This work was supported by R01 NS079623, P01 HL149630, and P01 HL095491.

The author declares that the protocol follows the guidelines of the Institutional Animal Care and Use Committee at Beth Israel Deaconess Medical Center.
1. Preparation of surgical instruments and stereotaxic frame
NOTE: The surgery is performed under aseptic conditions. Sterility is maintained using the sterile tip technique.
<ol>
	<li>Install the stereotaxic arm with a micropipette or syringe filled with an injectable of choice (adeno-associated virus (AAV) or conventional tracer) on the stereotaxic frame and prepare the mouse adapter (Figure 2A).</li>
	<li>Prepare autoclaved surgical instruments (Table of materials) and place them on a sterile surface.</li>
</ol>
2. Anesthesia induction and mouse preparation
<ol>
	<li>Turn on O2 at 0.5 L/min and set the isoflurane vaporizer at 4.0, making sure that O2 flow is to the induction box. 
	CAUTION: Make sure that the isoflurane induction box is placed in a hood and that the isoflurane is scavenged away from the surgical site.</li>
	<li>Place the mouse (10-week-old male C57BL/6J) in the induction chamber.</li>
	<li>Once breathing has slowed, open the induction chamber, and lift the mouse slightly. Use clippers to remove hair from head to shoulders.</li>
</ol>
3. Positioning of the mouse in the stereotaxic frame
<ol>
	<li>Move the mouse to the stereotaxic frame and place the nose in a flexible nose cone. At this stage, make sure the O2 flow is now directed to the nose cone.</li>
	<li>Place the mouse in the stereotaxic frame using ear bars only. 
	NOTE: Make sure the ear bars are even and the head is level.</li>
	<li>Anteroflex the head of the mouse to a 90&#176; angle by manually guiding the nose. To secure this position, place a plastic barrier between the ear bar pillars of the mouse adapter, parallel to the pillars. The flat part of the skull serves as the reference, similar to the flat skull approach in conventional stereotaxic surgery. 
	NOTE: Do not over flex the head (i.e. beyond an angle of 90&#176; between the plane of the frontal skull bone and the plane of the surface of the table) as this impedes airflow through the upper airway. If airflow is impeded, reposition the mouse, making sure the body is supported underneath the trunk and a plastic card is set at 90&#176; between the plane of the frontal skull bone and the plane of the surface of the table as outlined in Figure 2A,C.</li>
	<li>Place the heating pad underneath the mouse, and then make sure that the neck and the rest of the body are positioned at the same level (i.e., at approximately 180&#176; or parallel to the table). The toolbox that holds the spring scissors can be used to lift the body to this position. 
	NOTE: This step is important as the caudal brainstem and upper cervical cord move depending on position, in contrast to more rostral parts of the CNS which are held in place by the skull.</li>
	<li>Inject a single dose of 4 mg/kg Meloxicam slow-release (SR) subcutaneously (s.c.) at a volume of 2 &#181;L/g body weight and place lubricant on the eyes.</li>
	<li>Clean the surgical incision site first with a 70% alcohol prep pad, then with a betadine prep pad, and then again with an alcohol prep pad and let dry.</li>
	<li>Place a drape underneath the body.</li>
	<li>Disinfect hands and put on sterile gloves.</li>
	<li>Place a drape at the surgical site.</li>
</ol>
4. Surgery to access the cisterna magna
<ol>
	<li>Make sure the mouse is appropriately anesthetized by pinching the toes or checking the corneal reflex.</li>
	<li>Reduce isoflurane to maintenance levels (2.0).</li>
	<li>Make a 1-1.2 cm incision with surgical blade #10 from the edge of the occipital bone toward the shoulders in one smooth movement.</li>
	<li>Make an incision in the midline raphe of the trapezius muscle. This exposes the paired longus capitis muscles. 
	NOTE: In mice, the trapezius muscle is a very thin, almost transparent muscle. Make sure to stay in the midline and do not cut into the underlying muscles as this will cause unnecessary bleeding.</li>
	<li>Place both retractor hooks in between the paired longus capitis muscles, one oriented to the left and the other to the right. The weight of the hemostats provides tension to the retractor hooks which can be modified by re-adjusting the position of the hemostats.</li>
	<li>Position the surgical microscope in place to better visualize the surgical field.</li>
	<li>Use the blunt laminectomy forceps to separate the left and right bellies of the paired longus capitis muscle, starting from the occiput, where the midline is readily visible. Guide the blunt forceps across the bone of the occiput in the midline down to where it meets the cisternal dura mater, and then continue across the dura mater to the atlas. 
	NOTE: There is no need to cut through the paired longus capitis muscles as nothing holds them together in the midline; doing so will cause unnecessary bleeding.</li>
	<li>Reposition the retractors and adjust the tension by repositioning the hemostats, opening the view of the cisterna magna.</li>
	<li>Use the blunt laminectomy forceps to separate the muscles further in the midline to get a good viewing window of the brainstem and cerebellum.</li>
