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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
The study was performed in six three-month-old male and female pigs (German Landrace) weighing approximately 40 kg. Animals were brought to the animal care facilities at least 7 days prior to the experiments and were housed in accordance to animal welfare recommendations. Animals were provided food and water ad libitum, and their health status was regularly assessed by the responsible veterinarian. A fasting time of 12 h was maintained prior to the experiments. The entire experimental procedure and handling of the animals was supervised by the responsible veterinarian.
1. Anesthesia induction and maintenance of anesthesia
<ol>
	<li>For anesthesia induction and maintenance of anesthesia, premedicate the animals and deeply sedate them using an intramuscular injection followed by intravenous injections, if necessary, to perform endotracheal intubation. Thereafter, induce and maintain anesthesia by using a combination of a volatile anesthesia agent with a continuous opioid application complemented with an additional opioid bolus injection.</li>
	<li>Perform intramuscular injections of ketamine 20 mg&#183;kg-1, azaperone 4 mg&#183;kg-1, and midazolam 0.1 mg&#183;kg-1&#160;for premedication and sedation.</li>
</ol>
2. Probe placement
<ol>
	<li>Place the animal in the right lateral position and flex the animal&#39;s back to widen the space between the vertebrae.</li>
	<li>For percutaneous placement of cerebrospinal fluid drainage for measuring and controlling cerebrospinal pressure, identify the level of L 4/5 or L 5/6, puncture the skin and the subcutaneous space with the introducer needle, and remove the inlay needle.</li>
	<li>Place a saline-filled syringe on the needle, and carefully introduce the needle with constant pressure on the fluid-filled syringe.</li>
	<li>Once a loss of resistance is felt as evidence for epidural position, re-introduce the inlay needle, and introduce the needle 2-3 mm further to puncture the dura mater and remove the inlay needle.</li>
	<li>Verify intrathecal position by fast dripping of clear liquor. Introduce the drainage up to 20 cm depth, attach the Luer-lock adapter, and verify the position by careful aspiration of liquor.</li>
	<li>Carefully fix the drainage with sutures and connect it to the cerebrospinal fluid drainage system.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>LiquoGuard 7</td>
			<td>&#160;M&#246;ller Medical GmbH, Fulda, Germany</td>
			<td></td>
			<td>Cerebrospinal Fluid Drainage System</td>
		</tr>
		<tr>
			<td>Twinwarm</td>
			<td>Moeck &#38; Moeck GmbH, Hamburg, Germany</td>
			<td>12TW921DE</td>
			<td>Warming System</td>
		</tr>
		<tr>
			<td>Universal II Warming Blanket</td>
			<td>Moeck &#38; Moeck GmbH, Hamburg, Germany&#160;</td>
			<td>906</td>
			<td>Warming Blanket</td>
		</tr>
		<tr>
			<td>Zeus Dr&#228;ger Medical,&#160;</td>
			<td>&#160;L&#252;beck, Germany</td>
			<td></td>
			<td>Anesthesia Machine</td>
		</tr>
	</tbody>
</table>

intrathecal placement of lumbar drain: a technique to withdraw cerebrospinal fluid from the lumbar spine in porcine model

Source: Behem, C. R. et al., <a href="https://www.jove.com/v/62047">Real-Time Assessment of Spinal Cord Microperfusion in a Porcine Model of Ischemia/Reperfusion.</a> J. Vis. Exp. (2020).
This video describes the technique of intrathecal placement of a lumbar drain in a porcine ischemic model, which helps in draining the cerebrospinal fluid to a drainage system. The cerebrospinal fluid pressure is monitored by the drainage system, and fluid is drained to maintain a constant cerebrospinal fluid pressure.

Intrathecal Placement of Lumbar Drain: A Technique to Withdraw Cerebrospinal Fluid from the Lumbar Spine in Porcine Model

Source: Behem, C. R. et al., Real-Time Assessment of Spinal Cord Microperfusion in a Porcine Model of Ischemia/Reperfusion. J. Vis. Exp. (2020).
This ...

A lumbar puncture is performed by injecting a specialized needle into the spinal column&#39;s intrathecal space in the lower back region to withdraw a cerebrospinal fluid, or CSF, sample. To begin, place an anesthetized pig in a right lateral position on a surgical platform. Ensure the spine is flexed, which increases the gaps between the vertebrae.
Take a needle assembly consisting of an inlay needle fitted inside an introducer needle. Locate the space between the fourth and fifth lumbar vertebrae. Pierce the needle through the skin layers and deeper tissues.&#160;
Remove the inlay needle and connect a saline-filled syringe to the introducer needle. Push the needle inward while injecting the saline into the spinal column. This step facilitates an easy passage of the needle into the epidural space located around the spinal cord.&#160;
Replace the syringe with an inlay needle. Advance the needle to puncture the dura mater - the tissue layer covering the spinal cord. Remove the inlay needle and introduce a lumbar drain tube into the intrathecal space.
Aspirate a small amount of CSF sample to confirm the appropriate positioning of the tube and remove the needle. Suture the drain tube in its position and connect it to a CSF drainage system while monitoring its pressure.

intrathecal-placement-lumbar-drain-technique-to-withdraw

Research

JoVE Encyclopedia of Experiments

Biology

Large Animal Models

Porcine

1.5K Views. Source: Behem, C. R. et al., Real-Time Assessment of Spinal Cord Microperfusion in a Porcine Model of Ischemia/Reperfusion. J. Vis. Exp. (2020). This video describes the technique of intrathecal placement of a lumbar drain in a porcine ischemic model, which helps in draining the cerebrospinal fluid to a drainage system. The cerebrospinal fluid pressure is monitored by the drainage system, and fluid is drained to maintain a constant cerebrospinal fluid pressure. 

