No funding was received.

This protocol was approved by the local ethics committee in accordance with the Helsinki Declaration (Reg. No. A2019-0214). Informed consent was obtained from all participants. In the presented case involving a child, written informed consent was obtained from both parents.
NOTE: Fluorescein is a fluorescent dye that emits green light (520-530 nm) when stimulated with blue light (of wavelength ~485 nm). It is used for visualization of the tear film in ophthalmology. Its intrathecal use is off-label and based on an individual therapeutic agreement. Fluorescein-Natrium at a 10% concentration is available and regularly used in fluorescein angiography of the ocular fundus in ophthalmology.
1. Preparation for surgery
<ol>
	<li>Obtain audiological testing, high resolution computed tomography (HRCT), and magnetic resonance imaging (MRI) of the skull base.</li>
	<li>Obtain written, informed consent from the patient regarding off-label use of fluorescein.</li>
	<li>Perform a lumbar puncture and place a lumbar drain for later application of the dye.</li>
	<li>Use fluorescein sodium (10% concentration). For lumbar application, dilute the fluorescein to 5% concentration with water.</li>
	<li>Apply 0.1 mL of the dilution per 10 kg of body weight, with a maximum of 1 mL, intrathecally.</li>
	<li>Perform the application immediately before surgery to achieve intraoperative visualization. 
	NOTE: When applying the method to children, the application should be performed under general anesthesia.</li>
	<li>Perform general anesthesia according to the patient&#39;s individual clinical history and risk factors and according to the decision of the anesthesiologist.</li>
	<li>Cover the microscope with a blue light filter to visualize the fluorescein, or use a fully digital microscope.</li>
	<li>Use sterile draping to cover the patient.</li>
</ol>
2. Surgery
<ol>
	<li>For the transmastoid approach, perform a mastoidectomy and posterior tympanotomy.</li>
	<li>Expand the surgery according to the expected origin of the leak.</li>
	<li>As a first landmark, expose the dura to the middle cranial fossa.</li>
	<li>Thin the posterior canal wall of the outer ear canal.</li>
	<li>Expose the short incus process in the antrum and identify the lateral semicircular canal.</li>
	<li>Expose the chorda facial angle at which the chorda tympani leaves the facial nerve.</li>
	<li>Open the facial recess. Leave the facial nerve with a bony coverage.</li>
	<li>Access the middle ear through a posterior tympanotomy. The origin of the fluid leak can easily be identified in the stapes footplate (Figure 2).</li>
	<li>Use fascia from the temporal muscle and absorbable collagen fibrin sealant patches to seal the CSF leak until no more fluid leaves the footplate. The stapes suprastructure stabilizes the sealant.</li>
</ol>