	<li>Repeat steps 4.7-4.9 as needed until the cerebellum and brainstem come in view below the dura.</li>
	<li>Using blunt laminectomy forceps, clear the dura of the small strands of connective tissue by moving the forceps from the midline in a lateral direction, until there is a clear view of the brainstem and to create more lateral space, as needed for the target.</li>
</ol>
5. Opening of the cisternal membrane
<ol>
	<li>Use the angled Dumont forceps (#4/45) to grab the dura, which extends from the occipital bone to the atlas. Grab the dura near the occipital bone and use the spring scissors to make a small opening (~0.5 to 1.5 mm) in the dura. 
	NOTE: At this rostral location, the space between the brainstem and the overlying dura is widest, providing ample room for safe manipulation of the dura.</li>
	<li>Use the spring scissors to lift the dura and open the dura further. The size of the window depends on the target. 
	NOTE: A larger window will be needed when making multiple longitudinal injections or bilateral injections; a small window will be sufficient when making single unilateral or midline injections.</li>
	<li>Once the dura is opened, drain excess cerebrospinal fluid with a sterile cue tip.</li>
</ol>
6. Identification of landmarks and zero point
<ol>
	<li>View the dorsal surface of the brainstem with detailed landmarks through the open dura. The obex, the point where the central canal opens into the IV ventricle, is the standard anterior-posterior and mediolateral zero point.</li>
</ol>
7. Target coordinates
NOTE: For various targets, we have included a list of standard coordinates with anterior posterior (AP) and mediolateral (ML) coordinates relative to zero-point bregma and cisterna magna coordinates with AP and ML coordinates relative to zero point obex to facilitate the transition between methodologies (Table 1). Dorsoventral (DV) coordinates are relative to the surface of the brain or cerebellum (standard approach) or the surface of the brainstem or upper cervical cord (cisterna magna approach) at the point of AP and ML entry. Planning should be done prior to surgery.
<ol>
	<li>Use the three sets of coordinates to determine the target: AP, ML, and DV. Due to the head position, the relative orientation of brainstem structures varies by location.
	<ol>
		<li>For target distance &#62;0.4 mm from caudal to the obex (Figure 1B, green) perform the following.
		<ol>
			<li>AP: Use any standard stereotaxic reference atlas (e.g., Paxinos and Franklin atlas2) or tissue series cut in the transverse plane to estimate the AP distance between obex and the target.</li>
			<li>ML: Use any standard stereotaxic reference atlas or tissue series cut in the transverse plane to estimate the ML distance between obex and the target.</li>
			<li>DV: Estimate coordinates relative to the surface of the brain or cerebellum at the AP and ML target point. Use any standard stereotaxic reference atlas or tissue series cut in the transverse plane to estimate the distance between the brainstem surface at the desired AP and ML coordinates and the target.</li>
		</ol>
		</li>
		<li>For target distance &#60;0.4 mm from caudal to the obex (Figure 1B, orange) perform the following.
		<ol>
			<li>AP: Adjust coordinates to account for anteroflexion of the brainstem. For ventral and rostral coordinates, the AP brainstem entry point will be more caudal relative to the target AP coordinate in the standard plane.</li>
			<li>ML: Derive target coordinates from a standard stereotaxic reference atlas or tissue series cut in the transverse plane. Coordinates will be relative to the visualized midline at the target AP level.</li>
			<li>DV: Estimate coordinates relative to the surface of the brainstem at the AP and ML target point. Adjust DV to account for anteroflexion of the brainstem. For ventral and rostral coordinates, the DV coordinates will be larger than the distance from the dorsal surface of the brainstem in the standard plane.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
8. Injection of the target
<ol>
	<li>Lower the pipette or syringe to the target using the stereotaxic arm and inject solution as for standard stereotaxic approaches. Leave in place for 1-5 min after injection, to avoid a needle track when using volumes between 3-50 nL. Then, lift the pipette or syringe using the stereotaxic arm.</li>
	<li>Repeat step 8.1. for multiple targets.</li>
</ol>
9. Closure of the surgical field
<ol>
	<li>Remove the hooks carefully from the surgical field. The paired longus capitis muscles will fall back into a neutral position, fully covering the cisterna magna. Do not close the trapezius muscle and dura mater in the midline as they are too fragile to hold sutures.</li>
	<li>Close the skin with three nylon or polypropylene sutures (5-0 or 6-0).</li>
</ol>
10. Post-operative care
<ol>
	<li>Turn off isoflurane and remove the mouse from the stereotaxic frame. Place the mouse in a clean cage on a heating pad and observe until awake and moving.</li>
	<li>Monitor health status, weight, and sutures on post-operative days 1-3. Remove sutures on day 10 if not already removed.</li>
</ol>

