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 carrying R26R-tdTomato12 and CreER, the latter under the Cx30 promoter13. Labelling of this population of cells may be achieved through injection of viral constructs using the same procedure outlined below. Finally, this approach can be employed for tracking cerebrospinal fluid flow, by infusion of fluorescent tracers.
1. Preparation of the Injection System
NOTE: We recommend carrying out the procedure in a suitable surgical room, and in aseptic conditions. Surgical tools can be sterilized using heat (autoclave, glass bead sterilizer) or sanitized using a high-level chemical disinfectant if they are heat-sensitive. Rinse the instruments before use when employing chemical disinfection or allow them to cool down when sanitized with heat.
<ol>
	<li>Using forceps, bend the needle of the injection syringe to 30&#176; at 3 mm from the tip. 
	NOTE: Use Hamilton syringes with a 30 G beveled needle.</li>
	<li>Prepare 1 mg/mL of endoxifen solution, diluted in 10% DMSO and backfill the syringe with the bent needle. 
	NOTE: Administer 5 &#956;L of the compound to ensure widespread exposure of meningeal cells in the adult C57Bl/6j mouse (ca. 25-30 g), although pilot experiments that test different concentrations and injection volumes may be necessary when treating animals of different ages and strains.</li>
	<li>Adjust the mouse head holder so that the mouth piece is at approximately 30&#176; from the surface of the surgical table. 
	NOTE: A stereotactic frame with three-point fixation (i.e., ears and mouth) can also be used for this procedure. In this case, however, the animal will only be fixed with the mouth piece, whereas ear bars can be extended under the animal&#39;s forelimbs and be used to support the animal&#39;s body during the procedure.</li>
</ol>
2. Induction of Anesthesia
<ol>
	<li>For injectable anesthetics, use concentrations and modes of administration recommended by the veterinary unit at the local institution.</li>
	<li>For inhalational anesthetics, such as isoflurane, prepare the administration unit according to the manufacturer&#39;s specifications.</li>
	<li>With isoflurane, set concentration of the compound at 4% for induction of anesthesia.</li>
	<li>Deliver air at a rate of 400 mL/min.</li>
	<li>Allow the anesthesia unit to fill the chamber with the anesthetic for a few minutes and place the mouse in the chamber afterwards. 
	NOTE: For the present experiments, we have used adult (&#62;2 months old and approximately 30 g) male and female transgenic mice carrying Cx30-CreER and R26R-tdTomato, and bred on a C57Bl/6j background.</li>
	<li>Monitor the animal during induction of anesthesia. The breathing pattern should slow down when the mouse is under full anesthesia.</li>
	<li>Remove the animal from the chamber and check for suppression of the paw reflex by delicately pinching the hind paws. 
	NOTE: The reflex may still be present but delayed a couple of seconds. If that is the case, place the animal back in the chamber until complete suppression of the paw reflex has been achieved.</li>
	<li>Administer analgesic (e.g., Carprofen, 5 mg/kg through subcutaneous injection) to aid post-operative pain management.</li>
</ol>
3. Positioning of the Animal for the Procedure
<ol>
	<li>Fix the animal&#39;s head onto the head holder. To improve accessibility to the cisterna magna, position the animal&#39;s body at approximately 30&#176; from the surface of the table and the head titled downwards, to establish an angle of 120&#176; with the rest of the body and extend the back of the neck to facilitate access to the cisterna magna (Figure 1A).</li>
	<li>Add paper towels underneath the animal to support its body throughout the procedure.</li>
