Name: Mouse Inner Ear Extraction and Stria Vascularis Dissection
Uploaded: 2023-04-21T12:50:00
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mouse inner ear extraction and stria vascularis dissection

Single-nucleus sequencing 또는 immunostaining을 위한 Stria Vascularis 미세절개

The cochlea consists of three fluid filled chambers, the scala vestibuli, scala media, and scala tympani. The scala vestibuli and scala tympani each contain perilymph, which has a high concentration of sodium (138 mM) and a low concentration of potassium (6.8 mM)1. The scala media contains endolymph, which has a high concentration of potassium (154 mM) and a low concentration of sodium (0.91 mM)1,2,3. This difference in ion concentration can be referred to as the endocochlear potential (EP), and is primarily generated by the movement of potassium ions through various ion channels and gap junctions in the stria vascularis (SV) along the lateral wall of the cochlea4,5,6,7,8,9,10,11. The SV is a heterogenous, highly vascularized tissue that lines the medial aspect of the lateral wall of the cochlea and contains three main cell types: marginal, intermediate, and basal cells12 (Figure 1).
Marginal cells are connected by tight junctions to form the most medial surface of the SV. The apical membrane faces the endolymph of the scala media and contributes to potassium ion transport into the endolymph using various channels, including KCNE1/KCNQ1, SLC12A2, and Na+-K+-ATPase (NKA)5,10,13,14. Intermediate cells are pigmented cells that reside between marginal and basal cells and facilitate potassium transport through the SV using KCNJ10 (Kir 4.1)15,16. Basal cells lie in close proximity to the lateral wall of the cochlea and are closely associated with fibrocytes of the spiral ligament to promote potassium recycling from the perilymph12. Pathology of the SV has been implicated in numerous otologic disorders17,18. Mutations in genes expressed in the major SV cell types, such as Kcnq1, Kcne1, Kcnj10, and Cldn11, can cause deafness and SV dysfunction, including the loss of EP19,20,21,22,23. In addition to the three major cell types, there are other less-studied cell types in the SV, such as spindle cells22, root cells12,24, macrophages25, pericytes26, and endothelial cells27, that have incompletely defined roles involving ionic homeostasis and the generation of EP28.
In comparison to bulk RNA-sequencing, single-nuclei RNA-sequencing (sNuc-Seq) provides information about cell heterogeneity, rather than an average of mRNA across a group of cells29, and can be particularly useful when studying the heterogenous SV30. For example, sNuc-Seq has produced transcriptional analysis that suggests there may be a role for spindle and root cells in EP generation, hearing loss, and Meniere&#39;s disease18. Further transcriptional characterization of the various SV cell types can provide us with invaluable information on the pathophysiology underlying different mechanisms and subtypes of SV-related hearing fluctuation and hearing loss. The harvest of these delicate inner ear structures is of paramount importance to optimal tissue analysis.
In this study, the microdissection approach to access and isolate the stria vascularis from the adult mouse cochlea for sNuc-Seq or immunostaining is described. Dissection of the adult mouse SV is required to understand various SV cell types and further characterize their role in hearing.

저자 스포트라이트: Dissection of Adult Mouse Stria Vascularis for Single-Nucleus Sequencing or Immunostaining (단핵 염기서열분석 또는 면역염색을 위한 성체 마우스 줄무늬 혈관 절제)  

단일 핵 시퀀싱 또는 면역염색을 위한 성인 마우스 줄무늬 혈관의 해부

The stria vascularis functions to generate the endocochlear potential and regulate ion homeostasis in the cochlea. The auditory development and restoration program seeks to understand the underlying mechanisms connecting dysfunctional ion homeostasis with hearing and stability disorders, which are diseases where either fluctuation in or sudden hearing loss occurs. Work in my lab has established the transcriptional landscape of the stria vascularis and has contributed to our understanding of its importance in hearing.We have used these data to draw parallels to poorly-understood hearing and stability disorders, including Meniere's disease. This protocol enables the dissection and isolation of whole-mount stria vascularis for further molecular studies including immunohistochemistry. Given that stria vascularis cell types are intricately intertwined with one another, the use of nuclear isolation and single-nucleus RNA sequencing has facilitated our understanding of the distinct transcriptional machinery involved.