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The authors have nothing to disclose.

Standard stereotaxic surgery commonly relies on skull landmarks to calculate the coordinates of target sites in the CNS1. Target sites are then accessed via burr holes that are drilled through the skull1. This method is not ideal for the caudal brainstem as target sites are located distant from the skull landmarks in the anteroposterior and dorsoventral planes2 and as the anatomy of the skull and overlying muscles make access challenging<sup...

Standard stereotaxic surgery to target brain sites in mice1 commonly involves fixation of the skull using a set of ear bars and a mouth bar. Coordinates are then estimated based on reference atlases2,3, and skull landmarks, namely, bregma (the point where the sutures of the frontal and parietal bones come together) or lambda (the point where the sutures of the parietal and occipital bones come together; Figure 1A,B). Through a burr hole into the skull above the estimated target, the target region can then be reached, either for delivery of microinjections or instrumentation with cannulas or optic fibers. Due to variation in the anatomy of these sutures and errors in the localization of bregma or lambda4,5, the position of zero points in relation to the brain varies from animal to animal. While small errors in targeting, that result from this variability, are not a problem for large or nearby targets, their impact is greater for smaller areas of interest that are remote from the zero points in the anteroposterior or dorsoventral planes and/or when studying animals of varying size due to age, strain and/or sex. There are several additional challenges that are unique for the medulla oblongata and the upper cervical cord. First, small changes in anteroposterior coordinates are associated with significant changes in dorsoventral coordinates relative to the dura, due to the position and shape of the cerebellum (Figure 1Bi)2,6,7. Second, the upper cervical cord is not contained within the skull2. Third, the slanting position of the occipital bone and overlying layer of neck muscles2 makes the standard stereotaxic approach even more challenging for structures located near the transition between the brainstem and spinal cord (Figure 1Bi). Finally, many targets of interest in the caudal brainstem and cervical cord are small2, requiring precise and reproducible injections8,9.
An alternative approach through the cisterna magna circumvents these problems. The cisterna magna is a large space that extends from the occipital bone to the atlas (Figure 1A, i.e., the second vertebral bone)10. It is filled with cerebrospinal fluid and covered by dura mater10. This space between the occipital bone and the atlas opens when anteroflexing the head. It can be accessed by navigating in between the overlying paired bellies of the longus capitis muscle, exposing the dorsal surface of the caudal brainstem. Regions of interest can then be targeted based upon the landmarks of these regions themselves if they are located near the dorsal surface; or by using the obex, the point where the central canal opens into the IV ventricle, as a zero point for coordinates to reach deeper structures. This approach has been successfully used in a variety of species, including the rat11, cat12, mouse8,9, and non-human primate13 to target the ventral respiratory group, medullary medial reticular formation, the nucleus of the solitary tract, area postrema, or hypoglossal nucleus. However, this approach is not widely utilized as it requires knowledge of anatomy, a specialized toolkit, and more advanced surgical skills compared to the standard stereotaxic approach.
Here we describe a step-by-step surgical approach to reach the brainstem and upper cervical cord via the cisterna magna, visualize landmarks, set the zero point (Figure 2), and estimate and optimize target coordinates for stereotaxic delivery of microinjections into the discrete brainstem and spinal cord regions of interest (Figure 3). We then discuss the advantages and disadvantages related to this approach.