	<li>Secure anesthesia delivery through the mouth piece and reduce isoflurane concentration to 2.5% and air delivery to 200 ml/min.</li>
	<li>Apply ophthalmic ointment.</li>
</ol>
4. Exposure of the Cisterna Magna
<ol>
	<li>Shave the back of the animal&#39;s neck and sanitize the area with 70% ethanol and Betadine.</li>
	<li>Using surgical scissors, perform a midline incision (ca. 7 mm in length) starting at the level of the occipital bone and extending it posteriorly.</li>
	<li>Gently separate superficial connective tissue and neck muscles pulling sidewise from the midline with fine tip tweezers. This will expose the dural membrane overlaying the cisterna magna, shaped as an inverted triangle.</li>
	<li>Use sterile absorption spears or cotton swabs to control any resultant bleeding.</li>
	<li>Position a small surgical separator to maintain the neck muscles pulled aside and enable visualization of the cisterna magna throughout the procedure.</li>
</ol>
5. Intracisternal Injection
<ol>
	<li>To gain access to the cisterna magna, identify the caudal end of the occipital bone and insert the needle that was previously bent immediately underneath. 
	NOTE: There will be a sudden drop in resistance as the dural membrane is punctured. However, the tip of the needle will only penetrate slightly underneath the meningeal surface, thanks to its hooked shape.</li>
	<li>Once the dura has been perforated, allow the bent tip of the needle to penetrate underneath the dural surface by gently pulling the syringe upwards and parallel to the animal&#39;s body, in order to &#34;hook&#34; the needle to the skull (see Figure 1B). This will ensure better stability and will prevent the needle from penetrating deeper, thus avoiding the risk of damaging the underlying cerebellum or medulla.</li>
	<li>Inject the compound slowly to avoid interference with the cerebrospinal fluid&#39;s natural flow. 
	NOTE: Depending on the purpose of the experiment, the infusion rate may vary. If slow and steady infusion rate is required (e.g., when using the procedure to trace cerebrospinal fluid movement), it may be advisable to use an automatized microinfusion system in combination with the syringe.</li>
	<li>After the injection, let the needle rest in site for 1 min and carefully remove it. Use fine tip forceps to aid retraction of the needle from its hooked position. 
	NOTE: The use of a thin needle (e.g., 30 G) does not lead to substantial damage of the meningeal membrane, and consequent outflow of the cerebrospinal fluid. If larger needle sizes are required, we recommend&#160;stopping the leaking of fluid by applying pressure at the injection site using a sterile cotton tip.</li>
</ol>
6. Concluding the Procedure and Post-operative Care
<ol>
	<li>Remove the separator and allow the muscles to go back to their original position overlaying the dural membrane.</li>
	<li>Close the skin incision using a few drops of cyanoacrylate adhesive. 
	NOTE: Alternatively, use absorbable sutures (e.g., 5-0 vicryl sutures) and close the skin incision with interrupted stitches.</li>
	<li>Apply local anesthetic (e.g., 100 &#956;L of 5% lidocaine) at the incision site.</li>
	<li>Remove the animal from the holder and place in a clean cage positioned on a heating pad. Monitor the animal until it regains consciousness. 
	NOTE: The procedure is not expected to cause long-lasting pain or distress in the animal. Nevertheless, the mouse should be monitored closely for the first postoperative days and pain relief measures may be undertaken whenever deemed necessary, and in accordance with recommendations provided by the veterinary unit at your institution.</li>
</ol>

