교통 동물 유지. </li><li> 이미지는 반사 표지판을 확인하고 필요한 경우 추가 인공 눈물을 적용하기 전에. 두개골을 확보 할 수있는 stereotaxic 프레임에 동물을 탑재합니다. </li><li> 아래의 장소 동물10월 카메라와 광학 빔 (그림 2)에 따라 위치 TSCW합니다. 두개골과 뇌의 단면도는 이제 (그림 3) 시각화 할 수 있습니다. </li><li> 관심 지역이 위치한되면 데이터 수집이 시작 할 수 있습니다. 영상을 위해, 우리는 4.0 mm의 폭과 이미지 창을 달성하기 위해 갈보 거울을 사용합니다. 2mm의 이미지 깊이는 6 사고 전력 MW과 초점 얇게 두개골 아래의 1mm를 얻은 것입니다. 각 단면적 이미지 당 0.14 초의 수집 속도 2,048 축 스캔으로 구성. </li><li> 뇌의 용적 검사는 XY의 화살 방향으로 빔을 스캔 첫 번째 갈보 거울과 코로나에서 스캔 두 번째 갈보 거울과 스캔을위한 갈보 거울 두 개를 사용하여 2D 단면 일련의 이미지를 수집하여 구할 수 있습니다 방향. </li></ol>

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관심 없음 충돌이 선언 없습니다.

10월와 얇게-두개골 영상은 최근 15, 16, 조사 된 소설 신경 영상 기술이다. 우리의 실험에서, 우리는 생체 내 마우스 모델에서 TSCW을 통해 SD 10 월 영상의 가능성을 보여 주었다. 결과에서 두개골이 약 55 μm로 얇게되어 있고 침투 깊이는 각각의 축 방향 및 측 방향에서 8 μm 및 20 μm의 이미지 해상도로 약 1mm에서 얻어진다. 정상적인 두개골 (그림 4)에 비해 신호 강도 프로파...

초기 1990 년대의 도입 이후, 10 월은 조직 구조와 기능 2 생물 이미징 용도로 사용되었습니다. 10월는 광섬유 켈슨 간섭계 2,4과 낮은 일관성 광원을 구현하여 backscattered 조명 4 반향 시간 지연을 측정하여 단면 이미지를 생성합니다. 또한 푸리에 도메인 10월 (FD 10 월)로 알려진 SD 10 월은, 첫째 1995 5 소개 및 전통적인 시간 도메인 10월 (TD -10 월)에 비해 우수한 이미징 양상을 제공합니다. SD 10 월에서 참조 암은 높은 속도와 초 고해상도 이미지 획득 6-9의 결과로 고정 유지됩니다. 현재 TSCW 모델은 전통적인 craniotomy 대신에 두 광자 현미경의 생체 뇌 이미징 응용 프로그램에 주로 사용되었습니다. 이러한 TSCW은 추가 IMA을 제공하기 위해 사용자 정의 두개골 판이나 유리 커버 슬립 10-13와 동시에 사용되어왔다안정성을 ging. 우리의 연구에서 우리는 TSCW을 사용하는 경우 다음과 같은 액세서리는 10월 이미징에 필요한 아니라는 것을 관찰했다. 그들은 광학 빔을 방해하고 10월 이미지를 변경 할 수 있으므로 따라서, 두개골 판이나 유리 커버 용지의 부족은 이미지 창 크기의 넓은 범위의 할 수 있습니다. 얇게-두개골 준비 두 광자 현미경 10-13를 사용하여 뇌의 이미징 연구에 도움이 될 입증되었습니다. 우리의 실험에서, 우리는 TSCW을 통해 생체 내 이미지 피질에 SD 10 월 시스템을 사용합니다. 우리의 사용자 정의 SD 10 월 영상 설정은 각각 8 μm 및 20 μm의 축 방향 및 측 방향 해상도의 결과로 97 nm의 대역폭과 1,295 nm의 중심이 superluminescent 다이오드 (SLD), 14로 구성된 광대역, 낮은 간섭 광원을 포함 . 우리의 광학 이미징 장치와 함께, 우리는 TSCW을 통해 이미지가 O에 구조와 기능을 파악하고 시각화에 큰 가능성이 있다는 구상ptically 조밀 한 뇌 조직.