<table><tbody><tr><td>Alcohol pad</td><td>Med-Vet International</td><td>SKU: MDS090735Z</td><td>skin preparation for the prevention of surgical site infection</td></tr><tr><td>Angled forceps, Dumont #5/45</td><td>FST</td><td>11251-35</td><td>only to grab dura</td></tr><tr><td>Betadine pad</td><td>Med-Vet International</td><td>SKU:PVP-PAD</td><td>skin preparation for the prevention of surgical site infection</td></tr><tr><td>Cholera toxin subunit-b, Alexa Fluor 488/594 conjugate</td><td>Thermo Fisher Scientific</td><td>488: C34775, 594: C22842</td><td>Fluorescent tracer</td></tr><tr><td>Clippers</td><td>Wahl</td><td>Model MC3, 28915-10</td><td>for shaving fur at surgical site</td></tr><tr><td>Electrode holder with corner clamp</td><td>Kopf</td><td>1770</td><td>to hold glass pipette</td></tr><tr><td>Flowmeter</td><td>Gilmont instruments</td><td>model # 65 MM</td><td>to regulate flow of isoflurane and oxygen to mouse on the surgical plane</td></tr><tr><td>Fluorescent microspheres, polystyrene</td><td>Thermo Fisher Scientific</td><td>F13080</td><td>Fluorescent tracer</td></tr><tr><td>Heating pad</td><td>Stoelting</td><td>53800M</td><td>thermoregulation</td></tr><tr><td>Induction chamber with port hook up kit</td><td>Midmark Inc</td><td>93805107 92800131</td><td>chamber providing initial anasthesia</td></tr><tr><td>Insulin Syringe</td><td>Exelint International</td><td>26028</td><td>to administer saline and analgesic</td></tr><tr><td>Isoflurane</td><td>Med-Vet International</td><td>SKU:RXISO-250</td><td>inhalant anesthetic</td></tr><tr><td>Isoflurane Matrix VIP 3000 vaporizer</td><td>Midmark Inc</td><td>91305430</td><td>apparatus for inhalant anesthetic delivery</td></tr><tr><td>Laminectomy forceps, Dumont #2</td><td>FST</td><td>11223-20</td><td>only to clean dura</td></tr><tr><td>Medical air, compressed</td><td>Linde</td><td>UN 1002</td><td>used with stimulator &amp;amp; PicoPump for providing air for precision solution injection</td></tr><tr><td>Meloxicam SR</td><td>Zoo Pharm LLC</td><td>Lot # MSR2-211201</td><td>analgesic</td></tr><tr><td>Microhematocrit borosilicate glass pre calibrated capillary tube</td><td>Globe Scientific Inc</td><td>51628</td><td>for transfection of material to designated co-ordinates</td></tr><tr><td>Mouse adaptor</td><td>Stoelting</td><td>0051625</td><td>&amp;nbsp;adapting rat stereotaxic frame for mouse surgery</td></tr><tr><td>Needle holder, Student Halsted- Mosquito Hemostats</td><td>FST</td><td>91308-12</td><td>for suturing</td></tr><tr><td>Oxygen regulator</td><td>Life Support Products</td><td>S/N 909328, lot 092109</td><td>regulate oxygen levels from oxygen tank</td></tr><tr><td>Oxygen tank, compressed</td><td>Linde</td><td>USP UN 1072</td><td>provided along with isoflurane anasthesia</td></tr><tr><td>Plastic card</td><td>not applicable</td><td>not applicable</td><td>any firm plastic card, cut to fit the stereotactic frame (e.g. ID card)</td></tr><tr><td>Pneumatic PicoPump ( or similar)</td><td>World Precision Instruments (WPI)</td><td>SYS-PV820</td><td>For precision solution injection</td></tr><tr><td>Saline, sterile</td><td>Mountainside Medical Equipment</td><td>H04888-10</td><td>to replace body fluids lost during surgery</td></tr><tr><td>Scalpel handle, #3</td><td>FST</td><td>10003-12</td><td>to hold scalpel</td></tr><tr><td>Scissors, Wagner</td><td>FST</td><td>14070-12</td><td>to cut polypropylene suture</td></tr><tr><td>Spring scissors, Vannas 2.5mm with accompanying box</td><td>FST</td><td>15002-08</td><td>scissors only to open dura, box to elevate body</td></tr><tr><td>Stereotactic micromanipulator</td><td>Kopf</td><td>1760-61</td><td>attached to electrode holder to adjust position based on co-ordinates</td></tr><tr><td>Stereotactic &#039;U&#039; frame assembly and intracellular base plate</td><td>Kopf</td><td>1730-B, 1711</td><td>frame for surgery</td></tr><tr><td>Sterile cotton tipped applicators</td><td>Puritan</td><td>25-806 10WC</td><td>absorbing blood from surgical field</td></tr><tr><td>Sterile non-fenestrated drapes</td><td>Henry Schein</td><td>9004686</td><td>for sterile surgical field</td></tr><tr><td>Sterile opthalmic ointment</td><td>Puralube</td><td>P1490</td><td>ocular lubricant</td></tr><tr><td>Stimulator &amp;amp; Tubing</td><td>Grass Medical Instruments</td><td>S44</td><td>to provide controlled presurred air for precision solution injection</td></tr><tr><td>Surgical Blade #10</td><td>Med-Vet International</td><td>SKU: 10SS</td><td>for skin incision</td></tr><tr><td>Surgical forceps, Extra fine Graefe</td><td>FST</td><td>11153-10</td><td>to hold skin</td></tr><tr><td>Surgical gloves</td><td>Med-Vet International</td><td>MSG2280Z</td><td>for asceptic surgery</td></tr><tr><td>Surgical microscope</td><td>Leica</td><td>Model M320/ F12</td><td>for 5X-40X magnification of surgical site</td></tr><tr><td>Suture 5-0 polypropylene</td><td>Oasis</td><td>MV-8661</td><td>to close the skin</td></tr><tr><td>Tegaderm</td><td>3M</td><td>3M ID 70200749250</td><td>provides sterile barrier</td></tr><tr><td>Universal Clamp and stand post</td><td>Kopf</td><td>1725</td><td>attached to stereotactic U frame and intracellular base plate</td></tr><tr><td>Wound hook with hartman hemostats</td><td>FST</td><td>18200-09, 13003-10</td><td>to separate muscles and provide surgical window</td></tr></tbody></table>