<ol><li>Vanlandewijck, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+molecular+atlas+of+cell+types+and+zonation+in+the+brain+vasculature%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">A molecular atlas of cell types and zonation in the brain vasculature.</a> Nature. 554 (7693), 475-480 (2018).</li>
<li>Whish, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+inner+CSF-brain+barrier%3A+developmentally+controlled+access+to+the+brain+via+intercellular+junctions%5BTitle%5D)+AND+%22Frontiers+in+Neuroscience%22%5BJournal%5D)">The inner CSF-brain barrier: developmentally controlled access to the brain via intercellular junctions.</a> Frontiers in Neuroscience. 9, 16(2015).</li>
<li>Weller, R. O., Sharp, M. M., Christodoulides, M., Carare, R. O., Mollgard, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+meninges+as+barriers+and+facilitators+for+the+movement+of+fluid%2C+cells+and+pathogens+related+to+the+rodent+and+human+CNS%5BTitle%5D)+AND+%22Acta+Neuropathologica%22%5BJournal%5D)">The meninges as barriers and facilitators for the movement of fluid, cells and pathogens related to the rodent and human CNS.</a> Acta Neuropathologica. 135 (3), 363-385 (2018).</li>
<li>Choe, Y., Siegenthaler, J. A., Pleasure, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+cascade+of+morphogenic+signaling+initiated+by+the+meninges+controls+corpus+callosum+formation%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">A cascade of morphogenic signaling initiated by the meninges controls corpus callosum formation.</a> Neuron. 73 (4), 698-712 (2012).</li>
<li>Siegenthaler, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Retinoic+acid+from+the+meninges+regulates+cortical+neuron+generation%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Retinoic acid from the meninges regulates cortical neuron generation.</a> Cell. 139 (3), 597-609 (2009).</li>
<li>Bifari, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neurogenic+Radial+Glia-like+Cells+in+Meninges+Migrate+and+Differentiate+into+Functionally+Integrated+Neurons+in+the+Neonatal+Cortex%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">Neurogenic Radial Glia-like Cells in Meninges Migrate and Differentiate into Functionally Integrated Neurons in the Neonatal Cortex.</a> Cell Stem Cell. 20 (3), 360-373 (2017).</li>
<li>De Bock, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+new+angle+on+blood-CNS+interfaces%3A+a+role+for+connexins%3F%5BTitle%5D)+AND+%22FEBS+Letters%22%5BJournal%5D)">A new angle on blood-CNS interfaces: a role for connexins?</a> FEBS Letters. 588 (8), 1259-1270 (2014).</li>
<li>Ramos, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cisterna+Magna+Injection+in+Rats+to+Study+Glymphatic+Function%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Cisterna Magna Injection in Rats to Study Glymphatic Function.</a> Methods in Molecular Biology. 1938, 97-104 (2019).</li>
<li>Xavier, A. L. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cannula+Implantation+into+the+Cisterna+Magna+of+Rodents%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Cannula Implantation into the Cisterna Magna of Rodents.</a> Journal of Visualized Experiments. (135), (2018).</li>
<li>Iliff, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+paravascular+pathway+facilitates+CSF+flow+through+the+brain+parenchyma+and+the+clearance+of+interstitial+solutes%2C+including+amyloid+beta%5BTitle%5D)+AND+%22Science+Translational+Medicine%22%5BJournal%5D)">A paravascular pathway facilitates CSF flow through the brain parenchyma and the clearance of interstitial solutes, including amyloid beta.</a> Science Translational Medicine. 4 (147), (2012).</li>
<li>Coureuil, M., Lecuyer, H., Bourdoulous, S., Nassif, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+journey+into+the+brain%3A+insight+into+how+bacterial+pathogens+cross+blood-brain+barriers%5BTitle%5D)+AND+%22Nature+Reviews+Microbiology%22%5BJournal%5D)">A journey into the brain: insight into how bacterial pathogens cross blood-brain barriers.</a> Nature Reviews Microbiology. 15 (3), 149-159 (2017).</li>
<li>Madisen, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transgenic+mice+for+intersectional+targeting+of+neural+sensors+and+effectors+with+high+specificity+and+performance%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Transgenic mice for intersectional targeting of neural sensors and effectors with high specificity and performance.</a> Neuron. 85 (5), 942-958 (2015).</li>
<li>Slezak, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transgenic+mice+for+conditional+gene+manipulation+in+astroglial+cells%5BTitle%5D)+AND+%22Glia%22%5BJournal%5D)">Transgenic mice for conditional gene manipulation in astroglial cells.</a> Glia. 55 (15), 1565-1576 (2007).</li>
<li>Hardy, S. J., Christodoulides, M., Weller, R. O., Heckels, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interactions+of+Neisseria+meningitidis+with+cells+of+the+human+meninges%5BTitle%5D)+AND+%22Molecular+Microbiology%22%5BJournal%5D)">Interactions of Neisseria meningitidis with cells of the human meninges.</a> Molecular Microbiology. 36 (4), 817-829 (2000).</li>
<li>Colicchio, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+meningococcal+ABC-Type+L-glutamate+transporter+GltT+is+necessary+for+the+development+of+experimental+meningitis+in+mice%5BTitle%5D)+AND+%22Infection+and+Immunity%22%5BJournal%5D)">The meningococcal ABC-Type L-glutamate transporter GltT is necessary for the development of experimental meningitis in mice.</a> Infection and Immunity. 77 (9), 3578-3587 (2009).</li>
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</ol>