<table border="1"><tbody><tr><td> 자료 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 </td></tr><tr><td> 케타민 </td><td> 피닉스 제약 </td><td> 57319-542-02 </td><td></td></tr><tr><td> Xylazine </td><td> Akorn 주식회사 </td><td> 139-236 </td><td></td></tr><tr><td> 인공 눈물 연고 </td><td> 럭비 </td><td> 0536-6550-91 </td><td></td></tr><tr><td> Nair </td><td> 교회 &amp; 드와이트 (주) 주식회사 </td><td> 4010130 </td><td></td></tr><tr><td> 멸균 알코올 준비 패드 </td><td> 켄달 의료 </td><td> 6818 </td><td></td></tr><tr><td> 면화는 Applicators 줬 </td><td> Fisherbrand </td><td> 23-400-115 </td><td></td></tr><tr><td> Betadine 솔루션 Swabstick </td><td> 퍼듀 제품 &lt;/ TD&gt; <td> 67618-153-01 </td><td></td></tr><tr><td> 생리 식염수, 0.9 % </td><td> 피닉스 제약 </td><td> 57319-555-08 </td><td></td></tr><tr><td> Stereotactic 프레임 </td><td> Stoelting </td><td></td><td></td></tr><tr><td> 고속 외과 핸드 드릴 </td><td> Foredom </td><td></td><td> 38,000 RPM </td></tr><tr><td> 초경 라운드 버 </td><td> Stoelting </td><td></td><td> 0.75 mm </td></tr><tr><td> 경질 - 그린 스톤 </td><td> Shofu </td><td></td><td> 생크 : HP 모양 : BA1 </td></tr><tr><td> CompoMaster 거친 &amp; CompoMaster 폴리 셔 </td><td> Shofu </td><td></td><td> 모양 : 미니 PT. </td></tr><tr><td> SpaceDrapes </td><td> Braintree 과학 주식회사 </td><td></td><td></td></tr></tbody></table>

thinned-skull cortical window technique for in vivo optical coherence tomography imaging

광 간섭 단층 촬영 (OCT)는 높은 공간적 - 시간적 해상도 바이오 메디컬 이미징 기술이다. 그 최소한 침략 방식으로 10월는 피부과 안과, 그리고 gastroenterology 1-3에서 널리 사용되었습니다. 얇게 - 두개골 대뇌 피질의 창 (TSCW)를 사용, 우리는 생체 내 이미지 피질을하는 도구로 스펙트럼 도메인 10월 (SD -10 월) 양상을 채용하고 있습니다. 더 다양한 기능을 제공으로 일반적으로, 오픈 - 머리가 신경 이미징에 사용 된 그러나, TSCW의 접근 방식은 덜 침습적이며, neuropathology의 연구에 장기 이미징을위한 효과적인 의미입니다. 여기, 우리는 대뇌 피질의 생체 10월 이미지에서의 마우스 모델에서 TSCW를 만드는 방법을 제시한다.

대뇌 피질 위에 얇게 창을 만든 후 vasculature는 이제 더 시각적으로 눈에 잘 띄는해야합니다 (그림 1)과 (최대 1 ㎜) 깊이 이미징 깊이있게됩니다. 140 μm (그림 1)에서 측정 및 더 큰 광학 선명도를 제공하는 일반 두개골에 비해 오른쪽 피질은 약 55 μm로 얇게되어 있습니다. 또한 10-15 μm로 썼음하면 11 그러나 유리 커버 용지와 두개골 플레이트의 사용 등 필요한는 <stro...

Watch this Scientific Journal Video about Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging at JoVE.com

Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging

우리는을위한 마우스 모델에서 얇게 - 두개골 대뇌 피질의 창 (TSCW)를 생성하는 방법을 제시<em&gt; 생체 내</em대뇌 피질의&gt; 10월 영상.

에 얇게 - 두개골 두피 창 기술<em&gt; 생체 내</em&gt; 광 결맞음 단층 촬영 영상

Optical coherence tomography (OCT) is a biomedical imaging technique  with high spatial-temporal resolution. With its minimally invasive approach OCT  has ...

thinned-skull-cortical-window-technique-for-vivo-optical-coherence

Research

JoVE Journal

Neuroscience

Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging

15.0K 조회수.  University of California, Riverside. 우리는을위한 마우스 모델에서 얇게 - 두개골 대뇌 피질의 창 (TSCW)를 생성하는 방법을 제시 생체 내 10월 영상.