stereotaxic surgical approach to microinject the caudal brainstem and upper cervical spinal cord via the cisterna magna in mice

Stereotaxic surgery to target brain sites in mice is commonly guided by skull landmarks. Access is then obtained via burr holes drilled through the skull. This standard approach can be challenging for targets in the caudal brainstem and upper cervical cord due to specific anatomical challenges as these sites are remote from skull landmarks, leading to imprecision. Here we outline an alternative stereotaxic approach via the cisterna magna that has been used to target discrete regions of interest in the caudal brainstem and upper cervical cord. The cisterna magna extends from the occipital bone to the atlas (i.e., the second vertebral bone), is filled with cerebrospinal fluid, and is covered by dura mater. This approach provides a reproducible route of access to select central nervous system (CNS) structures that are otherwise hard to reach due to anatomical barriers. Furthermore, it allows for direct visualization of brainstem landmarks in close proximity to the target sites, increasing accuracy when delivering small injection volumes to restricted regions of interest in the caudal brainstem and upper cervical cord. Finally, this approach provides an opportunity to avoid the cerebellum, which can be important for motor and sensorimotor studies.

The cisterna magna approach makes it possible to target caudal brainstem and upper cervical cord structures that are otherwise hard to reach via standard stereotaxic approaches or are prone to inconsistent targeting. The surgery to reach the cisterna magna requires incisions of the skin, a thin layer of trapezius muscle, and opening of the dura mater and is therefore well tolerated by mice. It is especially efficient and less invasive when targeting multiple (longitudinally dispersed or bilateral) sites, as it d...