<ol>
	<li>Gacek, R. R., Gacek, M. R., Tart, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+spontaneous+cerebrospinal+fluid+otorrhea:+diagnosis+and+management.">Adult spontaneous cerebrospinal fluid otorrhea: diagnosis and management.</a> The American Journal of Otology. 20 (6), 770-776 (1999).</li><li>Schraven, S. P., Bisdas, S., Wagner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synchronous+spontaneous+cerebrospinal+fluid+leaks+in+the+nose+and+ear.">Synchronous spontaneous cerebrospinal fluid leaks in the nose and ear.</a> The Journal of Laryngology and Otology. 126 (11), 1186-1188 (2012).</li><li>Raza, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitivity+and+specificity+of+intrathecal+fluorescein+and+white+light+excitation+for+detecting+intraoperative+cerebrospinal+fluid+leak+in+endoscopic+skull+base+surgery:+a+prospective+study.">Sensitivity and specificity of intrathecal fluorescein and white light excitation for detecting intraoperative cerebrospinal fluid leak in endoscopic skull base surgery: a prospective study.</a> Journal of Neurosurgery. 124 (3), 621-626 (2016).</li><li>Englhard, A. S., Volgger, V., Leunig, A., Messmer, C. S., Ledderose, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+nasal+cerebrospinal+fluid+leaks:+management+of+24+patients+over+11+years.">Spontaneous nasal cerebrospinal fluid leaks: management of 24 patients over 11 years.</a> European Archives of Oto-rhino-laryngology. 275 (10), 2487-2494 (2018).</li><li>Sieskiewicz, A., Lyson, T., Rogowski, M., Mariak, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoscopic+management+of+cerebrospinal+fluid+rhinorhea.">Endoscopic management of cerebrospinal fluid rhinorhea.</a> The Polish Otolaryngology. 63 (4), 343-347 (2009).</li><li>Liu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+topical+intranasal+fluorescein+in+endoscopic+endonasal+repair+of+cerebrospinal+fluid+rhinorrhea.">The use of topical intranasal fluorescein in endoscopic endonasal repair of cerebrospinal fluid rhinorrhea.</a> Surgical Neurology. 72 (4), 341-345 (2009).</li><li>Javadi, S. A. H., Samimi, H., Naderi, F., Shirani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+low-+dose+intrathecal+fluorescein+in+endoscopic+repair+of+cerebrospinal+fluid+rhinorrhea.">The use of low- dose intrathecal fluorescein in endoscopic repair of cerebrospinal fluid rhinorrhea.</a> Archives of Iranian Medicine. 16 (5), 264-266 (2013).</li><li>Cheng, E., Grande, D., Leonetti, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Management+of+spontaneous+temporal+bone+cerebrospinal+fluid+leak:+A+30-year+experience.">Management of spontaneous temporal bone cerebrospinal fluid leak: A 30-year experience.</a> American Journal of Otolaryngology. 40 (1), 97-100 (2019).</li><li>Tandon, S., Singh, S., Sharma, S., Lahiri, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Intrathecal+Fluorescein+in+Recurrent+Meningitis+after+Cochlear+Implantation.">Use of Intrathecal Fluorescein in Recurrent Meningitis after Cochlear Implantation.</a> Iranian Journal of Otorhinolaryngology. 28 (86), 221-226 (2016).</li><li>Warnecke, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+relevance+of+beta2-transferrin+for+the+detection+of+cerebrospinal+fluid+fistulas.">Diagnostic relevance of beta2-transferrin for the detection of cerebrospinal fluid fistulas.</a> Archives of Otolaryngology - Head &amp; Neck Surgery. 130 (10), 1178-1184 (2004).</li><li>Keerl, R., Weber, R. K., Draf, W., Wienke, A., Schaefer, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+sodium+fluorescein+solution+for+detection+of+cerebrospinal+fluid+fistulas:+an+analysis+of+420+administrations+and+reported+complications+in+Europe+and+the+United+States.">Use of sodium fluorescein solution for detection of cerebrospinal fluid fistulas: an analysis of 420 administrations and reported complications in Europe and the United States.</a> The Laryngoscope. 114 (2), 266-272 (2004).</li><li>Keerl, R., Weber, R. K., Draf, W., Radziwill, R., Wienke, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complications+of+lumbar+administration+of+5%+sodium+fluorescein+solution+for+detection+of+cerebrospinal+fluid+fistula.">Complications of lumbar administration of 5% sodium fluorescein solution for detection of cerebrospinal fluid fistula.</a> Laryngo-rhino-otologie. 82 (12), 833-838 (2003).</li><li>Harley, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoscopic+transnasal+repair+of+two+cases+of+spontaneous+cerebrospinal+fluid+fistula+in+the+foramen+rotundum.">Endoscopic transnasal repair of two cases of spontaneous cerebrospinal fluid fistula in the foramen rotundum.</a> Journal of Clinical Neuroscience: Official Journal of the Neurosurgical Society of Australia. 59, 350-352 (2019).</li><li>Wolf, G., Greistorfer, K., Stammberger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoscopic+detection+of+cerebrospinal+fluid+fistulas+with+a+fluorescence+technique.+Report+of+experiences+with+over+925+cases.">Endoscopic detection of cerebrospinal fluid fistulas with a fluorescence technique. Report of experiences with over 925 cases.</a> Laryngo-rhino-otologie. 76 (10), 588-594 (1997).</li></ol>

The authors declare no financial disclosures.

While many reports concerning CSF leaks focus on transnasal endoscopic approaches in skull base surgery, a standardized method of visualizing CSF leaks of the temporal bone via microscopy5,6,7 is lacking. This protocol describes an adaption of existing literature reviews for use in microscopic ear surgery.
The application of fluorescein has been described as a safe method in skull base surgery and use...