비디오: Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or thinned-skull 3 preparations. While the open-skull technique is very versatile, it is not optimal for studying microglia because it is invasive and can cause microglial activation. Even though the thinned-skull approach is minimally invasive, the repeated re-thinning of skull required for chronic imaging increases the risks of tissue injury and microglial activation and allows for a limited number of imaging sessions. Here we present a chronic thin-skull window method for monitoring murine microglia in vivo over an extended period of time using two-photon microscopy. We demonstrate how to prepare a stable, accessible, thinned-skull cortical window (TSCW) with an apposed glass coverslip that remains translucent over the course of three weeks of intermittent observation. This TSCW preparation is far more immunologically inert with respect to microglial activation than open craniotomy or repeated skull thinning and allows an arbitrary number of imaging sessions during a time period of weeks. We prepare TSCW in CX3CR1 GFP/+ mice 4 to visualize microglia with enhanced green fluorescent protein to &le;150 &mu;m beneath the pial surface. We also show that this preparation can be used in conjunction with stereotactic brain injections of the HIV-1 neurotoxic protein Tat, adjacent to the TSCW, which is capable of inducing durable microgliosis. Therefore, this method is extremely useful for examining changes in microglial morphology and motility over time in the living brain in models of HIV Associated Neurocognitive Disorder (HAND) and other neurodegenerative diseases with a neuroinflammatory component.

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

We describe a method for repeatedly visualizing murine microglia and circulating monocytes in vivo over hours, days or weeks using transcranial two-photon microscopy. We demonstrate how to prepare a thinned-skull window that allows intermittent observation of quiescent microglia that can be activated by adjacent stereotactic injection of the HIV-1 regulatory protein Tat.

만성 두 광자를위한 박막 해골 창 기법 생체내에서</emNeuroinflammation의 모델 Murine Microglia의> 이미징

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or ...

연구

신경과학

Chronic Imaging of Mouse Visual Cortex Using a Thinned-skull Preparation

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The technique presented here allows the imaging of the same area of the brain at several time points (chronic imaging) with microscopic resolution allowing the tracking of dendritic spines which are the small structures that represent the majority of postsynaptic excitatory sites in the CNS. The ability to clearly resolve fine cortical structures over several time points has many advantages, specifically in the study of brain plasticity in which morphological changes at synapses and circuit remodeling may help explain underlying mechanisms. In this video and supplementary material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation. The thinned-skull preparation is a minimally invasive approach, which avoids potential damage to the dura and/or cortex, thus reducing the onset of an inflammatory response. When this protocol is performed correctly, it is possible to clearly monitor changes in dendritic spine characteristics in the intact brain over a prolonged period of time.

In this video and supplemental material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation.

Thinned - 해골 준비를 사용하여 마우스 시각 피질의 만성 이미징

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The ...

A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form a polished and reinforced thinned skull (PoRTS) window in the mouse skull that spans several millimeters in diameter and is stable for months. The skull is thinned to 10 to 15 &mu;m in thickness with a hand held drill to achieve optical clarity, and is then overlaid with cyanoacrylate glue and a cover glass to: 1) provide rigidity, 2) inhibit bone regrowth and 3) reduce light scattering from irregularities on the bone surface. Since the skull is not breached, any inflammation that could affect the process being studied is greatly reduced. Imaging depths of up to 250 &mu;m below the cortical surface can be achieved using two-photon laser scanning microscopy. This window is well suited to study cerebral blood flow and cellular function in both anesthetized and awake preparations. It further offers the opportunity to manipulate cell activity using optogenetics or to disrupt blood flow in targeted vessels by irradiation of circulating photosensitizers.

We present a method to form an imaging window in the mouse skull that spans millimeters and is stable for months without inflammation of the brain. This method is well suited for longitudinal studies of blood flow, cellular dynamics, and cell/vascular structure using two-photon microscopy.

마우스 뇌의 장기 이미징을위한 세련된와 철근 Thinned-해골 창

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

두피 뉴런에서 생체 내 두 - 광자의 영상은 경험에 의존 분자 변경

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

두 광자<em&gt; 생체</em&gt; 시닝 두개골 준비를 사용하여 마우스 피질 돌기 쪽의 영상

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in vivo&#8212;rapidly, repetitively, and at a high resolution. This protocol describes OCT imaging in the mouse retina as a powerful tool to study optic neuropathies (OPN). The OCT system is an interferometry-based, non-invasive alternative to common post mortem histological assays. It provides a fast and accurate assessment of retinal thickness, allowing the possibility to track changes, such as retinal thinning or thickening. We present the imaging process and analysis with the example of the Opa1delTTAG mouse line. Three types of scans are proposed, with two quantification methods: standard and homemade calipers. The latter is best for use on the peripapillary retina during radial scans; being more precise, is preferable for analyzing thinner structures. All approaches described here are designed for retinal ganglion cells (RGC) but are easily adaptable to other cell populations. In conclusion, OCT is efficient in mouse model phenotyping and has the potential to be used for the reliable evaluation of therapeutic interventions.

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

This manuscript describes a protocol for the in vivo imaging of the mouse retina with high-resolution spectral domain optical coherence tomography (SD-OCT). It focuses on retinal ganglion cells (RGC) in the peripapillary region, with several scanning and quantifying approaches described.