Watch this Scientific Journal Video about Stereotaxic Surgical Approach to Microinject the Caudal Brainstem and Upper Cervical Spinal Cord via the Cisterna Magna in Mice at JoVE.com

Stereotaxic Surgical Approach to Microinject the Caudal Brainstem and Upper Cervical Spinal Cord via the Cisterna Magna in Mice

Stereotaxic surgery to target brain sites in mice commonly involves access through the skull bones and is guided by skull landmarks. Here we outline an alternative stereotaxic approach to target the caudal brainstem and upper cervical spinal cord via the cisterna magna that relies on direct visualization of brainstem landmarks.

Stereotaxic Surgical Approach to Microinject the Caudal Brainstem and Upper Cervical Spinal Cord via the Cisterna Magna in Mice

Stereotaxic surgery to target brain sites in mice is commonly guided by skull landmarks. Access is then obtained via burr holes drilled through the skull. ...

stereotaxic-surgical-approach-to-microinject-caudal-brainstem-upper

Research

JoVE Journal

Neuroscience

9.4K Views.  Harvard Medical School. Stereotaxic surgery to target brain sites in mice commonly involves access through the skull bones and is guided by skull landmarks. Here we outline an alternative stereotaxic approach to target the caudal brainstem and upper cervical spinal cord via the cisterna magna that relies on direct visualization of brainstem landmarks.

Video: Stereotaxic Surgical Approach to Microinject the Caudal Brainstem and Upper Cervical Spinal Cord via the Cisterna Magna in Mice

Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are restricted to two specific neurogenic niches: the subgranular zone of the dentate gyrus in the hippocampus and the ventricular-subventricular zone (V-SVZ; also called subependymal zone or SEZ) in the walls of the lateral ventricles. The development of in vivo gene transfer strategies for adult stem cell populations (i.e. those of the mammalian brain) resulting in long-term expression of desired transgenes in the stem cells and their derived progeny is a crucial tool in current biomedical and biotechnological research. Here, a direct in vivo method is presented for the stable genetic modification of adult mouse V-SVZ cells that takes advantage of the cell cycle-independent infection by LVs and the highly specialized cytoarchitecture of the V-SVZ niche. Specifically, the current protocol involves the injection of empty LVs (control) or LVs encoding specific transgene expression cassettes into either the V-SVZ itself, for the in vivo targeting of all types of cells in the niche, or into the lateral ventricle lumen, for the targeting of ependymal cells only. Expression cassettes are then integrated into the genome of the transduced cells and fluorescent proteins, also encoded by the LVs, allow the detection of the transduced cells for the analysis of cell autonomous and non-autonomous, niche-dependent effects in the labeled cells and their progeny.

Here we describe a procedure based on the use of lentiviral particles for the long-term genetic modification of neural stem cells and/or their adjacent ependymal cells in the adult ventricular-subventricular neurogenic niche which allows the separate analysis of cell autonomous and non-autonomous, niche-dependent effects on neural stem cells.

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are ...

Developmental Biology

Survivable Stereotaxic Surgery in Rodents 

The ability to measure extracellular basal levels of neurotransmitters in the brain of awake animals allows for the determination of effects of different systemic challenges (pharmacological or physiological) to the CNS. For example, one can directly measure how the animal's midbrain dopamine projections respond to dopamine-releasing drugs like d-amphetamine or natural stimuli like food. In this video, we show you how to implant guide cannulas targeting specific sites in the rat brain, how to insert and implant a microdialysis probe and how to use high performance liquid chromatography coupled with electrochemical detection (HPLC-EC) to measure extracellular levels of oxidizable neurotransmitters and metabolites. Local precise introduction of drugs through the microdialysis probe allows for refined work on site specificity in a compound s mechanism of action. This technique has excellent anatomical and chemical resolution but only modest time resolution as microdialysis samples are usually processed every 20-30 minutes to ensure detectable neurotransmitter levels. Complementary ex vivo tools (i.e., slice and cell culture electrophysiology) can assist with monitoring real-time neurotransmission.

Survivable Stereotaxic Surgery in Rodents

The monitoring of extracellular neurotransmitter levels in distinct brain regions of freely moving animals offers insights on the link between neurotransmitter release and behavior. In vivo microdialysis coupled with electrochemical detection provides excellent anatomical and chemical resolution; and information on how basal neurotransmission is altered by pharmacological or physiological manipulations.