The authors declare no competing interests.

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><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 (30 G 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&amp;amp;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 ...

induction-leptomeningeal-cells-modification-via-intracisternal

Research

JoVE Journal

Neuroscience

8.0K Views.  Karolinska Institute. Onze chirurgische ingreep maakt het mogelijk specifieke genbewerking van leptomeningeale cellen om hun functie te bestuderen in vele fysiologische en pathologische aandoeningen zoals neuroontwikkeling en de verspreiding van bacteriële meningitis. Om schade tijdens de intracisternale levering van endoxifen te minimaliseren, gebruiken we een gebogen naald die tegen de staartrand van de schedel kan worden bevestigd om te voorkomen dat het dieper in het weefsel doordringt. De procedure kan worde...

Video: Induction of Leptomeningeal Cells Modification Via Intracisternal Injection

Isolation of Brain-infiltrating Leukocytes

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine encephalomyelitis virus (TMEV) via a rapid intracranial injection technique and how to purify a leukocyte-enriched population of infiltrating cells from whole brain. Briefly, mice are anesthetized with isoflurane in a closed chamber and are free-hand injected with a Hamilton syringe into the frontal cortex. Mice are then killed at various times after infection by isoflurane overdose and whole brains are extracted and homogenized in RPMI with a Tenbroeck tissue grinder. Brain homogenates are centrifuged through a continuous 30% Percoll gradient to remove the myelin and other cell debris. The cell suspension is then strained at 40 &mu;m, washed and centrifuged on a discontinuous Ficoll-Paque Plus gradient to select and purify the leukocytes. The leukocytes are then washed and resuspended in appropriate buffers for immunophenotyping by flow cytometry. Flow cytometry reveals a population of innate immune cells at the early stages of infection in C57BL/6 mice. At 24 hours post infection, multiple subsets of immune cells are present in the BILs, with an enriched population of Gr1+, CD11b+ and F4/80+cells. Therefore, this method is useful in characterizing the immune response to acute infection in the brain.

A rapid method to obtain infiltrating leukocytes from the murine brain is described. This method utilizes a continuous Percoll gradient and discontinuous Ficoll gradient to select and purify the leukocyte-enriched layer. Isolated leukocytes may then be characterized by flow cytometric measurements.

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine ...

Immunology and Infection

A Murine Ommaya Xenograft Model to Study Direct-Targeted Therapy of Leptomeningeal Disease

Leptomeningeal disease (LMD) is an uncommon type of central nervous system (CNS) metastasis to the cerebral spinal fluid (CSF). The most common cancers that cause LMD are breast and lung cancers and melanoma. Patients diagnosed with LMD have a very poor prognosis and generally survive for only a few weeks or months. One possible reason for the lack of efficacy of systemic therapy against LMD is the failure to achieve therapeutically effective concentrations of drug in the CSF because of an intact and relatively impermeable blood-brain barrier (BBB) or blood-CSF barrier across the choroid plexus. Therefore, directly administering drugs intrathecally or intraventricularly may overcome these barriers. This group has developed a model that allows for the effective delivery of therapeutics (i.e., drugs, antibodies, and cellular therapies) chronically and the repeated sampling of CSF to determine drug concentrations and target modulation in the CSF (when the tumor microenvironment is targeted in mice). The model is the murine equivalent of a magnetic resonance imaging-compatible Ommaya reservoir, which is used clinically. This model, which is affixed to the skull, has been designated as the &#34;Murine Ommaya.&#34; As a therapeutic proof of concept, human epidermal growth factor receptor 2 antibodies (clone 7.16.4) were delivered into the CSF via the Murine Ommaya to treat mice with LMD from human epidermal growth factor receptor 2-positive breast cancer. The Murine Ommaya increases the efficiency of drug delivery using a miniature access port and prevents the wastage of excess drug; it does not interfere with CSF sampling for molecular and immunological studies. The Murine Ommaya is useful for testing novel therapeutics in experimental models of LMD.