Watch this Scientific Journal Video about Mouse Inner Ear Extraction and Stria Vascularis Dissection at JoVE.com

Mouse Inner Ear Extraction and Stria Vascularis Dissection  

마우스 내이 추출 및 줄무늬 혈관 박리

시작하려면 안락사된 쥐의 측두골 내에서 내이를 식별합니다. 55번 집게를 사용하여 뇌신경을 긁어냅니다. 그런 다음 55번 집게를 사용하여 수포와 캡슐을 해부하여 주변 뼈에서 분리합니다.내이 표면에 남아 있는 연조직을 제거합니다. SV 절제의 경우 55번 집게를 사용하여 달팽이관이 위를 향하도록 하여 전정 부분으로 표본을 잡습니다. 동일한 집게를 사용하여 정점의 달팽이관 뼈를 뚫습니다.정점 회전을 따라 긁어내고 부드러운 힘을 가하여 바깥쪽 뼈층의 작은 조각을 떼어냅니다. 뼈를 부드럽게 들어 올려 측면 벽에서 분리하십시오. 55번 집게를 사용하여 정점과 중간 회전에서 달팽이관 뼈 조각을 제거합니다.측면 벽을 부드럽게 아래로 밀고 뼈 벽 조각을 중간 회전쪽으로 들어 올려 제거하여 정점 및 중간 회전의 측면 벽을 노출시킵니다. 정점 회전 측벽을 옆으로 밀어 나선형 신경절을 노출시킵니다. 바깥 쪽 유모 세포층을 따라 나선형 신경절에서 정점과 중간 회전의 측면 벽을 분리합니다.그런 다음 달팽이관 내부에서 나선형 신경절 조각을 제거합니다. 집게를 원형 및 타원형 창에 삽입하고 기저부의 측면 벽에 더 잘 접근할 수 있도록 현관 쪽으로 아래로 밀어 측두골의 전정 부분에서 달팽이관을 분리합니다. 달팽이관을 분리한 후 내이의 전정 부분을 제거합니다.가능한 한 많은 길 잃은 사람을 보존하는 것을 우선시하십시오. 중간 회전의 뼈가 SV에서 분리될 때까지 계속 제거합니다. 다음으로, 기초 회전을 덮고있는 나머지 달팽이관 뼈 조각을 제거하십시오. 이것은 뼈 아래의 측벽 층을 밀어 분리함으로써 달성 될 수 있습니다.조직을 들어 올려 부드럽게 당겨 측면 벽의 가장 먼 기저 부분을 제거하고 아래의 연조직이 손상되지 않도록 주의하십시오. 측면 벽의 기저 부분을 분리한 상태에서 측면 벽을 따라 집게를 추적하여 나머지 달팽이관에서 측면 벽을 분리합니다. 뼈와 나머지 측벽 사이의 집게가 정점에 도달할 때까지 부드럽게 빗질합니다.그런 다음 측벽을 새 PBS로 옮깁니다. 측면 벽을 평평하게 놓아 SV를 측면 벽의 안쪽에 더 어두운 조직층으로 드러냅니다. SV 레이어를 측면 벽에서 부드럽게 들어 올립니다.분리된 SV를 옆으로 밀고 SV를 쥐지 않으려고 하나의 긴 리본으로 SV를 분리하려고 측면 벽을 따라 집게를 전진시킵니다. 티슈 스쿱을 사용하여 깨지기 쉬운 조직을 채취하십시오. 달팽이관은 출생 후 30일 이상의 성인 마우스에서 해부되었습니다. 주변 측두골에서 내이를 적출했습니다.SV는 중간 턴을 덮고 있는 뼈를 제거한 후 노출되었다. SV는 해부 후 측벽에서 완전히 분리되었습니다.

mouse-inner-ear-extraction-and-stria-vascularis-dissection

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JoVE Journal

Biology

Mouse Inner Ear Extraction and Stria Vascularis Dissection

Dissection of Adult Mouse Stria Vascularis for Single-Nucleus Sequencing or Immunostaining

Endocochlear potential, which is generated by the stria vascularis, is essential to maintain an environment conducive to appropriate hair cell mechanotransduction and ultimately hearing. Pathologies of the stria vascularis can result in a decreased hearing. Dissection of the adult stria vascularis allows for focused single-nucleus capture and subsequent single-nucleus sequencing and immunostaining. These techniques are used to study stria vascularis pathophysiology at the single-cell level. 
Single-nucleus sequencing can be used in the setting of transcriptional analysis of the stria vascularis. Meanwhile, immunostaining continues to be useful in identifying specific populations of cells. Both methods require proper stria vascularis dissection as a prerequisite, which can prove to be technically challenging.

The stria vascularis is vital to the generation of endocochlear potential. Here, we present the dissection of the adult mouse stria vascularis for single-nucleus sequencing or immunostaining.

Endocochlear potential, which is generated by the stria vascularis, is essential to maintain an environment conducive to appropriate hair cell ...