광학 일관성 단층 촬영: 마우스 망막 신경 절 세포에서 Vivo에서 화상 진 찰

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

Vivo에서 신생아 쥐에 있는 대뇌 피 질의 뉴런의 두 광자 영상

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

Using Optical Coherence Tomography and Optokinetic Response As Structural and Functional Visual System Readouts in Mice and Rats

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for the investigation of processes of neurodegeneration, neuroprotection and neuro-repair involving the visual system, as these often correlate well with retinal changes. As a functional readout, visually evoked compensatory eye and head movements are commonly used in experimental models involving the visual function. Combining both techniques allows a quantitative in vivo investigation of structure and function, which can be used to investigate the pathological conditions or to evaluate the potential of novel therapeutics. A great benefit of the presented techniques is the possibility to perform longitudinal analyses allowing the investigation of dynamic processes, reducing variability and cuts down the number of animals needed for the experiments. The protocol described aims to provide a manual for acquisition and analysis of high quality retinal scans of mice and rats using a low cost customized holder with an option to deliver inhalational anesthesia. Additionally, the proposed guide is intended as an instructional manual for researchers using optokinetic response (OKR) analysis in rodents, which can be adapted to their specific needs and interests.

A detailed protocol for the assessment of structural and visual readouts in rodents by optical coherence tomography and optokinetic response is presented. The results provide valuable insights for ophthalmologic as well as neurologic research.

쥐와 쥐에 구조와 기능적 시각 시스템 판독으로 광학 일관성 단층 촬영 및 Optokinetic 응답을 사용 하 여

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for ...

Retinal Vascular Reactivity as Assessed by Optical Coherence Tomography Angiography

The vascular supply to the retina has been shown to dynamically adapt through vasoconstriction and vasodilation to accommodate the metabolic demands of the retina. This process, referred to as retinal vascular reactivity (RVR), is mediated by neurovascular coupling, which is impaired very early in retinal vascular diseases such as diabetic retinopathy. Therefore, a clinically feasible method of assessing vascular function may be of significant interest in both research and clinical settings. Recently, in vivo imaging of the retinal vasculature at the capillary level has been made possible by the FDA approval of optical coherence tomography angiography (OCTA), a noninvasive, minimal risk and dyeless angiography method with capillary level resolution. Concurrently, physiological and pathological changes in RVR have been shown by several investigators. The method shown in this manuscript is designed to investigate RVR using OCTA with no need for alterations to the clinical imaging procedures or device. It demonstrates real time imaging of the retina and retinal vasculature during exposure to hypercapnic or hyperoxic conditions. The exam is easily performed with two personnel in under 30 min with minimal subject discomfort or risk. This method is adaptable to other ophthalmic imaging devices and the applications may vary based on the composition of the gas mixture and patient population. A strength of this method is that it allows for an investigation of retinal vascular function at the capillary level in human subjects in vivo. Limitations of this method are largely those of OCTA and other retinal imaging methods including imaging artifacts and a restricted dynamic range. The results obtained from the method are OCT and OCTA images of the retina. These images are amenable to any analysis that is possible on commercially available OCT or OCTA devices. The general method, however, can be adapted to any form of ophthalmic imaging.

This article describes a method for measuring retinal vasculature reactivity in vivo with human subjects using a gas breathing provocation technique to deliver vasoactive stimuli while acquiring retinal images.

광학 일관성 단층 조영술 혈관 조영술에 의해 평가된 망막 혈관 반응성

The vascular supply to the retina has been shown to dynamically adapt through vasoconstriction and vasodilation to accommodate the metabolic demands of ...

Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice

To fully understand the cellular physiology of neurons and glia in behaving animals, it is necessary to visualize their morphology and record their activity in vivo in behaving mice. This paper describes a method for the implantation of a chronic cranial window to allow for the longitudinal imaging of brain cells in awake, head-restrained mice. In combination with genetic strategies and viral injections, it is possible to label specific cells and regions of interest with structural or physiological markers. This protocol demonstrates how to combine viral injections to label neurons in the vicinity of GCaMP6-expressing astrocytes in the cortex for simultaneous imaging of both cells through a cranial window. Multiphoton imaging of the same cells can be performed for days, weeks, or months in awake, behaving animals. This approach provides researchers with a method for viewing cellular dynamics in real time and can be applied to answer a number of questions in neuroscience.

Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice

Presented here is a protocol for the implantation of a chronic cranial window for the longitudinal imaging of brain cells in awake, head-restrained mice.

깨어있는 마우스 에서 반복되는 생체 내 이미징을위한 두개골 창 이식

To fully understand the cellular physiology of neurons and glia in behaving animals, it is necessary to visualize their morphology and record their ...