This study was supported by grants from the Swedish Research Council, the Swedish Cancer Society, the Swedish Foundation for Strategic Research, Knut och Alice Wallenbergs Stiftelse and the Strategic Research Programme in Stem Cells and Regenerative Medicine at Karolinska Institutet (StratRegen).

The surgical procedures hereby presented were approved by Stockholms Norra Djurf&#246;rs&#246;ksetiska N&#228;mnd and carried out in agreement with specifications provided by the research institute (Karolinska Institute, Sweden).
NOTE: Intracisternal injection can be flexibly adapted for multiple research purposes. We present below a procedure developed to efficiently label leptomeningeal cells for fate mapping based on injection of endoxifen in a transgenic mouse line carryin...

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The protocol outlined here presents a straightforward and reproducible procedure to label leptomeningeal cells for fate mapping. We use intracisternal injection of endoxifen, an active metabolite of Tamoxifen, to induce expression of tdTomato fluorescent reporter in Cx30-CreER; R26R-tdTomato mice12,13.
Compared to other protocols used for gaining access to the cerebrospinal fluid through the cisterna magna9, our...

Leptomeningeal cells are a fibroblast-like population of cells organized in a thin layer overlaying the brain and expressing genes implicated in collagen crosslinking (e.g., Dcn and Lum), and in the establishment of a brain-meningeal barrier (e.g., Cldn11)1,2. Leptomeningeal cells are implicated in a wide range of physiological functions, from strict control over the cerebrospinal fluid drainage3 to guidance of neural progenitors in the developing brain4,5. A recent study has also proposed that leptomeninges in the newborn may harbor radial glia-like cells that migrate into the brain parenchyma and develop into functional cortical neurons6.
Leptomeningeal cells are located in close proximity to surface astrocytes and share with them, as well as other parenchymal astroglia, expression of connexin-30 (Cx30)7. The surgical procedure outlined below allows widespread and specific labelling of these meningeal cells via a one-time delivery of endoxifen into the cisterna magna of transgenic mice conditionally expressing tdTomato in Cx30+ cells (i.e., using a CreER-loxP system for fate mapping). Endoxifen is an active metabolite of Tamoxifen and induces recombination of CreER-expressing cells in the same way as Tamoxifen does. It is, however, the recommended solution for topical application because it dissolves in 5-10% DMSO, instead of high concentrations of ethanol. Additionally, endoxifen does not cross the brain-meningeal barrier, thereby enabling specific recombination of leptomeningeal cells, without labelling of the underlying Cx30+ astroglial population (see Representative Results).
The technique presented here aims at manually and safely injecting the compound in the cerebrospinal fluid, via direct access to the cisterna magna. Unlike other, more invasive procedures requiring craniotomy, this approach allows to infuse compounds without causing damage to the skull or the brain parenchyma. Thus, it is not associated with the induction of inflammatory reactions triggered by activation of parenchymal glia cells. Similar to other injection strategies described before8,9,10, the present approach relies on the surgical exposure of the atlanto-occipital dural membrane covering the cisterna magna, after blunt dissection of the overlaying neck muscles. However, unlike for other procedures, we recommend the use of a needle bent at the tip, which can be stabilized against the occipital bone during administration. This will prevent the risk of the needle penetrating too deep and damaging the underlying cerebellum and medulla.
This surgical procedure is compatible with lineage tracing investigations that aim at mapping changes in cell identities and migration routes through parenchymal layers. It can also be adapted to genetic ablation studies that intend to probe the role of leptomeningeal cells in health and disease, such as their contribution to cortical development5 or the spreading of bacterial meningitis3,11. Finally, it can be utilized to track cerebrospinal fluid movement when combined with delivery of fluorescent tracers in wildtype animals.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anesthesia unit</td>
			<td>Univentor 410</td>
			<td>8323102</td>
			<td>Complete of vaporizer, chamber, and tubing that connects to chamber and mouse head holder</td>
		</tr>
		<tr>
			<td>Anesthesia (Isoflurane)</td>
			<td>Baxter Medical AB</td>
			<td>000890</td>
			<td></td>
		</tr>
		<tr>
			<td>Betadine</td>
			<td>Sigma-Aldrich</td>
			<td>PVP1</td>
			<td></td>
		</tr>
		<tr>
			<td>Carprofen</td>
			<td>Orion Pharma AB</td>
			<td>014920</td>
			<td>Commercial name Rymadil</td>
		</tr>
		<tr>
			<td>Cyanoacrylate glue</td>
			<td>Carl Roth</td>
			<td>0258.1</td>
			<td>Use silk 5-0 sutures, in alternative</td>
		</tr>
		<tr>
			<td>Medbond Tissue Glue</td>
			<td>Stoelting</td>
			<td>50479</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Endoxifen</td>
			<td>Sigma-Aldrich</td>
			<td>E8284</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 70%</td>
			<td>Histolab</td>
			<td>01370</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton syringe (30G beveled needle)</td>
			<td>Hamilton</td>
			<td>80300</td>
			<td></td>
		</tr>
		<tr>
			<td>Lidocaine</td>
			<td>Aspen Nordic</td>
			<td>520455</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse head holder</td>
			<td>Narishige International</td>
			<td>SGM-4</td>
			<td>With mouth piece for inhalational anhestetics. Alternatively, use a stereotactic frame</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Fine Science Tools</td>
			<td>15009-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaver</td>
			<td>Aesculap</td>
			<td>GT420</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile absorption spears</td>
			<td>Fine Science Tools</td>
			<td>18105-01</td>
			<td>Sterile cotton swabs are also a good option</td>
		</tr>
		<tr>
			<td>Surgical separator</td>
			<td>World Precision Instrument</td>
			<td>501897</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Dumont</td>
			<td>11251-35</td>
			<td></td>
		</tr>
		<tr>
			<td>Viscotears</td>
			<td>Bausch&#38;Lomb Nordic AB</td>
			<td>541760</td>
			<td></td>
		</tr>
	</tbody>
</table>

