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 ...

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Respiratory-related+outputs+of+glutamatergic,+hypercapnia-responsive+parabrachial+neurons+in+mice.">Respiratory-related outputs of glutamatergic, hypercapnia-responsive parabrachial neurons in mice.</a> Journal of Comparative Neurology. 523 (6), 907-920 (2015).</li><li>Anselmi, C., 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=Ultrasonographic+anatomy+of+the+atlanto-occipital+region+and+ultrasound-guided+cerebrospinal+fluid+collection+in+rabbits+(Oryctolagus+cuniculus).">Ultrasonographic anatomy of the atlanto-occipital region and ultrasound-guided cerebrospinal fluid collection in rabbits (Oryctolagus cuniculus).</a> Veterinary Radiology &amp; Ultrasound. 59 (2), 188-197 (2018).</li><li>Herbert, H., Moga, M. M., Saper, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connections+of+the+parabrachial+nucleus+with+the+nucleus+of+the+solitary+tract+and+the+medullary+reticular+formation+in+the+rat.">Connections of the parabrachial nucleus with the nucleus of the solitary tract and the medullary reticular formation in the rat.</a> The Journal of Comparative Neurology. 293 (4), 540-580 (1990).</li><li>Vanderhorst, V. G., Holstege, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caudal+medullary+pathways+to+lumbosacral+motoneuronal+cell+groups+in+the+cat:+evidence+for+direct+projections+possibly+representing+the+final+common+pathway+for+lordosis.">Caudal medullary pathways to lumbosacral motoneuronal cell groups in the cat: evidence for direct projections possibly representing the final common pathway for lordosis.</a> The Journal of Comparative Neurology. 359 (3), 457-475 (1995).</li><li>Vanderhorst, V. G., Terasawa, E., Ralston, H. J., Holstege, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monosynaptic+projections+from+the+nucleus+retroambiguus+to+motoneurons+supplying+the+abdominal+wall,+axial,+hindlimb,+and+pelvic+floor+muscles+in+the+female+rhesus+monkey.">Monosynaptic projections from the nucleus retroambiguus to motoneurons supplying the abdominal wall, axial, hindlimb, and pelvic floor muscles in the female rhesus monkey.</a> The Journal of Comparative Neurology. 424 (2), 233-250 (2000).</li><li>Wall, N. R., Wickersham, I. R., Cetin, A., De La Parra, M., Callaway, E. 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J., 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=Rapid,+reversible+activation+of+AgRP+neurons+drives+feeding+behavior+in+mice.">Rapid, reversible activation of AgRP neurons drives feeding behavior in mice.</a> The Journal of Clinical Investigation. 121 (4), 1424-1428 (2011).</li><li>Ganchrow, D., 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=Nucleus+of+the+solitary+tract+in+the+C57BL/6J+mouse:+Subnuclear+parcellation,+chorda+tympani+nerve+projections,+and+brainstem+connections.">Nucleus of the solitary tract in the C57BL/6J mouse: Subnuclear parcellation, chorda tympani nerve projections, and brainstem connections.</a> The Journal of Comparative Neurology. 522 (7), 1565-1596 (2014).</li><li>Ung, K., Arenkiel, B. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#38; 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>&#160;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 &#39;U&#39; 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 &#38; 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. ...