연구

생물학

To begin, identify the inner ear within the temporal bone of a euthanized mouse. Scrape away the cranial nerves using number 55 forceps. Then use number 55 forceps to dissect out the bulla and capsule, detaching them from surrounding bone.Remove any remaining soft tissue from the inner ear surface. For SV dissection, use number 55 forceps to hold the specimen by the vestibular portion with the cochlear facing up. Pierce through the cochlear bone at the apex using the same forceps.Scrape along the apical turn, applying gentle force to break away small pieces of the outer bone layer. Gently lift the bone and detach it from the lateral wall. Using number 55 forceps remove cochlear bone pieces from the apical and middle turns.Gently pushing down the lateral wall, pry and remove the bone wall pieces towards the middle turn to expose the lateral wall of the apical and middle turns. Push the apical turn lateral wall aside to expose the spiral ganglion. Detach the lateral wall of the apical and middle turns from the spiral ganglion along the outer hair cell layer.Then remove pieces of spiral ganglion from inside the cochlear. Detach the cochlear from the vestibular portion of the temporal bone by inserting the forceps into the round and oval window and pushing down towards the vestibule to have better access to the lateral wall of the basal turn. After the cochlear is detached remove the vestibular portion of the inner ear.Prioritize preserving as much of the strayer as possible. Continue removing til the bone of the middle turn is detached from the SV.Next, remove pieces of the remaining cochlear bone covering the basal turn. This can be accomplished by pushing the lateral wall layer under the bone to detach it.Pry and gently pull the tissue to remove the farthest basal part of the lateral wall being careful not to damage the soft tissue below. With the basal part of the lateral wall detached, separate the lateral wall from the remaining cochlear by tracing the forceps along the lateral wall. Gently brush the forceps between the bone and the remaining lateral wall until it reaches the apex.Then move the lateral wall to a fresh PBS. Lay the lateral wall flat revealing the SV as a darker layer of tissue on the internal side of the lateral wall. Gently pry the SV layer away from the lateral wall.Push aside the detached SV and advance the forceps along the lateral wall attempting to detach the SV as one long ribbon, trying not to squeeze the SV.Use a tissue scoop to collect the fragile tissue. Cochlear were dissected from adult mouse aged greater than or equal to postnatal day 30. Inner ear was extracted from the surrounding temporal bone.SV was exposed after the removal of bone covering the middle turn. The SV was fully separated from the lateral wall after dissection.

Preparation of Stria Vascularis Single-Nucleus Suspension

Homogenize the dissected stria vascularis tissue from mouse ear in a two-milliliter Dounce homogenizer, with 10 to 20 strokes on ice. Lyse the tissue on ice for 25 minutes. Then, filter the tissue through a 30-micron filter and spin the filtrate at 500G for five minutes at four degrees Celsius.Remove the supernatant and resuspend the cell pellet in one milliliter of nuclei wash and resuspension buffer. Next, filter the cells through a 10-micron filter and centrifuge at 500G for five minutes at four degrees Celsius. Remove the supernatant and resuspend in 50 microliters of nuclei wash and resuspension buffer.After Trypan Blue staining, dilute five microliters of the cell suspension into 50 microliters of 1X PBS and count the cells. To prepare single nuclei captures, load the sample with the desired nuclear density onto the chip.

Stria Vascularis Single-Nucleus Suspension의 제조

Immunostaining of Mouse Stria Vascularis and Tissue Mounting

Prepare for immunostaining by dissecting the stria vascularis from the mouse and add the tissue to a 24-well plate containing 200 microliters of 4%paraformaldehyde in 1X PBS. Incubate the tissue for 20 minutes at room temperature. perform two short washes of 1X PBS on an orbital shaker at low RPM.Remove the PBS, then perform permeabilization and blocking for a minimum of one hour at room temperature in 300 microliters of PBST solution. Next, stain the tissue with the primary antibody overnight at four degrees Celsius. Then add a secondary antibody and incubate for two hours at room temperature on an orbital shaker at low RPM.Cover the plate to protect the fluorescently tagged secondary antibody from light. Next, prepare a larger microslide by placing two small streaks of glue along the 25 mm axis, just smaller than the 18 by 18 millimeter glass cover slip. Place one drop of mounting reagent between the glue streaks.Using number 55 forceps, gab as little tissue as possible on one end of the SV, and transfer it from PBS to the mounting reagent. Place one end of the cover slip on the slide where one streak of glue was placed. Then gently release the cover slip to avoid creating air bubbles.Seal the mount by dabbing a drop of transparent nail polish on each corner of the mount. Label the specimen on the glass cover slip, away from the visualization field. Visualize the SV tissue under a dissection microscope to ensure it is lying flat and not near any air bubbles.If the SV tissue is not oriented correctly, try to push out air bubbles or carefully remove the smaller glass cover slip and reposition the SV.A whole mount of SV from P30 mouse was prepared and confocal imaging was performed. ZsGreen expression was observed in the intermediate cells, GSIB4 labeled endothelial cells, and DAPI labeled the nuclei.