induction of leptomeningeal cells modification via intracisternal injection

The protocol outlined here describes how to safely and manually inject solutions through the cisterna magna while eliminating the risk of damage to the underlying parenchyma. Previously published protocols recommend using straight needles that should be lowered to a maximum of 1-2 mm from the dural surface. The sudden drop in resistance once the dural membrane has been punctured makes it difficult to maintain the needle in a steady position. Our method, instead, employs a needle bent at the tip that can be stabilized against the occipital bone of the skull, thus preventing the syringe from penetrating into the tissue after perforation of the dural membrane. The procedure is straightforward, reproducible, and does not cause long-lasting discomfort in the operated animals. We describe the intracisternal injection strategy in the context of genetic fate mapping of vascular leptomeningeal cells. The same technique can, furthermore, be utilized to address a wide range of research questions, such as probing the role of leptomeninges in neurodevelopment and the spreading of bacterial meningitis, through genetic ablation of genes putatively implicated in these phenomena. Additionally, the procedure can be combined with an automatized infusion system for a constant delivery and used for tracking cerebrospinal fluid movement via injection of fluorescently labelled molecules.

Intracisternal injection of endoxifen in transgenic mice expressing CreER under the Cx30 promoter13 and an inducible fluorescent reporter allows for specific recombination of leptomeningeal cells without labelling the neighboring Cx30-expressing surface and parenchymal astrocytes in the cortex (Figure 1). In order to gain access to the cisterna magna, the anesthetized animal is positioned with its body and its head at an angle of appro...

Watch this Scientific Journal Video about Induction of Leptomeningeal Cells Modification Via Intracisternal Injection at JoVE.com

Induction of Leptomeningeal Cells Modification Via Intracisternal Injection

We describe an intracisternal injection that employs a needle bent at the tip that can be stabilized to the skull, thus eliminating the risk of damage to the underlying parenchyma. The approach can be used for genetic fate mapping and manipulations of leptomeningeal cells and for tracking cerebrospinal fluid movement.

The protocol outlined here describes how to safely and manually inject solutions through the cisterna magna while eliminating the risk of damage to the ...