Here, we describe a murine xenograft model that functionally resembles an Ommaya reservoir in patients. We developed the Murine Ommaya to study novel therapeutics for the universally fatal leptomeningeal disease.

Leptomeningeal disease (LMD) is an uncommon type of central nervous system (CNS) metastasis to the cerebral spinal fluid (CSF). The most common cancers ...

Cancer Research

Systemic Injection of Neural Stem/Progenitor Cells in Mice with Chronic EAE

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative neurology. This directive has arisen from the extensive evidence that brain repair is achieved after focal or systemic NPC transplantation in several preclinical models of neurological diseases.
These experimental data have identified the cell delivery route as one of the main hurdles of restorative stem cell therapies for brain diseases that requires urgent assessment. Intraparenchymal stem cell grafting represents a logical approach to those pathologies characterized by isolated and accessible brain lesions such as spinal cord injuries and Parkinson's disease. Unfortunately, this principle is poorly applicable to conditions characterized by a multifocal, inflammatory and disseminated (both in time and space) nature, including multiple sclerosis (MS). As such, brain targeting by systemic NPC delivery has become a low invasive and therapeutically efficacious protocol to deliver cells to the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system (CNS).
This alternative method of cell delivery relies on the NPC pathotropism, specifically their innate capacity to (i) sense the environment via functional cell adhesion molecules and inflammatory cytokine and chemokine receptors; (ii) cross the leaking anatomical barriers after intravenous (i.v.) or intracerebroventricular (i.c.v.) injection; (iii) accumulate at the level of multiple perivascular site(s) of inflammatory brain and spinal cord damage; and (i.v.) exert remarkable tissue trophic and immune regulatory effects onto different host target cells in vivo.
Here we describe the methods that we have developed for the i.v. and i.c.v. delivery of syngeneic NPCs in mice with experimental autoimmune encephalomyelitis (EAE), as model of chronic CNS inflammatory demyelination, and envisage the systemic stem cell delivery as a valuable technique for the selective targeting of the inflamed brain in regenerative neurology.

The transplantation of neural stem/progenitor cells (NPCs) holds great promises in regenerative neurology. The systemic delivery of NPCs has turned into effective, low invasive, and therapeutically very efficacious protocol to deliver stem cells in the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system.

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative ...

Rat Model of Widespread Cerebral Cortical Demyelination Induced by an Intracerebral Injection of Pro-Inflammatory Cytokines

Multiple sclerosis (MS) is the most common immune-mediated disease of the central nervous system (CNS) and progressively leads to physical disability and death, caused by white matter lesions in the spinal cord and cerebellum, as well as by demyelination in grey matter. Whilst conventional models of experimental allergic encephalomyelitis are suitable for the investigation of the cell-mediated inflammation in the spinal and cerebellar white matter, they fail to address grey matter pathologies. Here, we present the experimental protocol for a novel rat model of cortical demyelination allowing the investigation of the pathological and molecular mechanisms leading to cortical lesions. The demyelination is induced by an immunization with low-dose myelin oligodendrocyte glycoprotein (MOG) in an incomplete Freund&#39;s adjuvant followed by a catheter-mediated intracerebral delivery of pro-inflammatory cytokines. The catheter, moreover, enables multiple rounds of demyelination without causing injection-induced trauma, as well as the intracerebral delivery of potential therapeutic drugs undergoing a preclinical investigation. The method is also ethically favorable as animal pain and distress or disability are controlled and relatively minimal. The expected timeframe for the implementation of the entire protocol is around 8 - 10 weeks.

The protocol presented here allows the reproduction of a widespread grey matter demyelination of both cortical hemispheres in adult male Dark Agouti rats. The method comprises of intracerebral implantation of a catheter, subclinical immunization against myelin oligodendrocyte glycoprotein, and intracerebral injection of a pro-inflammatory cytokine mixture through the implanted catheter.