CSF leaks can be caused by trauma, preexist congenitally, or appear spontaneously. Clinically, they appear via otoliquorrhea or rhinoliquorrhea and can be confirmed by positive &#946;-transferrin secretion1,2. In cases of CSF leaks, reliable detection of its origin is needed to seal the leak sufficiently and prevent complications, such as meningitis.
Intrathecal fluorescein application (IFA) has been known to be highly sensitive in detecting CSF leaks after neurosurgical skull base operations3. However, there is no common consensus on its exact applications (e.g., concentration, amount of fluid, addition of other drugs such as dexamethasone)3,4. A majority of cases describe transnasal endoscopic skull base surgery, while a standardized method of visualizing CSF leaks in the temporal bone via microscopy5,6,7 is missing. These leaks frequently occur at multiple localizations and bear the risk of recurrence if their whole extent is not visualized during surgery8. Specifically, a transmastoid approach only has been found to bear the risk of recurrent leaks8.
This protocol presents the use of intrathecal administered fluorescein via a transmastoid approach. A clinical case of bilateral congenital ear malformation (right ear: cochlear aplasia, left ear: incomplete partition type I), in which minor trauma led to perforation of the left stapes footplate in the middle ear, is also presented. A similar case has only been reported once9.

<table><tbody><tr><td>Fluorescein ALCON 10% injection solution</td><td>NOVARTIS</td><td>1467007</td><td></td></tr><tr><td>External lumbar drain catheter Silverline</td><td>Spiegelberg</td><td>ELD33.010.02</td><td></td></tr><tr><td>Otologic instruments (round knifes, hooks, curette, sickle knife, microscissors and microforceps)</td><td>Spiggle und Theis</td><td></td><td>No further specification; instruments can vary between the clinics and their use depends on the surgeon&#039;s preferation</td></tr><tr><td>Surgical microscope ARRISCOPE 1.0</td><td>ARRI Medicals</td><td>NA</td><td></td></tr><tr><td>&lt;strong&gt;Consumables&lt;/strong&gt;</td><td></td><td></td><td>No further specification; material of the standard equipment of the operating clinic can be used</td></tr><tr><td>Water to rinse</td><td></td><td></td><td></td></tr><tr><td>Antifog solution</td><td></td><td></td><td></td></tr><tr><td>Cotton pads</td><td></td><td></td><td></td></tr><tr><td>Cottonoid pledges</td><td></td><td></td><td></td></tr><tr><td>Gown</td><td></td><td></td><td></td></tr><tr><td>Gloves</td><td></td><td></td><td></td></tr><tr><td>Mask</td><td></td><td></td><td></td></tr><tr><td>Suction tubes</td><td></td><td></td><td></td></tr><tr><td>Blade</td><td></td><td></td><td></td></tr></tbody></table>

intrathecal application of a fluorescent dye for the identification of cerebrospinal fluid leaks in cochlear malformation

In cases of cerebrospinal fluid (CSF) leaks, reliable detection of their origins is needed to seal the leak sufficiently and prevent complications, such as meningitis. A method is presented here using intrathecal administered fluorescein in a clinical case of bilateral congenital ear malformation. A fluorescent dye is administered intrathecally to achieve intraoperative visualization of CSF leaks. The dye is applied 20 min before surgery, and concentration of 5% is used. Per every 10 kg of body weight, 0.1 mL of the fluid is applied intrathecally. The fluorescein is visualized using a fully digital microscope. The origin of the fluid leak is identified in the stapes footplate. During primary surgery, it is sealed, and cochlea implantation is performed for hearing restoration. In this specific case, 6 weeks later, the implant was explanted due to acute meningitis, and the electrode array was left as a spacer. Postoperatively, in the aural smear, &#946;-transferrin was detected. During a revision mastoidectomy, dislocated coverage of the leak was found. The stapes was removed and oval window sealed. Five days after revision surgery, no &#946;-transferrin was detected in the aural smear. During the revision of cochlea implantation 6 months later, intact coverage of the oval niche was observed. Thus, intrathecal fluorescein application proves to be a reliable tool for the detection of CSF leaks. It facilitates the orientation in malformations and complicated or unknown surgical situs. In the literature, its use is described for CSF fistulas in endonasal surgery but is rarely described in skull base and mastoid surgeries. The method has been used successfully in several cases with CSF leaks, and the results confirm the feasibility of safely accessing the origin of the leak.

In the presented case, minor trauma led to a CSF leak from the nose in a 10 month-old child. Magnetic resonance imaging (MRI) revealed a bilateral temporal bone malformation with aplasia of the right cochlea and dilation of the left cochlea and vestibule with absent interscalar septum identical to an incomplete partition type one. Accordingly, brainstem-evoked audiometry was performed (Figure 1) and confirmed bilateral deafness.
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Intrathecal Application of a Fluorescent Dye for the Identification of Cerebrospinal Fluid Leaks in Cochlear Malformation

Intrathecally applied fluorescein is used to achieve intraoperative visualization of CSF leaks. This protocol describes a lumbar puncture, the application of 5% fluorescein, and intraoperative visualization using a fully digital microscope.