Video: Intrathecal Placement of Lumbar Drain: A Technique to Withdraw Cerebrospinal Fluid from the Lumbar Spine in Porcine Model - Concept

Isolation of Cerebrospinal Fluid from Rodent Embryos for use with Dissected Cerebral Cortical Explants

The CSF is a complex fluid with a dynamically varying proteome throughout development and in adulthood. During embryonic development, the nascent CSF differentiates from the amniotic fluid upon closure of the anterior neural tube. CSF volume then increases over subsequent days as the neuroepithelial progenitor cells lining the ventricles and the choroid plexus generate CSF. The embryonic CSF contacts the apical, ventricular surface of the neural stem cells of the developing brain and spinal cord. CSF provides crucial fluid pressure for the expansion of the developing brain and distributes important growth promoting factors to neural progenitor cells in a temporally-specific manner. To investigate the function of the CSF, it is important to isolate pure samples of embryonic CSF without contamination from blood or the developing telencephalic tissue. Here, we describe a technique to isolate relatively pure samples of ventricular embryonic CSF that can be used for a wide range of experimental assays including mass spectrometry, protein electrophoresis, and cell and primary explant culture. We demonstrate how to dissect and culture cortical explants on porous polycarbonate membranes in order to grow developing cortical tissue with reduced volumes of media or CSF. With this method, experiments can be performed using CSF from varying ages or conditions to investigate the biological activity of the CSF proteome on target cells.

The ventricular cerebrospinal fluid (CSF) bathes the neuroepithelial and cerebral cortical progenitor cells during early brain development in the embryo. Here we describe the method developed to isolate ventricular CSF from rodent embryos of different ages in order to investigate its biological function. In addition, we demonstrate our cerebral cortical explant dissection and culture technique that allows for explant growth with minimal volumes of culture medium or CSF.

The CSF is a complex fluid with a dynamically varying proteome throughout development and in adulthood. During embryonic development, the nascent CSF ...

JoVE Journal

Neuroscience

An Improved Method for Collection of Cerebrospinal Fluid from Anesthetized Mice

The cerebrospinal fluid (CSF) is a valuable body fluid for analysis in neuroscience research. It is one of the fluids in closest contact with the central nervous system and thus, can be used to analyze the diseased state of the brain or spinal cord without directly accessing these tissues. However, in mice it is difficult to obtain from the cisterna magna due to its closeness to blood vessels, which often contaminate samples. The area for CSF collection in mice is also difficult to dissect to and often only small samples are obtained (maximum of 5-7 &#181;L or less). This protocol describes in detail a technique that improves on current methods of collection to minimize contamination from blood and allow for the abundant collection of CSF (on average 10-15 &#181;L can be collected). This technique can be used with other dissection methods for tissue collection from mice, as it does not impact any tissues during CSF extraction. Thus, the brain and spinal cord are not affected with this technique and remain intact. With greater CSF sample collection and purity, more analyses can be used with this fluid to further aid neuroscience research and better understand diseases affecting the brain and spinal cord.

This protocol describes an improved technique for the abundant collection of cerebrospinal fluid (CSF) with no contamination from blood. With greater sample collection and purity, more analyses can be performed using CSF to further our understanding of diseases that affect the brain and spinal cord.

The cerebrospinal fluid (CSF) is a valuable body fluid for analysis in neuroscience research. It is one of the fluids in closest contact with the central ...

Direct Cannula Implantation in the Cisterna Magna of Pigs

The glymphatic system is a waste clearance system in the brain that relies on the flow of cerebrospinal fluid (CSF) in astrocyte-bound perivascular spaces and has been implicated in the clearance of neurotoxic peptides such as amyloid-beta. Impaired glymphatic function exacerbates disease pathology in animal models of neurodegenerative diseases, such as Alzheimer's, which highlights the importance of understanding this clearance system. The glymphatic system is often studied by cisterna magna cannulations (CMc), where tracers are delivered directly into the cerebrospinal fluid (CSF). Most studies, however, have been carried out in rodents. Here, we demonstrate an adaptation of the CMc technique in pigs. Using CMc in pigs, the glymphatic system can be studied at a high optical resolution in gyrencephalic brains and in doing so bridges the knowledge gap between rodent and human glymphatics.

This article presents a step-by-step protocol for the direct cannula implantation in the cisterna magna of pigs.

The glymphatic system is a waste clearance system in the brain that relies on the flow of cerebrospinal fluid (CSF) in astrocyte-bound perivascular spaces ...

Intrathecal Placement of Lumbar Drain: A Technique to Withdraw Cerebrospinal Fluid from the Lumbar Spine in Porcine Model

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