Multiple sclerosis (MS) is the most common immune-mediated disease of the central nervous system (CNS) and progressively leads to physical disability and ...

Inducing Meningococcal Meningitis Serogroup C in Mice via Intracisternal Delivery

Neisseria meningitidis (meningococcus) is a narrow-host-range microorganism, globally recognized as the leading cause of bacterial meningitis. Meningococcus is a transient colonizer of human nasopharynx of approximately 10% of healthy subject. In particular circumstances, it acquires an invasive ability to penetrate the mucosal barrier and invades the bloodstream causing septicaemia. In the latest case, fulminating sepsis could arise even without the consequent development of meningitis. Conversely, bacteria could poorly multiply in the bloodstream, cross the blood brain barrier, reach the central nervous system, leading to fulminant meningitis. The murine models of bacterial meningitis represent a useful tool to investigate the host-pathogen interactions and to analyze the pathogenetic mechanisms responsible for this lethal disease. Although, several experimental model systems have been evaluated over the last decades, none of these were able to reproduce the characteristic pathological events of meningococcal disease. In this experimental protocol, we describe a detailed procedure for the induction of meningococcal meningitis in a mouse model based on the intracisternal inoculation of bacteria. The peculiar signs of human meningitis were recorded in the murine host through the assessment of clinical parameters (e.g., temperature, body weight), evaluation of survival rate, microbiological analysis and histological examination of brain injury. When using intracisternal (i.cist.) inoculum, meningococci complete delivery directly into cisterna magna, leading to a very efficient meningococcal replication in the brain tissue. A 1,000-fold increase of viable count of bacteria is observed in about 18 h. Moreover, meningococci are also found in the spleen, and liver of infected mice, suggesting that the liver may represent a target organ for meningococcal replication.

Here, we describe a method to induce meningococcal meningitis through an intracisternal route of infection in adult mice. We present a step by step protocol of meningococcal infection from the preparation of inoculum to the intracisternal infection; then record the animal survival and evaluate the bacterial loads in murine tissues.

Neisseria meningitidis (meningococcus) is a narrow-host-range microorganism, globally recognized as the leading cause of bacterial meningitis. ...

A Comprehensive Protocol for Delivering Intrathecal Injections in Neonatal Mice

Intrathecal Injection of Newborn Mouse for Genome Editing and Drug Delivery

Intrathecal injection is a commonly employed procedure in both pediatric and adult clinics, serving as an effective means to administer medications and treatments. By directly delivering medications and treatments into the cerebrospinal fluid of the central nervous system, this method achieves higher localized drug concentrations while reducing systemic side-effects compared to other routes such as intravenous, subcutaneous, or intramuscular injections. Its importance extends beyond clinical settings, as intrathecal injection plays a vital role in preclinical studies focused on treating neurogenetic disorders in rodents and other large animals, including non-human primates. However, despite its widespread application, intrathecal injection in young, particularly neonatal pups, poses significant technical challenges due to their small size and fragile nature. Successful and reliable administration of intrathecal injections in newborn mice requires meticulous attention to detail and careful consideration of various factors. Thus, there is a crucial need for a standardized protocol that not only provides instructions but also highlights key technical considerations and good laboratory practices to ensure procedural consistency, as well as the safety and welfare of the animals.
To address this unmet need, we present a detailed and comprehensive protocol for performing intrathecal injections specifically in newborn pups on postnatal day 1 (P1). By following the step-by-step instructions, researchers can confidently perform intrathecal injections in neonatal pups, enabling the accurate delivery of drugs, antisense oligos, and viruses for gene replacement or genome editing-based treatments. Furthermore, the importance of adhering to good laboratory practices is emphasized to maintain the well-being of animals and ensure reliable experimental outcomes. This protocol aims to address the technical challenges associated with intrathecal injections in neonatal mice, ultimately facilitating advances in the field of neurogenetic research that aims to develop potential therapeutic interventions.