The ability to measure extracellular basal levels of neurotransmitters in the brain of awake animals allows for the determination of effects of different ...

Biology

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

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central nervous system. We demonstrate a stereotaxic injection technique that allows targeted gene expression or silencing in the dorsal horn of the mouse spinal cord. The surgical procedure is brief. It requires laminectomy of a single vertebra, providing for quick recovery of the animal and unimpaired motility of the spine. Controlled injection of a small vector suspension volume at low speed and use of a microsyringe with beveled glass cannula minimize the tissue lesion. The local immune response to the vector depends on the intrinsic properties of the virus employed; in our experience, it is minor and short-lived when a recombinant adeno-associated virus is used. A reporter gene such as enhanced green fluorescent protein facilitates monitoring spatial distribution of the vector, and the efficacy and cellular specificity of the transfection.

Viral vectors allow for targeted gene manipulation. We demonstrate a method for conditional gene expression or ablation in the mouse spinal cord, using stereotaxic injection of a viral vector into the dorsal horn, a prominent site of synaptic contact between primary somatosensory afferents and neurons of the central nervous system.

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central ...

Developing Efficient and Precise Mouse Model for Spinal Cord Glioma

Surgical Transplantation of Tumor Cells into the Spinal Cord of Mice

Spinal cord gliomas are commonly malignant tumors of the spinal cord, leading to a high rate of disability. However, uniform treatment guidelines and comprehensive data on spinal cord gliomas remain limited due to the lack of suitable preclinical animal models. Developing a simple and reproducible animal model has become essential for advancing basic and translational research. A murine model is ideal, as the murine spinal cord shares structural similarities with the human spinal cord. This protocol describes the generation of a reproducible murine model of spinal cord glioma by directly injecting tumor cells into the intervertebral space using the spinous process of the seventh cervical vertebra as a guide. Compared to other methods, this approach is more effective and convenient, involving a smaller incision, reduced invasiveness and blood loss, faster recovery, and more stable tumor formation. This model is expected to advance the understanding of disease mechanisms, optimize surgical strategies, and support the development of therapeutic drugs for spinal cord gliomas.

The present protocol describes the development of a reproducible murine model of spinal cord glioma by injecting tumor cells into the intervertebral space, offering a more effective and less invasive approach for research and therapeutic development.

Spinal cord gliomas are commonly malignant tumors of the spinal cord, leading to a high rate of disability. However, uniform treatment guidelines and ...

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 ...

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 ...

Stereotaxic Viral Injection and Gradient-Index Lens Implantation for Deep Brain In Vivo Calcium Imaging

A miniature fluorescence microscope (miniscope) is a potent tool for in vivo calcium imaging from freely behaving animals. It offers several advantages over conventional multi-photon calcium imaging systems: (1) compact; (2) light-weighted; (3) affordable; and (4) allows recording from freely behaving animals. This protocol describes brain surgeries for deep brain in vivo calcium imaging using a custom-developed miniscope recording system. The preparation procedure consists of three steps, including (1) stereotaxically injecting the virus at the desired brain region of a mouse brain to label a specific subgroup of neurons with genetically encoded calcium sensor; (2) implantation of gradient-index (GRIN) lens that can relay calcium image from deep brain region to the miniscope system; and (3) affixing the miniscope holder over the mouse skull where miniscope can be attached later. To perform in vivo calcium imaging, the miniscope is fastened onto the holder, and neuronal calcium images are collected along with simultaneous behavior recordings. The present surgery protocol is compatible with any commercial or custom-built single-photon and two-photon imaging systems for deep brain in vivo calcium imaging.

Stereotaxic Viral Injection and Gradient-Index Lens Implantation for Deep Brain In Vivo Calcium Imaging

Miniscope in vivo calcium imaging is a powerful technique to study neuronal dynamics and microcircuits in freely behaving mice. This protocol describes performing brain surgeries to achieve good in vivo calcium imaging using a miniscope.

A miniature fluorescence microscope (miniscope) is a potent tool for in vivo calcium imaging from freely behaving animals. It offers several advantages ...