In cases of cerebrospinal fluid (CSF) leaks, reliable detection of their origins is needed to seal the leak sufficiently and prevent complications, such ...

intrathecal-application-fluorescent-dye-for-identification

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

8.0K Views.  University Medical Center Rostock. Intrathecally applied fluorescein is used to achieve intraoperative visualization of CSF leaks. This protocol describes a lumbar puncture, the application of 5% fluorescein, and intraoperative visualization using a fully digital microscope.

Video: Intrathecal Application of a Fluorescent Dye for the Identification of Cerebrospinal Fluid Leaks in Cochlear Malformation

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Neuroscience

Retrograde Loading of Nerves, Tracts, and Spinal Roots with Fluorescent Dyes

Retrograde labeling of neurons is a standard anatomical method1,2 that has also been used to load calcium and voltage-sensitive dyes into neurons3-6. Generally, the dyes are applied as solid crystals or by local pressure injection using glass pipettes. However, this can result in dilution of the dye and reduced labeling intensity, particularly when several hours are required for dye diffusion. Here we demonstrate a simple and low-cost technique for introducing fluorescent and ion-sensitive dyes into neurons using a polyethylene suction pipette filled with the dye solution. This method offers a reliable way for maintaining a high concentration of the dye in contact with axons throughout the loading procedure.

We describe a simple and low cost technique for introducing high concentration of fluorescent and calcium-sensitive dyes into neurons or any neuronal tract using a polyethylene suction pipette.

Retrograde labeling of neurons is a standard anatomical method1,2 that has also been used to load calcium and voltage-sensitive dyes into neurons3-6. ...

An In Vivo Duo-color Method for Imaging Vascular Dynamics Following Contusive Spinal Cord Injury

Spinal cord injury (SCI) causes significant vascular disruption at the site of injury. Vascular pathology occurs immediately after SCI and continues throughout the acute injury phase. In fact, endothelial cells appear to be the first to die after a contusive SCI. The early vascular events, including increased permeability of the blood-spinal cord barrier (BSCB), induce vasogenic edema and contribute to detrimental secondary injury events caused by complex injury mechanisms. Targeting the vascular disruption, therefore, could be a key strategy to reduce secondary injury cascades that contribute to histological and functional impairments after SCI. Previous studies were mostly performed on postmortem samples and were unable to capture the dynamic changes of the vascular network. In this study, we have developed an in vivo duo-color two-photon imaging method to monitor acute vascular dynamic changes following contusive SCI. This approach allows detecting blood flow, vessel diameter, and other vascular pathologies at various sites of the same rat pre- and post-injury. Overall, this method provides an excellent venue for investigating vascular dynamics.

An In Vivo Duo-color Method for Imaging Vascular Dynamics Following Contusive Spinal Cord Injury

We introduce an in vivo imaging method using two different fluorescent dyes to track dynamic spinal vascular changes following a contusive spinal cord injury in adult Sprague-Dawley rats.

Spinal cord injury (SCI) causes significant vascular disruption at the site of injury. Vascular pathology occurs immediately after SCI and continues ...

Cannula Implantation into the Cisterna Magna of Rodents

Cisterna magna cannulation (CMc) is a straightforward procedure that enables direct access to the cerebrospinal fluid (CSF) without operative damage to the skull or the brain parenchyma. In anesthetized rodents, the exposure of the dura mater by blunt dissection of the neck muscles allows the insertion of a cannula into the cisterna magna (CM). The cannula, composed either by a fine beveled needle or borosilicate capillary, is attached via a polyethylene (PE) tube to a syringe. Using a syringe pump, molecules can then be injected at controlled rates directly into the CM, which is continuous with the subarachnoid space. From the subarachnoid space, we can trace CSF fluxes by convective flow into the perivascular space around penetrating arterioles, where solute exchange with the interstitial fluid (ISF) occurs. CMc can be performed for acute injections immediately following the surgery, or for chronic implantation, with later injection in anesthetized or awake, freely moving rodents. Quantitation of tracer distribution in the brain parenchyma can be performed by epifluorescence, 2-photon microscopy, and magnetic resonance imaging (MRI), depending on the physico-chemical properties of the injected molecules. Thus, CMc in conjunction with various imaging techniques offers a powerful tool for assessment of the glymphatic system and CSF dynamics and function. Furthermore, CMc can be utilized as a conduit for fast, brain-wide delivery of signaling molecules and metabolic substrates that could not otherwise cross the blood brain barrier (BBB).