Our surgical procedure enables specific gene editing of leptomeningeal cells to study their function in many physiological and pathological conditions such as neurodevelopment and the spreading of bacterial meningitis. To minimize damage during the intracisternal delivery of endoxifen, we utilize a bent needle that can be secured against the caudal edge of the skull to prevent it from penetrating deeper in the tissue. The procedure can be used to probe the role of genes expressing leptomeningeal cells through loss and gain of function experiments.It can also be adopted to track the cerebrospinal fluid flow. Visual demonstration of this method will ensure successful identification of the injection site for intracisternal injection and understanding on how to secure the needle against the occipital bone. Begin by preparing a Hamilton syringe with a 30 gauge beveled needle for injection.Use forceps to bend the needle to 30 degrees three millimeters from the tip. Next, dilute endoxifen in 10%DMSO to a concentration of one milligram per milliliter and backfill the syringe. Anesthetize the animal in the isoflurane chamber.Then adjust the mouse head holder so that the mouthpiece is approximately at a 30 degree angle from the surface of the surgical table and fix the animal's head onto the holder. To improve accessibility to the cisterna magna, position the animal's body at approximately 30 degrees from the surface of the table with the head tilted downward which will establish an angle of 120 degrees with the rest of the body and extend the back of the neck to facilitate access to the cisterna magna. Once the animal is properly positioned, apply ophthalmic ointment, shave the back of its neck, and sanitize the area with alcohol wipes and Betadine.Use surgical scissors to make a midline incision starting at the level of the occipital bone and extending posteriorly. Gently separate the superficial connective tissue and neck muscles by pulling sideways from the midline with fine tip tweezers which will expose the dural membrane overlying the cisterna magna. Position a small surgical separator to enable visualization of the cisterna magna throughout the procedure.Identify the caudal end of the occipital bone and insert the previously bent needle immediately underneath. Once the dura has been perforated, allow the bent tip of the needle to penetrate underneath the surface by gently pulling the syringe upward and parallel to the animal's body which will ensure better stability. Inject the compound slowly to avoid interference with cerebrospinal fluid's natural flow.After the injection, let the needle rest inside for one minute, then carefully remove it with the help of fine tip forceps. Close the skin with a few drops of cyanoacrylate adhesive and apply local anesthetic at the injection site. Remove the animal from the holder and place it in a clean cage on a heating pad.Then make sure to monitor the animal until it regains consciousness. Intracisternal injection of endoxifen in transgenic mice expressing CreER under the Cx30 promoter, an inducible fluorescent reporter allows for specific recombination of leptomeningeal cells without labeling the neighboring Cx30 expressing surface astrocytes and parenchymal astrocytes in the cortex. This injection protocol takes advantage of the physiological movement of the cerebrospinal fluid so the endoxifen solution is distributed throughout the subarachnoid space to efficiently recombine leptomeningeal cells overlaying the olfactory bulbs, cortex, and cerebellum.The solution does not cross the brain meningeal barrier or come in contact with astroglial cells of the parenchyma as opposed to systemic administration through oral gavage. Recombination of leptomeningeal cells after intracisternal injection was identified through Pdgfr-alpha reactivity while surface and parenchymal astrocytes expressing Gfap remained unlabeled. When attempting this experiment, it is important to locate the injection site for intracisternal delivery.This visual demonstration provides the technical know-how to carry out the procedure while minimizing the risk of damaging the neural tissue under it. Gene ablation studies can be performed with this technique to investigate the role of leptomeningeal cells in corticogenesis and regulation of cerebrospinal fluid formation and to identify additional sites for spreading of bacteria in the subarachnoid space.

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7.7K 조회수.  Karolinska Institute. 우리의 외과 적 절차는 렙토멘닝 세포의 특정 유전자 편집을 가능하게신경 발달과 세균성 뇌막염의 확산과 같은 많은 생리적 및 병리학 적 조건에서 자신의 기능을 연구할 수 있습니다. 엔독시펜의 인트라디에이터리 전달 중 손상을 최소화하기 위해, 우리는 두개골의 caudal 가장자리에 대해 고정 될 수있는 구부러진 바늘을 사용하여 조직 깊숙이 침투하는 것을 방지합니다. ?...