Dolichoectasia: Vascular Dementia and Alzheimer&#39;s Disease Models

A Visual Approach for Inducing Dolichoectasia in Mice to Model Large Vessel-Mediated Cerebrovascular Dysfunction

The blood-brain (BBB) is a crucial system that regulates selective brain circulation with the periphery, as an example, allowing necessary nutrients to enter and expel excessive amino acids or toxins from the brain. To model how the BBB can be compromised in diseases like vascular dementia (VaD) or Alzheimer's disease (AD), researchers developed novel methods to model vessel dilatation. A compromised BBB in these disease states can be detrimental and result in the dysregulation of the BBB leading to untoward and pathological consequences impacting brain function. We were able to modify an existing technique that enabled us to inject directly into the Cisterna magna (CM) to induce dilatation of blood vessels using elastase, and disrupt the tight junctions (TJ) of the BBB. With this method, we were able to see various metrics of success over previous techniques, including consistent blood vessel dilatation, reduced mortality or improved recovery, and improving the fill/opacifying agent, a silicone rubber compound, delivery for labeling blood vessels for dilatation analysis. This modified minimally invasive method has had promising results, with a 19%-32% increase in sustained dilatation of large blood vessels in mice from 2 weeks to 3 months post-injection. This improvement contrasts with previous studies, which showed increased dilatation only at the 2 week mark. Additional data suggests sustained expansion even after 9.5 months. This increase was confirmed by comparing the diameter of blood vessels of the elastase and the vehicle-injected group. Overall, this technique is valuable for studying pathological disorders that affect the central nervous system (CNS) using animal models.

Author Spotlight: Modeling Vascular Contributions to Alzheimer's Disease in Transgenic Mice

We demonstrate chemically inducing large blood vessel dilatation in mice as a model for investigating cerebrovascular dysfunction, which can be used for vascular dementia and Alzheimer's disease modeling. We also demonstrate visualizing the vasculature by injecting silicone rubber compound and providing clear visual guidance for measuring changes in blood vessel size.

The blood-brain (BBB) is a crucial system that regulates selective brain circulation with the periphery, as an example, allowing necessary nutrients to ...

Optimizing Stereotaxic Microinjection Accuracy in Mice

Preliminary Validation of Stereotaxic Injection Coordinates via Cryosectioning

Stereotaxic injection of a specific brain region constitutes a fundamental experimental technique in basic neuroscience. Researchers commonly base their choice of stereotaxic injection parameters on mouse brain atlases or published materials that employed various populations/ages of mice and different stereotaxic equipment, necessitating further validation of the stereotaxic coordinate parameters. The efficacy of calcium imaging, chemogenetic, and optogenetic manipulations relies on the precise expression of reporter genes within the region of interest, often requiring several weeks of effort. Thus, it is a time-consuming task if the coordinates of the target brain region are not verified in advance. Using an appropriate dye instead of a virus and implementing cryosectioning, researchers can observe the injection site immediately following dye administration. This facilitates timely adjustments to coordinate parameters in cases where discrepancies exist between the actual injection site and the theoretical position. Such adjustments significantly enhance the accuracy of viral expression within the target region in subsequent experiments.

Author Spotlight: Accelerating Cognitive Impairment Research in Mice Through Stereotaxic Injection and Cost-Effective Dyes

The present protocol describes a practical strategy to expedite the verification step of stereotaxic injection coordinates before conducting viral tracing using dyes and frozen sections.

Stereotaxic injection of a specific brain region constitutes a fundamental experimental technique in basic neuroscience. Researchers commonly base their ...

Stereotaxic Surgical Approach to Microinject the Caudal Brainstem and Upper Cervical Spinal Cord via the Cisterna Magna in Mice

Summary

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Chapters in this video

Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

Survivable Stereotaxic Surgery in Rodents

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

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Surgical Transplantation of Tumor Cells into the Spinal Cord of Mice

Cannula Implantation into the Cisterna Magna of Rodents

Induction of Leptomeningeal Cells Modification Via Intracisternal Injection

Stereotaxic Viral Injection and Gradient-Index Lens Implantation for Deep Brain In Vivo Calcium Imaging

A Visual Approach for Inducing Dolichoectasia in Mice to Model Large Vessel-Mediated Cerebrovascular Dysfunction

Preliminary Validation of Stereotaxic Injection Coordinates via Cryosectioning