Here we describe a protocol to perform cisterna magna cannulation (CMc), a minimally invasive way to deliver tracers, substrates and signaling molecules into the cerebrospinal fluid (CSF). Combined with different imaging modalities, CMc enables glymphatic system and CSF dynamics assessment, as well as brain-wide delivery of various compounds.

Cisterna magna cannulation (CMc) is a straightforward procedure that enables direct access to the cerebrospinal fluid (CSF) without operative damage to ...

Three-Dimensional Imaging of the Vertebral Lymphatic Vasculature and Drainage using iDISCO+ and Light Sheet Fluorescence Microscopy

The lymphatic system associated with the central nervous system (CNS) includes the lymphatic vasculature that spins around the brain, the spinal cord, and its associated LNs. The CNS-associated lymphatic system is involved in the drainage of CSF macromolecules and meningeal immune cells toward CNS-draining LNs, thereby regulating waste clearance and immune surveillance within CNS tissues. Presented is a novel approach to obtain three-dimensional (3D) and cellular resolution images of CNS-associated lymphatics while preserving the integrity of their circuits within surrounding tissues. The iDISCO+ protocol is used to immunolabel lymphatic vessels in decalcified and cleared whole mount preparations of the vertebral column that are subsequently imaged with light sheet fluorescence microscopy (LSFM). The technique reveals the 3D structure of the lymphatic network connecting the meningeal and epidural spaces around the spinal cord to extravertebral lymphatic vessels. Provided are 3D images of the drainage circuits of molecular tracers previously injected into either the CSF via the cisterna magna or the thoracolumbar spinal parenchyma. The iDISCO+/LSFM approach brings unprecedented opportunities to explore the structure and function of the CNS-associated lymphatic system in neurovascular biology, neuroimmunology, brain and vertebral cancer, or vertebral bone and joint biology.

Three-Dimensional Imaging of the Vertebral Lymphatic Vasculature and Drainage using iDISCO+ and Light Sheet Fluorescence Microscopy

A protocol is presented combining tissue clearing with light sheet fluorescence microscopy (LSFM) to obtain three-dimensional and cellular resolution images of the lymphatic vessels and lymph nodes (LNs) collecting the cerebrospinal fluid (CSF) and spinal epidural fluid.

The lymphatic system associated with the central nervous system (CNS) includes the lymphatic vasculature that spins around the brain, the spinal cord, and ...

Immunology and Infection

In Vivo Imaging of Cerebrospinal Fluid Transport through the Intact Mouse Skull using Fluorescence Macroscopy

Cerebrospinal fluid (CSF) flow in rodents has largely been studied using ex vivo quantification of tracers. Techniques such as two-photon microscopy and magnetic resonance imaging (MRI) have enabled in vivo quantification of CSF flow but they are limited by reduced imaging volumes and low spatial resolution, respectively. Recent work has found that CSF enters the brain parenchyma through a network of perivascular spaces surrounding the pial and penetrating arteries of the rodent cortex. This perivascular entry of CSF is a primary driver of the glymphatic system, a pathway implicated in the clearance of toxic metabolic solutes (e.g., amyloid-&#946;). Here, we illustrate a new macroscopic imaging technique that allows real-time, mesoscopic imaging of fluorescent CSF tracers through the intact skull of live mice. This minimally-invasive method facilitates a multitude of experimental designs and enables single or repeated testing of CSF dynamics. Macroscopes have high spatial and temporal resolution and their large gantry and working distance allow for imaging while performing tasks on behavioral devices. This imaging approach has been validated using two-photon imaging and fluorescence measurements obtained from this technique strongly correlate with ex vivo fluorescence and quantification of radio-labeled tracers. In this protocol, we describe how transcranial macroscopic imaging can be used to evaluate glymphatic transport in live mice, offering an accessible alternative to more costly imaging modalities.

Transcranial optical imaging allows wide-field imaging of cerebrospinal fluid transport in the cortex of live mice through an intact skull.

Cerebrospinal fluid (CSF) flow in rodents has largely been studied using ex vivo quantification of tracers. Techniques such as two-photon microscopy and ...

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.

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 ...