Author Spotlight: Intrathecal Injection &#8211; An Efficient and Reliable Delivery Method to Test the Efficacy of Gene Editing in Neonatal Mouse Brains

The present protocol outlines step-by-step instructions for performing intrathecal injections in neonatal mice for gene editing and drug delivery.

Intrathecal injection is a commonly employed procedure in both pediatric and adult clinics, serving as an effective means to administer medications and ...

Intraspinal Cavity Injection of Human Mesenchymal Stem Cells and Tracking their Migration into the Rat Brain

Mesenchymal stem cells (MSCs) have been studied for the treatment of various diseases. In neurodegenerative diseases involving defects in both the brain and the spinal cord, the route of administration is very important, because MSCs must migrate to both the brain and the spinal cord. This paper describes a method for administering MSCs into the spinal canal (intraspinal cavity injection) that can target the brain and spinal cord in a rat model. One million MSCs were injected into the spinal canals of rats at the level of lumbar vertebrae 2-3. After administration, the rats were euthanized at 0, 6, and 12 h post-injection. Optical imaging and quantitative real-time polymerase chain reaction (qPCR) were used to track the injected MSCs. The results of the present study demonstrated that MSCs administered via the spinal cavity could be detected subsequently in both the brain and spinal cord at 12 h. Intraspinal cavity injection has the advantage of not requiring general anesthesia and has few side effects. However, the drawback of the low migration rate of MSCs to the brain must be overcome.

Several routes of administration can be used to deliver mesenchymal stem cells (MSCs) to the brain. In the present study, MSCs were delivered throughout the neuraxis and brain via intra-spinal cavity injection. MSCs were injected into the spinal cavities of rats, and stem cell migration was tracked and quantified.

Mesenchymal stem cells (MSCs) have been studied for the treatment of various diseases. In neurodegenerative diseases involving defects in both the brain ...

Biology

Intracisternal Delivery of Meningococci into Mouse Models to Induce Meningococcal Meningitis

Source: Pagliuca, C., et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/60047">Inducing Meningococcal Meningitis Serogroup C in Mice via Intracisternal Delivery.</a> J. Vis. Exp. (2019)
In this video, we demonstrate the intracisternal delivery of Neisseria meningitidis into a mouse model to generate meningitis infection.

Source: Pagliuca, C., et al., Inducing Meningococcal Meningitis Serogroup C in Mice via Intracisternal Delivery. J. Vis. Exp. (2019)
In this video, we ...

JoVE Encyclopedia of Experiments

Immunology

Immune Response

Host-Pathogen Interaction

Surgical Cisterna Magna Injection: A Method for Administering Tumor Cells Directly into Central Nervous System of Murine Model

Source: Law, V. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/62033/a-murine-ommaya-xenograft-model-to-study-direct-targeted-therapy">A Murine Ommaya Xenograft Model to Study Direct-Targeted Therapy of Leptomeningeal Disease.</a> J. Vis. Exp. (2021)
In this video, we describe the procedure to deliver human-derived tumor cells into the cisterna magna of a mouse&#8217;s brain. The orthotopic mouse model can be used to study metastatic spread of cancer to the cranial meninges.

Source: Law, V. et al. A Murine Ommaya Xenograft Model to Study Direct-Targeted Therapy of Leptomeningeal Disease. J. Vis. Exp. (2021)
In this video, we ...

Cancers of the Nervous System

In vivo studies

Intracerebroventricular Injection in Mouse: An Ommaya Mediated Direct Drug Administration Method to Deliver Drugs to Cerebral Ventricles of Mouse Brain

Source: Law, V. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/62033/a-murine-ommaya-xenograft-model-to-study-direct-targeted-therapy">A Murine Ommaya Xenograft Model to Study Direct-Targeted Therapy of Leptomeningeal Disease.</a> J. Vis. Exp. (2021)
This video demonstrates ommaya mediated intracerebroventricular injection in a mouse model. The method enables localized delivery of potential drug candidates and evades the blood-brain barrier.

Induction of Leptomeningeal Cells Modification Via Intracisternal Injection

Samenvatting

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