This protocol provides step-by-step guidelines for a reproducible route of access to regions of interest in the caudal brainstem and the upper cervical cord. This technique increases precision when delivering small injection volumes to restricted regions of interest in the caudal brainstem and upper cervical cord. This technique has been and can be applied to other animal models.Dr.Veronique VanderHorst, an associate professor of neurology and principal investigator of the VanderHorst Laboratory, will demonstrate this procedure. Make sure that the oxygen flow is directed to the nose cone. Move the mouse to the stereotaxic frame and place the nose in a flexible nose cone.Place the mouse in the stereotaxic frame using ear bars only. Place lubricant on the eyes. Anteroflex the mouse's head to a 90 degree 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 with the flat part of the skull serving as the reference. Place the heating pad underneath the mouse and make sure that the neck and the rest of the body are positioned parallel to the table by elevating the body with a small box. Place a drape underneath the body.Inject a single dose of four milligrams per kilogram Meloxicam slow release subcutaneously at a volume of two microliters per gram of body weight. Clean the surgical incision site first with a 70%alcohol prep pad, then with a Betaine prep pad, and then again with an alcohol prep pad and let it dry. Disinfect hands and put on sterile gloves, then place a drape at the surgical site.Ensure that the mouse is appropriately anesthetized by pinching the toes or checking the corneal reflex. Reduce isoflurane to maintain the levels at 2.0. Make a one to 1.2 centimeter incision with a surgical 10 blade from the edge of the occipital bone toward the shoulders in one smooth movement.Carefully make an incision in the midline raphe of the trapezius muscle, exposing the paired longus capitis muscles. Place both retractor hooks between the paired longus capitis muscles, one oriented to the left and the other to the right. 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. Reposition the retractors and adjust the tension by repositioning the hemostats. Use the blunt laminectomy forceps to separate the muscles further in the midline to obtain a good view of the brainstem and cerebellum.Repeat the above procedure as needed until the cerebellum and brainstem appear below the dura. 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, creating more lateral space. View the dorsal surface of the brainstem with detailed landmarks through the open dura.Use the angled forceps to grab the dura extending from the occipital bone to the atlas, then use the spring scissors to make a small opening of approximately 0.5 to 1.5 millimeters in the dura. Once the dura is opened, drain excess cerebrospinal fluid with a sterile Q-tip. The obex, the point where the central canal opens into the fourth ventricle, is the standard anterior-posterior and mediolateral zero point.Position the pipette or syringe to the target using the stereotaxic arm. Lower the arm of the dorsal onto the dorsal surface which forms from the dorsoventral zero point. Then lower the pipette onto the brainstem and inject the solution.Leave the needle in place for one to five minutes after injection to avoid a needle track when using volumes between three to 50 nanoliters. Then lift the pipette or syringe using the stereotaxic arm and repeat this for multiple targets. 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. Close the skin with 3 nylon or polypropylene sutures.The cisterna magna approach makes it possible to target the caudal brainstem and upper cervical cord structures that are otherwise hard to reach via standard stereotaxic approaches or are prone to inconsistent targeting. In mice, structures such as the hypoglossal nucleus, ventral respiratory group and adjacent reticular formation in the caudal brainstem have been routinely targeted using the cisterna magna approach as illustrated here for the hypoglossal nucleus and the ventromedial medulla. In order to determine the accuracy of the cisterna magna approach versus the standard approach, the distance between the intended and actual target sites in the anteroposterior, mediolateral, and dorsoventral planes for ventral and dorsal regions was measured.The results show significantly smaller errors in the anteroposterior, mediolateral, and dorsoventral planes compared to the standard approach, highlighting the enhanced accuracy of the cisterna magna approach for these targets. When attempting this protocol, it is critical to make sure that the anteroflexion of the head and the elevation of the body is performed as described. Next, it is important to recognize key landmarks before manipulating the muscle or the dura mater.If these landmarks are not recognized or if they're lost, it will be challenging to stay oriented and execute the procedure as intended. This technique helped address conceptual questions related to the functional anatomical organization within the caudal brainstem and upper cervical cord.

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

Research

JoVE Journal

Neuroscience

8.2K Views.  Harvard Medical School. This protocol provides step-by-step guidelines for a reproducible route of access to regions of interest in the caudal brainstem and the upper cervical cord. This technique increases precision when delivering small injection volumes to restricted regions of interest in the caudal brainstem and upper cervical cord. This technique has been and can be applied to other animal models.Dr.Veronique VanderHorst, an associate professor of neurology and principal investigator of the VanderHorst ...