Measurement of Strial Blood Flow in Mouse Cochlea Utilizing an Open Vessel-Window and Intravital Fluorescence Microscopy

Transduction of sound is metabolically demanding, and the normal function of the microvasculature in the lateral wall is critical for maintaining endocochlear potential, ion transport, and fluid balance. Different forms of hearing disorders are reported to involve abnormal microcirculation in the cochlea. Investigation of how cochlear blood flow (CoBF) pathology affects hearing function is challenging due to the lack of feasible interrogation methods and the difficulty in accessing the inner ear. An open vessel-window in the lateral cochlear wall, combined with fluorescence intravital microscopy, has been used for studying CoBF changes in vivo, but mostly in guinea pig and only recently in the mouse. This paper and the associated video describe the open vessel-window method for visualizing blood flow in the mouse cochlea. Details include 1) preparation of the fluorescent-labeled blood cell suspension from mice; 2) construction of an open vessel-window for intravital microscopy in an anesthetized mouse, and 3) measurement of blood flow velocity and volume using an offline recording of the imaging. The method is presented in video format to show how to use the open window approach in mouse to investigate structural and functional changes in the cochlear microcirculation under normal and pathological conditions.

An open vessel-window approach using fluorescent tracers provides sufficient resolution for cochlear blood flow (CoBF) measurement. The method facilitates the study of structural and functional changes in CoBF in mouse under normal and pathological conditions.

Transduction of sound is metabolically demanding, and the normal function of the microvasculature in the lateral wall is critical for maintaining ...

Lumbar Puncture Delivery of AAV9 in Juvenile Rats for CNS Gene Therapy

Intrathecal Vector Delivery in Juvenile Rats via Lumbar Cistern Injection

Gene therapy is a powerful technology to deliver new genes to a patient for the treatment of disease, be it to introduce a functional gene, inactivate a toxic gene, or provide a gene whose product can modulate the biology of the disease. The delivery method for the therapeutic vector can take many forms, ranging from intravenous infusion for systemic delivery to direct injection into the target tissue. For neurodegenerative disorders, it is often desirable to skew transduction towards the brain and/or spinal cord. The least invasive approach to target the entire central nervous system involves injection into the cerebrospinal fluid (CSF), allowing the therapeutic to reach a large fraction of the central nervous system. The safest approach to deliver a vector into the CSF is the lumbar intrathecal injection, where a needle is introduced into the lumbar cistern of the spinal cord. This technique, also known as a lumbar puncture, has been widely used in neonatal and adult rodents and in large animal models. While the technique is similar across species and developmental stages, subtle differences in size, structure, and elasticity of tissues surrounding the intrathecal space require accommodations in the approach. This article describes a method for performing lumbar puncture in juvenile rats to deliver an adeno-associated serotype 9 vector. Here, 25-35 &#181;L of vector were injected into the lumbar cistern, and a green fluorescent protein (GFP) reporter was used to evaluate the transduction profile resulting from each injection. The benefits and challenges of this approach are discussed.

Author Spotlight: Assessing Intrathecal Gene Therapy Efficacy in Juvenile Rats

A surgical procedure is described to perform injections into the lumbar cistern of the juvenile rat. This approach has been used for the intrathecal delivery of gene therapy vectors, but it is anticipated that this approach can be used for a variety of therapeutics, including cells and drugs.

Gene therapy is a powerful technology to deliver new genes to a patient for the treatment of disease, be it to introduce a functional gene, inactivate a ...

Tracer Molecule Delivery in the Cisterna Magna: A Method to Deliver Fluorescent Tracer Molecules via Direct Cannulation of the Cisterna Magna in Pig

Source: B&#232;chet, N. B., et al. <a href="https://jove.unicartagenaproxy.elogim.com/v/62641">Direct Cannula Implantation in the Cisterna Magna of Pigs.</a> J. Vis. Exp. (2021).
This video describes the protocol for tracer molecule delivery via direct cannulation of the cisterna magna in a pig brain. This method helps visualize the distribution of cerebrospinal fluid in the glymphatic system.

Source: B&#232;chet, N. B., et al. Direct Cannula Implantation in the Cisterna Magna of Pigs. J. Vis. Exp. (2021).
This video describes the protocol for ...

Intrathecal Application of a Fluorescent Dye for the Identification of Cerebrospinal Fluid Leaks in Cochlear Malformation

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Cannula Implantation into the Cisterna Magna of Rodents

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Intrathecal Vector Delivery in Juvenile Rats via Lumbar Cistern Injection

Tracer Molecule Delivery in the Cisterna Magna: A Method to Deliver Fluorescent Tracer Molecules via Direct Cannulation of the Cisterna Magna in Pig